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WO2024235481A1 - Nanoparticules pour le transport de substances actives comprenant des groupes anioniques, leur procédé de production et leur utilisation - Google Patents

Nanoparticules pour le transport de substances actives comprenant des groupes anioniques, leur procédé de production et leur utilisation Download PDF

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WO2024235481A1
WO2024235481A1 PCT/EP2024/000028 EP2024000028W WO2024235481A1 WO 2024235481 A1 WO2024235481 A1 WO 2024235481A1 EP 2024000028 W EP2024000028 W EP 2024000028W WO 2024235481 A1 WO2024235481 A1 WO 2024235481A1
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lipid
lipids
lpl
lipid nanoparticles
radical
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Anja TRÄGER
Michael STREIBER
Ulrich S. Schubert
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Friedrich Schiller Universtaet Jena FSU
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Friedrich Schiller Universtaet Jena FSU
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    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • 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
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • 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
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • 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
    • A61K9/5192—Processes
    • 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

Definitions

  • Nanoparticles for the transport of active substances with anionic groups processes for their preparation and their use
  • the invention relates to the field of production and processing of nanoparticles that can be loaded with active substances, e.g. with genetic material. These nanoparticles can be advantageously used for transporting active substances in organisms, in particular for transferring nucleic acids into cells.
  • Vaccine antigens particularly purified or recombinant subunit vaccines, are often poorly immunogenic and require the use of adjuvants to stimulate protective immunity.
  • adjuvants to stimulate protective immunity.
  • adjuvants there remains a need for improved adjuvants and delivery systems that enhance the protective antibody response, particularly in populations that are poorly responsive to current vaccines.
  • Lipid nanoparticles represent an alternative to other particulate systems such as emulsions, liposomes, micelles, microparticles and/or polymeric nanoparticles for the administration of active substances such as oligonucleotides and low molecular weight drugs.
  • LNP and their use for the administration of active substances have already been described, for example in US 7,691,405, US 2006/0083780, US 2006/0240554, US 2008/0020058, US 2009/0263407, US 2009/0285881, WO 2009/086558, W02009/127060, W02009/132131, WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405 and WO2010/054406.
  • Lipid-based nanoparticles as carriers of pharmaceutical active ingredients have also been described, e.g. in Puri, A.; Loomis, K.; Smith, B.; Lee, J.H.; Yavlovich, A.; Heldman, E.; Blumenthal, R., Lipid-based nanoparticles as pharmaceutical drug carriers: from concepts to clinic. Crit. Rev. Ther. Drug Carrier Syst. 2009, 26, 523-80.
  • nucleic acids such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA or ASOs), which, due to their high molar mass and negative charge, require a transporter in contrast to classical drugs (Kulkarni, J. A.; Witzigmann, D.; Thomson, S. B.; Chen, S.; Leavitt, B. R.; Cullis, P. R.; van der Meel, R., The current landscape of nucleic acid therapeutics. Nat. Nanotechnol. 2021, 16, 630-643).
  • pDNA DNA
  • mRNA, siRNA, miRNA or ASOs RNA
  • lipid nanoparticles established themselves as an important drug form for the transport of genetic material (cf. Tenchov, R.; Bird, R.; Curtze, AE; Zhou, Q., Lipid nanoparticles - From liposomes to mRNA vaccine delivery, a landscape of research diversity and advancement.
  • ACS Nano 2021, 15, 16982-17015 In addition to the two clinically used COVID-19 vaccines, BNT162b and mRNA-1273, other promising therapeutic approaches have now been approved or are involved in clinical trials (cf.
  • Kulkarni JA; Witzigmann, D.; Thomson, SB; Chen, S.; Leavitt, BR; Cullis, PR; van der Meel, R., The current landscape of nucleic acid therapeutics. Nat.Nanotechnol. 2021, 16, 630-643; Hou, X.; Zaks, T.; Langer, R.; Dong, Y., Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 2021 , 6, 1078- 1094).
  • Fomivirsen is an antisense oligonucleotide (ASO) that targets specific sequences of cellular RNA and was approved in 1998 for the treatment of cytomegalovirus infections of the retina in AIDS patients (withdrawn in 2002) (Roberts, TC; Langer, R.; Wood, MJ
  • Patisiran (Onpattro) is the first approved representative of novel "first-in-class" drugs based on RNA interference in 2018 and is used to treat hereditary transthyretin amyloidosis in patients with stage 1 or 2 polyneuropathy (Buck, J.; Grossen, P.; Cullis, P. R.; Huwyler, J.; Witzigmann, D., Lipid-based DNA therapeutics: Hallmarks of non-viral gene delivery. ACS Nano 2019, 13, 3754-3782).
  • a plasmid encoding human hepatocyte growth factor has been approved for the treatment of patients with critical limb ischemia.
  • RNA-based systems such as /V-acetylgalactosamine (GalNAc)-siRNA conjugates: i) givosiran (Givlaari) for the treatment of acute intermittent hepatic porphyria, ii) lumasiran (Oxlumo) for the treatment of primary hyperoxaluria type 1, and iii) inclisiran (Leqvio), a subcutaneous therapeutic for the treatment of hypercholesterolemia.
  • GalNAc /V-acetylgalactosamine
  • RNA-based systems include lipid-based siRNA drugs and the mRNA vaccines against SARS-CoV-2 (Paunovska, K.; Loughrey, D.; Dahlman, JE, Drug delivery systems for RNA therapeutics. Nat. Rev. Genet. 2022, 23, 265-280).
  • the currently approved LNPs of the vaccine consist of four different lipid components and the genetic material (cf. Kulkarni, JA; Cullis, PR; van der Meel, R., Lipid nanoparticles enabling gene therapies: From concepts to clinical utility, Nucl. Acid Ther. 2018, 28, 146-157; and Schoenmaker, L.;
  • lipids used in the case of BNT162b (BioNTech) and mRNA-1273 (Moderna) fulfill different functions in the formulation of the LNP and are based on an intensive screening of lipids (Dolgin, E., The tangled history of mRNA vaccines. Nature 2021 , 597, 318-324).
  • lipids For transport, the conserved negatively charged phosphates in the RNA and DNA backbone can be used for interactions with lipids.
  • the cationic lipids play a key role here, as they are responsible for binding to the negatively charged genetic material. They also influence endosomal uptake (see Haid Albertsen, C.; Kulkarni, J. A.; Witzigmann, D., et al., The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv. Drug Del. Rev.
  • ionizable lipids bind to the genetic material through a pH-dependent formulation that starts at a lower pH to allow binding of the genetic material and slowly increases to the physiological pH.
  • the formulations are neutral upon administration, e.g.
  • a dimethylamino base showed high transfection efficiency (Mo, R.; Sun, Q.; Li, N.; Zhang, C., Intracellular delivery and antitumor effects of pH-sensitive liposomes based on zwitterionic oligopeptide lipids. Biomaterials 2013, 34, 2773-2786).
  • Influencing factors include, for example, the length of the alkyl chains, the type of ionizable group and the pK a value of the molecule (cf. Haid Albertsen, C.; Kulkarni, JA; Witzigmann, D., et al., The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv. Drug Del. Rev.
  • the stealth effect is caused by limiting the adhesion of immune-triggering proteins, such as opsonins and immunoglobulins, to the particle surface, which prevents opsonization and leads to a prolonged half-life in the bloodstream after systemic administration (see Friedl, JD; Nele, V. ; De Rosa, G.; Bernkop-Schnürch, A., Bioinert, Stealth or interactive: How surface chemistry of nanocarriers determines their fate in vivo, Adv. Func. Mater. 2021, 31, 2103347).
  • immune-triggering proteins such as opsonins and immunoglobulins
  • Camouflaged nanoparticles affect drug delivery, especially in cancer therapy.
  • Langer et al. presented PEG-grafted polymer nanoparticles that can circulate in the blood for longer due to the passivation effect of PEG.
  • PEGylation can reduce the nonspecific binding of serum proteins to the particles, thereby reducing their clearance by cells of the mononuclear phagocytic system (MPS).
  • MPS mononuclear phagocytic system
  • PEGylated compounds A number of alternatives to PEGylated compounds are known. In general, these show a prolonged systemic circulation time, sustained drug release kinetics and better tumor accumulation.
  • alternative polymers to PEG include poly(glycerine) (PG), poly(oxazoline) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(/V-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(A/,A/-dimethylacrylamide) (PDMA) and Poly(/V-acryloylmorpholine) (PAcM) (see Hoang Thi, TT; Pilkington, EH; Nguyen, DH; Lee, JS; Park, KD; Truong, NP, The Importance of poly(ethylene glycol) alternatives for overcoming PEG immunogenicity in drug delivery and bioconjugation. Polymers 2020,
  • PEG lipids have so far been the most important and widely used.
  • PEG lipids in the LNP formulation are responsible, among other things, for extending the residence time in the organism and they reduce the organism's immune response to the drug (cf. Nosova, AS; Koloskova, OO; Nikonova, AA; Simonova, VA; Smirnov, VV; Kudlay, D.; Khaitov, MR, Diversity of PEGylation methods of liposomes and their influence on RNA delivery.
  • helper lipids increase the encapsulation efficiency of the genetic material and the endosomal release of the LNP (cf. Kulkarni, J. A.; Witzigmann, D.; Leung, J.; Tam, Y. Y. C.; Cullis, P. R., On the role of helper lipids in lipid nanoparticle formulations of siRNA. Nanoscale 2019, 11, 21733- 21739).
