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

Abstract

The invention relates to lipid nanoparticles containing selected quantities 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 of greater than or equal to 3. These nanoparticles can be charged with active substances containing anionic groups, preferably with nucleic acids, and are suitable as highly efficient vehicles for transferring nucleic acids into cells.

Description

[0001] Description

[0002] Nanoparticles for the transport of active substances with anionic groups, processes for their preparation and their use

[0003] The invention relates to the field of production and processing of nanoparticles that can be loaded with active substances, e.g., genetic material. These nanoparticles can be advantageously used for the transport of active substances into organisms, in particular for the transfer of nucleic acids into cells.

[0004] Vaccine antigens, especially purified or recombinant subunit vaccines, are often poorly immunogenic and require the use of adjuvants to stimulate protective immunity. Despite the success of currently approved adjuvants, there remains a need for improved adjuvants and delivery systems that enhance protective antibody responses, particularly in populations with poor response to current vaccines.

[0005] Lipid nanoparticles (LNPs) represent an alternative to other particulate systems such as emulsions, liposomes, micelles, microparticles and / or polymeric nanoparticles for the delivery of active substances, such as oligonucleotides and low-molecular-weight drugs. LNPs and their use for the delivery of active substances have already been described, for example in US 7,691,405, US 2006 / 0083780, US 2006 / 0240554, US 2008 / 0020058, US 2009 / 0263407, US 2009 / 0285881, WO 2009 / 086558, WO2009 / 127060, WO2009 / 132131, WO2010 / 042877, WO2010 / 054384, WO2010 / 054401, WO2010 / 054405 and WO2010 / 054406. Lipid-based nanoparticles as carriers for pharmaceutical active ingredients have also been described, e.g., 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.

[0006] Gene therapy using siRNA and mRNA has gained increasing importance in recent years (cf. 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 efficacy 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).

[0007] This requires the delivery of various nucleic acids such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA, or ASOs). Due to their high molar mass and negative charge, these require a transporter, unlike conventional drugs (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). However, the application of gene transfer to cure, treat, or prevent a wide range of diseases is limited due to technical and biological obstacles. Due to the high renal clearance, instability, and immune-activating potential of the genetic material, carrier systems are required. For this purpose, various nanomaterials have been developed to protect nucleic acids from degradation and nonspecific immune reactions or to transport them specifically to application areas or cell types.

[0008] With the outbreak of the COVID-19 pandemic at the beginning of 2020, lipid nanoparticles (LNPs) established themselves as an important pharmaceutical form for the transport of genetic material (cf. Tenchov, R.; Bird, R.; Curtze, AE; Zhou, Q., Lipid nanoparticles - From liposomes to mRNA vaccine delivery, a landscape of research diversity and advancement. ACS Nano 2021, 15, 16982-17015). In addition to the two clinically used COVID-19 vaccines, BNT162b and mRNA-1273, other promising therapeutic approaches have now been approved or are involved in clinical trials (cf. Kulkarni, JA; Witzigmann, D.; Thomson, SB; Chen, S.; Leavitt, BR; Cullis, PR; van der Meel, R., The current landscape of nucleic acid therapeutics. Nat.Nanotechnol. 2021, 16, 630-643; Hou, X.; Zaks, T.; Langer, R.; Dong, Y., Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 2021 , 6, 1078- 1094).To name a few: Fomivirsen (Vitravene) is an antisense oligonucleotide (ASO) that targets specific sequences of cellular RNA and was approved in 1998 for the treatment of cytomegalovirus infections of the retina in AIDS patients (withdrawn in 2002) (Roberts, TC; Langer, R.; Wood, MJ

[0009] A., Advances in oligonucleotide drug delivery. Nat. Rev. Drug Discov. 2020, 19, 673-694); Mipomersen (Kynamro) is an ASO designated as an orphan drug for hypercholesterolemia. Patisiran (Onpattro) is the first approved first-in-class RNA-interference novel drug in 2018 and is used to treat hereditary transthyretin 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 critical limb ischemia.

[0010] The approval of novel RNA-based systems has further advanced the development of non-viral delivery systems such as / β-acetylgalactosamine (GalNAc)-siRNA conjugates: i) givosiran (Givlaari) for the treatment of acute intermittent hepatic porphyria, ii) lumasiran (Oxlumo) for the treatment of primary hyperoxaluria type 1, and iii) inclisiran (Leqvio), a subcutaneous therapeutic for the treatment of hypercholesterolemia. Other RNA-based systems include lipid-based siRNA drugs and the mRNA vaccines against SARS-CoV-2 (Paunovska, K.; Loughrey, D.; Dahlman, JE, Drug delivery systems for RNA therapeutics. Nat. Rev. Genet. 2022, 23, 265-280). The currently approved LNPs of the vaccine consist of four different lipid components and the genetic material (cf. Kulkarni, JA; Cullis, PR; van der Meel, R., Lipid nanoparticles enabling gene therapies: From concepts to clinical utility, Nucl. Acid Ther. 2018, 28, 146-157; and Schoenmaker, L.;

[0011] Witzigmann, D.; Kulkarni, JA, et al., mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 2021, 601, 120586).

[0012] These include an ionizable lipid, the helper lipids 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol, and a lipid conjugated with polyethylene glycol (PEG). The lipids used in BNT162b (BioNTech) and mRNA-1273 (Moderna) fulfill various functions in the formulation of the LNPs and are based on an intensive lipid screening (Dolgin, E., The tangled history of mRNA vaccines. Nature 2021 , 597, 318-324).

[0013] For transport, the conserved negatively charged phosphates in the RNA and DNA backbone can be used for interactions with lipids. Cationic lipids play a key role in this process, as they are responsible for binding to the negatively charged genetic material. They also influence endosomal uptake (see Haid Albertsen, C.; Kulkarni, JA; Witzigmann, D., et al., The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv. Drug Del. Rev. 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).

[0014] The development of ionizable lipids is considered a breakthrough for the widespread application of LNPs (Dolgin, E., The tangled history of mRNA vaccines. Nature 2021 , 597, 318-324). The original cationic lipids achieved high transfection efficiency in cell cultures but caused toxic effects. In vivo, they exhibited a short half-life in the bloodstream and nonspecific binding to cell surfaces. To overcome this obstacle, so-called ionizable lipids with a pKa value below 7 were developed. These lipids bind to the genetic material through a pH-dependent formulation that begins at a lower pH to allow binding of the genetic material and slowly increases to physiological pH. The formulations are neutral upon administration, e.g., intravenously or intramuscularly, and are charged under the acidic environment in the endosome, which offers advantages for endosomal release.Protonation in the endosome provides good interaction with the lipids of the endosomal membrane (Han, X.; Zhang, H.; Butowska, K.; Swingle, KL; Alameh, M.-G.; Weissman, D.; Mitchell, M.J., An ionizable lipid toolbox for RNA delivery. Nat. Commun. 2021, 12, 7233). After optimization, ionizable lipids showed promising results even with less genetic material. Regarding the ionizable headgroup, a dimethylamino base showed high transfection efficiency (Mo, R.; Sun, Q.; Li, N.; Zhang, C., Intracellular delivery and antitumor effects of pH-sensitive liposomes based on zwitterionic oligopeptide lipids. Biomaterials 2013, 34, 2773-2786).

[0015] There are a variety of different ionizable lipids that differ in their potential for gene transfer into LNP (Semple, SC; Akinc, A.; Chen,

[0016] J., et al., Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 2010, 28, 172-176). Influencing factors include the length of the alkyl chains, the type of ionizable group, and the pK a -Value of the molecule (see Haid Albertsen, C.; Kulkarni, JA; Witzigmann, D., et al., The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv. Drug Del. Rev. 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;

[0017] KA; Dorkin, JR; Vegas, AJ, et al., Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity. Nat. Commun. 2014, 5, 4277). To minimize unwanted interactions with the biological environment or the immune system, stealthy surface modifications of particles are crucial. The term "stealth," which dates back to initial studies in the 1980s, describes the "hiding" of particles by functionalizing them with hydrophilic molecules to evade detection and elimination by the immune system. The stealth effect is caused by the restriction of 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 circulation after systemic administration (cf. Friedl, JD; Nele, V.; De Rosa, G.; Bernkop-Schnürch, A., Bioinert, Stealth or interactive: How surface chemistry of nanocarriers determines their fate in vivo, Adv. Func. Mater. 2021, 31, 2103347).

