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WO2025003463A1 - Polyproline-based block copolymers - Google Patents

Polyproline-based block copolymers Download PDF

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Publication number
WO2025003463A1
WO2025003463A1 PCT/EP2024/068352 EP2024068352W WO2025003463A1 WO 2025003463 A1 WO2025003463 A1 WO 2025003463A1 EP 2024068352 W EP2024068352 W EP 2024068352W WO 2025003463 A1 WO2025003463 A1 WO 2025003463A1
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WO2025003463A9 (en
Inventor
María Jesús VICENT DOCÓN
Inmaculada CONEJOS SÁNCHEZ
Camilla PEGORARO
Ekaterina KARPOVA
Gloria SOGORB
Carles FELIP LEON
Vicent Josep Nebot Carda
Petra Schwille
Yusuf QUTBUDDIN
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
Polypeptide Therapeutic Solutions SL
Fundacion de la Comunidad Valenciana Centro de Investigacion Principe Felipe
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
Polypeptide Therapeutic Solutions SL
Fundacion de la Comunidad Valenciana Centro de Investigacion Principe Felipe
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Publication of WO2025003463A1 publication Critical patent/WO2025003463A1/en
Publication of WO2025003463A9 publication Critical patent/WO2025003463A9/en
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers

Definitions

  • This invention relates to polyproline-based block copolymers which comprise a polyproline moiety and a polycationic moiety. These polyproline-based copolymers can be used as non-viral vectors for delivery of active ingredients, including nucleic acids, and/or imaging agents to target cells or tissues.
  • Document W02023002012 discloses 3-arm star-shaped polycationically charged polymers consisting of a 1,3,5-benzenetricarboxamide related central core and 3 polypeptide backbone arms.
  • a polyproline-based block copolymer of formula (I) or a compound comprising a structural unit of formula (I') or (I”), or a salt thereof as defined herein are useful as a chemical-mediated vector for the delivery of molecules of interest, such as active ingredients or detection moieties. More particularly, the polyproline-based block copolymer of formula (I) or the compound comprising a structural unit of formula (I') or (I”), or a salt thereof as defined herein may act as cell penetrating peptide for the cellular delivery of attached cargoes.
  • polyproline-based block copolymer compound of formula (I) or the compound comprising a structural unit of formula (I') or (I”) of the present invention may allow the simultaneous delivery of several molecules of interest conjugated to the polymer, since the backbone of the polyproline-based block copolymer compound of the invention can be modified by including in the structural repeating units one or more active ingredients (such as pharmaceutically, veterinary, and cosmetic active ingredients), and/or detection agents (such as chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, transition metal and isotope mass tag moiety). Therefore, the polyproline-based block copolymer compounds herein defined may be used in lab research, therapy, cosmetic or diagnostic circumstances.
  • active ingredients such as pharmaceutically, veterinary, and cosmetic active ingredients
  • detection agents such as chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, transition
  • the polyproline-based block copolymer compound of the present invention is capable of successfully being internalized and colocalized in mitochondria of mammalian cell lines, including MDA-MB 231. In addition, it can be applied to many types of unicellular organisms, and to multicellular organisms.
  • a first aspect of the invention relates to a polyproline-based block copolymer compound of formula (I), a salt thereof, or any stereoisomer or mixtures of stereoisomers, either of the compound of formula (I), or of any of its salts, wherein the “*” denotes the attaching points; where the repetitive unit defined by square brackets with the numerical value m is denoted as PAA1; wherein though the main repeating units AA1, AA2 and AA3 are shown in a particular order for convenience of description, said AA1, AA2 and AA3 main repeating units may be present in any order and may be block or randomly present; and wherein each of the repeating units PAA1, AA1, AA2 and AA3, may comprise blocks of monomer units which may be the same or different between each other; wherein m is an integer selected from 5 to 100; n is an integer selected from 0 to 150; p is an integer selected from 0 to 150; q is an integer selected from 0 to
  • Li if present, is a biradical selected from the group consisting of wherein the wavy lines denote the attaching points; wherein each A and A' is independently selected from -O-, -CO- and -NH-; wherein y and z are integers independently ranging from 1 to 20; and wherein each Z is a biradical selected from the group consisting of -NH(Ci-C6)alkyl-O-,
  • L1 is attached to the AA1 , AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond when attached to the N-terminal end of AA1 , AA2 or AA3 repeating unit; or alternatively L1 is attached to the AA1 , AA3 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond when is attached to the C-terminal end of the AA1 , AA2 or AA3 repeating unit; wherein R 2 and R4, are each a biradical independently selected from the group consisting of -(Ci-C 6 )alkyl-, -(Ci-C 6 )alkyl-S-S-(Ci-C 6 )alkyl-, -(Ci-C 6 )alkyl-O-(Ci-C
  • R 2 and R4 are each independently optionally substituted by one or more substituents selected from the group consisting of -NH 2 and -(Ci-Ce)alkyl-NH 2 ; each R3 is a radical independently selected from the group consisting of H and -(Ci-Ce)alkyl; each W1 is independently selected from CH and N;
  • X2 in formula XVI is selected from -NH-, -COO-, and -O-; each R' 2 is selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -OAIkyl(Ci-C6), halogen, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO 2 , -CN, -OC(O)-(Ci-C 30 )alkyl, -OC(O)O(Ci-C 30 )alkyl, -
  • R' 2 , R'" 2 , R' v2 and R v2 are each independently selected from the group consisting of H, -(Ci-C3o)alky I, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl NH2, -(Ci-C3o)alkyl-N((Ci-C3o)alkyl)2, -(Ci-C3o)alkyl-NH(Ci-C3o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI)
  • R v ' 2 and R v ' 2 ’ are each independently selected from the group consisting of H, -(Ci-C3o)alky I, -(C 2 -C 3 o)alkenyl, -(C 2 -C 3 o)alkynyl, -OAIkyl(Ci-Ci 2 ), F, Cl, Br, I, -CF 3 , -OCF 3 , -NO 2 , -CN, -NH 2 , -(Ci-C 3 o)alkylNH 2 , -N((Ci-C 30 )alkyl) 2 , and-NH(Ci-C 30 )alkyl, wherein R vi ' 2 , RTM 2 , R ix2 , and R x2 are each independently selected from the group consisting of H, -(Ci-Ci 2 )alkyl, -(Ci-Ci 2 )alkylNH 2 , -(C
  • each R' 1 is independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2- C3o)alkynyl, -OAIkyl(Ci-C6), halogen, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO 2 , -CN, -OC(O)-(Ci-C 30 )alkyl, -OC(O)O(Ci-C 30 )alkyl, -OC(O)NH 2
  • R' 1 , ' i1 , R' v1 , R v1 and R vi1 are each independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl NH2, -(Ci-C3o)alkyl-N((Ci-C3o)alkyl)2, -(Ci-C3o)alkyl-NH(Ci-C3o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV),
  • T and T are each independently selected from -OH, -OCORx and -COORx;
  • Q and Q' are each independently selected from -OCORy and -COORy; each G is independently selected from -OCO-, -COO-, -NRz'CO-, and -CONRz'-; each Rz' is H or Rz; each Rx, Ry, and Rz is independently -(Ci-Ci8)alkyl or -(C2-Ci8)alkenyl; each a1 is independently an integer from 0 to 18; preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15, 16, 17, or 18; each b1 is independently an integer from 1 to 18; preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15, 16, 17, or 18; each b1 is independently an integer from 1 to 18; preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15,
  • each d is independently an integer from 0 to 18; preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15,
  • each d1 is independently an integer from 0 to 18; preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15,
  • each e1 is independently an integer from 0 to 18; preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15,
  • T1 , T2, T3, TT, T2' and T3' are each independently selected from the group consisting of hydrogen, fluorine, methyl, -CH2F, -CHF2, and -CF3; each of the dashed bonds — is independently a single bond or, alternatively, a double bond; each J is a biradical chain which comprises one or more moieties selected from the group consisting of - )-, , , l, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkylphenyl, and -phenyl(Ci-C3o)alkyl; wherein when J comprises a -CH2- moiety, the two hydrogen atoms attached to the carbon atom are optionally replaced by the ring: , , , wherein R2, R3, R4, R5, Re, and W1 are as defined above.
  • a third aspect of the present invention relates to a star-shaped polymer comprising a starshaped multifunctional linking agent and at least one compound comprising the structural unit of formula (I') or (I”) as defined in accordance with the second aspect and/or embodiments thereof.
  • the star-shaped multifunctional linking agent (central core of the star-shaped polymer) may be any known multifunctional moiety suitable for the preparation of chemical-mediated vectors for the delivery of molecules of interest.
  • star-shaped multifunctional moieties are those derived from a multi-arm star-shaped linking agent based on branched polyethylenimine (PEI-based); a four-arm star-shaped linking agent based on branched polyamidoamine (PAMAM-based) of formula (Pm); and a four-arm star-shaped linking agent of formula (Tz ) (Trizma-based).
  • the present invention encompasses star-shaped polymers having all the arms of the star-shaped multifunctional linking agent substituted with the same polyproline-based block copolymer of formula (I') or (I”); but also, configurations wherein at least one arm of the central core is different from the others.
  • a fourth aspect of the present invention relates to a self-assembled particle comprising the compound of formula (I) as defined herein, the compound comprising the structural unit of formula (I') or (I”) as defined herein, or the star-shaped polymer as defined herein, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • composition comprising compound of formula (I) as defined herein, the compound comprising the structural unit of formula (I') or (I”), the star-shaped polymer or alternatively, the self-assembled particle, together with one or more appropriate excipients or carriers.
  • Another aspect of the present invention relates to a therapeutic product which comprises: a) the compound of formula (I) as defined herein, wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively b) the compound comprising the structural unit of formula (I') or (I”) as defined herein, wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively c) the star-shaped polymer as defined herein, wherein at least one of L4 or R8 comprise a pharmaceutically active agent;or alternatively d) the self-assembled particle as defined herein, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively e) a composition containing the self-assembled particle d) as defined herein; for use in medicine.
  • a therapeutic product for use (i) as transfection reagent for transfecting at least one active agent into a cell; (ii) for use in the in vivo or ex vivo production of biologies encoding a recombinant protein, a peptide or an antibody, or in the production of recombinant virus; (iii) for use as a therapeutic or prophylactic vaccine against viral infections or as a therapeutic vaccine against cancers or infectious diseases; or (v) for use in genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
  • a diagnostic product which comprises: a') the compound of formula (I) as defined herein, wherein at least one of L4 or R8 comprise a diagnostically active agent; or alternatively b') the compound comprising the structural unit of formula (I') or (I”) as defined herein, wherein at least one of L4 or R8 comprises a diagnostically active agent;or alternatively c') the star-shaped polymer as defined herein, wherein at least one of L4 or R8 comprise a diagnostically active agent ; or alternatively d') a self-assembled particle as defined herein, which comprises diagnostically active agent, or alternatively e') a composition containing the self-assembled particle d') as defined herein; for use in diagnostics.
  • a cosmetic product which comprises: a") the compound of formula (I) as defined herein, wherein at least one of L4 or R8 comprise a cosmetically active agent;or alternatively b”) the compound comprising the structural unit of formula (I') or (I”) as defined herein, wherein at least one of L4 or R8 comprises a cosmetically active agent;or alternatively c”) the star-shaped polymer as defined herein, wherein at least one of L4 or R8 comprise a cosmetically active agent;or alternatively d”) the self-assembled particle as defined herein, which comprises a cosmetically active agent, or alternatively e”) a composition containing the self-assembled particle d”) as defined herein comprising a cosmetically active agent.
  • the compound of formula (I) as defined herein, wherein L4 and R8 are other than an active moiety the compound comprising the structural unit of formula (I') or (I”) as defined herein, wherein at least one of L4 or R8 is other than an active moiety; the star-shaped polymer as defined herein, wherein L4 and R8 are other than an active moiety; or alternatively the selfassembled particle as defined herein, for use as a carrier.
  • a therapeutic product which comprises: a) the compound of formula (I) as defined herein, wherein at least one of L4 or R8 comprise a pharmaceutically active agent;or alternatively b) the compound comprising the structural unit of formula (I') or (I”) as defined herein, wherein at least one of L4 or R8 comprises a pharmaceutically active agent; or alternatively c) the star-shaped polymer as defined herein, wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively d) a self-assembled particle as defined herein, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively e) a composition containing the self-assembled particle d) as defined herein; for use in a method for delivering a pharmaceutical active agent (i.e.
  • a nucleic acid or a drug into a target cell, which comprises administering a composition (i.e. solution) that contains any of a), b), c), d) or e) to an animal, including human, so that a), b), c), d) or e) is getting into physical contact with the target cell and thereby delivers the pharmaceutical active agent into the cell.
  • a composition i.e. solution
  • an animal including human
  • FIG. 1 Cardiolipin (CL)-based Supported Lipid Bilayers examined using a Zeiss Elyra 7 total internal reflection fluorescence microscope (TIRFM). The samples were observed both with and without incubation with compound 1a.
  • TIRFM total internal reflection fluorescence microscope
  • FIG. 2 Quantitative analysis of 11a interaction with POPC/CL and POPC/POPG (control) based Small Unilamellar Vescicles (SUVs) by Fluorescence (Cross) Correlation Spectroscopy (FCCS).
  • SUVs Small Unilamellar Vescicles
  • FCCS Fluorescence Correlation Spectroscopy
  • moiety refers to a specific segment or functional group of a molecule or compound.
  • the term "subject" refers to any mammal, including both human and other mammals.
  • compositions of formula (I) or (I') of the invention can be carried out by methods well-known in the art. Generally, such salts can be prepared by reacting the free acid or base form of a compound of formula (I) or (I') of the invention with a stoichiometric amount of an appropriate base or acid, respectively, in a suitable solvent such as water, an organic solvent or a mixture of them.
  • the salts of the repeating units of monomer PAA1, AA1, AA2 and AA3 encompasses acid addition salts formed from acceptable non-toxic acids including inorganic or organic acids, and base addition salts.
  • acceptable non-toxic acids including inorganic or organic acids, and base addition salts.
  • the salts There is no limitation regarding the salts, except that they must be therapeutically (pharmaceutically or veterinary), diagnostic or cosmetic acceptable when they are used for therapeutic (pharmaceutical or veterinary), diagnostic or cosmetic purposes, respectively.
  • Most of the acceptable salts are commercially available. If not, these salts can be prepared following the processes disclosed in the state of the art, which involves starting from acceptable non-toxic acids, including inorganic and organic acids.
  • Such acids include for instance inorganic acids e.g.
  • salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate acceptable base or acid in water or in an organic solvent or in a mixture of them.
  • the compounds of formula (I) or (I') of the invention and their salts may differ in some physical properties, but they are equivalent for the purposes of the present invention.
  • the term “pharmaceutically acceptable salts” used herein encompasses any salt formed from pharmaceutically acceptable non-toxic acids or bases as defined above.
  • veterinary acceptable salts used herein encompasses any salt formed from veterinary acceptable non-toxic acids or bases as defined above.
  • diagnostic acceptable salts encompasses any salt formed from diagnostic acceptable non-toxic acids or bases as defined above.
  • cosmetic acceptable salts used herein encompasses any salt formed from cosmetic acceptable non-toxic acids or bases as defined above.
  • alkyl refers to a saturated straight, or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims. Examples include, among others, the group methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl. As disclosed above, the alkenyl groups can be optionally substituted.
  • alkynyl refers to a straight or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims and having at least one carbon-carbon triple bond. Examples include, among others, ethynyl, 2-propynyl, and 3-hexynyl. As disclosed above, the alkynyl groups can be optionally substituted.
  • -(Ci-C x )alkyl refers to a saturated linear or branched hydrocarbon chain which contains from 1 to x carbon atoms and only single bonds.
  • alkyl groups may include without limitation methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, neopentyl, n-hexyl, decyl, undecyl, dodecyl, tetradecyl and hexadecyl.
  • alkenyl refers to an unsaturated branched or linear hydrocarbon chain which comprises from 2 to x carbon atoms and at least one or more double bonds.
  • alkenyl groups may include without limitation ethenyl (i.e. vinyl), allyl, propenyl, butenyl, pentenyl and hexenyl, dodecenyl, tetradecenyl and hexadecenyl.
  • alkoxy refers to an alkyl group as defined herein comprising 1 to 10 carbon atoms which is connected to the rest of the molecule via an oxygen atom.
  • alkoxy groups may include without limitation methoxy, ethoxy, 1 -propoxy, and 2-propoxy.
  • -(C5-Cio)aryl refers to an aromatic carbocyclic mono- or bicyclic ring system comprising 5 to 10 carbon ring atoms.
  • aryl groups may include without limitation phenyl, biphenyl, and naphthyl.
  • -(C6-Cio)aryloxy refers to an aryl group as defined herein comprising 6 to 10 carbon ring atoms, which is connected to the rest of the molecule via an oxygen atom.
  • aryloxy groups may include without limitation phenoxy and biphenyloxy.
  • -(C5-Cio)heteroaryl refers to an aromatic carbocyclic mono- or bicyclic ring system comprising 5 to 10 ring atoms, wherein at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, and S, the remaining ring atoms being carbon atoms.
  • heteroaryl groups may include without limitation pyridyl, azetidinyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl, isoquinolinyl, benzthiazolyl, benzimidazolyl, benzthiazolyl, quinolyl, and quinazolyl.
  • -(C6-Cio)aralkyl refers to a lower alkyl group as defined herein (e.g. -(Ci-Ce)alkyl) which is substituted by an aryl group (e.g. “-(Cs-C jaryl) as defined herein.
  • aralkyl groups may include without limitation benzyl, phenethyl, and methylbenzyl.
  • -(C6-Cio)aralkoxy refers to a lower alkoxy group as defined herein (e.g. -(Ci- Ce)alkoxy) which is substituted by an aryl group (e.g. “-(C5-Cio)aryl) as defined herein.
  • aralkoxy groups may include without limitation benzyloxy, phenethoxy, and methylbenzyloxy.
  • heteroaralkoxy refers to a lower alkoxy group as defined herein (e.g. -(Ci- Ce)alkoxy) which is substituted by an heteroaryl group (e.g. "-(C5-Cio)heteroaryl) as defined herein.
  • heteroaralkoxy groups may include without limitation furyloxy, pyridyloxy, and azetidinyloxy.
  • heterocycloalkyl refers to a 5-10 membered mono- or bicylic (fused or bridged) saturated o unsaturated ring structure, in which one or more of the ring atoms is a heteroatom selected from N, O, and S.
  • heterocycloalkyl groups may include without limitation include piperidyl, piperazyl, tetrahydropyranyl, tetrahydrofuryl, 1 ,4-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholyl.
  • alkenyl refers to a straight or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims and having at least one carbon-carbon double bond. Examples include, among others, ethenyl, 2-propenyl, and 3-hexenyl. As disclosed above, the alkenyl groups can be optionally substituted.
  • bivalent refers to a moiety that is bond to two other moieties.
  • -(Ci-Ci2)alkylene- refers to a bivalent saturated straight, or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims.
  • -(C2-Ci2)alky ny lene- refers to a bivalent straight or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims and having at least one carbon-carbon triple bond.
  • carrier ring refers to a known saturated, partially unsaturated; or aromatic ring system comprising one or more rings and having the number of carbon atoms specified in the description or claims, wherein all ring members are carbon atoms. As disclosed above, the ring members can be optionally substituted.
  • heterocyclic ring refers to a known saturated, partially unsaturated; or aromatic ring system comprising one or more rings and having the number of carbon atoms specified in the description or claims, wherein one or more of the ring members, preferably 1, 2, 3, or 4 ring members, are selected from NH, N, O, and S, and are chemically possible; and the remaining members of the ring are carbon atoms.
  • the ring may be attached to the rest of the molecule through the C or the N atom.
  • the ring members can be optionally substituted.
  • Non limiting examples of Cy 1 include, but not limited to, phenyl and naphthyl, furan-2-yl, furan- 3-yl, thiophen-2-yl, thiophen-2-yl, indol-2-yl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl, pyrazolyl, 2- pyridyl, 3-pyridyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, and triazolyl.
  • amine protecting group and “nitrogen protecting group” have the same meaning and are used interchangeable. They refer to a chemical compound which, when bound to an amino group of the polymer or of a starting material (reagent) for its preparation, prevents undesired reactions from occurring at this amino group and which can be removed by conventional chemical or enzymatic steps to re-establish the amino group.
  • amine protecting groups include carbamate protecting group including, without limitation, t-butyl carbamate, methyl carbamate, ethyl carbamate, 2,2,2-trichloroethyl carbamate, 2-(trimethylsilyl)ethyl carbamate, 1, 1 -dimethyl-2,2,2-trichloroethyl carbamate, benzyl carbamate, p-methoxybenzyl carbamate, p-nitrobenzylcarbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, and 2,4-dichlorobenzyl carbamate; -Fluorenylmethyl Carbamate (“Fmoc”), formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (“Cbz”), t-butoxycarbonyl (“BOO”), trimethylsilyl ("TMS”), 2- trimethylsilyl
  • alcohol protecting group and “oxygen protecting group” have the same meaning and are used interchangeable. They refer to a chemical compound which, when bound to an alcohol group of the polymer or of a starting material (reagent) for its preparation, prevents undesired reactions from occurring at this alcohol group and which can be removed by conventional chemical or enzymatic steps to re-establish the alcohol group.
  • alcohol protecting groups include 9- Fluorenylmethyl, methoxy methyl, methylthiomethyl, tetrahydrofuranyl, Methoxyethoxymethyl, 2- (Trimethylsilyl)ethoxymethyl, Benzyloxymethyl, Phenylacetoxymethyl,
  • substituted means that one or more hydrogen atoms on the designated atom or group are replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded. Combinations of substituents and/or variables are permissible.
  • optionally substituted means that the number of substituents can be equal to or different from zero. Unless otherwise indicated, it is possible that optionally substituted groups are substituted with as many optional substituents as can be accommodated by replacing a hydrogen atom with a non-hydrogen substituent on any available carbon or nitrogen atom. It is possible that groups in the conjugates according to the invention are substituted with one, two, three, four or five identical or different substituents, particularly with one, two or three substituents.
  • detection moiety refers to a moiety possessing a property or function which can be used for detection purposes. This term encompasses moieties selected from the group consisting of chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, and transition metal isotope mass tag moiety.
  • chromophore or “chromophore moiety” refers to a moiety that exhibits a detectable absorption of light such as for example (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) (BOPIPY).
  • Suitable fluorescent moieties are those known from the art of immunofluorescence technologies, e.g., flow cytometry or fluorescence microscopy, wherein, the compound labelled with this detection moiety is detected by exciting the detection moiety and detecting the resulting emission (photoluminescence).
  • Useful fluorescent moieties for the present invention include protein-based, such as phycobiliproteins, polymeric, such as polyfluorenes, small organic molecule dyes, such as xanthene, like fluorescein, or rhodamines, cyanine, oxazines, coumarins, acridines, oxadiazoles, pyrenes, pyrromethene, or metallo- organic complexes, such as Ru, Eu, Pt complexes.
  • Phosphorescent moieties include metallo- organic complexes, such as Pd, Pt, Tb, Eu complexes, or nanoparticles with incorporated phosphorescent pigments such as lanthanide doped SrAI2O4.
  • detection moiety is a radioactive label, wherein the compound labelled with this detection moiety is detected without prior excitation by irradiation. They can be in the form of radioisotope labelling by exchanging nonradioactive isotopes for their radioactive counterparts, such as tritium, 32P, 35S or 14C, or introducing covalently bound labels, such as 1251, which is bound to tyrosine, 18F within fluorodeoxyglucose, or metallo- organic complexes, i.e. 99Tc-DTPA.
  • Other group is a detection moiety capable of causing chemiluminescence, i.e. horseradish peroxidase label in the presence of luminol.
  • the labelled compound is detected by absorption of UV, visible light, or NIR radiation.
  • Suitable light-absorbing detection moieties are light absorbing dyes without fluorescence emission, such as small organic molecule quencher dyes like N-aryl rhodamines, azo dyes, and stilbenes.
  • Other detection moiety appropriate for the present invention are light-absorbing detection capable of generating a photoacoustic signal after irradiation by pulsed laser light.
  • the labelled compound is detected by mass spectrometric detection of a transition metal isotope.
  • Known in the art are isotope tags of lanthanides and adjacent late transition elements.
  • the term “active moiety” is a moiety possessing therapeutically (pharmaceutical or veterinary) or cosmetic activity. This term encompasses moieties selected from the group consisting of a pharmaceutically active agent, a veterinary active agent, and a cosmetic active agent.
  • active agent refers to any chemical compound or substance that has activity in the pharmaceutical, veterinary or cosmetic field.
  • pharmaceutically active agent refers to any substance or combination of substances used in a finished pharmaceutical product, intended to furnish pharmacological activity or to otherwise have direct effect in the cure, mitigation, treatment, or prevention of disease, or to have direct effect in restoring, correcting, or modifying physiological functions in human beings.
  • pharmaceutically active agents include low molecular weight drugs, peptides, antibodies, hormones, enzymes, nucleic acids, proteins, and combinations thereof.
  • nucleic acid refers to DNA or RNA.
  • the nucleic acid is an DNA/RNA hybrid, a short interfering RNA (siRNA), a microRNA (miRNA), a single guide RNA (sgRNA), a donorDNA, a self-amplyfing/replicating RNA, a circularRNA (oRNA), a plasmid DNA (pDNA), a closed- linear DNA (clDNA), a short hairpin RNA (shRNA), messenger RNA (mRNA), and antisense RNA (aRNA), a CRISPR guide RNA, an antisense nucleic acid, a decoy nucleic acid, an aptamer, and a ribozyme to name a few, and encompasses both the nucleotide sequence and any structural embodiments thereof, such as double stranded, single stranded, helical
  • the nucleic acid may be a poly- or oligonucleotide, such as oligo- or poly-double stranded RNA, oligo- or poly-double stranded DNA, oligo- or poly-single stranded RNA, oligo- or poly-single stranded DNA.
  • Each of the nucleotides contained in the nucleic acid may be a naturally occurring nucleotide or a chemically-modified, non-naturally occurring nucleotide.
  • the strand length of the nucleic acid is not particularly limited and the nucleic acid may have a short strand ranging from 10 to 200 bases, preferably from 20 to 180 bases, preferably from 25 to 100 bases, preferably from 30 to 50 bases; or the nucleic acid may have a relatively long strand of from 200 to 20000 bases, more preferably of from 250 to about 15000 bases.
  • the polynucleotide may be of any sequence.
  • the polynucleotide encodes a protein or peptide.
  • the encoded proteins may be enzymes, structural proteins, receptors, soluble receptors, ion channels, pharmaceutically active proteins, cytokines, interleukins, antibodies, antibody fragments, antigens, coagulation factors, albumin, growth factors, hormones, insulin, etc.
  • the polynucleotide may also comprise regulatory regions to control the expression of a gene. These regulatory regions may include, but are not limited to, promoters, enhancer elements, repressor elements, TATA box, ribosomal binding sites, stop site for transcription, etc.
  • the polynucleotide is not intended to encode a protein.
  • the polynucleotide may be used to fix an error in the genome of the cell being transfected.
  • the polynucleotide to be delivered comprises a sequence encoding an antigenic peptide or protein.
  • Nanoparticles containing these polynucleotides can be delivered to an individual to induce an immunologic response sufficient to decrease the chance of a subsequent infection and/or lessen the symptoms associated with such an infection.
  • the nucleic acid is closed-linear DNA (clDNA), i.e. molecules wherein the double stranded region is flanked and protected by two single stranded loops thereby generating dumbbell-shaped molecules.
  • clDNA closed-linear DNA
  • the clDNA consists of a stem region comprising a double stranded DNA sequence of interest covalently closed at both ends by hairpin loops, the clDNA comprising at least two modified nucleotides.
  • clDNA refers to a single stranded covalently closed DNA molecule that forms a “dumbbell” or “doggy-bone” shaped structure under conditions allowing nucleotide hybridization. Therefore, although the clDNA is formed by a single stranded DNA molecule, the formation of the "dumbbell” structure by the hybridization of two complementary sequences within the same molecule generates a structure consisting on a double-stranded middle segment flanked by two single-stranded loops. The skilled in the art know how to generate clDNA from open or closed double stranded DNA using routine molecular biology techniques.
  • a clDNA can be generated by attaching hairpin DNA adaptors, for instance, by the action of a ligase, to both ends of an open double stranded DNA.
  • Hairpin DNA adaptor refers to a single stranded DNA that forms a stem-loop structure by the hybridization of two complementary sequences, wherein the stem region formed is closed at one end by a single stranded loop and is open at the other end.
  • a "modified nucleotide” is any nucleotide (e.g., adenosine, guanosine, cytidine, uracil, and thymidine) that has been chemically modified -by modification of the base, the sugar or the phosphate group- or that incorporates a non-natural moiety in its structure.
  • the modified nucleotide may be naturally or non-naturally occurring depending on the modification.
  • peptide refers to molecules that comprise two or more consecutive amino acids linked to one another via peptide bonds.
  • peptide includes oligopeptides and polypeptides.
  • protein refers to large peptides, in particular peptides having at least about 50 amino acids.
  • proteins of interest include, without limitation cytokines, interleukins, tumor necrosis factor (TNF), interferons, integrins, chimeric antigen receptors (CARs), antibodies, hormones, growth factors, enzymes), collagen, fibrinogen, elastin, tubulin, thrombin, serum albumin, erythropoietin, granulocyte colony stimulating factor (G-CSF), colony stimulating factor (CSF), and the like.
  • TNF tumor necrosis factor
  • CARs chimeric antigen receptors
  • proteins of interest include, without limitation cytokines, interleukins, tumor necrosis factor (TNF), interferons, integrins, chimeric antigen receptors (CARs), antibodies, hormones, growth factors, enzymes), collagen, fibrinogen, elastin, tubulin, thrombin, serum albumin, erythropoietin, granulocyte colony stimulating factor (G-CSF), colony stimulating factor
  • veterinary active agent refers to any substance or combination of substances used in a finished veterinary product, intended to furnish pharmacological activity or to otherwise have direct effect in the cure, mitigation, treatment, or prevention of disease, or to have direct effect in restoring, correcting, or modifying physiological functions in animals.
  • cosmetic active agent refers to any substance or combination of substances used in a finished cosmetic product, intended to improve its appearance or to beautify, preserve, condition, cleanse, color or protect the skin, nails, or hair without non-medical application.
  • the term "cell-targeting agent” refers to any molecule, macromolecule, or biomacromolecule, displaying affinity for a molecule present in the human or animal body, which is able to direct the conjugates or the self-assembled particles thereof by directing them towards the target site for therapeutic treatment since e.g., it selectively binds to receptors that are expressed or overexpressed on specific cell types.
  • Cell-targeting groups are well known in the art. The term therefore includes ligands for specific receptors or antigens, such as antibodies for a specific antigen, folic acid for its receptor or sugars such as galactose for its hepatic receptors.
  • the targeting agent may be attached to the polyproline-based copolymer backbone.
  • targeting agents include a peptide, a protein, an enzyme, a nucleic acid, a fatty acid, a hormone, an antibody, a carbohydrate, mono-, oligo- or polysaccharides, a peptidoglycan, a glycopeptide, or the like.
  • a peptide a protein
  • an enzyme a nucleic acid
  • a fatty acid a hormone
  • an antibody a carbohydrate, mono-, oligo- or polysaccharides
  • a peptidoglycan a glycopeptide, or the like.
  • any of a number of different materials that bind to antigens on the surfaces of target cells can be employed.
  • Antibodies to target cell surface antigens will generally exhibit the necessary specificity for the target.
  • suitable immunoreactive fragments can also be employed, such as the Fab, Fab', F(ab')2 or scFv fragments or single-domain antibodies (e.g. camelids VHH fragments).
  • Fab, Fab', F(ab')2 or scFv fragments or single-domain antibodies e.g. camelids VHH fragments.
  • ligands for any receptors on the surface of the target cells can suitably be employed as targeting agent. These include any small molecule or biomolecule, natural or synthetic, which binds specifically to a cell surface receptor, protein or glycoprotein found at the surface of the desired target cell.
  • cell-targeting groups include, but are not limited to, galactosamine, folate, a Her- 2 binding peptide, TLR agonists, • -D-Glucose, Asn-Gly-Arg peptide, angiopep2, folic acid, aptamers (A- 9, A10, Anti-gp120, TTA1, sgc8, Anti MUC-1, AS1411), primaquine, zidovudine, superoxide dismutase, prednisolone, platinum, cisplatin, sulphamethoxazole, amoxicillin, etoposide, mesalzine, doxorubicin, paclitaxel, 5-amino salicylic acid, denosumab, docetaxel, calcitonin, proanthocyanidin, methotrexate, camptothecin, galactose, glycyrrhetinic acid, lactose, hyalur
  • RCPLSHSLICY laminin receptor binding peptide
  • laminin receptor binding peptide e.g. YIGSR
  • a skin homing peptide a retina homing peptide, a pancreas homing peptide, a liver homing peptide, a lymph node homing peptide, an adrenal gland homing peptide, a thyroid homing peptide, a bladder homing peptide, a breast homing peptide, a neuroblastoma homing peptide, a lymphoma homing peptide, a muscle homing peptide, a wound vasculature homing peptide, an adipose tissue homing peptide, a virus binding peptide, or a fusogenic peptide.
  • YIGSR laminin receptor binding peptide
  • penetration enhancing agent refers to moiety or compound that increases the permeability of an active agent, in particular selected from a pharmaceutically active agent, a cell-targeting agent, a cosmetically active agent, and a diagnostically active agent.
  • the penetration enhancing agent is also known as permeation enhancer.
  • Examples of penetration enhancing agents include, without limitation, cell penetrating peptides, surfactants, terpenes, sulfoxides, pyrrolidones, fatty acids, fatty alcohols, urea, azones, fatty alcohols, fatty acids, fatty esters; such as for example, lauryl sarcosine, octoxynol, phenylsulfonate, pluronic, sodium laurate, sodium oleate, sorbitan dilaurate, sorbitan dioleate, sorbitan trilaurate, sorbitan trioleate, sodium octyl sulfate, alkyl ammonium halides, decanol, dodecanol, linolenyl alcohol, oleyl alcohol, butyl acetate, cetyl lactate, lauryl lactate, myristyl lactate, diethyl sebacate, diethyl
  • labeling or imaging agent refers to any substance that is used as a label, or that enhances specific structures in any imaging technique.
  • An imaging agent hence, includes optical imaging agent, magnetic resonance imaging agent, radioisotope, and contrast agent. Imaging or labelling agents are well known in the art.
  • imaging or labelling agents are gases such as sterilized air, oxygen, argon, nitrogen, fluor, perfluorocarbons, carbon dioxide, nitrogen dioxide, sulfur hexafluoride, xenon and helium; commercially available agents used in positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI).
  • gases such as sterilized air, oxygen, argon, nitrogen, fluor, perfluorocarbons, carbon dioxide, nitrogen dioxide, sulfur hexafluoride, xenon and helium
  • PET positron emission tomography
  • CAT computer assisted tomography
  • single photon emission computerized tomography single photon emission computerized tomography
  • x-ray x-ray
  • fluoroscopy and magnetic resonance imaging
  • MRI magnetic resonance imaging
  • gadolinium chelates currently available, such as diethylene triamine pentaacetic acid (DTPA) and
  • Examples of materials useful for CAT and x-rays include iodine based materials for intravenous administration, such as ionic monomers typified by diatrizoate and iothalamate, non-ionic monomers such as iopamidol, isohexol, and ioversol, non-ionic dimers, such as iotrol and iodixanol, and ionic dimers, for example, ioxagalte.
  • Other useful materials include barium for oral use and non-soluble salts such as zinc acetate.
  • an imaging agent is a dye.
  • an imaging agent is a fluorescent moiety.
  • a fluorescent moiety is selected from: a fluorescent protein, a fluorescent peptide, a fluorescent dye, a fluorescent material or a combination thereof.
  • fluorescent dyes include, but are not limited to, xanthenes (e.g., rhodamines, rhodols and fluoresceins, and their derivatives); bimanes; coumarins and their derivatives (e.g., umbelliferone and aminomethyl coumarins); aromatic amines (e.g., dansyl; squarate dyes); benzofurans; fluorescent cyanines; indocarbocyanines; carbazoles; dicyanomethylene pyranes; polymethine; oxabenzanthrane; xanthene; pyrylium; carbostyl; perylene; acridone; quinacridone; rubrene; anthracene; coronene; phenanthrecene; pyrene; but
  • fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5- isothiocyanate, fluorescein-6-isothiocyanate and 6-carboxyfluorescein.
  • rhodamine dyes include, but are not limited to, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine, diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101 sulfonyl chloride (sold under the tradename of TEXAS RED(R)).
  • cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, IRDYE680, Alexa Fluor 750, IRDye800CW, ICG.
  • fluorescent peptides include GFP (Green Fluorescent Protein) or derivatives of GFP (e.g., EBFP, EBFP2, Azurite, mKalamal, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet). Fluorescent labels are detected by any suitable method.
  • a fluorescent label may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs), photomultipliers, etc.
  • CCDs charge coupled devices
  • photomultipliers etc.
  • the imaging agent is labeled with a positron-emitting isotope (e.g., 18F) for positron emission tomography (PET), gamma-ray isotope (e.g., 99mTc) for single photon emission computed tomography (SPECT), or a paramagnetic molecule or nanoparticle (e.g.,Gd3+ chelate or coated magnetite nanoparticle) for magnetic resonance imaging (MRI).
  • a positron-emitting isotope e.g., 18F
  • PET positron emission tomography
  • gamma-ray isotope e.g., 99mTc
  • SPECT single photon emission computed tomography
  • MRI magnetic resonance imaging
  • the imaging agent is labeled with: a gadolinium chelate, an iron oxide particle, a super paramagnetic iron oxide particle, an ultra small paramagnetic particle, a manganese chelate or gallium containing
  • gadolinium chelates include, but are not limited to diethylene triamine pentaacetic acid (DTPA), 1 ,4,7,10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid (DOTA), and 1,4,7- triazacyclononane-N, N',N"-triacetic acid (NOTA).
  • the imaging agent is a nearinfrared fluorophore for near-infra red (near-IR) imaging, a luciferase (firefly, bacterial, or coelenterate) or other luminescent molecule for bioluminescence imaging, or a perfluorocarbon-filled self-assembled particle for ultrasound.
  • the imaging agent is a nuclear probe. In some molecules, the imaging agent is a SPECT or PET radionuclide probe. In some molecules, the radionuclide probe is selected from: a technetium chelate, a copper chelate, a radioactive fluorine, a radioactive iodine, a indiuim chelate. Examples of Tc chelates include, but are not limited to HYNIC, DTPA, and DOTA.
  • the imaging agent contains a radioactive moiety, for example a radioactive isotope such as 211 At, 1311, 1251, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 64Cu radioactive isotopes of Lu, and others.
  • the diagnostically active agents agent may be attached to the polyproline-based block copolymer backbone and/or may be contained in the self-assembled particles formed from it.
  • the pharmaceutically active agent is not particularly limited, and any pharmaceutically active agent having desired activity can be used.
  • pharmaceutically active agents that are desired to be delivered intracellularly, eg, to mitochondria are used.
  • the physiological activity possessed by the pharmaceutically active agent may be any physiological activity capable of functioning as an active ingredient of a drug, and examples thereof include antitumor activity, immunostimulatory activity, antiviral activity, antibacterial activity, and antiinflammatory activity.
  • Drugs may be enzymes, hormones, vaccines, proteins such as antibodies, mRNA, pDNA, antisense, ribozymes, siRNA, decoy nucleic acids, nucleic acids such as aptamers, and macromolecular pharmaceuticals such as polysaccharides.
  • lipid-like moiety refers to a moiety which, while it cannot be considered to be a lipid as such, structurally and/or functionally resembles a lipid.
  • lipid-like moiety is intended to include moieties that are able to form amphiphilic layers.
  • lipid-like material lipid-like compound or “lipid-like molecule” relates to substances that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense.
  • the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties.
  • the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.
  • the term “lipid” is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.
  • lipids are fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, sterols, and the like, which are insoluble in water.
  • linker refers to a chemical unit which connects two other chemical units through strong chemical bonds, for example: covalent bonds.
  • a linker ensures a certain molecular distance and, at the same time, a connection between the two linked chemical units.
  • the linkers may be cleaved using a variety of physiological stimuli enabling tissue or disease-specific drug targeting.
  • physiological stimuli may include hydrolysis (ester containing linkers, carbonates, carbamates), pH-sensitive (imine, oxime, Schiff base and hydrazone containing linkers), redox sensitive (disulfide, diselenide, benzylboronic ester, thioketal, peroxalate, thioether, dithioether, arylboronic containing linkers), enzyme cleavable (peptide containing linkers), multi-stimuli responsive (amide containing linkers).
  • hydrolysis ester containing linkers, carbonates, carbamates
  • pH-sensitive imine, oxime, Schiff base and hydrazone containing linkers
  • redox sensitive disulfide, diselenide, benzylboronic ester, thioketal, peroxalate, thioether, dithioether, arylboronic containing linkers
  • enzyme cleavable peptide containing linkers
  • multi-stimuli responsive amide containing linkers
  • the linker may essentially be seen as a covalent linker which is used to attach/bind the compound of formula (I) to at least one inert moiety, at least one detection moiety, at least one active moiety, at least one lipid-like moiety Rw, to at least one pH, redox or protease responsive unit, or a mixture thereof.
  • linker related radicals may be many different suitable structures, for instance a structure comprising a, in a human cell in vivo, cleavable disulfide bond.
  • MW molecular weight
  • linkers are those derived from a biradical selected from the group consisting of -(Ci-C6)NH-CO-(Ci-C6)alkyl-O-, -(Ci-C6)NH-CO-(Ci-C6)alkyl-, -(Ci-C 6 )NH-CO-(Ci-C 6 )alkyl-NH-, -(Ci-C 6 )NH-CO-(Ci-C 6 )alkyl-CO -NH(Ci-C 6 )alkyl-O-, -NH(Ci-C 6 )alkyl-NH-, -O-(Ci-C 6 )alkyl-NH-, -O-(Ci-C 6 )alkyl-O-, a straight or branched -(CI-C 6 )-NH-CO- (Ci-C 3 o)alkylene-0-, -(Ci-C 6 )-NH-CO(
  • linkers are a biradical of formula (IV), (V), (VI), (VI I), (VIII), (IX), (X), or (XI) as defined above; a biradical of formula (L) or (LI); and a biradical selected from the group consisting of (XXVII), (XXIX), (XXX), (XXXIII), (XXXIV), (XXXV), (XXXVI) and (XXXVII) optionally attached by a chemically feasible bond to a biradical of formula (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI);
  • the polyproline-based block copolymers of formula (I), the compound comprising a structural unit of formula (I') or the compound comprising a structural unit of formula (I”) may comprise at least one lipid-like moiety R10 which is a radical derived from any known lipid, including fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, sterols, and the like, which are insoluble in water.
  • the first aspect of the invention is a copolymer (a polyproline-based block copolymer) whose structure is composed of multiple repeating units.
  • copolymer refers to polymers obtained by polymerization of two or more different kinds of monomers, thereby the repeating units are different.
  • the copolymers can be divided into “block copolymers” or “random copolymers”
  • block copolymer refers to a polymer comprising two or more homopolymer subunits linked by covalent bonds. Therefore, a block copolymer is made of blocks of different polymerized monomers.
  • random copolymer refers to a polymer comprising two or more monomers that are distributed randomly throughout the polymer without forming blocks.
  • weight average molecular weight “weight average molar weight” and the abbreviature “Mw” have the same meaning and they are used interchangeable. Mw is a way of determining the molecular weight of a polymer. Polymer molecules, even ones of the same type, come in different sizes (chain lengths, for linear polymers), so the average molecular weight will depend on the method of averaging. It is determined by summing all molecular weights of the fractions of the polymer multiplied by their weight fractions: wherein w, is the number of molecules having the molecular weight M/.
  • the weight average molecular weight of a polyproline-based block copolymer of the present invention can be determined by any method known in the state of the art for instance gel permeation chromatography (GPC), viscometry via the (Mark-Houwink equation), colligative methods such as vapor pressure osmometry, analytical size-exclusion chromatography, and refractive index detector (SEC/M ALS/RI ), and end-group determination or proton NMR.
  • GPC gel permeation chromatography
  • Mw weight average molecular weight
  • Mw weight average molecular weight of polyproline-based block copolymers were performed by analytical size-exclusion chromatography coupled with multi-angle light scattering and refractive index detector (SEC/MALS/RI).
  • Mn is a way of determining the molecular mass of a polymer. Polymer molecules, even ones of the same type, come in different sizes (chain lengths, for linear polymers), so the average molecular mass will depend on the method of averaging.
  • the number average molecular mass is the ordinary arithmetic means or average of the molecular masses of the individual macromolecules. It is determined by measuring the molecular mass of n polymer molecules, summing the masses, and dividing by n. The Mn is calculated by the following formula: wherein N, is the number of molecules of molecular mass M/.
  • the number average molecular mass of a polymer can be determined by gel permeation chromatography (GPC), viscometry via the (Mark-Houwink equation), colligative methods such as vapor pressure osmometry, end-group determination or proton NMR.
  • GPC gel permeation chromatography
  • Viscometry via the (Mark-Houwink equation)
  • colligative methods such as vapor pressure osmometry, end-group determination or proton NMR.
  • the Mn is measured by GPC.
