Nanoparticles for transporting active substances having anionic groups, method for production thereof and use thereof

Abstract

The invention relates to nanoparticles for transporting active substances having anionic groups, to a method for the production thereof, and to the use thereof. A lipid nanoparticle is described containing selected amounts of a) at least one cationic lipid, b) at least one phospholipid, and c) at least one selected stealth lipid, with the proviso that all the lipids contained in the lipid nanoparticle have an HLB value greater than or equal to 3. These nanoparticles can be loaded with an active material containing anionic groups, preferably with nucleic acids, and are suitable as efficient carriers for transferring nucleic acids into cells.

Description

Technical Field

[0001] This invention relates to the field of preparing and processing nanoparticles that can be loaded with active substances, such as genetic material. These nanoparticles can be advantageously used to transport active substances into organisms, particularly for transporting nucleic acids into cells. Background Technology

[0002] Vaccine antigens, especially purified or recombinant subunit vaccines, are often poorly immunogenic and require adjuvants to stimulate protective immunity. While currently approved adjuvants have been successful, improved adjuvants and delivery systems to enhance protective antibody responses are still needed, particularly in populations that do not respond well to current vaccines.

[0003] Lipid nanoparticles (LNPs) represent alternatives to other particulate systems such as emulsions, liposomes, micelles, microparticles, and / or polymer nanoparticles for the delivery of active substances such as oligonucleotides and low molecular weight drugs. LNPs and their uses for the administration of active substances have been described, for example in US 7,691,405, US 2006 / 0083780, US 2006 / 0240554, US2008 / 0020058, US 2009 / 0263407, US 2009 / 0285881, WO 2009 / 086558, WO2009 / 127060, WO2009 / 132131, WO2010 / 042877, WO2010 / 054384, WO2010 / 054401, WO2010 / 054405 and WO2010 / 054406. Lipid-based nanoparticles as carriers of pharmaceutically active substances are also described, for example in Puri, A.; Loomis, K.; Smith, B.; Lee, JH; 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.

[0004] In recent years, gene therapy using siRNA and mRNA has become increasingly important (see Akinc, A.; Maier, MA; Manoharan, M.; et al., The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat. Nanotechnol. 2019, 14, 1084-1087; Polack, FP; Thomas, SJ; Kitchin, N., et al., Safety and efficiency of the BNT162b2 mRNA Covid-19 vaccine. N. Engl. J. Med. 2020, 383, 2603-2615; and Baden, LR; El Sahly, HM; Essink, B., et al., Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 2021, 384, 403-416).

[0005] In this context, a wide variety of nucleic acids, such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA, or ASO), must be supplied. These differ from classical active substances due to their high molar mass and negative charge, necessitating transport proteins (Kulkarni, JA; Witzigmann, D.; Thomson, SB; Chen, S.; Leavitt, BR; Cullis, PR; van der Meel, R., The current landscape of nucleic acidtherapeutics. Nat. Nanotechnol. 2021, 16, 630-643). However, the application of gene transfer for curing, treating, or preventing a wide range of diseases is limited by technological and biological barriers. Due to the high renal clearance, instability, and immune activation potential of genetic material, carrier systems are required. Various nanomaterials have been developed for this purpose to protect nucleic acids from degradation and nonspecific immune responses or to purposefully transport them to application sites or cell types.

[0006] Recently, with the outbreak of the COVID-19 pandemic in early 2020, lipid nanoparticles (LNPs) have established themselves as an important drug form for the transport of genetic material (see 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 treatments have been approved or entered into clinical trials during this period (see 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). To name just a few: Fomivirsen (Vitravene) is an antisense oligonucleotide (ASO) that targets a specific sequence of cellular RNA and was approved in 1998 for the treatment of cytomegalovirus infection of the retinas in AIDS patients (withdrawn in 2002) (Roberts, TC; Langer, R.; Wood, MJA, Advances in oligonucleotide drug delivery. Nat. Rev. Drug Discov. 2020, 19, 673-694); Mipomersen (Kynamro) is an ASO that is being used as an orphan drug for hypercholesterolemia.Patisiran (Onpattro) is a representative of the first-in-class RNA interference-based novel drug approved in 2018 for the treatment of hereditary thyroxine transporter amyloidosis in patients with stage 1 or 2 polyneuropathy (Buck, J.; Grossen, P.; Cullis, PR; Huwyler, J.; Witzigmann, D., Lipid-based DNA therapeutics: Hallmarks of non-viral gene delivery. ACS Nano 2019, 13, 3754-3782). In addition, a plasmid encoding human hepatocyte growth factor has been approved for the treatment of patients with severe limb ischemia.

[0007] The approval of novel RNA-based systems has further advanced the development of non-viral administration systems, such as N-acetylgalactosamine (GalNAc)-siRNA conjugates: i) Givosiran (Givlaari) for the treatment of acute intermittent hepatic porphyria, ii) Lumasilan (Oxlumo) for the treatment of primary type 1 hyperoxaluria, and iii) Inclisiran (Leqvio), a subcutaneous treatment for hypercholesterolemia. Other RNA-based systems include lipid-based siRNA drugs and 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).

[0008] The LNPs of currently approved vaccines consist of four different lipid components and genetic material (see 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.; Witzigmann, D.; Kulkarni, JA, et al., mRNA-lipidnanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 2021, 601, 120586).

[0009] These are ionizable lipids, cofactor lipids 1,2-distearate-sn-glycerol-3-phosphocholine and cholesterol, and lipids conjugated with polyethylene glycol (PEG). The lipids used in the cases of BNT162b (BioNTech) and mRNA-1273 (Moderna) achieve various functions in LNP formulations and are based on intensive lipid screening (Dolgin, E., Thetangled history of mRNA vaccines. Nature 2021, 597, 318-324).

[0010] For transport, conserved negatively charged phosphate groups in the RNA and DNA backbones can be used for lipid interactions. Cationic lipids play a crucial role in this context because they are responsible for binding to negatively charged genetic material. Furthermore, they influence endosome uptake (see Hald 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. 2022, 188, 114416; and Paloncyova, M.; Cechova, P.; Srejber, M., et al., Role of ionizable lipids in SARS-CoV-2 vaccines as revealed by molecular dynamics simulations: From membrane structure to interaction with mRNA fragments. J. Phys. Chem. Lett. 2021, 12, 11199-11205).

[0011] The development of ionizable lipids is considered a breakthrough for the widespread use of LNPs (Dolgin, E., The tangled history of mRNA vaccines. Nature 2021, 597, 318-324). Initially, cationic lipids achieved high transfection efficiency in cell cultures but caused toxicity. In vivo, they have short half-lives in the bloodstream and non-specific binding to cell surfaces. To overcome this obstacle, so-called ionizable lipids with pKa values ​​below 7 were developed. These lipids bind to genetic material via pH-dependent formulations that begin at lower pH levels to allow binding and slowly increase to physiological pH. The formulations are neutral when administered, e.g., intravenously or intramuscularly, and are charged in the acidic environment of the endosomes, which facilitates endosome release. Protonation in endosomes facilitates good interaction with lipids in the endosome membrane (Han, X.; Zhang, H.; Butowska, K.; Swingle, KL; Alameh, M.-G.; Weissman, D.; Mitchell, MJ, An ionizable lipidtoolbox for RNA delivery. Nat. Commun. 2021, 12, 7233). After optimization, ionizable lipids showed promising results even with limited genetic material. Regarding ionizable head groups, dimethylamino bases showed high transfection efficiency (Mo, R.; Sun, Q.; Li, N.; Zhang, C., Intracellular delivery and antitumor effects of pH-sensitive liposomes based on zwitterionicoligopeptide lipids. Biomaterials 2013, 34, 2773-2786).

[0012] Various ionizable lipids exist, each with different gene transfer potential in LNPs (Semple, SC; Akinc, A.; Chen, J., et al., Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 2010, 28, 172-176). Influencing factors include, for example, the length of the alkyl chain, the type of ionizable group, and the molecule's pK. aValue (see Hald 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. 2022, 188, 114416; Zhang, Y.; Sun, C.; Wang, C.; Jankovic, KE; Dong, Y., Lipids and lipid derivatives for RNA delivery. Chem. Rev. 2021, 121, 12181-12277; and Whitehead, KA; Dorkin, JR; Vegas, AJ, et al., Degradable lipid nanoparticles withpredictable in vivo siRNA delivery activity. Nat. Commun. 2014, 5, 4277).

[0013] Stealth surface modification of particles is crucial for minimizing undesirable interactions with the biological environment or the immune system. The term "stealth," dating back to early research in the 1980s, describes the ability to "hide" particles by functionalizing them with hydrophilic molecules to evade recognition and elimination by the immune system. The stealth effect is achieved by restricting 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.; DeRosa, G.; Bernkop-Schnürch, A., Bioinert, Stealth or interactive: How surface chemistry of nanocarriers determines their fate in vivo, Adv. Func. Mater. 2021, 31, 2103347).

[0014] Stealthy nanoparticles impact drug delivery, particularly in cancer treatment. In 1994, Langer et al. presented PEG-grafted polymer nanoparticles that could circulate in the bloodstream for longer periods due to PEG passivation (Gref, R.; Minamitake, Y.; Peracchia, MT; Trubetskoy, V.; Torchilin, V.; Langer, R., Biodegradable long-circulating polymeric nanospheres. Science 1994, 263, 1600-1603.). By forming a hydration layer and spatial barrier, PEGylation reduces the non-specific binding of serum proteins to the particles, thereby reducing their excretion by the mononuclear phagocytic system (MPS). Due to the enhanced penetration and retention (EPR) phenomenon, these long-circulating nanoparticles have been shown to be beneficial for drug delivery to the tumor microenvironment (Suk, JS; Xu, Q.; Kim, N.; Hanes, J.; Ensign, LM, PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv. Drug Delivery Rev. 2016, 99, 28-51).

[0015] Many alternatives to PEGylated compounds are known. These typically exhibit prolonged systemic circulation, sustained active substance release kinetics, and better tumor accumulation. Examples of alternative polymers to PEG include poly(glycerol) (PG), poly(oxazoline) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethylacrylamide) (PDMA), and poly(N-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, 12, 298).

[0016] Among so-called stealth lipids, PEG lipids are by far the most important and widely used. PEGylated lipids are particularly responsible for prolonging retention time in organisms and reducing the immune response to drugs in LNP formulations (see 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. MedChemComm 2019, 10, 369-377; Bao, Y.; Jin, Y.; Chivukula, P.; Zhang, J.; Liu, Y.; Liu, J.; Clamme, JP; Mahato, RI; Ng, D.; Ying, W.; Wang, Y.; Yu, L., Effect of PEGylation on biodistribution and gene silencing of siRNA / lipid nanoparticle complexes. Pharm. Res. 2013, 30,342-351.; Suzuki, T.; Suzuki, Y.; Hihara, T.; Kubara, K.; Kondo, K.; Hyodo, K.; Yamazaki, K.; Ishida, T.; Ishihara, H., PEG shedding-rate-dependent bloodclearance of PEGylated lipid nanoparticles in mice: Faster PEG sheddingattenuates anti-PEG IgM production. Int. J. Pharm. 2020, 588, 119792).Furthermore, it was shown that they have a significant impact on the size and stability of the resulting nanoparticles and stabilize the formulation (see Mui, BL; Tam, YK; Jayaraman, M., et al., Influence of polyethylene glycol lipid desorption rates on pharmacokinetics and pharmacodynamics of siRNA lipid nanoparticles. Mol. Ther. Nucleic Acids 2013, 2, e139; Holland, JW; Hui, C.; Cullis, PR; Madden, TD, Poly(ethylene glycol)-lipid conjugates regulate the calcium-induced fusion of liposomes composed of phosphatidylethanolamine and phosphatidylserine. Biochemistry 1996, 35, 2618-2624; Kauffman, KJ; Dorkin, JR; Yang, JH; Heartlein, MW; DeRosa, F.; Mir, FF; Fenton, OS; Anderson, DG, Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Lett 2015, 15, 7300-7306; and Li, S.; Hu, Y.; Li, A.; Lin, J.; Hsieh, K.; Schneiderman, Z.; Zhang, P.; Zhu, Y.; Qiu, C.; Kokkoli, E.; Wang, T.-H.; Mao, H.-Q., Payload distribution and capacity of mRNA lipid nanoparticles. Nat. Commun. 2022, 13, 5561).However, antibody formation is a barrier (Hald Albertsen, C.; Kulkarni, JA; Witzigmann, D.; Lind, M.; Petersson, K.; Simonsen, JB, Therole of lipid components in lipid nanoparticles for vaccines and genetherapy. Adv. Drug Deliv. Rev. 2022, 188, 114416), which is why alternatives to PEG are being investigated (Bleher, S.; Buck, J.; Muhl, C.; Sieber, S.; Barnert, S.; Witzigmann, D.; Huwyler, J.; Barz, M.; Süss, R., Poly(sarcosine) surface modification imparts stealth-like properties to liposomes. Small 2019, 15, 1904716).

[0017] The remaining components in the form of helper lipids improve the encapsulation efficiency of genetic material and the release of LNPs from the nucleus (see Kulkarni, JA; Witzigmann, D.; Leung, J.; Tam, YYC; Cullis, PR, On the role of helper lipids in lipid nanoparticle formulations of siRNA. Nanoscale 2019, 11, 21733-21739).