[0018] Camouflaged nanoparticles influence drug delivery, particularly in cancer therapy. In 1994, Langer et al. (Gref, R.; Minamitake, Y.; Peracchia, MT; Trubetskoy, V.; Torchilin, V.; Langer, R., Biodegradable long-circulating polymeric nanospheres. Science 1994, 263, 1600-1603.) presented PEG-grafted polymer nanoparticles that can circulate in the blood longer due to the passivation effect of PEG. By forming a hydration layer and a steric barrier, PEGylation can reduce the nonspecific binding of serum proteins to the particles, thereby reducing their clearance by cells of the mononuclear phagocytic system (MPS). These long-circulating nanoparticles have been shown to be advantageous for drug delivery to the tumor microenvironment due to the enhanced permeation and retention (EPR) phenomenon (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).

[0019] Several alternatives to PEGylated compounds are known. These generally demonstrate prolonged systemic circulation time, sustained drug release kinetics, and improved tumor accumulation. Examples of alternative polymers to PEG include poly(glycerine) (PG), poly(oxazoline) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly( / V-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(A / ,A / -dimethylacrylamide) (PDMA) and Poly( / V-acryloylmorpholine) (PAcM) (see Hoang Thi, TT; Pilkington, EH; Nguyen, DH; Lee, JS; Park, KD; Truong, NP, The Importance of poly(ethylene glycol) alternatives for overcoming PEG immunogenicity in drug delivery and bioconjugation. Polymers 2020, 12, 298).

[0020] Among the so-called stealth lipids, PEG lipids have so far been the most important and widely used. PEG lipids in the LNP formulation are responsible, among other things, for extending the residence time in the organism and they reduce the organism’s immune response to the drug (cf. Nosova, AS; Koloskova, OO; Nikonova, AA; Simonova, VA; Smirnov, VV; Kudlay, D.; Khaitov, MR, Diversity of PEGylation methods of liposomes and their influence on RNA delivery. MedChemComm 2019, 10, 369-377; Bao, Y.; Jin, Y.; Chivukula, P.; Zhang, J.; Liu, Y.; Liu, J.; Clamme, JP; Mahato, R. L; 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.; and Suzuki, T.; Suzuki, Y.; Hihara, T.; Kubara, K.; Kondo, K.; Hyodo, K.; Yamazaki, K.; Ishida, T.; Ishihara, H., PEG shedding-rate-dependent blood clearance of PEGylated lipid nanoparticles in mice: Faster PEG shedding attenuates anti-PEG IgM production. Int. J. Pharm. 2020, 588, 119792). In addition, they were shown to have a significant influence on the size and stability of the resulting nanoparticles and stabilize the formulation (cf. 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, the formation of antibodies is a hindrance (Haid Albertsen, C.; Kulkarni, JA; Witzigmann, D.; Lind, M.; Petersson, K.; Simonsen, JB, The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv. Drug Deliv. Rev. 2022, 188, 114416), which is why alternatives to PEG are being researched (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).

[0021] The remaining components in the form of helper lipids increase the encapsulation efficiency of the genetic material and the endosomal release of the LNP (cf. 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).

[0022] Several LNPs and their preparation have already been described in the patent literature.

[0023] For example, a lipid-encapsulated nucleic acid is known from US Pat. No. 7,341,348 B2. The patent describes a particle consisting of a lipid layer surrounding a nucleic acid-containing central zone and containing an aminolipid with an amino group with a pKa value of 4 to 11 and a PEG-DAG conjugate.

[0024] From WO 2018 / 081480 A1 it is known in LNP that 40 to 50 mol % cationic

[0025] Contains lipid, neutral lipid, steroid, polymer-conjugated lipid, and a therapeutic agent encapsulated therein. WO 2019 / 089828 A1 discloses a LNP with a bilayer structure containing at least 40 mol% of a cationic lipid and a nucleic acid encapsulated therein.

[0026] EP 3 556 353 A2 describes LNPs composed of a cationic lipid, a PEG-lipid, and an antigen. These LNPs typically contain other components, such as cholesterol or phospholipids. The lipids are dissolved in ethanol to produce the LNPs.

[0027] The LNPs described in these documents contain increasingly large amounts of steroids, such as cholesterol. These LNPs are produced from an ethanolic-aqueous solution, as this is not possible in an aqueous solution.

[0028] In Nanoscale, 11, 2019, 18806-18824, Dongyu Chen et al. describe LNPs in which cholesterol has been replaced by a modified cholesterol DC-CHOL containing an ammonium group. This lipid has a Griffin HLB value of 5.8. In addition to the DC-CHOL component, the LNPs described in this document also contain a cationic lipid, a phospholipid, and a stealth lipid. The cationic lipids have Griffin HLB values ​​of less than 4. Information on the amount of lipids used in the LNPs is missing from this document.

[0029] LNPs with reduced cholesterol content are disclosed by Maho Kawatuchi et al. in J. of Pharmaceutical Sciences, 112 (2023) 1401-1410 and by Samuel T. LoPresti et al. in Journal of Controlled Release, 345 (2022) 819-831. The LNPs described in these documents both exhibit a reduction but not a replacement of cholesterol content. In both studies, the produced LNPs exhibit structural disadvantages compared to the commercial cholesterol-containing alternative. At the same time, the authors were unable to produce LNPs with a complete absence of pure cholesterol. Furthermore, both studies describe LNPs containing various lipids whose HLB value is not greater than or equal to 3.

[0030] Various methods can be used to produce LNPs. The most common are ultrasonication, 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-lipid nanoparticles for therapeutic applications. Methods Mol. Biol. 2014, 1141, 109-120). The latter technique has prevailed for commercial scale in the case of the COVID-19 vaccine. One of the advantages of microfluidics is its good reproducibility in production from batch to batch (cf. Maeki, M.; Uno, S.; Niwa, A.; Okada, Y.; Tokeshi, M., Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery. J. Control.Release 2022, 344, 80-96; and Shepherd, SJ; Issadore, D.; Mitchell, MJ, Microfluidic formulation of nanoparticles for biomedical applications. Biomaterials 2021, 274, 120826). The LNPs are formed by the rapid mixing of an organic lipid phase (usually based on ethanol) and an aqueous phase, the latter containing the genetic material. During this process, the lipids precipitate, and nanoparticles are formed. The organic solvent must then be removed from the formulation. In addition to evaporation, dialysis or cross-flow filtration are often used (cf. 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.; Mathre, 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.

[0031] 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 a design-of-experiments approach. Langmuir 2021, 37, 1120-1128). In the case of the described LNPs, the solvents are dialyzed against PBS. The formulation is directly adjusted to the physiological pH of 7.4.

[0032] The use of organic solvents for formulation represents a significant disadvantage. The genetic material becomes more unstable and prolonged storage can lead to degradation of the lipids (cf. 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.;

[0033] 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 the nucleic acids, rapid removal of the organic solvents is necessary. This leads to increased production costs.

[0034] The material from which the microfluidic chips are made can also be disadvantageous. Polydimethylsiloxane, for example, absorbs genetic material and tends to swell in 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).

[0035] Avoiding organic solvents for the production of lipid-based nanoparticles thus represents a good opportunity to overcome some of the disadvantages of conventional production. It has been shown that ethanol is not absolutely necessary for the encapsulation of genetic material (cf. Kulkarni, JA; Thomson, SB; Zaifman, J.; Leung, J.; Wagner, PK; Hill, A.; Tam, YYC; Cullis, PR; Petkau, TL; Leavitt, BR, Spontaneous, solvent-free entrapment of siRNA within lipid nanoparticles. Nanoscale 2020, 12, 23959-23966). One way to avoid the use of ethanol is to homogenize the lipids in an aqueous solution above their melting point. Alternating cooling and heating leads to the formation of LNPs (cf. 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 heat-stable, which, in the case of lipids and RNA, requires intensive quality control.

[0036] It has now been surprisingly discovered that LNPs can be produced without the use of organic solvents. Conventional processes required the use of organic solvents, such as ethanol, to incorporate the highly lipophilic portions of the lipids into the LNPs. The process according to the invention allows the production of LNPs that can be loaded at high concentrations with active ingredients (e.g., nucleic acids) containing anionic groups, such as phosphate groups.

[0037] The LNPs according to the invention (also referred to as BLNPs in this description) can be loaded with sensitive active substances in a gentle manner and allow, for example, the introduction and transport of genetic material into cells.

[0038] The BLNPs according to the invention are produced without the use of highly lipophilic steroids and thus contain fewer lipid components than previously known LNPs. Conventional LNPs contain large amounts of cholesterol. This easily diffuses between the LNPs and also serum components and lipids in the biological environment. Furthermore, cholesterol is not produced entirely synthetically but rather from natural sources, meaning fluctuating quality and impurities cannot be ruled out. Furthermore, a process with more steps and components is more complex for regulatory approval, and thus more expensive and less robust.