  • each Rs and L4 are independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C 2 -C 3 o)alkenyl, -(Ci-C 3 o)alkyl-R i1 , -(Ci-C3o)alkyl-COOR i1 , -(Ci-C 3 o)alkyl-0-R iii1 , -(Ci-C 3 o)alkyl-NR iv1 R v1 , -C(O)-R vi1 , -(Ci-Cis)alkyl-CO-NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety Rw, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting
  • I2, v2, w2, x2, y2, z2 and o2 are each independently an integer selected from 1 to 6; each R' 1 is independently selected from the group consisting of H, -(Ci-C 3 o)alkyl, -(C2-C 3 o)alkenyl, -(C2- Csojalkynyl, -OAIkyl(Ci-C6), halogen, -CF 3 , -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO 2 , -CN, -OC(O)-(Ci-C 30 )alkyl, -OC(O)O(Ci-C 30 )alkyl, -OC
  • R' 1 , R iii1 , R v1 , R v1 and R vi1 are each independently selected from the group consisting of H, -(Ci-C 3 o)alkyl, -(C2-C 3 o)alkenyl, -(Ci-C 3 o)alkyl NH2, -(Ci-C 3 o)alkyl-N((Ci-C 3 o)alkyl)2, -(Ci-C 3 o)alkyl-NH(Ci-C 3 o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein each R' 1 , R ii1 , R iii1 , R'
  • each Rs and L4 are independently selected from selected from the group consisting of H, -(Ci-Cis)alkyl, -(C 2 -Ci 8 )alkenyl, -(Ci-Cis)alkyl-R 1 , -(Ci-Cis)alkyl-COO i1 , -(Ci-Cis)alkyl-O-R" 1 , -(Ci-Ci 8 )alkyl-NR iv1 R v1 , -C(O)-R vi1 , -(Ci-Ci8)alkyl-CO-NH 2 , an inert moiety, a detection moiety, an active moiety, a lipid-like moiety R10, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker,
  • R' 1 , R iii1 , R v1 , R v1 and R vi1 are each independently selected from the group consisting of H, -(Ci-Cis)alkyl, -(C 2 -Cis)alkenyl, -(Ci-Cis)alkylNH 2 , -(Ci-Cis)alkyl-N((Ci-Cis)alkyl) 2 , -(Ci-Cis)alkyl-NH(Ci-Cis)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); R' 1 , R ii1 , R iii1 , R' v1 , R v1 , and
  • each Rs and L4 are independently selected from selected from the group consisting of H, -(Ci-Cis)alkyl, -(C 2 -Ci 8 )alkenyl, -(Ci-C 6 )alkyl-R 1 , -(Ci-C 6 )alkyl-COOR i1 , -(Ci-C 6 )alkyl-O-R" 1 , -(Ci-C 6 )alkyl-NR iv1 R v1 , -C(O)-R vi1 , -(Ci-C6)alkyl-CO-NH 2 , an inert moiety, a detection moiety, an active moiety, a lipid-like moiety R10, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker
  • R' 1 , R iii1 , R v1 , R v1 and R vi1 are each independently selected from the group consisting of H, -(Ci-Ci 8 )alkyl, -(C 2 -Ci 8 )alkenyl, -(Ci-Ci 8 )alkylNH 2 , -(Ci-Ci 8 )alkyl-N((Ci-Ci 8 )alkyl) 2 , -(Ci-Ci 8 )alkyl-NH(Ci-Ci 8 )alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI);
  • R 1 , R ii1 , R iii1 , R v1 , R v1 , and R vi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -O(Ci-Ci 8 )alkyl, -CF 3 , -OCF 3 , -NH 2 , -(Ci-Ci 8 )alkyl, -(C 2 -Ci 8 )alkenyl, -(C 2 -Ci 8 )alkynyl, -SH, -NHNH 2 , -NHCH 3 , -N(CH 3 ) 2 , -NCH(CH 3 ) 2 and -(Ci-Ci 8 )alkyl-OH; and Rs and L4 are optionally substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety Rw; or a linker; the linker being
  • each Rs and L4 are independently selected from selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, -(Ci-Cejalkyl-R' 1 , - (Ci-C 6 )alkyl-COOR ii1 , -(Ci-C 6 )alkyl-O-R" 1 , -(Ci-C 6 )alkyl-NR iv1 R v1 , -C(O)-R vi1 , -(Ci-C 6 )alkyl-CO-NH 2 , an inert moiety, a detection moiety, an active moiety, a lipid-like moiety Rw, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker
  • R' 1 , R iii1 , R v1 , R v1 and R vi1 are each independently selected from the group consisting of H, -(Ci-Ci 8 )alkyl, -(C 2 -Ci 8 )alkenyl, -(Ci-C 4 )alkylNH 2 , -(Ci-C 4 )alkyl-N((Ci-C 4 )alkyl) 2 , -(Ci-C 4 )alkyl-NH(Ci-C 4 )alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI);
  • R 1 , R ii1 , R iii1 , R v1 , R v1 , and R vi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, F, Cl, Br, -O(Ci-Ci 8 )alkyl, -CF3, -OCF3, -NH 2 , -(Ci-Ci 8 )alkyl, -(C 2 -Ci 8 )alkenyl, -(C 2 -Ci 8 )alkynyl, -SH, -NHNH 2 , -NHCH3, -N(CH 3 ) 2 , -NCH(CH 3 ) 2 and -(Ci-Ci 8 )alkyl-OH; and each R 8 and L4 are independently optionally substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety Rw; or a linker; the linker being attached
  • each Rs and L4 are independently selected from a detection moiety, an active moiety, a lipid-like moiety R10, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, H, methyl, ethyl, propyl, isopropyl, butyl, n-butyl, -(C2-Cis)alkenyl, -CH2SCH3,-CH2CH2SCH3, -CH2CH2SCH2CH3, -CH2SCH2CH3, -CH2SCH2CH3, -CH 2 SH, -CH 2 -SeH, -CH2CH2SH, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2NH2, -CH 2 CH(CH3)CH 2 NH 2 , -CH2NHCH3, -
  • each Rs and L4 are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, n-butyl, -CH2SCH3, -CH2CH2SCH3, -CH2CH2SCH2CH3, -CH2SCH2CH3, -CH 2 SH, -CH2CH2SH, -CH 2 SeH, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety R10, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XX
  • each Rs and L4 are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, n-butyl, -CH2SCH3, -CH2CH2SCH3, -CH2CH2SCH2CH3, -CH2SCH2CH3, -CH 2 SH, -CH2CH2SH, -CH 2 SeH, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety Rw, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XX
  • Li is a biradical selected from the group consisting of (II) and (III), wherein the biradical (II) or (III) is attached to the AA1 , AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond when attached to the N-terminal end of AA1 , AA2 or AA3 repeating unit; or alternatively the biradical (II) or (III) is attached to the AA1 , AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond when is attached to the C-terminal end of the AA1 , AA2 or AA3 repeating unit.
  • Li is a biradical selected from (II) and (III), which is attached to the C-terminal end of AA1 , AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond;
  • L4 is selected from the group consisting of an inert moiety; a detection moiety; an active moiety; a lipid- like moiety Rw; a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; wherein the inert moiety is selected from the group consisting of H, -N3, -C(O)H, -S(O)H, -NHCH(CH3)2, -(Ci-C 4 )alkylNH 2 , -(Ci-C4)alkylNHCH 3 , -(Ci-C 4 )alkylN(CH 3 )2, -O-(
  • L4 is a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof.
  • L1 is a biradical selected from (II) and (III), which is attached to the C-terminal end of AA1, AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond; and L4 is a lipid-like moiety Rw selected from the group consisting of - (Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, and a radical of formula (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII) or (XLIX).
  • the lipid-like moiety Rw is selected from the group consisting of -(Ci-C jalkyl, -(C2-Ci8)alkenyl, and a radical of formula (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII) or (XLIX) wherein:
  • T and T are independently selected from -OH, -OCORx and -COORx;
  • L1 is a biradical selected from (II) and (III), which is attached to the C-terminal end AA1, AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond; wherein L4 is a lipid-like moiety Rw; and each Rw is independently selected from the groups consisting of -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, and a radical of formula (XL), (XLI), (XLIII), (XLIV), (XLV), and (XLVI).
  • Li is a biradical selected from (II) and (III), which is attached to the C-terminal end of AA1, AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond; and L4 is selected from the group consisting of an inert moiety; a detection moiety; an active moiety; a lipid- like moiety Rw; or a linker; the linker being attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw; or alternatively attached to at least one pH, redox or protease responsive unit; and a mixture thereof; wherein the inert moiety is selected from the group consisting of H, -N3, -C(O)H, -S(O)H, -NHCH(CH3)2, -(Ci-C 4 )alkylNH 2 , -(Ci-C4)al
  • each Rw is independently selected from the groups consisting of -(Ci-C jalkyl, -(C2-Ci8)alkenyl, and a radical derived from (2S)-2,5- bis(3-aminopropylamino)- N-[2-(dioctadecyl-amino)acetyl]pentanamide (DOGS), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]-but
  • polyproline-based block copolymer of formula (I) are:
  • polyproline-based block copolymer of formula (I) are:
  • m is an integer selected from 4 to 40; preferably from 6 to 36; from 8 to 32; from 10 to 30, from 12 to 28; from 16 to 24; from 18 to 22; p is an integer selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; each R8 is independently a cationic moiety selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2NH2, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); wherein each Rs are optionally independently substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety R10; or a linker; the linker being attached to at least one inert moiety, or
  • L4 is selected from the group consisting of an inert moiety, a detection moiety, an active moiety, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a lipid-like moiety R10; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; each R8 is independently a cationic moiety selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2NH2, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII
  • polyproline-based block copolymer of formula (I) are those wherein
  • the polyproline-based block copolymers of the invention are polymeric structures containing, as repeating units, the following polyamino acid-based moieties PAA1, AA1, AA2 and AA3 wherein the “*” denote the attaching points of the PAA-based moiety to the rest of the molecule.
  • the main repeating units AA1, AA2 and AA3 are shown in a particular order for convenience of description, the main repeating units may be present in any order and may be block or randomly present; and each of the repeating units, may comprise blocks of monomer units which may be the same or different from each other.
  • the polyamino acid-based moieties PAA1, AA1, AA2 and AA3 have the formula (PAA'), (AAT), (AA2’) and (AA3’)
  • Processes for the preparation of the polyproline-based block copolymers as defined herein are also part of the invention.
  • the appropriate reagents and theirs amounts as well as the reaction conditions can be determined by those skilled in the art according to the polyproline-based block copolymer being prepared.
  • the above mentioned polyprolinebased block copolymers can be prepared according to polymerizing methods well known in the state of the art.
  • Polyproline based block copolymers were prepared by NCA polymerization techniques using the corresponding NCA monomers with adequate protecting groups when necessary, and primary amine initiators. Briefly, the block designed to be the polycation block was first polymerized using Schlenk techniques under Nitrogen atmosphere and in a suitable solvent using n-butylamine, or other suitable primary amine. Reactions usually proceed at low temperature (10°C) and monomer consumption was confirmed by IR usually after 16 hours. After IR verification, the proline NCA was polymerized either by adding the previous solution to the polyproline NCA suspended in acetonitrile or viceversa. The mixture was stirred at room temperature for 16 hours.
  • the reaction mixture is precipitated (commonly in diethyl ether).
  • the precipitate was isolated by centrifugation and dried under vacuum or resuspended and liophillized.
  • the block copolymer was isolated as a white solid.
  • Deprotection of the protecting groups i.e. to obtain ornithine, lysine and arginine block copolymers
  • backbone modifications i.e. to obtain oligoamine block copolymers
  • Drug conjugations were performed using the free amines of the polymer backbone with the carboxylic group activation of the drugs using DMTMM BF4.
  • Dye conjugations were performed using the form of NHS-Dyes and the free amines of the polymer backbone.
  • solvents for performing the preparation processes disclosed herein above and below for the polyproline-based block copolymers of the present invention include, but it is not limited to, (Ci-Csjalcohols such as ethanol and methanol; dimethylsulphoxide (DMSO) and dimethylformamide (DMF).
  • DMSO dimethylsulphoxide
  • DMF dimethylformamide
  • alcohol refers to an "alkane” wherein at least one hydrogen atom is substituted by a hydroxyl group and which contains the number of carbon atoms specified in the description or claims.
  • alkane refers to a saturated, branched, or linear hydrocarbon which contains the number of carbon atoms specified in the description or claims.
  • room temperature refers to a temperature of the environment, without heating or cooling, and it is generally comprised from 20 °C to 25 °C.
  • the processes disclosed in the present invention can be performed using ethanol as solvent at a temperature from 20 to 78°C; or in the sinus of dimethyl sulfoxide at a temperature from 20-140°C, for the appropriate time for obtaining the polyproline-based block copolymers of the present invention.
  • a process for the preparation of a polyproline-based block copolymer of the present invention having a weight average molecular weight (Mw) from 2000 to 30000 Da can be performed following any one of the methods A-C as defined above using; particularly in the sinus of (C1-C5) alcohol (particularly ethanol) as a solvent and at a temperature from 20-78°C.
  • stereoisomer refers to all isomers of individual polyproline-based block copolymers that differ only in the orientation of their atoms in space.
  • stereoisomer includes mirror image isomers (enantiomers), mixtures of mirror image isomers (racemates, racemic mixtures), geometric (cis/trans or syn/anti or E/Z) isomers, and isomers of polyproline-based block copolymers with more than one chiral center that are not mirror images of one another (diastereoisomers).
  • the present invention relates to each of these stereoisomers and also mixtures thereof.
  • the definition of the polyproline-based block copolymers of formula (I) is also intended to encompass all R- and S-isomers of a chemical structure in any ratio, e.g. with enrichment (i.e. enantiomeric excess or diastereomeric excess) of one of the possible isomers and corresponding smaller ratios of other isomers.
  • enrichment i.e. enantiomeric excess or diastereomeric excess
  • they may acquire L-configuration or D-configuration.
  • Diastereoisomers and enantiomers can be separated by conventional techniques such as chromatography or fractional crystallization. Optical isomers may be obtained using enantiospecific synthesis. Alternatively, optical isomers can be resolved by conventional techniques to give optically pure isomers. The resolution can be carried out on any chiral synthetic intermediates or on the polymers of the invention.
  • the pharmaceutically, cosmetically or diagnostically acceptable salts thereof and the stereoisomers or mixtures of stereoisomers, either of any of the polyproline-based block copolymers or of any of their pharmaceutically, cosmetically or diagnostically acceptable salts are always contemplated even if they are not specifically mentioned.
  • the polyproline-based block copolymers of the invention may be in crystalline form either as free solvation polyproline-based block copolymers or as solvates (e.g. hydrates). It is intended that all these forms are within the scope of the present invention. Methods of solvation are generally known within the art. For the purposes of the invention, the solvated forms with pharmaceutically, cosmetically or diagnostically acceptable solvents such as water, ethanol and the like are equivalent to the unsolvated form.
  • polyproline-based block copolymers of the present disclosure also include polyproline-based block copolymers that differ only in the presence of one or more isotope-enriched atoms.
  • isotope-enriched atoms without limitation, are deuterium, tritium, 13C or 14C, or a nitrogen atom enriched in 15N or a fluorine atom enriched in 18F.
  • the invention provides a conjugate which comprises the polyproline-based block copolymer of the first aspect of the invention and one or more molecules of interest.
  • conjugate and complex have the same meaning and are used interchangeable. They refer to the union of a polyproline-based block copolymer of the first aspect of the invention and one or more molecules of interest linked together.
  • the union between the polyproline-based block copolymer and the molecule of interest is "non-covalent”.
  • non- covalent refers to the bond between the polyproline-based block copolymer and the molecule of interest that involves weak interactions such as for example ionic interactions, electrostatic interactions, hydrogen bonding and/or van der Waals interactions. The type of interaction (union) mainly depends on the molecule of interest.
  • conjugate encompasses the term “poly plex” which refers to a specific conjugate of the present invention comprising a polyprolinebased block copolymer of the invention and one or more genetic material, including a nucleic acidcontaining compound, as a molecule of interest (also called "cargo”).
  • molecule of interest encompasses active ingredients, amino acid-containing compounds, nucleic acid-containing compounds, and mixtures thereof.
  • the conjugate of the invention is one wherein the one or more molecules of interest comprises at least one active ingredient as defined above.
  • the conjugate of the invention is one wherein the one or more molecules of interest comprises at least one amino acid-containing compound. In an embodiment, the conjugate of the invention is one wherein the one or more molecules of interest comprises at least one amino acidcontaining compound selected from the group consisting of a polypeptide, a protein and a mixture thereof.
  • the terms "peptide” and “polypeptide” have the same meaning and are used interchangeably. They refer to chains having from 2 to 50 amino acid residues, and the term “protein” refers to chains of more than 50 amino acid residues.
  • the conjugate of the invention is one wherein the one or more molecules of interest comprises at least an antibody or a fragment thereof as amino acidcontaining compound.
  • antibody or a fragment thereof' refers to any immunoglobulin or fragment thereof suitable to bind an epitope of the target protein. It includes monoclonal and polyclonal antibodies.
  • fragment thereof' encompasses any part of an antibody having the size and conformation suitable to bind an epitope of the target protein. Suitable fragments include F(ab), F(ab') and Fv.
  • An "epitope" is the part of the antigen being recognized by the immune system (B-cells, T-cells or antibodies). Particularly, the antibodies used for specific detection can be polyclonal or monoclonal. There are well known means in the state of the art for preparing and characterizing antibodies.
  • polyclonal antibodies Methods for generating polyclonal antibodies are well known in the prior art. Briefly, one prepares polyclonal antibodies by immunizing an animal with the protein; then, serum from the immunized animal is collected and the antibodies isolated. A wide range of animal species can be used for the production of the antiserum. Typically, the animal used for production of antisera can be a rabbit, mouse, rat, hamster, guinea pig or goat. Moreover, monoclonal antibodies (MAbs) can be prepared using well-known techniques. Typically, the procedure involves immunizing a suitable animal with the protein associated with the disease. The immunizing composition can be administered in an amount effective to stimulate antibody producing cells.
  • MAbs monoclonal antibodies
  • Methods for preparing monoclonal antibodies are initiated generally following the same lines as the polyclonal antibody preparation.
  • the immunogen is injected into animals as antigen.
  • the antigen may be mixed with adjuvants such as complete or incomplete Freund's adjuvant. At intervals of two weeks, approximately, the immunization is repeated with the same antigen.
  • the conjugate of the invention is one wherein the one or more molecules of interest comprises at least one nucleic acid-containing compound.
  • the conjugate of the invention is one wherein the one or more molecules of interest comprises at least one nucleic acidcontaining compound selected from the group consisting of single strand oligonucleotides such as DNA, RNA, PNA, LNA and analogues thereof; double-strand oligonucleotides such as siRNA, shRNA, decoy DNA; plasmids and analogues thereof.
  • the polyproline-based block copolymer of the present invention can be used as transfecting agent. In fact, it is shown that a polyplex comprising the polyproline-based block copolymer of the present invention is capable of conjugate until ten molecules of nucleic acid of interest for transfecting into cells.
  • the process for the preparation of the conjugates of the invention comprises contacting the polyproline-based block copolymer with the molecule of interest under such reaction conditions that allows the union between them.
  • conjugate of the second aspect of the invention obtainable by the processes disclosed above in the present application are also part of the invention. All embodiments disclosed above for the polyproline-based block copolymer of the first aspect of the invention, and the conjugate of the second aspect of the invention also apply here for the conjugate obtainable by its preparation process.
  • star-shaped polymers according to the present invention relates to at least three compounds comprising the structural unit of formula (I') or (I”) radiating from one single star-shaped multifunctional linking agent, usually called the core or the central nodule, and which can itself be polymeric.
  • the polyproline-based copolymers of the invention may be attached to a star-shaped multifunctional linking agent, thus forming a star-shaped polymer.
  • the star-shaped polymers thus obtained comprise at least two, preferably 2, 3, 4, 5 or 6 structural units of formula (I') or (I”) as defined above.
  • the star-shaped polymers of the present invention may comprise symmetric or asymmetric architecture depending on the presence of same or combination of different polymeric arms emanating from a shared core.
  • the star-shaped polymer comprises a symmetric architecture of structural units of formula (I') or (I”), as defined herein, as arms emanating from a shared core.
  • star-shaped multifunctional linking agent the core of the star-shaped polymers.
  • Star-shaped multifunctional linking agents suitable for the preparation of chemical-mediated vectors for the delivery of molecules of interest such as active ingredients or detection moieties acting as cell penetrating peptide for the cellular delivery of attached cargoes, are well known in the art.
  • the star-shaped multifunctional linking agent is selected from the group consisting of
  • E and E’ are each independently selected from -0- and -NH-; wherein the 3-arm star-shaped linking agent of formula (Pm) is attached to the L4 moiety of each of the at least one polyproline-based block copolymer of formula (I) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond; ill) a 4-arm star-shaped linking agent of formula (Tz) wherein each V is selected from -0- and -NH-;
  • Y, Y' and Y are each independently selected from -0- and -NH-; wherein the 4-arm star-shaped linking agent of formula (Tz) is attached to the L4 moiety of each of the at least one polyproline-based block copolymer of formula (I) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond.
  • the polyproline-based copolymers of the invention may have amphiphilic nature since they may comprise a hydrophilic polyamino acid-based moiety and at least a hydrophobic moiety. Thanks to their amphiphilic nature, the polyamino acid copolymers of the invention may form self-assembled particles in solution such as for example liposomes or lipid nanoparticles which may be used to encapsulate and deliver molecules of different nature.
  • the polyproline-based copolymers of the invention may also form polymer nanoparticles (PNP), including polyplexes.
  • PNP polymer nanoparticles
  • the compounds of the invention allow carrying and/or delivering different active agents at the same time, not only bonded to the structure of the compound but also contained in self-assembled particles formed from the compound.
  • the present invention also relates to a self-assembled particle comprising the polyproline-based block copolymers of formula (I) as defined according to the first aspect of the invention, the compound comprising the structural unit of formula (I') or the structural unit of formula (I”) as defined according to the second aspect of the invention, or the star-shaped polymers as defined according to the third aspect of the invention, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, penetration enhancing agents, cell-targeting agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • active agents selected from the group consisting of pharmaceutically active agents, penetration enhancing agents, cell-targeting agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • self-assembled particles refers to the arrangement of the polyproline-based block copolymers of formula (I), the compounds comprising the structural unit of formula (I') or (I”), or the star-shaped polymers in a solvent.
  • small, self-assembled particles may be enclosed structures of any shape, typically spherical and/or tubular.
  • self-assembled particles intends to encompass any of a number of structures that are known in the art to be formed from amphiphilic polymers.
  • Non-limiting examples of self-assembled particles include micelles (also being referred to interchangeably herein as micellar worms or simply "worms”), inverted micelles, planar bilayers, crystal nanoparticles, liposomes, microbubbles or lipid nanoparticles.
  • the self-assembled nanoparticles and microparticles can also form gels.
  • the polyprolinebased copolymers of formula (I), or the compounds comprising the structural unit of formula (I') or the compounds comprising the structural unit of formula (I”) of the invention may comprise at least one lipid- like moiety Rw,
  • at least one of the R8 and/or L4 comprise a lipid-like moiety Rw. Therefore, it is possible that the polyproline-based block copolymers of formula (I), or the compounds comprising the structural unit of formula (I') or the compounds comprising the structural unit of formula (I”) of the invention comprise at least one lipid-like moiety Rw, both in R8 and L4.
  • polyproline-based copolymers of formula (I) are those of formula (la)
  • polyproline-based copolymers of formula (I) are those of formula (lb)
  • polyproline-based copolymers of formula (I) are those of formula (Ic)
  • lipid-containing polyproline-based copolymers according to formula (la), (lb) and (Ic), have amphiphilic nature since they comprise a polyamine acid-based moiety (hydrophilic part) and at least one lipid-like moiety (hydrophobic part). Thanks to their amphiphilic nature, the lipid-containing polyproline-based copolymers of the invention may form self-assembled particles in solution, such as for example lipid nanoparticles (LNPs), and can be used as non-viral vectors for delivery of active ingredients, including nucleic acids, to cells.
  • LNPs lipid nanoparticles
  • the present invention also relates to a self-assembled particle comprising the lipid- containing polyproline-based copolymer as defined herein according to formula (la), (lb) or (Ic), the compound comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid-like moiety Rw; and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, penetration enhancing agents, cell-targeting agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • the self-assembled particles are arrangements of the lipid-containing polyproline-based copolymers in a solvent, in particular, small, self-assembled particle may be enclosed structures of any shape, typically spherical and/or tubular.
  • the term "self-assembled particles” intends to encompass any of a number of structures that are known in the art to be formed from amphiphilic polymers.
  • Non-limiting examples of self-assembled particles include micelles (also being referred to interchangeably herein as micellar worms or simply "worms”), inverted micelles, planar bilayers, crystal nanoparticles, liposomes, microbubbles or lipid nanoparticles.
  • the self-assembled nanoparticles and microparticles can also form gels.
  • the self-assembled particles of the invention are non-viral particles, which means that they are not able to virally infect cells.
  • the polyproline-based copolymers of formula (I) are those wherein R8 is independently substituted by at least one linker attached to at least one inert moiety, at least one detection moiety, at least one active moiety or at least one lipid-like moiety Rw.
  • the self-assembled particle is a lipid nanoparticle.
  • the self-assembled particle is a polymer nanoparticle, more particularly a polyplex.
  • the self-assembled particle further comprises one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids.
  • the self-assembled particle further comprises an ionizable lipid or a cationic lipid, a phospholipid, and a sterol.
  • the lipid nanoparticle further comprises at least one lipid selected from the group consisting of a non-cationic lipid, a sterol or a steroid precursor, and a PEG-modified lipid.
  • Examples of sterols include, without being limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.
  • the sterol is cholesterol.
  • Examples of sterol precursors include, without being limited to, a triterpene, a triterpenoid, or a steroid precursor of this kind.
  • Non-limiting examples of triterpenes, triterpenoids and other steroid precursors include squalene, achilleol, polypodatetrane, lanostane, cucurbitacin, hopane, oleanane, chamaecydin, lupine, and mixtures thereof.
  • the lipid nanoparticles comprise the compounds of formula (la), (lb) or (Ic), the compounds comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid-like moiety Rw of the present disclosure, a non-cationic lipid as defined above, and a sterol or a sterol precursor as defined above.
  • the lipid nanoparticles further comprise a PEG-modified lipid.
  • PEG-modified lipid refers to a lipid comprising a polyethylene moiety.
  • PEG-modified lipids include, without being limited to, a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified phosphatidylcholine, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG- DOMG), PEG-DSG, PEG-DPG, or a combination thereof.
  • the lipid nanoparticles comprise a lipid component comprising or consisting of the compounds of formula (la), (lb) or (Ic), the compounds comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid-like moiety Rw of the present disclosure, a non-cationic lipid, a sterol, and a PEG-modified lipid.
  • the self-assembled particle comprises: i) the polyproline-based block copolymer of formula (I), or alternatively the compound comprising the structural unit of formula (I') or (I”), in an amount from 0.1 mol% to 60 mol%; in some embodiments from 0.5 mol% to 50 mol%, in some embodiments from 5 mol% to 40 mol%; in some embodiments from 10 mol% to 20 mol%; ii) an ionizable or a cationic lipid in an amount from 30 mol% to 70 mol%, in some embodiments from 40 mol% to 60 mol%; in some embodiments from 45 mol% to 50 mol% iii) a phospholipid in an amount from 1 mol% to 20 mol%, in some embodiments from 3 mol% to 15 mol%; in some embodiments from 5 mol% to 10 mol%; and iv
  • the self-assembled particles may show a variety of sizes, in particular, they can be nanoparticles or microparticles.
  • a “nanoparticle”, as defined herein, is any particle of nanometric size, in particular having smallest end-to-end diameter of between 1 and 900, more particularly, between 1 and 700 nm, between 1 and 500 nm, between 1 and 300 nm, between 1 and 200 nm, and between 1 and 100 nm in size.
  • a "microparticle”, as defined herein, is typically any particle of micrometric size, having a smallest end-to-end between 1 and 100 pm in size.
  • the relevant diameter is the number average diameter.
  • the term "size" refers to a characteristic physical dimension.
  • the size of the particle corresponds to the diameter of the particle.
  • the size of the particle is determined by the diameter of the two relevant cross-section dimensions of the particle.
  • the size of the nanoparticle corresponds to the maximum edge length.
  • the set of particles can have a distribution of sizes around the specified size.
  • a size of a set of particles can refer to a mode of a distribution of sizes, such as a peak size of the distribution of sizes.
  • the term "diameter” refers to the average diameter and is also designated as Z- average or Z-ave.
  • the average diameter corresponds to the mean hydrodynamic diameter (Dh) and can be measured by dynamic light scattering (DLS) as shown in the examples below.
  • the self-assembled particles of the invention have a hydrodynamic diameter (Dh) in water from 2 to 1200 nm, more particularly, from 10 to 1100 nm, from 10 to 1000 nm, from 10 to 700, from 20 to 500 nm, from 20 to 400 nm, from 20 to 300 nm, from 30 to 200 nm, or from 50 to 150 nm.
  • the invention relates to a polymeric nanoparticles (PNP), including a polyplexes, comprising the polyproline-based copolymers of formula (I) as defined according to the first aspect of the invention, the compounds comprising a structural unit of formula (I') or (I”) as defined according to the second aspect of the invention, or the star-shaped polymer as defined according to the third aspect of the invention, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • PNP polymeric nanoparticles
  • the invention in another embodiment, optionally in combination with one or more features of the various embodiments described above or below, relates to a lipid nanoparticle (LNP) or a liposome comprising the lipid-containing polyproline-based copolymers of formula (la), (lb) or (Ic) as defined herein, or the compound comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid- like moiety Rw; and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • the liposomes and lipid nanoparticles disclosed herein may comprise any suitable lipids, including ionizable lipids, cationic lipids, zwitterionic lipids, neutral lipids, or anionic lipids.
  • Suitable ionizable lipids include, without limitation, (2S)-2,5- bis(3-aminopropylamino)- N-[2- (dioctadecyl-amino)acetyl]pentanamide (DOGS), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3- aminopropyl)amino]-butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 3 « -[N-(N « N «- dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), (1 , 2-d isteary loxy- N , N-d i methyl -3- aminopropane (DSDMA), 1 ,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DOGS),
  • Suitable cationic lipids include, without limitation, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDA13), N-(1-(2,3-dioleoyloxy)propyl)- N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)-propyl)-N,N,N-trimethylammonium chloride (DOTMA), N-[1 -(2,3,-ditetradecyloxy)propyll-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1 -(2, 3, dioleyloxy)-propyl]-N, N-dimethyl-N-hydroxy ethylammonium bromide (DORIE), 3« - [N-(N« N« N« N
  • anionic lipids include, but are not limited to, phosphatidylglycerol, diacylphosphatidylserine, diacylphosphatidic acid, N-Succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine cholesterol hemisuccinate (CHEMS), lysylphos-phatidylglycerol, N- dodecanoyl phosphatidyl ethanoloamine, cardiolipin, and combinations thereof.
  • phosphatidylglycerol diacylphosphatidylserine, diacylphosphatidic acid
  • N-Succinyl phosphatidylethanolamine N-glutaryl phosphatidylethanolamine cholesterol hemisuccinate (CHEMS)
  • CHEMS N-glutaryl phosphatidylethanolamine cholesterol hemisuccinate
  • lysylphos-phatidylglycerol N- dodecanoyl phosphat
  • Suitable neutral lipids may be uncharged or zwitterionic lipids and include, without limitation steroids, phospholipids, and combinations thereof.
  • steroids examples include, without limitation, cholesterol, progesterone, cortisone, aldosterone, estradiol, testosterone, and combinations thereof.
  • Examples of phospholipids include, but are not limited to, phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidylinositol (PI), dimyristoyl phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl gly
  • the liposomes and lipid nanoparticles disclosed herein the lipid-containing polyproline-based copolymers of formula (la), (lb) and (Ic), or the compound comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid-like moiety Rw; advantageously provides stealth properties.
  • the term "stealth” refers to the fact that the liposomes or lipid nanoparticles are not detected and sequestered and/or degraded by the immune system of the host to which they are administered, and consequently the length of time for which the liposomes or lipid nanoparticles can exist in vivo is increased.
  • the liposomes and lipid nanoparticles disclosed herein do not comprise a polyethyleneglycol (PEG)-lipid conjugate, that is a lipid containing polyethyleneglycol.
  • PEG polyethyleneglycol
  • the self-assembled particles of the invention do not comprise polyethyleneglycol (PEG).
  • PEG polyethyleneglycol
  • the invention in another embodiment, optionally in combination with one or more features of the various embodiments described above or below, relates to a lipid nanoparticle (LNP) or a liposome comprising the lipid-containing polyproline-based copolymers of formula (la), (lb) or (Ic), or the compound comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid-like moiety Rw, as defined herein, and one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids.
  • LNP lipid nanoparticle
  • a liposome comprising the lipid-containing polyproline-based copolymers of formula (la), (lb) or (Ic), or the compound comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid-like moiety Rw, as defined herein, and one or more lipids selected from the group consisting of
  • the lipid nanoparticle (LNP) or liposome comprises the lipid-containing polyproline-based copolymers of formula (la) or (lb) as defined herein, an ionizable lipid or a cationic lipid, a phospholipid, and a sterol.
  • the lipid nanoparticle (LNP) or liposome comprises: I) the polyproline-based block copolymer of formula (I) as defined herein in an amount from 0.1 to 10 mol%, more particularly from 1 to 5 mol% or from 1 to 6 mol%, even more particularly from 2 to 5% mol%; II) a ionizable lipid or a cationic lipid in an amount from 30 to 70 mol%, more particularly from 40 to 60 mol%; iii) a phospholipid in an amount from 1 to 20 mol%, more particularly from 5 to 15 mol%; and iv) a sterol in an amount from 20 to 60 mol%, more particularly from 30 to 50 mol%; wherein the percentages are expressed with respect to the sum of the mol% of the lipids and the lipid-containing polyproline-based copolymers of formula (la), (lb) or (Ic).
  • the invention in another embodiment, optionally in combination with one or more features of the various embodiments described above or below, relates to a lipid nanoparticle (LNP) or a liposome comprising the lipid-containing polyproline-based copolymers of formula (la), (lb) or (Ic) as defined herein, one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids, and one or more nucleic acids.
  • LNP lipid nanoparticle
  • a liposome comprising the lipid-containing polyproline-based copolymers of formula (la), (lb) or (Ic) as defined herein, one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids, and one or more nucleic acids.
  • the lipid nanoparticles and the liposomes containing one or more nucleic acids may be prepared by standard methods.
  • the process for the preparation of LNPs comprises: i) preparing a first alcoholic mixture comprising one or more lipids and the polyproline-based block copolymer of the invention in a suitable alcohol such as for example ethanol; ii) preparing a second aqueous composition comprising one or more nucleic acids and an aqueous solvent (an acidic buffer); and iii) mixing i) with ii) in a microfluidic mixing device.
  • the microfluidic mixing allows thorough and rapid mixing of the lipid phase and the nucleic acid phase in a microscale device.
  • the skilled person will be able to modulate the size of the LNPs.
  • composition comprising the polyproline-based block copolymer of formula (I) as defined herein, the compound comprising a structural unit of formula (I') or (I”) as defined according to the second aspect of the invention, the star-shaped polymer as defined according to the third aspect of the invention, or alternatively, the self-assembled particle, together with one or more appropriate excipients or carriers.
  • the compounds of the invention may be formulated in a variety of compositions, including pharmaceutical, cosmetic and diagnostic compositions, with excipients and carriers.
  • the compounds of the invention, self-assembled particles, and compositions thereof may be used in medicinal, cosmetic and diagnostic applications.
  • the composition is a therapeutic composition comprising a therapeutically (pharmaceutical or veterinary) effective amount of one or more active ingredients; and one or more therapeutically acceptable excipients or carriers.
  • the composition is a pharmaceutical composition comprising a therapeutically effective amount of: (a) the polyproline-based block copolymer of formula (I) as defined herein, wherein at least one of R8 or L4 comprises a pharmaceutically active agent; or alternatively (b) the compound comprising a structural unit of formula (I') or (I”) wherein at least on R8 comprises a pharmaceutically active agent; or alternatively (c) the star-shaped polymer as defined herein, wherein at least one of L4 or R8 is a pharmaceutically active agent or alternatively, or alternatively (d) a selfassembled particle containing (a), (b) or (c) as defined above, and one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof, together with one or more pharmaceutically acceptable excipients or carriers.
  • composition refers to a composition suitable for use in the pharmaceutical technology with medical use.
  • therapeutically effective amount of an active ingredient refers to the amount of a pharmaceutical active ingredient that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed.
  • the dose of the pharmaceutically active ingredient administered will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.
  • the pharmaceutical compositions of the present invention comprise one or more pharmaceutically acceptable excipients or carriers.
  • compositions of the present invention comprise one or more pharmaceutically acceptable excipients and/or carriers selected from the group consisting of diluent, binder, glidant, disintegrant, lubricant and mixtures thereof.
  • pharmaceutical compositions of the present invention may contain other ingredients, such as fragrances, colorants, and other components known in the state of the art.
  • polyproline-based block copolymer of formula (I), the compound comprising a structural unit of formula (I'), the compound comprising a structural unit of formula (I”), the star-shaped polymers, the self-assembled particles and pharmaceutical compositions containing them may be used jointly with other additional drugs, to provide combined therapy.
  • Said additional drugs may be a part of the same pharmaceutical composition or, alternatively, may be provided in the form of a separate composition for simultaneous or non-simultaneous administration.
  • the composition is a diagnostic composition comprising a diagnostically effective amount of: (a') the polyproline-based block copolymer as defined herein, wherein at least one of R8 or L4 comprises a diagnostically active agent or alternatively, (b’) the compound comprising a structural unit of formula (I') or (I”), wherein at least one of R8 comprises a diagnostically active agent; or alternatively (c’) a star-shaped polymer wherein at least one of L4 or R8 is a diagnostically active agent, or alternatively (d’) a self-assembled particle containing (a'), (b’) or (c’) as defined above, together with one or more diagnostically acceptable excipients or carriers.
  • diagnosis composition refers to a composition suitable for use in diagnostic, particularly in imaging diagnostic technology.
  • diagnostic composition refers to the effective amount of a detection compound that, when administered, is sufficient for the diagnosis of a disease or disorder; particularly as imaging diagnostic use as contrast imaging agent.
  • the dose of the detection compound administered will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being diagnosticated, and the similar considerations.
  • the diagnostic compositions of the present invention comprise one or more diagnostically acceptable excipients or carriers.
  • diagnosticstically acceptable refers to that excipients or carriers suitable for use in the diagnosing technology for preparing compositions with diagnostic use; particularly by imaging diagnostic use.
  • the detection of these diagnostic agents in the body of the patient can be carried out by the well-known techniques used such as in imaging diagnostic with magnetic resonance imaging (MRI) and X-ray.
  • MRI magnetic resonance imaging
  • X-ray X-ray
  • the composition is a cosmetic composition comprising a cosmetically effective amount of: (a”) the polyproline-based block copolymer as defined herein, wherein at least one of R8 or L4 comprises a cosmetically active agent or alternatively, or alternatively (b”) a compound comprising a structural unit of formula (I') or (I”) wherein at least one R8 comprises a cosmetically active agent, or alternatively, or alternatively (c”) a star-shaped polymer wherein at least one of L4 or R8 is a cosmetically active agent, or alternatively, (d”) a self-assembled particle containing (a”), (b”) or (c”) as defined herein and one or more cosmetically active agents, together with one or more cosmetically acceptable excipients or carriers.
  • cosmetic composition refers to a composition suitable for use in cosmetic for the body care.
  • cosmetically effective amount refers to the effective amount of a cosmetic active agent that, when administered, is intended to improve its appearance or to beautify, preserve, condition, cleanse, color or protect the skin, nails or hair without non-medical application.
  • the dose of the cosmetic active ingredient administered will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition and the similar considerations.
  • the cosmetic compositions of the present invention comprise one or more cosmetically acceptable excipients or carriers.
  • cosmetically effective amount refers to the effective amount of a cosmetically active agent that, when administered, is intended to improve its appearance or to beautify, preserve, condition, cleanse, color or protect the skin, nails or hair without a medical application.
  • cosmetically acceptable or “dermatologically acceptable” excipients or carriers is used interchangeably in this document and refer to components which are appropriate for use in human skin contact without toxicity, incompatibility, instability, inappropriate allergic response, among others.
  • composition can be in form of topical composition, oral composition, and injectable composition.
  • compositions of the invention may be in solid or liquid form.
  • solid forms include frozen forms, lyophilized forms and spray-dried forms.
  • excipients or carriers include, without limitation, diluents, binders, glidants, disintegrants, lubricants colorants, mixtures thereof, and other components known in the state of the art.
  • Any administration route may be used such as e.g. oral, topical, rectal or parenteral route (including subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous route, etc.).
  • the composition is an oral composition, particularly selected form liquid or solid oral composition.
  • the composition of the invention is a solid oral composition.
  • the oral solid compositions of the invention can be formulated in any form that includes any single unit dosage form and any multiple unit dosage forms.
  • the term “single unit” encompasses one entity such as a single tablet, a single granule, and a single pellet.
  • the term “single unit dosage form” defines a dosage form which consists only of one unit which contains the effective amount of the polyproline-based block copolymer of the present invention.
  • multiple unit dosage form defines a dosage from which consists of more than one unit which contains the effective amount of polyproline-based block copolymer of the present invention.
  • the multiple unit dosage forms are based on subunits such as granules, pellets or minitablets. They are usually delivered in hard gelatine capsules or transformed into tablets.
  • a unit dosage from which comprises the composition of the present invention is also part of the invention.
  • the unit dosage from which comprises the composition of the present invention is a single unit dosage form.
  • the unit dosage from which comprises the composition of the present invention is a multiple unit dosage form.
  • the composition is a topical composition.
  • the topical compositions of the invention can be formulated in several forms that include, but are not limited to, solutions, aerosols, and non-aerosol sprays, shaving creams, powders, mousses, lotions, gels, sticks, ointments, pastes, creams, shampoos, shower gel, body washes or face washes.
  • the composition is an injectable composition; particularly selected from the group consisting of intramuscular, subcutaneous, or intravenous application.
  • the compositions of the present invention are in form of parenteral compositions suitable for their injection, infusion, or implantation into the body.
  • parenteral compositions defined above should be sterile, and pyrogen-free, and they can be in form of liquid such as solutions, emulsions, or suspensions, or in solid form packaged in either single-dose or multidose containers suitably diluted before use.
  • Parenteral compositions can comprise appropriate excipients or carriers for parenteral administration that can be pharmaceutical or cosmetic excipients, including, but not limited to, solvents, suspending agents, buffering agents, substances to make the preparation isotonic with blood, stabilizers, or antimicrobial preservatives.
  • excipients should be kept to a minimum. When excipients are used, they should not adversely affect the stability, bioavailability, safety, or efficacy of the components, or cause toxicity or undue local irritation. There should not be any incompatibility between any of the components of the dosage form.
  • composition can be readily determinate by those skilled in the art according to its intended use.
  • excipients and/or carriers, and their amounts can readily be determined by those skilled in the art according to the type of formulation being prepared.
  • the polyproline-based block copolymers of the invention, self-assembled particles, and compositions thereof may be used in therapeutic applications.
  • they may be used as non- viral vectors of general use for biomedical applications, such as vaccines or gene therapy, being effective for transfection of hosts eukaryotic cells in culture, in vivoot ex vivo, monocellular parasites and bacteria, including gene editing using the CRISP/Cas9 methodology.
  • they may be used in protein-based therapy; particularly protein-based vaccine against viral infections or as a therapeutic protein-based vaccine against cancers or infectious diseases.
  • the invention relates to a therapeutic product which is or which comprises: a) the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively b) the compound comprising aa structural unit of formula (I') or (I”), wherein at least one of L4 or R8 comprises a pharmaceutically active agent; or alternatively c) the star-shaped polymer, wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively d) a self-assembled particle, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively e) a composition as defined herein, containing the self-assembled particle (d); for use in medicine.
  • This aspect may also be formulated as a method for treating or preventing a disease or disorder in a subject, more particularly a mammal, and even more particularly a human, comprising administering the therapeutic product as defined herein, and one or more pharmaceutically acceptable excipients or carriers.
  • Non-limiting examples of diseases that may be treated and/or prevented by the compounds of the present invention include neurodegenerative disorders, neurological diseases, cancer, infectious diseases, disorders related to aging, neuro-inflammation, demyelinating disorders, multiple sclerosis, ischemic disorders, ischemia-reperfusion damage, amyloidotic disease, cardiomyopathy, spinal cord injury, immune disorders, inflammatory disorders, rare diseases, wound healing, skin related diseases and lysosomal storage diseases.
  • Non-limiting examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cerebral ischaemia, post-encephalitic Parkinsonisms, dystonias, Tourette syndrome, periodic limb movement pathologies, restless legs syndrome, attention deficit hyperactivity disorders, Huntington's disease, progressive supranuclear palsy, Pick's disease, frontotemporal dementia and neuromuscular diseases.
  • the invention relates to a therapeutic product which is or which comprises: a) the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively b) the compound comprising a structural unit of formula (I') or (I”), wherein at least one of L4 or R8 comprises a pharmaceutically active agent; or alternatively c) the star-shaped polymer, wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively d) a self-assembled particle, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively e) a composition as defined herein, containing the self-assembled particle (d; for use in the treatment and/or prevention of cancer or an infectious disease.
  • disorder as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disease,” “syndrome,” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms.
  • the terms “treat”, “treating” and “treatment”, as used herein, refers to ameliorating symptoms associated with a disease or disorder, including preventing or delaying the onset of the disease or disorder symptoms, and/or lessening the severity or frequency of symptoms of the disease or disorder.
  • the invention relates to the therapeutic product as defined herein for use (I) as transfection reagent for transfecting at least one active agent into a cell; (ii) for use in the in vivoor ex vivo production of biologies encoding a recombinant protein, a peptide or an antibody, or in the production of recombinant virus; (ill) for use as a therapeutic or prophylactic vaccine against viral infections or as a therapeutic vaccine against cancers or infectious diseases; or (iv) for use in genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
  • the present invention relates to the therapeutic product as defined herein as transfection reagent for delivering one or more nucleic acids (regardless of size and structure, circular and linear nucleic acids) to target cells, in in vivo, in vitro or ex vivo.
  • the active agent is selected from the group consisting of low molecular weight drugs, peptides, proteins, antibodies, nucleic acids, aptamers, and combinations thereof.
  • the present invention also relates to a method for in vitro, ex vivo and in vivo transferring active agents comprising the step of administering to a subject (e.g. a mammal, such as a human) the therapeutic product as defined herein, in which administering involves contacting the cell with the therapeutic product, whereby the pharmaceutically active agent is delivered to the cell.
  • a subject e.g. a mammal, such as a human
  • administering involves contacting the cell with the therapeutic product, whereby the pharmaceutically active agent is delivered to the cell.
  • the therapeutic product is a self-assembled particle which comprises the polyprolinebased block copolymer of formula (I) or the compounds comprising the structural unit (I') or (I”) as defined herein, and one or more nucleic acids, or alternatively, a composition containing the selfassembled particle as defined herein and one or more nucleic acids.
  • transfection reagents of the invention are also useful for co-transfection of two or more active agents simultaneously, e.g. two or more nucleic acids, simultaneously.
  • Transfection compositions (such as kits), as well as methods of using the transfection reagents to deliver nucleic acid to target cells are also within the scope of the present invention.
  • the present invention also provides therapeutic products as defined herein for inducing a regulating effect on the expression of one or more target proteins responsible or involved in genetic hereditary diseases or complex genetic diseases, immune diseases, cancers, viral infections in various tissues/organs or tumors.
  • the present invention also relates to the in vitro or ex vivo use of therapeutic products as defined herein in the production of biologies, in particular biologies encoding a recombinant protein, a peptide or an antibody; or in the production of recombinant virus, such as adeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolytic virus, or baculovirus, or viral or virus-like particles, in particular said products comprising the polyproline-based block copolymers of the invention and at least one nucleic acid molecule for transfection.
  • AAV adeno-associated virus
  • LV lentivirus
  • adenovirus adenovirus
  • oncolytic virus or baculovirus
  • viral or virus-like particles in particular said products comprising the polyproline-based block copolymers of the invention and at least one nucleic acid molecule for transfection.
  • the term "biologies” refers to proteins or nucleic
  • the present invention also relates to an in vitro or ex vivo use of the therapeutic products as defined herein, in particular said products comprising the polyproline-based block copolymers of the invention and at least one nucleic acid molecule for transfection, for genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
  • compositions for transfecting cells comprise the therapeutic products as defined herein, in particular said products comprising the polyproline-based block copolymers of the invention and at least one nucleic acid molecule for transfection, and an acceptable excipient, buffering agent, cell culture medium, or transfection medium.
