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WO2015011675A1 - Complexes nano-assemblés d'acides nucléiques, d'avidine et de composés biotinylés utilisables en tant que supports d'administration intracellulaire - Google Patents

Complexes nano-assemblés d'acides nucléiques, d'avidine et de composés biotinylés utilisables en tant que supports d'administration intracellulaire Download PDF

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WO2015011675A1
WO2015011675A1 PCT/IB2014/063408 IB2014063408W WO2015011675A1 WO 2015011675 A1 WO2015011675 A1 WO 2015011675A1 IB 2014063408 W IB2014063408 W IB 2014063408W WO 2015011675 A1 WO2015011675 A1 WO 2015011675A1
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comprised
avidin
complexes according
biotin
nanoassembled complexes
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Margherita Morpurgo
Elisabetta CASARIN
Paolo BIGINI
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ANANAS Nanotech Srl
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    • 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
    • A61K47/6921—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 particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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

Definitions

  • the invention relates to the use of nanoassembled complexes as carriers for intracellular delivery of molecules, equal or different from one another, selected from molecules for diagnostic use and bioactive molecules for therapeutic use, such as for example chromophores or fluorophores, radiotracers, drugs, antibodies, peptides, proteins, enzymes, single or double-chained oligonucleotides and their analogues (PNA, LNA).
  • molecules for diagnostic use and bioactive molecules for therapeutic use such as for example chromophores or fluorophores, radiotracers, drugs, antibodies, peptides, proteins, enzymes, single or double-chained oligonucleotides and their analogues (PNA, LNA).
  • the objective pursued with the most recent research is to develop multi-functional transport nanosystems capable of transporting molecules having different functions (i.e. bioactive and/or tracer molecules) specifically to the desired anatomical site in a more efficient manner.
  • the technical problem relates to the possibility of charging onto the nanoparticles the various compounds to be carried, and of controlling their individual charge in a reproducible and convenient manner as well.
  • nanocomplexes based on the high-affinity interaction between avidin and biotin.
  • avidin's property of having an elevated and multiple affinity for biotin represents the basis for its use as a molecular instrument in a very large number of biotechnological applications. Owing to this property, avidin is in fact able to function as a molecular bridge for stably combining various biological and chemical units together with the proviso that they are covalently bound to a biotin molecule (Wilchek M and Bayer EA, 1988; Wilchek M and Bayer EA, 1990).
  • avidin-biotin technology fall within the scope of analysis, more precisely in detection and quantification systems usually based on the possibility of combining an antibody, or any other molecule having a high affinity to the analyte (ligand/antigen), with a signal system (a fluorophore, an enzyme capable of emitting light/colour, a radionuclide etc).
  • Other applications include the functionalisation of surfaces with specific chemical/biochemical entities, a procedure that is frequently performed using the molecular bridge consisting of the avidin-biotin complex; another application consists in the directing of parenterally administered drugs or diagnostic elements to specific sites of the body (Goldenberg DM et al., 2006).
  • biotin technology consists in the maximum number, which is equal to four, of biotins that may be combined with an individual molecule of avidin, which constitutes the central "nucleus" of the system, being avidin as known a tetrameric protein.
  • the possibility of having a central nucleus capable of binding a greater number of biotin molecules to itself should theoretically enable the potentiality of the system to be increased.
  • This increased capacity can be achieved by joining together a plurality of avidin molecules into a single unit definable as a poly-avidin unit.
  • the substantial impulse towards the development of a poly-avidin-type technology derives from the discovery of the ability of avidin to also bind to nucleic acids with high-affinity interactions with the nucleobases thereof (Morpurgo M, et al. 2004). This has contributed to the development of nanocomplexes formed of nucleic acids/avidin/biotin self-assembling themselves around a central nucleus consisting of a nucleic acid and a plurality of avidin units in a stoichiometric ratio between avidin and the nucleic acid base pairs of 1 :18 ⁇ 4. The avidin assembled on the nucleic acid can in turn bind biotin.
  • biotinylated compounds that is, compounds derived from covalent conjugation with biotin according to the previously cited known avidin : biotin technology.
  • nanoparticles derived from the double interaction of nucleic acid: avidin and of avidin: biotin, wherein the biotin binds with a covalent bond a hydrophilic polymer capable of ensuring protection at the surface of these nanocomplexes have been developed.