  • a nucleic acid encapsulated with lipids is known from US 7,341,348 B2.
  • the patent describes a particle made of a lipid layer that encloses a central zone containing nucleic acid and that contains an aminolipid with an amino group with a pKa value of 4 to 11 and a PEG-DAG conjugate.
  • lipid lipid, neutral lipid, steroid, polymer-conjugated lipid and a therapeutic agent encapsulated therein.
  • WO 2019/089828 A1 discloses a LNP having a bilayer structure containing at least 40 mol% of a cationic lipid and a nucleic acid encapsulated therein.
  • EP 3 556 353 A2 describes LNPs that are made up of a cationic lipid, a PEG lipid and an antigen. These LNPs usually contain other components, such as cholesterol or phospholipids. The lipids are dissolved in ethanol to produce the LNPs.
  • LNPs described in these documents contain increasing amounts of steroids such as cholesterol. These LNPs are produced from an ethanolic-aqueous solution, as this is not possible in an aqueous solution.
  • LNPs in which cholesterol has been replaced by a modified cholesterol DC-CHOL with an ammonium group.
  • This lipid has a Griffin HLB value of 5.8.
  • the LNPs described in this document also contain a cationic lipid, a phospholipid and a stealth lipid.
  • the cationic lipids have Griffin HLB values of less than 4. Information on the amount of lipids used in the LNPs is missing from this document.
  • LNP with reduced cholesterol content are disclosed by Maho Kawatuchi et al. in J. of Pharmaceutical Sciences, 112 (2023) 1401-1410 and by Samuel T. LoPresti et al. in Journal of Controlled Release, 345 (2022) 819-831.
  • the LNP described in these documents both show a reduction but not a replacement of the cholesterol content.
  • the LNP produced have structural disadvantages compared to the commercial cholesterol-containing alternative.
  • the authors were not able to produce LNP with an absolute absence of pure cholesterol.
  • Both studies describe LNP containing various lipids whose HLB value is not greater than or equal to 3.
  • LNPs liposomes containing natural and synthetic lipids.
  • microfluidics For the commercial scale, the latter technique prevailed in the case of the COVID-19 vaccine.
  • One of the advantages of microfluidics is its good reproducibility in production from batch to batch (cf. Maeki, M.; Uno, S.; Niwa, A.; Okada, Y.; Tokeshi, M., Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery. J. Control. Release 2022, 344, 80-96; and Shepherd, S. J.; Issadore, D.; Mitchell, M. J., Microfluidic formulation of nanoparticles for biomedical applications. Biomaterials 2021, 274, 120826).
  • the LNPs are created by rapidly mixing an organic lipid phase (usually based on ethanol) and an aqueous phase, the latter containing the genetic material. During this process, the lipids precipitate and nanoparticles form. The organic solvent must then be removed from the formulation. In addition to evaporation, dialysis or cross-flow filtration is often used (see Evers, M. J. W.; Kulkarni, J. A.; van der Meel, R.; Cullis, P. R.; Vader, P.; Schifflers, R. M., State-of-the-art design and rapid-mixing production techniques of lipid nanoparticles for nucleic acid delivery. Small Methods 2018, 2, 1700375; Mihaila, S., Wei, Z. Y., Shadel, S.; Cunningham, J. J.; Lipid nanoparticle purification by spin centrifugation-dialysis for small scale preparation of siRNA-lipid complexes, Int.
  • the material from which the microfluidic chips are made can also be disadvantageous.
  • Polydimethylsiloxane absorbs genetic material and tends to swell in the solvents (cf. Kwon, H. J.; Kim, S.; Kim, S.; Kim, J. H.; Lim, G., Controlled production of monodisperse polycaprolactone microspheres using flow-focusing microfluidic device. BioChip Journal 2017, 11, 214- 218; and Tsao, C.-W. Polymer microfluidics: Simple, low-cost fabrication process bridging academic lab research to commercialized production. Micromachines 2016, 7, 225).
  • ethanol is not absolutely necessary for the encapsulation of genetic material (cf. Kulkarni, JA; Thomson, SB; Zaifman, J.; Leung, J.; Wagner, PK; Hill, A.; Tam, YYC; Cullis, PR; Petkau, TL; Leavitt, BR, Spontaneous, solvent-free entrapment of siRNA within lipid nanoparticles. Nanoscale 2020, 12, 23959-23966).
  • One way to avoid the use of ethanol is to homogenize the lipids in an aqueous solution above the melting point.
  • LNP Long RNA-ionizable LNPs
  • a disadvantage is that only temperature-stable drugs can be used and all components must be heat-stable, which in the case of lipids and RNA requires intensive quality control.
  • LNP can be produced without the need to use organic solvents.
  • organic solvents such as ethanol, had to be used in order to be able to introduce the highly lipophilic portions of the lipids used into the LNP.
  • the process according to the invention allows the production of LNP that can be loaded in high concentrations with active substances (e.g. nucleic acids) containing anionic groups, such as phosphate groups.
  • LNPs according to the invention can be loaded with sensitive active substances in a gentle manner and allow, for example, the introduction and transport of genetic material into cells.
  • the BLNPs according to the invention are produced without the use of highly lipophilic steroids and thus contain a smaller number of lipid components than the previously known LNPs.
  • Conventional LNPs contain large amounts of cholesterol. This easily diffuses back and forth between the LNPs and also serum components and lipids in the biological environment.
  • Cholesterol is not produced entirely synthetically, but rather from natural sources, which means that fluctuating qualities and impurities cannot be ruled out.
  • a process with more steps and components for approval is more complex and therefore more expensive and less robust.
  • the lipophilic character of lipids can be described by the HLB value (HLB stands for hydrophilic-lipophilic balance).
  • HLB hydrophilic-lipophilic balance
  • the HLB value was introduced in 1954 by W. C. Griffin and describes the hydrophilic and lipophilic proportion of lipids.
  • the HLB value scale ranges from 0 (strongly lipophilic) to 20 (weakly lipophilic).
  • Griffin In addition to the Griffin method, there are other methods for calculating the HLB value. However, these are far less common.
  • One example is the method according to Davies, who in 1957 proposed calculating the HLB value from numerical values for the individual chemical groups of a molecule. The advantage of this method is that strongly interacting groups are given a higher weighting than less interacting ones. It can also be used to define the HLB value for cationic and anionic lipids.
  • An object of the present invention was to provide lipid nanoparticles which have a compact and simple structure, which can be loaded with a high content of active ingredient and which are excellently suited for the transport of active ingredients in organisms or cells, for example for the gene transfer of nucleic acids.
  • a further object of the present invention was to provide a simple process for the production of lipid nanoparticles, which can be carried out without the use of organic solvents.
  • the present invention relates to lipid nanoparticles containing a) 51 to 94.9 mol-% of at least one cationic/ionizable lipid, b) 5 to 40 mol-% of at least one phospholipid, and c) 0.1 to 10 mol-% of at least one stealth lipid selected from the group of lipids containing one or more poly(alkylene oxide) chains (hereinafter "PEG lipids”), lipids containing one or more poly(oxazoline) chains (hereinafter “POx lipids”), lipids containing one or more poly(glycerol) chains (hereinafter "PG lipids”), lipids containing one or more poly(hydroxyalkyl(meth)acrylate) chains (hereinafter "PHAA lipids”), lipids containing one or more poly(/V-(hydroxyalkyl)(meth)acrylamide) chains (hereinafter "PHAAA lipids”), lipids containing one or more poly(vinylpyr
  • lipid nanoparticles or “LNP” or “BLNP” are understood to mean particles whose diameter (z-average) is less than or equal to 900 nm and which consist mainly or completely of lipids from the above-mentioned groups a), b) and c). These BLNPs can be loaded with active ingredients containing anionic groups.
  • the BLNPs are generally characterized by a very high surface-to-volume ratio and thus offer very high chemical reactivity.
  • BLNPs can consist only of the above-mentioned lipids from groups a), b) and c) or additionally contain complexes of active ingredient and the cationic lipid from group a), or the BLNPs contain small amounts of other components in addition to the lipids and possibly complexes, such as excipients or additives e).
  • auxiliaries and additives are understood to mean substances that are added to a formulation in order to give it certain additional properties and/or to facilitate its processing. to facilitate.
  • excipients and additives are sugars such as sucrose, contrast media, carriers, fillers, pigments, dyes, perfumes, radiopharmaceuticals such as tracers, lubricants, UV stabilizers, polymers such as nitrogen-containing polymers, or antioxidants.
  • excipients and additives are understood to mean any substance useful for the intended application which is not a pharmaceutical or agrochemical active ingredient and is not a lipid, but which can be formulated together with an active ingredient in an active ingredient-lipid complex in order to influence, in particular to improve, the qualitative properties of the LNP.
  • the excipients and/or additives e) have no effect or, with regard to the intended treatment, no significant effect or at least no undesirable effect.
  • HLB value is understood to be a numerical value between 0 and 20, which is calculated according to the following formula
  • HLB 20 * (1 - M
  • the freely accessible software MarvinSketch 23.4 is used to determine the HLB value of the lipids (cf. https://docs.chemaxon.- com/display/docs/hlb-predictor.md#src-1806640-hlbpredictor-fig-1 ) and the HLB is determined according to Griffin, since the Davis method is not optimal for stealth lipids with many repeat units.