  • the present invention is also directed to the therapeutic products as defined herein for use as a therapeutic or prophylactic vaccine against viral infections, or a therapeutic vaccine against cancers.
  • the vaccine is delivered through direct administration such as systemic, intramuscular, intradermal, intraperitoneal, intratumoral, oral, topical, or sub-cutaneous administration, and, in said vaccine, the composition is in association with a pharmaceutically acceptable vehicle.
  • the vaccine can be injected directly into the body, in particular in a human individual, for inducing a cellular and/or a humoral response.
  • the cell targeting is achieved through different mechanisms and depends on the nature and properties of the transfection reagent, method or protocol composition or formulation and the route of administration.
  • polyproline-based block copolymers of the invention the compounds comprising the structural unit of formula (I') or (I”), the self-assembled particles, and compositions thereof may be used also in cosmetic and diagnostic applications.
  • a diagnostic product which is or which comprises: a') the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively b') the compound comprising the structural units of formula (I') or (I”), wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively c') the star-shaped polymer, wherein at least one of L4 or R8 comprise a diagnostically active agent; or alternatively d') a self-assembled particle as defined herein, which comprises a diagnostically active agent, or alternatively e') a composition as defined herein, comprising a diagnostically active agent; for use in diagnostics.
  • This aspect of the invention may also be formulated as the use of a') the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively b') the compound comprising the structural units of formula (I') or (I”), wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively c') the star-shaped polymer, wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively d') a self-assembled particle, which comprises diagnostically active agent, or alternatively e') a composition as defined herein, comprising a diagnostically active agent in diagnostics.
  • the diagnostic of a disease or condition comprising administering a') the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprise a diagnostically active agent; or alternatively b') the compound comprising the structural units of formula (I') or (I”), wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively c') the star-shaped polymer, wherein at least one of L4 or R8 comprise a diagnostically active agent; or alternatively d') a self-assembled particle, which comprises diagnostically active agent, or alternatively e') a composition as defined herein, comprising a diagnostically active agent; in a subject in need thereof, more particularly a mammal, and even more particularly a human.
  • the invention relates to microbubbles which (a'), (b'), (c'), (d’) or (e’) as defined herein and one or more diagnostically active agents, in particular contrast agents, for use in diagnostics.
  • imaging diagnostic techniques suitable for the present disclosure include, but not limited to, are magnetic resonance imaging (MRI), X-ray, positron emission tomography (PET), single-photon emission computed tomography (SPECT), fluorescence microscopy, and in vivo fluorescence.
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • fluorescence microscopy and in vivo fluorescence.
  • the invention relates to the use in cosmetics of a cosmetic product which is or which comprises: a") the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprise a cosmetically active agent; or alternatively b”) the compound comprising the structural units of formula (I') or (I”), wherein at least one of L4 or R8 comprises a cosmetically active agent; or alternatively c”) the star-shaped polymer, wherein at least one of L4 or R8 comprise a cosmetically active agent; or alternatively d”) the self-assembled particle, which comprises a cosmetically active agent, or alternatively e”) a composition as defined herein, comprising a cosmetically active agent
  • kits comprising a therapeutic, diagnostic, or cosmetic product as defined herein, and optionally means for its use.
  • the kit of the present invention comprises: the therapeutic, cosmetic or diagnostic product as defined herein; and optionally means to its administration.
  • Examples of appropriate means for their administration include reagents and/or solvents for its use, as well as equipment (such as syringe) and instructions for its use.
  • the use in therapy can be also drafted as a method for the prophylaxis and/or treatment of a disease which comprises administering to mammals in need of such treatment an effective amount of the polyproline-based block copolymer or the conjugate disclosed herein above in the fifth aspect of the invention, together with one or more appropriate pharmaceutically acceptable excipients or carriers.
  • the use in therapy can be also reformulated as the use of the polyproline-based block copolymer as defined herein above and below or the conjugate disclosed herein above and below in the fifth aspect of the invention for the preparation of a medicament for the prophylaxis and/or treatment of a disease or condition.
  • the type of disease or condition to be treated depend on the pharmacological activity of the active ingredient of the polymer and/or the molecule of interest of the molecule.
  • Chemicals and solvents are either A.R grade or purified by standard techniques.
  • Cell lines used in the present application are commercially available.
  • the triple-negative breast cancer cell line MDA-MB 231 luc is available from ATCC.
  • NMR spectra were recorded at 27 °C (300 K) on a 300 UltrashieldTM from Bruker (Billerica MA, USA). Data were processed with the software Topspin (Bruker GmbH, Düsseldorf, Germany). Samples were prepared at a concentration of 20 - 10 mg/mL approx, in the required solvent.
  • the secondary structure was determined by Circular Dichroism.
  • CD Spectroscopy was performed with a J-815 CD Spectrometer (JASCO Corporation) using a Peltier thermostated cell holder (PTC-423, JASCO15 Corporation) with a recirculating cooler (JULABO F250, JASCO Corporation).
  • a nitrogen flow ( ⁇ 1.5L/min) was lead through the spectrometer and controlled with a nitrogen flow monitor (Afriso Euro-lndex).
  • PicoQuant MT200 confocal microscope with a 60x Na1 .2 UPlanApo water immersion objective and excitation line 488 nm was used to perform the Fluorescence Correlation Spectroscopy (FCS) analysis.
  • FCS Fluorescence Correlation Spectroscopy
  • the signal is directed onto two single photon avalanche diode (SPAD) detectors arranged in Hanbury-Brown and Twiss (HBT) geometry (i ,e. , emitted light is split using a 50/50 beam splitter and sent to two SPAD detectors).
  • SCS Fluorescence Correlation Spectroscopy
  • NMR DOSY Diffusion-ordered NMR spectroscopy
  • FEI Tecnai G2 Spirit Biotwin 120 kw transmission electron microscope was used to determine the size of 1mg/mL solution in phosphate buffer 1 M pH 7.4. 1% solution of uranyl acetate dissolved in ddH2O was used for the negative staining of the sample.
  • FEI Tales Arctica Transmission Electron Microscope (University of Groningen, ND) was used the cryo-TEM analysis of the samples.
  • HEK293 cells employed in Example 9 were cultured in DMEM high glucose with Glutamax (Gibco- Thermo Fisher # 61965-059) supplemented with 10% of Fetal Bovine Serum (Hyclone # SV30160.03HI, provided by GE Healthcare Europe GmbH) and 1 % penicillin/streptomycin. Transfections were carried out on 96-well plates containing 30000 cells/well in a final volume of 100 pl, and cells were incubated 24 hours at 37 °C and 5% CO2. After 24 h from cell seeding, the medium was removed and refreshed with 90 pl of complete medium. 10 pl of each LNPs formulation were added to the cells. After 24 hours cells were recovered and processed.
  • ATPLite reagent ATPLite PerkinElmer #6016731
  • Luminiscence was read spectrophotometrically using VictorNivo (PerkinElmer) following manufacturer's instructions and data was represented as the percentage of cell viability, taken untreated control cells as 100%.
  • Luciferase Assay for transfection efficiency evaluation [00247] After 24h post-incubation with the LNPs formulation, 100 pl of BrightGlo reagent (Promega # E2620) was added in each well following manufacturer instructions. After 5 minutes of incubation at room temperature luciferase activity was measured using VictorNivo (PerkinElmer). Data was represented as luminescence relative to the percentage of transfection relative to the positive control of transfection.
  • the uptake studies were performed by confocal microscope (Leica TCS-SP8) equipped with 4 laser lines for fluorescence excitation (405 nm, 488 nm, 561 nm, and 638 nm), 4 detectors for simultaneous data acquisition, CO2 and temperature control, a resonant scanner for live-cell studies, high content screening automation (HCS A) module, and super-resolution module (HyVolution II). Pulse chase and time-lapse studies were performed at non-toxic concentration of the selected polymers and polymer-drug conjugates using the following fluorescent markers: MitoTrackerTM Red CM-H2Xros M7513 (mitochondria, abs/ems.
  • the early endosomes marker was added o/n, non-toxic concentrations of the polymers were added 2, 4, 6 h and o/n, and the mitotracker and lysotracker were added 1 h before the analysis.
  • the internalization and the mitochondria colocalization were observed by confocal microscope Leica TCS-SP8.
  • Ornithine Z or Lysine Z N-carboxyanhydride was added to a Schlenk tube fitted with a stirring bar and a stopper. After 3 cycles of vacuum/N2, the mixture was dissolved in anhydrous DMF. Then, the initiator (n-Butylamine) diluted in DMF (0.5 mL) and was added to the reaction mixture, which was stirred at 10 °C for 16 hours. Once NCA consumption was confirmed by IR the obtained solution was added to Proline NCA previously suspended in anhydrous acetonitrile. The mixture was stirred at room temperature for 16 hours. The reaction mixture was precipitated with diethyl ether. The precipitate was , into diethyl ether to precipitate the product.
  • Trifluoroacetyl-L-lysine N-carboxyanhydride (2 g, 7 mmol) was added to a Schlenk tube fitted with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, and dissolved in anhydrous DMF (8 mL). Then, the star initiator (tetradecylamine) was dissolved in DMF (2 mL) and was added to the reaction mixture. The mixture was stirred at 10 °C for 16 hours. The reaction mixture was poured into water to precipitate the product. The precipitate was isolated by filtration and freeze-dryedg. Tetradecyl- PlysfTFA] was isolated as a white solid. Yield: 70-90%
  • Bz-PProline is synthesized as described in example 1.1 via ring-opening polymerization by employing n-Benzylamine as initiator. Polymer was isolated as a white solid and used in the next step without further characterization. Yield: 70-80%. Homopolyproline was dissolved in TFE and left stirring for 16 hours. TFE was evaporated and polymer redissolved in water. Desired product was obtained after dialysis (Vivaspin®, 3000 MWCO Da or 500-1000 MWCO Float-A-Lyzer®). White powder was obtained after freeze-drying. Yield: 50-90%.
  • the total drug loading was calculated by 1 H-NMR analysis (TDL, mol %). Due to signal overlapping, TOS conjugates drug loading was performed by UV-Vis obtaining the %weight drug which later was back-calculated (b.c) to %mol. [00262] For all the synthesized diblock copolymers, the total drug loading (TDL) determination (%wt) by
  • UV-vis was established after the calibration curve preparation of the free drugs and matrix effect adjustment, defined as the naked polymer's influence on the conjugates abs. The measurements were performed considering the lonidamine abs at 298 nm and TOS at 285 nm.
  • Atto488 estimation was carried out by fluorescence (Atto488-NHS «ex:500 «em:520) after preparing the calibration curve in DMF:H2O. %wt was 1.7; while Cy5 estimation was carried out by fluorescence (Cy5- NHS «ex: 646 •em:662) after preparing the calibration curve in DMF:H2O. %wt was 0.5-1.8.
  • Table 11 Summary of the compounds obtained indicating the corresponding polymer precursor and dye.
  • Compound 16 was synthesized through a convergent process, which initially involved the synthesis of 1a-VC-Mal and 15-PD separately.
  • the VC functionalization was calculated by 1 H-NMR analysis (mol %) and UV-vis (% wt) after the calibration curve preparation of the free VC and matrix effect adjustment, defined as the naked polymer's influence on the conjugates absorbance. The measurements were performed considering the VC abs at 250nm.
  • the pH was adjusted at 8 with sodium bicarbonate 1 M. The reaction was left to proceed for 48 hours. The product was precipitated in cold Et 2 O while stirring, left at -20° for 1 hour, centrifuged and the pellet was lyophilized o/n. To convert the product to its salt form, NaHCOs 1 M solution was added drop by drop and mixing until pH 7/8 and the perfect dissolution at RT. After that, the product was purified by VivaspinTM 3,000 MWCO.
  • the PD functionalization was calculated by 1 H-NMR as described for VC. but with a calibration curve preparation of the free PD-NH 2 and matrix effect adjustment.
  • Compound 16 was derivatized with fluorophores Atto488-NHS (16b) or Cyanine5-NHS (Cy5- NHS) (16a). Briefly, in a one-neck bottom flask, 16 was dissolved in ddH2O (1 eq, 10 mg/mL). In a glass vial protected by light, the corresponding fluorophore was dissolved in DMSO and added to the polymer solution (0.02 eq, 2% mol). The pH was adjusted with sodium bicarbonate 1 M. The reaction was allowed to proceed o/n protected by light and checked by TLC (m.p MeOH). The products were purified by Sephadex G25 (PD10 column).
  • Atto488 estimation was carried out by fluorescence (Atto488-NHS «ex:500 «em:520) after preparing the calibration curve in DMF:H2O. %wt was ⁇ 1%; while Cy5 estimation was carried out by fluorescence (Cy5-NHS «ex: 646 •em:662) after preparing the calibration curve in DMF:H2O. %wt was ⁇ 1%.
  • the helix type II stability was also evaluated for the selected diblock copolymer 1a (0.25 mg/mL) Specifically, we verified that the secondary structure was maintained after different cycles of freezing (o/n) and defrosting and at 37° C in phosphate buffer 1 M pH 7 (1 h).
  • FCS Fluorescence Correlation Spectroscopy
  • the hydrodynamic radius was also determined by diffusion-ordered NMR spectroscopy (NMR DOSY). As FCS, this technique allows to obtain the D of the particles in solution. Compounds 1a, 9a_5 and 9b_6 were dissolved in dPBS 1 M pH 7.4 and the D were extracted from the 2D NMR spectra.
  • CMC of TOS conjugates was checked by fast fluorimetric method using pyrene as probe according to Li et al. Pyrene fluorescent pattern (band I, 371/372 nm and III, 383/383 nm) increases when migrating from hydrophilic to hydrophobic regions. Serial dilutions of 9b were prepared, and 10 pL of a 0.02 mg/mL pyrene solution was added to each concentration. After acetone evaporation (37° C, 1 h), the solutions were stabilized o/n. The samples were analyzed at the spectrofluorometer (exc. 335 nm; ems I 374 nm, ems III 385 nm).
  • MDA-MB-231 cells were initially seeded into two 12-well plates and allowed to adhere for 24 hours at 37°C. Subsequently, 0.15 mg/ml of compounds 10a and 16a were introduced at 0, 30, 60, and 180-minute time points in the two plates. One plate was maintained at 37°C, while the other was stored at 4°C during the respective incubation periods. Following this, the cells were scratched, and then harvested into separate tubes for each condition. Subsequently, they were incubated with propidium iodide (2ug/ml), a marker for cell viability. The resulting samples were then analyzed using flow cytometry. The findings indicate that both compounds 10a and 16a permeate the cell membrane within 30 minutes at 37°C. However, when incubated at 4°C, the direct cell membrane permeation of 10a was delayed compared to 16a. The results suggest that both compounds enter the cells using a predominantly endocytosis independent mechanism.
  • Example 4 In vitro validation of polypeptide-drug conjugates 9a and 9b and the corresponding free drugs (lonidamine and TOS).
  • the early endosomes marker was added overnight, 0.2 mg/mL of the conjugate solutions (12b 12% wt, 13b 6.7% wt) were added at 2,4,6 h and overnight, and the mitotracker and lysotracker were added 1 h before the analysis. Both compounds 12b and 13b colocalized with mitochondria at short times ( ⁇ 2h, Pearson r > 0.8) and this colocalization was slightly reduced when tested overnight, meaning that the drug conjugation improved the mitochondria tropism for the polymer PLO:PPro 6:22 (10a).
  • C1, C2, and C3 are the three concentrations tested for each of the compounds as:
  • conjugates 9a and 9b decrease the mitochondria membrane potential.
  • the Agilent Seahorse XF Mitostress Test measures critical parameters of the mitochondrial function by directly measuring the oxygen consumption rate of the cells after adding modulators of the respiration.
  • the modulators include oligomycin, an inhibitor of the ATP synthase; carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP), an uncoupling agent that collapses the proton gradient; and rotenone/antimycin, respectively, inhibitors of the complex I and III.
  • the cells were seeded in 96-well plates and incubated at 37° and 5% CO2.
  • Concentrated stock solutions of the polymers were prepared in phosphate buffer 1 M pH 7 and diluted in DMEM/F-12 medium (with 10% FBS and 1% P/S).
  • the polymer solutions were then sterilized (0.2 - m filter) and added to give a final concentration of - IC40 (C1) and diluted solution (C2; dilution factor 1.5).
  • their culture medium was substituted with the DMEM XF medium (with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose).
  • the inhibitors were loaded into the injection ports of the XFe96 Sensor Cartridge, previously hydrated for 24h with the XF calibrant solution (final concentration oligomycin 1 .5 iM, FCCP 1 iM, and Rot/AA 0.5 iM).
  • the results were normalized by Hoechst 3342 marking and the optimal seeding density for the MDA-MB 231 cell line was 2x10 4 cells/well, which were applied in the 24 hours protocol of lonidamine, TOS, 1a, 16, 9a_1 and 9b_1 treatment.
  • both drugs (data not shown) and the polymer 1a affected the cellular metabolism related to the mitochondria function. Specifically, lonidamine induced a reduction of the ATP-production coupled respiration and slight and non-significant of the spare respiration capacity. TOS induced a significant and non-concentration-dependent reduction of both ATP-production coupled respiration and spare respiration capacity. Interestingly, 1a induced a significant and non-concentration- dependent reduction of ATP-production coupled respiration, while it didn't affect the spare respiration capacity. This result suggests, together with the JC-1 data, that polymer 1a acts as a bioactive molecule itself. Treatment with compound 16 did not show any impact on the mitochondria respiration, proving its safer profile while maintaining its mitochondria targeting ability. The conjugates affected the cellular metabolism related to mitochondria respiration. Specifically, the conjugates maintain the ability to decrease the ATP-production coupled respiration and the spare respiration capacity for 9b_1, even at low drug concentrations in equivalents.
  • MDA-MB-231 cells were initially seeded into a 6-well plate and allowed to adhere for 24 hours at 37°C. Subsequently, 0.4 mg/ml of 10a and 16a, 0.08 mg/ml of 10h, and 0.06 mg/ml of 10i were added to separate wells for a 24-hour treatment. After washing with PBS, try psinization, and centrifugation, the cell pellets were resuspended in fresh DMEM/F-12 medium (with 10% FBS and 1% P/S), and then incubated with 5 ig/ml (final concentration) of 2,7-dichlorodihydrofluorescein (DCFH) for
  • the compound 3,6-di(azetidin-1 -yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide is an analog of 10-N-nonyl acridine orange (NAO) with increased photochemical properties, water solubility and cardiolipin (CL) affinity described by Dimitrijevs P. & Arsenyan P. This fluorescent probe was used in quantitative competitive binding studies to measure and compare the affinities of various substances for CL.
  • Cyt c was used as a positive control (CLDOPC liposomes ref. EC50 0.26 ⁇ 0.02; mitoplast ref. EC50 0.78 ⁇ 0.15
  • NonylTPP was used as a positive control (CLDOPC liposomes % fluorescence intensity
  • lipid compositions consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 18:1 cardiolipin (CL) at 3:1 molar ratio were used, with an addition of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) labeled with Atto-655 at the headgroup.
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • POPG 1 -palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
  • POPG has a negatively charged headgroup at neutral pH
  • CL has two negative charges at its headgroup due to two phosphate groups.
  • the lipid composition of the negative control was chosen to achieve equipotential membranes with CL and PG.
  • the molar amounts for the labeling varied for fluorescence spectroscopy and microscopy measurements.
  • the different lipid bilayer systems employed for the in vitro affinity studies were obtained by the following methods: PVA assisted swelling method for the formation of giant unilamellar vesicles (GUV); and lipid film rehydration method for the formation of small unilamellar vesicles (SUV) and supported lipid bilayers (SLB).
  • sample was also imaged on a PicoQuant MT200 confocal microscope with a 60x Na1.2 UPlanApo water immersion objective.
  • the membrane irregularities showed two components in the fluorescence lifetime histogram, with fitted values of T i, T 2 peaks at 3.0 and 4.5 ns respectively.
  • the presence of an increased lifetime hints at changes in the lipid packing locally where the polymers bind with the membrane.
  • FCCS Fluorescence Correlation Spectroscopy
  • PicoQuant MT200 microscope were used.
  • FCS autocorrelation curves for the individual channels were fitted with a two component 3D diffusion model, and the cross-correlation curve was fitted with a 3D diffusion model respectively.
  • the focal volume overlap was calculated using a DNA oligo sample with both 5' and 3' end labeled with the respective fluorophores.
  • the DNA oligomers used as a FCCS standard are double-stranded DNA, consisting of a labeled ssDNA with 5' Atto655 and 3' Atto488 labels on the sequence CTTTTCTTCTTTTCTTTCTT (SEQ ID NO. 1) which is mixed with it's complementary unlabeled ssDNA AAGAAAGAAAAGAAGAAAAG (SEQ ID NO. 2). It was observed that for both POPC/CL and POPC/POPG based vesicles, there was a cross-correlation curve present that indicates co-diffusion of both the labeled polymers and the labeled vesicles.
  • the vesicles with POPC/CL showed a higher molecular brightness that confirms a higher affinity of 11a for CL-based vesicles.
  • the Zeta potential of the POPC/CL (3.1) and POPC/POPG (1 :1) SUVs were measured using the Malvern ZetaSizer Nano incubating with PLO:PPro 6:22 (1a), PArg:Ppro 6:22 (3a), and PHis:PPro 7:26 (4).
  • the zeta potential measurements showed that there was a higher change in the zeta potential of the negatively charged POPC/CL vesicles than the POPC/POPG vesicles with the addition of all positively charged polymers (1a, 2a, 3a).
  • oligonucleotides used were: a mRNA purchased from CATUG Biotechnology, with reference FLuc mRNA (N 1 -Me-Pseudo UTP) Cat. number CT072; Lot number P23H065 expressing luciferase as reporter gene.
  • the desired amount of mRNA and the calculated amount of the cationic polymer at indicated charge-ratio (+/• ) or amine to phosphate ratio (N/P) were diluted in separate tubes in MES Buffer Glucose (MBG) 10 mM, Glucose 5% w/v pH 6.1. Only protonatable nitrogens, not amide nitrogens, were considered in the +/• ratio and N/P ratio calculations.
  • the cationic polymer solution and the genetic material solution were mixed by rapidly pipetting up and down (ten times) and incubated for 20 min at RT. Then the polyplexes formed were characterized by DLS to determine Z-average (nm) and PDI.
  • oligonucleotides used were: a mRNA purchased from CATUG Biotechnology, with reference FLuc mRNA (N 1 -Me-Pseudo UTP) Cat. number CT072; Lot number P23H065 expressing luciferase as reporter gene. Any other oligonucleotide with the characteristics above indicated could be used to carry out the experiments below.
  • the ionizable lipid DODMA, or 1 ,2-dioleyloxy-3-dimethylaminopropane was purchased from Avanti Polar Lipids. Shielding lipid 1 ,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) was purchased from Avanti Polar Lipids. Both polymers were used to form benchmark LNPs. Structural lipids used to form LNPs were: dioleoylphosphatidylethanolamine (DOPE, from Sigma- Aldrich) and Cholesterol (Sigma-Aldrich).
  • DOPE dioleoylphosphatidylethanolamine
  • Cholesterol Sigma-Aldrich
  • Lipids were dissolved in ethanol at 50-x/10/38.5/1 .5 molar ratio (DODMA/DOPE/Cholesterol/ DMG-PEG), where x is the molar ratio corresponding to the compound of interest (1a or 5c).
  • LNPs were further diluted in 10 mM Phosphate buffer saline (PBS) pH 7.4 to decrease ethanol concentration and purified with centrifugal concentrators (VivaspinTM 500, Sigma) to remove ethanol.
  • PBS Phosphate buffer saline
  • the formulation containing the polymer of the invention was prepared analogously with the difference that different compounds were used instead of ionizable lipid DODMA and the lipid ratio was different (shown below Table 26 and 27).
  • LNPs containing DODMA as ionizable lipid as well as the compounds of interest 1c and 5c, DMG-PEG as shielding lipid and mRNA as cargo were formulated at 50/10/38.5/1.5 molar ratios for DODMA/DOPE/Cholesterol/DMG-PEG obtaining the following results:
  • Table 25 shows the results obtained of LNPs containing 1a and 5c formulated at molar ratio of 10% containing mRNA as cargo.
  • oligonucleotides used were: a mRNA purchased from CATUG Biotechnology, with reference FLuc mRNA (N 1 -Me-Pseudo UTP) Cat. number CT072; Lot number P23H065 expressing luciferase as reporter gene.
  • the transfection efficiency and the cell viability of the LNPs formulations in HEK293 cells is reported in the following table.
  • the transfection data is represented as % of the positive control DODMA LNPs with the formulation reported 50-x/10/38.5/1 .5 molar ratios for DODMA-compound 1a/DOPE/Cholesterol/DMG-PEG being x the different molar ratios, and being the positive control 100% after 24h of treatment and cell viability is compared to non-treated (NT) cells, being the ATP content readout of NT (non-treated) cells equal to 100%.
  • NT non-treated
  • Li is a biradical selected from the group consisting of wherein the wavy lines denote the attaching points; wherein each A and A' is independently selected from -O-, -CO- and -NH-; wherein y and z are integers independently ranging from 1 to 20; and wherein each Z is a biradical selected from the group consisting of -NH(Ci-C6)alkyl-O-, -NH(Ci-C 6 )alkyl-NH-, -NH-(Ci-C 6 )alkyl-CO-, -O-(Ci-C 6 )alkyl-CO-, -O-(Ci-C 6 )alkyl-O-, -CO-(Ci-C6)alkyl-CO-, a straight or branched -NH-(Ci-C3o)alkylene-0-, -NH-(Ci-C3o)alkylene-NH-, -NH-(Ci-C3o)
  • R' 2 , R" 2 , R v2 and R v2 are each independently selected from the group consisting of H, -(Ci-C3o)alky I, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl NH2, -(Ci-C3o)alkyl-N((Ci-C3o)alkyl)2, -(Ci-C3o)alkyl-NH(Ci-C3o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI)
  • R v ' 2 and R v ' 2 ’ are each independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C 2 -C 3 o)alkenyl, -(C 2 -C 3 o)alkynyl, -OAIkyl(Ci-Ci 2 ), F, Cl, Br, I, -CF 3 , -OCF 3 , -NO 2 , -CN, -NH 2 , -(Ci-C 3 o)alkylNH 2 , -N((Ci-C 30 )alkyl) 2 , and-NH(Ci-C 30 )alkyl, wherein R vi ' 2 , RTM 2 , R ix2 , and R x2 are each independently selected from the group consisting of H, -(Ci-Ci 2 )alkyl, -(Ci-Ci 2 )alkylNH 2 , -(C
  • each R' 1 is independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2- C3o)alkynyl, -OAIkyl(Ci-C6), halogen, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO 2 , -CN, -OC(O)-(Ci-C 30 )alkyl, -OC(O)O(Ci-C 30 )alkyl, -OC(O)NH 2
  • R ii1 , R iii1 , R' v1 , R v1 and R vi1 are each independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl NH2, -(Ci-C3o)alkyl-N((Ci-C3o)alkyl)2, -(Ci-C3o)alkyl-NH(Ci-C3o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein R' 1 , R ii1 , R iii1 , R' v1 ,
  • T and T are each independently selected from -OH, -OCORx and -COORx;
  • each Rs is independently selected from selected from the group consisting of H, -(Ci-Cis)alkyl, -(C 2 -Ci 8 )alkenyl, -(Ci-Cis)alkyl-R 1 , -(Ci-Cis)alkyl-COOR i1 , -(Ci-Cis)alkyl-O-R" 1 , -(Ci-Ci 8 )alkyl-NR iv1 R v1 , -C(O)-R vi1 , -(Ci-Ci8)alkyl-CO-NH2, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XIXIII
  • R' 1 , R iii1 , R v1 , R v1 and R vi1 are each independently selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, -(Ci-Cis)alkylNH2, -(Ci-Cis)alkyl-N((Ci-Cis)alkyl)2, -(Ci-Cis)alkyl-NH(Ci-Cis)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein R 1 , R ii1 , R iii1 , R v1 , R v1 , and R vi1 are each independently
  • each Rs is independently selected from selected from the group consisting of H, -(Ci-Cis)alkyl, -(C 2 -Ci 8 )alkenyl, -(Ci-C 6 )alkyl-R i1 , -(Ci-C6)alkyl-COO i1 , -(Ci-C 6 )alkyl-O-R iii1 , -(Ci-C 6 )alkyl-NR iv1 R v1 , -C(O)-R vi1 , -(Ci-C6)alkyl-CO-NH2, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XVII), (XXVII),
  • R' 1 , R iii1 , R v1 , R v1 and R vi1 are each independently selected from the group consisting of H, -(Ci-Ci 8 )alkyl, -(C2-Cis)alkenyl, -(Ci-Cis)alkylNH 2 , -(Ci-Cis)alkyl-N((Ci-Cis)alkyl) 2 , -(Ci-Cis)alkyl-NH(Ci-Cis)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein R 1 , R ii1 , R iii1 , R v1 , R v1 , and
  • each R8 is a radical independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, n-butyl, -CH2SCH3, -CH2CH2SCH3, -CH2CH2SCH2CH3, -CH2SCH2CH3, -CH 2 SH, -CH2CH2SH, -CH 2 SeH, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2NH2, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); wherein each Rs is independently substituted by at least one detection moiety, at least one active moiety, at least one lipid-like moiety Rw, or a mixture thereof; wherein each Rw is independently selected from the groups consisting of -(Ci-C )alky I, -(
  • Li is a biradical selected from (II) and (III), which is attached to the C-terminal end AA1 , AA3 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond;
  • L4 is selected from the group consisting of an inert moiety, a detection moiety, an active moiety, and a lipid-like moiety Rw; wherein the inert moiety is selected from the group consisting of H, -N3, -C(O)H, -S(O)H, -NHCH(CH3)2, -(Ci-C 4 )alkylNH2, -(Ci-C4)alkylNHCH 3 , -(Ci-C4)alkylN(CH 3 ) 2 , -O-(Ci-C4)alkyl-NH 2 ,
  • the inert moiety is optionally substituted by a detection moiety, an active moiety, and a lipid-like moiety Rwor a mixture thereof;
  • the detection moiety is selected from the group consisting of chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, transition metal and isotope mass tag moiety;
  • the active moiety is selected from the group consisting of pharmaceutically active agents, veterinary active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures
  • each R8 is independently a cationic moiety selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2NH2, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); wherein each Rs are optionally independently substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety R10; or a linker; the linker being attached to at least one inert moiety, or alternatively optionally attached to at least one detection moiety, or alternatively optionally attached to at least one active moiety or at least one lipid-like moiety R10, or alternatively optionally attached to at least one pH, redox or protease responsive unit; or a
  • n 0;
  • L4 is selected from the group consisting of an inert moiety, a detection moiety, an active moiety, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a lipid-like moiety R10; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; each R8 is independently a cationic moiety selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2NH2, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII
  • a star-shaped polymer comprising a star-shaped multifunctional linking agent and at least one polyproline-based block copolymer of formula (I) according to any of clauses 1 -6 attached thereto; and wherein the star-shaped multifunctional linking agent is selected from the group consisting of
  • L is selected from K1-CH2CH2-S-S-CH2CH2-K2; K1-CH2CH2-K2; and a moiety of formula (St_a) wherein K, Ki and K2 are each independently selected from -0- and -NH-; p is an integer selected from 2-4; wherein in the moiety of formula (St_a) each wavy line in (St_a) denotes the attaching points to the polymer comprising the backbone of repeating structural units of formula (I); when L is (St_a), then (St_a) attaches to the CO moiety in (St) through the K moiety; when L is selected from K1-CH2CH2-S-S-CH2CH2-K2 or K1-CH2CH2-K2, then L attaches to the CO moiety in (St) through the K1 or K2 moieties, and the other K2 or K1 attaches to the polymer comprising the backbone of repeating structural units of formula (I); wherein
  • E and E’ are each independently selected from -0- and -NH-; wherein the 3-arm star-shaped linking agent of formula (Pm) is attached to the L4 moiety of each of the at least one polyproline-based block copolymer of formula (I) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond; iii) a 4-arm star-shaped linking agent of formula (Tz) wherein each V is selected from -0- and -NH-;
  • Y, Y' and Y are each independently selected from -0- and -NH-; wherein the 4-arm star-shaped linking agent of formula (Tz) is attached to the L4 moiety of each of the at least one polyproline-based block copolymer of formula (I) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond.
  • L is selected from K1-CH2CH2-S-S-CH2CH2-K2 and K1-CH2CH2-K2; wherein p, ' and p” are each independently an integer from 0 to 1; wherein Ki and K2 are each independently selected from -0- and -NH-; wherein L attaches to the CO moiety in (StP) through the K1 or K2 moieties, and the other K2 or K1 attaches to the polymer comprising the backbone of repeating structural units of formula (I); wherein the 3-arm star-shaped linking agent of formula (StP) is attached to the polymer comprising the backbone of repeating structural units of formula (I) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond.
  • a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an
  • a self-assembled particle comprising the polyproline-based block copolymer of formula (I) as defined in any of clauses 1-12, or the star-shaped polypeptide as defined in any of clauses 13-14, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • Clause 16 A composition comprising the polyproline-based block copolymer of formula (I) according to any of clauses 1-12, the star-shaped polymer as defined in any of clauses 13-14, or alternatively, the self-assembled particle of clause 15, together with one or more appropriate excipients or carriers.
  • a therapeutic product which comprises: a) the polyproline-based block copolymer of formula (I) as defined in any of the clauses 1-12, wherein at least one of L4 or R8 is a pharmaceutically active agent; or alternatively b) the star-shaped polymer as defined in any clauses 13-14, wherein at least one of L4 or R8 is a pharmaceutically active agent; or alternatively c) a self-assembled particle as defined in clause 15, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively d) a composition as defined in clause 16, comprising a pharmaceutically active agent; for use in medicine.
  • Clause 18 The therapeutic product for use according to clause 17, for use (i) as transfection reagent for transfecting at least one active agent into a cell; (ii) for use in the in vivo or ex vivo production of biologies encoding a recombinant protein, a peptide or an antibody, or in the production of recombinant virus; (iii) for use as a therapeutic or prophylactic vaccine against viral infections or as a therapeutic vaccine against cancers or infectious diseases; or (v) for use in genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
  • a diagnostic product which comprises: a') the polyproline-based block copolymer of formula (I) as defined in any of the clauses 1-12, wherein at least one of L4 or R8 is a diagnostically active agent; or alternatively b') the star-shaped polymer as defined in any of clauses 13-14, wherein at least one of L4 or R8 is a diagnostically active agent; or alternatively c') a self-assembled particle as defined in clause 15, which comprises diagnostically active agent, or alternatively d') a composition as defined in clause 16, comprising a diagnostically active agent; for use in diagnostics.
  • Clause 20 Use in cosmetics of a cosmetic product which comprises: a") the polyproline-based block copolymer of formula (I) as defined in any of the clauses 1-12, wherein at least one of L4 or R8 is a cosmetically active agent; or alternatively b”) the star-shaped polymer as defined in any of clauses 13-14, wherein at least one of L4 or R8 is a cosmetically active agent; or alternatively c”) the self-assembled particle as defined in clause 15, which comprises a cosmetically active agent, or alternatively d”) a composition as defined in clause 16 containing a cosmetically active agent.
  • Clause 21 Use of the polyproline-based block copolymer of formula (I) as defined in any of the clauses 1-12, wherein L4 and R8 are other than an active moiety; the starshaped polymer as defined in any of clauses 13-14, wherein L4 and R8 are other than an active moiety; or alternatively, a self-assembled particle as defined in clause 15, or a composition as defined in clause 16 containing either a polyproline-based block copolymer of formula (I) or the star-shaped polymer wherein L4 and R8 are other than an active moiety, as a carrier.

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Abstract

The present invention relates to polyproline-based block copolymers and compositions thereof, which e.g. are useful for delivering active ingredients, including nucleic acids, and/or imaging agents to target cells or tissues.

Description

Polyproline-based block copolymers
[0001] This application claims the benefit of the European Patent Application 23382668.4 filed on 29.06.2023.
[0002] This invention relates to polyproline-based block copolymers which comprise a polyproline moiety and a polycationic moiety. These polyproline-based copolymers can be used as non-viral vectors for delivery of active ingredients, including nucleic acids, and/or imaging agents to target cells or tissues.
Background art
[0003] It is well known that when a drug is administered systemically orally or by intravenous injection, the drug accumulates not only in the diseased area to which the drug is administered, but also in normal tissues. As a result, side effects due to drug administration are observed. In order to reduce side effects, drug delivery systems have been developed that selectively deliver drugs to affected areas. Recently, attention has turned to drug delivery to mitochondria in cells.
[0004] There exists a necessity to efficiently delivering drugs into cells, and more particularly to mitochondria.
[0005] Although endocytosis has generally been acknowledged as a major mode of uptake for cell penetrating peptides, only a few have been found to enter cells via a direct transport pathway, with sub- cellular localization at the mitochondria. A direct transport mechanism of cell entry is advantageous overall, as internalization of the cell penetrating peptides and its cargo would avoid endosomal entrapment, thereby increasing the possibility of direct sub-cellular localization.
[0006] Document W02023002012 discloses 3-arm star-shaped polycationically charged polymers consisting of a 1,3,5-benzenetricarboxamide related central core and 3 polypeptide backbone arms.
[0007] Document US2008125581 discloses a block copolypeptide polymer which is made from glutamic acid N-carboxyanhydride and proline N-carboxyanhydride.
[0008] Document US5204099 discloses a block copolypeptide polymer proline-glutamic acid.
Summary
[0009] The inventors have surprisingly found that a polyproline-based block copolymer of formula (I) or a compound comprising a structural unit of formula (I') or (I”), or a salt thereof as defined herein are useful as a chemical-mediated vector for the delivery of molecules of interest, such as active ingredients or detection moieties. More particularly, the polyproline-based block copolymer of formula (I) or the compound comprising a structural unit of formula (I') or (I”), or a salt thereof as defined herein may act as cell penetrating peptide for the cellular delivery of attached cargoes.
[0010] The use of the polyproline-based block copolymer compound of formula (I) or the compound comprising a structural unit of formula (I') or (I”) of the present invention may allow the simultaneous delivery of several molecules of interest conjugated to the polymer, since the backbone of the polyproline-based block copolymer compound of the invention can be modified by including in the structural repeating units one or more active ingredients (such as pharmaceutically, veterinary, and cosmetic active ingredients), and/or detection agents (such as chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, transition metal and isotope mass tag moiety). Therefore, the polyproline-based block copolymer compounds herein defined may be used in lab research, therapy, cosmetic or diagnostic circumstances.
[0011] As demonstrated in the experimental section, the polyproline-based block copolymer compound of the present invention is capable of successfully being internalized and colocalized in mitochondria of mammalian cell lines, including MDA-MB 231. In addition, it can be applied to many types of unicellular organisms, and to multicellular organisms.
[0012] Therefore, a first aspect of the invention relates to a polyproline-based block copolymer compound of formula (I), a salt thereof, or any stereoisomer or mixtures of stereoisomers, either of the compound of formula (I), or of any of its salts,
Figure imgf000003_0001
wherein the “*” denotes the attaching points; where the repetitive unit defined by square brackets with the numerical value m is denoted as PAA1;
Figure imgf000004_0001
wherein though the main repeating units AA1, AA2 and AA3 are shown in a particular order for convenience of description, said AA1, AA2 and AA3 main repeating units may be present in any order and may be block or randomly present; and wherein each of the repeating units PAA1, AA1, AA2 and AA3, may comprise blocks of monomer units which may be the same or different between each other; wherein m is an integer selected from 5 to 100; n is an integer selected from 0 to 150; p is an integer selected from 0 to 150; q is an integer selected from 0 to 100; wherein at least one of n, p or q is • 4 or alternatively n + p • 4; with the proviso that when p« 0, and R8 is not a cationic moiety, then n • 0; with the proviso that when n=0, then p« 0, and R8 is a cationic moiety; and with the proviso that when p=0, then n« 0; wherein Li may be present or absent;
Li, if present, is a biradical selected from the group consisting of
Figure imgf000004_0002
wherein the wavy lines denote the attaching points; wherein each A and A' is independently selected from -O-, -CO- and -NH-; wherein y and z are integers independently ranging from 1 to 20; and wherein each Z is a biradical selected from the group consisting of -NH(Ci-C6)alkyl-O-,
-NH(Ci-C6)alkyl-NH-, -NH-(Ci-C6)alkyl-CO-, -O-(Ci-C6)alkyl-CO-, -O-(Ci-C6)alkyl-O-, -CO-(Ci-C6)alkyl-CO-, a straight or branched -NH-(Ci-C3o)alkylene-0-, -NH-(Ci-C3o)alkylene-NH-, -NH-(Ci-C3o)alkylene-CO-, -0-(Ci-C3o)alkylene-0-, -0-(Ci-C3o)alkylene-CO-, -CO-(Ci-C3o)alkylene-CO- and a biradical of formula (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI)
Figure imgf000005_0001
wherein the “*” denotes the attaching points; wherein B and B’ are each a biradical independently selected from -O-, -NH-; and -CO-; wherein each D is a biradical selected from -0- and -NH-; wherein k is an integer selected from 1 to 12; preferably from 1 to 6; wherein the -NH-(Ci-C3o)alkylene-0-, -NH-(Ci-C3o)alkylene-NH-, -NH-(Ci-C3o)alkylene-CO-, - 0-(Ci-C3o)alkylene-0-, -0-(Ci-C3o)alkylene-CO-, -CO-(Ci-C3o)alkylene-CO- biradical of Z is optionally substituted with one or more radical selected from the group consisting of -OH, -NRaRb,-SH2, -NHNH2, -COORc, -CF3, -OCF3, and halogen; wherein Ra, Rb and Rc are each a radical independently selected from the group consisting of H, -phenyl, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkylphenyl, and -phenyl(Ci-C3o)alkyl. wherein L1 is attached to the AA1 , AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond when attached to the N-terminal end of AA1 , AA2 or AA3 repeating unit; or alternatively L1 is attached to the AA1 , AA3 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond when is attached to the C-terminal end of the AA1 , AA2 or AA3 repeating unit; wherein R2 and R4, are each a biradical independently selected from the group consisting of -(Ci-C6)alkyl-, -(Ci-C6)alkyl-S-S-(Ci-C6)alkyl-, -(Ci-C6)alkyl-O-(Ci-C6)alkyl-, and -(Ci-C6)alkyl-NH-(Ci-C6)alkyl-;
R2 and R4 are each independently optionally substituted by one or more substituents selected from the group consisting of -NH2 and -(Ci-Ce)alkyl-NH2; each R3 is a radical independently selected from the group consisting of H and -(Ci-Ce)alkyl; each W1 is independently selected from CH and N;
Rs and Re are each independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C30)alkenyl, -(C2-C30)alkynyl, -(Ci-C3o)alkyl-R2, -(Ci-C3o)aikyl-0-R"2, -(Ci-C3o)alkyl-NRiii2Riv2, -C(O)-Rv2, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII),
Figure imgf000006_0001
Figure imgf000007_0001
35 wherein denotes the attaching point; X2 in formula XVI is selected from -NH-, -COO-, and -O-; each R'2 is selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -OAIkyl(Ci-C6), halogen, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-C30)alkyl, -OC(O)O(Ci-C30)alkyl, -OC(O)NH2, -OC(0)N((Ci-C3o)alkyl)2, -SH, -S(Ci-C30)alkyl, -S(O)H, -S(O)(Ci-C30)alkyl, -S02(Ci-C3o)alkyl;
R'2, R'"2, R'v2 and Rv2 are each independently selected from the group consisting of H, -(Ci-C3o)alky I, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl NH2, -(Ci-C3o)alkyl-N((Ci-C3o)alkyl)2, -(Ci-C3o)alkyl-NH(Ci-C3o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI)
Rv'2 and Rv'2’ are each independently selected from the group consisting of H, -(Ci-C3o)alky I, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -OAIkyl(Ci-Ci2), F, Cl, Br, I, -CF3, -OCF3, -NO2, -CN, -NH2, -(Ci-C3o)alkylNH2, -N((Ci-C30)alkyl)2, and-NH(Ci-C30)alkyl, wherein Rvi'2, R™2, Rix2, and Rx2are each independently selected from the group consisting of H, -(Ci-Ci2)alkyl, -(Ci-Ci2)alkylNH2, -(Ci-Ci2)alkyl-N((Ci-Ci2)alkyl)2, -(Ci-Ci2)alkyl-NH(Ci-Ci2)alkyl, -O(Ci-Ci2)alkyl, -COH, -CO(Ci-Ci2)alkyl, and -C(C2-C3o)alkenyl, wherein R2, R'2, R"2, Riv2, Rv2, Rv'2, Rv'2’, Rvi'2, R™2, Rix2, and Rx2 are each optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -0(Ci-C3o)alkyl, -CF3, -OCF3, -NH2, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-C3o)alkyl-OH; b2, c2, d2, e2, f2, g2, h2, 12, j2, k2, b2', c2', d2', e2', and g2' are each independently an integer selected from 1 , 2, 3, 4, 5, and 6; r2, s2, t2 and t2' are each independently an integer selected from 0 to 200; preferably from 0 to 180, more preferably from 5 to 150, from 10 to 140; each Rz is independently selected from H and -CH3; each R9 is independently selected from H and -CH3; each Rs and L4 are independently selected from the group consisting of -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl-R1, -(Ci-C3o)alkyl-COORi1, -(Ci-C3o)alkyl-0-Riii1, -(Ci-C3o)alkyl-NRiv1Rv1, -C(O)-Rvi1, -(Ci-Ci8)alkyl-CO-NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety R10, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI) and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit, or a mixture thereof;
Figure imgf000009_0001
wherein denotes the attaching point;
12, v2, w2, x2, y2, z2 and o2 are each independently an integer selected from 1 to 6; each R'1 is independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2- C3o)alkynyl, -OAIkyl(Ci-C6), halogen, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-C30)alkyl, -OC(O)O(Ci-C30)alkyl, -OC(O)NH2,
-OC(0)N((Ci-C3o)alkyl)2, -SH, -S(Ci-C30)alkyl, -S(O)H, -S(O)(Ci-C30)alkyl, -S02(Ci-C3o)alkyl; R'1, 'i1, R'v1, Rv1 and Rvi1 are each independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl NH2, -(Ci-C3o)alkyl-N((Ci-C3o)alkyl)2, -(Ci-C3o)alkyl-NH(Ci-C3o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein R'1, Rii1, Riii1, R'v1, Rv1, and Rvi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -0(Ci-C3o)alkyl, -CF3, -OCF3, -NH2, -(Ci-C30)alkyl, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-C30)alkyl-OH; wherein each Rs and L4 are independently optionally substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety Rw; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; a1 is an integer selected from 0 to 1 ; with the proviso that R2 is absent when a1 =1 ; wherein L4 is attached to the nitrogen atom of the AA1 , AA2 or AA3 by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond; or alternatively L4 is attached to the carbonyl group of the AA1 , AA2 or AA3 by a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond; the inert moiety is selected from the group consisting of H, -N3, -NH2, -C(O)H, -S(O)H, -NHCH(CH3)2, -(Ci-C4)alkylNH2, -NH-(Ci-C4)alkyl, -(Ci-C4)alkylNHCH3, -(Ci-C4)alkylN(CH3)2, -(O-CH2-CH2)7r-(Ci-C4)alkyl, N3-(O-CH2-CH2)7r-(Ci-C4)alkyl, -O-(Ci-C4)alkyl-NH2, -O-(Ci-C4)alkyl-NHCH3, -O-(Ci-C4)alkyl-N(CH3)2, -(Ci-Ci2)alkyl, -(C2-Ci2)alkenyl, -(C2-C6)alkynyl, -Cy1 , -(Ci-Ci2)alkylene-Cy1 , -(C2-Ci2)alkenylene-Cy1, -(C2-Ci2)alkynylene-Cy1, -Cy2-(Ci-Ci2)alkyl, -Cy2-(C2-Ci2)alkenyl, and -Cy2-(C2-Ci2)alkynyl; being Cy 1 a known ring system independently selected from the group consisting of 5- to 7-membered saturated or partially unsaturated carbocyclic or heterocyclic ring; and 5- to 7-membered carbocyclic or heterocyclic aromatic ring; Cy2 a bivalent known ring system independently selected from the group consisting of 5- to 7-membered saturated or partially unsaturated carbocyclic or heterocyclic ring; and 5- to 7-membered carbocyclic or heterocyclic aromatic ring; and being each one optionally substituted by one or more groups consisting of OH, halogen, NH2, NHRd, NRdRe, -(Ci-C6)alkyl, NO2, N3, -(Ci-Ci2)alkyl, -O-(Ci-Ci2)alkyl, -CO-(Ci-Ci2)alkyl and -CO-O-(Ci-Ci2)alkyl; wherein it is an integer selected from 1 to 6; wherein Rd and Re are each independently selected from H, and -(Ci-C4)alkyl; wherein the inert moiety is optionally substituted by a detection moiety, an active moiety, a lipid-like moiety Rw, and a pH, redox or protease responsive unit, or a mixture thereof; the detection moiety is selected from the group consisting of chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, transition metal and isotope mass tag moiety; the active moiety is selected from the group consisting of pharmaceutically active agents, veterinary active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof; the lipid-like moiety R is selected from the group consisting of -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, and a
Figure imgf000011_0001
Figure imgf000012_0001
T and T are each independently selected from -OH, -OCORx and -COORx;
Q and Q' are each independently selected from -OCORy and -COORy; each G is independently selected from -OCO-, -COO-, -NRz'CO-, and -CONRz'-; each Rz' is H or Rz; each Rx, Ry, and Rz is independently -(Ci-Ci8)alkyl or -(C2-Ci8)alkenyl; each a1 is independently an integer from 0 to 18; preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15, 16, 17, or 18; each b1 is independently an integer from 1 to 18; preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15,
16, 17, or 18; each d is independently an integer from 0 to 18; preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15,
16, 17, or 18; each d1 is independently an integer from 0 to 18; preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15,
16, 17, or 18; each e1 is independently an integer from 0 to 18; preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15,
16, 17, or 18; each fl is 0 or 1; each f1' is 0 or 1 ;
T1 , T2, T3, TT, T2' and T3' are each independently selected from the group consisting of hydrogen, fluorine, methyl, -CH2F, -CHF2, and -CF3; each of the dashed bonds — is independently a single bond or, alternatively, a double bond; each J is a biradical chain which comprises one or more moieties selected from the group consisting of - )-,
Figure imgf000013_0001
, , l, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkylphenyl, and -phenyl(Ci-C3o)alkyl; wherein when J comprises a -CH2- moiety, the two hydrogen atoms attached to the carbon atom are optionally replaced by the ring:
Figure imgf000013_0002
, , ,
Figure imgf000014_0001
wherein R2, R3, R4, R5, Re, and W1 are as defined above.