[0039] The lipophilic character of lipids can be described by the HLB value (HLB stands for hydrophilic-lipophilic balance). The HLB value was introduced by WC Griffin in 1954 and describes the hydrophilic and lipophilic proportions of lipids. The HLB value scale ranges from 0 (strongly lipophilic) to 20 (weakly lipophilic). In addition to the Griffin method, there are other methods for calculating the HLB value. However, these are far less common. One example is the method according to Davies, who in 1957 proposed calculating the HLB value from numerical values ​​for the individual chemical groups of a molecule. The advantage of this method is the higher weighting of strongly interacting groups over less interacting ones. It can also be used to define the HLB value for cationic and anionic lipids.

[0040] An object of the present invention was to provide lipid nanoparticles which have a compact and simple structure, which can be loaded with a high content of active ingredient and which are excellently suited for the transport of active ingredients into organisms or cells, for example for the gene transfer of nucleic acids.

[0041] A further object of the present invention was to provide a simple method for producing lipid nanoparticles that can be carried out without the use of organic solvents.

[0042] The present invention relates to lipid nanoparticles containing a) 51 to 94.9 mol% of at least one cationic / ionizable lipid, b) 5 to 40 mol% of at least one phospholipid, and c) 0.1 to 10 mol% of at least one stealth lipid selected from the group of lipids containing one or more poly(alkylene oxide) chains (hereinafter "PEG lipids"), lipids containing one or more poly(oxazoline) chains (hereinafter "POx lipids"), lipids containing one or more poly(glycerol) chains (hereinafter "PG lipids"), lipids containing one or more poly(hydroxyalkyl(meth)acrylate) chains (hereinafter "PHAA lipids"), lipids containing one or more poly(V-(hydroxyalkyl)(meth)acrylamide) chains (hereinafter "PHAAA lipids"), the lipids containing one or more poly(vinylpyrrolidone) chains (hereinafter "PVP lipids"), the lipids containing one or more poly(A / ,A / -dialkyl(meth)acrylamide) chains (hereinafter "PDMAA lipids"),the lipids containing one or more poly(A / -(meth)acryloylmorpholine) chains (hereinafter "PAM lipids") or the lipids containing one or more poly(amino acid) chains (hereinafter "PAA lipids"), with the proviso that all lipids contained in the lipid nanoparticle have an HLB value greater than or equal to 3 and that the stated percentages refer to the total mass of the lipids contained in the lipid nanoparticle.

[0043] In the context of this description, “lipid nanoparticles” or “LNPs” or “BLNPs” are understood to mean particles whose diameter (z-average) is less than or equal to 900 nm and which consist mainly or entirely of lipids from the above-mentioned groups a), b) and c). These BLNPs can be loaded with active ingredients containing anionic groups. The BLNPs are generally characterized by a very high surface-to-volume ratio and thus offer very high chemical reactivity. BLNPs can consist only of the aforementioned lipids from groups a), b) and c) or can additionally contain complexes of the active ingredient and the cationic lipid from group a), or the BLNPs can contain, in addition to the lipids and optionally complexes, small amounts of other components, such as excipients or additives e).

[0044] For the purposes of this description, "auxiliaries and additives" are substances that are added to a formulation to impart certain additional properties and / or to facilitate its processing. Examples of excipients and additives include sugars such as sucrose, contrast agents, carriers, fillers, pigments, dyes, perfumes, radiopharmaceuticals such as tracers, lubricants, UV stabilizers, polymers such as nitrogen-containing polymers, or antioxidants. In particular, "auxiliaries and additives" are understood to mean any substance useful for the intended application that is neither a pharmaceutical or agrochemical active ingredient nor a lipid, but can be formulated together with an active ingredient in an active ingredient-lipid complex to influence, in particular improve, the qualitative properties of the LNP.Preferably, the excipients and / or additives e) have no effect or, with regard to the intended treatment, no significant effect or at least no undesirable effect.

[0045] In the context of this description, HLB value is understood to be a numerical value between 0 and 20, which is calculated according to the following formula

[0046] HLB = 20 * (1 - M| / M), where Mi is the molar mass of the lipophilic part of a molecule and M is the molar mass of the entire molecule.

[0047] In the context of the invention, the freely accessible software MarvinSketch 23.4 is used to determine the HLB value of the lipids (cf. https: / / docs.chemaxon.- com / display / docs / hlb-predictor.md#src-1806640-hlbpredictor-fig-1 ) and the HLB is determined according to Griffin, since the Davis method is not optimal for stealth lipids with many repeating units.

[0048] Highly lipophilic compounds generally have HLB values ​​of 1 to 3. These are hydrophilic (oil-soluble) lipids, such as antifoam agents. Compounds with significant hydrophilic content are dispersible in water and have HLB values ​​of 3 to 9. These include water-in-oil 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 oil-in-water 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.

[0049] Phospholipids typically have HLB values ​​of 4 to 5.

[0050] Preferably, all lipids in the nanoparticles according to the invention have HLB values ​​of 3 to 20, in particular of 3 to 18, in particular of greater than or equal to 4 and very particularly preferably of 4 to 17.5.

[0051] The BLNPs according to the invention can be loaded with active ingredients that contain at least one anionic group. The invention therefore also relates to the above-described LNPs loaded with active ingredients containing anionic groups.

[0052] In the BLNPs according to the invention, the molar fraction (mol%) of the cationic lipid a) or the combined amount of lipids a) is typically 51 to 94.9%, preferably 55 to 89.5%, particularly preferably 60 to 85% and most preferably 75 to 82%.

[0053] In the BLNPs according to the invention, the molar fraction (mol%) of the phospholipid b) or the combined amount of lipids b) is typically 5 to 40%, preferably 10 to 30%, particularly preferably 14 to 25% and most preferably 15 to 18%.

[0054] In the BLNPs according to the invention, the molar fraction (mol%) of the stealth lipid c) or the combined amount of lipids c) is typically 0.1 to 10%, preferably 0.5 to 5% and most preferably 1 to 3%.

[0055] The percentages given above refer to the total amount of lipids contained in the BLNP. If the BLNPs according to the invention contain additional lipids d) with HLB values ​​of at least 3 that do not belong to one of groups a) to c), the weight fraction of these lipids d) is at most 10%, preferably at most 5%, and in particular at most 1%.

[0056] If the BLNPs according to the invention contain auxiliaries or additives e), their total weight proportion is at most 5%, preferably at most 1% and in particular at most 0.5%.

[0057] The BLNPs according to the invention preferably contain no further lipids d) and no excipients or additives e).

[0058] Most preferably, the proportion of lipids with sterol residues in the BLNPs according to the invention is 0 to 15 mol%, in particular 0 to 10 mol%.

[0059] The cationic lipids a) for the production of the BLNPs according to the invention include all lipids with at least one cationic group, for example, an amino group. However, these do not include phospholipids, which are classified as lipids in group b). Cationic lipids a) preferably do not contain any phosphate residues.

[0060] Examples of cationic groups are amino groups, i.e. primary, secondary and tertiary amino groups or quaternary ammonium groups; guanidino groups and amide groups, i.e. groups with secondary, tertiary and quaternary amide groups; aminoalkanol groups, i.e. groups with primary, secondary, tertiary and quaternary amino groups; phosphane groups, i.e. primary, secondary and tertiary phosphane groups or quaternary phosphonium groups.

[0061] The term "cationic lipid" used here refers to lipids that exhibit one or more positive net charges at certain pH values, e.g., acidic pH values. Cationic lipids within the scope of this description also include ionizable cationic lipids. Ionizable cationic lipids are characterized by the weak basicity of their ionizable groups, which influences the charge of the lipid in a pH-dependent manner. As a result, these lipids will be positively charged at acidic pH values, but are nearly charge-neutral at physiological pH values.

[0062] The preferred cationic lipids a) for the preparation of the BLNPs according to the invention include all lipids with at least one amino group (which are not phospholipids).

[0063] The preferred cationic lipids a) for producing the BLNPs according to the invention also include ionizable lipids containing at least one amino group (which are not phospholipids). These have the property of developing a positive charge on the nitrogen atom via protonation in the acidic pH range of 4 to 7 and of being virtually neutral in the basic pH range above 7.

[0064] Particularly preferably, ionizable lipids a) are used for the preparation of the BLNPs according to the invention which contain at least one amino group (which are not phospholipids), in particular one to two amino groups and very particularly preferably one amino group, said amino group(s) having / having a pKa value of 7 to 9.