  • Highly lipophilic compounds generally have HLB values of 1 to 3. These are hydrophilic (oil-soluble) lipids, such as antifoam agents. Compounds with significant hydrophilic components are dispersible in water and have HLB values of 3 to 9. These include W/O emulsifiers with HLB values of 3 to 6 and wetting agents with HLB values of 7 to 9. Hydrophilic (water-soluble) lipids have HLB values of 9 to 18. These include O/W emulsifiers with HLB values of 8 to 18, detergents with HLB values of 13 to 15 and solubilizers with HLB values of 15 to 18.
  • Phospholipids usually have HLB values of 4 to 5.
  • all lipids in the nanoparticles according to the invention have HLB values of 3 to 20, in particular of 3 to 18, in particular of greater than or equal to 4 and very particularly preferably of 4 to 17.5.
  • the BLNPs according to the invention can be loaded with active substances that have at least one anionic group.
  • the invention therefore also relates to the LNPs described above that are loaded with active substances that have anionic groups.
  • the molar fraction (mol%) of the cationic lipid a) or the combined amount of lipids a) is typically 51 to 94.9%, preferably 55 to 89.5%, particularly preferably 60 to 85% and most preferably 75 to 82%.
  • the molar fraction (mol%) of the phospholipid b) or the combined amount of lipids b) is typically 5 to 40%, preferably 10 to 30%, particularly preferably 14 to 25% and most preferably 15 to 18%.
  • the molar proportion (mol%) of the stealth lipid c) or the combined amount of lipids c) is typically 0.1 to 10%, preferably 0.5 to 5% and most preferably 1 to 3%.
  • the percentages given above refer to the total amount of lipids contained in the BLNP. If the BLNPs according to the invention contain further lipids d) with HLB values of at least 3 which do not belong to one of the groups a) to c), the weight proportion of these lipids d) is at most 10%, preferably at most 5% and in particular at most 1%.
  • BLNPs according to the invention contain auxiliary substances or additives e), their total weight proportion is not more than 5%, preferably not more than 1% and in particular not more than 0.5%.
  • the BLNPs according to the invention contain no further lipids d) and no excipients or additives e).
  • the proportion of lipids with sterol residues in the BLNPs according to the invention is 0 to 15 mol-%, in particular 0 to 10 mol-%.
  • the cationic lipids a) for producing the BLNP according to the invention include all lipids with at least one cationic group, for example an amino group. However, these are not phospholipids, which are classified as lipids in group b). Cationic lipids a) preferably do not contain any phosphate residues.
  • cationic groups are amino groups, i.e. primary, secondary and tertiary amino groups or quaternary ammonium groups; guanidino groups and amide groups, i.e. groups with secondary, tertiary and quaternary amide groups; aminoalkanol groups, i.e. groups with primary, secondary, tertiary and quaternary amino groups; phosphane groups, i.e. primary, secondary and tertiary phosphane groups or quaternary phosphonium groups.
  • cationic lipid refers to lipids that have one or more net positive charge(s) at certain pH values, e.g., acidic pH values.
  • the cationic lipids within the scope of this description also include ionizable cationic lipids. Ionizable cationic lipids are characterized by the weak basicity of their ionizable groups, which influences the charge of the lipid in a pH-dependent manner. As a result, these lipids are positively charged at acidic pH, but are almost charge-neutral at physiological pH.
  • the preferred cationic lipids a) for the preparation of the BLNPs according to the invention include all lipids with at least one amino group (which are not phospholipids).
  • the preferred cationic lipids a) for producing the BLNPs according to the invention also include ionizable lipids containing at least one amino group (which are not phospholipids). These have the property that they form a positive charge on the nitrogen atom via protonation in the acidic pH range from 4 to 7 and that they are almost neutral in the basic pH range above 7.
  • ionizable lipids a) are used for the preparation of the BLNPs according to the invention which contain at least one amino group (which are not phospholipids), in particular one to two amino groups and very particularly preferably one amino group, wherein this amino group(s) has/have a pKa value of 7 to 9.
  • the cationic lipids a) have no phosphate residues and one or two nitrogen atoms, preferably one nitrogen atom, and at least one alkyl residue having six to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO- or an amide group -CO-NH- or -NH-CO-, and/or at least one alkylene residue having six to twenty carbon atoms and one, two or three non-directly adjacent neighboring double bonds.
  • These nitrogen atoms can be present as amino groups, amide groups or as alkanolamino groups, preferably as amino groups.
  • the cationic lipids particularly preferably have a) no phosphate residues and one or two nitrogen atoms and at least two alkyl residues having six to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O- CO- or an amide group -CO-NH- or -NH-CO-, or one or both of these alkyl residues are replaced by one or two alkylene residues having six to twenty carbon atoms and one, two or three double bonds which are not directly adjacent to one another.
  • Particularly preferred cationic lipids a) have the structure of formula (I)
  • R 3 R 1-N 1 -(CO) r -R 2 (I), in which R 1 is a radical of the formula R 4 R 5 N-(C m H 2m )-, CH 3 -(C n H 2 n) -O-(C o H 2 o)-, HO-(C m H 2m )-, HO-CH 2 -CH(OH)-CH 2 -, CH 3 -(CH 2 ) n -O-CO-(C m H 2m )-, CH 3 -(C n H 2n )-CO-O-(C m H 2m )-, NC-(C O H 2O )-, HO-CH 2 -CH((C 0 H 2O )-CH 3 )-, CH 3 -(C o H 2o )-CH(OH)-(CpH 2 p)-, CH 3 -(C n H 2 n)-CO-NH-(CoH 2o
  • cationic lipids a) include those of formula (I) in which R 2 and R 3 are independently selected from the residues of the group
  • R 2 and R 3 are independently selected from the radicals of the formula -(CH 2 ) V -O-CO-R 5 or -(CH 2 ) V -CO-OR 6 , v is an integer from 1 to 20, preferably from 5 to 12, and
  • R 5 and R 6 independently of one another represent alkyl radicals having 6 to 20 carbon atoms and/or alkenyl radicals having 6 to 20 carbon atoms and having one or preferably two ethylenically unsaturated bonds which are not directly adjacent to one another, in particular radicals selected from the group
  • the cationic lipids a) preferably form a positive charge on the nitrogen atom in the pH range from 5 to 8.
  • the compounds of formula (I) are then present as cationic compounds of formula (II)
  • R 1 , R 2 , R 3 and r have the meaning defined above, j is an integer which corresponds to the number of nitrogen atoms in the compound of formula (II), preferably 1 or 2, i is an integer from 1 to 5000, and
  • X represents an i-valent anion
  • Any inorganic or organic i-valent anions X can be used.
  • inorganic anions X' are halide ions, such as fluoride, chloride, bromide or iodide, or hydroxide ions or anions of inorganic acids, such as Phosphate, sulfate, nitrate, hexafluorophosphate, tetrafluoroborate, perchlorate, chlorate, hexafluoroantimonate, hexafluoroarsenate, cyanide.
  • halide ions such as fluoride, chloride, bromide or iodide
  • hydroxide ions or anions of inorganic acids such as Phosphate, sulfate, nitrate, hexafluorophosphate, tetrafluoroborate, perchlorate, chlorate, hexafluoroantimonate, hexafluoroarsenate, cyanide.
  • organic anions X 1 ' are anions of mono- or polybasic carboxylic acids or mono- or polybasic sulfonic acids, where these acids can be saturated or unsaturated.
  • examples of anions of organic acids are acetate, formate, trifluoroacetate, trifluoromethanesulfonate, pentafluoroethanesulfonate, nonofluorobutanesulfonate, butyrate, citrate, fumarate, glutarate, lactate, malate, malonate, oxalate, pyruvate or tartrate.
  • These anions can exist in the form of polyanions.
  • cationic lipids a) contain a quaternary ammonium group.
  • examples of such lipids are compounds of the formula (lla) (lla), wherein
  • R 1a , R 2a and R 3a are independently alkyl radicals having one to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO-, and/or alkylene radicals having two to twenty carbon atoms and one, two or three double bonds which are not directly adjacent to one another,
  • R 4a is an alkyl radical having six to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO-, and/or an alkylene radical having six to twenty carbon atoms and one, two or three double bonds which are not directly adjacent to one another, or in which two radicals R 1a and R 2a together with the common nitrogen atom form a pyrrolidine or piperidine radical, and X, i and j have the meaning defined above.
  • the nanoparticles according to the invention very particularly preferably contain cationic lipids which are selected from the group N, A/-dioleyl-/V, N-dimethylammonium chloride (DODAC); A/-(2,3-dioleyloxy)propyl)-A/,/V,A/-trimethylammonium chloride (DOTMA); A/,A/-distearyl-A/,A/-dimethylammonium bromide (DDAB); A/-(2,3-dioleoyloxy)propyl)-A/,A/,A/-trimethylammonium chloride (DOTAP); 3-(A/-(A/',A/'-dimethylaminoethane)-carbamoyl)-cholesterol (DC-Chol), /V-(1-(2,3-dioleoyloxy)propyl)/V-2-(spermine-carboxamido)ethyl)-/V,
  • the nanoparticles according to the invention contain only one cationic/ionizable lipid a).
  • the phospholipids b) used according to the invention are generally lipids which, in addition to at least one lipid residue, have an associated residue of a polyhydric alcohol, to which in turn a phosphate group is bound, which is linked to a head group via an ester bond.