[0013] A second aspect of the present invention relates to a compound comprising -a structural unit of formula (I') or a structural unit of formula (I”), a salt thereof formed from acceptable non-toxic acids, or any stereoisomer or mixtures of stereoisomers, either of the compound of formula (I') or (I”), or of any of its salts,
Figure imgf000014_0002
wherein m, p, R1 , R9, L1 , L4, AA1 , AA2 and AA3 are as defined above; with the proviso that when p« 0, and R8 is not a cationic moiety, then n • 0; with the proviso that when n=0, then p« 0, and R8 is a cationic moiety; and with the proviso that when p=0, then n« 0; wherein the “*” denotes the attaching point; wherein each R8' and L4' are independently selected from a radical derived from the group consisting of -(Ci-C30)alkyl-R1, -(Ci-C3o)alkyl-COORii1, -(Ci-C30)alkyl-O-R"1, -(Ci-C30)alkyl-NRiv1Rv1, -C(O)-Rvi1, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety Rw, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, (XXVII), (XXIX), (XXX), (XXXIII), and (XXXIV); wherein R1, Ri1, R"1, Rv1, Rv1, Rvi1, (XXVII), (XXIX), (XXX), (XXXIII), and (XXXIV) are as defined above; the linker being optionally attached to at least one inert moiety, or alternatively optionally attached to at least one detection moiety, or alternatively optionally attached to at least one active moiety, or alternatively optionally attached to at least one lipid-like moiety Rw, or alternatively optionally attached to at least one pH, redox or protease responsive unit, or a mixture thereof; and with the proviso that the structural unit of formula (I') or (I”) is not attached to a star-shaped multifunctional linking agent.
[0014] A third aspect of the present invention relates to a star-shaped polymer comprising a starshaped multifunctional linking agent and at least one compound comprising the structural unit of formula (I') or (I”) as defined in accordance with the second aspect and/or embodiments thereof.
[0015] The star-shaped multifunctional linking agent (central core of the star-shaped polymer) may be any known multifunctional moiety suitable for the preparation of chemical-mediated vectors for the delivery of molecules of interest. Particular examples of star-shaped multifunctional moieties are those derived from a multi-arm star-shaped linking agent based on branched polyethylenimine (PEI-based); a four-arm star-shaped linking agent based on branched polyamidoamine (PAMAM-based) of formula (Pm); and a four-arm star-shaped linking agent of formula (Tz ) (Trizma-based).
[0016] Different configurations regarding to the arms of the star-shaped multifunctional linking agent are possible. Thus, the present invention encompasses star-shaped polymers having all the arms of the star-shaped multifunctional linking agent substituted with the same polyproline-based block copolymer of formula (I') or (I”); but also, configurations wherein at least one arm of the central core is different from the others.
[0017] A fourth aspect of the present invention relates to a self-assembled particle comprising the compound of formula (I) as defined herein, the compound comprising the structural unit of formula (I') or (I”) as defined herein, or the star-shaped polymer as defined herein, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
[0018] In accordance with a fifth aspect of the present invention, it is provided a composition comprising compound of formula (I) as defined herein, the compound comprising the structural unit of formula (I') or (I”), the star-shaped polymer or alternatively, the self-assembled particle, together with one or more appropriate excipients or carriers.
[0019] Another aspect of the present invention relates to a therapeutic product which comprises: a) the compound of formula (I) as defined herein, wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively b) the compound comprising the structural unit of formula (I') or (I”) as defined herein, wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively c) the star-shaped polymer as defined herein, wherein at least one of L4 or R8 comprise a pharmaceutically active agent;or alternatively d) the self-assembled particle as defined herein, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively e) a composition containing the self-assembled particle d) as defined herein; for use in medicine.
[0020] In accordance with a particular embodiment, it is provided a therapeutic product as defined above, for use (i) as transfection reagent for transfecting at least one active agent into a cell; (ii) for use in the in vivo or ex vivo production of biologies encoding a recombinant protein, a peptide or an antibody, or in the production of recombinant virus; (iii) for use as a therapeutic or prophylactic vaccine against viral infections or as a therapeutic vaccine against cancers or infectious diseases; or (v) for use in genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
[0021] In another aspect, it is provided a diagnostic product which comprises: a') the compound of formula (I) as defined herein, wherein at least one of L4 or R8 comprise a diagnostically active agent; or alternatively b') the compound comprising the structural unit of formula (I') or (I”) as defined herein, wherein at least one of L4 or R8 comprises a diagnostically active agent;or alternatively c') the star-shaped polymer as defined herein, wherein at least one of L4 or R8 comprise a diagnostically active agent ; or alternatively d') a self-assembled particle as defined herein, which comprises diagnostically active agent, or alternatively e') a composition containing the self-assembled particle d') as defined herein; for use in diagnostics.
[0022] In a further aspect, it is provided the use in cosmetics of a cosmetic product which comprises: a") the compound of formula (I) as defined herein, wherein at least one of L4 or R8 comprise a cosmetically active agent;or alternatively b”) the compound comprising the structural unit of formula (I') or (I”) as defined herein, wherein at least one of L4 or R8 comprises a cosmetically active agent;or alternatively c”) the star-shaped polymer as defined herein, wherein at least one of L4 or R8 comprise a cosmetically active agent;or alternatively d”) the self-assembled particle as defined herein, which comprises a cosmetically active agent, or alternatively e”) a composition containing the self-assembled particle d”) as defined herein comprising a cosmetically active agent.
[0023] In another aspect, it is provided the compound of formula (I) as defined herein, wherein L4 and R8 are other than an active moiety; the compound comprising the structural unit of formula (I') or (I”) as defined herein, wherein at least one of L4 or R8 is other than an active moiety; the star-shaped polymer as defined herein, wherein L4 and R8 are other than an active moiety; or alternatively the selfassembled particle as defined herein, for use as a carrier.
[0024] In another aspect, it is provided a therapeutic product which comprises: a) the compound of formula (I) as defined herein, wherein at least one of L4 or R8 comprise a pharmaceutically active agent;or alternatively b) the compound comprising the structural unit of formula (I') or (I”) as defined herein, wherein at least one of L4 or R8 comprises a pharmaceutically active agent; or alternatively c) the star-shaped polymer as defined herein, wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively d) a self-assembled particle as defined herein, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively e) a composition containing the self-assembled particle d) as defined herein; for use in a method for delivering a pharmaceutical active agent (i.e. a nucleic acid or a drug) into a target cell, which comprises administering a composition (i.e. solution) that contains any of a), b), c), d) or e) to an animal, including human, so that a), b), c), d) or e) is getting into physical contact with the target cell and thereby delivers the pharmaceutical active agent into the cell.
Description of the drawings
[0025] Non-limiting examples of the present disclosure will be described in the following, with reference to the appended drawings, in which:
FIG. 1. Cardiolipin (CL)-based Supported Lipid Bilayers examined using a Zeiss Elyra 7 total internal reflection fluorescence microscope (TIRFM). The samples were observed both with and without incubation with compound 1a.
FIG. 2. Quantitative analysis of 11a interaction with POPC/CL and POPC/POPG (control) based Small Unilamellar Vescicles (SUVs) by Fluorescence (Cross) Correlation Spectroscopy (FCCS).
Detailed description
[0026] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply throughout the description and claims.
[0027] The term "about" or "around” as used herein refers to a range of values ± 10% of a specified value. For example, the expression "about 10" or "around 10” includes ± 10% of 10, i.e. from 9 to 11.
[0028] As used herein, the indefinite articles "a” and "an” are synonymous with "at least one” or "one or more.” Unless indicated otherwise, definite articles used herein, such as "the” also include the plural of the noun.
[0029] The term "moiety” refers to a specific segment or functional group of a molecule or compound.
[0030] As used herein, the term "subject" refers to any mammal, including both human and other mammals.
[0031] The term "pharmaceutically, cosmetically or diagnostically acceptable salt", embraces non-toxic salts commonly used. The preparation of pharmaceutically, cosmetically or diagnostically acceptable salts of the compounds of formula (I) or (I') of the invention can be carried out by methods well-known in the art. Generally, such salts can be prepared by reacting the free acid or base form of a compound of formula (I) or (I') of the invention with a stoichiometric amount of an appropriate base or acid, respectively, in a suitable solvent such as water, an organic solvent or a mixture of them. The salts of the repeating units of monomer PAA1, AA1, AA2 and AA3 encompasses acid addition salts formed from acceptable non-toxic acids including inorganic or organic acids, and base addition salts. There is no limitation regarding the salts, except that they must be therapeutically (pharmaceutically or veterinary), diagnostic or cosmetic acceptable when they are used for therapeutic (pharmaceutical or veterinary), diagnostic or cosmetic purposes, respectively. Most of the acceptable salts are commercially available. If not, these salts can be prepared following the processes disclosed in the state of the art, which involves starting from acceptable non-toxic acids, including inorganic and organic acids. Such acids include for instance inorganic acids e.g. hydrochloric, hydrobromic, sulfuric, nitric, hydroiodic, metaphosphoric, or phosphoric acid; and organic acids e.g. acetic, benzenesulfonic, benzoic, camphor sulfonic, citric, ethansulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, lactic, maleic, malic, mandelic, methanesulfonic, succinic, tartaric, p-toluensulfonic acid, and formic acid; trifluoroacetic, propionic, glycolic, camphorsulfuric, isothionic, mucic, gentisic, isonicotinic, saccharic, glucuronic, furoic, ascorbic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), ethanesulfonic, pantothenic, stearic, sulfinilic, alginic and galacturonic acid, benzenesulfonic, oxalic, methanesulfonic or naphthalenesulfonic acid; and base addition salts formed with alkali metals and alkaline earth metals and organic bases such as N, N-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), lysine and procaine; and internally formed salts. For instance, they can be prepared from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate acceptable base or acid in water or in an organic solvent or in a mixture of them. The compounds of formula (I) or (I') of the invention and their salts may differ in some physical properties, but they are equivalent for the purposes of the present invention.
[0032] For the purpose of the present invention, the term "pharmaceutically acceptable salts” used herein encompasses any salt formed from pharmaceutically acceptable non-toxic acids or bases as defined above. The term "veterinary acceptable salts” used herein encompasses any salt formed from veterinary acceptable non-toxic acids or bases as defined above. The term "diagnostic acceptable salts” used herein encompasses any salt formed from diagnostic acceptable non-toxic acids or bases as defined above. The term "cosmetic acceptable salts” used herein encompasses any salt formed from cosmetic acceptable non-toxic acids or bases as defined above.
[0033] The term "alkyl” refers to a saturated straight, or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims. Examples include, among others, the group methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl. As disclosed above, the alkenyl groups can be optionally substituted. The term "alkynyl” refers to a straight or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims and having at least one carbon-carbon triple bond. Examples include, among others, ethynyl, 2-propynyl, and 3-hexynyl. As disclosed above, the alkynyl groups can be optionally substituted.
[0034] The term -(Ci-Cx)alkyl refers to a saturated linear or branched hydrocarbon chain which contains from 1 to x carbon atoms and only single bonds. Examples of alkyl groups may include without limitation methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, neopentyl, n-hexyl, decyl, undecyl, dodecyl, tetradecyl and hexadecyl. The term -(C2-Cx)alkenyl refers to an unsaturated branched or linear hydrocarbon chain which comprises from 2 to x carbon atoms and at least one or more double bonds. Examples of alkenyl groups may include without limitation ethenyl (i.e. vinyl), allyl, propenyl, butenyl, pentenyl and hexenyl, dodecenyl, tetradecenyl and hexadecenyl.
[0035] The term “-(Ci-Cio)alkoxy” refers to an alkyl group as defined herein comprising 1 to 10 carbon atoms which is connected to the rest of the molecule via an oxygen atom. Examples of alkoxy groups may include without limitation methoxy, ethoxy, 1 -propoxy, and 2-propoxy.
[0036] The term “-(C5-Cio)aryl” refers to an aromatic carbocyclic mono- or bicyclic ring system comprising 5 to 10 carbon ring atoms. Examples of aryl groups may include without limitation phenyl, biphenyl, and naphthyl. [0037] The term "-(C6-Cio)aryloxy” refers to an aryl group as defined herein comprising 6 to 10 carbon ring atoms, which is connected to the rest of the molecule via an oxygen atom. Examples of aryloxy groups may include without limitation phenoxy and biphenyloxy.
[0038] The term "-(C5-Cio)heteroaryl” refers to an aromatic carbocyclic mono- or bicyclic ring system comprising 5 to 10 ring atoms, wherein at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, and S, the remaining ring atoms being carbon atoms. Examples of heteroaryl groups may include without limitation pyridyl, azetidinyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl, isoquinolinyl, benzthiazolyl, benzimidazolyl, benzthiazolyl, quinolyl, and quinazolyl.
[0039] The term "-(C6-Cio)aralkyl” refers to a lower alkyl group as defined herein (e.g. -(Ci-Ce)alkyl) which is substituted by an aryl group (e.g. “-(Cs-C jaryl) as defined herein. Examples of aralkyl groups may include without limitation benzyl, phenethyl, and methylbenzyl.
[0040] The term "-(C6-Cio)aralkoxy” refers to a lower alkoxy group as defined herein (e.g. -(Ci- Ce)alkoxy) which is substituted by an aryl group (e.g. “-(C5-Cio)aryl) as defined herein. Examples of aralkoxy groups may include without limitation benzyloxy, phenethoxy, and methylbenzyloxy.
[0041] The term "-(C5-Cio)heteroaralkoxy” refers to a lower alkoxy group as defined herein (e.g. -(Ci- Ce)alkoxy) which is substituted by an heteroaryl group (e.g. "-(C5-Cio)heteroaryl) as defined herein. Examples of heteroaralkoxy groups may include without limitation furyloxy, pyridyloxy, and azetidinyloxy.
[0042] The term "-(C5-Cio)heterocycloalkyl” refers to a 5-10 membered mono- or bicylic (fused or bridged) saturated o unsaturated ring structure, in which one or more of the ring atoms is a heteroatom selected from N, O, and S. Examples of a heterocycloalkyl groups may include without limitation include piperidyl, piperazyl, tetrahydropyranyl, tetrahydrofuryl, 1 ,4-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholyl.
[0043] The term "alkenyl” refers to a straight or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims and having at least one carbon-carbon double bond. Examples include, among others, ethenyl, 2-propenyl, and 3-hexenyl. As disclosed above, the alkenyl groups can be optionally substituted.
[0044] For the purpose of the invention, the term "bivalent” refer to a moiety that is bond to two other moieties.
[0045] The term “-(Ci-Ci2)alkylene- "refers to a bivalent saturated straight, or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims.
[0046] The term "-(C2-Ci2)alkenylene-“ refers to a bivalent straight or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims and having at least one carbon-carbon double bond.
[0047] The term “-(C2-Ci2)alky ny lene-" refers to a bivalent straight or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims and having at least one carbon-carbon triple bond.
[0048] The term "carbocyclic ring” refers to a known saturated, partially unsaturated; or aromatic ring system comprising one or more rings and having the number of carbon atoms specified in the description or claims, wherein all ring members are carbon atoms. As disclosed above, the ring members can be optionally substituted.
[0049] The term "heterocyclic ring” refers to a known saturated, partially unsaturated; or aromatic ring system comprising one or more rings and having the number of carbon atoms specified in the description or claims, wherein one or more of the ring members, preferably 1, 2, 3, or 4 ring members, are selected from NH, N, O, and S, and are chemically possible; and the remaining members of the ring are carbon atoms. In the case of a ring system containing a CH member and a NH member, the ring may be attached to the rest of the molecule through the C or the N atom. As disclosed above, the ring members can be optionally substituted.
[0050] Non limiting examples of Cy 1 include, but not limited to, phenyl and naphthyl, furan-2-yl, furan- 3-yl, thiophen-2-yl, thiophen-2-yl, indol-2-yl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl, pyrazolyl, 2- pyridyl, 3-pyridyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, and triazolyl.
[0051] The terms "amine protecting group” and "nitrogen protecting group" have the same meaning and are used interchangeable. They refer to a chemical compound which, when bound to an amino group of the polymer or of a starting material (reagent) for its preparation, prevents undesired reactions from occurring at this amino group and which can be removed by conventional chemical or enzymatic steps to re-establish the amino group. Examples of amine protecting groups include carbamate protecting group including, without limitation, t-butyl carbamate, methyl carbamate, ethyl carbamate, 2,2,2-trichloroethyl carbamate, 2-(trimethylsilyl)ethyl carbamate, 1, 1 -dimethyl-2,2,2-trichloroethyl carbamate, benzyl carbamate, p-methoxybenzyl carbamate, p-nitrobenzylcarbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, and 2,4-dichlorobenzyl carbamate; -Fluorenylmethyl Carbamate ("Fmoc"), formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("Cbz"), t-butoxycarbonyl ("BOO"), trimethylsilyl ("TMS"), 2- trimethylsilylethanesulfonyl, ("SES"), trityl and substituted trityl groups, allyloxycarbonyl, nitroveratryloxycarbonyl ("NVOC") , and allyloxycarbonyl (Alloc), (cf. Greene and Wuts, Protecting Groups in Organic Synthesis, chapter.: "Protection of amines”. Third Edition, John Wiley & Sons (1999) pages 494-653).
[0052] The terms "alcohol protecting group” and "oxygen protecting group" have the same meaning and are used interchangeable. They refer to a chemical compound which, when bound to an alcohol group of the polymer or of a starting material (reagent) for its preparation, prevents undesired reactions from occurring at this alcohol group and which can be removed by conventional chemical or enzymatic steps to re-establish the alcohol group. Examples of alcohol protecting groups include 9- Fluorenylmethyl, methoxy methyl, methylthiomethyl, tetrahydrofuranyl, Methoxyethoxymethyl, 2- (Trimethylsilyl)ethoxymethyl, Benzyloxymethyl, Phenylacetoxymethyl,
Triisopropylsilylmethyl, Cyanomethyl, Phenacyl, 2,2,2-Trichloroethyl, 2-(Trimethylsilyl)ethyl, 2- Methylthioethyl, 2-(pNitrophenylsulfenyl)ethyl, 2-(pToluenesulfonyl)ethyl, t-Butyl, 2,6-Dimethylphenyl, p(Methylthio)phenyl, Pentafluorophenyl, Benzyl, Nitrobenzyl, p-Nitrobenzyl, p-Methoxybenzyl, 4- Sulfobenzyl, Trimethylsilyl, t-Butyldimethylsilyl, i-Propyldimethylsilyl, Phenyldimethylsilyl, Di-t- butylmethylsilyl, 2-Alkyl-l ,3-oxazoline, and Tetraalkylammonium salts, (cf. Greene and Wuts, Protecting Groups in Organic Synthesis, chapter.: "Protection of carboxyl group”. Third Edition, John Wiley & Sons (1999) pages 369-451).
[0053] The term "substituted" means that one or more hydrogen atoms on the designated atom or group are replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded. Combinations of substituents and/or variables are permissible.
[0054] The term "optionally substituted" means that the number of substituents can be equal to or different from zero. Unless otherwise indicated, it is possible that optionally substituted groups are substituted with as many optional substituents as can be accommodated by replacing a hydrogen atom with a non-hydrogen substituent on any available carbon or nitrogen atom. It is possible that groups in the conjugates according to the invention are substituted with one, two, three, four or five identical or different substituents, particularly with one, two or three substituents.
[0055] The term "Conjugated" and "attached" refer to the covalent attachment of a group, and are used interchangeably herein.
[0056] Further, the term "detection moiety” refers to a moiety possessing a property or function which can be used for detection purposes. This term encompasses moieties selected from the group consisting of chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, and transition metal isotope mass tag moiety. The term "chromophore” or "chromophore moiety” refers to a moiety that exhibits a detectable absorption of light such as for example (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) (BOPIPY). Suitable fluorescent moieties are those known from the art of immunofluorescence technologies, e.g., flow cytometry or fluorescence microscopy, wherein, the compound labelled with this detection moiety is detected by exciting the detection moiety and detecting the resulting emission (photoluminescence). Useful fluorescent moieties for the present invention include protein-based, such as phycobiliproteins, polymeric, such as polyfluorenes, small organic molecule dyes, such as xanthene, like fluorescein, or rhodamines, cyanine, oxazines, coumarins, acridines, oxadiazoles, pyrenes, pyrromethene, or metallo- organic complexes, such as Ru, Eu, Pt complexes. Besides single molecule entities, clusters of fluorescent proteins or small organic molecule dyes, as well as nanoparticles, such as quantum dots, upconverting nanoparticles, gold nanoparticles, dyed polymer nanoparticles can also be used as fluorescent moieties. Another group of photoluminescent detection moieties are phosphorescent moieties with time-delayed emission of light after excitation. Phosphorescent moieties include metallo- organic complexes, such as Pd, Pt, Tb, Eu complexes, or nanoparticles with incorporated phosphorescent pigments such as lanthanide doped SrAI2O4. Other group of detection moiety is a radioactive label, wherein the compound labelled with this detection moiety is detected without prior excitation by irradiation. They can be in the form of radioisotope labelling by exchanging nonradioactive isotopes for their radioactive counterparts, such as tritium, 32P, 35S or 14C, or introducing covalently bound labels, such as 1251, which is bound to tyrosine, 18F within fluorodeoxyglucose, or metallo- organic complexes, i.e. 99Tc-DTPA. Other group is a detection moiety capable of causing chemiluminescence, i.e. horseradish peroxidase label in the presence of luminol. In other group of detection moiety, the labelled compound is detected by absorption of UV, visible light, or NIR radiation. Suitable light-absorbing detection moieties are light absorbing dyes without fluorescence emission, such as small organic molecule quencher dyes like N-aryl rhodamines, azo dyes, and stilbenes. Other detection moiety appropriate for the present invention are light-absorbing detection capable of generating a photoacoustic signal after irradiation by pulsed laser light. In other group of detection moiety, the labelled compound is detected by mass spectrometric detection of a transition metal isotope. Known in the art are isotope tags of lanthanides and adjacent late transition elements.
[0057] For the purpose of the invention, the term "active moiety” is a moiety possessing therapeutically (pharmaceutical or veterinary) or cosmetic activity. This term encompasses moieties selected from the group consisting of a pharmaceutically active agent, a veterinary active agent, and a cosmetic active agent. The term "active agent” refers to any chemical compound or substance that has activity in the pharmaceutical, veterinary or cosmetic field.
[0058] The term "pharmaceutically active agent” refers to any substance or combination of substances used in a finished pharmaceutical product, intended to furnish pharmacological activity or to otherwise have direct effect in the cure, mitigation, treatment, or prevention of disease, or to have direct effect in restoring, correcting, or modifying physiological functions in human beings.
[0059] For the purposes of the present invention, pharmaceutically active agents include low molecular weight drugs, peptides, antibodies, hormones, enzymes, nucleic acids, proteins, and combinations thereof.
[0060] As used herein, the term "nucleic acid” refers to DNA or RNA. In a particular embodiment, optionally in combination with any of the embodiments provided above or below, the nucleic acid is an DNA/RNA hybrid, a short interfering RNA (siRNA), a microRNA (miRNA), a single guide RNA (sgRNA), a donorDNA, a self-amplyfing/replicating RNA, a circularRNA (oRNA), a plasmid DNA (pDNA), a closed- linear DNA (clDNA), a short hairpin RNA (shRNA), messenger RNA (mRNA), and antisense RNA (aRNA), a CRISPR guide RNA, an antisense nucleic acid, a decoy nucleic acid, an aptamer, and a ribozyme to name a few, and encompasses both the nucleotide sequence and any structural embodiments thereof, such as double stranded, single stranded, helical, hairpin, etc, and may contain modified or unmodified bases. When distinct nucleic acids are provided, they may be all DNA molecules or all RNA molecules or may be mixtures of DNA and RNA molecules or molecules comprising an association of DNA and RNA strands.
[0061] The nucleic acid may be a poly- or oligonucleotide, such as oligo- or poly-double stranded RNA, oligo- or poly-double stranded DNA, oligo- or poly-single stranded RNA, oligo- or poly-single stranded DNA. Each of the nucleotides contained in the nucleic acid may be a naturally occurring nucleotide or a chemically-modified, non-naturally occurring nucleotide. The strand length of the nucleic acid is not particularly limited and the nucleic acid may have a short strand ranging from 10 to 200 bases, preferably from 20 to 180 bases, preferably from 25 to 100 bases, preferably from 30 to 50 bases; or the nucleic acid may have a relatively long strand of from 200 to 20000 bases, more preferably of from 250 to about 15000 bases.
[0062] The polynucleotide may be of any sequence. In certain embodiments, the polynucleotide encodes a protein or peptide. The encoded proteins may be enzymes, structural proteins, receptors, soluble receptors, ion channels, pharmaceutically active proteins, cytokines, interleukins, antibodies, antibody fragments, antigens, coagulation factors, albumin, growth factors, hormones, insulin, etc. The polynucleotide may also comprise regulatory regions to control the expression of a gene. These regulatory regions may include, but are not limited to, promoters, enhancer elements, repressor elements, TATA box, ribosomal binding sites, stop site for transcription, etc. In certain embodiments, the polynucleotide is not intended to encode a protein. For example, the polynucleotide may be used to fix an error in the genome of the cell being transfected.
[0063] In certain embodiments, the polynucleotide to be delivered comprises a sequence encoding an antigenic peptide or protein. Nanoparticles containing these polynucleotides can be delivered to an individual to induce an immunologic response sufficient to decrease the chance of a subsequent infection and/or lessen the symptoms associated with such an infection.
[0064] In accordance with a particular embodiment, optionally in combination with any of the embodiments provided above or below, the nucleic acid is closed-linear DNA (clDNA), i.e. molecules wherein the double stranded region is flanked and protected by two single stranded loops thereby generating dumbbell-shaped molecules.
[0065] In a more particular embodiment, optionally in combination with any of the embodiments provided above or below, the clDNA consists of a stem region comprising a double stranded DNA sequence of interest covalently closed at both ends by hairpin loops, the clDNA comprising at least two modified nucleotides.
[0066] As used herein, the term "closed linear DNA” or "clDNA” refers to a single stranded covalently closed DNA molecule that forms a "dumbbell” or "doggy-bone” shaped structure under conditions allowing nucleotide hybridization. Therefore, although the clDNA is formed by a single stranded DNA molecule, the formation of the "dumbbell” structure by the hybridization of two complementary sequences within the same molecule generates a structure consisting on a double-stranded middle segment flanked by two single-stranded loops. The skilled in the art know how to generate clDNA from open or closed double stranded DNA using routine molecular biology techniques. For instance, those skilled in the art knows that a clDNA can be generated by attaching hairpin DNA adaptors, for instance, by the action of a ligase, to both ends of an open double stranded DNA. "Hairpin DNA adaptor” refers to a single stranded DNA that forms a stem-loop structure by the hybridization of two complementary sequences, wherein the stem region formed is closed at one end by a single stranded loop and is open at the other end.
[0067] A "modified nucleotide” is any nucleotide (e.g., adenosine, guanosine, cytidine, uracil, and thymidine) that has been chemically modified -by modification of the base, the sugar or the phosphate group- or that incorporates a non-natural moiety in its structure. Thus, the modified nucleotide may be naturally or non-naturally occurring depending on the modification.
[0068] As used herein, the term "peptide" refers to molecules that comprise two or more consecutive amino acids linked to one another via peptide bonds. The term peptide includes oligopeptides and polypeptides. The term "protein" refers to large peptides, in particular peptides having at least about 50 amino acids.
[0069] Examples of proteins of interest include, without limitation cytokines, interleukins, tumor necrosis factor (TNF), interferons, integrins, chimeric antigen receptors (CARs), antibodies, hormones, growth factors, enzymes), collagen, fibrinogen, elastin, tubulin, thrombin, serum albumin, erythropoietin, granulocyte colony stimulating factor (G-CSF), colony stimulating factor (CSF), and the like.
[0070] The term "veterinary active agent” refers to any substance or combination of substances used in a finished veterinary product, intended to furnish pharmacological activity or to otherwise have direct effect in the cure, mitigation, treatment, or prevention of disease, or to have direct effect in restoring, correcting, or modifying physiological functions in animals.
[0071] The term "cosmetic active agent” refers to any substance or combination of substances used in a finished cosmetic product, intended to improve its appearance or to beautify, preserve, condition, cleanse, color or protect the skin, nails, or hair without non-medical application.
[0072] As used herein, the term "cell-targeting agent” refers to any molecule, macromolecule, or biomacromolecule, displaying affinity for a molecule present in the human or animal body, which is able to direct the conjugates or the self-assembled particles thereof by directing them towards the target site for therapeutic treatment since e.g., it selectively binds to receptors that are expressed or overexpressed on specific cell types. Cell-targeting groups are well known in the art. The term therefore includes ligands for specific receptors or antigens, such as antibodies for a specific antigen, folic acid for its receptor or sugars such as galactose for its hepatic receptors. The targeting agent may be attached to the polyproline-based copolymer backbone.
[0073] A variety of suitable targeting agents are known in the art. Non-limiting examples of targeting agents include a peptide, a protein, an enzyme, a nucleic acid, a fatty acid, a hormone, an antibody, a carbohydrate, mono-, oligo- or polysaccharides, a peptidoglycan, a glycopeptide, or the like. For example, any of a number of different materials that bind to antigens on the surfaces of target cells can be employed. Antibodies to target cell surface antigens will generally exhibit the necessary specificity for the target. In addition to antibodies, suitable immunoreactive fragments can also be employed, such as the Fab, Fab', F(ab')2 or scFv fragments or single-domain antibodies (e.g. camelids VHH fragments). Many antibody fragments suitable for use in forming the targeting mechanism are already available in the art. Similarly, ligands for any receptors on the surface of the target cells can suitably be employed as targeting agent. These include any small molecule or biomolecule, natural or synthetic, which binds specifically to a cell surface receptor, protein or glycoprotein found at the surface of the desired target cell. Thus examples of cell-targeting groups include, but are not limited to, galactosamine, folate, a Her- 2 binding peptide, TLR agonists, • -D-Glucose, Asn-Gly-Arg peptide, angiopep2, folic acid, aptamers (A- 9, A10, Anti-gp120, TTA1, sgc8, Anti MUC-1, AS1411), primaquine, zidovudine, superoxide dismutase, prednisolone, platinum, cisplatin, sulphamethoxazole, amoxicillin, etoposide, mesalzine, doxorubicin, paclitaxel, 5-amino salicylic acid, denosumab, docetaxel, calcitonin, proanthocyanidin, methotrexate, camptothecin, galactose, glycyrrhetinic acid, lactose, hyaluronic acid, octeotride, lactobionic acid, • - galactosyl moiety, arabino-galactan, chitosan, azo-based poly-phosphazene, azo group and 4-amino- benzyl-carbamate, succinate, 4,4’-dihydroxy azo benzene-3-carboxilic acid, cyclic RGD penta-peptide, Aspartic acid octapeptide, alendronate, transferrin, bisphosphonate adendronate, mono sialoganglioside GM1, gluthatione, E-selectin thioaptamer, poloxamer-407, a urokinase-type plasminogen activator receptor (uPAR) antagonist, a CXCR4 chemokine receptor antagonist, a GRP78 peptide antagonist, an RGD peptide, an RGD cyclic peptide, a luteinizing hormone-releasing hormone (LHRH) antagonist peptide, an aminopeptidase targeting peptide, a brain homing peptide, a kidney homing peptide, a heart homing peptide, a gut homing peptide, an integrin homing peptide, an angiogenic tumor endothelium homing peptide, an ovary homing peptide, a uterus homing peptide, a sperm homing peptide, a microglia homing peptide, a synovium homing peptide, a urothelium homing peptide, a prostate homing peptide, a lung homing peptide (e.g. RCPLSHSLICY), laminin receptor binding peptide (e.g. YIGSR) a skin homing peptide, a retina homing peptide, a pancreas homing peptide, a liver homing peptide, a lymph node homing peptide, an adrenal gland homing peptide, a thyroid homing peptide, a bladder homing peptide, a breast homing peptide, a neuroblastoma homing peptide, a lymphoma homing peptide, a muscle homing peptide, a wound vasculature homing peptide, an adipose tissue homing peptide, a virus binding peptide, or a fusogenic peptide.
[0074] The term "penetration enhancing agent" as used herein refers to moiety or compound that increases the permeability of an active agent, in particular selected from a pharmaceutically active agent, a cell-targeting agent, a cosmetically active agent, and a diagnostically active agent. The penetration enhancing agent is also known as permeation enhancer.
[0075] Examples of penetration enhancing agents include, without limitation, cell penetrating peptides, surfactants, terpenes, sulfoxides, pyrrolidones, fatty acids, fatty alcohols, urea, azones, fatty alcohols, fatty acids, fatty esters; such as for example, lauryl sarcosine, octoxynol, phenylsulfonate, pluronic, sodium laurate, sodium oleate, sorbitan dilaurate, sorbitan dioleate, sorbitan trilaurate, sorbitan trioleate, sodium octyl sulfate, alkyl ammonium halides, decanol, dodecanol, linolenyl alcohol, oleyl alcohol, butyl acetate, cetyl lactate, lauryl lactate, myristyl lactate, diethyl sebacate, diethyl succinate, diisopropyl sebacate, glycerol monolaurate, glycerol monooleate, glycerol monolinoleate, isopropyl isostearate, isopropyl linoleate, isopropyl myristate, isopropyl palmitate, methyl caprate, methyl laurate, methyl valerate, octyl acetate, oleyl oleate, sorbitan dilaurate, dodecyl acetate, sorbitan dioleate, sorbitan monolaurate, sorbitan trilaurate, sorbitan trioleate, capric acid, hexanoic acid, lactic acid, lauric acid, linoleic acid, linolenic acid, neodecanoic acid, oleic acid, palmitic acid, lecithin, phospholipids, sodium deoxycholate, and sodium taurocholate.
[0076] In the context of the present disclosure the expression, "diagnostically active agent”, also referred to as "labeling or imaging agent", refers to any substance that is used as a label, or that enhances specific structures in any imaging technique. An imaging agent, hence, includes optical imaging agent, magnetic resonance imaging agent, radioisotope, and contrast agent. Imaging or labelling agents are well known in the art. Particular examples or imaging or labelling agents are gases such as sterilized air, oxygen, argon, nitrogen, fluor, perfluorocarbons, carbon dioxide, nitrogen dioxide, sulfur hexafluoride, xenon and helium; commercially available agents used in positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI). Examples of suitable materials for use as contrast agents in MRI include the gadolinium chelates currently available, such as diethylene triamine pentaacetic acid (DTPA) and gadopentotate dimeglumine, as well as iron, magnesium, manganese, copper and chromium. Examples of materials useful for CAT and x-rays include iodine based materials for intravenous administration, such as ionic monomers typified by diatrizoate and iothalamate, non-ionic monomers such as iopamidol, isohexol, and ioversol, non-ionic dimers, such as iotrol and iodixanol, and ionic dimers, for example, ioxagalte. Other useful materials include barium for oral use and non-soluble salts such as zinc acetate. In some molecules, an imaging agent is a dye. In some molecules, an imaging agent is a fluorescent moiety. In some molecules, a fluorescent moiety is selected from: a fluorescent protein, a fluorescent peptide, a fluorescent dye, a fluorescent material or a combination thereof. Examples of fluorescent dyes include, but are not limited to, xanthenes (e.g., rhodamines, rhodols and fluoresceins, and their derivatives); bimanes; coumarins and their derivatives (e.g., umbelliferone and aminomethyl coumarins); aromatic amines (e.g., dansyl; squarate dyes); benzofurans; fluorescent cyanines; indocarbocyanines; carbazoles; dicyanomethylene pyranes; polymethine; oxabenzanthrane; xanthene; pyrylium; carbostyl; perylene; acridone; quinacridone; rubrene; anthracene; coronene; phenanthrecene; pyrene; butadiene; stilbene; porphyrin; pthalocyanine; lanthanide metal chelate complexes; rare-earth metal chelate complexes; and derivatives of such dyes. Examples of fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5- isothiocyanate, fluorescein-6-isothiocyanate and 6-carboxyfluorescein. Examples of rhodamine dyes include, but are not limited to, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine, diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101 sulfonyl chloride (sold under the tradename of TEXAS RED(R)). Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, IRDYE680, Alexa Fluor 750, IRDye800CW, ICG. Examples of fluorescent peptides include GFP (Green Fluorescent Protein) or derivatives of GFP (e.g., EBFP, EBFP2, Azurite, mKalamal, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet). Fluorescent labels are detected by any suitable method. For example, a fluorescent label may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs), photomultipliers, etc. In some molecules, the imaging agent is labeled with a positron-emitting isotope (e.g., 18F) for positron emission tomography (PET), gamma-ray isotope (e.g., 99mTc) for single photon emission computed tomography (SPECT), or a paramagnetic molecule or nanoparticle (e.g.,Gd3+ chelate or coated magnetite nanoparticle) for magnetic resonance imaging (MRI). In some molecules, the imaging agent is labeled with: a gadolinium chelate, an iron oxide particle, a super paramagnetic iron oxide particle, an ultra small paramagnetic particle, a manganese chelate or gallium containing agent. Examples of gadolinium chelates include, but are not limited to diethylene triamine pentaacetic acid (DTPA), 1 ,4,7,10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid (DOTA), and 1,4,7- triazacyclononane-N, N',N"-triacetic acid (NOTA). In some molecules, the imaging agent is a nearinfrared fluorophore for near-infra red (near-IR) imaging, a luciferase (firefly, bacterial, or coelenterate) or other luminescent molecule for bioluminescence imaging, or a perfluorocarbon-filled self-assembled particle for ultrasound. In some molecules, the imaging agent is a nuclear probe. In some molecules, the imaging agent is a SPECT or PET radionuclide probe. In some molecules, the radionuclide probe is selected from: a technetium chelate, a copper chelate, a radioactive fluorine, a radioactive iodine, a indiuim chelate. Examples of Tc chelates include, but are not limited to HYNIC, DTPA, and DOTA. In some molecules, the imaging agent contains a radioactive moiety, for example a radioactive isotope such as 211 At, 1311, 1251, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 64Cu radioactive isotopes of Lu, and others. The diagnostically active agents agent may be attached to the polyproline-based block copolymer backbone and/or may be contained in the self-assembled particles formed from it.
[0077] In accordance with the present invention, the pharmaceutically active agent is not particularly limited, and any pharmaceutically active agent having desired activity can be used. Preferably, pharmaceutically active agents that are desired to be delivered intracellularly, eg, to mitochondria, are used. The physiological activity possessed by the pharmaceutically active agent may be any physiological activity capable of functioning as an active ingredient of a drug, and examples thereof include antitumor activity, immunostimulatory activity, antiviral activity, antibacterial activity, and antiinflammatory activity. Drugs may be enzymes, hormones, vaccines, proteins such as antibodies, mRNA, pDNA, antisense, ribozymes, siRNA, decoy nucleic acids, nucleic acids such as aptamers, and macromolecular pharmaceuticals such as polysaccharides.
[0078] The term "lipid-like moiety" refers to a moiety which, while it cannot be considered to be a lipid as such, structurally and/or functionally resembles a lipid. The term lipid-like moiety is intended to include moieties that are able to form amphiphilic layers.
[0079] The term "lipid-like material", "lipid-like compound" or "lipid-like molecule" relates to substances that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids. As used herein, the term "lipid" is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.
[0080] Examples of lipids are fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, sterols, and the like, which are insoluble in water.
[0081] In the context of the present invention, the term "linker” refers to a chemical unit which connects two other chemical units through strong chemical bonds, for example: covalent bonds. Thus, a linker ensures a certain molecular distance and, at the same time, a connection between the two linked chemical units. The linkers may be cleaved using a variety of physiological stimuli enabling tissue or disease-specific drug targeting. Examples of physiological stimuli may include hydrolysis (ester containing linkers, carbonates, carbamates), pH-sensitive (imine, oxime, Schiff base and hydrazone containing linkers), redox sensitive (disulfide, diselenide, benzylboronic ester, thioketal, peroxalate, thioether, dithioether, arylboronic containing linkers), enzyme cleavable (peptide containing linkers), multi-stimuli responsive (amide containing linkers).
[0082] The linker may essentially be seen as a covalent linker which is used to attach/bind the compound of formula (I) to at least one inert moiety, at least one detection moiety, at least one active moiety, at least one lipid-like moiety Rw, to at least one pH, redox or protease responsive unit, or a mixture thereof.
[0083] As discussed above, these linker related radicals may be many different suitable structures, for instance a structure comprising a, in a human cell in vivo, cleavable disulfide bond. In short, the skilled person knows from the art numerous different suitable linker related structures of different molecular weight (MW) sizes.
[0084] Particular examples of linkers are those derived from a biradical selected from the group consisting of -(Ci-C6)NH-CO-(Ci-C6)alkyl-O-, -(Ci-C6)NH-CO-(Ci-C6)alkyl-, -(Ci-C6)NH-CO-(Ci-C6)alkyl-NH-, -(Ci-C6)NH-CO-(Ci-C6)alkyl-CO -NH(Ci-C6)alkyl-O-, -NH(Ci-C6)alkyl-NH-, -O-(Ci-C6)alkyl-NH-, -O-(Ci-C6)alkyl-O-, a straight or branched -(CI-C6)-NH-CO- (Ci-C3o)alkylene-0-, -(Ci-C6)-NH-CO(Ci-C3o)alkylene-NH-, -(Ci-C6)-NH-CO(Ci-C30)alkylene-CO -NH-(Ci-C3o)alkylene-0-, -NH-(Ci-C3o)alkylene-NH-, -NH-(Ci-C3o)alkylene-CO-, -0-(Ci-C3o)alkylene-0-, -0-(Ci-C3o)alkylene-CO-, -CO-(Ci-C3o)alkylene-CO-. Additional examples of linkers are a biradical of formula (IV), (V), (VI), (VI I), (VIII), (IX), (X), or (XI) as defined above; a biradical of formula (L) or (LI); and a biradical selected from the group consisting of (XXVII), (XXIX), (XXX), (XXXIII), (XXXIV), (XXXV), (XXXVI) and (XXXVII) optionally attached by a chemically feasible bond to a biradical of formula (IV), (V), (VI), (VII), (VIII), (IX), (X), and (XI);
Figure imgf000030_0001
[0085] Thus, according to some embodiments, the polyproline-based block copolymers of formula (I), the compound comprising a structural unit of formula (I') or the compound comprising a structural unit of formula (I”) may comprise at least one lipid-like moiety R10 which is a radical derived from any known lipid, including fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, sterols, and the like, which are insoluble in water. [0086] As it is mentioned above, the first aspect of the invention is a copolymer (a polyproline-based block copolymer) whose structure is composed of multiple repeating units. The term "copolymer” refers to polymers obtained by polymerization of two or more different kinds of monomers, thereby the repeating units are different. Particularly, the copolymers can be divided into "block copolymers” or "random copolymers” the term "block copolymer" refers to a polymer comprising two or more homopolymer subunits linked by covalent bonds. Therefore, a block copolymer is made of blocks of different polymerized monomers. Meanwhile, the "random copolymer” refers to a polymer comprising two or more monomers that are distributed randomly throughout the polymer without forming blocks.
[0087] The terms "weight average molecular weight”, "weight average molar weight” and the abbreviature "Mw” have the same meaning and they are used interchangeable. Mw is a way of determining the molecular weight of a polymer. Polymer molecules, even ones of the same type, come in different sizes (chain lengths, for linear polymers), so the average molecular weight will depend on the method of averaging. It is determined by summing all molecular weights of the fractions of the polymer multiplied by their weight fractions:
Figure imgf000031_0001
wherein w, is the number of molecules having the molecular weight M/.