[0065] The cationic lipids preferably have a) no phosphate residues and one or two nitrogen atoms, preferably one nitrogen atom, and at least one alkyl residue having six to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO- or an amide group -CO-NH- or -NH-CO-, and / or at least one alkylene residue having six to twenty carbon atoms and one, two, or three double bonds that are not directly adjacent to one another. These nitrogen atoms can be present as amino groups, amide groups, or alkanolamino groups, preferably as amino groups.

[0066] Particularly preferably, the cationic lipids a) have no phosphate residues and one or two nitrogen atoms and at least two alkyl residues having six to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O- CO- or an amide group -CO-NH- or -NH-CO-, or one or both of these alkyl residues are replaced by one or two alkylene residues having six to twenty carbon atoms and one, two or three double bonds which are not directly adjacent to one another.

[0067] Particularly preferred cationic lipids a) have the structure of formula (I)

[0068] R 3 R1-N 1 -(CO) r -R 2 (I), where R 1 a residue of the formula R 4 R 5 N-(C m H 2m )-, CH3-(C n H2n)-O-(C o H2o)-, HO-(C m H 2m )-, HO-CH2-CH(OH)-CH2-, CH3-(CH2) n -O-CO-(C m H 2m )-, CH3-(C nH 2n )-CO-O-(C m H 2m )-, NC-(C O H 2O )-, HO-CH2-CH((C0H 2O )-CH3)-, CH3-(C o H 2o )-CH(OH)-(CpH2p)-, CH3-(C n H2n)-CO-NH-(CoH 2o )-, (HO-CH((C q H 2q )-CH3)-CH((C o H 2o )-OH)- or C6H 10 (OH)-, R 2 and R 3 independently of one another are alkyl radicals having six to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO-, and / or alkylene radicals having six to twenty carbon atoms and one, two or three double bonds not directly adjacent to one another, R 4 and R 5 independently of one another are hydrogen or alkyl radicals having one to five carbon atoms or both radicals R 4 and R 5together with the common nitrogen atom form a pyrrolidine or piperidine residue, m is an integer from 2 to 6, n is an integer from 0 to 6, o and p are independently integers from 1 to 6, q is an integer from 2 to 16, and r is 0 or 1.

[0069] Very particular preference is given to cationic lipids a) of formula (I), wherein R 1 a residue of the formula R 4 R 5 N-(CH2) m - is, in particular those in which m is 2.

[0070] Extremely preferred are cationic lipids a) of formula (I), wherein R 1 a residue of the formula R 4 R 5 N-(CH2)2-, and R 4 and R 5 are independently hydrogen, methyl or ethyl or wherein or both radicals R 4 and R 5 together with the common nitrogen atom form a pyrrolidine or piperidine residue.

[0071] Other particularly preferred cationic lipids a) include those of formula (I), wherein R 2 and R 3 be selected independently from the remainder of the group

[0072] -(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), -(C s H 2s )-O-CO-CH((CtH 2t )-CH3)((C u H 2u )-CH3), -(C s H 2s )-CO-O-CH((C t H 2t )-CH3)(( C u H 2u )-CH3),

[0073] -(C S H 2S )-CO-O-( C t H 2t )-CH3, -(C s H 2s )-O-CO-(C t H 2t )-CH3, -(C s H 2s )-O-CO-CH2-CH((C t H 2t )-CH3)(( C u H 2u )-CH3), -(C s H 2s )-CO-O-CH2-CH((C t H 2t)-CH3)(( C u H 2u )-CH3), -CH((C s H 2s )-O-CO-CH2-CH((C t H 2t )-CH3)(( C u H 2u )-CH3))2, -CH((C s H 2s )-CO-O-CH2-CH((C t H 2t )-CH3)(( C u H 2u )-CH3))2, -CH((C s H 2s )-O-CO-CH((C t H 2t )-CH3)(( C u H 2u )-CH3))2, -CH((C s H 2s )-CO-O-CH((C t H 2t )-CH3)(( C u H 2u )-CH3))2, -(C t H 2t )-CH=CH-CH2-CH=CH-(C u H 2u )-CH3, -CH((C t H 2t )-CH=CH-CH2-CH=CH-( C u H 2u )-CH3)2,

[0074] -(C t H 2t )-CH=CH-( C U H 2U )-CH3und -CH((C t H 2t )-CH=CH-( C u H 2u )-CH3)2, -(C t H2t)-CH=C(CH3)-CH2-CH=CH-( C u H 2u )-CH3, -CH((C tH2t)-CH=C(CH3)-CH2-CH=CH-( C u H2u)-CH3)2, -(CtH2t)-CH=C(CH3)-(C u H 2u )-CH3 and -CH((C t H 2t )-CH=C(CH3)-( C u H 2u )-CH3)2, -(CtH2t)-CeHii and -C6H-IO-C(CH3)3, wherein s is an integer from 4 to 20, and t and u independently represent integers from 1 to 10.

[0075] Very particular preference is given to cationic lipids a) of formula (I), wherein R 1 a residue of the formula R 4 R 5 N-(CH2)2-, and R 4 and R 5 are independently hydrogen, methyl or ethyl or wherein or both radicals R 4 and R 5 together with the common nitrogen atom form a pyrrolidine or piperidine residue,

[0076] R 2 and R 3 are independently selected from the radicals of the formula -(CH2) V -O-CO-R 5 or -(CH2) V -CO-OR 6, v is an integer from 1 to 20, preferably from 5 to 12, and

[0077] R 5 and R 6 independently of one another are alkyl radicals having 6 to 20 carbon atoms and / or alkenyl radicals having 6 to 20 carbon atoms and having one or preferably two ethylenically unsaturated bonds not directly adjacent to one another, in particular radicals selected from the group

[0078] -(C V H 2V )-CH3, -(CH2)W-C6HH, -C6H 1O -C(CH3)3, -CH((CH2)x-CH3)((CH2) y -CH3), -CH2-CH((CH2)x-CH3)((CH2)y-CH3), -CH((CH2)xC(CH3)3)((CH2) y -C(CH3)3) , -CH2-CH((CH2)xC(CH3)3)((CH2) y -C(CH3)3) , -(CH2) w -CH((CH2)x-CH3)((CH2) y-CH=C(CH3)2), where v is an integer from 7 to 12, w is an integer from 1 to 4, x and y are independently integers from 0 to 12, and very particularly preferably radicals selected from the group -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) I CH((CH2)3-CH3)((CH2)3-CH3), -CH(CH3)-(CH2)9-CH3, -CH2-CH((CH2)5-CH3)((CH2)5-CH3),

[0079] -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-CH((CH2)5-CH3)((CH2)7-CH3),

[0080] -CH2-CH((CH2)3-CH3)((CH2)3-CH3), -(CH2)7-CH 3I -(CH2)IO-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,

[0081] -(CH2)2-C6HH and -C6HIO-C(CH3)3,

[0082] The cationic lipids a) preferably form a positive charge on the nitrogen atom in the pH range from 5 to 8. The compounds of formula (I) then exist as cationic compounds of formula (II).

[0083] R 3

[0084] R 1 -N + -(CO) r -R 2 (X'')j / i (II), where

[0085] R 1 , R 2 , R 3 and r have the meaning defined above, j is an integer corresponding to the number of nitrogen atoms in the compound of formula (II), preferably 1 or 2, i is an integer from 1 to 5000, and

[0086] X represents an i-valent anion.

[0087] Any inorganic or organic i-valent anions X can be used.

[0088] Examples of inorganic anions X' are halide ions, such as fluoride, chloride, bromide or iodide, or hydroxide ions or anions of inorganic acids, such as phosphate, sulfate, nitrate, hexafluorophosphate, tetrafluoroborate, perchlorate, chlorate, hexafluoroantimonate, hexafluoroarsenate, cyanide.

[0089] Examples of organic anions X 1 ' are anions of mono- or polybasic carboxylic acids or mono- or polybasic sulfonic acids, whereby these acids can be saturated or unsaturated. Examples of anions of organic acids are acetate, formate, trifluoroacetate, trifluoromethanesulfonate, pentafluoroethanesulfonate, nonofluorobutanesulfonate, butyrate, citrate, fumarate, glutarate, lactate, malate, malonate, oxalate, pyruvate, or tartrate.

[0090] These anions can be in the form of polyanions.

[0091] Further preferred cationic lipids a) contain a quaternary ammonium group. Examples of such lipids are compounds of the formula (IIa) (IIa), wherein

[0092] R 1a , R 2a and R 3a independently of one another are alkyl radicals having one to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO-, and / or alkylene radicals having two to twenty carbon atoms and one, two or three double bonds not directly adjacent to one another,

[0093] R 4a an alkyl radical having six to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO-, and / or an alkylene radical having six to twenty carbon atoms and one, two or three double bonds not directly adjacent to one another, or in which two radicals R 1a and R 2atogether with the common nitrogen atom form a pyrrolidine or piperidine residue, and X, i and j have the meaning defined above.