  • a phospholipid b) generally has a structure of the formula (III)
  • LP is a residue of a fatty acid
  • BG is a (np+1 )-valent bridging group
  • np is an integer from 1 to 5, preferably 1 or 2
  • Me is hydrogen, a monovalent or divalent metal cation or an ammonium cation
  • KG represents a head group which is an aliphatic radical containing at least one hydroxyl group, preferably an aliphatic radical having one hydroxyl group and one amino group, one hydroxyl group and one quaternary ammonium group or the radical of a carbohydrate having five to six hydroxyl groups, where
  • Residues LP can assume different meanings within a molecule within the given definitions.
  • the phospholipids b) of the formula (III) have one to five LP residues, preferably one or two LP residues, which are alkyl residues having six to twenty carbon atoms, and/or mono- to tri-ethylenically unsaturated alkenyl residues having six to twenty carbon atoms, and/or saturated or mono- to tri-ethylenically unsaturated fatty acid residues having six to twenty carbon atoms, wherein several double bonds in an alkenyl residue are not directly adjacent to one another.
  • LP residues preferably one or two LP residues, which are alkyl residues having six to twenty carbon atoms, and/or mono- to tri-ethylenically unsaturated alkenyl residues having six to twenty carbon atoms, and/or saturated or mono- to tri-ethylenically unsaturated fatty acid residues having six to twenty carbon atoms, wherein several double bonds in an alkenyl residue are not directly adjacent to one another.
  • the phospholipids b) of the formula (III) have one to five residues LP which are linked to the head group via a bridging group BG via a phosphate group, wherein the bridging group BG is the residue of a di- to hexavalent aliphatic or cycloaliphatic alcohol or a di- to hexavalent aliphatic or cycloaliphatic amino alcohol.
  • residues of di- to hexavalent aliphatic or cycloaliphatic alcohols are groups derived from ethylene glycol, propylene glycol, glycerin, propanetriol, pentaethylthritol or inositol.
  • residues of di- to hexavalent aliphatic or cycloaliphatic amino alcohols are groups derived from 2-aminoethanol, 3-aminopropanol, prolinol, alaninol, valinol, leucinol, phenylalaninol, phenylglycinol or sphingosine.
  • Preferred phospholipids b) have a residue derived from glycerol as a bridging group and have the structure of formula (IVa) or (IVb)
  • LP is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to each other,
  • phospholipids b) have a residue derived from sphingosine as a bridging group and have the structure of the formula (Va) or (Vb)
  • LP is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, KG and Me have the meanings defined above, mp is an integer from 2 to 8, in particular 6, and the radicals LP in the compound of formula (Vb) can assume different meanings within a molecule within the framework of the given definitions.
  • Further preferred phospholipids b) have as head group KG a residue derived from an aliphatic amino alcohol or a residue derived from inositol.
  • R 6 is hydrogen or Ci-C 5 alkyl
  • R 7 and R 8 are independently Ci-Ce-alkyl
  • XP means an ip-valent anion, and ip is an integer from 1 to 3, preferably 1 or 2.
  • phospholipids b) with head groups KG which are selected from the group choline, ethanolamine, serine and inositol.
  • the nanoparticles according to the invention very particularly preferably contain phospholipids b) which are selected from the group distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-sn-glycero-3-phosphoethanolamine-/V-(maleimidomethyl) sodium salt (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (
  • the stealth lipids c) used according to the invention are generally lipids which, in addition to at least one lipid residue, have at least one poly(alkylene oxide) residue, poly(oxazoline) residue, polyglycerol residue, poly(hydroxyalkyl(meth)acrylate) residue, poly(A/-(hydroxyalkyl)(meth)acrylamide) residue, poly(vinylpyrrolidone) residue, poly(/V,/V-dialkyl(meth)acrylamide) residue, poly(/V-(meth)acryloylmorpholine) residue or poly(amino acid) residue connected to it.
  • These residues can be connected by a covalent bond or preferably via a bridging group BG.
  • Preferred stealth lipids c) have no phosphate residues.
  • Preferred stealth lipids c) are PEG lipids. These are in particular lipids with the structure of formula (VII) (LPL) nl -(BGL) m i-(O-CH 2 -CH 2 ) O i-OR 9 (VII) wherein
  • LPL is an alkyl or alkenyl radical having 6-20 carbon atoms, a radical of a fatty acid, a fatty alcohol or a sterol radical
  • BGL is a (nl+1)-valent bridging group
  • nl is an integer from 1 to 5, preferably 1 or 2
  • ml is 0 or 1
  • ol is an integer from 5 to 500, preferably 10 to 200
  • R 9 represents hydrogen, alkyl having one to six carbon atoms or a radical LPL, preferably hydrogen, methyl ethyl or a sterol radical, where the radicals LPL can assume different meanings within a molecule within the framework of the given definitions.
  • the stealth lipids c) of the formula (VII) have one to five LPL residues, preferably one or two LPL residues, which are alkyl residues with six to twenty carbon atoms, and/or mono- to tri-ethylenically unsaturated alkenyl residues with six to twenty carbon atoms, and/or saturated or mono- to tri-ethylenically unsaturated fatty acid residues with six to twenty carbon atoms, and/or saturated or mono- to tri-ethylenically unsaturated fatty alcohol residues with six to twenty carbon atoms, and/or sterol residues, wherein several double bonds in an alkenyl residue are not directly adjacent to one another.
  • LPL residues preferably one or two LPL residues, which are alkyl residues with six to twenty carbon atoms, and/or mono- to tri-ethylenically unsaturated alkenyl residues with six to twenty carbon atoms, and/or saturated or mono- to tri-eth
  • the stealth lipids c) of formula (VII) have one to five LPL residues which are directly covalently linked to a PEG residue via an ester bond; or the stealth lipids c) of formula (VII) have one to five LPL residues which are linked to a stealth residue (PEG, POx or others) via a bridging group BGL, where the bridging group BGL is the residue of a di- to hexavalent aliphatic or cycloaliphatic alcohol, or the residue of a di- to hexavalent carboxylic acid or a carbamate residue or the residue of an amino alcohol.
  • residues of di- to hexavalent aliphatic or cycloaliphatic alcohols are groups derived from ethylene glycol, propylene glycol, glycerin, propanetriol, pentaethylthritol or inositol.
  • residues of di- to hexavalent carboxylic acids are groups derived from oxalic acid, maleic acid, fumaric acid, adipic acid, sebacic acid, succinic acid, tartaric acid, terephthalic acid, isophthalic acid, trimellitic acid, trimesic acid or pyromellitic acid.
  • carbamate residues are groups derived from residues of the formula >N-CO-O-, in which the PEG residue is bonded to the oxygen atom and one or two LPL residues to the nitrogen atom.
  • amino alcohol residues are groups derived from residues of the formula >N-R-O-, in which R is a divalent organic residue, preferably an alkylene residue, the PEG residue is bonded to the oxygen atom and one or two LPL residues are bonded to the nitrogen atom.
  • R is a divalent organic residue, preferably an alkylene residue
  • the PEG residue is bonded to the oxygen atom
  • one or two LPL residues are bonded to the nitrogen atom.
  • Other amino alcohol residues can have several amino groups and/or oxygen atoms, for example aminophenols with two hydroxy groups and/or amino groups.
  • sterol residues are residues derived from saturated or mono- or diethylenically unsaturated sterols (3-hydroxysterols), which are preferably substituted in the 17-position with an alkyl residue having one to ten carbon atoms, in particular with a 2,6-dimethylhexyl residue.
  • a particularly preferred sterol residue is a residue derived from cholesterol.
  • Preferred stealth lipids c) have a glycerol-derived residue as a bridging group and have the structure of the formula (Villa) or (Vlllb) O-CH 2 -CH2-(O-CH2-CH 2 )OI-I-OR 9
  • LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, or a sterol radical, R 9 and ol have the meanings defined above, and the LPL radicals can assume different meanings within a molecule within the framework of the given definitions.
  • Further preferred stealth lipids c) have a residue derived from carbamate as a bridging group and have the structure of the formula (IXa) or (IXb)
  • Further preferred stealth lipids c) have a residue derived from succinic acid as a bridging group and have the structure of formula (X)
  • LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, and R 9 and ol have the meanings defined above.
  • Preferably used stealth lipids c) have the structure of formula (Xa)
  • R a is an alkylene radical having one to eight carbon atoms, preferably an ethylene radical
  • Ster is a radical derived from saturated or mono- or diethylenically unsaturated 3-hydroxysterols, which is preferably substituted in the 17-position by an alkyl radical having one to ten carbon atoms, in particular by a 2,6-dimethylhexyl radical, and is very particularly preferably a radical derived from cholesterol.
  • the PEG lipids c) preferably used according to the invention include the substance listed below, wherein n is a number between 15 and 200, preferably between 18 and 70.
  • the nanoparticles according to the invention very particularly preferably contain PEG lipids which are selected from the group of pegylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy-(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropyl carbamate, such as e.g.
  • PEG-DAG pegylated diacylglycerol
  • PEG-DMG
  • POx lipids are POx lipids. These are generally polymers which, in addition to at least one lipid residue, have a polyoxazoline residue connected to it, the latter having been produced by the polymerization of oxazoline. The two residues can be connected by a covalent bond or via a bridging group BGL.