[0088] The weight average molecular weight of a polyproline-based block copolymer of the present invention can be determined by any method known in the state of the art for instance gel permeation chromatography (GPC), viscometry via the (Mark-Houwink equation), colligative methods such as vapor pressure osmometry, analytical size-exclusion chromatography, and refractive index detector (SEC/M ALS/RI ), and end-group determination or proton NMR. For the purpose of the present invention, the measurement of the weight average molecular weight (Mw) of polyproline-based block copolymers were performed by analytical size-exclusion chromatography coupled with multi-angle light scattering and refractive index detector (SEC/MALS/RI).
[0089] The terms "number average molecular weight”, "number average molar mass” and the abbreviature "Mn” have the same meaning and they are used interchangeable. Mn is a way of determining the molecular mass of a polymer. Polymer molecules, even ones of the same type, come in different sizes (chain lengths, for linear polymers), so the average molecular mass will depend on the method of averaging. The number average molecular mass is the ordinary arithmetic means or average of the molecular masses of the individual macromolecules. It is determined by measuring the molecular mass of n polymer molecules, summing the masses, and dividing by n. The Mn is calculated by the following formula:
Figure imgf000032_0001
wherein N, is the number of molecules of molecular mass M/.
[0090] The number average molecular mass of a polymer can be determined by gel permeation chromatography (GPC), viscometry via the (Mark-Houwink equation), colligative methods such as vapor pressure osmometry, end-group determination or proton NMR. For the purpose of the invention the Mn is measured by GPC.
[0091] In the embodiments of the invention where the substitution or unsubstitution of a certain group is not specified, i.e., a certain substitution for that group is not indicated, nor is it indicated that the group is unsubstituted, it has to be understood that the possible substitution of this group is the broadest one as defined herein.
[0092] In the polyproline-based block copolymers of the invention, each Rs and L4 are independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl-Ri1, -(Ci-C3o)alkyl-COORi1, -(Ci-C3o)alkyl-0-Riii1, -(Ci-C3o)alkyl-NRiv1Rv1, -C(O)-Rvi1, -(Ci-Cis)alkyl-CO-NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety Rw, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI) and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof;
I2, v2, w2, x2, y2, z2 and o2 are each independently an integer selected from 1 to 6; each R'1 is independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2- Csojalkynyl, -OAIkyl(Ci-C6), halogen, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-C30)alkyl, -OC(O)O(Ci-C30)alkyl, -OC(O)NH2, -OC(O)N((Ci-C30)alkyl)2, -SH, -S(Ci-C30)alkyl, -S(O)H, -S(O)(Ci-C30)alkyl, -SO2(Ci-C30)alkyl;
R'1, Riii1, Rv1, Rv1 and Rvi1 are each independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl NH2, -(Ci-C3o)alkyl-N((Ci-C3o)alkyl)2, -(Ci-C3o)alkyl-NH(Ci-C3o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein each R'1, Rii1, Riii1, R'v1, Rv1, and Rvi1 are independently optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -0(Ci-C3o)alkyl, -CF3, -OCF3, -NH2, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-C30)alkyl-OH; and each Rs and L4 are independently optionally substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety R10; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit, or a mixture thereof; or a mixture thereof.
[0093] According to an embodiment, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer each Rs and L4 are independently selected from selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Ci8)alkenyl, -(Ci-Cis)alkyl-R1, -(Ci-Cis)alkyl-COO i1, -(Ci-Cis)alkyl-O-R"1, -(Ci-Ci8)alkyl-NRiv1Rv1, -C(O)-Rvi1, -(Ci-Ci8)alkyl-CO-NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety R10, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXVII), (XXXIII), (XXXVI) and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; each R'1 is independently selected from the group consisting of H, -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, -(C2- Cis)alkynyl, -OAIkyl(Ci-C4), F, Cl, Br, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-Ci8)alkyl, -OC(O)O(Ci-Ci8)alkyl, -OC(O)NH2, -OC(O)N((Ci-Ci8)alkyl)2, -SH, -S(Ci-Ci8)alkyl, -S(O)H, -S(O)(Ci-Ci8)alkyl, -SO2(Ci-Cis)alkyl;
R'1, Riii1, Rv1, Rv1 and Rvi1 are each independently selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, -(Ci-Cis)alkylNH2, -(Ci-Cis)alkyl-N((Ci-Cis)alkyl)2, -(Ci-Cis)alkyl-NH(Ci-Cis)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); R'1, Rii1, Riii1, R'v1, Rv1, and Rvi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -O(Ci-Ci8)alkyl, -CF3, -OCF3, -NH2, -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, -(C2-Ci8)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-Ci8)alkyl-OH; each Rs and L4 are independently optionally substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety R10; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10; or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof.
[0094] According to another embodiment, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer each Rs and L4 are independently selected from selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Ci8)alkenyl, -(Ci-C6)alkyl-R1, -(Ci-C6)alkyl-COORi1, -(Ci-C6)alkyl-O-R"1, -(Ci-C6)alkyl-NRiv1Rv1, -C(O)-Rvi1, -(Ci-C6)alkyl-CO-NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety R10, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXVII), (XXXIII), (XXXVI) and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; each R'1 is independently selected from the group consisting of H, -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, -(C2- Ci8)alkynyl, -OAIkyl(Ci-C4), F, Cl, Br, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-Ci8)alkyl, -OC(O)O(Ci-Ci8)alkyl, -OC(O)NH2, -OC(O)N((Ci-Ci8)alkyl)2, -SH, -S(Ci-Ci8)alkyl, -S(O)H, -S(O)(Ci-Ci8)alkyl, -SO2(Ci-Ci8)alkyl;
R'1, Riii1, Rv1, Rv1 and Rvi1 are each independently selected from the group consisting of H, -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, -(Ci-Ci8)alkylNH2, -(Ci-Ci8)alkyl-N((Ci-Ci8)alkyl)2, -(Ci-Ci8)alkyl-NH(Ci-Ci8)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI);
R1, Rii1, Riii1, Rv1, Rv1, and Rvi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -O(Ci-Ci8)alkyl, -CF3, -OCF3, -NH2, -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, -(C2-Ci8)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-Ci8)alkyl-OH; and Rs and L4 are optionally substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety Rw; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof.
[0095] According to another embodiment, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer, each Rs and L4 are independently selected from selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, -(Ci-Cejalkyl-R'1 , - (Ci-C6)alkyl-COORii1, -(Ci-C6)alkyl-O-R"1, -(Ci-C6)alkyl-NRiv1Rv1, -C(O)-Rvi1, -(Ci-C6)alkyl-CO-NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety Rw, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXV), (XXVII), and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid -like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; each R'1 is independently selected from the group consisting of H, -(Ci-C )alkyl, -(C2-Ci8)alkenyl, -(C2- Cw)alkynyl, -OAIkyl(Ci-C4), F, Cl, Br, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-Ci8)alkyl, -OC(O)O(Ci-Ci8)alkyl, -OC(O)NH2, -OC(O)N((Ci-Ci8)alkyl)2, -SH, -S(Ci-Ci8)alkyl, -S(O)H, -S(O)(Ci-Ci8)alkyl, -SO2(Ci-Ci8)alkyl;
R'1, Riii1, Rv1, Rv1 and Rvi1 are each independently selected from the group consisting of H, -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, -(Ci-C4)alkylNH2, -(Ci-C4)alkyl-N((Ci-C4)alkyl)2, -(Ci-C4)alkyl-NH(Ci-C4)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI);
R1, Rii1, Riii1, Rv1, Rv1, and Rvi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, F, Cl, Br, -O(Ci-Ci8)alkyl, -CF3, -OCF3, -NH2, -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, -(C2-Ci8)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-Ci8)alkyl-OH; and each R8 and L4 are independently optionally substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety Rw; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof. [0096] According to another embodiment, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer each Rs and L4 are independently selected from a detection moiety, an active moiety, a lipid-like moiety R10, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, H, methyl, ethyl, propyl, isopropyl, butyl, n-butyl, -(C2-Cis)alkenyl, -CH2SCH3,-CH2CH2SCH3, -CH2CH2SCH2CH3, -CH2SCH2CH3, -CH2SH, -CH2-SeH, -CH2CH2SH, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -CH2CH(CH3)CH2NH2, -CH2NHCH3, -CH2NHCH2CH3, -CH2CH2NHCH3, -CH2CH2NHCH2CH3, -CH2CH2CH2NHCH3, -CH2CH2CH2NHCH2CH3, -CH2CH(CH3)CH2NHCH3, -CH2CH(CH3)CH2NHCH2CH3, -CH2CH2NHCH2CH2NH2, -CH2CH2CH2NHCH2CH2CH2NH2, -CH2CH2N((CH(CH3)2)), -CH2CH2CH2N((CH(CH3)2)), -CH2CH2NH(CH(CH3)2), -CH2CH2CH2NH(CH(CH3)2), -C(O)H, -C(O)OCH3, -C(O)OCH2CH3, -C(O)OCH(CH3)2, -C(O)CH2NH2, -C(O)CH2CH2NH2, -C(O)CH(CH3)CH2NH2, -C(O)CH2NHCH3, -C(O)CH2NHCH2CH3, -C(O)CH2CH2NHCH2CH3, -C(O)CH2CH2CH2NHCH2CH3, -C(O)CH2CH2CH2NHCH2CH2CH3, -C(O)CH2CH(CH3)CH2NHCH3, -C(O)CH2CH(CH3)CH2NHCH2CH3, -C(O)CH2CH2NHCH2CH2NH2, -C(O)CH2CH2CH2NHCH2CH2CH2NH2, -C(O)CH2CH2N((CH(CH3)2)), -C(O)CH2CH2CH2N((CH(CH3)2)), -C(O)CH2CH2NH(CH(CH3)2), -C(O)CH2CH2CH2NH(CH(CH3)2), -CH2COOCH3, -CH2CH2COOCH3, -CH2COOCH2CH3, -CH2CH2COOCH2CH3, -CH2COOCH(CH3)2, -CH2CONH2, -CH2CH2CONH2, -CH2CH2CH2CONH2, -CONH-oleic, -CONH-noneic, -CONH-lipoic, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; wherein each Rs and L4 are independently optionally substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety R10, or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10. or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof.
[0097] According to another embodiment, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer each Rs and L4 are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, n-butyl, -CH2SCH3, -CH2CH2SCH3, -CH2CH2SCH2CH3, -CH2SCH2CH3, -CH2SH, -CH2CH2SH, -CH2SeH, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety R10, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; wherein each Rs and L4 are independently optionally substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety Rw; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof.
[0098] According to another embodiment, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer each Rs and L4 are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, n-butyl, -CH2SCH3, -CH2CH2SCH3, -CH2CH2SCH2CH3, -CH2SCH2CH3, -CH2SH, -CH2CH2SH, -CH2SeH, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety Rw, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; wherein each Rs and L4 are independently substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety Rw; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety or at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; wherein each Rw is independently selected from the groups consisting of -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, and a radical of formula (XL), (XLI), (XLIII), (XLIV), (XLV), and (XLVI).
[0099] In the polyproline-based block copolymers of the invention,
Li is a biradical selected from the group consisting of (II) and (III), wherein the biradical (II) or (III) is attached to the AA1 , AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond when attached to the N-terminal end of AA1 , AA2 or AA3 repeating unit; or alternatively the biradical (II) or (III) is attached to the AA1 , AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond when is attached to the C-terminal end of the AA1 , AA2 or AA3 repeating unit.
[00100] According to one embodiment, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer
Li is a biradical selected from (II) and (III), which is attached to the C-terminal end of AA1 , AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond;
L4 is selected from the group consisting of an inert moiety; a detection moiety; an active moiety; a lipid- like moiety Rw; a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; wherein the inert moiety is selected from the group consisting of H, -N3, -C(O)H, -S(O)H, -NHCH(CH3)2, -(Ci-C4)alkylNH2, -(Ci-C4)alkylNHCH3, -(Ci-C4)alkylN(CH3)2, -O-(Ci-C4)alkyl-NH2, -O-(Ci-C4)alkyl-NHCH3, -O-(Ci-C4)alkyl-N(CH3)2, -(Ci-Ci2)alkyl, -(C2-Ci2)alkenyl, and -(C2-C6)alkynyl; wherein the inert moiety is optionally substituted by a detection moiety, an active moiety, and a lipid-like moiety Rwor a mixture thereof; the detection moiety is selected from the group consisting of chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, transition metal and isotope mass tag moiety; the active moiety is selected from the group consisting of pharmaceutically active agents, veterinary active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof; and the lipid-like moiety Rw is selected from the group consisting of -(Ci-C jalkyl, -(C2-Ci8)alkenyl, and a radical of formula (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII) or (XLIX).
[00101] According to one embodiment, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer, L4 is a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof.
[00102] According to one embodiment, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer
L1 is a biradical selected from (II) and (III), which is attached to the C-terminal end of AA1, AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond; and L4 is a lipid-like moiety Rw selected from the group consisting of - (Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, and a radical of formula (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII) or (XLIX).
[00103] In accordance with some embodiments, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer of the invention the lipid-like moiety Rw is selected from the group consisting of -(Ci-C jalkyl, -(C2-Ci8)alkenyl, and a radical of formula (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII) or (XLIX) wherein:
T and T are independently selected from -OH, -OCORx and -COORx;
Q and Q' are independently selected from -OCORy and -COORy; each G is independently selected from -OCO-, -COO-, -NRz'CO-, and -CONRz'-; each Rz' is H or Rz; each Rx, Ry, and Rz is independently -(Ci-Ci8)alkyl or -(C2-Ci8)alkenyl; each a1 is independently an integer from 0 to 18; each b1 is independently an integer from 1 to 18; each d is independently an integer from 0 to 18; each d1 is independently an integer from 0 to 18; each e1 is independently an integer from 0 to 18; f1 Is O or 1; fT Is O or 1; TT, T2 and T3 are independently selected from the group consisting of hydrogen, fluorine, methyl, -CH2F, -CHF2, and -CF3; each of the dashed bonds — is independently a single bond or, alternatively, a double bond; J is a biradical chain which comprises one or more moieties selected from the group consisting
Figure imgf000040_0002
wherein Rf, Rg and Rh are radicals independently selected from the group consisting of H, -phenyl, -(Ci- Csojalkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkylphenyl, and -phenyl (Ci-C3o)alkyl; wherein when J comprises a -CH2- moiety, the two hydrogen atoms attached to the carbon atom are optionally replaced by the ring:
Figure imgf000040_0001
[00104] According to one embodiment, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer
L1 is a biradical selected from (II) and (III), which is attached to the C-terminal end AA1, AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond; wherein L4 is a lipid-like moiety Rw; and each Rw is independently selected from the groups consisting of -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, and a radical of formula (XL), (XLI), (XLIII), (XLIV), (XLV), and (XLVI).
[00105] In accordance with some embodiments, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer
Li is a biradical selected from (II) and (III), which is attached to the C-terminal end of AA1, AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond; and L4 is selected from the group consisting of an inert moiety; a detection moiety; an active moiety; a lipid- like moiety Rw; or a linker; the linker being attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw; or alternatively attached to at least one pH, redox or protease responsive unit; and a mixture thereof; wherein the inert moiety is selected from the group consisting of H, -N3, -C(O)H, -S(O)H, -NHCH(CH3)2, -(Ci-C4)alkylNH2, -(Ci-C4)alkylNHCH3, -(Ci-C4)alkylN(CH3)2, -O-(Ci-C4)alkyl-NH2, -O-(Ci-C4)alkyl-NHCH3, -O-(Ci-C4)alkyl-N(CH3)2, -(Ci-Ci2)alkyl, -(C2-Ci2)alkenyl, and -(C2-C6)alkynyl; wherein the inert moiety is optionally substituted by a detection moiety, an active moiety, and a lipid-like moiety Rwor a mixture thereof; the detection moiety is selected from the group consisting of chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, transition metal and isotope mass tag moiety; the active moiety is selected from the group consisting of pharmaceutically active agents, veterinary active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof; and the lipid-like moiety Rw is selected from the group consisting of -(Ci-C jalkyl, -(C2-Ci8)alkenyl, and a radical of formula (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII) or (XLIX).
[00106] According to one embodiment, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymer of formula (I) as defined according to the first aspect of the invention, the compound comprising a structural unit of formula (I') or the compound comprising a structural unit of formula (I”) as defined according to the second aspect of the invention, or the star-shaped polymer as defined according to the third aspect of the invention, each Rw is independently selected from the groups consisting of -(Ci-C jalkyl, -(C2-Ci8)alkenyl, and a radical derived from (2S)-2,5- bis(3-aminopropylamino)- N-[2-(dioctadecyl-amino)acetyl]pentanamide (DOGS), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]-butylcarboxamido)ethyl]-3,4- di[oleyloxy]-benzamide (MVL5), 3« -[N-(N’ N«- dimethylaminoethane)-carbamoyl]cholesterol (DC- Cholesterol), (1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1 ,2-dilinoleyloxy-N,N-dimethyl- 3-ami nopropane (DLin-DMA), 1 ,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 1 ,2-di-y- linolenyloxy-N,N-dimethylaminopropane (’-DLenDMA), 2-dilinoleyl-4-dimethyl-aminomethyl-[1,3]- dioxolane (DLin-K-DMA), 2,2- dilinoleyl-4- dimethylaminoethyl-[1 ,3]- dioxolane (DLin- KC2-DMA, also known as DLin-C2K-DMA, XTC2, and C2K), 2,2-Dilinoleyl-4-(3-dimethylaminopropyl)-[1 ,3]-dioxolane (DLin-KC3-DMA), 2,2-Dilinoleyl-4-(4-dimethylaminopropyl)-[1 ,3]-dioxolane (DLin-KC4-DMA), 1,2- dilinoleny loxy-4-(2-dimethylaminoethyl)-1 ,3-dioxolane (DLen-C2K-DMA), 1,2-di- y-linolenyloxy-4-(2- dimethylaminoethyl)-1 ,3-dioxolane (y-DLen-C2K-DMA), (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31 - tetraen-19-yl 3-(dimethylamino)propanoate (DLin-M-C2-DMA, also known as MC2), (6Z,9Z,28Z,31 Z)- heptatriaconta-6,9,28,31 - tetraen-19- yl 4-(dimethylamino)-butanoate (DLin-M-C3-DMA, also known as MC3), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (DLin-MP-DMA, also known as 1-B11), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N- distearyl-N,N-dimethylammonium bromide (DDA13), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)-propyl)-N,N,N-trimethylammonium chloride (DOTMA), N-[1 -(2,3,-ditetradecyloxy)propyll-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1-(2,3,dioleyloxy)-propyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide (DORIE), 3« -[N-(N’ N«- dimethylamino-ethane)-carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB), 2,3- dioleyloxy- N-[2-(spermine-carboxamido)ethyl]- N,N- dimethyl-1 - propanaminium trifluoroacetate (DOSPA), ethylphosphatidylcholine, (ePC), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ([(4- hydroxybutyl)azanediyl]di(hexane-6,1 -diyl) bis(2-hexyldecanoate)) (ALC-0315), phosphatidylglycerol, diacylphosphatidylserine, diacylphosphatidic acid, N-Succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine cholesterol hemisuccinate (CHEMS), lysylphos-phatidylglycerol, N- dodecanoyl phosphatidyl ethanoloamine, cardiolipin, steroids (such as cholesterol, progesterone, cortisone, aldosterone, estradiol, testosterone), phospholipids (such as phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidylinositol (PI), dimyristoyl phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl serine (DSPS), dioleoyl phosphatidyl serine (DOPS), dipalmitoyl phosphatidyl serine (DPPS), dioleoyl phosphatidyl ethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyp-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidyl-ethanolamine (DSPE); lysophosphatidyl choline, lysophosphatidylethanolamine, 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), dilauryloylphosphatidylcholine (DLPC), 1-myristoyl- 2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1- palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1 ,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), and 1,2-dieicosenoyl-sn-glycero3-phosphocholine (DEPC)).
[00107] In the polyproline-based block copolymers of formula (I), m is an integer selected from 4 to 100; preferably from 6 to 80; preferably from 6 to 60, more preferably from 6 to 40; n is an integer selected from 0 to 150; preferably from 0 to 140, more preferably from 20 to 125; p is an integer selected from 0 to 150; preferably from 0 to 120, preferably from 0 to 100, more preferably from 0 to 80, from 4 to 80, from 4 to 50; q is an integer selected from 0 to 100; preferably from 0 to 80, preferably from 0 to 70, more preferably from 0 to 60; wherein at least one of n, p or q is • 4 or alternatively n + p • 4; with the proviso that when p« 0, and R8 is not a cationic moiety, then n • 0; with the proviso that when n=0, then p« 0; and R8 is a cationic moiety; and with the proviso that when p=0, then n« 0.
[00108] In accordance with some embodiments, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymers of formula (I) m is an integer selected from 6 to 80; p is an integer selected from 4 to 50; q is an integer selected from 0 to 80 n is an integer selected from 0 to 140 at least one of n, p or q is • 6 or alternatively n + p • 6; and m>p; with the proviso that when p« 0, and R8 is not a cationic moiety, then n • 0; with the proviso that when n=0, then p« 0; and R8 is a cationic moiety; and with the proviso that when p=0, then n« 0.
[00109] Particular examples of the polyproline-based block copolymer of formula (I) according to this embodiment are:
(1a) Ppro22POrn6
(1c) Ppro30POrn16
(lf) Ppro12POrn6
(lg) pPro7POrn6
(lh) Ppro54POrn18
(li) Ppro64POrn22
(2a) Ppro19POrn6
[00110] In accordance with some embodiments, optionally in combination with any of the embodiments provided above or below, in the polyproline-based block copolymers of formula (I) m is an integer selected from 4 to 40; p is an integer selected from 4 to 80; q is an integer selected from 0 to 80; n is an integer selected from 0 to 140; at least one of n, p or q is • 6 or alternatively n + p • 6; and p>m; with the proviso that when p« 0, and R8 is not a cationic moiety, then n • 0; with the proviso that when n=0, then p« 0; and R8 is a cationic moiety; and with the proviso that when p=0, then n« 0.
[00111] Particular examples of the polyproline-based block copolymer of formula (I) according to this embodiment are:
(1b) Ppro52POrn10
(ld) pPro5POrn35
(le) Ppro15POrn35
(5c) pPro5PLys56
(7a) PProl 0-(Plys7-PArg24-PVal 10)
[00112] In one embodiment, optionally in combination with any of the embodiments provided above or below, m is an integer selected from 4 to 40; n is an integer selected from 20 to 140; q is an integer selected from 0 to 80; p is an integer selected from 0 to 80; at least one of n, p or q is • 6 or alternatively n + p • 6; and n>m; with the proviso that when p« 0, and R8 is not a cationic moiety, then n • 0; with the proviso that when n=0, then p« 0; and R8 is a cationic moiety; and with the proviso that when p=0, then n« 0.
[00113] In one embodiment, optionally in combination with any of the embodiments provided above or below, m is an integer selected from 4 to 40; preferably from 6 to 36; from 8 to 32; from 10 to 30, from 12 to 28; from 16 to 24; from 18 to 22; p is an integer selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; each R8 is independently a cationic moiety selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); wherein each Rs are optionally independently substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety R10; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety or at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof. [00114] In one embodiment, optionally in combination with any of the embodiments provided above or below, m is an integer selected from 4 to 40; preferably from 6 to 36; from 8 to 32; from 10 to 30, from 12 to 28; from 16 to 24; from 18 to 22; p is an integer selected from 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, and 20; n=0;
L4 is selected from the group consisting of an inert moiety, a detection moiety, an active moiety, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a lipid-like moiety R10; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; each R8 is independently a cationic moiety selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); wherein each Rs are optionally independently substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety R10; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; wherein the inert moiety is selected from the group consisting of H, -N3, -C(O)H, -S(O)H, -NHCH(CHs)2, -(Ci-C4)alkylNH2, -(Ci-C^alkylNHCHs, -(Ci-C4)alkylN(CH3)2, -O-(Ci-C4)alkyl-NH2, -O-(Ci-C4)alkyl-NHCH3, -O-(Ci-C4)alkyl-N(CH3)2, -(Ci-Ci2)alkyl, -(C2-Ci2)alkenyl, and -(C2-C6)alkynyl; wherein the inert moiety is optionally substituted by a detection moiety, an active moiety, and a lipid-like moiety Rwor a mixture thereof; the detection moiety is selected from the group consisting of a chromophore moiety, a fluorescent moiety, a phosphorescent moiety, a luminescent moiety, a light absorbing moiety, a radioactive moiety, a transition metal and an isotope mass tag moiety; the active moiety is selected from the group consisting of pharmaceutically active agents, veterinary active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof; and the lipid-like moiety Rw is selected from the group consisting of - (Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, and a radical of formula (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII) or (XLIX).
[00115] Particular examples of the polyproline-based block copolymer of formula (I) according to this embodiment are those wherein
(16) Ppro22POrn6ValCitrulinePGA-PEG-Azide
Figure imgf000046_0001
[00116] The polyproline-based block copolymers of the invention are polymeric structures containing, as repeating units, the following polyamino acid-based moieties PAA1, AA1, AA2 and AA3
Figure imgf000046_0002
Figure imgf000047_0001
wherein the “*” denote the attaching points of the PAA-based moiety to the rest of the molecule. [00117] In the formula (I) as depicted above, the main repeating units AA1, AA2 and AA3 are shown in a particular order for convenience of description, the main repeating units may be present in any order and may be block or randomly present; and each of the repeating units, may comprise blocks of monomer units which may be the same or different from each other.
[00118] The orientation of the polyamino acid-based moiety does not influence the amphiphilic behavior of the polymers described herein, and consequently the activity displayed is maintained.
[00119] According to one embodiment, optionally in combination with any of the embodiments provided above or below, the polyamino acid-based moieties PAA1, AA1, AA2 and AA3 have the formula (PAA'), (AAT), (AA2’) and (AA3’)
Figure imgf000047_0002
(AA3‘) Preparation processes
[00120] Processes for the preparation of the polyproline-based block copolymers as defined herein are also part of the invention. The appropriate reagents and theirs amounts as well as the reaction conditions (for example temperature, time, and solvents), can be determined by those skilled in the art according to the polyproline-based block copolymer being prepared. The above mentioned polyprolinebased block copolymers can be prepared according to polymerizing methods well known in the state of the art.
[00121] Polyproline based block copolymers were prepared by NCA polymerization techniques using the corresponding NCA monomers with adequate protecting groups when necessary, and primary amine initiators. Briefly, the block designed to be the polycation block was first polymerized using Schlenk techniques under Nitrogen atmosphere and in a suitable solvent using n-butylamine, or other suitable primary amine. Reactions usually proceed at low temperature (10°C) and monomer consumption was confirmed by IR usually after 16 hours. After IR verification, the proline NCA was polymerized either by adding the previous solution to the polyproline NCA suspended in acetonitrile or viceversa. The mixture was stirred at room temperature for 16 hours. For purification, the reaction mixture is precipitated (commonly in diethyl ether). The precipitate was isolated by centrifugation and dried under vacuum or resuspended and liophillized. The block copolymer was isolated as a white solid. Deprotection of the protecting groups (i.e. to obtain ornithine, lysine and arginine block copolymers) and/or backbone modifications (i.e. to obtain oligoamine block copolymers) where performed in a second step. Drug conjugations were performed using the free amines of the polymer backbone with the carboxylic group activation of the drugs using DMTMM BF4. Dye conjugations were performed using the form of NHS-Dyes and the free amines of the polymer backbone.
[00122] Examples of appropriate solvents for performing the preparation processes disclosed herein above and below for the polyproline-based block copolymers of the present invention include, but it is not limited to, (Ci-Csjalcohols such as ethanol and methanol; dimethylsulphoxide (DMSO) and dimethylformamide (DMF). The term "alcohol” refers to an "alkane” wherein at least one hydrogen atom is substituted by a hydroxyl group and which contains the number of carbon atoms specified in the description or claims. The term "alkane" refers to a saturated, branched, or linear hydrocarbon which contains the number of carbon atoms specified in the description or claims.
[00123] These processes can be performed at a temperature from room temperature to below the boiling point of the solvent. The term "room temperature” refers to a temperature of the environment, without heating or cooling, and it is generally comprised from 20 °C to 25 °C. For example, the processes disclosed in the present invention can be performed using ethanol as solvent at a temperature from 20 to 78°C; or in the sinus of dimethyl sulfoxide at a temperature from 20-140°C, for the appropriate time for obtaining the polyproline-based block copolymers of the present invention. By way of example, a process for the preparation of a polyproline-based block copolymer of the present invention having a weight average molecular weight (Mw) from 2000 to 30000 Da can be performed following any one of the methods A-C as defined above using; particularly in the sinus of (C1-C5) alcohol (particularly ethanol) as a solvent and at a temperature from 20-78°C.
[00124] It is also part of the invention a polyproline-based block copolymer characterized by its preparation process. Therefore, the polyproline-based block copolymers of the present invention obtainable by the processes disclosed above in the present application are also part of the invention. For the purposes of the invention the expressions "obtainable", "obtained" and equivalent expressions are used interchangeably, and in any case, the expression "obtainable" encompasses the expression "obtained".
[00125] Some of the polyproline-based block copolymers of the invention can have chiral centers that can give rise to various stereoisomers. As used herein, the term "stereoisomer" refers to all isomers of individual polyproline-based block copolymers that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers (enantiomers), mixtures of mirror image isomers (racemates, racemic mixtures), geometric (cis/trans or syn/anti or E/Z) isomers, and isomers of polyproline-based block copolymers with more than one chiral center that are not mirror images of one another (diastereoisomers). The present invention relates to each of these stereoisomers and also mixtures thereof. Hence, the definition of the polyproline-based block copolymers of formula (I) is also intended to encompass all R- and S-isomers of a chemical structure in any ratio, e.g. with enrichment (i.e. enantiomeric excess or diastereomeric excess) of one of the possible isomers and corresponding smaller ratios of other isomers. In the particular case of amino acids, they may acquire L-configuration or D-configuration.
[00126] Diastereoisomers and enantiomers can be separated by conventional techniques such as chromatography or fractional crystallization. Optical isomers may be obtained using enantiospecific synthesis. Alternatively, optical isomers can be resolved by conventional techniques to give optically pure isomers. The resolution can be carried out on any chiral synthetic intermediates or on the polymers of the invention.
[00127] In all embodiments of the invention referring to the polyproline-based block copolymers, the pharmaceutically, cosmetically or diagnostically acceptable salts thereof and the stereoisomers or mixtures of stereoisomers, either of any of the polyproline-based block copolymers or of any of their pharmaceutically, cosmetically or diagnostically acceptable salts are always contemplated even if they are not specifically mentioned.
[00128] The polyproline-based block copolymers of the invention may be in crystalline form either as free solvation polyproline-based block copolymers or as solvates (e.g. hydrates). It is intended that all these forms are within the scope of the present invention. Methods of solvation are generally known within the art. For the purposes of the invention, the solvated forms with pharmaceutically, cosmetically or diagnostically acceptable solvents such as water, ethanol and the like are equivalent to the unsolvated form.
[00129] Except as otherwise specified, the polyproline-based block copolymers of the present disclosure also include polyproline-based block copolymers that differ only in the presence of one or more isotope-enriched atoms. Examples of isotope-enriched atoms, without limitation, are deuterium, tritium, 13C or 14C, or a nitrogen atom enriched in 15N or a fluorine atom enriched in 18F.
[00130] In accordance with an additional aspect, the invention provides a conjugate which comprises the polyproline-based block copolymer of the first aspect of the invention and one or more molecules of interest.
[00131] For the purpose of the invention, the terms "conjugate” and "complex” have the same meaning and are used interchangeable. They refer to the union of a polyproline-based block copolymer of the first aspect of the invention and one or more molecules of interest linked together. The union between the polyproline-based block copolymer and the molecule of interest is "non-covalent”. The term "non- covalent” refers to the bond between the polyproline-based block copolymer and the molecule of interest that involves weak interactions such as for example ionic interactions, electrostatic interactions, hydrogen bonding and/or van der Waals interactions. The type of interaction (union) mainly depends on the molecule of interest. In particular, the type of union between the polyproline-based block copolymer and the molecule of interest can be readily determined by those skilled in the art according to the chemical structure and the physico-chemical properties of the molecule of interest used for the preparation of the conjugate. For the purpose of the invention, the term "conjugate” encompasses the term “poly plex” which refers to a specific conjugate of the present invention comprising a polyprolinebased block copolymer of the invention and one or more genetic material, including a nucleic acidcontaining compound, as a molecule of interest (also called "cargo”).
[00132] For the purpose of the present invention, the term "molecule of interest” encompasses active ingredients, amino acid-containing compounds, nucleic acid-containing compounds, and mixtures thereof.
[00133] In an embodiment, the conjugate of the invention is one wherein the one or more molecules of interest comprises at least one active ingredient as defined above.
[00134] In an embodiment, the conjugate of the invention is one wherein the one or more molecules of interest comprises at least one amino acid-containing compound. In an embodiment, the conjugate of the invention is one wherein the one or more molecules of interest comprises at least one amino acidcontaining compound selected from the group consisting of a polypeptide, a protein and a mixture thereof. The terms "peptide” and "polypeptide” have the same meaning and are used interchangeably. They refer to chains having from 2 to 50 amino acid residues, and the term "protein” refers to chains of more than 50 amino acid residues. In an embodiment, the conjugate of the invention is one wherein the one or more molecules of interest comprises at least an antibody or a fragment thereof as amino acidcontaining compound. The term "antibody or a fragment thereof' refers to any immunoglobulin or fragment thereof suitable to bind an epitope of the target protein. It includes monoclonal and polyclonal antibodies. The term "fragment thereof' encompasses any part of an antibody having the size and conformation suitable to bind an epitope of the target protein. Suitable fragments include F(ab), F(ab') and Fv. An "epitope" is the part of the antigen being recognized by the immune system (B-cells, T-cells or antibodies). Particularly, the antibodies used for specific detection can be polyclonal or monoclonal. There are well known means in the state of the art for preparing and characterizing antibodies. Methods for generating polyclonal antibodies are well known in the prior art. Briefly, one prepares polyclonal antibodies by immunizing an animal with the protein; then, serum from the immunized animal is collected and the antibodies isolated. A wide range of animal species can be used for the production of the antiserum. Typically, the animal used for production of antisera can be a rabbit, mouse, rat, hamster, guinea pig or goat. Moreover, monoclonal antibodies (MAbs) can be prepared using well-known techniques. Typically, the procedure involves immunizing a suitable animal with the protein associated with the disease. The immunizing composition can be administered in an amount effective to stimulate antibody producing cells. Methods for preparing monoclonal antibodies are initiated generally following the same lines as the polyclonal antibody preparation. The immunogen is injected into animals as antigen. The antigen may be mixed with adjuvants such as complete or incomplete Freund's adjuvant. At intervals of two weeks, approximately, the immunization is repeated with the same antigen.
[00135] In an embodiment, the conjugate of the invention is one wherein the one or more molecules of interest comprises at least one nucleic acid-containing compound. In an embodiment, the conjugate of the invention is one wherein the one or more molecules of interest comprises at least one nucleic acidcontaining compound selected from the group consisting of single strand oligonucleotides such as DNA, RNA, PNA, LNA and analogues thereof; double-strand oligonucleotides such as siRNA, shRNA, decoy DNA; plasmids and analogues thereof. As it is demonstrated in the experimental section, the polyproline-based block copolymer of the present invention can be used as transfecting agent. In fact, it is shown that a polyplex comprising the polyproline-based block copolymer of the present invention is capable of conjugate until ten molecules of nucleic acid of interest for transfecting into cells.
[00136] All embodiments disclosed above for the polyproline-based block copolymer also applies for the conjugate of this aspect of the invention.
[00137] It is also a part of the invention a process for the preparation of the conjugates of the invention. The appropriate reagents and theirs amounts as well as the reaction conditions, can be determined by those skilled in the art according to the conjugate being prepared. Commonly, the process for the preparation of the conjugates comprises contacting the polyproline-based block copolymer with the molecule of interest under such reaction conditions that allows the union between them.
[00138] Further, it is also part of the invention a conjugate characterized by its preparation process. Therefore, the conjugate of the second aspect of the invention obtainable by the processes disclosed above in the present application are also part of the invention. All embodiments disclosed above for the polyproline-based block copolymer of the first aspect of the invention, and the conjugate of the second aspect of the invention also apply here for the conjugate obtainable by its preparation process.
Star-shaped polymers
[00139] The term "star-shaped polymers” according to the present invention relates to at least three compounds comprising the structural unit of formula (I') or (I”) radiating from one single star-shaped multifunctional linking agent, usually called the core or the central nodule, and which can itself be polymeric.
[00140] The polyproline-based copolymers of the invention may be attached to a star-shaped multifunctional linking agent, thus forming a star-shaped polymer. The star-shaped polymers thus obtained comprise at least two, preferably 2, 3, 4, 5 or 6 structural units of formula (I') or (I”) as defined above.
[00141] The star-shaped polymers of the present invention may comprise symmetric or asymmetric architecture depending on the presence of same or combination of different polymeric arms emanating from a shared core.
[00142] In accordance with an embodiment, the star-shaped polymer comprises a symmetric architecture of structural units of formula (I') or (I”), as defined herein, as arms emanating from a shared core.
[00143] In the context of the present invention, the core of the star-shaped polymers is also referred herein as "star-shaped multifunctional linking agent”.
[00144] Star-shaped multifunctional linking agents suitable for the preparation of chemical-mediated vectors for the delivery of molecules of interest, such as active ingredients or detection moieties acting as cell penetrating peptide for the cellular delivery of attached cargoes, are well known in the art.
[00145] In accordance with some embodiments, the star-shaped multifunctional linking agent is selected from the group consisting of
I) a multi-arm star-shaped linking agent based on branched polyethylenimine;
II) a 3-arm star-shaped linking agent based on branched polyamidoamine of formula (Pm)
Figure imgf000052_0001
wherein each U is selected from -0- and -NH-;
E and E’ are each independently selected from -0- and -NH-; wherein the 3-arm star-shaped linking agent of formula (Pm) is attached to the L4 moiety of each of the at least one polyproline-based block copolymer of formula (I) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond; ill) a 4-arm star-shaped linking agent of formula (Tz)
Figure imgf000053_0001
wherein each V is selected from -0- and -NH-;
Y, Y' and Y” are each independently selected from -0- and -NH-; wherein the 4-arm star-shaped linking agent of formula (Tz) is attached to the L4 moiety of each of the at least one polyproline-based block copolymer of formula (I) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond.
Self-assembled particles
[00146] The polyproline-based copolymers of the invention may have amphiphilic nature since they may comprise a hydrophilic polyamino acid-based moiety and at least a hydrophobic moiety. Thanks to their amphiphilic nature, the polyamino acid copolymers of the invention may form self-assembled particles in solution such as for example liposomes or lipid nanoparticles which may be used to encapsulate and deliver molecules of different nature. The polyproline-based copolymers of the invention may also form polymer nanoparticles (PNP), including polyplexes. Thus, the compounds of the invention allow carrying and/or delivering different active agents at the same time, not only bonded to the structure of the compound but also contained in self-assembled particles formed from the compound.
[00147] Therefore, the present invention also relates to a self-assembled particle comprising the polyproline-based block copolymers of formula (I) as defined according to the first aspect of the invention, the compound comprising the structural unit of formula (I') or the structural unit of formula (I”) as defined according to the second aspect of the invention, or the star-shaped polymers as defined according to the third aspect of the invention, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, penetration enhancing agents, cell-targeting agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
[00148] As used herein, the term "self-assembled particles” refers to the arrangement of the polyproline-based block copolymers of formula (I), the compounds comprising the structural unit of formula (I') or (I”), or the star-shaped polymers in a solvent. In particular, small, self-assembled particles may be enclosed structures of any shape, typically spherical and/or tubular. The term "self-assembled particles” intends to encompass any of a number of structures that are known in the art to be formed from amphiphilic polymers. Non-limiting examples of self-assembled particles include micelles (also being referred to interchangeably herein as micellar worms or simply "worms”), inverted micelles, planar bilayers, crystal nanoparticles, liposomes, microbubbles or lipid nanoparticles. The self-assembled nanoparticles and microparticles can also form gels.
[00149] As mentioned above, in accordance with some embodiments of the invention, the polyprolinebased copolymers of formula (I), or the compounds comprising the structural unit of formula (I') or the compounds comprising the structural unit of formula (I”) of the invention may comprise at least one lipid- like moiety Rw, Thus, in accordance with some embodiments, at least one of the R8 and/or L4 comprise a lipid-like moiety Rw. Therefore, it is possible that the polyproline-based block copolymers of formula (I), or the compounds comprising the structural unit of formula (I') or the compounds comprising the structural unit of formula (I”) of the invention comprise at least one lipid-like moiety Rw, both in R8 and L4.
[00150] In accordance with some embodiments, optionally in combination with any of the embodiments provided above or below, the polyproline-based copolymers of formula (I) are those of formula (la)
Figure imgf000054_0001
[00151] In accordance with some embodiments, optionally in combination with any of the embodiments provided above or below, the polyproline-based copolymers of formula (I) are those of formula (lb)
Figure imgf000055_0001
[00152] In accordance with some embodiments, optionally in combination with any of the embodiments provided above or below, the polyproline-based copolymers of formula (I) are those of formula (Ic)
Figure imgf000055_0002
[00153] Thus, in those embodiments, lipid-containing polyproline-based copolymers, according to formula (la), (lb) and (Ic), have amphiphilic nature since they comprise a polyamine acid-based moiety (hydrophilic part) and at least one lipid-like moiety (hydrophobic part). Thanks to their amphiphilic nature, the lipid-containing polyproline-based copolymers of the invention may form self-assembled particles in solution, such as for example lipid nanoparticles (LNPs), and can be used as non-viral vectors for delivery of active ingredients, including nucleic acids, to cells.
[00154] Therefore, the present invention also relates to a self-assembled particle comprising the lipid- containing polyproline-based copolymer as defined herein according to formula (la), (lb) or (Ic), the compound comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid-like moiety Rw; and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, penetration enhancing agents, cell-targeting agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
[00155] In some embodiments, the self-assembled particles are arrangements of the lipid-containing polyproline-based copolymers in a solvent, in particular, small, self-assembled particle may be enclosed structures of any shape, typically spherical and/or tubular. The term "self-assembled particles” intends to encompass any of a number of structures that are known in the art to be formed from amphiphilic polymers. Non-limiting examples of self-assembled particles include micelles (also being referred to interchangeably herein as micellar worms or simply "worms”), inverted micelles, planar bilayers, crystal nanoparticles, liposomes, microbubbles or lipid nanoparticles. The self-assembled nanoparticles and microparticles can also form gels. The self-assembled particles of the invention are non-viral particles, which means that they are not able to virally infect cells.
[00156] In accordance with some embodiments, optionally in combination with any of the embodiments provided above or below, the polyproline-based copolymers of formula (I) are those wherein R8 is independently substituted by at least one linker attached to at least one inert moiety, at least one detection moiety, at least one active moiety or at least one lipid-like moiety Rw.
[00157] In accordance with some particular embodiments, the self-assembled particle is a lipid nanoparticle. In accordance with other embodiments, the self-assembled particle is a polymer nanoparticle, more particularly a polyplex.
[00158] In accordance with some embodiments, optionally in combination with any of the embodiments provided above or below, the self-assembled particle further comprises one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids.
[00159] In accordance with some embodiments, optionally in combination with any of the embodiments provided above or below, the self-assembled particle further comprises an ionizable lipid or a cationic lipid, a phospholipid, and a sterol.
[00160] In an embodiment, optionally in combination with one or more features of the various embodiments described above, the lipid nanoparticle further comprises at least one lipid selected from the group consisting of a non-cationic lipid, a sterol or a steroid precursor, and a PEG-modified lipid.
[00161] Examples of sterols include, without being limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above, the sterol is cholesterol. Examples of sterol precursors include, without being limited to, a triterpene, a triterpenoid, or a steroid precursor of this kind. Non-limiting examples of triterpenes, triterpenoids and other steroid precursors include squalene, achilleol, polypodatetrane, lanostane, cucurbitacin, hopane, oleanane, chamaecydin, lupine, and mixtures thereof.
[00162] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the lipid nanoparticles comprise the compounds of formula (la), (lb) or (Ic), the compounds comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid-like moiety Rw of the present disclosure, a non-cationic lipid as defined above, and a sterol or a sterol precursor as defined above. [00163] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the lipid nanoparticles further comprise a PEG-modified lipid. The term "PEG-modified lipid refers to a lipid comprising a polyethylene moiety. Examples of PEG-modified lipids include, without being limited to, a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified phosphatidylcholine, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG- DOMG), PEG-DSG, PEG-DPG, or a combination thereof.
[00164] In an embodiment, optionally in combination with one or more features of the various embodiments described above, the lipid nanoparticles comprise a lipid component comprising or consisting of the compounds of formula (la), (lb) or (Ic), the compounds comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid-like moiety Rw of the present disclosure, a non-cationic lipid, a sterol, and a PEG-modified lipid.
[00165] In accordance with some embodiments, optionally in combination with any of the embodiments provided above or below, the self-assembled particle comprises: i) the polyproline-based block copolymer of formula (I), or alternatively the compound comprising the structural unit of formula (I') or (I”), in an amount from 0.1 mol% to 60 mol%; in some embodiments from 0.5 mol% to 50 mol%, in some embodiments from 5 mol% to 40 mol%; in some embodiments from 10 mol% to 20 mol%; ii) an ionizable or a cationic lipid in an amount from 30 mol% to 70 mol%, in some embodiments from 40 mol% to 60 mol%; in some embodiments from 45 mol% to 50 mol% iii) a phospholipid in an amount from 1 mol% to 20 mol%, in some embodiments from 3 mol% to 15 mol%; in some embodiments from 5 mol% to 10 mol%; and iv) a sterol in an amount from 20 mol% to 60 mol%, in some embodiments from 30 mol% to 50 mol%; in some embodiments from 35 mol% to 45 mol%; wherein the percentages are expressed with respect to the sum of the mol% of the lipids and the polyproline-based block copolymer of formula (I) or the compound comprising the structural unit of formula (I’) or (I”).
[00166] The self-assembled particles may show a variety of sizes, in particular, they can be nanoparticles or microparticles. A "nanoparticle”, as defined herein, is any particle of nanometric size, in particular having smallest end-to-end diameter of between 1 and 900, more particularly, between 1 and 700 nm, between 1 and 500 nm, between 1 and 300 nm, between 1 and 200 nm, and between 1 and 100 nm in size. A "microparticle”, as defined herein, is typically any particle of micrometric size, having a smallest end-to-end between 1 and 100 pm in size. Typically, in a composition comprising a plurality of particles, the relevant diameter is the number average diameter.