[0094] Ganz besonders bevorzugt enthalten die erfindungsgemäßen Nanopartikel kationischen Lipide, die ausgewählt sind aus der Gruppe N, A / -Dioleyl- / V, N- dimethylammoniumchlorid (DODAC); A / -(2,3-Dioleyloxy)propyl)-A / , / V,A / - trimethylammoniumchlorid (DOTMA); A / ,A / -Distearyl-A / ,A / -dimethylammoniumbromid (DDAB); A / -(2,3-Dioleoyloxy)propyl)-A / ,A / ,A / -trimethylammoniumchlorid (DOTAP); 3- (A / -(A / ',A / '-Dimethylaminoethan)-carbamoyl)-cholesterol (DC-Chol), / V-(1 -(2,3- Dioleoyloxy)propyl) / V-2-(spermin-carboxamido)ethyl)- / V, / V-dimethylammonium- trifluoroacetat (DOSPA), 1 ,2-Dioleoyl-sn-3phosphoethanolamin (DOPE), Diocta- decylamidoglycylcarboxyspermin (DOGS), 1 ,2-Dioleoyl-3-dimethylammonium- propan (DODAP), A / ,A / -Dimethyl-2,3-dioleoyloxy)propylamin (DODMA), A / -(1 ,2- Dimyristyloxyprop-3-yl)- / V, / V-dirnethyl- / V-hydroxyethylammoniurnbromid (DMRIE). 1 ,2-Dilinoleyloxy- / V,A / -dimethylaminopropan (DLinDMA), und 1 ,2-Dilinolenyloxy- / V,A / - dimethylaminopropan (DLenDMA).

[0095] Most preferably, the nanoparticles according to the invention contain only one cationic / ionizable lipid a).

[0096] The phospholipids b) used according to the invention are generally lipids which, in addition to at least one lipid residue, have an associated residue of a polyhydric alcohol, to which in turn a phosphate group is bound, which is linked to a head group via an ester bond.

[0097] A phospholipid b) generally has a structure of formula (III)

[0098] (LP) np -BG-OP(O)(OMe)-O-KG (III) wherein

[0099] LP is a residue of a fatty acid, BG is a (np+1 )-valent bridging group, np is an integer from 1 to 5, preferably 1 or 2,

[0100] Me is hydrogen, a monovalent or divalent metal cation or an ammonium cation,

[0101] KG represents a head group which is an aliphatic radical containing at least one hydroxyl group, preferably an aliphatic radical having one hydroxyl group and one amino group, one hydroxyl group and one quaternary ammonium group, or the radical of a carbohydrate having five to six hydroxyl groups, where

[0102] Residues LP can take on different meanings within a molecule within the given definitions.

[0103] In general, the phospholipids b) of formula (III) have one to five LP residues, preferably one or two LP residues, which are alkyl residues having six to twenty carbon atoms, and / or mono- to tri-ethylenically unsaturated alkenyl residues having six to twenty carbon atoms, and / or saturated or mono- to tri-ethylenically unsaturated fatty acid residues having six to twenty carbon atoms, wherein several double bonds in an alkenyl residue are not directly adjacent to one another.

[0104] In general, the phospholipids b) of formula (III) have one to five LP residues which are linked to the head group via a bridging group BG via a phosphate group, wherein the bridging group BG is the residue of a di- to hexavalent aliphatic or cycloaliphatic alcohol or a di- to hexavalent aliphatic or cycloaliphatic amino alcohol.

[0105] Examples of residues of di- to hexavalent aliphatic or cycloaliphatic alcohols are groups derived from ethylene glycol, propylene glycol, glycerol, propanetriol, pentaethylthritol, or inositol. Examples of residues of di- to hexavalent aliphatic or cycloaliphatic amino alcohols are groups derived from 2-aminoethanol, 3-aminopropanol, prolinol, alaninol, valinol, leucinol, phenylalaninol, phenylglycinol, or sphingosine.

[0106] Preferred phospholipids b) have a glycerol-derived residue as a bridging group and have the structure of formula (IVa) or (IVb)

[0107] OP(O)(OMe)-O-KG

[0108] I

[0109] LP-CO-O-CH2-CH-CH2-O-CO-LP (IVa)

[0110] O-CO-LP I

[0111] LP-CO-O-CH2-CH-CH2-OP(O)(OMe)-O-KG (IVb) wherein

[0112] LP is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to each other,

[0113] KG and Me have the meanings defined above, and the residues LP can take on different meanings within a molecule within the given definitions.

[0114] Further preferred phospholipids b) have a residue derived from sphingosine as a bridging group and have the structure of formula (Va) or (Vb)

[0115] NH-CO-LP

[0116] I

[0117] CH3-(CH2-CH2) mp -CH=CH-CH(OH)-CH-OP(O)(OMe)-O-KG (Va) LP-N-CO-LP

[0118] CH3-(CH2-CH2) mp -CH=CH-CH(OH)-CH-OP(O)(OMe)-O-KG (Vb) where

[0119] LP is a saturated or mono- to tri-ethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, KG and Me have the meanings defined above, mp is an integer from 2 to 8, in particular 6, and the radicals LP in the compound of formula (Vb) can assume different meanings within one molecule within the framework of the given definitions.

[0120] Further preferred phospholipids b) have as head group KG a residue derived from an aliphatic amino alcohol or a residue derived from inositol.

[0121] Particularly preferred are phospholipids b) with head groups KG of the formula (Via), (Vlb) or (Vic)

[0122] -O-(CppH 2p p)-NH2(Via) -O-(CppH 2p p)-N + R 6 R 7 R 8 (XP ip -)i / ip (Vlb)

[0123] -O-(Cpp.1H 2p p.1)-CH(NH2)(COOH) (Vic) where pp is an integer from 2 to 6, preferably 2,

[0124] R 6 hydrogen or Ci-C5-alkyl,

[0125] R 7 and R 8 are independently Ci-Ce-alkyl, and

[0126] XP represents an ip-valent anion, and ip is an integer from 1 to 3, preferably 1 or 2.

[0127] Phospholipids b) with head groups KG, which are selected from the group choline, ethanolamine, serine and inositol, are particularly preferred.

[0128] The nanoparticles according to the invention very particularly preferably contain phospholipids b) which are selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-sn-glycero-3-phosphoethanolamine- / V-(maleimidomethyl) sodium salt (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-I-trans PE, 1-Stearioyl-2-oleoylphosphatidyethanolamine (SOPE), and 1,2-Dielaidoyl-sn-glycero-3-phosphoethanolamine (transDOPE).

[0129] The stealth lipids c) used in the invention are generally lipids which, in addition to at least one lipid residue, have at least one associated poly(alkylene oxide) residue, poly(oxazoline) residue, polyglycerol residue, poly(hydroxyalkyl(meth)acrylate) residue, poly(α-(hydroxyalkyl)(meth)acrylamide) residue, poly(vinylpyrrolidone) residue, poly(α-dialkyl(meth)acrylamide) residue, poly(α-(meth)acryloylmorpholine) residue, or poly(amino acid) residue. These residues can be linked by a covalent bond or, preferably, via a bridging group BG.

[0130] Preferred stealth lipids c) do not have any phosphate residues.

[0131] Preferred stealth lipids c) are PEG lipids. These are, in particular, lipids with the structure of formula (VII) (LPL) nl -(BGL) m i-(O-CH2-CH2) O i-OR 9 (VII) wherein

[0132] LPL is an alkyl or alkenyl radical having 6-20 carbon atoms, a radical of a fatty acid, a fatty alcohol or a sterol radical, BGL is a (nl+1)-valent bridging group, nl is an integer from 1 to 5, preferably 1 or 2, ml is 0 or 1, preferably 1, ol is an integer from 5 to 500, preferably 10 to 200, and

[0133] R 9 represents hydrogen, alkyl having one to six carbon atoms or a radical LPL, preferably hydrogen, methyl ethyl or a sterol radical, where the radicals LPL can assume different meanings within a molecule within the framework of the given definitions.

[0134] In general, the stealth lipids c) of formula (VII) have one to five LPL residues, preferably one or two LPL residues, which are alkyl residues having six to twenty carbon atoms, and / or mono- to tri-ethylenically unsaturated alkenyl residues having six to twenty carbon atoms, and / or saturated or mono- to tri-ethylenically unsaturated fatty acid residues having six to twenty carbon atoms, and / or saturated or mono- to tri-ethylenically unsaturated fatty alcohol residues having six to twenty carbon atoms, and / or sterol residues, wherein several double bonds in an alkenyl residue are not directly adjacent to one another.