[0018] Several types of LNPs and their preparation have been described in patent literature.

[0019] For example, nucleic acids encapsulated with lipids are known from US 7,341,348 B2. This patent describes a particle composed of a lipid layer surrounding a central region containing nucleic acids and containing pK... a Amino lipids with amino groups having values ​​of 4 to 11 and PEG-DAG conjugates.

[0020] According to WO 2018 / 081480 A1, LNP contains 40 to 50 mol% cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and therapeutic agents encapsulated therein.

[0021] WO 2019 / 089828 A1 discloses an LNP with a bilayer structure containing at least 40 mol% cationic lipids and nucleic acids encapsulated therein.

[0022] LNPs composed of cationic lipids, PEG lipids, and antigens are described in EP 3 556 353 A2. These LNPs often contain other components, such as cholesterol or phospholipids. The lipids are dissolved in ethanol to prepare the LNPs.

[0023] The LNPs described in these documents always contain an increasingly larger proportion of steroids, such as cholesterol. These LNPs are prepared using an aqueous solution of ethanol, as this is not possible in aqueous solution.

[0024] In Nanoscale, 11, 2019, 18806-18824, Dongyu Chen et al. described an LNP in which cholesterol was replaced by modified cholesterol DC-CHOL with an ammonium group. The lipid had an HLB value of 5.8 according to Griffin. In addition to the component DC-CHOL, the LNP described in this literature also contains cationic lipids, phospholipids, and occult lipids. The cationic lipids had an HLB value of less than 4 according to Griffin. No information regarding the amount of lipids used in the LNP is provided in this document.

[0025] Maho Kawatuchi et al. disclosed cholesterol-reduced LNPs in the *Journal of Pharmaceutical Sciences*, 112 (2023) 1401-1410, and Samuel T. LoPresti et al. in the *Journal of Controlled Release*, 345 (2022) 819-831. The LNPs described in these publications all showed reduced cholesterol content without substitution. In both papers, the resulting LNPs had structural disadvantages compared to commercially available cholesterol-containing substitutes. Furthermore, the authors were unable to prepare LNPs completely devoid of pure cholesterol. In addition, both papers described LNPs containing various lipids with HLB values ​​not exceeding 3.

[0026] Various methods can be used to prepare LNPs. The most common in this context are sonication, extrusion, and microfluidics (see Chatterjee, S.; Banerjee, DK, Preparation, isolation, and characterization of liposomes containing natural and synthetic lipids. Methods Mol. Biol. 2002, 199, 3-16; Mozafari, MR, Nanoliposomes: preparation and analysis. Methods Mol. Biol. 2010, 605, 29-50; and Walsh, C.; Ou, K.; Belliveau, NM, et al., Microfluidic-based manufacture of siRNA-lipidnanoparticles for therapeutic applications. Methods Mol. Biol. 2014, 1141, 109-120). In the case of COVID-19 vaccines, the latter technology has dominated on a commercial scale. Among other things, microfluidics is characterized by its excellent reproducibility in batch-to-batch manufacturing (see 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, SJ; Issadore, D.; Mitchell, MJ, Microfluidic formulation of nanoparticles for biomedical applications. Biomaterials 2021, 274, 120826). In this case, LNPs are produced by rapidly mixing an organic lipid phase (typically based on ethanol) and an aqueous phase (the latter containing genetic material). This process results in lipid precipitation and the formation of nanoparticles. The organic solvent must then be removed from the formulation.In addition to evaporation, dialysis or cross-flow filtration is typically applied in this case (see Evers, MJW; Kulkarni, JA; van der Meel, R.; Cullis, PR; Vader, P.; Schiffelers, RM, State-of-the-art design and rapid-mixing production techniques of lipid nanoparticles for nucleic acid delivery. Small Methods 2018, 2, 1700375; Mihaila, R.; Chang, S.; Wei, AT; Hu, ZY; Ruhela, D.; Shadel, TR; Duenwald, S.; Payson, E.; Cunningham, JJ; Kuklin, N.; Mathr, DJ, Lipid nanoparticle purification by spin centrifugation-dialysis (SCD): A facile and high-throughput approach for small-scale preparation of siRNA-lipid complexes, Int. J. Pharm. 2011, 420). 118-121; and Terada, T.; Kulkarni, JA; Huynh, A.; Chen, S.; van der Meel, R.; Tam, YYC; Cullis, PR, Characterization of lipid nanoparticles containing ionizable cationic lipids using design-of-experiments approach. Langmuir 2021, 37, 1120-1128). In the case of the LNP described herein, the solvent is dialyzed relative to PBS. In this case, the formulation is directly adjusted to a physiological pH of 7.4.

[0027] The use of organic solvents for formulation is a significant drawback. Genetic material becomes more unstable, and prolonged storage can lead to lipid degradation (see Evers, MJW; Kulkarni, JA; van der Meel, R.; Cullis, PR; Vader, P.; Schiffelers, RM, State-of-the-art design and rapid-mixing production techniques of lipid nanoparticles for nucleic acid delivery. Small Methods 2018, 2, 1700375; and Roces, CB; Lou, G.; Jain, N.; Abraham, S.; Thomas, A.; Halbert, GW; Perrie, Y. Manufacturing considerations for the development of lipid nanoparticles using microfluidics, Pharmaceutics 2020, 12, 1095). To ensure the stability of nucleic acids, the organic solvent needs to be removed rapidly. This increases production costs.

[0028] The materials used to fabricate microfluidic chips can also be disadvantageous. For example, polydimethylsiloxane absorbs genetic material and tends to swell when exposed to solvents (see Kwon, HJ; Kim, S.; Kim, S.; Kim, JH; 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).

[0029] Therefore, abandoning organic solvents in the preparation of lipid-based nanoparticles is a promising possibility for overcoming some of the aforementioned drawbacks of conventional preparations. It has been shown that ethanol is not essential for the encapsulation of genetic material (see 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 possibility for abandoning the use of ethanol is to homogenize the lipids in an aqueous solution above their melting point. LNP formation is induced by alternating cooling and heating (see De, A.; Ko, YT, Single pot organic solvent-free thermocycling technology for siRNA-ionizable LNPs: A proof-of-concept approach for alternative to microfluidics. Drug Delivery 2022, 29, 2644-2657). A disadvantage is that only temperature-stable drugs can be used, and all components must be thermally stable, requiring stringent quality control in the case of lipids and RNA.

[0030] It has now been surprisingly discovered that LNPs can be prepared without the need for organic solvents. Conventional methods require the use of organic solvents, such as ethanol, to introduce the highly lipophilic portion of the lipids used into the LNP. The method according to the invention allows for the preparation of LNPs loaded at high concentrations with active substances (e.g., nucleic acids) containing anionic groups such as phosphate groups.

[0031] The LNP (also referred to as BLNP in this specification) according to the present invention can load sensitive active substances in a mild manner and allows, for example, the introduction and transport of genetic material into cells.

[0032] The BLNP according to the invention is prepared without the use of highly lipophilic steroids, and therefore contains a smaller amount of lipid component than LNPs known to date. Conventional LNPs contain a large amount of cholesterol. This readily diffuses between LNPs and between serum components and lipids in the biological environment. Furthermore, cholesterol is not prepared entirely synthetically but is obtained from natural sources, meaning that fluctuations in quality and impurities cannot be excluded. In addition, methods with more approval steps and components are more complex, and therefore more expensive and less robust.

[0033] The lipophilic properties of lipids can be described by their HLB values ​​(HLB stands for hydrophilic-lipophilic balance). Introduced by W.C. Griffin in 1954, the HLB value describes the hydrophilic and lipophilic content of lipids. The HLB value scales from 0 (strong lipophilicity) to 20 (weak lipophilicity). Besides Griffin's method, there are other methods for calculating HLB values; however, these are far less commonly used. One method worth mentioning is that of Davies, who proposed in 1957 to calculate HLB values ​​from the numerical values ​​of individual chemical groups in the molecule. The advantage of this method is that it assigns greater weight to strongly interacting groups than to weakly interacting groups. Furthermore, it can define HLB values ​​for both cationic and anionic lipids. Summary of the Invention

[0034] One object of the present invention is to provide lipid nanoparticles having a compact and simple structure, capable of loading high amounts of active substances, and excellently suited for transporting active substances into organisms or cells, for example for gene transfer of nucleic acids.

[0035] Another object of the present invention is to provide an easy method for preparing lipid nanoparticles, which can be carried out without the use of organic solvents.

[0036] This invention relates to lipid nanoparticles, which contain...

[0037] a) 51 to 94.9 mol% of at least one cationic / ionizable lipid,

[0038] b) 5 to 40 mol% of at least one phospholipid, and

[0039] c) 0.1 to 10 mol% of at least one occult lipid, said occult lipid being selected from the group consisting of lipids comprising one or more poly(olefin oxide) chains (hereinafter referred to as "PEG lipids"), lipids comprising one or more poly(oxazoline) chains (hereinafter referred to as "POx lipids"), lipids comprising one or more poly(glycerol) chains (hereinafter referred to as "PG lipids"), lipids comprising one or more poly(hydroxyalkyl(meth)acrylate) chains (hereinafter referred to as "PHAA lipids"), and lipids comprising one or more poly(N-(hydroxyalkyl)(meth)acrylamide) chains (hereinafter referred to as "PHAAA lipids"). Lipids containing one or more poly(vinylpyrrolidone) chains (hereinafter referred to as "PVP lipids"), lipids containing one or more poly(N,N-dialkyl(meth)acrylamide) chains (hereinafter referred to as "PDMAA lipids"), lipids containing one or more poly(N-(meth)acryloylmorpholine) chains (hereinafter referred to as "PAM lipids"), or lipids containing one or more poly(amino acid) chains (hereinafter referred to as "PAA lipids"), provided that all lipids contained in the lipid nanoparticles have an HLB value greater than or equal to 3, and the percentages given are based on the total mass of lipids contained in the lipid nanoparticles.

[0040] In the context of this specification, "lipid nanoparticles" or "LNP" or "BLNP" are understood to be particles with a diameter (z-mean) less than or equal to 900 nm, which are mainly or entirely composed of lipids from groups a), b), and c) above. These BLNPs may be loaded with active substances containing anionic groups. BLNPs are typically characterized by a very high surface-to-volume ratio, thus providing very high chemical reactivity. BLNPs may consist only of the lipids mentioned in groups a), b), and c), or they may additionally contain a complex of an active substance and cationic lipids from group a), or BLNPs may contain small amounts of other components besides lipids and optional complexes, such as excipients or additives e).

[0041] In the context of this specification, "excipients and additives" are understood to be substances added to a formulation to impart certain additional properties and / or facilitate its processing. Examples of excipients and additives include sugars such as sucrose, contrast agents, carriers, fillers, pigments, dyes, fragrances, radiopharmaceuticals such as tracers, lubricants, UV stabilizers, polymers such as nitrogen-containing polymers, or antioxidants. In particular, "excipients and additives" are understood to be any substance that can be used for the intended application purpose, which is not a pharmaceutically or agriculturally active chemical substance and is not a lipid, but can be formulated with the active substance in an active substance-lipid complex to influence and, in particular, improve the qualitative properties of the LNP. Preferably, the excipients and / or additives e) have no or no significant or at least no undesirable effect with respect to the intended treatment.

[0042] In the context of this specification, the HLB value is understood to be a value between 0 and 20, calculated according to the following formula:

[0043] HLB = 20 * (1 - M l / M),

[0044] Where M l Let be the molar mass of the lipophilic portion of the molecule, and M be the molar mass of the entire molecule.

[0045] Within the scope of this invention, to determine the HLB value of lipids, the freely available software MarvinSketch 23.4 (see https: / / docs.chemaxon.-com / display / docs / hlb-predictor.md#src-1806640-hlbpredictor-fig-1) was used, and the HLB was determined according to Griffin, because the method according to Davis is not optimal for hidden lipids with many repeating units.

[0046] Strongly lipophilic compounds typically have HLB values ​​of 1 to 3. These are hydrophilic (oil-soluble) lipids, such as defoamers. Compounds with a significant hydrophilic moiety 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 typically have HLB values ​​of 4 to 5.

[0047] Preferably, all lipids in the nanoparticles according to the invention have an HLB value of 3 to 20, especially 3 to 18, especially greater than or equal to 4, and very particularly preferably 4 to 17.5.

[0048] The BLNP according to the present invention can be loaded with an active substance having at least one anionic group. Therefore, the present invention also relates to the above-described LNP loaded with an active substance having an anionic group.

[0049] In the BLNP according to the invention, the molar ratio (molar percentage) of the cationic lipid a) or the combination of lipids a) is generally 51 to 94.9%, preferably 55 to 89.5%, particularly preferably 60 to 85%, and very particularly preferably 75 to 82%.

[0050] In the BLNP according to the invention, the molar ratio (molar percentage) of the combined amount of phospholipids (b) or lipids (b) is generally 5 to 40%, preferably 10 to 30%, particularly preferably 14 to 25%, and very particularly preferably 15 to 18%.

[0051] In the BLNP according to the invention, the molar percentage (molar percentage) of the amount of the hidden lipid c) or the combination of lipid c) is generally 0.1 to 10%, preferably 0.5 to 5%, and very particularly preferably 1 to 3%.

[0052] The percentages given above are based on the total mass of lipids contained in BLNP.