[00167] As used herein, the term "size" refers to a characteristic physical dimension. For example, in the case of a particle that is substantially spherical, the size of the particle corresponds to the diameter of the particle. In the case of a particle that is substantially rod-shaped with a substantially circular crosssection, such as wire or a tube, the size of the particle is determined by the diameter of the two relevant cross-section dimensions of the particle. In the case of a particle that is substantially box-shaped, such as a cube, a box, or a cage, the size of the nanoparticle corresponds to the maximum edge length. When referring to a set of particles as being of a particular size, it is contemplated that the set of particles can have a distribution of sizes around the specified size. Thus, as used herein, a size of a set of particles can refer to a mode of a distribution of sizes, such as a peak size of the distribution of sizes.
[00168] As used herein, the term "diameter” refers to the average diameter and is also designated as Z- average or Z-ave. The average diameter corresponds to the mean hydrodynamic diameter (Dh) and can be measured by dynamic light scattering (DLS) as shown in the examples below. In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the self-assembled particles of the invention have a hydrodynamic diameter (Dh) in water from 2 to 1200 nm, more particularly, from 10 to 1100 nm, from 10 to 1000 nm, from 10 to 700, from 20 to 500 nm, from 20 to 400 nm, from 20 to 300 nm, from 30 to 200 nm, or from 50 to 150 nm.
[00169] In some embodiments, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a polymeric nanoparticles (PNP), including a polyplexes, comprising the polyproline-based copolymers of formula (I) as defined according to the first aspect of the invention, the compounds comprising a structural unit of formula (I') or (I”) as defined according to the second aspect of the invention, or the star-shaped polymer as defined according to the third aspect of the invention, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
[00170] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a lipid nanoparticle (LNP) or a liposome comprising the lipid-containing polyproline-based copolymers of formula (la), (lb) or (Ic) as defined herein, or the compound comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid- like moiety Rw; and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
[00171] The liposomes and lipid nanoparticles disclosed herein may comprise any suitable lipids, including ionizable lipids, cationic lipids, zwitterionic lipids, neutral lipids, or anionic lipids. [00172] Suitable ionizable lipids include, without limitation, (2S)-2,5- bis(3-aminopropylamino)- N-[2- (dioctadecyl-amino)acetyl]pentanamide (DOGS), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3- aminopropyl)amino]-butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 3« -[N-(N« N«- dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), (1 , 2-d isteary loxy- N , N-d i methyl -3- aminopropane (DSDMA), 1 ,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA), 1 ,2- dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 1 ,2-di-y-linolenyloxy-N,N- dimethylaminopropane (’ -DLenDMA), 2-dilinoleyl-4-dimethyl-aminomethyl-[1,3]-dioxolane (DLin-K- DMA), 2,2- dilinoleyl-4- dimethylaminoethyl-[1 ,3]- dioxolane (DLin- KC2-DMA, also known as DLin-C2K- DMA, XTC2, and C2K), 2,2-Dilinoleyl-4-(3-dimethylaminopropyl)-[1 ,3]-dioxolane (DLin-KC3-DMA), 2,2- Dilinoleyl-4-(4-dimethylaminopropyl)-[1 ,3]-dioxolane (DLin-KC4-DMA), 1 ,2-dilinoleny loxy-4-(2- dimethy I ami noethy l)-1 ,3-dioxolane (DLen-C2K-DMA), 1 ,2-di- y-li noleny loxy-4-(2-d i methyl aminoethyl)- 1 ,3-dioxolane (y-DLen-C2K-DMA), (6Z,9Z,28Z,31 Z)-Heptatriaconta-6,9,28,31 -tetraen-19-yl 3- (dimethylamino)propanoate (DLin-M-C2-DMA, also known as MC2), (6Z,9Z,28Z,31Z)- heptatriaconta- 6,9,28,31 - tetraen-19- yl 4-(dimethylamino)-butanoate (DLin-M-C3-DMA, also known as MC3) and 3- ((6Z,9Z,28Z, 31 Z)-heptatriaconta-6, 9, 28, 31 -tetraen-19-yloxy)-N,N-dimethylpropan-1 -amine (DLin-MP- DMA, also known as 1-B11).
[00173] Suitable cationic lipids include, without limitation, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDA13), N-(1-(2,3-dioleoyloxy)propyl)- N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)-propyl)-N,N,N-trimethylammonium chloride (DOTMA), N-[1 -(2,3,-ditetradecyloxy)propyll-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1 -(2, 3, dioleyloxy)-propyl]-N, N-dimethyl-N-hydroxy ethylammonium bromide (DORIE), 3« - [N-(N« N«-dimethylamino-ethane)-carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB), 2,3-dioleyloxy- N-[2-(spermine-carboxamido)ethyl]- N,N- dimethyl-1 - propanaminium trifluoroacetate (DOSPA), ethylphosphatidylcholine, (ePC), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and ([(4-hydroxybutyl)azanediyl]di(hexane-6,1 -diyl) bis(2-hexyldecanoate)) (ALC-0315).
[00174] Examples of anionic lipids include, but are not limited to, phosphatidylglycerol, diacylphosphatidylserine, diacylphosphatidic acid, N-Succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine cholesterol hemisuccinate (CHEMS), lysylphos-phatidylglycerol, N- dodecanoyl phosphatidyl ethanoloamine, cardiolipin, and combinations thereof.
[00175] Suitable neutral lipids may be uncharged or zwitterionic lipids and include, without limitation steroids, phospholipids, and combinations thereof.
[00176] Examples of steroids include, without limitation, cholesterol, progesterone, cortisone, aldosterone, estradiol, testosterone, and combinations thereof.
[00177] Examples of phospholipids include, but are not limited to, phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidylinositol (PI), dimyristoyl phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl serine (DSPS), dioleoyl phosphatidyl serine (DOPS), dipalmitoyl phosphatidyl serine (DPPS), dioleoyl phosphatidyl ethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyp-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidyl-ethanolamine (DSPE); lysophosphatidyl choline, lysophosphatidylethanolamine, 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), dilauryloylphosphatidylcholine (DLPC), 1-myristoyl- 2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1- palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1 ,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero3-phosphocholine (DEPC), and combinations thereof.
[00178] In the liposomes and lipid nanoparticles disclosed herein the lipid-containing polyproline-based copolymers of formula (la), (lb) and (Ic), or the compound comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid-like moiety Rw; advantageously provides stealth properties. As used herein, the term "stealth” refers to the fact that the liposomes or lipid nanoparticles are not detected and sequestered and/or degraded by the immune system of the host to which they are administered, and consequently the length of time for which the liposomes or lipid nanoparticles can exist in vivo is increased.
[00179] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the liposomes and lipid nanoparticles disclosed herein do not comprise a polyethyleneglycol (PEG)-lipid conjugate, that is a lipid containing polyethyleneglycol.
[00180] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the self-assembled particles of the invention, in particular liposomes and lipid nanoparticles, do not comprise polyethyleneglycol (PEG).
[00181] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a lipid nanoparticle (LNP) or a liposome comprising the lipid-containing polyproline-based copolymers of formula (la), (lb) or (Ic), or the compound comprising the structural unit of formula (I') or (I”) wherein Rs comprises a lipid-like moiety Rw, as defined herein, and one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids. More particularly, the lipid nanoparticle (LNP) or liposome comprises the lipid-containing polyproline-based copolymers of formula (la) or (lb) as defined herein, an ionizable lipid or a cationic lipid, a phospholipid, and a sterol. Even more particularly, the lipid nanoparticle (LNP) or liposome comprises: I) the polyproline-based block copolymer of formula (I) as defined herein in an amount from 0.1 to 10 mol%, more particularly from 1 to 5 mol% or from 1 to 6 mol%, even more particularly from 2 to 5% mol%; II) a ionizable lipid or a cationic lipid in an amount from 30 to 70 mol%, more particularly from 40 to 60 mol%; iii) a phospholipid in an amount from 1 to 20 mol%, more particularly from 5 to 15 mol%; and iv) a sterol in an amount from 20 to 60 mol%, more particularly from 30 to 50 mol%; wherein the percentages are expressed with respect to the sum of the mol% of the lipids and the lipid-containing polyproline-based copolymers of formula (la), (lb) or (Ic).
[00182] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a lipid nanoparticle (LNP) or a liposome comprising the lipid-containing polyproline-based copolymers of formula (la), (lb) or (Ic) as defined herein, one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids, and one or more nucleic acids.
[00183] The lipid nanoparticles and the liposomes containing one or more nucleic acids may be prepared by standard methods. Typically, the process for the preparation of LNPs comprises: i) preparing a first alcoholic mixture comprising one or more lipids and the polyproline-based block copolymer of the invention in a suitable alcohol such as for example ethanol; ii) preparing a second aqueous composition comprising one or more nucleic acids and an aqueous solvent (an acidic buffer); and iii) mixing i) with ii) in a microfluidic mixing device. The microfluidic mixing allows thorough and rapid mixing of the lipid phase and the nucleic acid phase in a microscale device. Depending on the process parameters, and in particular on the total flow rate, the skilled person will be able to modulate the size of the LNPs.
Figure imgf000061_0001
[00184] As mentioned above, in accordance with an aspect of the present invention, it is provided a composition comprising the polyproline-based block copolymer of formula (I) as defined herein, the compound comprising a structural unit of formula (I') or (I”) as defined according to the second aspect of the invention, the star-shaped polymer as defined according to the third aspect of the invention, or alternatively, the self-assembled particle, together with one or more appropriate excipients or carriers.
[00185] The compounds of the invention may be formulated in a variety of compositions, including pharmaceutical, cosmetic and diagnostic compositions, with excipients and carriers.
[00186] The compounds of the invention, self-assembled particles, and compositions thereof may be used in medicinal, cosmetic and diagnostic applications.
[00187] The appropriate excipients and carriers and their amounts can readily be determined by those skilled in the art according to the type of composition being prepared.
[00188] In an embodiment, the composition is a therapeutic composition comprising a therapeutically (pharmaceutical or veterinary) effective amount of one or more active ingredients; and one or more therapeutically acceptable excipients or carriers.
[00189] In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the composition is a pharmaceutical composition comprising a therapeutically effective amount of: (a) the polyproline-based block copolymer of formula (I) as defined herein, wherein at least one of R8 or L4 comprises a pharmaceutically active agent; or alternatively (b) the compound comprising a structural unit of formula (I') or (I”) wherein at least on R8 comprises a pharmaceutically active agent; or alternatively (c) the star-shaped polymer as defined herein, wherein at least one of L4 or R8 is a pharmaceutically active agent or alternatively, or alternatively (d) a selfassembled particle containing (a), (b) or (c) as defined above, and one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof, together with one or more pharmaceutically acceptable excipients or carriers.
[00190] The term "pharmaceutical composition” refers to a composition suitable for use in the pharmaceutical technology with medical use. The term "therapeutically effective amount of an active ingredient” as used herein, refers to the amount of a pharmaceutical active ingredient that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed. The dose of the pharmaceutically active ingredient administered will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations. The pharmaceutical compositions of the present invention comprise one or more pharmaceutically acceptable excipients or carriers. The term "pharmaceutically acceptable excipients or carriers” refers to that excipients or carriers suitable for use in the pharmaceutical technology for preparing compositions with medical use. Each component should be "acceptable" in the sense of being compatible with the other ingredients of the composition. When used in contact with the tissue or organ of humans and animals should not have excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. In an embodiment, the compositions of the present invention comprise one or more pharmaceutically acceptable excipients and/or carriers selected from the group consisting of diluent, binder, glidant, disintegrant, lubricant and mixtures thereof. Additionally, the pharmaceutical compositions of the present invention may contain other ingredients, such as fragrances, colorants, and other components known in the state of the art.
[00191] The polyproline-based block copolymer of formula (I), the compound comprising a structural unit of formula (I'), the compound comprising a structural unit of formula (I”), the star-shaped polymers, the self-assembled particles and pharmaceutical compositions containing them may be used jointly with other additional drugs, to provide combined therapy. Said additional drugs may be a part of the same pharmaceutical composition or, alternatively, may be provided in the form of a separate composition for simultaneous or non-simultaneous administration. [00192] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the composition is a diagnostic composition comprising a diagnostically effective amount of: (a') the polyproline-based block copolymer as defined herein, wherein at least one of R8 or L4 comprises a diagnostically active agent or alternatively, (b’) the compound comprising a structural unit of formula (I') or (I”), wherein at least one of R8 comprises a diagnostically active agent; or alternatively (c’) a star-shaped polymer wherein at least one of L4 or R8 is a diagnostically active agent, or alternatively (d’) a self-assembled particle containing (a'), (b’) or (c’) as defined above, together with one or more diagnostically acceptable excipients or carriers.
[00193] The term "diagnostic composition” refers to a composition suitable for use in diagnostic, particularly in imaging diagnostic technology. The term "diagnostically effective amount of a detection moiety” as used herein, refers to the effective amount of a detection compound that, when administered, is sufficient for the diagnosis of a disease or disorder; particularly as imaging diagnostic use as contrast imaging agent. The dose of the detection compound administered will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being diagnosticated, and the similar considerations. The diagnostic compositions of the present invention comprise one or more diagnostically acceptable excipients or carriers. The term "diagnostically acceptable” refers to that excipients or carriers suitable for use in the diagnosing technology for preparing compositions with diagnostic use; particularly by imaging diagnostic use. The detection of these diagnostic agents in the body of the patient can be carried out by the well-known techniques used such as in imaging diagnostic with magnetic resonance imaging (MRI) and X-ray.
[00194] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the composition is a cosmetic composition comprising a cosmetically effective amount of: (a”) the polyproline-based block copolymer as defined herein, wherein at least one of R8 or L4 comprises a cosmetically active agent or alternatively, or alternatively (b”) a compound comprising a structural unit of formula (I') or (I”) wherein at least one R8 comprises a cosmetically active agent, or alternatively, or alternatively (c”) a star-shaped polymer wherein at least one of L4 or R8 is a cosmetically active agent, or alternatively, (d”) a self-assembled particle containing (a”), (b”) or (c”) as defined herein and one or more cosmetically active agents, together with one or more cosmetically acceptable excipients or carriers.
[00195] The term "cosmetic composition” refers to a composition suitable for use in cosmetic for the body care. The term "cosmetically effective amount” as used herein, refers to the effective amount of a cosmetic active agent that, when administered, is intended to improve its appearance or to beautify, preserve, condition, cleanse, color or protect the skin, nails or hair without non-medical application. The dose of the cosmetic active ingredient administered will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition and the similar considerations. The cosmetic compositions of the present invention comprise one or more cosmetically acceptable excipients or carriers. The term "cosmetically effective amount” as used herein, refers to the effective amount of a cosmetically active agent that, when administered, is intended to improve its appearance or to beautify, preserve, condition, cleanse, color or protect the skin, nails or hair without a medical application. The term "cosmetically acceptable" or "dermatologically acceptable" excipients or carriers is used interchangeably in this document and refer to components which are appropriate for use in human skin contact without toxicity, incompatibility, instability, inappropriate allergic response, among others.
[00196] The composition can be in form of topical composition, oral composition, and injectable composition.
[00197] In accordance with some embodiments, the compositions of the invention may be in solid or liquid form. Non-limiting examples of solid forms include frozen forms, lyophilized forms and spray-dried forms. Depending on the desired purpose, the skilled person will know the most appropriate formulation and excipients to be used. Examples of excipients or carriers include, without limitation, diluents, binders, glidants, disintegrants, lubricants colorants, mixtures thereof, and other components known in the state of the art. Any administration route may be used such as e.g. oral, topical, rectal or parenteral route (including subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous route, etc.).
[00198] In an embodiment, the composition is an oral composition, particularly selected form liquid or solid oral composition. In an embodiment, the composition of the invention is a solid oral composition. The oral solid compositions of the invention can be formulated in any form that includes any single unit dosage form and any multiple unit dosage forms. The term "single unit” encompasses one entity such as a single tablet, a single granule, and a single pellet. The term "single unit dosage form” defines a dosage form which consists only of one unit which contains the effective amount of the polyproline-based block copolymer of the present invention. The term "multiple unit dosage form” defines a dosage from which consists of more than one unit which contains the effective amount of polyproline-based block copolymer of the present invention. Usually, the multiple unit dosage forms are based on subunits such as granules, pellets or minitablets. They are usually delivered in hard gelatine capsules or transformed into tablets. Thus, it is also part of the invention a unit dosage from which comprises the composition of the present invention. In an embodiment, the unit dosage from which comprises the composition of the present invention is a single unit dosage form. In an embodiment, the unit dosage from which comprises the composition of the present invention is a multiple unit dosage form.
[00199] In an embodiment, the composition is a topical composition. The topical compositions of the invention can be formulated in several forms that include, but are not limited to, solutions, aerosols, and non-aerosol sprays, shaving creams, powders, mousses, lotions, gels, sticks, ointments, pastes, creams, shampoos, shower gel, body washes or face washes. [00200] In an embodiment, the composition is an injectable composition; particularly selected from the group consisting of intramuscular, subcutaneous, or intravenous application. In an embodiment, the compositions of the present invention are in form of parenteral compositions suitable for their injection, infusion, or implantation into the body. The parenteral compositions defined above should be sterile, and pyrogen-free, and they can be in form of liquid such as solutions, emulsions, or suspensions, or in solid form packaged in either single-dose or multidose containers suitably diluted before use. Parenteral compositions can comprise appropriate excipients or carriers for parenteral administration that can be pharmaceutical or cosmetic excipients, including, but not limited to, solvents, suspending agents, buffering agents, substances to make the preparation isotonic with blood, stabilizers, or antimicrobial preservatives. The addition of excipients should be kept to a minimum. When excipients are used, they should not adversely affect the stability, bioavailability, safety, or efficacy of the components, or cause toxicity or undue local irritation. There should not be any incompatibility between any of the components of the dosage form.
[00201] The appropriate form of the composition can be readily determinate by those skilled in the art according to its intended use. Furthermore, the excipients and/or carriers, and their amounts, can readily be determined by those skilled in the art according to the type of formulation being prepared.
Uses
[00202] The polyproline-based block copolymers of the invention, self-assembled particles, and compositions thereof may be used in therapeutic applications. In particular, they may be used as non- viral vectors of general use for biomedical applications, such as vaccines or gene therapy, being effective for transfection of hosts eukaryotic cells in culture, in vivoot ex vivo, monocellular parasites and bacteria, including gene editing using the CRISP/Cas9 methodology. Further, they may be used in protein-based therapy; particularly protein-based vaccine against viral infections or as a therapeutic protein-based vaccine against cancers or infectious diseases.
[00203] Thus, the invention relates to a therapeutic product which is or which comprises: a) the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively b) the compound comprising aa structural unit of formula (I') or (I”), wherein at least one of L4 or R8 comprises a pharmaceutically active agent; or alternatively c) the star-shaped polymer, wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively d) a self-assembled particle, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively e) a composition as defined herein, containing the self-assembled particle (d); for use in medicine. [00204] This aspect may also be formulated as a method for treating or preventing a disease or disorder in a subject, more particularly a mammal, and even more particularly a human, comprising administering the therapeutic product as defined herein, and one or more pharmaceutically acceptable excipients or carriers.
[00205] Non-limiting examples of diseases that may be treated and/or prevented by the compounds of the present invention include neurodegenerative disorders, neurological diseases, cancer, infectious diseases, disorders related to aging, neuro-inflammation, demyelinating disorders, multiple sclerosis, ischemic disorders, ischemia-reperfusion damage, amyloidotic disease, cardiomyopathy, spinal cord injury, immune disorders, inflammatory disorders, rare diseases, wound healing, skin related diseases and lysosomal storage diseases.
[00206] Non-limiting examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cerebral ischaemia, post-encephalitic Parkinsonisms, dystonias, Tourette syndrome, periodic limb movement pathologies, restless legs syndrome, attention deficit hyperactivity disorders, Huntington's disease, progressive supranuclear palsy, Pick's disease, frontotemporal dementia and neuromuscular diseases.
[00207] In one embodiment, optionally in combination with any of the embodiments provided above or below, the invention relates to a therapeutic product which is or which comprises: a) the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively b) the compound comprising a structural unit of formula (I') or (I”), wherein at least one of L4 or R8 comprises a pharmaceutically active agent; or alternatively c) the star-shaped polymer, wherein at least one of L4 or R8 comprise a pharmaceutically active agent; or alternatively d) a self-assembled particle, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively e) a composition as defined herein, containing the self-assembled particle (d; for use in the treatment and/or prevention of cancer or an infectious disease.
[00208] The term "disorder" as used herein is intended to be generally synonymous, and is used interchangeably with, the terms "disease," "syndrome," and "condition" (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms.
[00209] In the context of the present invention, the terms "treat", "treating" and "treatment", as used herein, refers to ameliorating symptoms associated with a disease or disorder, including preventing or delaying the onset of the disease or disorder symptoms, and/or lessening the severity or frequency of symptoms of the disease or disorder.
[00210] According to one embodiment, optionally in combination with any of the embodiments provided above or below, the invention relates to the therapeutic product as defined herein for use (I) as transfection reagent for transfecting at least one active agent into a cell; (ii) for use in the in vivoor ex vivo production of biologies encoding a recombinant protein, a peptide or an antibody, or in the production of recombinant virus; (ill) for use as a therapeutic or prophylactic vaccine against viral infections or as a therapeutic vaccine against cancers or infectious diseases; or (iv) for use in genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
[00211] In a particular embodiment, optionally in combination with any of the embodiments provided above or below, the present invention relates to the therapeutic product as defined herein as transfection reagent for delivering one or more nucleic acids (regardless of size and structure, circular and linear nucleic acids) to target cells, in in vivo, in vitro or ex vivo. More particularly, the active agent is selected from the group consisting of low molecular weight drugs, peptides, proteins, antibodies, nucleic acids, aptamers, and combinations thereof.
[00212] The present invention also relates to a method for in vitro, ex vivo and in vivo transferring active agents comprising the step of administering to a subject (e.g. a mammal, such as a human) the therapeutic product as defined herein, in which administering involves contacting the cell with the therapeutic product, whereby the pharmaceutically active agent is delivered to the cell.
[00213] In a particular embodiment, optionally in combination with any of the embodiments provided above or below, the therapeutic product is a self-assembled particle which comprises the polyprolinebased block copolymer of formula (I) or the compounds comprising the structural unit (I') or (I”) as defined herein, and one or more nucleic acids, or alternatively, a composition containing the selfassembled particle as defined herein and one or more nucleic acids.
[00214] The transfection reagents of the invention are also useful for co-transfection of two or more active agents simultaneously, e.g. two or more nucleic acids, simultaneously. Transfection compositions (such as kits), as well as methods of using the transfection reagents to deliver nucleic acid to target cells are also within the scope of the present invention.
[00215] The present invention also provides therapeutic products as defined herein for inducing a regulating effect on the expression of one or more target proteins responsible or involved in genetic hereditary diseases or complex genetic diseases, immune diseases, cancers, viral infections in various tissues/organs or tumors.
[00216] The present invention also relates to the in vitro or ex vivo use of therapeutic products as defined herein in the production of biologies, in particular biologies encoding a recombinant protein, a peptide or an antibody; or in the production of recombinant virus, such as adeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolytic virus, or baculovirus, or viral or virus-like particles, in particular said products comprising the polyproline-based block copolymers of the invention and at least one nucleic acid molecule for transfection. As used herein, the term "biologies” refers to proteins or nucleic acids or combinations thereof, living entities such as cells or viruses, cell compartments, organoids, and tissues.
[00217] The present invention also relates to an in vitro or ex vivo use of the therapeutic products as defined herein, in particular said products comprising the polyproline-based block copolymers of the invention and at least one nucleic acid molecule for transfection, for genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
[00218] The compositions for transfecting cells comprise the therapeutic products as defined herein, in particular said products comprising the polyproline-based block copolymers of the invention and at least one nucleic acid molecule for transfection, and an acceptable excipient, buffering agent, cell culture medium, or transfection medium.
[00219] The present invention is also directed to the therapeutic products as defined herein for use as a therapeutic or prophylactic vaccine against viral infections, or a therapeutic vaccine against cancers. Generally, in this aspect, the vaccine is delivered through direct administration such as systemic, intramuscular, intradermal, intraperitoneal, intratumoral, oral, topical, or sub-cutaneous administration, and, in said vaccine, the composition is in association with a pharmaceutically acceptable vehicle. In other words, the vaccine can be injected directly into the body, in particular in a human individual, for inducing a cellular and/or a humoral response.
[00220] The cell targeting is achieved through different mechanisms and depends on the nature and properties of the transfection reagent, method or protocol composition or formulation and the route of administration.
[00221] The polyproline-based block copolymers of the invention, the compounds comprising the structural unit of formula (I') or (I”), the self-assembled particles, and compositions thereof may be used also in cosmetic and diagnostic applications.
[00222] Accordingly, another aspect of the invention relates to a diagnostic product which is or which comprises: a') the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively b') the compound comprising the structural units of formula (I') or (I”), wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively c') the star-shaped polymer, wherein at least one of L4 or R8 comprise a diagnostically active agent; or alternatively d') a self-assembled particle as defined herein, which comprises a diagnostically active agent, or alternatively e') a composition as defined herein, comprising a diagnostically active agent; for use in diagnostics.
[00223] This aspect of the invention may also be formulated as the use of a') the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively b') the compound comprising the structural units of formula (I') or (I”), wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively c') the star-shaped polymer, wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively d') a self-assembled particle, which comprises diagnostically active agent, or alternatively e') a composition as defined herein, comprising a diagnostically active agent in diagnostics.
[00224] Further, it may also be formulated as a method for the diagnostic of a disease or condition, comprising administering a') the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprise a diagnostically active agent; or alternatively b') the compound comprising the structural units of formula (I') or (I”), wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively c') the star-shaped polymer, wherein at least one of L4 or R8 comprise a diagnostically active agent; or alternatively d') a self-assembled particle, which comprises diagnostically active agent, or alternatively e') a composition as defined herein, comprising a diagnostically active agent; in a subject in need thereof, more particularly a mammal, and even more particularly a human.
[00225] In a particular embodiment, the invention relates to microbubbles which (a'), (b'), (c'), (d’) or (e’) as defined herein and one or more diagnostically active agents, in particular contrast agents, for use in diagnostics.
[00226] The detection of these imaging agents can be carried out by well-known techniques such as imaging diagnostic techniques. Examples of imaging diagnostic techniques suitable for the present disclosure include, but not limited to, are magnetic resonance imaging (MRI), X-ray, positron emission tomography (PET), single-photon emission computed tomography (SPECT), fluorescence microscopy, and in vivo fluorescence.
[00227] According to another aspect, the invention relates to the use in cosmetics of a cosmetic product which is or which comprises: a") the polyproline-based block copolymer of formula (I), wherein at least one of L4 or R8 comprise a cosmetically active agent; or alternatively b”) the compound comprising the structural units of formula (I') or (I”), wherein at least one of L4 or R8 comprises a cosmetically active agent; or alternatively c”) the star-shaped polymer, wherein at least one of L4 or R8 comprise a cosmetically active agent; or alternatively d”) the self-assembled particle, which comprises a cosmetically active agent, or alternatively e”) a composition as defined herein, comprising a cosmetically active agent
[00228] It is also a part of the invention a kit comprising a therapeutic, diagnostic, or cosmetic product as defined herein, and optionally means for its use.
[00229] In an embodiment, the kit of the present invention comprises: the therapeutic, cosmetic or diagnostic product as defined herein; and optionally means to its administration.
[00230] Examples of appropriate means for their administration include reagents and/or solvents for its use, as well as equipment (such as syringe) and instructions for its use.
[00231] The use in therapy can be also drafted as a method for the prophylaxis and/or treatment of a disease which comprises administering to mammals in need of such treatment an effective amount of the polyproline-based block copolymer or the conjugate disclosed herein above in the fifth aspect of the invention, together with one or more appropriate pharmaceutically acceptable excipients or carriers. Further, the use in therapy can be also reformulated as the use of the polyproline-based block copolymer as defined herein above and below or the conjugate disclosed herein above and below in the fifth aspect of the invention for the preparation of a medicament for the prophylaxis and/or treatment of a disease or condition. The type of disease or condition to be treated depend on the pharmacological activity of the active ingredient of the polymer and/or the molecule of interest of the molecule.
[00232] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise” encompasses the case of "consisting of'. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Reference signs related to drawings and placed in parentheses in a claim, are solely for attempting to increase the intelligibility of the claim, and shall not be construed as limiting the scope of the claim. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.
EXAMPLES
[00233] Chemicals and solvents are either A.R grade or purified by standard techniques. Cell lines used in the present application are commercially available. The triple-negative breast cancer cell line MDA-MB 231 luc is available from ATCC.
NMR spectroscopy:
[00234] NMR spectra were recorded at 27 °C (300 K) on a 300 UltrashieldTM from Bruker (Billerica MA, USA). Data were processed with the software Topspin (Bruker GmbH, Karlsruhe, Germany). Samples were prepared at a concentration of 20 - 10 mg/mL approx, in the required solvent.
Size Exclusion Chromatography (SEO
[00235] For SEC measurements in dimethylformamide (DMF) containing 0.1 % (w/w) of lithium bromide as an additive, a GPC max (Malvern Instruments) autosampler was used with a flow rate of 0.7 mL/min at 60 °C with TSKgel Alpha-4000 column from Tosoh Bioscience. ViscotekTDA-302 was used as an integrated detection system. The system was calibrated with polymethyl methacrylate (PMMA) (Mw = 65 kDa; PDI = 1.05) from PSS. For this Mw determination an integrated triple detection system was used [Refractive Index, Light Scattering (two angles: 7 and 90 °) and Ultraviolet-Visible detector],
[00236] For SEC measurements in aqueous containing 0.1 M of sodium nitrate and 0.005% of sodium azide as an additive, a GPC max (Malvern Instruments) pump and autosampler was used with a flow rate of 0.7 mL/min at 25 °C with TSKGel PWXL G5000 column from Tosoh Bioscience. Viscotek TDA- 305 was used as an integrated detection system. The system was calibrated with polyethylene oxide (PEC) (Mw = 24 kDa; PDI = 1.01) from Malvern Panalytical. For this Mw determination an integrated triple detection system was used [Refractive Index, Light Scattering (two angles: 7 and 90 °) and Ultraviolet-Visible detector].
Circular Dichroism
[00237] The secondary structure was determined by Circular Dichroism. CD Spectroscopy was performed with a J-815 CD Spectrometer (JASCO Corporation) using a Peltier thermostated cell holder (PTC-423, JASCO15 Corporation) with a recirculating cooler (JULABO F250, JASCO Corporation). A nitrogen flow (~1.5L/min) was lead through the spectrometer and controlled with a nitrogen flow monitor (Afriso Euro-lndex). The samples were dissolved in under different conditions (ddH2O, PBS, different temperature; cone. 0.25 mg/mL). Samples were measured repeatedly (n= 3) in a quartz cuvette with d=0.1cm.
Size Determination
[00238] The size distribution was assessed by different techniques (example 2.2).
[00239] PicoQuant MT200 confocal microscope with a 60x Na1 .2 UPlanApo water immersion objective and excitation line 488 nm was used to perform the Fluorescence Correlation Spectroscopy (FCS) analysis. The signal is directed onto two single photon avalanche diode (SPAD) detectors arranged in Hanbury-Brown and Twiss (HBT) geometry (i ,e. , emitted light is split using a 50/50 beam splitter and sent to two SPAD detectors).
[00240] Diffusion-ordered NMR spectroscopy (NMR DOSY) was recorded on a Bruker Avance 500 MHz and performed with a stimulated echo sequence using bipolar gradient pulses. Delay lengths were held constant at • = 100 ms, and 32 spectra of 128 scans were acquired with the diffusion gradient strength varying between 5 % and 95 %. The samples were prepared in deuterated phosphate buffer 1 M pH 7.4 (5mg in 0.6 mL of dPBS). Data were processed with the software Mestrenova (Bruker GmbH, Karlsruhe, Germany).
[00241] FEI Tecnai G2 Spirit Biotwin 120 kw transmission electron microscope was used to determine the size of 1mg/mL solution in phosphate buffer 1 M pH 7.4. 1% solution of uranyl acetate dissolved in ddH2O was used for the negative staining of the sample. FEI Tales Arctica Transmission Electron Microscope (University of Groningen, ND) was used the cryo-TEM analysis of the samples.
Cell Viability
[00242] Cell viability was evaluated in assessed in the triple negative breast cancer cell line MDA-MB 231 . 96-well plate was seeded with 5000 cells and incubated for 24h at 37° and 5% carbon dioxide. Concentrated stock solutions of the polymers were prepared in phosphate buffer 1 M pH 7 and diluted in DMEM/F-12 medium (with 10% FBS and 1% P/S; final DMSO 0.5%) starting from 0.5 mg/mL (dilution factor d.f 1 .5). MTS ([3-(4,5-dimethy lthiazol-2-y l)-5-(3-carboxymethoxypheny l)-2-(4-sulfopheny l)-2H- tetrazolium] colorimetric agent was added, and after 4 hours the absorbance at 490 nm was measured spectrophotometrically using a plate reader. Values represent Mean ± SD (n=3).
[00243] HEK293 cells employed in Example 9 were cultured in DMEM high glucose with Glutamax (Gibco- Thermo Fisher # 61965-059) supplemented with 10% of Fetal Bovine Serum (Hyclone # SV30160.03HI, provided by GE Healthcare Europe GmbH) and 1 % penicillin/streptomycin. Transfections were carried out on 96-well plates containing 30000 cells/well in a final volume of 100 pl, and cells were incubated 24 hours at 37 °C and 5% CO2. After 24 h from cell seeding, the medium was removed and refreshed with 90 pl of complete medium. 10 pl of each LNPs formulation were added to the cells. After 24 hours cells were recovered and processed.
[00244] A TP Evaluation for Cell Toxicity evaluation
[00245] After 24h post-incubation with the LNPs formulation, the medium was aspirated and 50 pl/well of ATPLite reagent (ATPLite PerkinElmer #6016731) were added. The plate was incubated 10 minutes at room temperature in the dark. Luminiscence was read spectrophotometrically using VictorNivo (PerkinElmer) following manufacturer's instructions and data was represented as the percentage of cell viability, taken untreated control cells as 100%.
[00246] Luciferase Assay for transfection efficiency evaluation [00247] After 24h post-incubation with the LNPs formulation, 100 pl of BrightGlo reagent (Promega # E2620) was added in each well following manufacturer instructions. After 5 minutes of incubation at room temperature luciferase activity was measured using VictorNivo (PerkinElmer). Data was represented as luminescence relative to the percentage of transfection relative to the positive control of transfection.
Uptake studies
[00248] The uptake studies were performed by confocal microscope (Leica TCS-SP8) equipped with 4 laser lines for fluorescence excitation (405 nm, 488 nm, 561 nm, and 638 nm), 4 detectors for simultaneous data acquisition, CO2 and temperature control, a resonant scanner for live-cell studies, high content screening automation (HCS A) module, and super-resolution module (HyVolution II). Pulse chase and time-lapse studies were performed at non-toxic concentration of the selected polymers and polymer-drug conjugates using the following fluorescent markers: MitoTracker™ Red CM-H2Xros M7513 (mitochondria, abs/ems. 579/599 nm; 0.5 uM); LysoTracker® Blue DND22 (lysosomes, abs/ems. 373/422; 0.2 uM); Cyanine5 (polymer): abs/ems. 646/662; CELLLIGHT EARLY ENDOSOMESGFP (early endosomes, abs/ems 488/510; 133 ppc). 1500 cells/well (triple negative breast cancer cell line MDA-MB 231) were seeded in 384-well black optical-bottom plate. The early endosomes marker was added o/n, non-toxic concentrations of the polymers were added 2, 4, 6 h and o/n, and the mitotracker and lysotracker were added 1 h before the analysis. The internalization and the mitochondria colocalization were observed by confocal microscope Leica TCS-SP8.
Example 1 : Synthesis of different block copolymers containing PPro of formula I:
1.1 General procedure for the polymerization of nBuPOrnZ-b-PPro and nBuPLysZ-b-PPro
Figure imgf000073_0001
[00249] Ornithine Z or Lysine Z N-carboxyanhydride was added to a Schlenk tube fitted with a stirring bar and a stopper. After 3 cycles of vacuum/N2, the mixture was dissolved in anhydrous DMF. Then, the initiator (n-Butylamine) diluted in DMF (0.5 mL) and was added to the reaction mixture, which was stirred at 10 °C for 16 hours. Once NCA consumption was confirmed by IR the obtained solution was added to Proline NCA previously suspended in anhydrous acetonitrile. The mixture was stirred at room temperature for 16 hours. The reaction mixture was precipitated with diethyl ether. The precipitate was
Figure imgf000074_0001
, into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. Next, the product was obtained after dialysis against water (Vivaspin®, 3000 MWCO Da) and subsequent freeze-drying. Yield: 70-80%. nBuPIO-b-PPro (1)
1H NMR (500 MHz, D2O) • 4.77 - 4.65 (m, • mH), 4.48 - 4.32 (m, • nH), 3.96 - 3.54 (m, 2mH), 4.27 (t, J = 7.0 Hz, 1 H, NH), 4.00 - 3.39 (m, 2mH), 3.31 - 3.17 (m, 2H, NHCH2), 3.07 (s, 2mH), 2.66 - 1.69 (m, 4mH+4nH), 1.52 (p, J = 7.0 Hz, 1 H, CH2CH2CH3), 1.34 (h, J = 7.3 Hz, 2H, CH2CH3), 0.92 (t, J = 7.3 Hz, 3H). nBuPLys-b-PPro (2)
1H NMR (300 MHz, D2O) • 4.58 - 4.19 (m, • mH), 4.00 - 3.42 (m, 2mH), 3.08 (s, 2mH), 2.54 - 1.30 (m, 4mH+6nH+CH2CH2CH3), 0.96 (t, J = 7.3 Hz, 3H). • nH covered with D2O Table 1. Targeted diblock copolymers according to the formula 1 and 2.
Figure imgf000075_0002
Figure imgf000075_0003
a Determined by NMR. bDetermined by SEC wherein the cited DP numbers are subject to a reasonable uncertainty within the range ±20%.
1 .3 General procedure for synthesis pArg-PPro block copolymers (3)
Figure imgf000075_0001
[00251] 1 H-1 ,2,4-Triazole-l-carboxamidine hydrochloride (1 eq. each Orn group) was dissolved in 450 piL of MQ water and 5M solution of KOH was added to reach pH=9.5. Separately the polymer was dissolved in 200 piL of MQ water and the triazole solution was added. Mixture left stirring for 16 hours, terminated by addition of 1 M HOI to reach pH = 5. Next, the mixture was filtered and purified by dialysis (MWCO 100-500Da). White powder was obtained after freeze-drying. Yield: 70%. 1H NMR (500 MHz, D2O) • 4.77 - 4.71 (m, m« H), 4.50 - 4.20 (m, n« H), 3.98 - 3.40 (m, 2mH), 3.24 (s, 2nH), 3.06 (s, NHCH2, 2H), 2.45 - 2.26 (m, 1 mH), 2.19 - 1.58 (m, 3m+4nH), 1.54 - 1.45 (m, CH2CH2CH3, 2H), 1.39 - 1.28 (m, CH2CH3, 2H), 0.91 (t, J = 7.3 Hz, 3H). Table 2. Targeted diblock copolymers according to the formula
Figure imgf000076_0002
uncertainty within the range ±20%.
1 .4 General procedure for synthesis pHis-PPro block copolymers (4)
Figure imgf000076_0001
[00252] The procedure to synthesize pHis-PPro block copolymers follows the general procedure described in Example 1.1 but replacing the monomers Ornithine Z or Lysine Z N-carboxyanhydride by Histidine DNP N-carboxyanhydride. Block copolymer nBuHisDnP-b-PPro was isolated as an orange solid. Yield: 87-90%.
1H NMR (500 MHz, TFA-d1) • 9.18 (s, nH, Ar), 9.08 - 8.97 (m, nH, Ar), 8.87 - 8.75 (m, nH, Ar), 8.14 - 8.00 (m, nH, im-CH), 7.80 - 7.63 (m, nH, im-CH), 5.47 - 5.13 (m, m« H), 5.05 - 4.69 (m, n« H), 4.21 - 3.31 (m, 2mH+2nH), 2.59 - 1.99 (m, 4mH), 1.63 - 1.54 (m, CH2CH2CH3, 2H), 1.44 - 1.34 (m, CH2CH3, 2H), 0.94 (t, J = 7.3 Hz, 3H).
[00253] The block copolymer of nBuHisDnP-b-PPro was suspended in dry DMF and mercaptoethanol (10 eq) added dropwise. After finishing the addition mixture was stirred at room temperature for 16 hours. The reaction mixture was precipitated into diethyl ether and was washed with diethyl ether and acetone. Precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. Dry deprotected copolymer was suspended in dry 1,4-dioxane under anhydrous conditions and excess of HCI in 1,4-dioxane added. Reaction mixture was stirred for 3 hours and poured in diethyl ether. The precipitate was isolated by centrifugation (4400 rpm, 4 min) and dried under vacuum. Desired product was obtained after dialysis (100-500 MWCO Da). Yellow powder was obtained after freeze-drying compound 4. DP Phis block: 7, DP PPro block: 26
Yield: 60%.
1H NMR (500 MHz, TFA-d1) • 8.92 - 8.53 (m, nH, im-CH), 7.80 - 7.38 (m, nH, im-CH), 5.33 - 4.77 (m, m« H+ n« H), 4.24 - 3.33 (m, 2nH+2mH), 2.84 - 2.04 (m, 4nH), 1 .67 - 1 .57 (m, CH2CH2CH3, 2H), 1 .48 - 1.33 (m, CH2CH3, 2H), 1.02 (t, J = 7.3 Hz, 3H). 1.5 General procedure for synthesis Tetradecylamine -Plys(Br-)-co-PPro block copolymers (5)
Figure imgf000077_0001
[00254] Trifluoroacetyl-L-lysine N-carboxyanhydride (2 g, 7 mmol) was added to a Schlenk tube fitted with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, and dissolved in anhydrous DMF (8 mL). Then, the star initiator (tetradecylamine) was dissolved in DMF (2 mL) and was added to the reaction mixture. The mixture was stirred at 10 °C for 16 hours. The reaction mixture was poured into water to precipitate the product. The precipitate was isolated by filtration and freeze-dryedg. Tetradecyl- PlysfTFA] was isolated as a white solid. Yield: 70-90%
1H NMR (TFA): • = 0.91 (t, J= 6.9 Hz, 3H, CH3), 1.34 (brs, 2H, CH2), 1.40 (brs, 2H, CH2), 1.44-2.08 (m, 2H, CH2), 3.52 (brs, 2H, CH2), 4.67 (s, 1 H, CH).
Table 3. Targeted polymers according to the formula
Figure imgf000077_0003
a Determined by NMR. b Determined by SEC wherein the cited DP numbers are subject to a reasonable uncertainty within the range ±20%.
Synthesis of block-copolymer:
Figure imgf000077_0002
[00255] The synthesis of the block-copolymer - Tetradecylamine -Plys[tFA]-PPro (5b) was synthesized as described in Example 1.1. for the polymerization of L-Proline. Tetradecyl-Plys[TFa]-b-PPro was isolated as a white solid. Yield: 50-60%
1H NMR (TFA): • = 0.80 (t, J= 6.7 Hz, 3H, CH3), 1.24 (brs, 2H, CH2), 1.30 (brs, 2H, CH2), 1.37-1.94 (m, 2H, CH2), 2.10-2.27 (m, 2H, CH2), 2.38-2.53 (m, 2H, CH2), 3.42 (brs, 2H, CH2), 3.82 (m, 1 H, CH), 4.67 (s, 1 H, CH).
Table 4. Targeted diblock copolymers according to the formula
Figure imgf000078_0002
uncertainty within the range ±20%.
Deprotection step:
Figure imgf000078_0001
[00256] Tetradecyl-Plys[TFa]-b-PPro block copolymer (400 mg, 0.02 mmol) was added to a round botton flask with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, and dissolved in a mixture of MeOH:THF (1 : 1) (4 mL). Then, NaOH (1.5 eq, 106 mg, 0.03 mmol) was dissolved in water (1 mL) and was added to the reaction mixture. The mixture was stirred at 10°C for 16 hours. After this time, the reaction mixture was acidified with HBr 48% to pH=2 at 10°C. The mixture was precipitated in acetone (ratio 1 :10). The obtained solid was washed with acetone (x3) and dried in a high vacuum pump.
[00257] After the drying process the neutralization of the polymer was carried out: Tetradecylamine- PLys[Br]-b-PPro was redissolved in water at 500 mg/mL and 0.1 M NaOH was added to ph=4-5, the resulting solution is filtered through 0.22 m and lyophilized. Tetradecylamine-PLys[Br]-b-PPro (5c) was isolated as a white solid. Yield: 70-80%
1H NMR (TFA): • = 0.81 (t, J= 6.9 Hz, 3H, CH3), 1.22 (brs, 2H, CH2), 1.29 (brs, 2H, CH2), 1.34-1.85 (m, 2H, CH2), 1.99 (m, 2H, CH2), 2.98 (brs, 2H, CH2), 3.56 (m, 1 H, CH), 4.29 (s, 1 H, CH).
Table 5. Targeted diblock copolymers according to the formula
Figure imgf000079_0002
a Determined by NMR. b Determined by SEC wherein the cited DP numbers are subject to a reasonable uncertainty within the range ±20%.
1.7 General procedure for synthesis random polymer nBu-PLys-HomoPArg-Ppro-Pval (7)
[00258] The synthesis of the random polymer nBu-Plys-homoPArg-Ppro-Pval was obtained as described in Example 1.1, 1.5. and 1.3 but for the polymerization step all NCAs (Trifluoroacetyl-L-lysine NCA, L-Proline NCA and Valine-NCA) are mixed at the same time to obtain random polymer.
Figure imgf000079_0001
Table 7. Targeted diblock copolymers according to the formula
Figure imgf000080_0002
a Determined by NMR. b Determined by SEC wherein the cited DP numbers are subject to a reasonable uncertainty within the range ±20%.
1 .8 General procedure for synthesis homo-PPro polymers (8)
Figure imgf000080_0001
[00259] Bz-PProline is synthesized as described in example 1.1 via ring-opening polymerization by employing n-Benzylamine as initiator. Polymer was isolated as a white solid and used in the next step without further characterization. Yield: 70-80%. Homopolyproline was dissolved in TFE and left stirring for 16 hours. TFE was evaporated and polymer redissolved in water. Desired product was obtained after dialysis (Vivaspin®, 3000 MWCO Da or 500-1000 MWCO Float-A-Lyzer®). White powder was obtained after freeze-drying. Yield: 50-90%.
1 H NMR (300 MHz, D2O) • 7.56 - 7.32 (m, 5H), 4.62 - 4.29 (m, 2H), 3.99 - 3.80 (m, 2nH), 3.77 - 3.57 (m, 2nH), 2.58 - 2.22 (m, nH), 2.22 - 1 .85 (m, 3nH). • nH covered with D2O
Table 8. Homo-PPro polymers.
Figure imgf000080_0003
a Determined by NMR. bDetermined by SEC wherein the cited DP numbers are subject to a reasonable uncertainty within the range ±20%.