[0135] In general, the stealth lipids c) of formula (VII) have one to five LPL residues which are covalently linked directly to a PEG residue via an ester bond; or the stealth lipids c) of formula (VII) have one to five LPL residues which are linked to a stealth residue (PEG, POx or others) via a bridging group BGL, wherein the bridging group BGL is the residue of a di- to hexavalent aliphatic or cycloaliphatic alcohol, or the residue of a di- to hexavalent carboxylic acid, or a carbamate residue, or the residue of an amino alcohol.

[0136] Examples of residues of di- to hexavalent aliphatic or cycloaliphatic alcohols are groups derived from ethylene glycol, propylene glycol, glycerol, propanetriol, pentaethylthritol or inositol.

[0137] Examples of residues of di- to hexavalent carboxylic acids are groups derived from oxalic acid, maleic acid, fumaric acid, adipic acid, sebacic acid, succinic acid, tartaric acid, terephthalic acid, isophthalic acid, trimellitic acid, trimesic acid or pyromellitic acid.

[0138] Examples of carbamate residues are groups derived from residues of the formula >N-CO-O-, in which the PEG residue is bonded to the oxygen atom and one or two LPL residues to the nitrogen atom.

[0139] Examples of amino alcohol residues are groups derived from residues of the formula >NRO-, where R is a divalent organic residue, preferably an alkylene residue, the PEG residue is bonded to the oxygen atom, and one or two LPL residues are bonded to the nitrogen atom. Other amino alcohol residues can contain multiple amino groups and / or oxygen atoms, for example, aminophenols with two hydroxyl groups and / or amino groups.

[0140] Examples of sterol residues are residues derived from saturated or mono- or diethylenically unsaturated sterols (3-hydroxysterols), which are preferably substituted in the 17-position with an alkyl residue having one to ten carbon atoms, in particular with a 2,6-dimethylhexyl residue. A particularly preferred sterol residue is a residue derived from cholesterol.

[0141] Preferred stealth lipids c) have a glycerol-derived residue as a bridging group and have the structure of the formula (VIIIa) or (VIIIb) O-CH2-CH2-(O-CH2-CH2)OI-I-OR 9

[0142] I

[0143] LPL-CO-O-CH2-CH-CH2-O-CO-LPL (Villa)

[0144] O-CO-LPL

[0145] I

[0146] LPL-CO-O-CH2-CH-CH2-O-CH2-CH2-(O-CH2-CH2)OI-I-OR 9 (Vlllb) wherein

[0147] LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to each other, or a sterol radical, R 9 and ol have the meanings defined above, and the residues LPL can assume different meanings within a molecule within the given definitions.

[0148] Further preferred stealth lipids c) have a carbamate-derived residue as a bridging group and have the structure of formula (IXa) or (IXb)

[0149] LPL-NH-CO-O-CH2-CH2-(O-CH2-CH2) O II-OR 9 (IXa)

[0150] LPL

[0151] I

[0152] LPL-N-CO-O-CH2-CH2-(O-CH2-CH2)0I-I-OR 9(IXb) wherein LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, wherein several double bonds are not directly adjacent to each other, R 9 and ol have the meanings defined above, and the LPL residues in the compound of formula (IXb) can assume different meanings within one molecule within the framework of the given definitions.

[0153] Further preferred stealth lipids c) have a residue derived from succinic acid as a bridging group and have the structure of formula (X)

[0154] LPL-O-OC-CH2-CH2-CO-O-CH2-CH2-(O-CH2-CH2) O II-OR 9 (X) wherein

[0155] LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, wherein several double bonds are not directly adjacent to each other, and R 9and ol have the meanings defined above.

[0156] Preferred stealth lipids c) have the structure of formula (Xa)

[0157] H-[O-CH2-CH2] O iO-CO-R a -CO-O-ster (Xa), wherein ol has the meaning defined above, R a an alkylene radical having one to eight carbon atoms, preferably an ethylene radical, and

[0158] Ster is a residue derived from saturated or mono- or diethylenically unsaturated 3-hydroxysterols, which is preferably substituted in the 17-position by an alkyl residue having one to ten carbon atoms, in particular by a 2,6-dimethylhexyl residue, and is most preferably a residue derived from cholesterol. The PEG lipids c) preferably used according to the invention include the substance listed below, wherein n is a number between 15 and 200, preferably between 18 and 70.

[0159] The nanoparticles according to the invention very particularly preferably contain PEG lipids which are selected from the group consisting of pegylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy-(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate, such as w-methoxy(polyethoxy)ethyl-A / -(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-Di(tetradecanoxy)propyl-A / -(w-methoxy-(polyethoxy)ethyl)carbamate.

[0160] Other particularly preferred stealth lipids c) are POx lipids. These are generally polymers that, in addition to at least one lipid residue, have a polyoxazoline residue linked to it, the latter being generated by the polymerization of oxazoline. The two residues can be connected by a covalent bond or via a bridging group (BGL).

[0161] Polymers produced by the polymerization of oxazoline monomers have the recurring structural element of formula (XI) CO-R

[0162] I

[0163] -N-CRH-CRH- (XI), wherein R is hydrogen or a monovalent organic radical.

[0164] A preferred POx lipid has a structure of formula (XII)

[0165] CO-R 11

[0166] I

[0167] (LPL) n i-(BGL) m iO-(CH2-CH2-N) o i-OR 10 (XII) wherein

[0168] LPL is an alkyl or alkenyl residue with 6-20 carbon atoms, a residue of a fatty acid, a fatty alcohol or a sterol residue,

[0169] BGL is a (nl+1 )-valent bridging group, nl is an integer from 1 to 5, preferably 1 or 2, ml is 0 or 1, preferably 1, ol is an integer from 5 to 500, preferably 10 to 200,

[0170] R 10 Hydrogen, alkyl with one to six carbon atoms or a radical

[0171] LPL represents, preferably hydrogen, methyl ethyl or a sterol residue, and

[0172] R 11 hydrogen or Ci-C4-alkyl, where

[0173] residues LPL, and R 11 can take on different meanings within a molecule within the given definitions.

[0174] In general, the POx lipids of the formula (XII) have one to five LPL residues, preferably one or two LPL residues, which are alkyl residues having six to twenty carbon atoms, and / or mono- to tri-ethylenically unsaturated alkenyl residues having six to twenty carbon atoms, and / or saturated or mono- to tri-ethylenically unsaturated fatty acid residues having six to twenty carbon atoms, and / or saturated or mono- to tri-ethylenically unsaturated fatty alcohol residues having six to twenty carbon atoms, and / or sterol residues, wherein several double bonds in an alkenyl residue are not directly adjacent to one another.

[0175] In general, the POx lipids of formula (XII) have one to five LPL residues which are covalently linked directly to a POx residue via an ether or ester bond; or the POx lipids of formula (XII) have one to five LPL residues which are linked to a POx residue via a bridging group BGL, wherein the bridging group BGL is the residue of a di- to hexavalent aliphatic or cycloaliphatic alcohol, or the residue of a di- to hexavalent carboxylic acid, or a carbamate residue, or the residue of an amino alcohol.

[0176] Examples of residues of di- to hexavalent aliphatic or cycloaliphatic alcohols, residues of di- to hexavalent carboxylic acids, carbamate residues and amino alcohol residues are listed above in the description of PEG lipids.

[0177] Preferred POx lipids have a glycerol-derived residue as a bridging group and have the structure of the formula (XI Ia) or (XIIIb)

[0178] COR 11

[0179] I

[0180] O-(CH2-CH2-N)0|-OR 10

[0181] LPL-CO-O-CH2-CH-CH2-O-CO-LPL (Xllla)

[0182] O-CO-LPL COR 11

[0183] II

[0184] LPL-CO-O-CH2-CH-CH2-O-(CH2-CH2-N)oi-OR 10 (Xlllb) wherein

[0185] LPL is a saturated or mono- to tri-ethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to each other,

[0186] R 10 , R 11 and ol have the meanings defined above, and the residues LPL can assume different meanings within a molecule within the given definitions.

[0187] Further preferred POx lipids have a residue derived from succinic acid as a bridging group and have the structure of formula (XIV)

[0188] COR 11 I LPL-O-OC-CH2-CH2-CO-O-(CH2-CH2-N) O I-OR 10 (XIV) wherein

[0189] LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, wherein several double bonds are not directly adjacent to each other, and

[0190] R 10 , R 11 and ol have the meanings defined above.