  • NB nucleobase
  • Av avidin
  • B biotin
  • X linker
  • PA hydrophilic polymer
  • nanoparticles consisting of these nanocomplexes, which are soluble in an aqueous environment and stable, and which have a highly defined composition, being obtainable by means of high-affinity interactions on the basis of stoichiometric ratios between nucleobase: avidin : biotin.
  • nanocomplexes have been compared with analogue complexes based on the conventional avidin: biotin interaction in an in vitro analytical study based on immunoassay, and have been demonstrated to be much more efficient in detection of the analyte (Morpurgo M et al., 2012).
  • hydrophilic polymers on the surface casts doubt on this possible capability, because it is known that the biological barriers, such as cell membranes, are thereby more impermeable or barely permeable to hydrophilic structures and that this is the main reason for their poor absorption and inefficient intracellular transport.
  • the poor trans-membrane permeability due to elevated hydrophilicity greatly limits the intracellular transport of macro- molecules used as carriers for bioactive molecules, such as polymeric conjugates and nanoparticles.
  • bioactive molecules such as polymeric conjugates and nanoparticles.
  • nanoparticle systems it is generally accepted that cellular internalisation can occur only following endocytic processes, which, however, need to be triggered by an initial reaction of the nanoparticle with specific surface receptors (receptor-induced internalisation).
  • nanoassembled complexes comprising a nucleus obtained by means of a high-affinity interaction between one or more units of avidin and one or more molecules of a nucleic acid and wherein said nucleus is stabilised by a biotinylated surface-protecting agent represented by the general formula (I)
  • NB are the single nucleobases of a single or double-stranded nucleic acid selected from among any sequence of a single or double-stranded deoxyribonucleic acid (DNA), any sequence of a ribonucleic acid (RNA) in form of a single strand or hybridised with an RNA or complementary DNA chain, or a sequence of the two in which part of or all the bases have been chemically modified;
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • Av is a tetrameric avidin unit
  • B-Xg-PAfc is a biotinylated surface-protecting agent, wherein PA is a polymer unit having one or two functionalisable residues, one of which binds, by a covalent bond either directly or through an X linker, a biotin residue B by means of the carboxyl functional group thereof, selected from among polyethylene glycol (PEG), optionally substituted, and a polyethylene glycol and polyoxypropylene copolymer (PEG-PPO);
  • n is a number higher than 16 and up to 100,000;
  • y is an integer equal to or higher than 1 and being relative to n is comprised from (0.0001 ) ⁇ to (0.0357) ⁇ with the proviso that, if (0.0001 -0.0357) ⁇ is less than 1, y is equal to 1 ;
  • z is an integer equal to or higher than 1 and being related to y is comprised from (0.02)*y to (4) » y with the proviso that, if (0.02-4) » y is less than 1 , z is equal to 1 ;
  • - a is a number comprised from 0 to 5;
  • b is a number comprised from 1 to 9,
  • molecules for use as carriers in the intracellular delivery of molecules, equal or different one from the other, selected from molecules for diagnostic use and bioactive molecules for therapeutic use.
  • FIG. 1 The figure shows the overview of the biotinylated fluorescent compounds described in examples 1 and 2: (A) compound 2 of example 1
  • Figure 2 The figure shows the stability of the nanoassembly of example 3 plasmid DNA:Av:[B-C 6 -Lys-(PEG-fluorescein) 2 , B-C 6 -Lys-Alexa 633 ] following incubation under different conditions simulating the physiological environment.
  • FIG. 3 The figure shows the images by confocal microscopy that are representative of HeLa cells incubated with the nanocomplex of example 3 plasmid DNA :Av:[B-C 6 -Lys-(PEG-fluorescein) 2 , B-C 6 -Lys-Alexa 633 ] (30 pg/ml in a medium containing 10% foetal calf serum). Similar results have been obtained at lower concentrations (6 pg/ml).
  • the panels A - E show the superimposition of the images taken in the three channels (red for the Alexa 633 channel, green for the fluorescein signal, yellow for the two superimposed signals red and green, and blue for the nuclei coloured with the Hoechst 33258 chromophore).
  • the panel A shows a representative image of the control cells (treated only with medium).
  • the panels B, C and D show the cells after 2 hours (B), 6 hours (C) e 24 hours (D) of treatment with the nanocomplexes.