1 .9 General procedure for the synthesis of 1a conjugated with lonidamine (9a) and • -tocopherol succinate (TOS) (9b)
Figure imgf000081_0001
[00260] In a two-neck bottom flask, PLO:PPro 6:22 (1a) was dissolved in 5:1 DMF:H2O (1 eq, 10 mg/mL). The reaction also occurs at a lower ratio (>2: 1 ), however, the solution is turbid (not optimal conditions). In a glass vial, the drug was dissolved in 0.5 mL of DMF (i.e 0.40 eq for 10% mol drug modification) and the carboxylic group was activated with DMTMM BF4 (i.e 0.10 eq for 10% mol modification) dissolved in the 0.5 mL (DFM). The activation was allowed to proceed for 20 minutes. After that time, the drug solution was added to the polymer solution. The pH was adjusted at 8 with sodium bicarbonate 1 M. The reaction was left to proceed for 48 hours. After DMF elimination by rotavapor and freeze-drying, the products were purified by Sephadex G25 (PD10 columns) and dialysis Float-A-Lyzer MWCO 500-1000 Da.
[00261] The total drug loading was calculated by 1H-NMR analysis (TDL, mol %). Due to signal overlapping, TOS conjugates drug loading was performed by UV-Vis obtaining the %weight drug which later was back-calculated (b.c) to %mol. [00262] For all the synthesized diblock copolymers, the total drug loading (TDL) determination (%wt) by
UV-vis was established after the calibration curve preparation of the free drugs and matrix effect adjustment, defined as the naked polymer's influence on the conjugates abs. The measurements were performed considering the lonidamine abs at 298 nm and TOS at 285 nm.
Yields: 70%; conjugation efficiency: 50-80%. Table 9. TDL determination by 1H-NMR and UV-vis of different batches of 9a, conjugates of lonidamine .
Figure imgf000082_0002
Table 10. TDL determination by 1H-NMR and UV-vis of different batches of 9b, conjugates of TOS.
Figure imgf000082_0003
1.10 General procedure for the conjugation of dyes to compounds 1, 9a and 9b to yield compounds 10-
Figure imgf000082_0001
[00263] Compounds of families 1, 9a and 9b were derivatized with fluorophores Atto488-NHS or Cyanine5-NHS (Cy5-NHS) (Table 11) . Briefly, in a one-neck bottom flask, PLO:Pro-based compounds were dissolved in ddH2O (1 eq, 10 mg/mL). In a glass vial protected by light, the corresponding fluorophore was dissolved in DMSO and added to the polymer solution (0.02 eq, 2% mol). The pH was adjusted with sodium bicarbonate 1 M. The reaction was allowed to proceed o/n protected by light and checked by TLC (m.p MeOH). The products were purified by Sephadex G25 (PD10 column).
Atto488 estimation was carried out by fluorescence (Atto488-NHS «ex:500 «em:520) after preparing the calibration curve in DMF:H2O. %wt was 1.7; while Cy5 estimation was carried out by fluorescence (Cy5- NHS «ex: 646 •em:662) after preparing the calibration curve in DMF:H2O. %wt was 0.5-1.8. Table 11. Summary of the compounds obtained indicating the corresponding polymer precursor and dye.
Figure imgf000083_0002
1.11 General procedure for the synthesis of compound 16
Figure imgf000083_0001
Figure imgf000084_0002
Compound 16 was synthesized through a convergent process, which initially involved the synthesis of 1a-VC-Mal and 15-PD separately.
Compound 1a-VC-Mal
Figure imgf000084_0001
[00264] In a two-neck bottom flask, 1a was dissolved in 5:1 DMF:H2O (1 eq, 10 mg/mL). In a glass vial, the Valine-Citrulline linker derivative (Maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p- nitrophenyl carbonate, VC-Mal) (purchased in Iris Biotech, ADC1110) was dissolved in 1 mL of DMF (i.e 0.03 eq for 3% mol VC modification) and added to the polymer solution, followed by triethylamine (TEA, i.e 0.03 eq). The reaction was left 24 hours. After DMF elimination by rotavapor and freeze-drying, the products were purified by Sephadex G25 (PD10 columns) and Vivaspin™ 2,000 MWCO.
1H NMR (500 MHz, D2O) • 7.6-7.3 (4H), 6.8 (2H,), 4.77 - 4.71 (2H,), 4.50 - 4.20 (4H), 3.96 - 3.30 (4H, ), 3.24 -3.06 (4H,), 251 - 1.23 (21 H , 0.9 (s, 6H ) 0.82 (t, 3H,).
[00265] The VC functionalization was calculated by 1H-NMR analysis (mol %) and UV-vis (% wt) after the calibration curve preparation of the free VC and matrix effect adjustment, defined as the naked polymer's influence on the conjugates absorbance. The measurements were performed considering the VC abs at 250nm.
%y ield = 70; %mol VC 2.8; %wt VC = 3.8
Compound 15-PD
Figure imgf000085_0001
[00266] In a two-neck bottom flask, 15 (N3-PEG3-PGIu(COOH)5o) (purchased in Iris Biotech, PGA1130) was dissolved in anhydrous DMSO (1 eq, 10 mg/mL) and the carboxylic group was activated with DMTMM BF4 (i.e 0.1 eq for 5% mol PD-NH2 modification) dissolved in the 0.5 mL of anhydrous DMSO. The activation was allowed to proceed for 20 minutes. After that time, the (S)-2-Pyridylthio cysteamine hydrochloride (PD-NH2, i.e 0.05 eq for 5% mol modification) solution was added to the polymer solution. The pH was adjusted at 8 with sodium bicarbonate 1 M. The reaction was left to proceed for 48 hours. The product was precipitated in cold Et2O while stirring, left at -20° for 1 hour, centrifuged and the pellet was lyophilized o/n. To convert the product to its salt form, NaHCOs 1 M solution was added drop by drop and mixing until pH 7/8 and the perfect dissolution at RT. After that, the product was purified by Vivaspin™ 3,000 MWCO.
1 H NMR (500 MHz, D2O) • 8.40 (1 H), 7.80 (2H,), 7.25 (1 H), 4.40 - 4.20 (1 H), 3.80 - 3.30 (12H), 3.17 - 2.66 (4H) 2.51 - 1.66 (4H)
[00267] The PD functionalization was calculated by 1H-NMR as described for VC. but with a calibration curve preparation of the free PD-NH2 and matrix effect adjustment.
%yield = 60; %mol PD-NH2 = 4; %wt PD-NH2 = 5.8
Compound 16
Figure imgf000085_0002
[00268] In a one-neck bottom flask 15-PD was dissolved in ddH2O (1 eq, 10 mg/ml). In a glass vial, 1a- VC-Mal (i.e 0.02 eq for 2% mol 1a-VC modification) was dissolved in 0.5 mL of ddH2O and added to the 15-PD solution, followed by the reducing agent tris(2-carboxyethyl)phosphine (TCEP i.e 0.0025 eq for 2% mol 1a-VC modification). The reaction was left to proceed for 48 hours and the product was purified by Sephadex G25 (PD10 column).
1H NMR (500 MHz, D2O) • 8.40 (1 H), 7.80 (2H), 7.25 (1 H), 4.77 - 4.71 (2H), 4.40 - 4.20 (5H, ), 3.83 -
3.30 (22H,), 3.20 -2.66 (8H) 2.51 - 1.66 (29H), 0.9 (s, 6H, ) 0.82 (t„ 3H)
%yield = 50; %wt 1a-VC (back calculated) = 3.3
General procedure for the conjugation of dyes to compound 16
Figure imgf000086_0001
[00269] Compound 16 was derivatized with fluorophores Atto488-NHS (16b) or Cyanine5-NHS (Cy5- NHS) (16a). Briefly, in a one-neck bottom flask, 16 was dissolved in ddH2O (1 eq, 10 mg/mL). In a glass vial protected by light, the corresponding fluorophore was dissolved in DMSO and added to the polymer solution (0.02 eq, 2% mol). The pH was adjusted with sodium bicarbonate 1 M. The reaction was allowed to proceed o/n protected by light and checked by TLC (m.p MeOH). The products were purified by Sephadex G25 (PD10 column).
[00270] Atto488 estimation was carried out by fluorescence (Atto488-NHS «ex:500 «em:520) after preparing the calibration curve in DMF:H2O. %wt was <1%; while Cy5 estimation was carried out by fluorescence (Cy5-NHS «ex: 646 •em:662) after preparing the calibration curve in DMF:H2O. %wt was <1%.
Example 2. Physico-chemical characterization of compounds of formula 1 and conjugates of formula 9a, 9b, 10-13.
2.1. Circular Dichroism
[00271] Compounds of formula 1 (1a-1e) were characterized by Circular Dichroism (CD) to evaluate their secondary structure, (i.e. • -helix, • -sheets and poly-L-proline helices). This study aimed to verify the influence of the PLO block in the typical poly-L-proline II (PPII) spectra, characterized by a robust negative band at 205 nm and a weak positive band at 228 nm. The experiments demonstrated that the higher the PPpro/PLO ratio in residues, the more accentuated the PPII conformation is. Therefore, PLO:Ppro 6:22 (1a) is the one with the most evident CD shape among the synthesized diblock copolymers. In the case of PLO:Ppro 35:6 (1d) and 35:15 (1e) a random coil conformation was evident. Therefore, we could conclude that the PPI I, with a strong negative band at 205nm and a weak positive band at 228nm, was prevalent in the synthesized diblock copolymers in the following order: PLO:Ppro 6:22 (1a)»PLO:PPro 16:30 (1c)»PLO:PPro 10:5 (1b)=PLO:Ppro 35:15(1e)»PLO:PPro 35:6 (1d).
[00272] The helix type II stability was also evaluated for the selected diblock copolymer 1a (0.25 mg/mL) Specifically, we verified that the secondary structure was maintained after different cycles of freezing (o/n) and defrosting and at 37° C in phosphate buffer 1 M pH 7 (1 h).
[00273] The helix type II formation of drug conjugates 9a and 9b was also evaluated at 0.25 mg/mL in ddH20 concluding that the drug conjugation did not affect the secondary structure of the PLO:Ppro based polymer, independently from the drug loading.
2.2. Size determination
[00274] The hydrodynamic radius was determined using Fluorescence Correlation Spectroscopy (FCS)). FCS is based on the analysis of time correlations in fluorescence fluctuation emitted when fluorescently labeled molecules are diffusing in and out of a tiny observation volume (focused light). The main remarkable advantages are the measurable time ranges (ns to s) and the high sensitivity, up to a single-molecule level. As FCS is a fluorescent-based technique, it was applied to compounds of formula 1a, 9a_2 and 9b_2 labeled with Atto488 (named as 11a, 12a and 13a) and dissolved in PBS 1 M pH 7.4 after adjusting the structural parameters with the free dye.
Table 12. Size determination by FCS of Atto488 labelled polymers.
Figure imgf000087_0001
[00275] The size results were consistent for all the compounds, proving that the nanoparticle formed independently from the tested concentrations.
[00276] The hydrodynamic radius was also determined by diffusion-ordered NMR spectroscopy (NMR DOSY). As FCS, this technique allows to obtain the D of the particles in solution. Compounds 1a, 9a_5 and 9b_6 were dissolved in dPBS 1 M pH 7.4 and the D were extracted from the 2D NMR spectra.
Higher concentrations were tested for sensitivity reasons. Table 13. Size determination by NMR DOSY.
Figure imgf000088_0001
[00277] A cryo-TEM analysis performed, observing the micelle-like formation for 9b_7 (4-5 nm), while 9a_6 and 1a could not be observed. These results were in line with the results obtained previously. The micelle formation for 9b_7 was further studied by a critical micelle concentration fluorescence assay.
2.3. Critical Micelle Concentration (CMC)
[00278] CMC of TOS conjugates was checked by fast fluorimetric method using pyrene as probe according to Li et al. Pyrene fluorescent pattern (band I, 371/372 nm and III, 383/383 nm) increases when migrating from hydrophilic to hydrophobic regions. Serial dilutions of 9b were prepared, and 10 pL of a 0.02 mg/mL pyrene solution was added to each concentration. After acetone evaporation (37° C, 1 h), the solutions were stabilized o/n. The samples were analyzed at the spectrofluorometer (exc. 335 nm; ems I 374 nm, ems III 385 nm). From the graphic obtained plotting the FIII/FI ratio and the log(conc) the CMC of 9b was obtained as: for 5.8% wt (9b_5), 0.12 mg/mL and for 10% wt (9b_4), 0.03 mg/mL. The micelle formation could explain the IC50 value increase at higher DL, supposing that when TOS is hidden inside the hydrophobic core, it cannot contribute to the conjugate toxicity.
Example 3. Validation of compounds of formula 1 and 16 as carriers and mitochondria targeting vectors
3.1 Cell viability
[00279] Cell viability studies for the diblock copolymers PLO:PPro (formula 1) were assessed in the triple negative breast cancer cell line MDA-MB 231The diblock copolymer PLO:PPro 6:22 (1a) resulted to be the least toxic compound (IC50 72h 0.4 mg/mL). The zwitterionic derivative 16 resulted to be nontoxic in all the tested concentrations (IC50 > 1 mg/ml).
Table 14. IC50 values (72h) for PLO:PPro diblock copolymer
Figure imgf000088_0002
Figure imgf000089_0001
3.2 Internalization studies by flow cytometry
[00280] MDA-MB-231 cells were initially seeded into two 12-well plates and allowed to adhere for 24 hours at 37°C. Subsequently, 0.15 mg/ml of compounds 10a and 16a were introduced at 0, 30, 60, and 180-minute time points in the two plates. One plate was maintained at 37°C, while the other was stored at 4°C during the respective incubation periods. Following this, the cells were scratched, and then harvested into separate tubes for each condition. Subsequently, they were incubated with propidium iodide (2ug/ml), a marker for cell viability. The resulting samples were then analyzed using flow cytometry. The findings indicate that both compounds 10a and 16a permeate the cell membrane within 30 minutes at 37°C. However, when incubated at 4°C, the direct cell membrane permeation of 10a was delayed compared to 16a. The results suggest that both compounds enter the cells using a predominantly endocytosis independent mechanism..
Table 15. Internalization studies of compounds 10a and 16 at different times at 37° (endocytic- dependent pathway) and 4° (endocytic-independent pathway). Units in % cellular uptake (Cy5+ %)
Figure imgf000089_0002
3.3. Internalization studies by confocal microscopy
[00281] The mitochondria colocalization of the different synthesized diblock copolymers PLO:Ppro was evaluated by confocal fluorescence microscopy. For this, all diblock copolymers PLO:Ppro tested were conjugated with Cyanine5 as described in Example 1.10.
Among the tested diblock copolymers (PLO:PPro 16:30 (10c), 10:5 (10b) 35:15 (10e), 35:5 (10d) Pearson r (mitochondria) < 0.4), PLO:Ppro 6:22 (10a) resulted in the mitochondria colocalization at short times (~ 4h, Pearson r (mitochondria) > 0.5) and also scaled-up diblock copolymers PLO:PPro 18:54 (10h) and 22:64 (101) (PLO:PPro ratio 2.8 and 3 respectively) preserve the mitochondria colocalization (~ 2h, Pearson r (mitochondria) > 0.6). The mitochondria colocalization of compound 16a was also evaluated by confocal fluorescence microscopy. The mitochondria colocalization was observed shortly after the cell membrane permeation (~ 1 h, Pearson r (mitochondria) 0.8).
Example 4. In vitro validation of polypeptide-drug conjugates 9a and 9b and the corresponding free drugs (lonidamine and TOS).
4.1 Cell viability
[00282] Cell viability study after the treatment with the free drugs lonidamine and TOS against the breast cancer cell line MDA-MB 231 was performed at 24h, 48h 72h. Concentrated stock solutions of lonidamine and TOS were prepared in 100% DMSO and diluted in DMEM/F-12 medium (with 10% FBS and 1% P/S; final DMSO 0.5%). we obtained a high IC50 for lonidamine (1050=130 iM, 72h), while TOS induced cytotoxicity at lower concentrations (1050=49 iM, 72h).
Table 16. IC50 values (24, 48, 72h) for lonidamine and TOS
Figure imgf000090_0001
[00283] The toxicity of the conjugates 9a and 9b is remarkably higher than both the drugs and the polymer naked in eq, suggesting that the conjugation might generate a synergistic effect.
Table 17. IC50 values (72h) for 9a and 9b
Figure imgf000090_0002
*non-toxic at tested concentrations
4.2 Internalization studies by confocal microscopy
[00284] The mitochondria colocalization of the synthesized polypeptide-drug conjugates was evaluated by confocal fluorescence microscopy Leica TCS-SP8 using the compounds 9a and 9b labeled with cy5 obtained as described in Example 1.10 (compounds 12b, 13b respectively)
[00285] The early endosomes marker was added overnight, 0.2 mg/mL of the conjugate solutions (12b 12% wt, 13b 6.7% wt) were added at 2,4,6 h and overnight, and the mitotracker and lysotracker were added 1 h before the analysis. Both compounds 12b and 13b colocalized with mitochondria at short times (~ 2h, Pearson r > 0.8) and this colocalization was slightly reduced when tested overnight, meaning that the drug conjugation improved the mitochondria tropism for the polymer PLO:PPro 6:22 (10a).
4.3 Mitochondria membrane potential analysis
[00286] The effect of the free drugs, polymer 1a, and conjugates 9a_1 (4.4% wt) and 9b_1 (7% wt). in mitochondria potential was analyzed by fluorescent microplate reader (CLARIOstar) according to the protocol described in Sivandzade et al., using 5,5,6,6'-tetrachloro-1 ,T,3,3' tetraethylbenzimi- dazoylcarbocyanine iodide (JC-1), a lipophilic cation dye that can be used to assess the mitochondria membrane potential (• • M).
[00287] For that, MDA-MB-231 cells were seeded in 96-well plates and treated after 24 hours with carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 50 uM) as positive control and the different compounds at different concentrations for 2,6,24 and 48h (< IC50 24h). After each chosen time, the medium was gently removed, the cells were washed twice with PBS and were incubated for 30 minutes with 2piM JC-1 solution in cell medium. The JC-1 solution was removed and the cells were washed twice with PBS. The fluorescence was simultaneously measured at abs/ems 550/605 red and 484/528 nm green. For the data analysis, red/green fluorescence ratio was considered after each time and treatment compared to untreated controls, representing 100% of cell viability. Values represent Mean ± SD (n=3).
[00288] From the analysis, free drug Lonidamine induced a significant mitochondria depolarization at all tested concentrations at 24h (p-value <0.01) while TOS induced an increasing and significant mitochondria depolarization starting at short times (2h, p-value <0.001). Interestingly, 1a affected the mitochondria membrane potential, prompting a significant (p-value <0.01) depolarization at all tested concentrations at 6h and a slight hyperpolarization at longer times. These results suggest that the polymer can act not only as a carrier but also as a bioactive compound.
[00289] C1, C2, and C3 are the three concentrations tested for each of the compounds as:
1a (IC50 24h 0.55 mg/mL): C1= 0.28 mg/mL, C2= 0.22 mg/mL, C3= 0.18 mg/mL
9a_1 (IC50 24h 0.37 mg/mL, 51 iM lonidamine eq, 0.35 mg/mL pol.eq.): C1= 0.3 mg/mL, C2= 0.24 mg/mL, C3= 0.19 mg/mL
9b_1 (IC50 24h 0.23 mg/mL, 30 piM TOS eq, 0.21 mg/mL pol.eq.): C1 = 0.2 mg/mL, C2= 0.16 mg/mL, C3= 0.13 mg/mL Table 18. Mitochondria membrane potential analysis after treatment with the studied compounds
Figure imgf000092_0001
*Normalized red/green fluorescence intensity.
Sign. = significance: * p value <0.05, ** p value <0.01, *** p value <0.001, **** p value <0.0001
[00290] From the data analysis, it is observed that the conjugates 9a and 9b, as the free drugs (data not shown), decrease the mitochondria membrane potential. The effect of the conjugates, with a relatively low drug loading compared to the lonidamine and TOS tested concentrations, is significant. Indeed, the polymer itself is capable of reducing the electro gradient compared to the untreated cells, thus explaining the additive or synergistic effect of the drug-conjugates.
4.4 Oxygen Consumption rate (OCR) analysis by Seahorse XF Analyzers
[00291] The Agilent Seahorse XF Mitostress Test measures critical parameters of the mitochondrial function by directly measuring the oxygen consumption rate of the cells after adding modulators of the respiration. The modulators include oligomycin, an inhibitor of the ATP synthase; carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP), an uncoupling agent that collapses the proton gradient; and rotenone/antimycin, respectively, inhibitors of the complex I and III.
[00292] For the experiment, the cells were seeded in 96-well plates and incubated at 37° and 5% CO2. Concentrated stock solutions of the polymers were prepared in phosphate buffer 1 M pH 7 and diluted in DMEM/F-12 medium (with 10% FBS and 1% P/S). The polymer solutions were then sterilized (0.2 - m filter) and added to give a final concentration of - IC40 (C1) and diluted solution (C2; dilution factor 1.5). After 24 hours, their culture medium was substituted with the DMEM XF medium (with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose). The inhibitors were loaded into the injection ports of the XFe96 Sensor Cartridge, previously hydrated for 24h with the XF calibrant solution (final concentration oligomycin 1 .5 iM, FCCP 1 iM, and Rot/AA 0.5 iM). The results were normalized by Hoechst 3342 marking and the optimal seeding density for the MDA-MB 231 cell line was 2x104 cells/well, which were applied in the 24 hours protocol of lonidamine, TOS, 1a, 16, 9a_1 and 9b_1 treatment.
1a (IC50 24h 0.55 mg/mL): C1= 0.4 mg/mL, C2= 0.3 mg/mL
16 (IC50> 1 mg/ml): C1= 0.4 mg/mL
9a_1 (IC50 24h 0.37 mg/mL, 51 iM lonidamine eq, 0.35 mg/mL pol.eq.): C1= 0.3 mg/mL, C2= 0.24 mg/mL, C3= 0.19 mg/mL
9b_1 (IC50 24h 0.23 mg/mL, 30 piM TOS eq, 0.21 mg/mL pol.eq.): C1= 0.2 mg/mL, C2= 0.16 mg/mL
[00293] From the analysis, both drugs (data not shown) and the polymer 1a affected the cellular metabolism related to the mitochondria function. Specifically, lonidamine induced a reduction of the ATP-production coupled respiration and slight and non-significant of the spare respiration capacity. TOS induced a significant and non-concentration-dependent reduction of both ATP-production coupled respiration and spare respiration capacity. Interestingly, 1a induced a significant and non-concentration- dependent reduction of ATP-production coupled respiration, while it didn't affect the spare respiration capacity. This result suggests, together with the JC-1 data, that polymer 1a acts as a bioactive molecule itself. Treatment with compound 16 did not show any impact on the mitochondria respiration, proving its safer profile while maintaining its mitochondria targeting ability. The conjugates affected the cellular metabolism related to mitochondria respiration. Specifically, the conjugates maintain the ability to decrease the ATP-production coupled respiration and the spare respiration capacity for 9b_1, even at low drug concentrations in equivalents.
Table 19. Oxygen consumption rate analysis after 24h treatment with the studied compounds (control= 100%; n = 3, mean ± SEM)
Figure imgf000093_0001
Figure imgf000094_0001
[00294] *Normalized from control (100%), OCR in pmol/min/2.104 cells
4.5 ROS production (DFCH)
[00295] MDA-MB-231 cells were initially seeded into a 6-well plate and allowed to adhere for 24 hours at 37°C. Subsequently, 0.4 mg/ml of 10a and 16a, 0.08 mg/ml of 10h, and 0.06 mg/ml of 10i were added to separate wells for a 24-hour treatment. After washing with PBS, try psinization, and centrifugation, the cell pellets were resuspended in fresh DMEM/F-12 medium (with 10% FBS and 1% P/S), and then incubated with 5 ig/ml (final concentration) of 2,7-dichlorodihydrofluorescein (DCFH) for
30 minutes at 37°C. Subsequently, propidium iodide (2ug/ml), a marker for cell viability, was added, and the resulting samples were analyzed using flow cytometry. The analysis revealed that all diblock copolymers with a ratio of 1 :3 (10a, 10h, and 10i) induced a significant increase in ROS production after the 24-hour treatment, while 16a did not impact ROS production, demonstrating its safer profile while maintaining its mitochondria-targeting ability.
10a (IC50 24h 0.55 mg/mL): C= 0.4 mg/mL 10h (IC50 24h 0.1 mg/mL) C= 0.08 mg/mL
10i (IC50 24h 0.07 mg/mL): C= 0.06 mg/mL
16a (IC50> 1 mg/ml): C= 0.4 mg/mL
Table 20. ROS production analysis after 24h treatment with the studied compounds
Figure imgf000094_0002
Figure imgf000095_0001
Example 5. Cardiolipin affinity studies
5.1. Fluorescent competitive binding assay
[00296] The compound 3,6-di(azetidin-1 -yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide is an analog of 10-N-nonyl acridine orange (NAO) with increased photochemical properties, water solubility and cardiolipin (CL) affinity described by Dimitrijevs P. & Arsenyan P. This fluorescent probe was used in quantitative competitive binding studies to measure and compare the affinities of various substances for CL. The affinity of PLO:Ppro 6:22 (1a), 6:7 (1g), and 6:12 (1f) and the derivatives 9a-5 and 9b-5 and the homopolymers Pprol 4 (8a) and Ppro25 (8b) for cardiolipin was determined after their incubation with cardiolipin containing liposomes and mitoplast isolated from rat heart (20 mM HEPES, pH 6.8 at 37 •C). Interestingly, the homopolymers showed no significant affinity for the mitochondria membranes, meaning that the cationic part of the diblock copolymer plays a crucial role in the interaction with the phospholipid. However, comparing PLO:Ppro 6:7 (1g) and 6:12 (1f) to 6:22 (1a) diblock copolymer, it was possible to observe a slight increase in cardiolipin affinity when the Ppro tail is elongated. PLO:Ppro 6:22 (1a) was also compared to other polycationicPpro diblock copolymers with the same composition (PArg:PPro-6:22 (3a) and Plys: Ppro-6: 19 (2a)). The results confirmed that the positively charged part of the diblock copolymer is essential for the cardiolipin interaction.
Table 21. EC50 (uM) values of the studied compounds in different CL-containing membranes
Figure imgf000095_0002
** not tested
Cyt c was used as a positive control (CLDOPC liposomes ref. EC50 0.26±0.02; mitoplast ref. EC50 0.78±0.15
5.2. Intercalation studies in the hydrophobic regions of cardiolipin containing liposomes
[00297] Quenching of 1 ,6-Diphenyl-1 ,3,5-hexatriene (DPH) fluorescence was used to study the intercalation of PLO:Ppro 6:22 (1a) and the derivatives conj-lonidamine (9a-5) and conj-TOS (9b-5), PLO:Ppro 6:7 (1g) and 6:12 (1h) and the homopolymers Pprol 4 (8a) and Ppro25 (8b) in the hydrophobic regions of cardiolipin containing liposomes and zwitterionic control liposomes (20 mM HEPES, pH 6.8 at 37 «C). All PLO-containing polymers and conjugates intercalated into the hydrophobic region of the CLDOPC liposomes regardless of the polyproline tail length, while the interaction with DOPC liposomes is much less pronounced. Ppro-homopolymers showed a slightly increased interaction with CLDOPC compared to DOPC liposomes. Nevertheless, these studies confirmed the significance of the diblock's cationic part for interacting with mitochondria-like membranes.
Table 22. DPH fluorescence quenching in CL-containing and control membranes
Figure imgf000096_0001
NonylTPP was used as a positive control (CLDOPC liposomes % fluorescence intensity
76.2 and 47.9 at 10 and 100 piM respectively; DOPC liposomes % fluorescence intensity
95.4 and 82.8 at 10 and 100 piM respectively)
Example 6. Physical studies
6.1 CL based in vitro systems for affinity studies [00298] Giant unilamellar vesicles, supported lipid bilayers and small unilamellar vesicles were formulated to examine the binding affinity of the PLO:Ppro 6:22 (1a) with cardiolipin (CL) comprising vesicles following procedures from Weinberger et al. For the CL based vesicles, lipid compositions consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 18:1 cardiolipin (CL) at 3:1 molar ratio were used, with an addition of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) labeled with Atto-655 at the headgroup. As a negative control, lipid compositions consisting of POPC and 1 -palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) at 1 :1 molar ratio were used with the same amount of labeled DOPE. POPG has a negatively charged headgroup at neutral pH, whereas CL has two negative charges at its headgroup due to two phosphate groups. The lipid composition of the negative control was chosen to achieve equipotential membranes with CL and PG. The molar amounts for the labeling varied for fluorescence spectroscopy and microscopy measurements. The different lipid bilayer systems employed for the in vitro affinity studies were obtained by the following methods: PVA assisted swelling method for the formation of giant unilamellar vesicles (GUV); and lipid film rehydration method for the formation of small unilamellar vesicles (SUV) and supported lipid bilayers (SLB).
6.2 In vitro affinity studies with cardiolipin (CL) based lipid bilayer systems.
[00299] CL Giant unilamellar vesicles were incubated with 5 iM PLO:Ppro 6:22 labeled with Atto-488 (11a) for 30 min. Images of lipid vesicle samples were taken by a Zeiss LSM780 confocal laser scanning microscope using a C-Apochromat 40x/1 .20 water-immersion objective (Carl Zeiss AG). Fluorophores were excited using a 488nm Argon laser (for Atto-488), and 633nm He-Ne laser (for ATTO655). Images were typically acquired with 512x512-pixel resolution.
[00300] Significant colocalization was observed with GUVs labeled with Atto655-DOPE. To verify if the interaction was dependent on the membrane surface potential, GUVs composed of POPG were also incubated with 5 iM of 11a labeled with Atto-488 for 30 min. As in the case of CL based vesicles, PG based vesicles also showed colocalization of the polymer on the surface of the membrane. Thus, verifying that the kinetics of the binding are driven through the charge of the polymer. However, the distribution of the polymer between the two samples were not similar.
[00301] In addition, 11a was incubated with POPC/CL and POPC/POPG SLBs showing that at increasing concentrations of the polymer, the deformations on the membrane of CL based bilayers, in form of membrane irregularities and alterations in membrane packing, were more clearly observed confirming the interaction. The crowding effect causing smaller membrane irregularities (i.e patches) in both SLBs (non CL-dependent) was also confirmed for PGA-PEG-VC-PP labeled with Atto-488 (16b), indicating that they are essential for the membrane binding (c.f Fig. 1).
[00302] These membrane irregularities in the membrane packing were observed by using the Flipper- TR® probe (Spirochrome) which can report on membrane tension or membrane packing by the change in the fluorescence lifetime of the fluorescent probe. The sample was imaged with a total internal reflection fluorescence microscope (TIRFM) Zeiss Elyra 7 with an alpha Plan-Apochromat 63x / 1.46 Oil objective and excitation wavelength at 488 nm (c.f. Fig. 2).
[00303] Furthermore, sample was also imaged on a PicoQuant MT200 confocal microscope with a 60x Na1.2 UPlanApo water immersion objective.
[00304] The membrane irregularities showed two components in the fluorescence lifetime histogram, with fitted values of Ti, T2 peaks at 3.0 and 4.5 ns respectively. The presence of an increased lifetime hints at changes in the lipid packing locally where the polymers bind with the membrane.
[00305] To quantitatively verify the interaction of 11a with POPC/CL and POPC/POPG based SUVs, Fluorescence (Cross) Correlation Spectroscopy (FCCS) and PicoQuant MT200 microscope were used. The FCS autocorrelation curves for the individual channels were fitted with a two component 3D diffusion model, and the cross-correlation curve was fitted with a 3D diffusion model respectively.
[00306] For FCCS analysis, the focal volume overlap was calculated using a DNA oligo sample with both 5' and 3' end labeled with the respective fluorophores. The DNA oligomers used as a FCCS standard are double-stranded DNA, consisting of a labeled ssDNA with 5' Atto655 and 3' Atto488 labels on the sequence CTTTTCTTCTTTTCTTTCTT (SEQ ID NO. 1) which is mixed with it's complementary unlabeled ssDNA AAGAAAGAAAAGAAGAAAAG (SEQ ID NO. 2). It was observed that for both POPC/CL and POPC/POPG based vesicles, there was a cross-correlation curve present that indicates co-diffusion of both the labeled polymers and the labeled vesicles.
[00307] The vesicles with POPC/CL showed a higher molecular brightness that confirms a higher affinity of 11a for CL-based vesicles. The Zeta potential of the POPC/CL (3.1) and POPC/POPG (1 :1) SUVs were measured using the Malvern ZetaSizer Nano incubating with PLO:PPro 6:22 (1a), PArg:Ppro 6:22 (3a), and PHis:PPro 7:26 (4). The zeta potential measurements showed that there was a higher change in the zeta potential of the negatively charged POPC/CL vesicles than the POPC/POPG vesicles with the addition of all positively charged polymers (1a, 2a, 3a). This further qualitatively confirms that, firstly the interaction is driven through ionic interactions between the negatively charged vesicles and positively charged polymers, and secondly, there is a higher affinity of the polymer for CL based vesicles, (c.f. Fig. 2). The increase in zeta potential with the presence of compound 16 is less when compared with compound 1a. The surface zeta potential of the negatively charged vesicles increases in both cases, which corresponds to the polymers binding with the membranes, although the higher increase for compound 1a indicates a higher affinity of this polymer towards the negatively charged membranes. Table 23 - Zeta potential measurements of POPC/CL and POPC/POPG (control) membranes after incubation with the studies compounds
Figure imgf000099_0001
[00308] Overall, the examples described in Sections 5 and 6 re-assure the interaction of compounds of formula I as described in the claims, with lipid-based nano-assemblies, thus, being an indirect proof of their suitability as components in lipid-based nano-assemblies such as liposomes or LNPs.
Example 7. Polymeric Nanoparticles (PNPs) formulation
7.1 PNPs formulation with compounds 1a, 5c and 9b.
[00309] In the following examples the oligonucleotides used were: a mRNA purchased from CATUG Biotechnology, with reference FLuc mRNA (N1-Me-Pseudo UTP) Cat. number CT072; Lot number P23H065 expressing luciferase as reporter gene.
[00310] Polyplex formulations to study size and PDI were prepared in-situ (mixing in a pipette) as follows:
The desired amount of mRNA and the calculated amount of the cationic polymer at indicated charge-ratio (+/• ) or amine to phosphate ratio (N/P) were diluted in separate tubes in MES Buffer Glucose (MBG) 10 mM, Glucose 5% w/v pH 6.1. Only protonatable nitrogens, not amide nitrogens, were considered in the +/• ratio and N/P ratio calculations. The cationic polymer solution and the genetic material solution were mixed by rapidly pipetting up and down (ten times) and incubated for 20 min at RT. Then the polyplexes formed were characterized by DLS to determine Z-average (nm) and PDI.
Table 24 - Z-average (nm) and PDI of the PNPs containing the compounds of formula 1a, 5c and
9b with mRNA.
Figure imgf000100_0001
Example 8. LNPs formulation
8.1 LNPs formulation with compound of formula 1a and 5c.
[00311] In the following examples the oligonucleotides used were: a mRNA purchased from CATUG Biotechnology, with reference FLuc mRNA (N1-Me-Pseudo UTP) Cat. number CT072; Lot number P23H065 expressing luciferase as reporter gene. Any other oligonucleotide with the characteristics above indicated could be used to carry out the experiments below.
[00312] The ionizable lipid DODMA, or 1 ,2-dioleyloxy-3-dimethylaminopropane was purchased from Avanti Polar Lipids. Shielding lipid 1 ,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) was purchased from Avanti Polar Lipids. Both polymers were used to form benchmark LNPs. Structural lipids used to form LNPs were: dioleoylphosphatidylethanolamine (DOPE, from Sigma- Aldrich) and Cholesterol (Sigma-Aldrich).
[00313] Benchmark LNPs and tested LNPs were formulated with NanoScaler (KNAUER) as follows:
• Lipids were dissolved in ethanol at 50-x/10/38.5/1 .5 molar ratio (DODMA/DOPE/Cholesterol/ DMG-PEG), where x is the molar ratio corresponding to the compound of interest (1a or 5c).
• mRNA was diluted in acetate buffer 25 mM at pH 4
• Amine to phosphate ratio (N/P) was 4
• Flow ratio was 1 :3 (ethanol :H2O)
• Total flow rate for LNPs was 4.5 mL/min (3mL/min undiluted LNPs + 1 .5mL/min in -line dilution with PBS)
After formulation, LNPs were further diluted in 10 mM Phosphate buffer saline (PBS) pH 7.4 to decrease ethanol concentration and purified with centrifugal concentrators (Vivaspin™ 500, Sigma) to remove ethanol. The formulation containing the polymer of the invention was prepared analogously with the difference that different compounds were used instead of ionizable lipid DODMA and the lipid ratio was different (shown below Table 26 and 27).
8.2 Size and polydispersity
[00314] Size and polydispersity (PDI) of the formulated LNPs containing mRNA at different N/P ratios (positively-chargeable polymer amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P)), different molar ratios and shielding polymers were performed using a Malvern ZetasizerNanoZS instrument, equipped with a 532 nm laser at a fixed scattering angle of 173°. 50 pl of the samples were measured using a quartz glass high performance cuvette (Hellma Analytics). Size distribution was measured (diameter, nm) with n • 3 measurements.
[00315] LNPs containing DODMA as ionizable lipid as well as the compounds of interest 1c and 5c, DMG-PEG as shielding lipid and mRNA as cargo, were formulated at 50/10/38.5/1.5 molar ratios for DODMA/DOPE/Cholesterol/DMG-PEG obtaining the following results:
Table 25 shows the results obtained of LNPs containing 1a and 5c formulated at molar ratio of 10% containing mRNA as cargo.
Figure imgf000101_0001
[00316] Results suggest that introducing 10% of PLO-PPro compounds 1a and 5c, are consistent in all replicates, showing low PDI values and appropriate sizes.
8.3 Free oligonucleotide detection by gel electrophoresis
[00317] The possible presence of free mRNA after LNPs formulation were assessed using an electrophoresis gel as first screening method. To perform the electrophoresis, E-gel Power Snap Electrophoresis Device and E-Gel Power Snap Camera (Invitrogen) was used. 2% agarose gels prepared that include the SYBR Gold DNA marker (E-Gel™ EX Agarose Gels, 2%, Invitrogen) were used following manufacturer's instructions. The LNPs (20 pl) were valuated, and also the disassembly of the LNPs in the presence of 10% triton X-100 (Scharlab.S.L). Once the gel was loaded (20pl/well), the equipment protocol was selected according to the type of gel used (in our case, protocol approximately 10 min, although the time can be modified according to the samples).
[00318] In all cases, no free mRNA was observed when different polymers were tested. Example 9. In vitro biological studies in HEK293 cells
[00319] In the following examples the oligonucleotides used were: a mRNA purchased from CATUG Biotechnology, with reference FLuc mRNA (N1-Me-Pseudo UTP) Cat. number CT072; Lot number P23H065 expressing luciferase as reporter gene.
9.1. Biological activity of LNPs in HEK293 cells
[00320] The transfection efficiency and the cell viability of the LNPs formulations in HEK293 cells is reported in the following table. The transfection data is represented as % of the positive control DODMA LNPs with the formulation reported 50-x/10/38.5/1 .5 molar ratios for DODMA-compound 1a/DOPE/Cholesterol/DMG-PEG being x the different molar ratios, and being the positive control 100% after 24h of treatment and cell viability is compared to non-treated (NT) cells, being the ATP content readout of NT (non-treated) cells equal to 100%.
Table 26. Different parameters of LNPs formulated with PLO-PPro 1a Molar ratio was 50- x/10/38.5/1.5, where x is the compound 1a Total flow used was 3+1.5mL/min. N/P ratio was 4 and nucleic acid content was 50, 150 and 300 ng mRNA/well. % Transfection is calculated considering the formulation DODMA/DOPE/Cholesterol/DMG-PEG 50/10/38.5/1.5:
Figure imgf000102_0001
[00321] According to the results, the transfection obtained from LNPs containing 10% of 1a was comparable or higher than those LNPs containing only DODMA LNPs.
[00322] Therefore, it can be concluded that by adding 1a into LNPs, the following is obtained: i) stable nanoparticles with acceptable size and polydispersity; ii) non-cytotoxic formulation, and iii) high transfection efficiency. These results demonstrate that this addition of a polycationic PLO-PPro (1a) presented in this application can be an improvement of the standard LNP formulations in the prior art.
REFERENCES CITED IN THE APPLICATION
Sivandzade, F., Bhalerao, A. & Cucullo, L. Analysis of the Mitochondrial Membrane Potential
Using the Cationic JC-1 Dye as a Sensitive Fluorescent Probe. Bio-Protocol 9, 1-13 (2019).
Li, H., Hu, D., Liang, F., Huang, X. & Zhu, Q. Influence factors on the critical micelle concentration determination using pyrene as a probe and a simple method of preparing samples. R. Soc. Open Sci. 7, (2020).
Dimitrijevs, P. & Arsenyan, P. Cardiolipin in the spotlight: Quantitative analysis and fluorescence-based competitive binding assay. Sensors Actuators B Chem. 346, 130537 (2021).
Andreas Weinberger et al. Gel-Assisted formation of giant unilamellar vesicles. Biophys J. 105(1): 154-64 (2013).
[00323] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:
[00324] Clause 1. A polyproline-based block copolymer of formula (I), a salt thereof, or any stereoisomer or mixtures of stereoisomers, either of the compound of formula (I), or of any of its salts,
Figure imgf000103_0001
wherein the “*” denotes the attaching points; where the repetitive unit defined by square brackets with the numerical value m is denoted as PAA1;
Figure imgf000103_0002
wherein though the main repeating units AA1 , AA2 and AA3 are shown in a particular order for convenience of description, the main repeating units may be present in any order and may be block or randomly present; and wherein each of the repeating units PAA1 , AA1 , AA2 and AA3, may comprise blocks of monomer units which may be the same or different between each other; wherein m is an integer selected from 4 to 100; n is an integer selected from 0 to 150; p is an integer selected from 0 to 150; q is an integer selected from 0 to 100; wherein at least one of n, p or q is • 4 or alternatively n + p • 4 wherein Li may be present or absent;
Li is a biradical selected from the group consisting of
Figure imgf000104_0001
wherein the wavy lines denote the attaching points; wherein each A and A' is independently selected from -O-, -CO- and -NH-; wherein y and z are integers independently ranging from 1 to 20; and wherein each Z is a biradical selected from the group consisting of -NH(Ci-C6)alkyl-O-, -NH(Ci-C6)alkyl-NH-, -NH-(Ci-C6)alkyl-CO-, -O-(Ci-C6)alkyl-CO-, -O-(Ci-C6)alkyl-O-, -CO-(Ci-C6)alkyl-CO-, a straight or branched -NH-(Ci-C3o)alkylene-0-, -NH-(Ci-C3o)alkylene-NH-, -NH-(Ci-C3o)alkylene-CO-, -0-(Ci-C3o)alkylene-0-, -0-(Ci-C3o)alkylene-CO-, -CO-(Ci-C3o)alkylene-CO-
Figure imgf000104_0002
Figure imgf000105_0001
wherein the “*” denotes the attaching points; wherein B and B’ are each a biradical independently selected from -O-, -NH-; and -CO-; wherein each D is a biradical selected from -0- and -NH-; wherein k is an integer selected from 1 to 12; preferably from 1 to 6; wherein the -NH-(Ci-C3o)alkylene-0-, -NH-(Ci-C3o)alkylene-NH-, -NH-(Ci-C3o)alkylene-CO-, - 0-(Ci-C3o)alkylene-0-, -0-(Ci-C3o)alkylene-CO-, -CO-(Ci-C3o)alkylene-CO- biradical of Z is optionally substituted with one or more radical selected from the group consisting of -OH, -NRaRb,-SH2, -NHNH2, -COORc, -CF3, -OCF3, and halogen; wherein Ra, Rb and Rc are each a radical independently selected from the group consisting of H, -phenyl, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkylphenyl, and -phenyl(Ci-C3o)alkyl; wherein R2 and R4, are each a biradical independently selected from the group consisting of
-(Ci-C6)alkyl-, -(Ci-C6)alkyl-S-S-(Ci-C6)alkyl-, -(Ci-C6)alkyl-O-(Ci-C6)alkyl-, and
-(Ci-C6)alkyl-NH-(Ci-C6)alkyl-; each R2 and R4 are independently optionally substituted by one or more substituents selected from the group consisting of -NH2 and -(Ci-C6)alkyl-NH2; each R3 is a radical independently selected from the group consisting of H and -(Ci-Ce)alkyl; each W1 is independently selected from CH and N;
Figure imgf000106_0001
Figure imgf000107_0001
wherein denotes the attaching point; each R'2 is selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -OAIkyl(Ci-C6), halogen, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-C30)alkyl, -OC(O)O(Ci-C30)alkyl, -OC(O)NH2, -OC(0)N((Ci-C3o)alkyl)2, -SH, -S(Ci-C30)alkyl, -S(O)H, -S(O)(Ci-C30)alkyl, -S02(Ci-C3o)alkyl;
R'2, R"2, Rv2 and Rv2 are each independently selected from the group consisting of H, -(Ci-C3o)alky I, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl NH2, -(Ci-C3o)alkyl-N((Ci-C3o)alkyl)2, -(Ci-C3o)alkyl-NH(Ci-C3o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI)
Rv'2 and Rv'2’ are each independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -OAIkyl(Ci-Ci2), F, Cl, Br, I, -CF3, -OCF3, -NO2, -CN, -NH2, -(Ci-C3o)alkylNH2, -N((Ci-C30)alkyl)2, and-NH(Ci-C30)alkyl, wherein Rvi'2, R™2, Rix2, and Rx2are each independently selected from the group consisting of H, -(Ci-Ci2)alkyl, -(Ci-Ci2)alkylNH2, -(Ci-Ci2)alkyl-N((Ci-Ci2)alkyl)2, -(Ci-Ci2)alkyl-NH(Ci-Ci2)alkyl, -O(Ci-Ci2)alkyl, -COH, -CO(Ci-Ci2)alkyl, and -0(C2-C3o)alkenyl, wherein R2, R'2, R"2, Riv2, Rv2, Rv'2, Rv'2’, Rvi'2, R™2, Rix2, and Rx2 are each optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -0(Ci-C3o)alkyl, -CF3, -OCF3, -NH2, -(Ci-C30)alkyl, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-C30)alkyl-OH; b2, c2, d2, e2, f2, g2, h2, I2, j2, k2, b2', c2', d2', e2', and g2' are each independently an integer selected from 1 to 6;
Figure imgf000108_0001
wherein denotes the attaching point;
12, v2, w2, x2, y2, z2 and o2 are each independently an integer selected from 1 to 6; each R'1 is independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2- C3o)alkynyl, -OAIkyl(Ci-C6), halogen, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-C30)alkyl, -OC(O)O(Ci-C30)alkyl, -OC(O)NH2,
-OC(0)N((Ci-C3o)alkyl)2, -SH, -S(Ci-C30)alkyl, -S(O)H, -S(O)(Ci-C30)alkyl, -S02(Ci-C3o)alkyl;
Rii1, Riii1, R'v1, Rv1 and Rvi1 are each independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl NH2, -(Ci-C3o)alkyl-N((Ci-C3o)alkyl)2, -(Ci-C3o)alkyl-NH(Ci-C3o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein R'1, Rii1, Riii1, R'v1, Rv1, and Rvi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -0(Ci-C3o)alkyl, -CF3, -OCF3, -NH2, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-C30)alkyl-OH; wherein each Rs is independently optionally substituted by at least one detection moiety, at least one active moiety, at least one lipid-like moiety R10, or a mixture thereof; a1 is an integer selected from 0 to 1 ; with the proviso that R2 is absent when a1 =1 ; wherein L4 is selected from the group consisting of an inert moiety, a detection moiety, an active moiety, and a lipid-like moiety R10; wherein L4 is attached to the nitrogen atom of the AA1 , AA2 or AA3 by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond; or alternatively L4 is attached to the carbonyl group of the AA1 , AA2 or AA3 by a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond; the inert moiety is selected from the group consisting of H, -N3, -NH2, -C(O)H, -S(O)H, -NHCH(CH3)2, -(Ci-C4)alkylNH2, -(Ci-C4)alkylNHCH3, -(Ci-C4)alkylN(CH3)2, -O-(Ci-C4)alkyl-NH2, -O-(Ci-C4)alkyl-NHCH3, -O-(Ci-C4)alkyl-N(CH3)2, -(Ci-Ci2)alkyl, -(C2-Ci2)alkenyl, -(C2-C6)alkynyl, -Cy1 , -(Ci-Ci2)alkylene-Cy1 , -(C2-Ci2)alkenylene-Cy1, -(C2-Ci2)alkynylene-Cy1, -Cy2-(Ci-Ci2)alkyl, -Cy2-(C2-Ci2)alkenyl, and -Cy2-(C2-Ci2)alkynyl; being Cy 1 a known ring system independently selected from the group consisting of 5- to 7-membered saturated or partially unsaturated carbocyclic or heterocyclic ring; and 5- to 7-membered carbocyclic or heterocyclic aromatic ring; Cy2 a bivalent known ring system independently selected from the group consisting of 5- to 7-membered saturated or partially unsaturated carbocyclic or heterocyclic ring; and 5- to 7-membered carbocyclic or heterocyclic aromatic ring; and being each one optionally substituted by one or more groups consisting of OH, halogen, NH2, NHRd, NRd e, -(Ci-C6)alkyl, NO2, N3, -(Ci-Ci2)alkyl, -O-(Ci-Ci2)alkyl, -CO-(Ci-Ci2)alkyl and -CO-O-(Ci-Ci2)alkyl; being each one optionally substituted by one or more groups consisting of OH, halogen, NH2, NHRd, NRdRe, -(Ci-C6)alkyl, NO2, N3, -(Ci-Ci2)alkyl, -O-(Ci-Ci2)alkyl, -CO-(Ci-Ci2)alkyl and -CO-O-(Ci-Ci2)alkyl; wherein Rd and Re are each independently selected from H, and -(Ci-C4)alkyl; wherein the inert moiety is optionally substituted by a detection moiety, an active moiety, and a lipid-like moiety Rw or a mixture thereof; the detection moiety is selected from the group consisting of chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, transition metal and isotope mass tag moiety; the active moiety is selected from the group consisting of pharmaceutically active agents, veterinary active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof; the lipid-like moiety R is selected from the group consisting of -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, and a radical of formula (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII)
Figure imgf000110_0001
Figure imgf000111_0001
I l l wherein:
T and T are each independently selected from -OH, -OCORx and -COORx;
Q and Q' are each independently selected from -OCORy and -COORy; each G is independently selected from -OCO-, -COO-, -NRz'CO-, and -CONRz'-; each Rz' is H or Rz; each Rx, Ry, and Rz is independently -(Ci-Ci8)alkyl or -(C2-Ci8)alkenyl; each a1 is independently an integer from 0 to 18; each b1 is independently an integer from 1 to 18; each d is independently an integer from 0 to 18; each d1 is independently an integer from 0 to 18; each e1 is independently an integer from 0 to 18; each fl is 0 or 1; each f1' is 0 or 1 ;
T1 , T2, T3, TT, T2' and T3' are each independently selected from the group consisting of hydrogen, fluorine, methyl, -CH2F, -CHF2, and -CF3; each of the dashed bonds — is independently a single bond or, alternatively, a double bond; each J is a biradical chain which comprises one or more moieties selected from the group consisting of -CH=CH-, -C« C-, -CH2-, -N=CH-, -CH=NH-, -NH-, -NH-NH-, -NH-N=CH-, -O-, S-,
Figure imgf000112_0002
wherein Rf, Rg and Rh are each radicals independently selected from the group consisting of H, -phenyl, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkylphenyl, and -phenyl(Ci-C3o)alkyl; wherein when J comprises a -CH2- moiety, the two hydrogen atoms attached to the carbon atom are optionally replaced by the ring:
Figure imgf000112_0001
and wherein each R1 is independently selected from the group consisting of H, OH, an amine protecting group and a radical selected from the group consisting of (AA1 b), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXVI), (XXVIII), (XXIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI) and (XXXVII)
Figure imgf000113_0001
, , , , , W1 are as defined above.