  • a preferred POx lipid has a structure of formula (XII)
  • LPL is an alkyl or alkenyl residue with 6-20 carbon atoms, a residue of a fatty acid, a fatty alcohol or a sterol residue,
  • BGL is a (nl+1 )-valent bridging group
  • nl is an integer from 1 to 5, preferably 1 or 2
  • ml is 0 or 1
  • ol is an integer from 5 to 500, preferably 10 to 200
  • R 10 is hydrogen, alkyl having one to six carbon atoms or a radical
  • LPL preferably hydrogen, methyl ethyl or a sterol residue
  • R 11 is hydrogen or Ci-C 4 alkyl, where
  • Residues LPL and R 11 can have different meanings within a molecule within the given definitions.
  • the POx lipids of formula (XII) have one to five LPL residues, preferably one or two LPL residues, which are alkyl residues with six to twenty carbon atoms, and/or mono- to tri-ethylenically unsaturated alkenyl residues with six to twenty carbon atoms, and/or saturated or mono- to tri-ethylenically unsaturated fatty acid residues with six to twenty carbon atoms, and/or saturated or mono- to tri-ethylenically unsaturated fatty alcohol residues with six to twenty carbon atoms, and/or sterol residues, where several double bonds in an alkenyl residue are not directly adjacent to each other.
  • LPL residues preferably one or two LPL residues, which are alkyl residues with six to twenty carbon atoms, and/or mono- to tri-ethylenically unsaturated alkenyl residues with six to twenty carbon atoms, and/or saturated or mono- to tri-ethylenically
  • the POx lipids of formula (XII) have one to five LPL residues which are covalently linked directly to a POx residue via an ether or ester bond; or the POx lipids of formula (XII) have one to five LPL residues which are linked to a POx residue via a bridging group BGL, where the bridging group BGL is the residue of a di- to hexavalent aliphatic or cycloaliphatic alcohol, or the residue of a di- to hexavalent carboxylic acid, or a carbamate residue, or the residue of an amino alcohol.
  • residues of di- to hexavalent aliphatic or cycloaliphatic alcohols residues of di- to hexavalent carboxylic acids, carbamate residues and amino alcohol residues are listed above in the description of PEG lipids.
  • Preferred POx lipids have a glycerol-derived residue as a bridging group and have the structure of the formula (XI I la) or (Xlllb)
  • LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to each other,
  • R 10 , R 11 and ol have the meanings defined above, and the LPL residues can assume different meanings within a molecule within the given definitions.
  • POx lipids have a residue derived from succinic acid as a bridging group and have the structure of formula (XIV)
  • LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to each other, and
  • R 10 , R 11 and ol have the meanings defined above.
  • Preferably used stealth lipids c) have the structure of formula (XlVa)
  • R c [N(CO-R b )-CH 2 -CH 2 ] oi-O-CO-R a -CO-O-Ster(XIVa), wherein ol, R a and Ster have the meanings defined above, R b is methyl or ethyl, and
  • R c is a radical derived from an initiator of the cationic polymerization, preferably hydrogen or a monovalent organic radical, in particular an alkyl, cycloalkyl, aryl, aralkyl or heterocyclyl radical.
  • Particularly preferred stealth lipids of the formula (XlVa) have a residue ster which is substituted in the 17-position with an alkyl residue having one to ten carbon atoms, in particular with a 2,6-dimethylhexyl residue, and which is very particularly preferably a residue derived from cholesterol.
  • the POx lipids preferably used according to the invention include those with the following general structure, in which n is a number between 15 and 200, preferably between 18 and 70, and linker is a divalent bridging group.
  • LPL is an alkyl or alkenyl residue with 6-20 carbon atoms, a residue of a fatty acid, a fatty alcohol or a sterol residue,
  • BGL is a (nl+1 )-valent bridging group
  • nl is an integer from 1 to 5, preferably 1 or 2
  • ml is 0 or 1, preferably 1 and
  • POLY is a radical of the formulas (XVa), (XVIb), (XVIc), (XVId), (XVIe), (XVIf), (XVIg) or (XVIh) OR 12 Pyr
  • R 13 is hydrogen or a monovalent organic radical such as alkyl, cycloalkyl, aryl or aralkyl,
  • R 14 is hydrogen or alkyl having one to six carbon atoms, in particular hydrogen or methyl,
  • R 15 and R 16 independently of one another are hydrogen or alkyl having one to six carbon atoms, in particular alkyl having one to four carbon atoms
  • R 17 is hydrogen, alkyl having one to six carbon atoms, which is optionally substituted by a hydroxyl, amine, phenyl, hydroxyphenyl, carboxyl or amide radical,
  • NMORPH means a morphonyl radical which is connected to the carbonyl group via the ring nitrogen atom, r, s, t and u are numbers greater than or equal to 1, preferably between 1 and 5000, where the LPL residues can assume different meanings within a molecule within the given definitions.
  • Index r is preferably a number between 1 and 10, in particular a number between 1 and 4.
  • Index s is preferably a number between 10 and 5000, in particular a number between 40 and 5000.
  • Index t is preferably a number between 10 and 5000, in particular a number between 50 and 5000.
  • Index u is preferably a number between 5 and 500, in particular a number between 10 and 100.
  • Particularly preferred stealth lipids c) of the formula (XV) are those in which POLY is a radical of the formula (XVIf), in which R 14 is methyl and R 17 is hydrogen.
  • Preferably used stealth lipids c) have the structure of formula (XVIi)
  • R d -O-[CH 2 -CH] O iOR a -CO-O-Ster (XVIi), I CO-NMORPH wherein ol, R a , Ster and NMORPH have the meanings defined above and R d is hydrogen or a monovalent organic radical, in particular an alkyl, cycloalkyl, aryl, aralkyl or heterocyclyl radical.
  • Particularly preferred stealth lipids of formula (XVIi) have a residue ster which is in the 17-position with an alkyl residue having one to ten carbon atoms, in particular substituted by a 2,6-dimethylhexyl radical, and which is most preferably a radical derived from cholesterol.
  • R d [NR b -CO-CH 2 -] oi-O-CO-R a -CO-O-Ster (XVIj), wherein ol, R a , R b , Ster and R d have the meanings defined above.
  • Particularly preferred stealth lipids of the formula (XVIj) have a residue Ster which is substituted in the 17-position with an alkyl residue having one to ten carbon atoms, in particular with a 2,6-dimethylhexyl residue, and which is very particularly preferably a residue derived from cholesterol.
  • the BLNPs according to the invention can be loaded with active ingredients f) containing anionic groups.
  • the anionic groups include carboxyl groups, sulfonic acid groups and phosphate or phosphoric acid ester residues. These are characterized by the fact that they form complexes with the cationic lipids a) in an aqueous medium and are encapsulated in the BLNP or associated with it.
  • the active substances f) may include any pharmaceutical and agrochemical active substances, provided that they have at least one anionic group per molecule.
  • Preferred active ingredients f) are nucleic acids. In the context of the present description, this refers to naturally occurring nucleic acids including modified derivatives thereof. Modified nucleic acids can contain modified nucleotides or be modified in other ways, for example by introducing chemical modifications. Nucleic acids form The phosphate ester residues present therein form complexes with the cationic lipids a).
  • nucleic acids form the basis for interaction with carrier materials, and small changes in the sequence can lead to different biological effects and allow rapid adaptation to different indications without having to adapt the entire formulation process.
  • nucleic acids are biopolymers with a higher molar mass (about 333 Da per nucleotide) and a strong negative charge, which leads to good water solubility.
  • their stability in the presence of degradation enzymes is low and they can trigger an immune response based on evolutionarily optimized mechanisms that protect the organism from viral gene manipulation.
  • nucleic acids allow modulation of gene expression, whereas classical drugs often have no causal effect.
  • DNA usually exists in the double-stranded variant (dsDNA), where two single-stranded DNA chains (ssDNA) are linked together by hydrogen bonds and hydrophobic interactions between the complementary base sequences, resulting in the familiar double helix conformation.
  • dsDNA is a semi-flexible polymer with a high negative charge density.
  • DNA is often encoded in plasmids (pDNA) containing a few thousand base pairs (bp) for therapeutic applications. When pDNA enters cell nuclei, it can affect gene expression. By repairing defective genes, whether congenital or acquired, gene therapy currently offers highly specific and potentially even curative therapies for diseases with no treatment or cure.
  • siRNA short interfering RNA
  • ASO antisense oligonucleotides
  • miRNAs regulate the expression of target genes by degrading mRNA or inhibiting translation.
  • mRNA is also a single-stranded nucleotide. It contains several hundred nucleotides and is more flexible than DNA or siRNA and ssDNA. Since the bases are accessible, the mRNAs have a stronger amphiphilic character, which allows hydrophobic interactions with potential transport molecules. Unmodified mRNA is more labile to nucleases due to its single-stranded nature and has a higher immunogenicity than DNA. Therefore, modified nucleotides were proposed and chemical modifications were introduced. Both siRNA and mRNA are active in the cytoplasm and thus bypass the nuclear membrane barrier.
  • DNA and/or RNA or their modifications are preferably used as active ingredients f) in the BLNPs according to the invention.
  • DNA Any type of DNA can be used. Examples include A-DNA, B-DNA, Z-DNA, mtDNA, antisense DNA, bacterial DNA, viral DNA and especially plasmids.
  • immunomodulatory elements such as TRL antagonists, CpG motifs and other functional nucleic acids can be used.