[0053] If the BLNP according to the invention contains additional lipids d) that do not belong to any of groups a) to c) with an HLB value of at least 3, then the weight percentage of these lipids d) is at most 10%, preferably at most 5%, and especially at most 1%.

[0054] If the BLNP according to the invention contains excipients or additives (e), their total weight percentage is at most 5%, preferably at most 1%, and especially at most 0.5%.

[0055] Preferably, the BLNP according to the present invention does not contain additional lipids (d) and does not contain excipients or additives (e).

[0056] Very particularly preferred, the proportion of lipids having sterol groups in the BLNP according to the invention is 0 to 15 mol%, especially 0 to 10 mol.

[0057] The cationic lipids a) used to prepare the BLNP according to the invention comprise all lipids having at least one cationic group, such as an amino group. However, these are not phospholipids, which are classified as lipids in group b). The cationic lipids a) preferably do not contain any phosphate groups.

[0058] Examples of cationic groups are amino groups, namely primary, secondary and tertiary amino groups or quaternary ammonium groups; guanidine and amide groups, namely groups having secondary, tertiary and quaternary amide groups; aminoalkanol groups, namely groups having primary, secondary, tertiary and quaternary amino groups; and phosphonyl groups, namely primary, secondary and tertiary phosphonyl groups or quaternary phosphonium groups.

[0059] The term “cationic lipid” as used in this article refers to lipids that have one or more positive net charges at certain pH values, such as at acidic pH values.

[0060] The cationic lipids covered in this specification also include ionizable cationic lipids. Ionizable cationic lipids are characterized by the weak basicity of their ionizable groups, which affects the lipid's charge in a pH-dependent manner. Therefore, these lipids will carry a positive charge at acidic pH values, but will be almost charge-neutral at physiological pH values.

[0061] Preferred cationic lipids a) for preparing the BLNP according to the invention comprise all lipids having at least one amino group (which are not phospholipids).

[0062] Preferred cationic lipids a) for preparing the BLNP according to the invention also include ionizable lipids (which are not phospholipids) containing at least one amino group. They have the property that they form a positive charge at the nitrogen atom by protonation in an acidic pH range of 4 to 7, and that they are almost neutral in an alkaline pH range above 7.

[0063] Particularly preferred are ionizable lipids (a) used to prepare BLNPs according to the invention, which contain at least one amino group (which is not a phospholipid), especially one or two amino groups, and very particularly preferred are one amino group, wherein these amino groups have a pKs value of 7 to 9.

[0064] Preferably, the cationic lipid a) lacks a phosphate group and has one or two nitrogen atoms, preferably one nitrogen atom, and at least one alkyl group having six to twenty carbon atoms, said alkyl group 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 group having six to twenty carbon atoms and one, two or three double bonds that are not directly adjacent to each other. These nitrogen atoms may be present as amino groups, amide groups or as alkanolic amino groups, preferably as amino groups.

[0065] Particularly preferred, the cationic lipid a) does not have a phosphate ester group and an alkyl group having one or two nitrogen atoms and at least two alkyl groups having six to twenty carbon atoms, the alkyl groups being optionally interrupted by an ester group -CO-O- or -O-CO- or an amide group -CO-NH- or -NH-CO-, or one or two of these alkyl groups being replaced by one or two alkylene groups having six to twenty carbon atoms and one, two or three double bonds that are not directly adjacent to each other.

[0066] The particularly preferred cationic lipid a) has the structure of formula (I).

[0067]

[0068] in

[0069] R1 Group R is a group of the following formula: 4 R 5 N-(C m H 2m )-、CH3-(C n H 2n )-O-(C o H 2o )-、HO-(C m H 2m )-, HO-CH2-CH(OH)-CH2-, CH3-(CH2) n -O-CO-(C m H 2m )-、CH3-(C n H 2n )-CO-O-(C m H 2m )-、NC-(C o H 2o )-、HO-CH2-CH((C o H 2o )-CH3 )-、CH3-(C o H 2o )-CH(OH)-(C p H 2p )-、CH3-(C n H 2n )-CO-NH-(C o H 2o )-、(HO-CH((C q H 2q )-CH3)-CH((C o H 2o )-OH)- or C6H 10 (OH)-,

[0070] R 2 and R 3 Each group is an alkyl group having six to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO-, and / or an alkylene group having six to twenty carbon atoms and one, two or three double bonds that are not directly adjacent to each other.

[0071] R 4 and R 5 Each of the following is independently hydrogen or an alkyl group having one to five carbon atoms, or a group R 4 and R 5 Both, together with a common nitrogen atom, form a pyrrolidinyl group or a piperidine group.

[0072] m is an integer from 2 to 6.

[0073] n represents an integer from 0 to 6.

[0074] o and p are independent integers from 1 to 6.

[0075] q is an integer from 2 to 16, and

[0076] r is 0 or 1.

[0077] Very particularly preferred are cationic lipids of formula (I) a), wherein R 1 For formula R 4 R 5 N-(CH2) m - groups, especially those where m is 2.

[0078] The most preferred is the cationic lipid a) of formula (I), wherein R 1 For formula R 4 R 5 The group N-(CH2)2-, and R 4 and R 5 Each is independently hydrogen, methyl, or ethyl, or contains a group R. 4 and R 5 Both, together with a common nitrogen atom, form a pyrrolidinyl group or a piperidine group.

[0079] Other particularly preferred cationic lipids a) include those of formula (I), wherein R 2 and R 3 Groups selected independently from the following group:

[0080] -(C s H 2s -CH3, -CH2-CH((C t H 2t )-CH3)((C u H 2u -CH3), -CH((C t H 2t )-CH3)((C u H 2u )-CH3),

[0081] -(C s H 2s )-O-CO-CH((C t H 2t )-CH3)((C u H 2u )-CH3),

[0082] -(C s H 2s )-CO-O-CH((C tH 2t )-CH3)((C u H 2u )-CH3)、

[0083] -(C s H 2s )-CO-O-(C t H 2t )-CH3、-(C s H 2s )-O-CO-(C t H 2t )-CH3,

[0084] -(C s H 2s )-O-CO-CH2-CH((C t H 2t )-CH3)((C u H 2u )-CH3)、

[0085] -(C s H 2s )-CO-O-CH2-CH((C t H 2t )-CH3)((C u H 2u )-CH3)、

[0086] -CH((C s H 2s )-O-CO-CH2-CH((C t H 2t )-CH3)((C u H 2u )-CH3))2、

[0087] -CH((C s H 2s )-CO-O-CH2-CH((C t H 2t )-CH3)((C u H 2u )-CH3))2、

[0088] -CH((C s H 2s )-O-CO-CH((C t H 2t )-CH3)((C u H 2u )-CH3))2、

[0089] -CH((C s H2s )-CO-O-CH((C t H 2t )-CH3)((C u H 2u )-CH3))2,

[0090] -(C t H 2t )-CH=CH-CH2-CH=CH-(C u H 2u )-CH3,

[0091] -CH((C t H 2t )-CH=CH-CH2-CH=CH-(C u H 2u )-CH3)2,

[0092] -(C t H 2t )-CH=CH-(C u H 2u )-CH3 and -CH((C t H 2t )-CH=CH-(C u H 2u )-CH3)2,

[0093] -(C t H 2t )-CH=C(CH3)-CH2-CH=CH-(C u H 2u )-CH3,

[0094] -CH((C t H 2t )-CH=C(CH3)-CH2-CH=CH-(C u H 2u )-CH3)2,

[0095] -(C t H 2t )-CH=C(CH3)-(C u [[ID=8l]]H 2u )-CH3 and -CH((C t H 2t )-CH=C(CH3)-(C u H 2u )-CH3)2, -(C t H 2t )-C6H 11 and -C6H 10 -C(CH3)3,

[0096] in

[0097] s is an integer from 4 to 20, and

[0098] t and u represent integers from 1 to 10 independently of each other.

[0099] Very particularly preferred are cationic lipids of formula (I) a), wherein R 1 For formula R 4 R 5 The N-(CH2)2- group, and R 4 and R 5 Each of the groups is independently hydrogen, methyl, or ethyl, or one or more of them R 4 and R 5 Together with a common nitrogen atom, they form a pyrrolidinyl group or a piperidine group.

[0100] R 2 and R 3 Groups selected independently from the following formula

[0101] -(CH2) v -O-CO-R 5 Or -(CH2) v -CO-OR 6 ,

[0102] v is an integer from 1 to 20, preferably from 5 to 12, and

[0103] R 5 and R 6 Alkyl groups having 6 to 20 carbon atoms and / or alkenyl groups having 6 to 20 carbon atoms and having one or more preferably two alkene unsaturated bonds that are not directly adjacent to each other, especially groups selected from the group consisting of:

[0104] -(C v H 2v -CH3, -(CH2) w -C6H 11 -C6H 10 -C(CH3)3、-CH((CH2) x -CH3)((CH2) y -CH3), -CH2-CH((CH2) x -CH3)((CH2) y -CH3), -CH((CH2) x -C(CH3)3)((CH2) y -C(CH3)3), -CH2-CH((CH2) x -C(CH3)3)((CH2) y-C(CH3)3), -(CH2) w -CH((CH2) x -CH3)((CH2) y -CH=C(CH3)2),

[0105] in

[0106] v is an integer between 7 and 12.

[0107] w is an integer from 1 to 4.

[0108] x and y independently represent integers from 0 to 12, and are particularly preferably selected from the following group:

[0109] -CH(CH2-CH3)((CH2)3-CH3), -CH((CH2)5-CH3)((CH2)7-CH3), -CH((CH2)5-CH3)((CH2)5-CH3 ), -CH((CH2)7-CH3)((CH2)7-CH3), -CH((CH2)5-CH3)((CH2)3-CH3), -CH((CH2)9-CH3)((CH2) 11 -CH3), -CH((CH2)9-CH3)((CH2)7-CH3), -CH((CH2)2-CH3)((CH2)2-CH3), -CH((CH2)5-CH3)((CH2)7-CH3), CH((CH2)3-CH3)((CH2)3-CH3), -CH(CH3)-(CH 2)9-CH3, -CH2-CH((CH2)5-CH3)((CH2)5-CH3), -CH2-CH((CH2)7-CH3)((CH2)7-CH3), -CH2-CH((CH2)5-CH3)((CH2)3-CH3), -CH2-CH((CH2)9-CH3)((CH2) 11 -CH3), -CH2-CH((CH2)9-CH3)((CH2)7-CH3), -CH2-CH((CH2)2-CH3)((CH2)2-CH3), -CH2-C H((CH2)5-CH3)((CH2)7-CH3), -CH2-CH((CH2)3-CH3)((CH2)3-CH3), -(CH2)7-CH3, -(CH2) 10 -CH3, -(CH2) 11-CH3, -CH2-CH=CH-(CH2)7-CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4-CH3, -CH2-CH=C(CH3)-(CH2)3-CH(CH3)-(CH2)3-CH(CH3)-(CH2)3-CH(CH3)2, -(CH2)2-C6H 11 and -C6H 10 -C(CH3)3,

[0110] Cationic lipids a) preferably form a positive charge at the nitrogen atom in a pH range of 5 to 8. The compound of formula (I) then exists as a cationic compound of formula (II):

[0111]

[0112] in,

[0113] R 1 R 2 R 3 r has the meaning defined above.

[0114] The meaning of j corresponds to the integer number of nitrogen atoms in the compound of formula (II), preferably 1 or 2.

[0115] i is an integer from 1 to 5000, and

[0116] X is an i-valent anion.

[0117] Any inorganic or organic i-valent anion X can be used.

[0118] Inorganic anion X i- Examples include halide ions such as fluoride, chloride, bromide, or iodide ions, or hydroxide ions or anions of inorganic acids such as phosphate, sulfate, nitrate, hexafluorophosphate, tetrafluoroborate, perchlorate, chlorate, hexafluoroantimonate, hexafluoroarsenate, and cyanide ions.

[0119] Organic anion X i- Examples of anions of organic acids are mono- or polycarboxylic acids or mono- or polysulfonic acids, wherein these acids may be saturated or unsaturated. Examples of anions of organic acids are acetate, formate, trifluoroacetate, trifluoromethanesulfonate, pentafluoroethanesulfonate, nonafluorobutyrate, butyrate, citrate, fumarate, glutarate, lactate, malate, malonic acid, oxalate, pyruvate, or tartrate.

[0120] These anions can exist in the form of polyanions.

[0121] Another preferred cationic lipid a) contains a quaternary ammonium group. Examples of such lipids are compounds of formula (IIa).

[0122]

[0123] in,

[0124] R 1a R 2a and R 3a Each group is an alkyl group having one to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO-, and / or an alkylene group having two to twenty carbon atoms and one, two or three double bonds that are not directly adjacent to each other.

[0125] R 4a An alkyl group having six to twenty carbon atoms, said alkyl group may optionally be interrupted by an ester group -CO-O- or -O-CO-, and / or an alkylene group having six to twenty carbon atoms and one, two or three double bonds that are not directly adjacent to each other, or wherein

[0126] Two groups R 1a and R 2a Together with a common nitrogen atom, they form a pyrrolidinyl group or a piperidine group, and

[0127] X, i, and j have the meanings defined above.