[0191] Preferred stealth lipids c) have the structure of formula (XlVa)

[0192] R c -[N(CO-R b )-CH2-CH2]oi-O-CO-R a -CO-O-Ster(XIVa), where ol, R a and Ster have the meanings defined above, R b methyl or ethyl, and

[0193] R cis a radical derived from a cationic polymerization initiator, preferably hydrogen or a monovalent organic radical, in particular an alkyl, cycloalkyl, aryl, aralkyl, or heterocyclyl radical. Particularly preferred stealth lipids of formula (XIVa) have a Ster radical substituted in the 17-position by an alkyl radical having one to ten carbon atoms, in particular by a 2,6-dimethylhexyl radical, and which is most preferably a radical derived from cholesterol.

[0194] The POx lipids preferably used according to the invention include those having the following general structure, in which n is a number between 15 and 200, preferably between 18 and 70, and linker is a divalent bridging group.

[0195] Further preferred stealth lipids c) have a structure of formula (XV)

[0196] (LPL) n (BGL) m |-POLY (XV) where

[0197] LPL is an alkyl or alkenyl residue with 6-20 carbon atoms, a residue of a fatty acid, a fatty alcohol or a sterol residue,

[0198] BGL is a (nl+1 )-valent bridging group, nl is an integer from 1 to 5, preferably 1 or 2, ml is 0 or 1, preferably 1, and

[0199] POLY is a radical of the formulas (XVa), (XVIb), (XVIc), (XVId), (XVIe), (XVIf), (XVIg) or (XVIh) OR 12 Pyr

[0200] I

[0201] (-O-CH2-CH-CH2)r-OR 12 (XVIa), (-CH-CH2) S -R 13 (XVIb),

[0202] (-CH2-CR 14 )tR 13 (XVIc), (-CH2-CR 14 ) t -R 13 (XVId)

[0203] COO-CH2-CH2-OH CO-NH-CH2-CH2-OH

[0204] (-CH2-CR 14 ) t -R 13 (XVIe), (-NR 14 -CH-CO) U -R 13 (XVIf)

[0205] (-CH2-CR 14 ) t -R13 (XVIg), (-CO-CH-NR 14 ) U -R 13 (XVIh) where R 12 Hydrogen or LPL means

[0206] R 13 represents hydrogen or a monovalent organic radical such as alkyl, cycloalkyl, aryl or aralkyl,

[0207] R 14 is hydrogen or alkyl having one to six carbon atoms, in particular hydrogen or methyl,

[0208] R 15 and R 16 independently of one another represent hydrogen or alkyl having one to six carbon atoms, in particular alkyl having one to four carbon atoms, R 17 Hydrogen, alkyl having one to six carbon atoms, optionally substituted by a hydroxyl, amine, phenyl, hydroxyphenyl, carboxyl or amide radical,

[0209] NMORPH means a morphonyl radical which is linked to the carbonyl group via the ring nitrogen atom, r, s, t and u are numbers greater than or equal to 1, preferably between 1 and 5000, where the LPL radicals can have different meanings within a molecule within the framework of the given definitions.

[0210] Index r is preferably a number between 1 and 10, in particular a number between 1 and 4.

[0211] Index s is preferably a number between 10 and 5000, in particular a number between 40 and 5000.

[0212] Index t is preferably a number between 10 and 5000, in particular a number between 50 and 5000.

[0213] Index u is preferably a number between 5 and 500, in particular a number between 10 and 100.

[0214] Particularly preferred stealth lipids c) of formula (XV) are those in which POLY is a radical of formula (XVIf), wherein R 14 Methyl means and R17 Hydrogen is.

[0215] Preferred stealth lipids c) have the structure of formula (XVIi)

[0216] R d -O-[CH2-CH] O iOR a -CO-O-Ster (XVIi), I CO-NMORPH where ol, R a , Ster and NMORPH have the meanings defined above and R d is hydrogen or a monovalent organic radical, in particular an alkyl, cycloalkyl, aryl, aralkyl or heterocyclyl radical.

[0217] Particularly preferred stealth lipids of formula (XVIi) have a residue ster which is substituted in the 17-position with an alkyl residue having one to ten carbon atoms, in particular with a 2,6-dimethylhexyl residue, and which is most preferably a residue derived from cholesterol.

[0218] Other preferred stealth lipids c) have the structure of formula (XVIj)

[0219] R d -[NR b -CO-CH2-]oi-O-CO-Ra -CO-O-ster (XVIj), where ol, R a , R b , Ster and R d have the meanings defined above.

[0220] Particularly preferred stealth lipids of formula (XVIj) have a residue ster which is substituted in the 17-position with an alkyl residue having one to ten carbon atoms, in particular with a 2,6-dimethylhexyl residue, and which is most preferably a residue derived from cholesterol.

[0221] The BLNPs according to the invention can be loaded with active ingredients f) containing anionic groups. These anionic groups include carboxyl groups, sulfonic acid groups, and phosphate or phosphoric acid ester residues. These are characterized by the fact that they form complexes with the cationic lipids a) in an aqueous environment and are encapsulated or associated with the BLNPs.

[0222] The active substances f) may include any pharmaceutical and agrochemical active substances, provided that they have at least one anionic group per molecule.

[0223] Preferred active ingredients f) are nucleic acids. For the purposes of the present description, these are naturally occurring nucleic acids, including modified derivatives thereof. Modified nucleic acids may contain modified nucleotides or be modified in other ways, for example, by the introduction of chemical modifications. Nucleic acids form complexes with the cationic lipids a) via the phosphate ester residues present therein.

[0224] The physicochemical properties of nucleic acids form the basis for their interaction with carrier materials, and small changes in the sequence can lead to different biological effects, enabling rapid adaptation to different indications without the need to adapt the entire formulation process. Compared to conventional pharmaceuticals, nucleic acids are biopolymers with a higher molar mass (approximately 333 Da per nucleotide) and a strong negative charge, which results in good water solubility. Furthermore, their stability in the presence of degradation enzymes is low, and they can trigger an immune response based on evolutionarily optimized mechanisms that protect the organism from viral genetic manipulation. Despite these hurdles, nucleic acids enable the modulation of gene expression, whereas conventional drugs often lack a causal effect.Some successful, approved systems for the application of nucleic acids are shown in Figure 1. DNA typically exists in the double-stranded form (dsDNA), in which two single-stranded DNA chains (ssDNA) are linked by hydrogen bonds and hydrophobic interactions between the complementary base sequences, resulting in the familiar double-helix conformation. dsDNA is a semi-flexible polymer with a high negative charge density. For therapeutic applications, DNA is often encoded in plasmids (pDNA) containing a few thousand base pairs (bp). When pDNA enters the cell nuclei, it can influence gene expression. By repairing defective genes, whether congenital or acquired, gene therapy currently offers highly specific and potentially even curative therapies for diseases with no treatment or cure. In addition to DNA, short interfering RNA (siRNA) is a double-stranded RNA that is typically 19–25 bp long.Due to their different sugar backbones, they exhibit a higher linear charge density and stiffness than DNA. Therapeutic siRNA temporarily inactivates genes (knockdown) by impeding translation of the target mRNA. In contrast, single-stranded antisense oligonucleotides (ASOs) can inactivate translation by binding to the corresponding mRNA. Both types of RNA are already in clinical use, as shown in Figure 1. MicroRNAs (miRNA) are small (approximately 23 nucleotides), single-stranded, non-coding RNAs derived from primary miRNAs. miRNAs regulate the expression of target genes by degrading mRNA or inhibiting translation. mRNA is also a single-stranded nucleotide. It contains several hundred nucleotides and is more flexible than DNA, siRNA, and ssDNA.Because the bases are accessible, mRNAs have a stronger amphiphilic character, allowing hydrophobic interactions with potential transport molecules. Unmodified mRNA, due to its single-stranded nature, is more labile to nucleases and exhibits 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.

[0225] DNA and / or RNA or modifications thereof are preferably used as active ingredients f) in the BLNPs according to the invention.

[0226] 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.

[0227] Any immunomodulatory elements such as TRL antagonists, CpG motifs and other functional nucleic acids can be used.

[0228] Any type of RNA can be used. Examples include hnRNA, mRNA, tRNA, rRNA, mtRNA, snRNA, snoRNA, scRNA, siRNA, miRNA, ncRNA, saRNA, antisense RNA, bacterial RNA, and viral RNA. Combinations of DNA and RNA can also be used in the BLNPs according to the invention.