[00325] Clause 2. The polyproline-based block copolymer of formula (I) according to clause 1 , wherein each Rs is independently selected from selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Ci8)alkenyl, -(Ci-Cis)alkyl-R1, -(Ci-Cis)alkyl-COORi1, -(Ci-Cis)alkyl-O-R"1, -(Ci-Ci8)alkyl-NRiv1Rv1, -C(O)-Rvi1, -(Ci-Ci8)alkyl-CO-NH2, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXVII), (XXXIII), (XXXVI) and (XXXVII); each R1 is independently selected from the group consisting of H, -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, -(C2- Cis)alkynyl, -OAIkyl(Ci-C4), F, Cl, Br, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-Ci8)alkyl, -OC(O)O(Ci-Cis)alkyl, -OC(O)NH2, -OC(O)N((Ci-Cis)alkyl)2, -SH, -S(Ci-Cis)alkyl, -S(O)H, -S(O)(Ci-Cis)alkyl, -SO2(Ci-Cis)alkyl;
R'1, Riii1, Rv1, Rv1 and Rvi1 are each independently selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, -(Ci-Cis)alkylNH2, -(Ci-Cis)alkyl-N((Ci-Cis)alkyl)2, -(Ci-Cis)alkyl-NH(Ci-Cis)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein R1, Rii1, Riii1, Rv1, Rv1, and Rvi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -O(Ci-Cis)alkyl, -CF3, -OCF3, -NH2, -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, -(C2-Cis)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-Cis)alkyl-OH; wherein each Rs is independently optionally substituted by at least one detection moiety, at least one active moiety, at least one lipid-like moiety Rw, or a mixture thereof.
[00326] Clause 3. The polyproline-based block copolymer of formula (I) according to any of clauses 1-
2, wherein each Rs is independently selected from selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Ci8)alkenyl, -(Ci-C6)alkyl-Ri1, -(Ci-C6)alkyl-COO i1, -(Ci-C6)alkyl-O-Riii1, -(Ci-C6)alkyl-NRiv1Rv1, -C(O)-Rvi1, -(Ci-C6)alkyl-CO-NH2, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXVII), (XXXIII), (XXXVI) and (XXXVII); each R'1 is independently selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Ci8)alkenyl, -(C2- Cis)alkynyl, -OAIkyl(Ci-C4), F, Cl, Br, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-Cis)alkyl, -OC(O)O(Ci-Cis)alkyl, -OC(O)NH2, -OC(O)N((Ci-Ci8)alkyl)2, -SH, -S(Ci-Ci8)alkyl, -S(O)H, -S(O)(Ci-Ci8)alkyl, -SO2(Ci-Ci8)alkyl;
R'1, Riii1, Rv1, Rv1 and Rvi1 are each independently selected from the group consisting of H, -(Ci-Ci8)alkyl, -(C2-Cis)alkenyl, -(Ci-Cis)alkylNH2, -(Ci-Cis)alkyl-N((Ci-Cis)alkyl)2, -(Ci-Cis)alkyl-NH(Ci-Cis)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein R1, Rii1, Riii1, Rv1, Rv1, and Rvi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -O(Ci-Cis)alkyl, -CF3, -OCF3, -NH2, -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, -(C2-Cis)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-Cis)alkyl-OH; wherein Rs is optionally substituted by at least one detection moiety, at least one active moiety, at least one lipid-like moiety Rw, or a mixture thereof.
[00327] Clause 4. The polyproline-based block copolymer of formula (I) according to any of clauses 1-
3, wherein each R8 is a radical independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, n-butyl, -CH2SCH3, -CH2CH2SCH3, -CH2CH2SCH2CH3, -CH2SCH2CH3, -CH2SH, -CH2CH2SH, -CH2SeH, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); wherein each Rs is independently substituted by at least one detection moiety, at least one active moiety, at least one lipid-like moiety Rw, or a mixture thereof; wherein each Rw is independently selected from the groups consisting of -(Ci-C )alky I, -(C2-C )alkeny I, and a radical of formula (XL), (XLI), (XLIII), (XLIV), (XLV), and (XLVI).
[00328] Clause 5. The polyproline-based block copolymer of formula (I) according to any of clauses 1 -
4, wherein
Li is a biradical selected from (II) and (III), which is attached to the C-terminal end AA1 , AA3 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond;
L4 is selected from the group consisting of an inert moiety, a detection moiety, an active moiety, and a lipid-like moiety Rw; wherein the inert moiety is selected from the group consisting of H, -N3, -C(O)H, -S(O)H, -NHCH(CH3)2, -(Ci-C4)alkylNH2, -(Ci-C4)alkylNHCH3, -(Ci-C4)alkylN(CH3)2, -O-(Ci-C4)alkyl-NH2,
-O-(Ci-C4)alkyl-NHCH3, -O-(Ci-C4)alkyl-N(CH3)2, -(Ci-Ci2)alkyl, -(C2-Ci2)alkenyl, and -(C2-C6)alkynyl; wherein the inert moiety is optionally substituted by a detection moiety, an active moiety, and a lipid-like moiety Rwor a mixture thereof; the detection moiety is selected from the group consisting of chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, transition metal and isotope mass tag moiety; the active moiety is selected from the group consisting of pharmaceutically active agents, veterinary active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof; and the lipid-like moiety Rw is selected from the group consisting of -(Ci-C )alkyl, -(C2-C )alkenyl, and a radical of formula (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII) or (XLIX).
[00329] Clause 6. The polyproline-based block copolymer of formula (I) according to any of clauses 1 -
5, wherein m is an integer selected from 5 to 80; n is an integer selected from 0 to 130; p is an integer selected from 0 to 100; q is an integer selected from 0 to 80; wherein at least one of n, p or q is • 6 or alternatively n + p • 6.
[00330] Clause 7. The polyproline-based block copolymer of formula (I) according to any of clauses 1 - 6, with the proviso that when p« 0 and R8 is not a cationic moiety, then n • 0; with the proviso that when n=0, then p« 0, and R8 is a cationic moiety; and with the proviso that when p= 0, then n • 0.
[00331] Clause 8. The polyproline-based block copolymer of formula (I) according to any of clauses 1 -
7, wherein p« 0, and R8 is a cationic moiety.
[00332] Clause 9. The polyproline-based block copolymer of formula (I) according to any of clauses 1 -
8, wherein p=0 and n • 0.
[00333] Clause 10. The polyproline-based block copolymer of formula (I) according to any of clauses 1-
9, wherein m is an integer selected from 5 to 80; p is an integer selected from 4 to 80; and each R8 is independently a cationic moiety selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); wherein each Rs are optionally independently substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety R10; or a linker; the linker being attached to at least one inert moiety, or alternatively optionally attached to at least one detection moiety, or alternatively optionally attached to at least one active moiety or at least one lipid-like moiety R10, or alternatively optionally attached to at least one pH, redox or protease responsive unit; or a mixture thereof.
[00334] Clause 11. The polyproline-based block copolymer of formula (I) according to any of clauses 1-
10, wherein m is an integer selected from 4 to 40; preferably from 6 to 36; from 8 to 32; from 10 to 30, from 12 to 28; from 16 to 24; from 18 to 22; p is an integer selected from 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, and 20; n=0;
L4 is selected from the group consisting of an inert moiety, a detection moiety, an active moiety, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a lipid-like moiety R10; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; each R8 is independently a cationic moiety selected from the group consisting of -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); wherein each Rs are optionally independently substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety R10; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; wherein the inert moiety is selected from the group consisting of H, -N3, -C(O)H, -S(O)H, -NHCH(CHs)2, -(Ci-C4)alkylNH2, -(Ci-C4)alkylNHCH3, -(Ci-C4)alkylN(CH3)2, -O-(Ci-C4)alkyl-NH2, -O-(Ci-C4)alkyl-NHCH3, -O-(Ci-C4)alkyl-N(CH3)2, -(Ci-Ci2)alkyl, -(C2-Ci2)alkenyl, and -(C2-C6)alkynyl; wherein the inert moiety is optionally substituted by a detection moiety, an active moiety, and a lipid-like moiety Rwor a mixture thereof; the detection moiety is selected from the group consisting of a chromophore moiety, a fluorescent moiety, a phosphorescent moiety, a luminescent moiety, a light absorbing moiety, a radioactive moiety, a transition metal and an isotope mass tag moiety; the active moiety is selected from the group consisting of pharmaceutically active agents, veterinary active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof; and the lipid-like moiety R10 is selected from the group consisting of - (Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, and a radical of formula (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII) or (XLIX).
[00335] Clause 12. The polyproline-based block copolymer of formula (I) according to any of clauses 1- 11 , which is selected from:
(1a) Ppro22POrn6;
(1c) Ppro30POrn16;
(lf) Ppro12POrn6;
(lg) pPro7POrn6;
(lh) Ppro54POrn18;
(li) Ppro64POrn22; (2a) Ppro19POrn6;
(1b) Ppro52POrn10;
(ld) pPro5POrn35;
(le) Ppro15POrn35;
(5c) pPro5PLys56;
(7a) PPro10-(Plys7-PArg24-PVal10); and
(16) Ppro22POrn6ValCitrulinePGA-PEG-Azide
[00336] Clause 13. A star-shaped polymer comprising a star-shaped multifunctional linking agent and at least one polyproline-based block copolymer of formula (I) according to any of clauses 1 -6 attached thereto; and wherein the star-shaped multifunctional linking agent is selected from the group consisting of
I) a 3-arm star-shaped linking agent of formula (St)
Figure imgf000118_0001
wherein the “*” denoted the attaching point to a polymer comprising a backbone of repeating structural units of formula (I); wherein p, ' and p” are an integer from 0 to 1;
L is selected from K1-CH2CH2-S-S-CH2CH2-K2; K1-CH2CH2-K2; and a moiety of formula (St_a)
Figure imgf000119_0001
wherein K, Ki and K2 are each independently selected from -0- and -NH-; p is an integer selected from 2-4; wherein in the moiety of formula (St_a) each wavy line in (St_a) denotes the attaching points to the polymer comprising the backbone of repeating structural units of formula (I); when L is (St_a), then (St_a) attaches to the CO moiety in (St) through the K moiety; when L is selected from K1-CH2CH2-S-S-CH2CH2-K2 or K1-CH2CH2-K2, then L attaches to the CO moiety in (St) through the K1 or K2 moieties, and the other K2 or K1 attaches to the polymer comprising the backbone of repeating structural units of formula (I); wherein the 3-arm star-shaped linking agent of formula (St) is attached to the polymer comprising the backbone of repeating structural units of formula (I) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond; i) a multi-arm star-shaped linking agent based on branched polyethylenimine; ii) a 3-arm star-shaped linking agent based on branched polyamidoamine of formula (Pm)
Figure imgf000119_0002
wherein each U is selected from -0- and -NH-;
E and E’ are each independently selected from -0- and -NH-; wherein the 3-arm star-shaped linking agent of formula (Pm) is attached to the L4 moiety of each of the at least one polyproline-based block copolymer of formula (I) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond; iii) a 4-arm star-shaped linking agent of formula (Tz)
Figure imgf000120_0001
wherein each V is selected from -0- and -NH-;
Y, Y' and Y” are each independently selected from -0- and -NH-; wherein the 4-arm star-shaped linking agent of formula (Tz) is attached to the L4 moiety of each of the at least one polyproline-based block copolymer of formula (I) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond.
[00337] Clause 14. The star-shaped polymer according to clause 13, wherein the star-shaped multifunctional linking agent is a 3-arm star-shaped linking agent of formula (St), and the star-shaped polymer is of formula (StP):
Figure imgf000120_0002
wherein (Pro), (Pro') and (Pro”) are each independently selected from a polyproline-based block copolymer of formula (I) as described herein;
L is selected from K1-CH2CH2-S-S-CH2CH2-K2 and K1-CH2CH2-K2; wherein p, ' and p” are each independently an integer from 0 to 1; wherein Ki and K2 are each independently selected from -0- and -NH-; wherein L attaches to the CO moiety in (StP) through the K1 or K2 moieties, and the other K2 or K1 attaches to the polymer comprising the backbone of repeating structural units of formula (I); wherein the 3-arm star-shaped linking agent of formula (StP) is attached to the polymer comprising the backbone of repeating structural units of formula (I) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond.
[00338] Clause 15. A self-assembled particle comprising the polyproline-based block copolymer of formula (I) as defined in any of clauses 1-12, or the star-shaped polypeptide as defined in any of clauses 13-14, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
[00339] Clause 16. A composition comprising the polyproline-based block copolymer of formula (I) according to any of clauses 1-12, the star-shaped polymer as defined in any of clauses 13-14, or alternatively, the self-assembled particle of clause 15, together with one or more appropriate excipients or carriers.
[00340] Clause 17. A therapeutic product which comprises: a) the polyproline-based block copolymer of formula (I) as defined in any of the clauses 1-12, wherein at least one of L4 or R8 is a pharmaceutically active agent; or alternatively b) the star-shaped polymer as defined in any clauses 13-14, wherein at least one of L4 or R8 is a pharmaceutically active agent; or alternatively c) a self-assembled particle as defined in clause 15, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively d) a composition as defined in clause 16, comprising a pharmaceutically active agent; for use in medicine.
[00341] Clause 18. The therapeutic product for use according to clause 17, for use (i) as transfection reagent for transfecting at least one active agent into a cell; (ii) for use in the in vivo or ex vivo production of biologies encoding a recombinant protein, a peptide or an antibody, or in the production of recombinant virus; (iii) for use as a therapeutic or prophylactic vaccine against viral infections or as a therapeutic vaccine against cancers or infectious diseases; or (v) for use in genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
[00342] Clause 19. A diagnostic product which comprises: a') the polyproline-based block copolymer of formula (I) as defined in any of the clauses 1-12, wherein at least one of L4 or R8 is a diagnostically active agent; or alternatively b') the star-shaped polymer as defined in any of clauses 13-14, wherein at least one of L4 or R8 is a diagnostically active agent; or alternatively c') a self-assembled particle as defined in clause 15, which comprises diagnostically active agent, or alternatively d') a composition as defined in clause 16, comprising a diagnostically active agent; for use in diagnostics.
[00343] Clause 20. Use in cosmetics of a cosmetic product which comprises: a") the polyproline-based block copolymer of formula (I) as defined in any of the clauses 1-12, wherein at least one of L4 or R8 is a cosmetically active agent; or alternatively b”) the star-shaped polymer as defined in any of clauses 13-14, wherein at least one of L4 or R8 is a cosmetically active agent; or alternatively c”) the self-assembled particle as defined in clause 15, which comprises a cosmetically active agent, or alternatively d”) a composition as defined in clause 16 containing a cosmetically active agent.
[00344] Clause 21 . Use of the polyproline-based block copolymer of formula (I) as defined in any of the clauses 1-12, wherein L4 and R8 are other than an active moiety; the starshaped polymer as defined in any of clauses 13-14, wherein L4 and R8 are other than an active moiety; or alternatively, a self-assembled particle as defined in clause 15, or a composition as defined in clause 16 containing either a polyproline-based block copolymer of formula (I) or the star-shaped polymer wherein L4 and R8 are other than an active moiety, as a carrier.

Claims

1. A polyproline-based block copolymer compound of formula (I), a salt thereof, or any stereoisomer or mixtures of stereoisomers, either of the compound of formula (I), or of any of its salts,
Figure imgf000123_0001
wherein the “*” denotes the attaching points; where the repetitive unit defined by square brackets with the numerical value m is denoted as PAA1;
Figure imgf000123_0002
wherein though the main repeating units AA1, AA2 and AA3 are shown in a particular order for convenience of description, said AA1, AA2 and AA3 main repeating units may be present in any order and may be block or randomly present; and wherein each of the repeating units PAA1, AA1, AA2 and AA3, may comprise blocks of monomer units which may be the same or different between each other; wherein m is an integer selected from 4 to 100; n is an integer selected from 0 to 150; p is an integer selected from 0 to 150; q is an integer selected from 0 to 100; wherein at least one of n, p or q is • 4 or alternatively n + p • 4; with the proviso that when p« 0 and R8 is not a cationic moiety, then n • 0; with the proviso that when n=0, then p« 0, and R8 is a cationic moiety; with the proviso that when p= 0, then n • 0; wherein Li may be present or absent;
Li, if present, is a biradical selected from the group consisting of
Figure imgf000124_0001
wherein the wavy lines denote the attaching points; wherein each A and A' is independently selected from -O-, -CO- and -NH-; wherein y and z are integers independently ranging from 1 to 20; and wherein each Z is a biradical selected from the group consisting of -NH(Ci-C6)alkyl-O-,
Figure imgf000124_0002
Figure imgf000125_0001
wherein the “*” denotes the attaching points; wherein B and B’ are each a biradical independently selected from -O-, -NH-; and -CO-; wherein each D is a biradical selected from -0- and -NH-; wherein k is an integer selected from 1 to 12; preferably from 1 to 6; wherein the -NH-(Ci-C3o)alkylene-0-, -NH-(Ci-C3o)alkylene-NH-, -NH-(Ci-C3o)alkylene-CO-, -0-(Ci-C3o)alkylene-0-, -0-(Ci-C3o)alkylene-CO-, -CO-(Ci-C3o)alkylene-CO- biradical of Z is optionally substituted with one or more radical selected from the group consisting of -OH, -NRaRb,-SH2, -NHNH2, -COORc, -CF3, -OCF3, and halogen; wherein Ra, Rb and Rc are each a radical independently selected from the group consisting of H, -phenyl, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkylphenyl, and -phenyl(Ci-C3o)alkyl; wherein L1 is attached to the AA1, AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond when attached to the N-terminal end of AA1, AA2 or AA3 repeating unit; or alternatively the biradical (II) or (III) is attached to the AA1, AA3 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond when is attached to the C-terminal end of the AA1, AA2 or AA3 repeating unit; wherein R2 and R4, are each a biradical independently selected from the group consisting of
-(Ci-C6)alkyl-, -(Ci-C6)alkyl-S-S-(Ci-C6)alkyl-, -(Ci-C6)alkyl-O-(Ci-C6)alkyl- and
-(Ci-C6)alkyl-NH-(Ci-C6)alkyl-; each R2 and R4 are independently optionally substituted by one or more substituents selected from the group consisting of -NH_ and -(Ci-C6)alkyl-NH2; each R3 is a radical independently selected from the group consisting of -H and -(Ci-Ce)alky I; each W1 is independently selected from CH and N;
Rs and Re are each independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C30)alkenyl, -(C2-C30)alkynyl, -(Ci-C3o)alkyl-R2, -(Ci-C3o)alkyi-0-R"2, -(Ci-C3o)alkyl-NRiii2Riv2, -C(O)-Rv2, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII),
Figure imgf000126_0001
Figure imgf000127_0001
wherein denotes the attaching point;
X2 in formula XVI is selected from -NH-, -COO-, and -O-; each R'2 is selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -OAIkyl(Ci-C6), halogen, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-C30)alkyl, -OC(O)O(Ci-C30)alkyl, -OC(O)NH2, -OC(0)N((Ci-C3o)alkyl)2, -SH, -S(Ci-C30)alkyl, -S(O)H, -S(O)(Ci-C30)alkyl, -S02(Ci-C3o)alkyl;
R'2, R"2, Rv2 and Rv2 are each independently selected from the group consisting of H, -(Ci-C3o)alky I, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl NH2, -(Ci-C3o)alkyl-N((Ci-C3o)alkyl)2, -(Ci-C3o)alkyl-NH(Ci-C3o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI)
Rv'2 and Rv'2’ are each independently selected from the group consisting of H, -(Ci-C3o)alky I, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -OAIkyl(Ci-Ci2), F, Cl, Br, I, -CF3, -OCF3, -NO2, -CN, -NH2, -(Ci-C3o)alkylNH2, -N((Ci-C30)alkyl)2, and-NH(Ci-C30)alkyl, wherein Rvi'2, R™2, R'x2, and Rx2are each independently selected from the group consisting of H, -(Ci-Ci2)alkyl, -(Ci-Ci2)alkylNH2, -(Ci-Ci2)alkyl-N((Ci-Ci2)alkyl)2, -(Ci-Ci2)alkyl-NH(Ci-Ci2)alkyl, -O(Ci-Ci2)alkyl, -COH, -CO(Ci-Ci2)alkyl, and -C(C2-C3o)alkenyl, wherein R'2, R'2, R'"2, Riv2, Rv2, Rv'2, Rv'2’, Rvi'2, R™2, R'x2, and Rx2 are each optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -0(Ci-C3o)alkyl, -CF3, -OCF3, -NH2, -(Ci-C3o)alkyl, -(C2-C30)alkenyl, -(C2-C30)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)- and -(Ci-C3o)alkyl-OH; b2, c2, d2, e2, f2, g2, h2, 12, j2, k2, b2', c2', d2', e2', and g2' are each independently an integer selected from 1 to 6; r2, s2, t2 and t2' are each independently an integer selected from 0 to 200; each Rz is independently selected from H and -CH3; each R9 is independently selected from H and -CH3; each Rs and L4 are independently selected from the group consisting of -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl-R1, -(Ci-C3o)alkyl-COORi1, -(Ci-C3o)alkyl-0-Riii1, -(Ci-C3o)alkyl-NRiv1Rv1, -C(O)-Rvi1, -(Ci-Ci8)alkyl-CO-NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety R10, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI) and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety R10, or alternatively attached to at least one pH, redox or protease responsive unit, or a mixture thereof;
Figure imgf000129_0001
wherein “*” denotes the attaching point;
12, v2, w2, x2, y2, z2 and o2 are each independently an integer selected from 1 to 6; preferably selected from 1 , 2, 3, 4, 5 or 6 each R'1 is independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(C2- C3o)alkynyl, -OAIkyl(Ci-C6), halogen, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-C30)alkyl, -OC(O)O(Ci-C30)alkyl, -OC(O)NH2, -OC(0)N((Ci-C3o)alkyl)2, -SH, -S(Ci-C30)alkyl, -S(O)H, -S(O)(Ci-C30)alkyl, -SO2(Ci-C30)alkyl;
Rii1, Riii1, R'v1, Rv1 and Rvi1 are each independently selected from the group consisting of H, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkyl NH2, -(Ci-C3o)alkyl-N((Ci-C3o)alkyl)2, -(Ci-C3o)alkyl-NH(Ci-C3o)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein Ri1, Rii1, Riii1, R'v1, Rv1, and Rvi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -0(Ci-C3o)alkyl, -CF3, -OCF3, -NH2, -(Ci-C30)alkyl, -(C2-C3o)alkenyl, -(C2-C3o)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-C30)alkyl-OH; wherein each Rs and L4 are independently optionally substituted by at least one detection moiety, at least one active moiety, at least one lipid-like moiety Rw, or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; a1 is an integer selected from 0 to 1 ; with the proviso that R2 is absent when a1 =1 ; wherein L4 is attached to the nitrogen atom of the AA1 , AA2 or AA3 by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond; or alternatively L4 is attached to the carbonyl group of the AA1 , AA2 or AA3 by a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond; the inert moiety is selected from the group consisting of H, -N3, -NH2, -C(O)H, -S(O)H, -NHCH(CH3)2, -(Ci-C4)alkyl-NH2, -NH-(Ci-C4)alkyl, -(Ci-C4)alkyl-NHCH3, -(Ci-C4)alkyl-N(CH3)2, -(O-CH2-CH2)7r-(Ci-C4)alkyl, N3-(O-CH2-CH2)7r-(Ci-C4)alkyl, -O-(Ci-C4)alkyl-NH2, -O-(Ci-C4)alkyl-NHCH3, -O-(Ci-C4)alkyl-N(CH3)2, -(Ci-Ci2)alkyl, -(C2-Ci2)alkenyl, -(C2-C6)alkynyl, -Cy1 , -(Ci-Ci2)alkylene-Cy1 , -(C2-Ci2)alkenylene-Cy1, -(C2-Ci2)alkynylene-Cy1, -Cy2-(Ci-Ci2)alkyl, -Cy2-(C2-Ci2)alkenyl, and -Cy2-(C2-Ci2)alkynyl; being Cy 1 a known ring system independently selected from the group consisting of 5- to 7-membered saturated or partially unsaturated carbocyclic or heterocyclic ring; and 5- to 7-membered carbocyclic or heterocyclic aromatic ring; Cy2 a bivalent known ring system independently selected from the group consisting of 5- to 7-membered saturated or partially unsaturated carbocyclic or heterocyclic ring; and 5- to 7-membered carbocyclic or heterocyclic aromatic ring; and being each one optionally substituted by one or more groups consisting of OH, halogen, NH2, NHRd, NRdRe, -(Ci-C6)alkyl, NO2, N3, -(Ci-Ci2)alkyl, -O-(Ci-Ci2)alkyl, -CO-(Ci-Ci2)alkyl and -CO-O-(Ci-Ci2)alkyl; wherein TT is an integer selected from 1 , 2, 3, 4, 5 or 6; wherein Rd and Re are each independently selected from H- and -(Ci-C4)alkyl; wherein the inert moiety is optionally substituted by a detection moiety, an active moiety, a lipid-like moiety Rw, and a pH, redox or protease responsive unit, or a mixture thereof; the detection moiety is selected from the group consisting of chromophore moiety, fluorescent moiety, phosphorescent moiety, luminescent moiety, light absorbing moiety, radioactive moiety, transition metal and isotope mass tag moiety; the active moiety is selected from the group consisting of pharmaceutically active agents, veterinary active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof; the lipid-like moiety Rw is selected from the group consisting of -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, and a
Figure imgf000131_0001
Figure imgf000132_0001
wherein:
T and T are each independently selected from -OH, -OCORx and -COORx;
Q and Q' are each independently selected from -OCORy and -COORy; each G is independently selected from -OCO-, -COO-, -NRz'CO-, and -CONRz'-; each Rz' is H or Rz; each Rx, Ry, and Rz is independently -(Ci-Ci8)alkyl or -(C2-Ci8)alkenyl; each a1 is independently an integer from 0 to 18; preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15, 16, 17, or 18; each b1 is independently an integer from 1 to 18; preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14, 15, 16, 17, or 18; each d is independently an integer from 0 to 18; preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,
13,14, 15, 16, 17, or 18; each d1 is independently an integer from 0 to 18; preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,
13,14, 15, 16, 17, or 18; each e1 is independently an integer from 0 to 18; preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,
13,14, 15, 16, 17, or 18; each fl is 0 or 1; each f1' is 0 or 1 ;
T1 , T2, T3, TT, T2' and T3' are each independently selected from the group consisting of hydrogen, fluorine, methyl, -CH2F, -CHF2, and -CF3; each of the dashed bonds — is independently a single bond or, alternatively, a double bond; each J is a biradical chain which comprises one or more moieties selected from the group consisting of -CH=CH-, -C« C-, -CH2-, -N=CH-, -CH=NH-, -NH-, -NH-NH-, -NH-N=CH-, -O-, S-,
Figure imgf000133_0001
wherein Rf, Rg and Rh are each radicals independently selected from the group consisting of H, -phenyl, -(Ci-C3o)alkyl, -(C2-C3o)alkenyl, -(Ci-C3o)alkylphenyl, and -phenyl(Ci-C3o)alkyl; wherein when J comprises a -CH2- moiety, the two hydrogen atoms attached to the carbon atom are optionally replaced by the ring:
Figure imgf000133_0002
, , ,
Figure imgf000134_0001
wherein R2, R3, R4, R5, Re, and W1 are as defined above.
2. The polyproline-based block copolymer of formula (I) compound according to claim 1 , wherein each Rs and L4 are independently selected from selected from the group consisting of -(Ci-Cis)alkyl, -(C2-Ci8)alkenyl, -(Ci-Ci8)alkyl-R1, -(Ci-Ci8)alkyl-COORi1, -(Ci-Ci8)alkyl-O-R"1, -(Ci-Ci8)alkyl-NRiv1Rv1, -C(O)-Rvi1, -(Ci-Ci8)alkyl-CO-NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety Rw, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXVII), (XXXIII), (XXXVI) and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; each R'1 is independently selected from the group consisting of H, -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, -(C2- Ci8)alkynyl, -OAIkyl(Ci-C4), F, Cl, Br, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-Ci8)alkyl, -OC(O)O(Ci-Ci8)alkyl, -OC(O)NH2, -OC(O)N((Ci-Ci8)alkyl)2, -SH, -S(Ci-Ci8)alkyl, -S(O)H, -S(O)(Ci-Ci8)alkyl, -SO2(Ci-Ci8)alkyl;
R'1, Riii1, Rv1, Rv1 and Rvi1 are each independently selected from the group consisting of H, -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, -(Ci-Ci8)alkylNH2, -(Ci-Ci8)alkyl-N((Ci-Ci8)alkyl)2, -(Ci-Ci8)alkyl-NH(Ci-Ci8)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein R1, Rii1, Riii1, Rv1, Rv1, and Rvi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -O(Ci-Ci8)alkyl, -CF3, -OCF3, -NH2, -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, -(C2-Ci8)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)2 and -(Ci-Ci8)alkyl-OH; wherein each Rs and L4 are independently optionally substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety Rw; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit, or a mixture thereof; or a mixture thereof.
3. The polyproline-based block copolymer of formula (I) compound according to any of claims 1-2, wherein each Rs and L4 are independently selected from the group consisting of -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, -(Ci-C6)alkyl-R1, -(Ci-C6)alkyl-COORi1, -(Ci-C6)alkyl-O-R"1, -(Ci-C6)alkyl-NRiv1Rv1, -C(O)-Rvi1, -(Ci-C6)alkyl-CO-NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety Rw, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXVII), (XXXIII), (XXXVI) and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; each R'1 is independently selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, -(C2- Cis)alkynyl, -OAIkyl(Ci-C4), F, Cl, Br, -CF3, -OCF3, isoxazole, oxazole, furan, oxolane, thiole, thiophene, N-methylpyrrole, pyrrole, pyrrolidine, pyrane, pyridine, piperidine, thiazole, dioxane, morpholine, pyrimidine, -NO2, -CN, -OC(O)-(Ci-Ci8)alkyl, -OC(O)O(Ci-Ci8)alkyl, -OC(O)NH2, -OC(O)N((Ci-Ci8)alkyl)2, -SH, -S(Ci-Ci8)alkyl, -S(O)H, -S(O)(Ci-Ci8)alkyl, -SO2(Ci-Cis)alkyl;
R'1, Riii1, R'v1, Rv1 and Rvi1 are each independently selected from the group consisting of H, -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, -(Ci-Cis)alkylNH2, -(Ci-Cis)alkyl-N((Ci-Cis)alkyl)2, -(Ci-Cis)alkyl-NH(Ci-Cis)alkyl, and a radical selected from the group consisting of (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXVI); wherein R'1, Rii1, Riii1, R'v1, Rv1, and Rvi1 are each independently optionally substituted by one or more substituents selected from the group consisting of H, OH, halogen, -O(Ci-Cis)alkyl, -CF3, -OCF3, -NH2, -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, -(C2-Cis)alkynyl, -SH, -NHNH2, -NHCH3, -N(CH3)2, -NCH(CH3)- and -(Ci-Ci8)alkyl-OH; wherein each Rs and L4 are independently optionally substituted by at least one detection moiety; at least one active moiety; at least one lipid-like moiety Rw; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof.
4. The polyproline-based block copolymer of formula (I) compound according to any of claims 1-3, wherein each R8 and L4 are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, n-butyl, -CH2SCH3, -CH2CH2SCH3, -CH2CH2SCH2CH3, -CH2SCH2CH3, -CH2SH, -CH2CH2SH, -CH2SeH, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety Rw, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, and a radical selected from the group consisting of (XIII), (XVII), (XXII), (XXV), (XXVII), and (XXXVII); the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; wherein each Rs and L4 are independently optionally substituted by at least one inert moiety, at least one detection moiety; at least one active moiety; at least one lipid-like moiety Rw; or a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof; wherein each Rw is independently selected from the groups consisting of -(Ci-Cis)alkyl, -(C2-Cis)alkenyl, and a radical of formula (XL), (XLI), (XLIII), (XLIV), (XLV), and (XLVI).
5. The polyproline-based block copolymer of formula (I) compound according to any of claims 1-4, wherein
Li is a biradical selected from (II) and (III), which is attached to the C-terminal end of AA1 , AA2 or AA3 repeating unit through a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond;
L4 is selected from the group consisting of an inert moiety, a detection moiety, an active moiety, a pH responsive unit, a redox responsive unit, a protease responsive unit, a linker, a lipid-like moiety Rw; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit; or a mixture thereof wherein the inert moiety is selected from the group consisting of H, -N3, -C(O)H, -S(O)H, -NHCH(CH3)2, -(Ci-C4)alkylNH2, -(Ci-C4)alkylNHCH3, -(Ci-C4)alkylN(CH3)2, -O-(Ci-C4)alkyl-NH2, -O-(Ci-C4)alkyl-NHCH3, -O-(Ci-C4)alkyl-N(CH3)2, -(Ci-Ci2)alkyl, -(C2-Ci2)alkenyl, and -(C2-C6)alkynyl; wherein the inert moiety is optionally substituted by a detection moiety, an active moiety, and a lipid-like moiety Rwor a mixture thereof; the detection moiety is selected from the group consisting of a chromophore moiety, a fluorescent moiety, a phosphorescent moiety, a luminescent moiety, a light absorbing moiety, a radioactive moiety, a transition metal and an isotope mass tag moiety; the active moiety is selected from the group consisting of pharmaceutically active agents, veterinary active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof; and the lipid-like moiety Rw is selected from the group consisting of - (Ci-C jalkyl, -(C2-Ci8)alkenyl, and a radical of formula (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII) or (XLIX).
6. The polyproline-based block copolymer of formula (I) compound according to any of claims 1-5, wherein m is an integer selected from 6 to 80; p is an integer selected from 4 to 50; q is an integer selected from 0 to 80 n is an integer selected from 0 to 140 at least one of n, p or q is • 6 or alternatively n + p • 6; and m>p; with the proviso that when p« 0, and R8 is not a cationic moiety, then n • 0; with the proviso that when n=0, then p« 0; and R8 is a cationic moiety; and with the proviso that when p=0, then n« 0.
7. The polyproline-based block copolymer of formula (I) compound according to any of claims 1-5, wherein m is an integer selected from 4 to 40; p is an integer selected from 4 to 80; q is an integer selected from 0 to 80; n is an integer selected from 0 to 140; at least one of n, p or q is • 6 or alternatively n + p • 6; and p>m; with the proviso that when p« 0, and R8 is not a cationic moiety, then n • 0; with the proviso that when n=0, then p« 0; and R8 is a cationic moiety; and with the proviso that when p=0, then n« 0.
8. The polyproline-based block copolymer of formula (I) compound according to any of claims 1-5, wherein m is an integer selected from 4 to 40; n is an integer selected from 20 to 140; q is an integer selected from 0 to 80; p is an integer selected from 0 to 80; at least one of n, p or q is • 6 or alternatively n + p • 6; and n>m; with the proviso that when p« 0, and R8 is not a cationic moiety, then n • 0; with the proviso that when n=0, then p« 0; and R8 is a cationic moiety; and with the proviso that when p=0, then n« 0.
9. A compound comprising a structural unit of formula (I') or a structural unit of formula (I”),
A salt thereof formed from acceptable non-toxic acids, or any stereoisomer or mixtures of stereoisomers of the compound of formula (I'), of the compound of formula (I”), or of any of its salts,
Figure imgf000138_0001
Figure imgf000139_0001
wherein m, p, R1 , R9, L1 , L4, AA1 , AA2 and AA3 are as defined in any of claims 1 -8; wherein the “*” denotes the attaching point; wherein each R8' and L4' are independently selected from a radical derived from the group consisting of -(Ci-C3o)alkyl-R1, -(Ci-C3o)alkyl-COO i1, -(Ci-C3o)alkyl-0-R"1, -(Ci-C3o)alkyl-NRv1Rv1, -C(O)-Rvi1, an inert moiety, a detection moiety, an active moiety, a lipid-like moiety Rw, a pH responsive unit, a redox responsive unit, a protease responsive unit, (XXVII), (XXIX), (XXX), (XXXIII), and (XXXIV); wherein R1, Ri1, Riii1, Rv1, Rv1, Rvi1, (XXVII), (XXIX), (XXX), (XXXIII), and (XXXIV) are as defined above; wherein each R8' may be optionally substituted by a linker; the linker being attached to at least one inert moiety, or alternatively attached to at least one detection moiety, or alternatively attached to at least one active moiety, or alternatively attached to at least one lipid-like moiety Rw, or alternatively attached to at least one pH, redox or protease responsive unit, or a mixture thereof; with the proviso that the structural unit of formula (I') or (I”) is not attached to a star-shaped multifunctional linking agent.
10. A star-shaped polymer comprising a star-shaped multifunctional linking agent and at least one compound comprising the structural unit of formula (I') or (I”) as defined in claim 9 attached thereto; and wherein the star-shaped multifunctional linking agent is selected from the group consisting of
I) a multi-arm star-shaped linking agent based on branched polyethylenimine;
II) a 3-arm star-shaped linking agent based on branched polyamidoamine of formula (Pm)
Figure imgf000140_0001
wherein each U is selected from -0- and -NH-;
E and E’ are each independently selected from -0- and -NH-; wherein the 3-arm star-shaped linking agent of formula (Pm) is attached to the compounds comprising the structural unit of formula (I') or (I”) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond through the U, E and E' moieties; wherein the chemically feasible-bond is selected from amine, carbamate and urea when is attached through U, and the chemically feasible-bond is selected from ester and amide when is attached through E, E' or U; ill) a 4-arm star-shaped linking agent of formula (Tz)
Figure imgf000140_0002
wherein each V is selected from -0- and -NH-;
Y, Y' and Y” are each independently selected from -0- and -NH-; wherein the 4-arm star-shaped linking agent of formula (Tz) is attached to the compounds comprising the structural unit of formula (I') or (I”) by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, an amine bond, and an ester bond, through the V, Y, Y' and Y” moieties; wherein the chemically feasible-bond is selected from amide, carbamate and urea when is attached through V, and the chemically feasible-bond is selected from amide and ester when is attached through Y, Y' or Y”.
11 . A self-assembled particle comprising the polyproline-based block copolymer compound of formula (I) as defined in any of claims 1-8, the compound comprising the structural unit of formula (I') or (I”) as defined in claim 9, or the star-shaped polymer as defined in claim 10, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
12. The self-assembled particle according to claim 11, wherein in the compound of formula (I), the compound comprising the structural unit of formula (I') or in the compound comprising the structural unit of formula (I”), at least one R8 or L4 are a lipid-like moiety R10 or alternatively are substituted by a lipid- like moiety R10.
13. The self-assembled particle according to any of the claims 11-12, which is a lipid nanoparticle.
14. The self-assembled particle according to any of the claims 11-13, which further comprises one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids.
15. The self-assembled particle according to any of the claims 11-14, which further comprises a ionizable lipid or a cationic lipid, a phospholipid, and a sterol.
16. The self-assembled particle according to claim 13, which comprises: i) the polyproline-based block copolymer of formula (I) as defined in any of the claims 1 to 8, or alternatively the compound comprising the structural unit of formula (I') or (I”) as defined in claim 9, in an amount from 0.1 mol% to 60 mol%; ii) an ionizable or a cationic lipid in an amount from 30 mol% to 70 mol%; iii) a phospholipid in an amount from 1 mol% to 20 mol%; and iv) a sterol in an amount from 20 mol% to 60 mol%; wherein the percentages are expressed with respect to the sum of the mol% of the lipids and the polyproline-based block copolymer of formula (I) or the compound comprising the structural unit of formula (I’) or (I”).
17. The self-assembled particle according to claim 11, which is a polymer nanoparticle.
18. A composition comprising the polyproline-based block copolymer compound of formula (I) according to any of claims 1- 8, the compound comprising the structural unit of formula (I') or (I”) as defined in claim 9, the star-shaped polymer as defined in claim 10, or alternatively, the self-assembled particle of claim 11, together with one or more appropriate excipients or carriers.
19. A therapeutic product which comprises: a) the polyproline-based block copolymer compound of formula (I) as defined in any of the claims 1- 8, wherein at least one of L4 or R8 comprises a pharmaceutically active agent; or alternatively b) the compound comprising the structural units of formula (I') or (I”) as defined in claim 9, wherein R8 comprises a pharmaceutically active agent; or alternatively c) the star-shaped polymer as defined in claim 10 wherein R8 comprises a pharmaceutically active agent; or alternatively d) a self-assembled particle as defined in claim 11, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively e) a composition as defined in claim 18 containing the self-assembled particle d), for use in medicine.
20. The therapeutic product according to claim 18, for use (I) as transfection reagent for transfecting at least one active agent into a cell; (II) for use in the in vivo or ex vivo production of biologies encoding a recombinant protein, a peptide or an antibody, or in the production of recombinant virus; (ill) for use as a therapeutic or prophylactic vaccine against viral infections or as a therapeutic vaccine against cancers or infectious diseases; or (v) for use in genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
21 . A diagnostic product which comprises: a') the polyproline-based block copolymer compound of formula (I) as defined in any of the claims 1- 8, wherein at least one of L4 or R8 comprises a diagnostically active agent; or alternatively b') the compound comprising the structural unit of formula (I') or (I”) as defined in claim 9, wherein R8 comprises a diagnostically active agent; or alternatively c') the star-shaped polymer as defined in claim 10, wherein R8 comprises a diagnostically active agent; or alternatively d') a self-assembled particle as defined in claim 11, which comprises diagnostically active agent, or alternatively e') a composition as defined in claim 18 containing the self-assembled particle d'); for use in diagnostics.
22. Use in cosmetics of a cosmetic product which comprises: a") the polyproline-based block copolymer compound of formula (I) as defined in any of the claims1-8, wherein at least one of L4 or R8 comprises a cosmetically active agent; or alternatively b”) the compound comprising the structural unit of formula (I') or (I”) as defined in claim 9, wherein R8 comprises a cosmetically active agent; or alternatively c”) the star-shaped polymer as defined in claim 10, wherein R8 comprises a cosmetically active agent; or alternatively d”) the self-assembled particle as defined in claim 11, which comprises a cosmetically active agent, or alternatively e”) a composition as defined in claim 18 containing the self-assembled particle d”).
PCT/EP2024/068352 2023-06-29 2024-06-28 Polyproline-based block copolymers Pending WO2025003463A1 (en)

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