  • RNA any type of RNA can also be used. Examples include hnRNA, mRNA, tRNA, rRNA, mtRNA, snRNA, snoRNA, scRNA, siRNA, miRNA, ncRNA, saRNA, antisense RNA, bacterial RNA and viral RNA. Combinations of DNA and RNA can also be used in the BLNPs according to the invention.
  • Modified nucleic acids also called xenonucleic acids (XNA)
  • XNA xenonucleic acids
  • XNA can exhibit increased biostability and, in addition, can increasingly be developed in vitro, accelerating lead discovery (Duffy, K.; Arangundy-Franklin, S.; Holliger, P., Modified nucleic acids: replication, evolution, and next-generation therapeutics. BMC Biol. 2020,18, 112).
  • Preferred nanoparticles according to the invention are characterized by a high content of active ingredient f), preferably nucleic acid.
  • the weight proportion of active ingredient f) in the LNP according to the invention is typically 1 to 10%, and preferably 2 to 8%, in particular 3 to 7%, particularly preferably 5 to 6%, based on the mass of the LNP loaded with active ingredient.
  • the nanoparticles according to the invention can be characterized by their particle diameter.
  • Typical particle diameters are in the range of less than or equal to 900 nm, preferably less than or equal to 500 nm, particularly preferably between 30 and 500 nm, very particularly preferably between 40 and 250 nm and in particular between 50 and 200 nm.
  • the particle diameters are determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, United Kingdom).
  • the intensity-weighted mean diameter was determined by means of cumulant analysis of the correlation function (ISO13321, ISO22412). For size determination, a refractive index of 1.33 was assumed for ultrapure water.
  • Particle diameters can alternatively be determined by other methods, for example by nanosize tracking analysis (NTA), or by electron microscopy, e.g. by transmission electron microscopy or by scanning electron microscopy.
  • NTA nanosize tracking analysis
  • electron microscopy e.g. by transmission electron microscopy or by scanning electron microscopy.
  • Particle diameters (z-average) of preferred LNPs according to the invention are in the range between 30 and 500 nm, determined by dynamic light scattering (DLS).
  • the BLNPs according to the invention can also be characterized by their polydispersity index (or PDI).
  • PDI indicates the width of the distribution of the particle sizes of particles. Values between 0 (monodisperse) and 1 (polydisperse) can be assumed.
  • the PDI value is determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, United Kingdom). PDI was determined by means of cumulant analysis of the correlation function.
  • the PDI value of the particle size distribution of the nanoparticles according to the invention typically ranges between 0.01 and 0.4, preferably between 0.02 and 0.3 and particularly preferably between 0.05 and 0.2.
  • the nanoparticles according to the invention can be further characterized by their N/P ratio. This is the molar ratio of nitrogen atoms in the cationic lipid a) to phosphate groups in the active ingredient e), for example in the nucleic acid.
  • the N/P ratio in the nanoparticles according to the invention can vary within wide ranges. Typically, the N/P ratio in the nanoparticles according to the invention is between 1 and 100, preferably between 1.5 and 50, particularly preferably between 2 and 25, and most preferably between 3 and 15.
  • Preferred nanoparticles according to the invention have diameters determined by means of DLS (z-average) between 40 and 250 nm, in particular between 50 and 200 nm, and a polydispersity index of the particle diameters between 0.05 and 0.3.
  • Very particularly preferred nanoparticles according to the invention have diameters determined by means of DLS (z-average) between 40 and 250 nm, in particular between 50 and 200 nm, and a polydispersity index of the particle diameters between 0.05 and 0.2 and an N/P ratio between 3 and 15.
  • nanoparticles according to the invention contain, in addition to the nucleic acid-lipid complexes described above, additional polymers or additional complexes of nucleic acids with additional polymers, these further components are present only in small amounts, for example their weight proportion is 10% or less, in particular less than 5%.
  • the nanoparticles according to the invention do not contain any further complexes of active ingredients with other polymers in addition to the active ingredient-lipid complexes described above.
  • the BLNPs of the invention may be in solid form as a powder or they may form a dispersion and be dispersed in aqueous solvents, the particles being in solid form in the dispersing medium.
  • the BLNPs according to the invention form a disperse phase in water or in an aqueous buffer solution.
  • the BLNPs according to the invention can be produced by assembly.
  • the lipids used according to the invention are dispersed in water or in an aqueous buffer solution.
  • One dispersion can be produced for each lipid or all lipids are dispersed together.
  • the pH of the aqueous dispersion is set to 3 to 8, preferably 4 to 7.5, e.g. by using an acetate buffer or another suitable buffer such as citrate buffer, lactate buffer, phosphate buffer and phosphate-citrate buffer.
  • the active ingredients f) containing anionic groups are dissolved or dispersed in water, the pH of the aqueous active ingredient solution or dispersion preferably being set to a value between 3 and 8, particularly preferably to a value between 4 and 7.5.
  • a buffer solution containing acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer, HEPES, TRIS, or just salts is particularly suitable for this purpose.
  • the aqueous dispersions of the lipids and the active ingredient solution or dispersion are combined with one another, with the amounts of active ingredient and cationic lipid a) being selected so that a desired active ingredient/lipid ratio, e.g.
  • a desired N/P ratio is established.
  • the mixture is agitated, for example for a short time between 2 and 120 seconds. This can be done by stirring and/or by vortexing and/or by sonication with ultrasound.
  • the resulting nanoparticles are preferably left to stand for some time before further use, for example between 5 and 20 minutes, to enable a bond between lipid a) and active ingredient e) (hereinafter referred to as "incubation").
  • the LNPs are preferably neutralized, for example by mixing with phosphate buffered saline (PBS) to pH 7.4.
  • PBS phosphate buffered saline
  • the nanoparticles according to the invention can then be lyophilized from the dispersion medium or remain in the dispersion medium.
  • the BLNPs according to the invention can be produced by nanoprecipitation, whereby the BLNPs initially contain only lipids and the active ingredient f) is added in a subsequent step.
  • the BLNPs are produced as described above, but without adding the solution or dispersion of the active ingredient f).
  • the solution or dispersion of the active ingredient f) is then prepared as described above.
  • aqueous dispersion of the BLNP and the active ingredient solution or dispersion are then combined with one another, with the amounts being chosen so that a desired active ingredient/lipid ratio, e.g. a desired N/P ratio, is achieved.
  • the mixture is agitated, for example for a short time between 2 and 120 seconds. This can be done by stirring and/or vortexing and/or by sonicating with ultrasound.
  • the resulting active ingredient-loaded nanoparticles are left to stand for some time before further use, for example between 5 and 20 minutes, to enable a bond between lipid a) and active ingredient e) (hereinafter referred to as "incubation").
  • acidic pH e.g.
  • the BLNPs are preferably neutralized, for example by mixing with phosphate-buffered saline (PBS) to pH 7.4.
  • PBS phosphate-buffered saline
  • the nanoparticles according to the invention can then be lyophilized from the dispersion medium or remain in the dispersion medium.
  • auxiliary and additive substances e) may be present in the dispersion medium during their nanoprecipitation.
  • these auxiliary and additive substances e) can be added after the nucleic acid copolymer complex has been dispersed in the aqueous phase. Water is used as a dispersing medium. Buffer substances, salts, sugars or acids and bases can be added to this in order to adjust the desired pH value or osmolarity.
  • the processes according to the invention are characterized by the fact that the use of organic solvents such as ethanol can be dispensed with during the production of the lipid nanoparticles. A subsequent separation of the solvent can therefore be omitted.
  • the invention also relates to a process for producing the BLNP described above, comprising the following measures: i) initial charging of aqueous dispersions of the lipids a), b) and c) in buffers in the pH range from 3 to 8, preferably 4 to 7.5 ii) combination of the aqueous dispersions from step i); and iii) treatment of the combined aqueous dispersions from step ii) with a mixing process selected from the group consisting of ultrasound, dual centrifugation, nanoprecipitation, microfluidics or in a vortex mixer, whereby the nanoparticles are formed.
  • the invention further relates to a process for producing the above-described BLNPs loaded with anionic group-containing active ingredient f), comprising the following measures: iv) initial charge of the LNP-containing aqueous dispersion from step iii) according to the above process, v) initial charge of an aqueous solution or dispersion of an active ingredient f) with anionic groups in a buffer in the pH range from 3 to 8, preferably 4 to 7.5, vi) combination of the aqueous dispersions or solutions from steps iv) and v), and vii) treatment of the combined aqueous dispersions or solutions from step vi) with ultrasound, microfluidics, dual centrifugation, ultrasound, Nanoprecipitation or in a vortex mixer, whereby the active ingredient-loaded LNPs are formed.
  • the invention relates to a process for the preparation of the above-described BLNPs loaded with anionic groups containing active ingredient f), comprising the following measures:
  • step IV) Treatment of the combined aqueous dispersions or solutions from step III) with ultrasound, microfluidics, dual centrifugation, nanoprecipitation or in a vortex mixer, whereby the drug-loaded LNPs are formed.
  • this includes the following measures:
  • step iii) introducing the LNP-containing aqueous dispersion having a pH value between 3 to 8, preferably 4 to 7.5 from step iii) prepared according to the above process,
  • the aqueous dispersions of the lipids a), b) and c) for steps i) or I) of the processes according to the invention preferably contain a buffer, in particular an acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer or mixtures thereof.