  • the panel E shows a size-enlarged image (2D) of the sample treated for 24 hours, and demonstrates how the nanoparticles localise spot-wise within the cytoplasm, close to the nuclei, and also as macroaggregates partly associated to the cell membrane.
  • panel F one can see a 3D reconstruction of an image obtained from the same sample at 24 h.
  • nanocomplexes are used to indicate the compounds obtained by double self-assembly between the nucleobases (NB) of an oligonucleotide sequence and the tetrameric protein avidin (Av), in what follows also referred to as an avidin unit, and between the avidin and biotin (B) of an agent covering the central nucleus obtained from the first NB:Av self-assembly.
  • NB nucleobases
  • Av tetrameric protein avidin
  • B biotin
  • avidin is intended to define both the native tetrameric protein originating from hens' eggs or from some other analogous source (birds' eggs in general), and the recombinant one, and in the glycosylated and the deglycosylated form. Also intended are other, chemically or genetically modified, forms of avidin, provided that they are capable of assembling on a single or double-stranded nucleic acid as previously defined.
  • dendrimer means a symmetrical macromolecular compound consisting of repeated ramifications around a central core consisting of a smaller molecule or a polymeric nucleus.
  • the functional groups present on the outside of the dendrimer, the number of which is dependent upon the number of its ramifications, are in turn functionalisable with other molecules including, for example, the PA polymers.
  • nucleic acid(s) is used to mean single or double-chain nucleic acids of DNA or RNA as defined previously, and which can occur in a linear or circular form, in a relaxed, coiled or supercoiled state.
  • the nanoassembly complexes of the general formula (I) NBnAvyiB-Xg-PAbJz can be used as such and can transport molecules, equal or different from one another, selected from molecules for diagnostic use and bioactive molecules for therapeutic use, bound by means of a covalent bond or equivalent bonds of hydrazonic type or a shift base to the polymer PA.
  • This feature is very important for diagnostic and therapeutic applications, in that it enables nanoparticles to be designed and prepared according to the target diagnostic and/or therapeutic requirements.
  • a first embodiment can be prepared ⁇ : ⁇ : ⁇ - ⁇ 3 - ⁇ ⁇ nanoparticles, where the polymer PA is not functionalised with molecules for diagnostic and/or therapeutic use, and the binding sites of biotin present on the avidin units are not saturated by the B-Xg-PAi, covering agent and, then, the free biotin binding sites bind biotinyiated compounds of molecules equal or different from one another for diagnostic and/or therapeutic use.
  • nanoparticles having a double label can be prepared by binding a tracer with a bond to the polymer of the protecting agent B- X a -PAi, and a second tracer to a further biotin different from that of the protecting agent.
  • This second biotinyiated tracer may be taken up again by processes of absorption favoured by the high affinity between avidin and biotin in the nanoassembly formed by ⁇ : ⁇ : ⁇ - ⁇ 9 - ⁇ ,.
  • the high affinity between the free avidin of the NB:Av central nucleus and the biotin of the biotinylated tracer enables its self-assembly thereon.
  • the nanoparticles can, instead, transport bioactive molecules, equal or different from one another, bound to the PA polymer of the B-X a -PA b protecting agent and directly to a further biotin.
  • the nanoparticles can be used as multiple and/or differentiated-release vectors of bioactive molecules bound respectively to the polymer of the protecting agent or directly to a biotin different from that of the cover agent B-X a -PA b .
  • the different localisation of the bioactive molecules bound to the polymer of the protecting agent B-X a -PA b or to a biotin different from that of B-X a -PAi, respect to the nanoparticle surface can produce a different susceptibility to degradation-type process with a differentiated release thereof.
  • nanoparticles which are able to act as carriers for bioactive molecules for therapeutic use that are bound to the polymer PA of the B-X a -PAt, protecting agent and molecules for diagnostic use, such as for example a tracer, bound to a biotin different from that of the B-X a - PA covering agent.
  • nanoparticles can be variously functionalised in order to direct them in a targeted manner towards specific organs and/or cells by functionalising either the polymer PA of the B-X a -PA b protecting agent or another biotin different from that of the B- Xa-PA b , protecting agent or both.
  • nanoparticles having predefined compositions are to be preferred, wherein the components NB,,, X a and PA b and y and z have the meanings given below.
  • NB n is a nucleic acid wherein n is the number of the base and is comprised between 3,000 and 50,000 and consequently y, which identifies the number of the avidin units Av, is comprised between 30 and 1785 and z, which identifies the number of the B-X a -PA 6 protecting agents, is comprised between 43 and 7140.