[0128] Very particularly preferred, the nanoparticles according to the invention contain cationic lipids selected from the group consisting of: N,N-diolenoyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-diolenoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearate-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)-cholesterol (DC-Chol); N-(1-(2,3-dioleoyloxy)propyl)N-2-(spermine-formylamino)ethyl N,N-dimethyltrifluoroacetate ammonium (DOSPA), 1,2-dioleoyl-sn-3-phosphate ethanolamine (DOPE), bis(octadecylaminoglycylcarboxylic acid)spermine (DOGS), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), and 1,2-dilinyloxy-N,N-dimethylaminopropane (DLenDMA).

[0129] Very particularly preferred, the nanoparticles according to the invention contain only one type of cation / ionizable lipid a).

[0130] The phospholipids b) used according to the present invention are typically lipids that, in addition to at least one lipid group, have a polyol group attached thereto, which is further bonded to a phosphate group, the phosphate group being connected to the head group via an ester bond.

[0131] Phospholipids b) typically have the structure of formula (III):

[0132] (LP) np -BG-OP(O)(OMe)-O-KG(III)

[0133] in,

[0134] LP stands for fatty acid group.

[0135] BG is an (np+1) valence bridging group.

[0136] np is an integer from 1 to 5, preferably 1 or 2.

[0137] Me can be hydrogen, a monovalent or divalent metal cation, or an ammonium cation.

[0138] KG represents a head group, which signifies an aliphatic group containing at least one hydroxyl group, preferably an aliphatic group having both a hydroxyl and an amino group, an aliphatic group having both a hydroxyl and a quaternary ammonium group, or a carbohydrate group having five to six hydroxyl groups.

[0139] The group LP can have different meanings within a molecule within a given definition.

[0140] Typically, the phospholipids of formula (III) b) have one to five groups LP, preferably one or two groups LP, wherein it is an alkyl group having six to twenty carbon atoms, and / or a mono- or ...

[0141] Typically, phospholipids of formula (III) b) have one to five groups LP, which are connected to the head group via a bridging group BG through a phosphate group, wherein the bridging group BG is a group of a binary to a six-membered aliphatic or alicyclic alcohol or a binary to a six-membered aliphatic or alicyclic amino alcohol.

[0142] Examples of groups in di- to hexa-membered aliphatic or alicyclic alcohols include groups derived from ethylene glycol, propylene glycol, glycerol, glycerol, pentaerythritol, or inositol.

[0143] Examples of groups of di- to hexa-membered aliphatic or alicyclic amino alcohols include groups derived from 2-aminoethanol, 3-aminopropanol, proline, alanine alcohol, valine, leucine, phenylpropanol, phenylglycine, or sphingosine.

[0144] Preferred phospholipids (b) have glycerol-derived groups as bridging groups and have a structure of formula (IVa) or (IVb):

[0145]

[0146]

[0147] in,

[0148] LP is a saturated or mono-alkenyl to triene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other.

[0149] KG and Me have the meanings defined above, and

[0150] The group LP can have different meanings within a molecule within a given definition.

[0151] Another preferred phospholipid b) has a bridging group derived from sphingosine and has a structure of formula (Va) or (Vb):

[0152]

[0153]

[0154] in,

[0155] LP is a saturated or mono-alkenyl to triene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other.

[0156] KG and Me have the meanings defined above.

[0157] mp is an integer from 2 to 8, especially 6, and

[0158] In compounds of formula (Vb), the group LP can have different meanings within the molecule within a given definition.

[0159] Another preferred phospholipid (b) has a group derived from an aliphatic amino alcohol or a group derived from inositol as a head group KG.

[0160] Particularly preferred are phospholipids b) having a head group KG of formula (VIa), (VIb), or (VIc).

[0161]

[0162] in,

[0163] pp is an integer from 2 to 6, preferably 2.

[0164] R 6 Indicates hydrogen or C1-C5-alkyl.

[0165] R 7 and R 8 Independently, they are C1-C6-alkyl groups, and

[0166] XP represents the IP valence anion, and

[0167] IP can be an integer from 1 to 3, preferably 1 or 2.

[0168] Phospholipids b) having a head group KG selected from the group consisting of choline, ethanolamine, serine and inositol are particularly preferred.

[0169] Very particularly preferred, the nanoparticles according to the invention contain phospholipids selected from the group consisting of: distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), dioleoyl-sn-glycerol-3 Sodium DOPE-mal, dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-ditransoleoyl-sn-glycerol-3-phosphatidylethanolamine (transDOPE).

[0170] The stealth lipids (c) used according to the present invention are generally lipids having at least one poly(olefin oxide) group, poly(oxazoline) group, polyglycerol group, poly(hydroxyalkyl(meth)acrylate) group, poly(N-(hydroxyalkyl)(meth)acrylamide) group, poly(vinylpyrrolidone) group, poly(N,N-dialkyl(meth)acrylamide) group, poly(N-(meth)acryloylmorpholine) group, or poly(amino acid) group, in addition to at least one lipid group. The linkage of these groups can be achieved by covalent bonds or preferably by bridging groups (BG).

[0171] The preferred stealth lipid (c) does not have any phosphate groups.

[0172] Preferred stealth lipids (c) are PEG lipids. These are especially lipids having the structure of formula (VII):

[0173] (LPL) nl -(BGL) ml -(O-CH2-CH2) ol -OR 9 (VII)

[0174] in,

[0175] LPL is an alkyl or alkenyl group, a fatty acid group, a fatty alcohol group, or a sterol group having 6-20 carbon atoms.

[0176] BGL is a (nl+1) valence bridging group.

[0177] n1 is an integer from 1 to 5, preferably 1 or 2.

[0178] ml represents 0 or 1, preferably 1.

[0179] ol is an integer from 5 to 500, preferably from 10 to 200, and

[0180] R 9 The LPL group represents hydrogen, an alkyl group having one to six carbon atoms, preferably hydrogen, methyl, ethyl or sterol groups, wherein the LPL group may have different meanings within the molecule within a given definition.

[0181] Typically, the occult lipid c) of formula (VII) has one to five LPL groups, preferably one or two LPL groups, wherein it is an alkyl group having six to twenty carbon atoms, and / or a mono- or ...

[0182] Typically, the occult lipid c) of formula (VII) has one to five LPL groups, which are directly covalently linked to the PEG group via ester bonds; or the occult lipid c) of formula (VII) has one to five LPL groups, which are linked to the occult group (PEG, POx or others) via a bridging group BGL, wherein the bridging group BGL is a di- to hexa-membered aliphatic or alicyclic alcohol, or a di- to hexavalent carboxylic acid, or a carbamate group, or an amino alcohol.

[0183] Examples of groups in di- to hexa-membered aliphatic or alicyclic alcohols include groups derived from ethylene glycol, propylene glycol, glycerol, glycerol, pentaerythritol, or inositol.

[0184] Examples of di- to hexacarboxylic acid groups are those derived from oxalic acid, maleic acid, fumaric acid, adipic acid, sebacic acid, succinic acid, tartaric acid, terephthalic acid, isophthalic acid, trimellitic acid, pyromellitic acid, or pyromellitic acid.

[0185] Examples of urethane groups are groups derived from the formula >N-CO-O-, wherein the PEG group is attached to an oxygen atom and one or two LPL groups are attached to a nitrogen atom.

[0186] Examples of amino alcohol groups are groups derived from the formula >NRO-, where R is a divalent organic group, preferably an alkylene group, the PEG group is attached to an oxygen atom, and one or two LPL groups are attached to a nitrogen atom. Other amino alcohol groups may have multiple amino groups and / or oxygen atoms, such as aminophenols having two hydroxyl groups and / or amino groups.

[0187] Examples of sterol groups are those derived from saturated or mono- or diene-unsaturated sterols (3-hydroxysterols), preferably substituted at the 17-position with an alkyl group having one to ten carbon atoms, particularly with a 2,6-dimethylhexyl group. Groups derived from cholesterol are particularly preferred as sterol groups.

[0188] Preferred stealth lipid c) has a glycerol-derived group as a bridging group and has a structure of formula (VIIIa) or (VIIIb):

[0189]

[0190]

[0191] in,

[0192] LPL is a saturated or mono-alkenyl to triene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other, or a sterol group.

[0193] R 9 "and ol" has the additional meanings defined above, and

[0194] The group LPL can have different meanings within a molecule within a given definition.

[0195] Another preferred stealth lipid c) has a carbamate-derived group as a bridging group and has a structure of formula (IXa) or (IXb):

[0196]

[0197] in,

[0198] LPL is a saturated or mono-alkenyl to triene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other.

[0199] R 9 "and ol" has the same meaning as defined above, and

[0200] In compounds of formula (IXb), the group LP1 can have different meanings within the molecule within a given definition.

[0201] Another preferred stealth lipid c) has a succinic acid-derived group as a bridging group and has the structure of formula (X):

[0202] LPL-O-OC-CH2-CH2-CO-O-CH2-CH2-(O-CH2-CH2)ol-1 -OR 9 (X),

[0203] in,

[0204] LPL is a saturated or mono-alkenyl to triene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other, and

[0205] R 9 "ol" has the meaning as defined above.

[0206] The preferred stealth lipid c) has the structure of formula (Xa):

[0207] H-[O-CH2-CH2] ol -O-CO-R a -CO-O-Ster(Xa),

[0208] Here, ol has the meaning defined above.

[0209] R a An alkylene group having one to eight carbon atoms, preferably an ethylene group, and

[0210] Ster is a group derived from saturated or mono- or diene-unsaturated 3-hydroxysterols, preferably substituted at the 17-position by an alkyl group having one to ten carbon atoms, especially by a 2,6-dimethylhexyl group, and very particularly preferably a group derived from cholesterol.

[0211] The PEG lipids (c) preferred for use according to the present invention include the substances listed below, wherein n is a number between 15 and 200, preferably between 18 and 70.

[0212]

[0213] Very particularly preferred, the nanoparticles according to the invention contain PEG lipids selected from the group consisting of: PEGylated diacylglycerols (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG-succinate-diacylglycerols (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecyloxy)propyl-1-O-(ω-methoxy-(polyethoxy)ethyl)succinate (PEG-S-DMG), PEGylated ceramides (PEG-cer), or PEG-dialkoxypropylcarbamates, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-(ω-methoxy-(polyethoxy)ethyl)carbamate.

[0214] Another particularly preferred stealth lipid, c), is a POx lipid. These are typically polymers that, in addition to at least one lipid group, have a polyoxazoline group linked to it, wherein the latter is generated by the polymerization of oxazoline. The linkage between the two groups can be achieved via a covalent bond or via a bridging group BGL.

[0215] Polymers produced by the polymerization of oxazoline monomers have repeating structural elements of formula (XI):

[0216]

[0217] Where R is hydrogen or a monovalent organic group.

[0218] Preferred POx-lipids have the structure of formula (XII):

[0219]

[0220] in,

[0221] LPL is an alkyl or alkenyl group, a fatty acid group, a fatty alcohol group, or a sterol group having 6-20 carbon atoms.

[0222] BGL is a (nl+1)- valent bridging group.

[0223] n1 is an integer from 1 to 5, preferably 1 or 2.

[0224] ml represents 0 or 1, preferably 1.

[0225] ol is an integer ranging from 5 to 500, preferably from 10 to 200.

[0226] R 10The groups representing hydrogen, alkyl groups or groups having one to six carbon atoms (LPL), preferably hydrogen, methyl, ethyl or sterol groups, and

[0227] R 11 Indicates hydrogen or C1-C4 alkyl, wherein

[0228] Groups LPL and R 11 Within a given definition, different meanings can be used within a molecule.

[0229] Typically, POx lipids of formula (XII) have one to five groups LPL, preferably one or two groups LPL, wherein the groups LPL are alkyl groups having six to twenty carbon atoms, and / or mono- or ...

[0230] Typically, the POx lipid of formula (XII) has one to five groups LPL, which are directly covalently linked to the POx group via ether or ester bonds; or the POx lipid of formula (XII) has one to five groups LPL, which are linked to the POx group via bridging groups BGL, wherein the bridging groups BGL are groups of di- to hexa-membered aliphatic or alicyclic alcohols, or groups of di- to hexa-membered carboxylic acids, or groups of carbamates, or groups of amino alcohols.

[0231] Examples of groups of di- to hexa-membered aliphatic or alicyclic alcohols, di- to hexa-membered carboxylic acids, carbamate groups, and amino alcohol groups are also listed in the description of PEG lipids above.

[0232] Preferred POx lipids have glycerol-derived groups as bridging groups and have a structure of formula (XIIIa) or (XIIIb):

[0233]

[0234]

[0235] in,

[0236] LPL is a saturated or mono-alkenyl to triene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other.

[0237] R 10 R 11 "and ol" has the additional meanings defined above, and

[0238] The group LPL can have different meanings within a molecule within a given definition.

[0239] Another preferred POx lipid has a succinic acid-derived group as a bridging group and has the structure of formula (XIV):

[0240]

[0241] in,

[0242] LPL is a saturated or mono-alkenyl to triene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other, and

[0243] R 10 R 11 "ol" has the meaning as defined above.

[0244] The preferred occult lipid c) has the structure of formula (XIVa):

[0245] R c -[N(CO-R b )-CH2-CH2] ol -O-CO-R a -CO-O-Ster(XIVa),

[0246] Among them, ol and R a And Ster has the meaning defined above.

[0247] R b Indicates methyl or ethyl, and

[0248] R c The group is derived from the cationic polymerization initiator, preferably hydrogen or a monovalent organic group, especially an alkyl group, a cycloalkyl group, an aryl group, an aralkyl group or a heterocyclic group.