  • the aqueous solution or dispersion of a nucleic acid for steps v), II) or VI) of the process according to the invention preferably has a pH of 3 to 8, preferably 4 to 7.5.
  • the aqueous solution or dispersion of the nucleic acid for steps v), II) or V) of the process according to the invention preferably contains a buffer, in particular an acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer, HBG, HEPES or TRIS buffer.
  • a buffer in particular an acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer, HBG, HEPES or TRIS buffer.
  • the agitation in steps iii), vii), IV) or VIII) of the process according to the invention is preferably carried out by stirring or vortexing.
  • the treatment time in this step is usually between 1 and 120 seconds, in particular between 2 and 60 seconds.
  • step IX) of the process according to the invention is usually carried out by simply allowing the resulting mixture to stand, for example for a period of 5 to 60 minutes, preferably 5 to 20 minutes.
  • the mixture can also be incubated in a refrigerator or heating cabinet, for example at temperatures between 1 °C and 80 °C.
  • the nanoparticles can be separated from the aqueous phase in different ways. Examples are crossflow filtration, centrifugation, ultrafiltration or dialysis.
  • the dispersion of the nanoparticles can also be used preferably directly after production without further processing. By cleaning by filtration, particles such as aggregates, but also excess auxiliary materials or impurities can be separated from the dispersion. The particle concentration can change in the process.
  • Dissolved molecules can be separated from the dispersion by cleaning using dialysis/crossflow filtration. The process is largely independent of the particle size with regard to the dispersed particles.
  • Dissolved molecules can also be separated from the dispersion by cleaning using centrifugation. However, this process also reduces the concentration of the dispersed particles. In addition, only dispersions with nanoparticles of larger diameter, e.g. more than 150 nm, can be treated and the particles can be damaged. Furthermore, redispersing the particles obtained in this way can be difficult.
  • the active ingredient-loaded BLNPs according to the invention are ideally suited as vehicles for the transport of pharmaceutical and agrochemical active ingredients.
  • the BLNPs loaded with active substances according to the invention are suitable for gene transfer into cells, i.e. for introducing nucleic acids and their functional release into cells.
  • the LNPs containing nucleic acids are added to individual cells, tissues or a cell culture and taken up by the cells through endocytosis.
  • it has been shown that high levels of nucleic acids can be transferred into cells using the BLNPs according to the invention.
  • the invention therefore also relates to a method for gene transfer into cells, which comprises the following steps: A) contacting cells, tissues or cell cultures with an aqueous dispersion containing the LNPs containing the nucleic acids described above, and
  • the invention relates to a method for gene transfer into cells, which comprises the following steps:
  • the gene transfer method according to the invention can be carried out using different cells, for example by using single cells, tissues or cell cultures.
  • the BLNPs loaded with nucleic acids according to the invention can be combined with prokaryotic or eukaryotic cells, with tissues from eukaryotic cells or with cell cultures. These can be plant cells or, preferably, animal cells, including human cells.
  • the application of the LNP loaded with nucleic acids according to the invention can be carried out in vivo, for example under the skin or in the muscle, or the application can also be carried out ex vivo, for example with immune cells, as in CAR-T therapy. It can also be an RNA vaccination or another vaccination.
  • parts are understood to mean the smallest living units of organisms. These can be cells of single- or multicellular organisms, which originate from prokaryotes or eukaryotes.
  • the cells can be microorganisms or individual cells. Cells can be of prokaryotic, plant or animal origin or even come from fungi. Eukaryotic cells are preferably used, especially those that were originally isolated from tissue and can be permanently cultivated, i.e. that are immortalized.
  • tissues are understood to mean collections of differentiated cells including their extracellular matrix.
  • cell cultures refers to combinations of cells or tissues and cell culture medium, whereby the cells or tissues are cultivated in the cell culture medium outside the organism.
  • Cell lines are used, i.e. cells of a tissue type that can divide during the course of cultivation. Both immortalized cell lines and primary cells (primary culture) can be cultivated.
  • Primary culture is usually understood to mean a non-immortalized cell culture that was obtained directly from a tissue.
  • the cell cultures used according to the invention can be produced and cultivated according to standard methods.
  • primary cultures can be created from different tissues, for example from tissues of individual organs such as skin, heart, kidney or liver, or from tumor tissue.
  • the tissue cells can be isolated using methods known per se, e.g. by treatment with a protease, which breaks down the proteins that maintain the cell association. It may also be appropriate to stimulate certain cell types to divide by adding growth factors or, in the case of poorly growing cell types, to use feeder cells, basement membrane-like matrices or recombinant components of the extracellular matrix.
  • the cells used according to the invention can also be genetically modified by introducing a plasmid as a vector.
  • the cells used according to the invention can have a limited lifespan or they can be immortal cell lines with the ability to divide infinitely. These can be generated by random mutation, e.g. in tumor cells, or by targeted modification, for example by the artificial expression of the telomerase gene.
  • the cells used according to the invention can be adherent (on surfaces) growing cells, such as fibroblasts, endothelial cells or cartilage cells, or they can be suspension cells that grow freely floating in the nutrient medium, such as lymphocytes.
  • Culture conditions and cell culture media are selected depending on the individual cells being cultured.
  • the different cell types prefer different nutrient media, which are specifically composed.
  • different pH values are set and the individual nutrient media can contain different amino acids and/or other nutrients in different concentrations.
  • the cells transfected according to the invention can be used in various areas, for example biotechnology, research or medicine, veterinary medicine. This can involve the production of (recombinant) proteins, virus and/or virus particle production, investigation of metabolism, division and other cellular processes. Furthermore, the cells transfected according to the invention can be used as test systems, for example in the investigation of the effect of substances on cell properties, such as signal transduction or toxicity. Other cells preferably used to produce the cells transfected according to the invention are stem cells. These are body cells that can differentiate into different cell types or tissues.
  • the invention also relates to the use of the LNPs containing the nucleic acids described above for gene transfer into cells, i.e. for introducing nucleic acids and their functional release into cells.
  • the property of the cationic lipids to form a homogeneous, disperse system in acidic aqueous solution e.g. 20 mM NaOAc, pH 5.5
  • the phospholipid e.g. DSPC
  • the stealth lipids such as PEG lipids or POx lipids, can also be transferred to the formulation in this way, since they are soluble in the buffer mentioned above. Cholesterol was omitted, since this molecule is insoluble in water and is not required for the construction of the lipid nanoparticles according to the invention.
  • the particles formed by ultrasound treatment differ significantly in shape and size from lipid nanoparticles that are produced by simply mixing the mixture.
  • the unloaded lipid nanoparticles can be loaded with the desired active ingredient, e.g. the desired genetic material, in a subsequent process step.
  • the desired active ingredient e.g. the desired genetic material
  • both pDNA and RNA were used.
  • the genetic material is diluted in e.g. 20 mM NaOAc buffer, pH 5.5 (MM).
  • MM mM NaOAc buffer
  • nanoparticle suspension and MM were combined after a defined incubation period by quickly transferring them into one another and mixing them using a vortexer.
  • the functionality of the lipid nanoparticles loaded with genetic material according to the invention was demonstrated with regard to protein expression. After it was shown that the lipid nanoparticles according to the invention are basically suitable for transfections with pDNA, the formulation was further optimized with regard to biocompatibility.
  • the main advantage of the lipid nanoparticles according to the invention is that the method according to the invention enables the production of lipid nanoparticles for gene transfer without the use of organic solvents. This leads to a reduction in production costs, since the rapid removal of the solvents, for example by dialysis, is no longer necessary. In addition, the genetic material is protected.
  • Figures 1A to 1D show the structures of the lipids used in the LNPs used by BioNTech and Moderna.
  • Figure 1A shows the structure of the phospholipid DSPC.
  • Figure 1B shows the structures of the ionizable cationic lipids ALC-0315 and SM-102.
  • Figure 1C shows the structures of the ethoxylated lipids PEG-DMG and ALC-0159.
  • Figure 1D shows the structure of the lipid cholesterol.
  • SM-102 has the molecular formula C44H87NO5 and a molecular weight of 710.18.
  • DSPC has the molecular formula C44H 88 NO 8 P and a molecular weight of 790.16.
  • PEG-DMG has the molecular formula C124H246O51 and a molecular weight of 2553.28.
  • the HLB value calculated according to Griffin is 17.13.
  • Cholesterol has the molecular formula C 2 7H 46 O and a molecular mass of 386.66.
  • composition of the LNP used by BioNTech corresponds to 46.3 mol% ALC-0315, 9.4 mol% DSPC, 42.7 mol% cholesterol and 1.6 mol% ALC-0159.
  • the composition of the LNP used by Moderna corresponds to 50 mol % SM-102, 10 mol % DSPC, 38.5 mol % cholesterol and 1.5 mol % PEG-DMG (see Schoenmaker, L.; Witzigmann, D.; Kulkarni, J. A.; Verbeke, R.; Kersten, G.; Jiskoot, W.; Crommelin, D. J. A., mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 2021 , 601, 120586).
  • Figure 2 shows the schematic structure of an apparatus used for microfluidics (cf. Maeki, M.; Uno, S.; Niwa, A.; Okada, Y.; Tokeshi, M., Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery. J. Control. Release 2022, 344, 80-96).
  • This is an example of a standard microfluidic apparatus for the production of conventional lipid nanoparticles.
  • Figure 3 shows the schematic structure of a commonly used LNP composed of the known lipids in known proportions.