  • linker X which binds the biotin to the hydrophilic polymer PA, is bifunctional it is a spacer of the general formula (II) Y-R-Y', wherein:
  • Y, , equal or different from one another, are -COO-, -NH-, -0-; -S02-, -S-
  • -SO-, -CO-, -COS-; -NH-CO-, -NH-COO-, -HN-SO-NH-, -HN-N CH- ; - R can be an alkyl, an alkenyl, an alkynyl, a cycloalkyl and an aryl having a number of carbon atoms between 1 and 20, and preferably between 5 and
  • the bond between the linker X and the biotin B, and that between the linker X and the hydrophilic polymer PA can be, without distinction, an amide bond, an amine bond, a carbamide bond, an ester bond, a ketone bond, an ether bond, a thioesteric bond, a thioether bond, an urea bond, a thiourea, sulphonic, sulphoxide and hydrazonic bond.
  • the linker X is a spacer group wherein Y and Y' are -NH-CO- and -NH-COO- and R is a linear alkyl having 6 carbon atoms.
  • PA represents a hydrophilic polymer consisting of at least 2 or more polymeric units
  • the latter are combined with one another by a different linker X having a number of derivatisable functionalities equal to or higher than 3 (> 3), one of which binds a first spacer X or directly the biotin B by means of its own carboxyl group, when a is equal to 0, and the others bind the polymeric units PA.
  • This diverse linker X can be selected from lysine, glutamic acid, aspartic acid, cysteine and a dendrimer.
  • this further polyfunctional linker is preferably lysine.
  • polymers in which the polymeric unit has a molecular weight between 400 and 20,000 and more preferably between 1 ,000 e 5,000 are to be preferred.
  • the most preferable polymer unit is polyethylene glycol (PEG), optionally substituted, or a copolymer thereof having a molecular weight between 2,000 and 5,000 and with b comprised from 2 to 5.
  • R 1 , R 2 , R 3 and R 4 can be, independently of one another, residues equal to hydrogen, alkyl, cycloalkyl, aryl, alkenyl, alkynyl, alkoxy, thio alkoxy, aryloxy, and thio aryloxy,
  • m is a number comprised from 2 to 500.
  • various compounds can be carried within the cells both by derivatisation of the polymer PA of the biotinylated surface protecting agent B-X a -PA b and by derivatisation of a further biotin different from that of the protecting agent B-Xa-PA*, of the nanoassemblies.
  • biotinylated compounds different from the B-Xa-PAb protecting agent of the nanocomplexes, represented by the general formula of (I) NBnAvy(B-Xa-
  • PAb)z can be comprised therein in consequence of the high-affinity interactions between the biotin and avidin of the central nucleus NB:Av.
  • - B is biotin
  • - X is a bifunctional or polyfunctional linker as previously defined
  • - A is a compound selected from molecules for diagnostic use and bioactive molecules for therapeutic use, and
  • a is an integer comprised from 0 to 5 and b is an integer comprised from 1 to 9.
  • compounds for diagnostic use can be carried, such as chromophores or fluorophores, radiotracers and chelating agents therefor, and bioactive molecules such as drugs, antibodies, peptides, proteins, enzymes, sugars, single or double-chained oligonucleotides and their analogues (PNA, LNA).
  • bioactive molecules such as drugs, antibodies, peptides, proteins, enzymes, sugars, single or double-chained oligonucleotides and their analogues (PNA, LNA).
  • the derivative biotinamidohexylamido-L-Lys (HCI) 2 was synthesised on the basis of the description in the literature (Pignatto M et al. 2010, ref. cit.). The compound was then dissolved in anhydrous dimethylformamide (DMF) and 2 equivalents of a-N-hydroxisuccinimidyl carboxy.co-Fmoc-amino-PEGsKDa (RAPP Polymere, Germany), followed by 4 equivalents of triethylamine (TEA), were added. After 1 hour, the quantitative modification of the primary amines was confirmed by ninhydrin assay and the product was isolated by precipitating with anhydrous diethyl ether. The protective group Fmoc was removed by treating with 20% piperidine, and the end product (compound 1 ) was again isolated by precipitating with diethyl ether.