[0249] The particularly preferred occult lipid of formula (XIVa) has a Stere group, which is replaced at the 17-position by an alkyl group having one to ten carbon atoms, especially by a 2,6-dimethylhexyl group, and very particularly preferably by a group derived from cholesterol.

[0250] The POx lipids preferably used according to the present invention include those having the following general structure, wherein n represents a number between 15 and 200, preferably between 18 and 70, and Linker represents a divalent bridging group.

[0251]

[0252] Another preferred stealth lipid c) has the structure of formula (XV):

[0253] (LPL) nl -(BGL) ml -POLY (XV)

[0254] in

[0255] LPL is an alkyl or alkenyl group, a fatty acid group, a fatty alcohol group, or a sterol group having 6-20 carbon atoms.

[0256] BGL is a bridging group with a valence of (nl+1).

[0257] n1 is an integer from 1 to 5, preferably 1 or 2.

[0258] ml represents 0 or 1, preferably 1, and

[0259] POLY is a group of the formula (XVa), (XVIb), (XVIc), (XVId), (XVIe), (XVIf), (XVIg), or (XVIh):

[0260]

[0261] Among them, R 12 Indicates hydrogen or LPL,

[0262] R 13 This indicates hydrogen or a monovalent organic group, such as alkyl, cycloalkyl, aryl, or aralkyl.

[0263] R 14 It is hydrogen or an alkyl group having one to six carbon atoms, especially hydrogen or methyl.

[0264] R 15 and R 16 Each of these can independently represent hydrogen or an alkyl group having one to six carbon atoms, especially an alkyl group having one to four carbon atoms.

[0265] R 17 It is a hydrogen-containing alkyl group having one to six carbon atoms, optionally substituted with a hydroxyl group, an amino group, a phenyl group, a hydroxyphenyl group, a carboxyl group, or an amide group.

[0266] NMORPH represents a morpholinyl group linked to a carbonyl group via a cyclic nitrogen atom.

[0267] r, s, t, and u represent numbers greater than or equal to 1, preferably between 1 and 5000.

[0268] The group LPL can have different meanings within a molecule within a given defined range.

[0269] The exponent r is preferably a number between 1 and 10, especially between 1 and 4.

[0270] The index s is preferably a number between 10 and 5000, especially a number between 40 and 5000.

[0271] The exponent t is preferably a number between 10 and 5000, especially a number between 50 and 5000.

[0272] The exponent u is preferably a number between 5 and 500, especially between 10 and 100.

[0273] Particularly preferred formula (XV) stealth lipids c) are those in which POLY is a group of formula (XVIf), wherein R 14 Indicates methyl and R 17 It is hydrogen.

[0274] The preferred stealth lipid c) has the structure of formula (XVIi):

[0275]

[0276] Among them, ol, R a Ster and NMORPH have the meanings defined above, and

[0277] R d It is hydrogen or a monovalent organic group, especially an alkyl group, a cycloalkyl group, an aryl group, an aralkyl group or a heterocyclic group.

[0278] The particularly preferred formula (XVIi) stealth lipid has a Stere group, which is replaced at the 17-position by an alkyl group having one to ten carbon atoms, especially by 2,6-dimethylhexyl, and is very particularly preferred to be a group derived from cholesterol.

[0279] Another preferred use of the hidden lipid c) has the structure of formula (XVIj):

[0280] R d -[NR b -CO-CH2-] ol -O-CO-R a -CO-O-Ster(XVIj),

[0281] Among them, ol and R a R b , Ster and R d It has the meaning defined above.

[0282] The particularly preferred occult lipid of formula (XVIj) has a Stere group, which is an alkyl group with one to ten carbon atoms at the 17-position, especially substituted with 2,6-dimethylhexyl, and is very particularly preferred to be a group derived from cholesterol.

[0283] The BLNP according to the present invention can be loaded with an active substance f) containing anionic groups. The anionic groups include carboxyl groups, sulfonic acid groups, and phosphate groups or phosphate ester groups. These are characterized in that they form complexes with cationic lipids a) in an aqueous medium and are encapsulated in or associated with the BLNP.

[0284] Active ingredient f) may include any pharmaceutical and agrochemically active substance, provided that each molecule of it has at least one anionic group.

[0285] Preferred active substance f) is a nucleic acid. In the context of this specification, this is understood to mean naturally occurring nucleic acids, including their modified derivatives. Modified nucleic acids may contain modified nucleotides or be otherwise modified, for example, by introducing chemical modifications. The nucleic acid forms a complex with the cationic lipid a) via the phosphate ester groups present therein.

[0286] The physicochemical properties of nucleic acids form the basis for their interaction with carrier materials. Small changes in sequence can lead to different biological effects and allow for rapid adaptation to different indications without requiring adjustments to the entire formulation process. Compared to conventional drugs, nucleic acids are biopolymers with a higher molar mass (approximately 333 Da per nucleotide) and a strong negative charge, resulting in good water solubility. Furthermore, they exhibit low stability in the presence of degrading enzymes, and they can trigger immune responses based on evolutionarily optimized mechanisms that protect organisms from viral gene manipulation. Despite these obstacles, nucleic acids can regulate gene expression, whereas classic active substances typically lack causal effects. Some successful, approved systems for the application of nucleic acids are shown in Figure 1. DNA typically exists in the form of a double-stranded variant (dsDNA), where two single-stranded DNA strands (ssDNA) are linked together by hydrogen bonds and hydrophobic interactions between complementary base sequences, resulting in the familiar double-helix conformation. dsDNA is a semi-flexible polymer with a high negative charge density. For therapeutic applications, DNA is typically encoded in plasmids (pDNA) containing thousands of base pairs (bp). When pDNA enters the cell nucleus, it can influence gene expression. By repairing defective genes, whether congenital or acquired, gene therapy currently offers highly specific and even potentially curative treatments for diseases for which there was no other treatment or cure. In addition to DNA, short interfering RNAs (siRNAs) are typically 19-25 bp long double-stranded RNAs. Due to their distinct sugar backbones, they have a higher linear charge density and stiffness than DNA. Therapeutic siRNAs temporarily shut down (knock down) genes by inhibiting the translation of target mRNAs. Conversely, single-stranded antisense oligonucleotides (ASOs) can shut down translation by binding to their corresponding mRNAs. Both types of RNA are already in clinical use, as shown in Figure 1. MicroRNAs (miRNAs) are small (approximately 23 nucleotides), single-stranded, non-coding RNAs derived from primary miRNAs. miRNAs regulate the expression of target genes by degrading mRNAs or inhibiting translation.

[0287] mRNA is also a single-stranded nucleotide. It contains hundreds of nucleotides and is more flexible than DNA, siRNA, or ssDNA. Due to the accessibility of its bases, mRNA exhibits stronger amphiphilic properties, enabling hydrophobic interactions with potential transport molecules. Unmodified mRNA is less stable to nucleases due to its single-stranded nature and possesses higher immunogenicity than DNA. Therefore, modified nucleotides have been proposed and chemical modifications have been introduced. Both siRNA and mRNA are active in the cytoplasm, thus bypassing the nuclear membrane barrier.

[0288] As the active substance f), DNA and / or RNA or their modifications are preferably used in the BLNP according to the invention.

[0289] 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.

[0290] Any immunomodulatory element, such as TRL antagonists, CpG motifs, and other functional nucleic acids, can be used.

[0291] Any RNA type can be used. Examples include hnRNA, mRNA, tRNA, rRNA, mtRNA, snRNA, snoRNA, scRNA, siRNA, miRNA, ncRNA, saRNA, antisense RNA, bacterial RNA, and viral RNA.

[0292] A combination of DNA and RNA can also be used in the BLNP according to the present invention.

[0293] Modified nucleic acids, also known as xenobiotic nucleic acids (XNAs), offer a range of advantages for biotechnological applications and overcome some of the limitations of first-generation nucleic acid therapeutics. In fact, several modified nucleic acid-based therapeutics have recently been approved, and many more are in clinical trials. XNAs can exhibit greater biostability and are increasingly being developed in vitro, accelerating the discovery of lead structures (Duffy, K.; Arangundy-Franklin, S.; Holliger, P., Modified nucleic acids: replication, evolution, and next-generation therapeutics. BMC Biol. 2020, 18, 112).

[0294] Preferred nanoparticles according to the invention are characterized by a high content of active substance f), preferably nucleic acid. Based on the mass of the LNP loaded with active substance, the weight fraction of active substance f) in the LNP according to the invention is typically 1 to 10%, and preferably 2 to 8%, especially 3 to 7%, and particularly preferably 5 to 6%.

[0295] The nanoparticles according to the invention can be characterized by their particle size. Typical particle sizes (e.g., z-average) are less than or equal to 900 nm, preferably less than or equal to 500 nm, particularly preferably between 30 nm and 500 nm, very particularly preferably between 40 nm and 250 nm, and especially in the range between 50 nm and 200 nm. For the purposes of this specification, the particle size is determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, United Kingdom). The intensity-weighted average diameter (e.g., z-average) is determined by cumulant analysis of correlation functions (ISO 13321, ISO 22412). For size determination, it is assumed that the refractive index of ultrapure water is 1.33.

[0296] Particle size can also be determined by other methods, such as by nanoscale tracking analysis (NTA) or by electron microscopy, for example by transmission electron microscopy or scanning electron microscopy.

[0297] Determined by dynamic light scattering (DLS), the preferred LNP according to the invention has a particle size (z-average) in the range between 30 nm and 500 nm.

[0298] The BLNPs according to the invention can also be characterized by their polydispersity index (or PDI). PDI represents the width of the particle size distribution. A value between 0 (monodisperse) and 1 (polydisperse) can be assumed here. For the purposes of this specification, the PDI value was determined using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, United Kingdom) by dynamic light scattering (DLS). The PDI was determined using cumulative analysis of the correlation function.

[0299] The PDI value of the particle size distribution of the nanoparticles according to the invention is typically between 0.01 and 0.4, preferably between 0.02 and 0.3, and particularly preferably between 0.05 and 0.2.

[0300] When using an active substance f) having phosphate groups, the nanoparticles according to the invention can also be characterized by their N / P ratio. This is understood as the molar ratio of nitrogen atoms in cationic lipids a) to phosphate groups in active substances e), such as nucleic acids.

[0301] The N / P ratio in the nanoparticles according to the invention can vary over a wide range. 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 very particularly preferably between 3 and 15.

[0302] Preferred nanoparticles according to the invention have a diameter (z-mean) between 40 and 250 nm, particularly between 50 and 200 nm, as determined by DLS, and a polydispersity index between 0.05 and 0.3 for the particle size.

[0303] The nanoparticles according to the invention are particularly preferred to have a diameter (z-average) between 40 and 250 nm, especially between 50 and 200 nm, as determined by DLS, a polydispersity index between 0.05 and 0.2, and an N / P ratio between 3 and 15.

[0304] In the case where the nanoparticles according to the invention contain additional polymers or additional complexes of nucleic acids and additional polymers in addition to the above-described nucleic acid-lipid complexes, these additional components are present only in small amounts, for example, in a weight ratio of 10% or less, especially less than 5%.

[0305] Particularly preferably, the nanoparticles according to the present invention do not contain any other complexes of active substances and other polymers other than the above-mentioned active substance-lipid complex.

[0306] The BLNPs according to the invention can exist as powders in solid form, or they can form dispersions and be dispersed in an aqueous solvent, whereby the particles exist in the dispersion medium in solid form.

[0307] In a preferred embodiment, the BLNP according to the invention forms a dispersed phase in water or an aqueous buffer solution.

[0308] The BLNP according to the invention can be prepared by assembly. For this purpose, the lipids used according to the invention are dispersed in water or an aqueous buffer solution. Here, dispersions can be prepared for each lipid separately, or all lipids can be dispersed together. Here, the pH of the aqueous dispersion is adjusted to 3 to 8, preferably 4 to 7.5, for example by using an acetate buffer or another suitable buffer such as citrate buffer, lactate buffer, phosphate buffer, and phosphate-citrate buffer. Furthermore, the active substance f) containing anionic groups, such as nucleic acid, is dissolved or dispersed in water, thereby preferably adjusting the pH of the aqueous solution of the active substance or the aqueous dispersion to a value between 3 and 8, particularly preferably between 4 and 7.5. Buffer solutions containing acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer, HEPES, TRIS, or salt only are particularly suitable for this purpose. The aqueous dispersion of lipids and the solution or dispersion of the active substance are combined with each other, thereby selecting the amount of active substance and cationic lipid a) such that the desired active substance / lipid ratio, for example, the desired N / P ratio, is adjusted. After mixing the aqueous dispersion or solution, 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. Preferably, the resulting nanoparticles are allowed to stand for a period of time, for example, between 5 and 20 minutes, before further use to allow binding between lipid a) and active substance e) (hereinafter referred to as "incubation"). After preparation in an acidic pH (e.g., acetate, pH 5.5), LNP is preferably neutralized, for example by mixing with phosphate-buffered saline (PBS) to pH 7.4. The nanoparticles according to the invention can then be lyophilized from the dispersion medium or retained in the dispersion medium.

[0309] In an alternative method, the BLNP according to the invention can be prepared by nanoprecipitation, whereby the BLNP initially contains only lipids, and active substance f is added in a downstream step. The BLNP is prepared as described above, but without the addition of active substance f) in a solution or dispersion.