  • FIG. 4 shows a scheme of the method according to the invention.
  • a first approach to production was based on a simple mixing process (vortexing), in which various ionizable lipids were mixed with genetic material and tested for transfection. It turned out that the cationic lipid SM-102 gave the best results, but these were rather below average.
  • the helper lipid DSPC and a stealth lipid (here Chol-POx) were added to the formulation.
  • Chol-POx is the polymer shown below
  • Figure 5 shows the schematic structure of an LNP according to the invention composed of cationic lipid, helper lipid and stealth lipid, which contains genetic material as an active ingredient.
  • Figure 6 shows the measurement results obtained by DLS for the LNP produced by vortexing.
  • the production was carried out according to the procedure outlined in Figure 4.
  • the influence of the individual components of the formulation on the particle size was investigated.
  • the combinations were produced from
  • FIG 7 shows the results of the transfection experiments.
  • the nanoparticles used are LNPs according to the invention, which were produced analogously to the particles in Figure 6. These particles were transferred to cells to measure the transfection. The particles with stealth lipid and phospholipid are the most efficient. After weighing up the material usage, a formulation with N/P 6 turned out to be optimal.
  • Figure 8 outlines another method for producing the LNPs according to the invention.
  • the aim was to reduce the size of the resulting LNPs and to further improve the efficiency of gene transport.
  • treatment with ultrasound turned out to be the most suitable.
  • the lipid mixture is treated with ultrasound and significantly smaller, still unloaded lipid particles are created, which are then loaded with genetic material.
  • Step 1) Lipid suspensions are prepared, 2) LNP assembly by ultrasound treatment, 3) dilution of the genetic material, 5 seconds vortexing, 4) 10 minutes incubation at room temperature, combining lipids and genetic material, 5) rapid vortexing.
  • Figures 9 and 10 show particle diameters and PDI values of LNP.
  • the LNPs investigated contain different lipids or lipid combinations as described for Figure 6.
  • LNPs produced using the methods outlined in Figures 4 (vortexing) and 8 (ultrasonic treatment) are compared. It turned out that the particles produced using the latter method are significantly smaller.
  • All measurement data for the LNP shown below refer to a molar composition of 82.3 mol% SM-102, 16.5 mol% DSPC and 1.2 mol% Stealth Lipid or 81.3 mol% SM-102, 16.3 mol% DSPC and 2.4 mol% Stealth Lipid.
  • the transfection experiments shown were carried out using HEK293T cells. The incubation period is 24 h at 37 °C and the measured values were recorded using flow cytometry.
  • Figure 11 shows the raw data from the measurement using a flow cytometer after transfection.
  • the LNPs according to the invention using the method shown in Figure 4 (vortexing) (A-C) are compared with the LNPs according to the invention using the method shown in Figure 8 (ultrasound treatment) (D-F).
  • Figure 11 B 7.0.05%), the LNPs produced using ultrasound show a significantly higher transfection efficiency of 88.92% ( Figure 11 E).
  • Figure 12 shows the relative mean fluorescence intensities to the raw data from Figure 11.
  • the horizontally striped bars represent the LNPs that were produced using the method shown in Figure 8.
  • the LNPs according to the invention show clear fluorescence values (400x - 900x more than the negative control).
  • a polyplex made of polyethyleneimine (PEI) was compared with the LNPs to better classify the results. It can be seen that the LNPs produced using ultrasound are superior to the PEI polyplex.
  • the LNPs produced using the method shown in Figure 8 (ultrasound) are clearly superior to the LNPs produced using the method shown in Figure 4 (vortexing).
  • Figure 13 shows results obtained with LNPs prepared using different buffers.
  • the results shown in the previous figures were obtained with LNPs prepared at pH 4.0 in citrate buffer (50 mM). Under these circumstances, 50 mM citrate buffer can only be adjusted to pH 7.4 with great effort (e.g. dialysis). neutralize. To facilitate this process, it is necessary to change the buffer systems used. Therefore, other buffer systems were investigated. TRIS, PBS (pH 7.4) and NaOAc (pH 5.5) were tested as solvents for the lipid suspensions. At the same time, the genetic material was dispersed in various buffers (MM).
  • MM buffers
  • HBG (20 mM, pH 7.4), TRIS (20 mM, pH 7.4), PBS (pH 7.4) and NaOAc (20 mM, pH 5.5) were used.
  • LNPs were prepared for this series of experiments by vortexing.
  • the advantage of using 20 mM NaOAc is that it is much easier to neutralize than citrate buffer. This solvent combination was used for the further experiments.
  • Figures 14 and 15 show the particle diameters (columns) and PDI values (points) of LNP.
  • Figure 14 once again illustrates the advantage of the ultrasound method, as significantly smaller particle sizes can be achieved.
  • the following study examined whether changing the buffer system from citrate buffer to NaOAc produces similar particle sizes.
  • Figure 15 shows that this is the case (similar sizes).
  • the buffer systems were varied and the nanoparticles were measured in the same way using DLS after production.
  • Figure 16 shows the transfection efficiency (relative mean fluorescence intensity) of the LNPs produced with NaOAc buffer.
  • the buffer was acidified once with an equimolar amount of hydrochloric acid (HCl) and adjusted to pH 4.0 to test whether the specific pH value of the formulation influences the effectiveness. It can be seen that the values are highest for pH 5.5. This means that further adjustment of the buffer system is unnecessary and for further optimization, work was continued with 20 mM NaOAc at pH 5.5. For further application, it is imperative that the formulation can be neutralized from pH 5.5 to pH 7.4.
  • Figure 17 shows how the pH 5.5 formulation can be neutralized without affecting the transfection efficiency.
  • a preferred preparation of the LNP therefore involves preparing the nanoparticles at pH 5.5 using 20 mM NaOAc followed by neutralization by adding PBS or TBS.
  • Figure 18 shows the results of the toxicity measurements.
  • unloaded LNPs were produced according to the method in Figure 8.
  • Two different stealth polymers were used for this purpose.
  • the test was carried out on L929 cells and these were incubated with LNP suspensions in various concentrations up to 1000 pM for 24 hours and then evaluated using the Presto-Blue assay. It can be seen that the LNPs according to the invention do not exhibit any toxicity in the tested range.
  • Figures 19 and 20 show DLS measurements (particle sizes) of the LNP, produced analogously to the description for Figure 18. Unloaded particles were measured (BLANK) as well as particles loaded with mRNA and pDNA. The latter were measured both at pH 5.5 (NaOAc) and after neutralization with PBS (pH 7.4). It can be seen that the choice of genetic material has no influence on the size. Neutralization leads to a slight increase in the sizes.
  • Figures 21 and 22 show the PDI values of the LNPs described in Figures 19 and 20.
  • Figure 23 shows the proportion of EGFP positive cells in a transfection experiment depending on the amount of genetic material used.
  • BLNP with the stealth lipid Chol-POx were produced as shown in Figure 8 and loaded with mRNA and pDNA. It can be seen that the BLNP according to the invention are clearly superior to PEI.
  • Figure 24 lists possible and already used stealth lipids.
  • BLNPs containing 81.3 mol% ionizable/cationic lipid (SM-102), 16.3 mol% phospholipid (DSPC), and 2.4 mol% stealth lipid were used.
  • Figure 25 shows that the polymers CHEMS-PEtOxn, CHEMS-PEGn, CHEMS- PMeOxn listed above are very well suited for use in BLNPs. This was demonstrated by the high transfection efficiencies. BLNPs were prepared by sonication as described above and the transfection efficiency was measured using mRNA.
  • Figure 26 shows that the polymers CHEMS-PEtOx n , CHEMS-PEGn, CHEMS-PMeOx n and DMG-PEG listed above differ significantly in their biocompatibility.
  • the PMeOx and PEtOx variants of the CHEMS-Stealth lipid are significantly less cytotoxic than the PEG variants.
  • PEG is more cytotoxic than POx.
  • the test carried out is based on ISO 10993-5 and measures the cytotoxicity represented by the relative metabolic activity of L929 cells after 24 h of incubation with the corresponding material in the specified concentration.
  • Figure 27 shows another specific application of BLNP.
  • BLNP were loaded with a Cas9 mRNA and a GFP specific gRNA and applied to GFP + HEK-293T cells.
  • KO knock-out
  • Figure 28 shows that BLNPs are also suitable for use on primary human immune cells.
  • BLNPs were applied to isolated human primary leukocytes and incubated for 18 hours. The cells were then examined using antibody staining via flow cytometry. It was demonstrated that transfection only occurred in the CD45/CD14 positive cells and thus in monocytes.
  • Figure 29 combines the expertise of the experiments from Figures 27 and 28.

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Abstract

L'invention concerne des nanoparticules lipidiques contenant des quantités sélectionnées d'a) au moins un lipide cationique, b) au moins un phospholipide, et c) au moins un lipide furtif sélectionné, à condition que tous les lipides contenus dans la nanoparticule lipidique aient une valeur HLB supérieure ou égale à 3. Ces nanoparticules peuvent être chargées avec des substances actives contenant des groupes anioniques, de préférence avec des acides nucléiques, et sont appropriées en tant que véhicules hautement efficaces pour le transfert des acides nucléiques dans des cellules.
PCT/EP2024/000028 2023-05-12 2024-05-10 Nanoparticules pour le transport de substances actives comprenant des groupes anioniques, leur procédé de production et leur utilisation Pending WO2024235481A1 (fr)

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