  • DMF dimethylformamide
  • TEA triethylamine
  • Compound 1 was then dissolved in 0.1 M borate buffer pH 8.5 and 3 equivalents of fluorescein isothiocyanate (FITC) were added. After incubating in the dark overnight, the product (compound 2) was purified of the excess of fluorophore by gel filtration on a Sephadex G25 (GE Healthcare) column and brought to dryness by lyophilisation. The amount of fluoroscein bound to biotinylated PEG in the end product was determined by UV-Vis spectrophotometric analysis. The biotin and PEG content in the end product were determined by testing with HABA (Green NM, 1965) and iodine (Morpurgo M et al., 2004), respectively.
  • FITC fluorescein isothiocyanate
  • the succinimidyl ester of Alexa Fluor ® 633 was obtained by Molecular probes ® (Invitrogen).
  • the derivative biotinamidohexylamine was synthesised as described in the literature (Pignatto M et al., 2010).
  • the conjugation of Alexa 633 - V/-/S and biotinamidohexilamine was conducted in anhydrous DMSO, by mixing in two reagents in equimolar ratios, in the presence of 2 equivalents of triethylamine (TEA).
  • TAA triethylamine
  • Example 3 Preparation of the nanocomplexes plasmid DNA: avidin:[B-C6-Lys- (PEG-fluorescein) 2 , B-C 6 -Alexa 633 ] Doubly fluorescent nanoparticles (by means of fluorescein and Alexa 3) were prepared in accordance with a protocol analogous to the one reported in the literature (Pignatto M et al. 2010; Morpurgo M et al., 2012).
  • a "basic" composition was prepared containing only the fluorophore fluorescein (Avidin:B- C6-Lys-(PEG-fluorescein)2) by mixing avidin (Belovo Chemical), compound 2 of example 1 and plasmid DNA (pEGFP-C1 , 4,7kb, Clonetech) in the ratios NB:Av:B- C6-Lys-(PEG-fluorescein) 2 corresponding to 9.400:1.000:460. After stabilisation, the nanoassemblies were purified of the excess of avidin by ultrafiltration or size exclusion chromatography.
  • the "basic" composition was added with biotinamido-hexylamido-Alexa 633 (B-C6-Alexa 633 added, compound 3, example 2) and was purified again as described above.
  • the number of fluorophores stably attached to the nanoparticles before and after addition of the compound 3 ⁇ -Ce- Alexa 633 was determined by spectrophotometry on the basis of their absorption at 280nm (contribution of avidin, DNA and fluorescein), 495nm and 631 nm (contribution of fluorescein and Alexa 633 , respectively).
  • the number of B-C 6 -Alexa 633 stably bound in the final composition was equal to 281 .
  • the average size of the nanoparticles measured by means of dynamic light scattering was 1 10nm.
  • Example 4 Stability of the nanoassembiy plasmid DNAiavidin [B-Ce-Lys-(PEG- fluorescein) 2 , ⁇ -Ce-Alexa 633 ] in plasma, and liver and spleen homogenates
  • the nanoassembiy of example 3 (120 pg/rnl in PBS) was incubated (15 h, 37°C) with 0.1 volumes of plasma or liver or spleen homogenates (Lazzari SJ et al., 2012), and PBS buffer as the control. The samples were then centrifuged (16600 g) for 2 min (4°C) and the supernatants were analysed by size exclusion chromatography using an FPLC Akta ® purifier (GE) integrated with a SuperoseTM-6 column, by monitoring the eluate at 280 nm, 495 nm and 631 nm. The chromatograms of the mixtures were compared with that of the nanoassembiy prior to treatment.
  • GE FPLC Akta ® purifier
  • the data in panel B shows that the intensity of the peak relative to the nanoassembly after treatment in the liver and spleen homogenates is similar to that recorded following treatment in phosphate buffer (PBS), indicating a good stability in these environments.
  • PBS phosphate buffer
  • the peak of the nanoassembly treated in plasma is lower than that in PBS.
  • the recorded chromatographic drop is due to the treatment of the assembly on the side of the column prefilter, a phenomenon which indicates the formation of aggregates of larger size than the original nanoassembly, probably secondary to interaction with the plasma proteins.
  • the data are therefore in accordance in saying that the nanoassembly slowly disintegrates in the media studied. In every case, the component which seems to separate off the most rapidly from the nanoparticles following the treatment is the one bound to PEG fluorescein (panel C).