[0310] Then, as described above, a solution or dispersion of the active substance f) is prepared.

[0311] The aqueous dispersion of BLNP and the active substance solution or dispersion are then combined, wherein the amounts are chosen such that the desired active substance / lipid ratio, such as the desired N / P ratio, is adjusted. After mixing the aqueous dispersion or solution, the mixture is stirred, 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. Here, also, before further use, the resulting nanoparticles loaded with active substance are allowed to stand for a period of time, for example, between 5 and 20 minutes, to allow binding between lipid a) and active substance e) (hereinafter referred to as "incubation"). After preparation in an acidic pH (e.g., acetate, pH 5.5), the BLNP is preferably neutralized, for example by mixing with a phosphate-buffered saline solution (PBS) to pH 7.4. The nanoparticles according to the invention can then be lyophilized from the dispersion medium or retained in the dispersion medium.

[0312] In addition to cationic lipids a), other lipids b) and c), and active substances f), one or more excipients and additives e) may be added during their nanoprecipitation in the dispersion medium. Alternatively, these excipients and additives e) may be added to the aqueous phase after the nucleic acid-copolymer complex has been dispersed.

[0313] Water is used as a dispersion medium. Buffers, salts, sugars, or acids and bases can be added to adjust the desired pH or osmotic pressure.

[0314] The method according to the invention is characterized in that the use of organic solvents such as ethanol can be omitted during the preparation of lipid nanoparticles. Therefore, downstream solvent separation is eliminated.

[0315] The present invention also relates to a method for preparing the above-mentioned BLNP, comprising the following measures:

[0316] i) Prepare aqueous dispersions of lipids a), b) and c) in a buffer solution with a pH range of 3 to 8, preferably 4 to 7.5.

[0317] ii) Combine the aqueous dispersion from step i); and

[0318] iii) The combined aqueous dispersion from step ii) is processed by a mixing method selected from the group consisting of ultrasound, double centrifugation, nanoprecipitation, and microfluidics, or in a vortex mixer, thereby forming nanoparticles.

[0319] In a first variant, the present invention also relates to a method for preparing the above-mentioned BLNP loaded with an active substance f) containing an anionic group, comprising the following steps:

[0320] iv) Prepare an aqueous dispersion containing LNP from step iii) of the method described above.

[0321] v) Prepare an active substance having anionic groups; f) An aqueous solution or dispersion in a buffer solution with a pH range of 3 to 8, preferably 4 to 7.5.

[0322] vi) Combine the aqueous dispersions or solutions from steps iv) and v); and

[0323] vii) The combined aqueous dispersion or solution from step vi) is treated by sonication, microfluidics, double centrifugation, sonication, nanoprecipitation, or vortex mixer to form lipid nanoparticles loaded with active substances.

[0324] In a second variation, the present invention relates to a method for preparing the above-mentioned BLNP loaded with an anionic group f), comprising the following steps:

[0325] I) Prepare aqueous dispersions of lipids a), b) and c) in a buffer solution with a pH range of 3 to 8, preferably 4 to 7.5.

[0326] II) Preparation of active substances with anionic groups f) in an aqueous solution or dispersion in a buffer solution with a pH range of 3 to 8, preferably 4 to 7.5.

[0327] III) Combine the aqueous dispersions or solutions from steps I) and II); and

[0328] IV) The combined aqueous dispersion or solution from step III) is treated by sonication, microfluidics, double centrifugation, nanoprecipitation, or vortex mixer to form an LNP loaded with active material.

[0329] In a preferred embodiment of a first variant of the method according to the invention, the method includes the following measures:

[0330] V) Prepare an aqueous dispersion containing LNP with a pH between 3 and 8, prepared according to step iii) of the aforementioned method.

[0331] VI) Prepare nucleic acids in an aqueous solution or dispersion in a buffer solution with a pH range of 3 to 8, preferably 4 to 7.5;

[0332] VII) The two dispersions or solutions from steps V) and VI) are mixed in a selected ratio of nucleic acid to lipid a) to obtain a desired molar N / P ratio of nitrogen atoms in lipid a) to phosphate groups in nucleic acid, preferably an N / P ratio between 1 and 200.

[0333] VIII) Stir the mixture from step VII); and

[0334] IX) Optionally, the mixture obtained is then incubated.

[0335] The aqueous dispersions of lipids a), b) and c) used in step i) or I) of the method according to the invention preferably contain buffers, especially acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer or mixtures thereof.

[0336] The aqueous solution or dispersion of the nucleic acid used in step v), II) or VI) of the method according to the invention preferably has a pH value of 3 to 8, more preferably 4 to 7.5.

[0337] The aqueous solution or dispersion of the nucleic acid used in step v), II), or V) of the method according to the invention preferably contains a buffer solution, especially an acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer, HBG buffer, HEPES buffer, or TRIS buffer.

[0338] The agitation in steps iii), vii), IV), or VIII) of the method according to the invention is preferably carried out by stirring or vortexing. The processing duration in this step is typically between 1 and 120 seconds, especially between 2 and 60 seconds.

[0339] In step IX of the method according to the invention, the incubation is typically carried out by simply allowing the obtained mixture to stand for a time span of, for example, 5 to 60 minutes, preferably 5 to 20 minutes. The mixture may also be incubated in a refrigerator or heating cabinet, for example, at a temperature between 1°C and 80°C.

[0340] The separation of nanoparticles from the aqueous phase can be achieved in various ways. Examples include cross-flow filtration, centrifugation, ultrafiltration, and dialysis. However, the nanoparticle dispersion can also preferably be used immediately after preparation without further processing.

[0341] Purification by filtration can separate particulates, such as aggregates, as well as excess excipients or impurities, from a dispersion. This allows for the alteration of particle concentration.

[0342] Dissolved molecules can be separated from the dispersion by purification using dialysis / cross-flow filtration. This method is largely independent of the particle size of the dispersed particles.

[0343] Dissolved molecules can also be separated from a dispersion by purification using centrifugation. However, this method also reduces the concentration of dispersed particles. Furthermore, only dispersions of nanoparticles with relatively large diameters (e.g., greater than 150 nm) can be processed, and the particles may be damaged in the process. Moreover, redispersing particles obtained in this manner can be difficult.

[0344] BLNPs loaded with the active substances according to the present invention are excellently suited as carriers for transporting pharmaceutical and agricultural chemical active substances.

[0345] In particular, the BLNPs loaded with active substances according to the invention are suitable for gene transfer into cells, i.e., for introducing nucleic acids and functionally releasing them into cells. For this purpose, BLNPs containing nucleic acids are added to single cells, tissues, or cell cultures and are absorbed by the cells via endocytosis. It has been surprisingly shown that high concentrations of nucleic acids can be transferred into cells using the BLNPs according to the invention.

[0346] Therefore, the present invention also relates to a method for transferring genes into cells, comprising the following steps:

[0347] A) Contact cells, tissues, or cell cultures with an aqueous dispersion containing the aforementioned LNP containing nucleic acids, and

[0348] B) Subsequent warming.

[0349] Preferably, the present invention relates to a method for transferring genes into cells, comprising the following steps:

[0350] C) Provide cell cultures in a bioreactor or incubator.

[0351] D) Add an aqueous dispersion containing LNPs with the above-mentioned nucleic acids.

[0352] E) Distribute the aqueous dispersion in the cell culture, and

[0353] F) Then nurture.

[0354] The gene transfer method according to the present invention can be performed using different cells, for example, by using single cells, tissues or cell cultures.

[0355] Therefore, the nucleic acid-loaded BLNP according to the present invention can be combined with prokaryotic or eukaryotic cells, with tissues derived from eukaryotic cells, or with cell cultures. These can be plant cells or preferably animal cells, including human cells.

[0356] The administration of the nucleic acid-loaded LNP according to the present invention can be performed in vivo, such as under the skin or in muscle, or it can also be administered ex vivo, such as with immune cells, as in CAR-T therapy. It can also be an RNA vaccination or another type of vaccination.

[0357] In the context of this specification, "cell" is understood as the smallest unit of life in an organism. It can be a cell from a single-celled or multicellular organism, and can originate from prokaryotes or eukaryotes. A cell can be a microorganism or a single cell. Cells can be of prokaryotic, plant, or animal origin, or fungal origin. Eukaryotic cells are preferred, especially those initially isolated from tissues and capable of permanent culture, i.e., immortalized cells.

[0358] In the context of this specification, “tissue” is understood as a collection of differentiated cells, including their extracellular matrix.

[0359] In the context of this specification, "cell culture" refers to a combination of cells or tissues and a cell culture medium, wherein the cells or tissues are cultured in a cell culture medium outside of an organism. Cell lines, i.e., tissue-type cells that can divide during culture, may be used here. Both immortalized (undying) cell lines and primary cells (primary cultures) can be cultured. Primary cultures are generally understood to be non-immortalized cell cultures obtained directly from tissues.

[0360] The cell cultures used in this invention can be generated and cultured according to standard methods.

[0361] For example, primary cultures can be generated from different tissues, such as tissues from a single organ like skin, heart, kidney, or liver, or from tumor tissue. Tissue cells can be isolated by methods known per se, such as by treatment with proteases, thereby breaking down the proteins that hold the cells together. It may also be suitable to purposefully stimulate the division of certain cell types by adding growth factors, or, in the case of poorly growing cell types, to use recombinant components of feeder cells, basement membrane-like matrix, or extracellular matrix. Cells used according to the invention can also be genetically altered by introducing plasmids as vectors.

[0362] The cells used in this invention can have a finite lifespan or can be immortalized cell lines with unlimited division capacity. These can be generated through random mutations (e.g., in tumor cells) or through targeted alterations (e.g., through artificial expression of the telomerase gene).

[0363] The cells used in this invention can be adherent (on a surface) cells, such as fibroblasts, endothelial cells or chondrocytes, or they can be suspension cells that grow freely in a culture medium, such as lymphocytes.

[0364] The culture conditions and cell culture medium are selected based on the individual cells being cultured. Different cell types prefer different nutrient media in this case, which are specially formulated. Thus, for example, different pH values ​​are set, and each nutrient medium may contain different concentrations of different amino acids and / or other nutrients.

[0365] Cells transfected according to the present invention can be used in various fields, such as biotechnology, research, medicine, and veterinary medicine. This may involve the production of (recombinant) proteins, viruses and / or viral particles, and the study of metabolism, division, and other cellular processes. Furthermore, cells transfected according to the present invention can be used as assay systems, for example, to study the effects of substances on cell properties such as signal transduction or toxicity. Another preferred cell type for preparing cells transfected according to the present invention is stem cells. These are known to be somatic cells capable of differentiating into different cell types or tissues.

[0366] The present invention also relates to the use of the above-mentioned nucleic acid-containing LNP for gene transfer into cells, that is, for introducing nucleic acids and functionally releasing them into cells.

[0367] In the method according to the invention described in the following examples, the property of cationic lipids to form a homogeneous dispersion in an acidic aqueous solution (e.g., 20 mM NaOAc, pH 5.5) is utilized. Phospholipids, such as DSPC, exhibit the same behavior. This makes it possible to extract reproducible volumes from the adjusted stock solution. Latent lipids, such as PEG lipids or POx lipids, can also be transferred to the formulation in this manner, as they are soluble in the aforementioned buffer solution. Cholesterol is omitted because this molecule is insoluble in water and is not necessary for constructing the lipid nanoparticles according to the invention. In view of this, the molar composition of the lipid nanoparticles (cationic lipid / auxiliary lipid / latent lipid = 81.3 / 16.3 / 2.4) is adjusted for the following examples; however, the ratio of the components to each other in the original Moderna formulation (50 / 10 / 1.5) is retained. Controlled mixing of the particles by means of ultrasonic treatment and subsequent shearing results in the formation of homogeneous, unloaded lipid nanoparticles. The particles formed by ultrasonic treatment are significantly different in shape and size from lipid nanoparticles prepared by simple mixing of added materials.

[0368] In subsequent process steps, unloaded lipid nanoparticles can be loaded with desired active substances, such as desired genetic material. Both pDNA and RNA were used in the following experiments. The genetic material was diluted, for example, in 20 mM NaOAc, pH 5.5 (mM) buffer (MM). After a defined incubation period, the mixture was then combined by rapidly transferring equal volumes of the nanoparticle suspension and MM to each other and mixing them using a vortex mixer. The functionality of the genetically loaded lipid nanoparticles according to the invention in protein expression can be demonstrated using an in vitro testing system. After demonstrating that the lipid nanoparticles according to the invention are substantially suitable for transfection with pDNA, the formulation was further optimized for biocompatibility. Various buffer systems for forming unloaded and loaded lipid nanoparticles were tested for this purpose. Data showed that an acidic pH was essential for positive transfection. Furthermore, mRNA was used as an alternative genetic material. Experiments showed that the lipid nanoparticles according to the invention are also suitable for transfection with mRNA. Additionally, various camouflage polymers can be used as alternatives to commercially available PEG-DMG.

[0369] A key advantage of the lipid nanoparticles according to the invention is that the method according to the invention enables the preparation of lipid nanoparticles for gene transfer without the use of organic solvents. This leads to a reduction in production costs because rapid solvent removal (e.g., by dialysis) is no longer required. Furthermore, genetic material is protected. Attached Figure Description

[0370] The embodiments and accompanying drawings described below illustrate the invention but do not limit its scope.