  • Example 5 In vitro cellular internalisation assay of the nanoassembly plasmid DNA:avidin:[B-C 6 -Lys-(PEG-Fluorescein) 2 , B-C 6 -Alexa 633 ]
  • HeLa cells (in DMEM with addition of 10% foetal bovine serum, 2 mM l-glutamine, 100 U penicillin/0.1 mg/ml streptomycin) were seeded onto glass cover plates positioned inside of a 24-well plate (10,000 cells/well). After 24 hours of incubation at 37°C, 5% CO2, the culture medium was removed and replaced with a solution containing the nanoassembly doubly labelled with fluorescein and Alexa 633 of example 3 (30 pg/rnl in the same culture medium). At predetermined times (2, 6 and 24 h), the cells were fixed with 4% paraformaldehyde and the nuclei were stained with the fluorescing blue chromophore Hoechst 33258.
  • Two- and three-dimensional images were obtained ( Figure 3).
  • the superimposition of the images taken in the three channels mentioned above was performed automatically by the software of the Olympus fluoview.
  • the 3-D reconstruction was performed using the Imaris 5.0 (Bitplane) software.
  • the internalisation within the cells is shown to be efficient and with a time-dependent kinetics. Indeed, in panel A one can see control cells (treated with medium only) in which the nucleus appears coloured blue, while panels B-D shows cells following treatment with the nanocomplex at 2, 6 and 24 h: in panel B, no colours other than the blue of the nuclei were ascertained; in panel C, a slight yellow and red colouration is already appearing alongside the blue colour; and in panel D there is a very intense yellow colouration around the blue nucleus.
  • the panel E (2D) shows an enlarged image of the sample treated for 24 hours and demonstrates the localisation of the nanoparticles at spot within the cytoplasm in proximity to the nuclei (blue colouration of the nuclei with an intense yellow colouration around it), but also macroaggregates in regions associated with the cell membrane.
  • panel F a 3D reconstruction can be seen of an image obtained from the same sample at 24 h with red and green macroaggregates.
  • Morpurgo M Cropurgo A, Bayer EA, and Wilchek M, J. Mol. Rec. 2004, 17, 558-566. Morpurgo M, Pignatto M, Teoli D, WO 2009/003951.

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Abstract

Nano-assemblages d'acides nucléiques, d'avidine et de composés biotinylés utilisables en tant que supports d'administration intracellulaire. Ces nano-assemblages sont composés de noyaux centraux de polyavidines, c'est-à-dire de noyaux formés par auto-assemblage d'une pluralité de motifs tétramères d'avidine sur des acides nucléiques, sur lesquels des composés biotinylés s'auto-assemblent encore grâce aux interactions de haute affinité entre l'avidine du noyau central et la biotine des composés biotinylés.
PCT/IB2014/063408 2013-07-26 2014-07-25 Complexes nano-assemblés d'acides nucléiques, d'avidine et de composés biotinylés utilisables en tant que supports d'administration intracellulaire Ceased WO2015011675A1 (fr)

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IT201600090019A1 (it) * 2016-09-06 2018-03-06 Univ Degli Studi Padova Peptide per la diagnosi differenziale della Rettocolite ulcerosa
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITUB20152058A1 (it) * 2015-07-10 2017-01-10 Jointherapeutics S R L Complessi nanoassemblati di acidi nucleici, avidina e composti biotinilati impiegabili per il trasporto di farmaci
WO2017009215A1 (fr) * 2015-07-10 2017-01-19 Jointherapeutics S.R.L. Complexes nano-assemblés d'acides nucléiques, d'avidine et de composés biotinylés utilisables en tant que supports de médicaments
US10940208B2 (en) 2015-07-10 2021-03-09 Uvera Srl Nanoassembled complexes of nucleic acids, avidin and biotinylated compounds for use as drug carriers
IT201600090019A1 (it) * 2016-09-06 2018-03-06 Univ Degli Studi Padova Peptide per la diagnosi differenziale della Rettocolite ulcerosa
WO2018047068A1 (fr) * 2016-09-06 2018-03-15 Universita' Degli Studi Di Padova Peptide utile dans le diagnostic différentiel de la rectocolite hémorragique
IT201900002679A1 (it) * 2019-02-25 2020-08-25 Livera S R L Complessi nanoassemblati di acidi nucleici, avidina e composti biotinilati per l’uso nel trattamento patologie epatiche

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