[0371] Figures 1A to 1D The structure of the lipids used in LNPs employed by BioNTech and Moderna is shown. Figure 1A The structure of phospholipid DSPC is shown. Figure 1B The structures of ionizable cationic lipids ALC-0315 and SM-102 are shown. Figure 1C The structures of ethoxylated lipids PEG-DMG and ALC-0159 are shown. Figure 1D The structure of cholesterol (Cholesterol) is shown.

[0372] SM-102 has C 44 H 87 The general formula for NO5 is given, and it has a molar mass of 710.18. The HLB value calculated by Griffin is 5.70.

[0373] DSPC has C 44 H 88The general formula for NO8P is given, and it has a molar mass of 790.16. The HLB value calculated by Griffin is 20*(1-(617.28 / 790.16)=4.38).

[0374] PEG-DMG has C 124 H 246 O 51 The total formula is given, and the molar mass is 2553.28. The HLB value calculated by Griffin is 17.13.

[0375] Cholesterol has C 27 H 46 The total formula for O is given, and it has a molar mass of 386.66. The HLB value calculated by Griffin is 20*(1-((386.66-17) / 386.66)=0.88.

[0376] The LNP used by BioNTech is composed of 46.3 mol% ALC-0315, 9.4 mol% DSPC, 42.7 mol% cholesterol, and 1.6 mol% ALC-0159.

[0377] The LNP used in Moderna is composed of 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG (see Schoenmaker, L.; Witzigmann, D.; Kulkarni, JA; Verbeke, R.; Kersten, G.; Jiskoot, W.; Crommelin, DJA, mRNA-lipidnanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 2021, 601, 120586).

[0378] Other occult lipids used in the following experiments have the following structures:

[0379] HEMS-PEtOx n

[0380]

[0381] CHEMS-PEG n

[0382]

[0383] CHEMS-PMeOx n

[0384]

[0385] CHEMS-PNAM

[0386]

[0387] CHEMS-PSar

[0388]

[0389] Figure 2 A schematic construction of a device for microfluidics is shown (see 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 device for the preparation of conventional lipid nanoparticles.

[0390] Figure 3 A schematic structure of a conventionally used LNP consisting of known proportions of lipids is shown.

[0391] Figure 4 A schematic diagram of the method according to the invention is shown. The initial preparation method is based on a simple mixing process (vortexing), in which various ionizable lipids are mixed with genetic material and transfection is tested. The cationic lipid SM-102 was found to provide the best results, but these results were quite below average. To improve transfection, the helper lipid DSPC and the stealth lipid (Chol-POx in this case) were added to the formulation. Steps: 1) Prepare the lipid suspension, 2) Dilute the genetic material and vortex for 5 seconds, 3) Incubate at room temperature for 10 min, combine the lipids and genetic material, and 4) Vortex rapidly.

[0392] Chol-POx is a polymer as shown below.

[0393]

[0394] Chol-POx-52 (n=52) has C 240 H 416 N 52 O 56 The overall formula is given, and the molar mass is 4926.28. R represents methyl. The HLB value calculated by Griffin is 15.27.

[0395] Figure 5The schematic structure of the LNP according to the present invention is shown, which is composed of cationic lipids, auxiliary lipids and stealth lipids, and contains genetic material as an active substance.

[0396] Figure 6 The results of measurements of the LNP prepared by vortexing, obtained using DLS, are shown. According to... Figure 4 The preparation was carried out using the methods outlined in [the document]. The effect of individual components of the formulation on particle size was investigated. The following combinations were prepared:

[0397] F1. Cationic lipids + genetic material

[0398] F2. Cationic lipids + accessory lipids + genetic material

[0399] F3. Cationic lipids + stealth lipids + genetic material, and

[0400] F4. The BLNP according to the present invention consists of all four components.

[0401] These particles are arranged from left to right and were prepared at three different N / P ratios (3, 6, and 9 from left to right). The cationic lipids alone have formed measurable lipid complexes (complexes of lipids and genetic material) with the genetic material. Clearly, particle size varies with the N / P ratio. However, the addition of accessory lipids (DSPC) and elusive lipids (Chol-POx) results in small, uniform particles, as can be seen from the lower PDI values ​​of these LNPs.

[0402] exist Figure 7 The results of the transfection experiment are shown in the figure. The nanoparticles used are LNPs according to the present invention, which are similar to those made from... Figure 6 Prepared particles. These particles were transferred to cells to measure transfection. Particles containing hidden lipids and phospholipids showed the highest efficiency in this study. Considering the material input, a formulation with an N / P ratio of 6 proved to be optimal.

[0403] Figure 8 Another method for preparing LNPs according to the present invention is summarized. The aim is to reduce the size of the resulting LNPs and further improve their efficiency in gene transport. Among the possible methods, sonication has proven to be the most suitable. By sonicating a lipid mixture, significantly smaller unloaded lipid particles are produced, which are then loaded with genetic material. Steps: 1) preparing a lipid suspension, 2) assembling LNPs by sonication, 3) diluting the genetic material and vortexing for 5 seconds, 4) incubating at room temperature for 10 min to combine the lipids and genetic material, and 5) rapid vortexing.

[0404] Figure 9 and 10 ( Figure 10 Showing from Figure 9The magnified view of the results shows the particle size and PDI value of the LNPs. The studied LNPs contained different lipids or lipid combinations, such as... Figure 6 The above. Will be used. Figure 4 (vortex) and Figure 8 The LNPs produced by the methods outlined in (ultrasonic processing) were compared with each other. It was found that the particles produced using the latter method were significantly smaller.

[0405] All LNP measurements shown below involve molar compositions of 82.3 mol% SM-102, 16.5 mol% DSPC, and 1.2 mol% cryptic lipids, or 81.3 mol% SM-102, 16.3 mol% DSPC, and 2.4 mol% cryptic lipids. Transfection experiments were performed using HEK293T cells. Measurements were obtained by flow cytometry after an incubation period of 24 h at 37°C.

[0406] exist Figure 11 The image shows the raw data measured by flow cytometry after transfection. (The data will be processed according to...) Figure 4 The method (vortex) (AC) according to the LNP of the present invention and according to Figure 8 The method (ultrasonic processing) (DF) was compared with the LNP according to the present invention. Figure 11 Compared to B (70.05%), LNPs prepared by ultrasound had a significantly higher transfection efficiency of 88.92%. Figure 11 E).

[0407] exist Figure 12 The text shows the relationship with sources. Figure 11 The relative average fluorescence intensity is compared to the original data. The stripes in this case are indicated by... Figure 8 LNPs were prepared using the method described above. It can be seen that the pure lipid complexes composed of cationic lipids with anionic activity showed almost no fluorescence in both methods. In contrast, the LNPs according to the present invention showed significant fluorescence values ​​(400x to 900x higher than the negative control). Furthermore, to better understand the results, a polymeric complex made of polyethyleneimine (PEI) was compared with the LNPs. It can be seen that the LNPs prepared by sonication were superior to the PEI polymeric complex. Furthermore, according to... Figure 8 LNPs prepared by the method (ultrasound) are significantly superior to those prepared by the method according to the method. Figure 4 LNPs were prepared by a method (vortex).

[0408] Figure 13The results of LNPs prepared using different buffers are shown. The results shown in the previous figure were obtained using LNPs prepared in citrate buffer (50 mM) at pH 4.0. Neutralizing 50 mM citrate buffer to pH 7.4 under these conditions is extremely difficult (e.g., dialysis is required). To facilitate this process, it is necessary to change the buffer system used. Therefore, other buffer systems were investigated. TRIS, PBS (pH 7.4), and NaOAc (pH 5.5) were tested as solvents for the lipid suspension. Meanwhile, genetic material was dispersed in various buffers (MM). 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 here. Thus, in addition to the initial combination of 50 mM citrate buffer and 20 mM HBG, 15 new buffer combinations were obtained. In the absence of citrate buffer, the combination of 20 mM NaOAc pH 5.5 (LNP) and 20 mM NaOAc pH 5.5 (MM) yielded the only positive results. LNPs for this study sequence were prepared using vortexing. The advantage of using 20 mM NaOAc is that it is significantly easier to neutralize than citrate buffer. This solvent combination was continued for subsequent experiments.

[0409] exist Figure 14 and 15 The image shows the particle size (column) and PDI value (point) of LNP. Figure 14 This further clarifies the advantages of the sonication method, as it allows for significantly smaller particle sizes. The following investigation examines whether switching the buffer system from citrate buffer to NaOAc results in similar particle sizes. Figure 15 This confirms the situation (similar sizes). The buffer system was changed, and the nanoparticles were measured using DLS after preparation in the same manner.

[0410] Figure 16 The transfection efficiency (relative average fluorescence intensity) of LNPs prepared with NaOAc buffer is shown. The buffer was acidified once with an equimolar amount of hydrochloric acid (HCl) and adjusted to pH 4.0 to test whether a specific pH value of the preparation affects efficacy. It can be seen that the highest value is achieved at pH 5.5. Therefore, no further adjustment of the buffer system is necessary, and further optimization can be performed using 20 mM NaOAc at pH 5.5.

[0411] For further application, it is essential that the formulation be neutralized from pH 5.5 to pH 7.4. Figure 17The figure shows how to neutralize the pH 5.5 formulation without compromising transfection efficiency. However, before contacting the LNP with the cell culture medium, the sample is added 1+1 to the corresponding buffer shown. The figure shows that transfection of the neutralized sample is surprisingly even better than that of the unneutralized (pH 5.5) sample. Therefore, the preferred preparation of LNP involves preparing nanoparticles at pH 5.5 using 20 mM NaOAc, followed by neutralization with the addition of PBS or TBS.

[0412] exist Figure 18 The results of toxicity measurements were plotted. Therefore, according to... Figure 8 Unloaded LNPs were prepared using a method described above. Two different stealth polymers were used for this purpose: Chol-Pox and PEG-DMG. Experiments were performed on L929 cells, which were incubated for 24 h with LNP suspensions of various concentrations up to 1000 μM, followed by evaluation using a Presto-Blue assay. It can be seen that the LNPs according to the present invention are non-toxic within the test range.

[0413] exist Figure 19 and 20 It describes something similar to targeting Figure 18 DLS measurements (particle size) of the prepared LNPs are described. Measurements were taken of unloaded particles (blank) and particles loaded with mRNA and pDNA. The latter were measured after neutralization with pH 5.5 (NaOAc) and PBS (pH 7.4). It can be seen that the choice of genetic material has no effect on size. Neutralization resulted in a slight increase in size.

[0414] Figure 21 and 22 Shown in Figure 19 and 20 The PDI value of LNP as described in [the document].

[0415] Figure 23 The proportion of EGFP-positive cells in the transfection experiment is shown as a function of the amount of genetic material used. Therefore, according to... Figure 8 BLNPs containing the stealthy lipid Chol-POx were prepared and loaded with mRNA and pDNA. It can be seen that the BLNPs according to the present invention are significantly superior to PEI.

[0416] Figure 24 The list includes possible and already used stealth lipids.

[0417] exist Figure 25 and 27In the experiments shown up to 29, BLNP containing 81.3 mol% ionizable / cationic lipids (SM-102), 16.3 mol% phospholipids (DSPC), and 2.4 mol% occult lipids was used.

[0418] exist Figure 25 The above-listed polymer CHEMS-PEtOx is shown in the figure. n CHEMS-PEG n CHEMS-PMeOx n It is very well suited for use with BLNPs. The high transfection efficiency demonstrates this. BLNPs were prepared by sonication as described above, and the transfection efficiency was measured using mRNA.

[0419] exist Figure 26 The above-listed polymer CHEMS-PEtOx is described in the text. n CHEMS-PEG n CHEMS-PMeOx n Furthermore, DMG-PEG differs significantly in their biocompatibility. The PmeOx and PEtOx variants of the CHEMS stealth lipids exhibited significantly lower cytotoxicity than the PEG variants. Generally, PEG is more cytotoxic than POx. The assays performed were based on ISO 10993-5, and cytotoxicity was measured as a measure of the relative metabolic activity of L929 cells after incubation with the corresponding material at a given concentration for 24 h.

[0420] exist Figure 27 This demonstrates another specific application possibility of BLNP. BLNP was loaded onto Cas9 mRNA and GFP-specific gRNA and applied to GFP. + HEK-293T cells. Nearly 100% knockout (KO) efficiency was demonstrated in the model assay. Nanoparticle application was performed once, followed by a complete replacement of the culture medium after 24 h. Cells were then incubated for an additional 14 days, and the reduction in GFP was measured using flow cytometry.

[0421] exist Figure 28 The study demonstrated that BLNP is also applicable to primary human immune cells. BLNP was applied to isolated primary human leukocytes and incubated for 18 h. Subsequently, the cells were examined by flow cytometry using antibody staining. It was shown that transfection occurred only in CD45 / CD14 positive cells, and therefore in monocytes.

[0422] Figure 29 Combining from Figure 27 and 28 The findings of the experiment. Large peritoneal macrophages were extracted from genetically modified mice and treated in vitro with CRISPR-Cas9 BLNPs. Figure 29 The results showed that mice stably produced GFP (NC). Furthermore, it was confirmed that the combined application of Cas9 mRNA and anti-GFP gRNA induced knockout (KO). This was demonstrated by a decrease in GFP signaling (right panel, top and bottom).

Claims

1. Lipid nanoparticles, which contain a) 51 to 94.9 mol% of at least one cationic / ionizable lipid, b) At least one phospholipid, comprising 5 to 40 mol% of its components, and c) 0.1 to 10 mol% of at least one occult lipid, said occult lipid being selected from the group consisting of lipids containing one or more poly(olefin oxide) chains (PEG-lipids), lipids containing one or more poly(oxazoline) chains (POx-lipids), lipids containing one or more polyglycerol chains (PG-lipids), lipids containing one or more poly(hydroxyalkyl(meth)acrylate) chains (PHAA-lipids), lipids containing one or more poly(N-(hydroxyalkyl)(meth)acrylamide) chains (PHAAA-lipids), lipids containing one or more poly(vinylpyrrolidone) chains (PVP-lipids), lipids containing one or more poly(N,N-dialkyl(methyl)acrylamide) chains (PDMAA-lipids), lipids containing one or more poly(N-(meth)acryloylmorpholine) chains (PAM-lipids), or lipids containing one or more poly(amino acid) chains (PAA-lipids), provided that all lipids contained in the lipid nanoparticles have an HLB value of 3 or greater, and the percentages given are based on the total mass of lipids contained in the lipid nanoparticles.

2. The lipid nanoparticles according to claim 1, characterized in that, The lipid nanoparticles are loaded with active substances having anionic groups.

3. The lipid nanoparticles according to claim 1, characterized in that, The cationic lipid a) has a molar mass fraction of 55 to 89.5%, the phospholipid b) has a molar mass fraction of 10 to 30%, and the occult lipid c) has a molar mass fraction of 0.5 to 5%, wherein the percentages given are based on the total mass of lipids contained in the LNP.

4. The lipid nanoparticles according to at least one of claims 1 to 3, characterized in that, The lipid nanoparticles, apart from lipids a), b), and c), do not contain any other lipids d) and do not contain any excipients or additives e).

5. The lipid nanoparticles according to at least one of claims 1 to 4, characterized in that, Cationic lipids a) do not contain phosphate groups and contain at least one amino group.

6. The lipid nanoparticles according to claim 5, characterized in that, The cationic lipid a) is an ionizable lipid that forms a positive charge at the nitrogen atom through protonation in an acidic pH range of 4 to 7 and exists almost neutrally in an alkaline pH range above 7.

7. The lipid nanoparticles according to claim 6, characterized in that, The cationic lipid a) contains one or two amino groups with a pK value of 7 to 9.

8. The lipid nanoparticles according to at least one of claims 1 to 7, characterized in that, The cationic lipid a) has the structure of formula (I). in, R 1 For formula R 4 R 5 N-(C m H 2m )-、CH3-(C n H 2n )-O-(C o H 2o )-、HO-(C m H 2m )-, HO-CH2-CH(OH)-CH2-, CH3-(CH2) n -O-CO-(C m H 2m )-、CH3-(C n H 2n )-CO-O-(C m H 2m )-、NC-(C o H 2o )-、HO-CH2-CH((C o H 2o )-CH3 )-、CH3-(C o H 2o )-CH(OH)-(C p H 2p )-、CH3-(C n H 2n )-CO-NH-(C o H 2o )-、(HO-CH((C q H 2q )-CH3)-CH((C o H 2o )-OH)- or C6H 10 (OH)- groups, R 2 and R 3 Each is an alkyl group having six to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO-, and / or an alkylene group having six to twenty carbon atoms and one, two or three double bonds that are not directly adjacent to each other. R 4 and R 5 Each of the following is independently hydrogen or an alkyl group having one to five carbon atoms, or a group R 4 and R 5 Both, together with a common nitrogen atom, form a pyrrolidinyl group or a piperidine group. m is an integer from 2 to 6. n represents an integer from 0 to 6. o and p are independent integers from 1 to 6. q is an integer from 2 to 16, and r represents 0 or 1.

9. The lipid nanoparticles according to at least one of claims 1 to 8, characterized in that, The phospholipid b) has a structure of formula (IVa) or (IVb). in, LP is a saturated or mono-alkene to tri-alkene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other. Me can be hydrogen, a monovalent or divalent metal cation, or an ammonium cation. KG indicates a head group, representing an aliphatic group containing at least one hydroxyl group, preferably an aliphatic group having both a hydroxyl and an amino group, an aliphatic group having both a hydroxyl and a quaternary ammonium group, or a carbohydrate group having five to six hydroxyl groups. The group LP can have different meanings within a molecule within a given definition.

10. The lipid nanoparticles according to at least one of claims 1 to 8, characterized in that, The phospholipid b) has a structure of formula (Va) or (Vb). in, LP is a saturated or mono-alkene to tri-alkene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other. Me can be hydrogen, a monovalent or divalent metal cation, or an ammonium cation. KG indicates a head group, representing an aliphatic group containing at least one hydroxyl group, preferably an aliphatic group having both a hydroxyl group and an amino group, an aliphatic group having both a hydroxyl group and a quaternary ammonium group, or a carbohydrate group having five to six hydroxyl groups. mp is an integer from 2 to 8, especially 6, and In compounds of formula (Vb), the group LP can have different meanings within the molecule within a given definition.

11. The lipid nanoparticles according to at least one of claims 1 to 10, characterized in that, The occult lipid c) has a structure of formula (VIIIa) or (VIIIb). in, LPL is a saturated or mono-alkenyl to triene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other, or it is a sterol group. ol is an integer from 5 to 500, preferably from 10 to 200. R 9 Representing hydrogen, an alkyl or group having one to six carbon atoms, preferably hydrogen, methyl, ethyl, or sterol groups, and The group LPL can have different meanings within a molecule within a given definition.

12. The lipid nanoparticles according to at least one of claims 1 to 10, characterized in that, The elusive lipid c) has a structure of formula (IXa) or (IXb). LPL-NH-CO-O-CH2-CH2-(O-CH2-CH2) ol-1 -OR 9 (IXa) in, LPL is a saturated or mono-alkenyl to triene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other, or a sterol group. ol is an integer from 5 to 500, preferably from 10 to 200. R 9 Representing hydrogen, an alkyl or group having one to six carbon atoms, preferably hydrogen, methyl, ethyl, or sterol groups, and In compounds of formula (IXb), the group LP1 can have different meanings within the molecule within a given definition.

13. The lipid nanoparticles according to at least one of claims 1 to 11, characterized in that, The hidden lipid c) is a POx-lipid.

14. The lipid nanoparticles according to claim 13, characterized in that, The POx-lipid has a structure of formula (XIIIa) or (XIIIb). in, LPL is a saturated or mono-alkenyl to triene unsaturated alkyl or alkenyl group having six to twenty carbon atoms, wherein multiple double bonds are not directly adjacent to each other, or it is a sterol group. ol is an integer from 5 to 500, preferably from 10 to 200. R 10 Representing hydrogen, an alkyl or group having one to six carbon atoms, preferably hydrogen, methyl, ethyl, or sterol groups, and R 11 Indicates hydrogen or C1-C4 alkyl, wherein The group LPL can have different meanings within a molecule within a given definition.

15. The lipid nanoparticles according to at least one of claims 1 to 14, characterized in that, Its lipids consist only of cationic lipids (a), phospholipids (b), and occult lipids (c). The cationic lipid a) is selected from the group consisting of: N,N-diolenoyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-diolenoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearate-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)-cholesterol (DC-Chol); N-(1-(2,3-dioleoyloxy)propyl)-N-2-(spermine-formylamino)ethyl)-N,N-dimethyl Ammonium trifluoroacetate (DOSPA), 1,2-dioleoyl-sn-3-phosphate ethanolamine (DOPE), bis(octadecylaminoglycyl-carboxyspermine) (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinolenicoyloxy-N,N-dimethylaminopropane (DLenDMA), The phospholipid b) is selected from the group consisting of: distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidyl-glycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-sn-glycerol-3-phosphate ethanolamine-N- Sodium (maleiminomethyl) (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-trans-PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE) or 1,2-ditransoleoyl-sn-glycerol-3-phosphoethanolamine (transDOPE), and The elusive lipid c) is selected from the group consisting of: PEGylated diacylglycerol (PEG-DAG), especially 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG-succinate-diacylglycerol (PEG-S-DAG), especially 4-O-(2',3'-di(tetradecyloxy)propyl-1-O-(ω-methoxy-(polyethoxy)ethyl)succinate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG-dialkoxypropylcarbamate, especially ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)-carbamate or 2,3-di(tetradecyloxy)propyl-N-(ω-methoxy-(polyethoxy)ethyl)-carbamate.

16. The lipid nanoparticles according to claim 2, characterized in that, The active substance is a nucleic acid, preferably DNA and / or RNA, especially nucleic acids selected from the group consisting of: A-DNA, B-DNA, Z-DNA, mtDNA, bacterial DNA, antisense DNA, viral DNA, plasmid, hnRNA, mRNA, tRNA, rRNA, mtRNA, snRNA, snoRNA, scRNA, siRNAa, miRNA, antisense RNA, bacterial RNA, and viral RNA.

17. The lipid nanoparticles according to at least one of claims 1 to 16, characterized in that, The particle size (z-average) of the lipid nanoparticles is between 30 and 500 nm, particularly preferably between 40 and 250 nm, and especially between 50 and 200 nm, wherein the particle size is determined by dynamic light scattering (DLS).

18. The lipid nanoparticles according to at least one of claims 1 to 17, characterized in that, The lipid nanoparticles have a PDI with a particle size distribution between 0.01 and 0.4, preferably between 0.02 and 0.3, and particularly preferably between 0.05 and 0.

2. TG The polydispersity index was determined by dynamic light scattering (DLS).

19. The lipid nanoparticles according to claim 16, characterized in that, The lipid nanoparticles have a molar ratio (N / P ratio) of nitrogen atoms in cationic lipid a) to phosphate groups in nucleic acids that is between 1 and 100, preferably between 1.5 and 50, particularly preferably between 2 and 25, and very particularly preferably between 3 and 15.

20. The lipid nanoparticles according to claim 19, characterized in that, The lipid nanoparticles have a diameter (z-average) between 40 and 250 nm, particularly between 50 and 200 nm, as determined by DLS, a polydispersity index between 0.05 and 0.2, and an N / P ratio between 3 and 15.

21. A method for preparing lipid nanoparticles according to any one of claims 1, 3 to 15, or 17 to 18, wherein... Including the following measures: i) Prepare aqueous dispersions of the lipids a), b) and c) according to claim 1 in a buffer solution with a pH range of 3 to 8, preferably 4 to 7.

5. ii) Combine the aqueous dispersions from step i); and iii) The combined aqueous dispersion from step ii) is processed by a mixing method selected from the group consisting of ultrasound, double centrifugation, nanoprecipitation, and microfluidics, or by mixing in a vortex mixer, thereby forming nanoparticles.

22. A method for preparing lipid nanoparticles according to any one of claims 2, 16, or 19 to 20, comprising the following steps: iv) Prepare an aqueous dispersion containing lipid nanoparticles from step iii) of the method according to claim 21. v) Prepare an aqueous solution or dispersion of the active substance having anionic groups in a buffer solution with a pH range of 3 to 8, preferably 4 to 7.

5. vi) Combine the aqueous dispersions or solutions from steps iv) and v); and vii) The combined aqueous dispersion or solution from step vi) is treated by sonication, microfluidics, double centrifugation, nanoprecipitation, or vortex mixer to form lipid nanoparticles loaded with active substances.

23. A method for preparing lipid nanoparticles according to any one of claims 2, 16, or 19 to 20, comprising the following steps: I) Prepare aqueous dispersions of the lipids a), b) and c) according to claim 1 in a buffer solution with a pH range of 3 to 8, preferably 4 to 7.

5. II) Prepare an aqueous solution or dispersion of the active substance having anionic groups in a buffer solution with a pH range of 3 to 8, preferably 4 to 7.

5. III) Combine the aqueous dispersions or solutions from steps I) and II); and IV) The combined aqueous dispersion or solution from step III) is treated by sonication, microfluidics, double centrifugation, nanoprecipitation, or vortex mixer to form lipid nanoparticles loaded with active substances.

24. The method of claim 22, further comprising the following measures: V) Prepare an aqueous dispersion containing lipid nanoparticles with a pH between 3 and 8 from step iii) of the method according to claim 21. VI) Prepare nucleic acids in an aqueous solution or dispersion in a buffer solution with a pH range of 3 to 8, preferably 4 to 7.5; VII) The two dispersions or solutions from steps V) and VI) are mixed in a selected ratio of nucleic acid to lipid a) to obtain a desired molar N / P ratio of nitrogen atoms in lipid a) to phosphate groups in nucleic acid, preferably an N / P ratio between 1 and 100. VIII) Stir the mixture from step VII); and IX) Optionally, the mixture obtained is then incubated.

25. A method for transferring a gene into a cell, comprising the following steps: A) Contacting cells, tissues, or cell cultures with an aqueous dispersion containing nucleic acid-containing lipid nanoparticles according to claim 16, and B) Subsequent warming.

26. The method of claim 25, further comprising the following steps: C) Provide cell cultures in a bioreactor or incubator. D) Add an aqueous dispersion containing nucleic acid-containing lipid nanoparticles according to claim 16. E) Distribute the aqueous dispersion in the cell culture, and F) Subsequent warming.

27. Use of the lipid nanoparticles according to any one of claims 2, 16 or 19 to 20 for transferring genes into cells.

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