[0229] Modified nucleic acids, also called xenonucleic acids (XNA), offer several advantages for biotechnological applications and address some of the limitations of first-generation nucleic acid therapeutics. Indeed, several therapeutics based on modified nucleic acids have recently been approved, and many more are in clinical trials. XNAs can exhibit greater biostability and, moreover, can increasingly be developed in vitro, accelerating lead discovery (Duffy, K.; Arangundy-Franklin, S.; Holliger, P., Modified nucleic acids: replication, evolution, and next-generation therapeutics. BMC Biol. 2020, 18, 112).

[0230] Preferred nanoparticles according to the invention are characterized by a high content of active ingredient f), preferably nucleic acid. The weight fraction of active ingredient f) in the LNPs according to the invention is typically 1 to 10%, and preferably 2 to 8%, in particular 3 to 7%, particularly preferably 5 to 6%, based on the mass of the active ingredient-loaded LNPs.

[0231] The nanoparticles according to the invention can be characterized by their particle diameter. Typical particle diameters (for example z-average) are in the range of less than or equal to 900 nm, preferably less than or equal to 500 nm, more preferably between 30 and 500 nm, most preferably between 40 and 250 nm, and in particular between 50 and 200 nm. For the purposes of the present description, the particle diameters are determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, United Kingdom). The intensity-weighted mean diameter (for example z-average) was determined using cumulant analysis of the correlation function (ISO13321, ISO22412). For size determination, a refractive index of 1.33 for ultrapure water was assumed.

[0232] Particle diameters can alternatively be determined by other methods, for example by nanosize tracking analysis (NTA), or by electron microscopy, e.g. by transmission electron microscopy or by scanning electron microscopy.

[0233] Particle diameters (z-average) of preferred LNPs according to the invention are in the range between 30 and 500 nm, determined by dynamic light scattering (DLS).

[0234] The BLNPs according to the invention can be further characterized by their polydispersity index (or PDI). The PDI indicates the breadth of the particle size distribution. Values ​​between 0 (monodisperse) and 1 (polydisperse) can be assumed. For the purposes of this description, the PDI value is determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, United Kingdom). PDI was determined using cumulant analysis of the correlation function.

[0235] The PDI value of the particle size distribution of the nanoparticles according to the invention typically ranges between 0.01 and 0.4, preferably between 0.02 and 0.3 and particularly preferably between 0.05 and 0.2.

[0236] When using active ingredients f) with phosphate groups, the nanoparticles according to the invention can be further characterized by their N / P ratio. This is the molar ratio of nitrogen atoms in the cationic lipid a) to phosphate groups in the active ingredient e), for example, in the nucleic acid. The N / P ratio in the nanoparticles according to the invention can vary within wide ranges. Typically, the N / P ratio in the nanoparticles according to the invention is between 1 and 100, preferably between 1.5 and 50, particularly preferably between 2 and 25, and most preferably between 3 and 15.

[0237] Preferred nanoparticles according to the invention have diameters (z-average) determined by DLS between 40 and 250 nm, in particular between 50 and 200 nm, and a polydispersity index of the particle diameters between 0.05 and 0.3.

[0238] Very particularly preferred nanoparticles according to the invention have diameters determined by DLS (z-average) between 40 and 250 nm, in particular between 50 and 200 nm, and a polydispersity index of the particle diameters between 0.05 and 0.2 and an N / P ratio between 3 and 15.

[0239] In the event that the nanoparticles according to the invention contain, in addition to the nucleic acid-lipid complexes described above, additional polymers or additional complexes of nucleic acids with additional polymers, these further components are present only in small amounts, for example their weight fraction is 10% or less, in particular less than 5%.

[0240] Particularly preferably, the nanoparticles according to the invention do not contain any further complexes of active ingredients with other polymers in addition to the active ingredient-lipid complexes described above.

[0241] The BLNPs according to the invention can be in solid form as powders, or they can form a dispersion and be dispersed in aqueous solvents, with the particles being in solid form in the dispersion medium. In a preferred embodiment, the BLNPs according to the invention form a dispersed phase in water or in an aqueous buffer solution.

[0242] The BLNPs according to the invention can be produced by assembly. For this purpose, the lipids used according to the invention are dispersed in water or in an aqueous buffer solution. One dispersion can be produced for each lipid, or all lipids are dispersed together. The pH of the aqueous dispersion is adjusted to a value of 3 to 8, preferably 4 to 7.5, e.g. by using an acetate buffer or another suitable buffer such as citrate buffer, lactate buffer, phosphate buffer, and phosphate-citrate buffer. In addition, the active ingredients f) containing anionic groups, for example the nucleic acids, are dissolved or dispersed in water, the pH of the aqueous active ingredient solution or dispersion preferably being adjusted to a value between 3 and 8, particularly preferably to a value between 4 and 7.5.A buffer solution containing acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer, HEPES, TRIS, or only salts is particularly suitable for this purpose. The aqueous dispersions of the lipids and the active ingredient solution or dispersion are combined, whereby the amounts of active ingredient and cationic lipid a) are selected such that a desired active ingredient / lipid ratio, e.g., a desired N / P ratio, is achieved. After mixing the aqueous dispersions or solutions, the mixture is agitated, for example, for a short time between 2 and 120 seconds. This can be done by stirring and / or vortexing and / or ultrasonication. Preferably, the resulting nanoparticles are allowed to stand for some time before further use, for example, between 5 and 20 minutes, to allow binding between lipid a) and active ingredient e) (hereinafter referred to as "incubation"). After preparation in acidic pH (e.g.Acetate, pH 5.5), the LNPs are 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 remain in the dispersion medium. In an alternative process, the BLNPs according to the invention can be produced by nanoprecipitation, whereby the BLNPs initially contain only lipids and the active ingredient f) is added in a subsequent step. The BLNPs are produced as described above, but without adding the solution or dispersion of the active ingredient f).

[0243] The solution or dispersion of the active ingredient f) is then prepared as described above.

[0244] The aqueous dispersion of the BLNPs and the active ingredient solution or dispersion are then combined, with the amounts being selected to achieve a desired active ingredient / lipid ratio, e.g., a desired N / P ratio. After mixing the aqueous dispersions or solutions, the mixture is agitated, for example, for a short time between 2 and 120 seconds. This can be done by stirring and / or vortexing and / or ultrasonication. Here, too, the resulting drug-loaded nanoparticles are left to stand for some time before further use, for example, between 5 and 20 minutes, to allow binding between lipid a) and active ingredient e) (hereinafter referred to as “incubation”). After preparation at an acidic pH (e.g., acetate, pH 5.5), the BLNPs are 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 dispersing medium or remain in the dispersing medium.

[0245] In addition to the cationic lipid a), the other lipids b) and c), and the active ingredient f), one or more excipients and additives e) may be present in the dispersion medium during their nanoprecipitation. Alternatively, these excipients and additives e) can be added after dispersing the nucleic acid copolymer complex in the aqueous phase. Water is used as the dispersion medium. Buffer substances, salts, sugars, or acids and bases can be added to adjust the desired pH or osmolarity.

[0246] The processes according to the invention are characterized by the fact that the use of organic solvents such as ethanol can be dispensed with during the production of the lipid nanoparticles. Subsequent solvent removal is therefore unnecessary.

[0247] The invention also relates to a process for producing the BLNPs described above, comprising the following measures: i) initial charging of aqueous dispersions of the lipids a), b) and c) in buffers in the pH range from 3 to 8, preferably 4 to 7.5 ii) combination of the aqueous dispersions from step i); and iii) treatment of the combined aqueous dispersions from step ii) with a mixing process selected from the group consisting of ultrasound, dual centrifugation, nanoprecipitation, microfluidics or in a vortex mixer, whereby the nanoparticles are formed.

[0248] In a first variant, the invention further relates to a process for producing the above-described BLNPs loaded with anionic groups containing active ingredient f), comprising the following measures: iv) initial charge of the LNP-containing aqueous dispersion from step iii) according to the above process, v) initial charge of an aqueous solution or dispersion of an active ingredient f) with anionic groups in a buffer in the pH range from 3 to 8, preferably 4 to 7.5 vi) combination of the aqueous dispersions or solutions from steps iv) and v), and vii) treatment of the combined aqueous dispersions or solutions from step vi) with ultrasound, microfluidics, dual centrifugation, ultrasound, nanoprecipitation or in a vortex mixer, whereby the LNPs loaded with active ingredient are formed.

[0249] In a second variant, the invention relates to a process for the preparation of the above-described BLNPs loaded with anionic groups containing active ingredient f) comprising the following measures:

[0250] I) Preparation of aqueous dispersions of lipids a), b) and c) in buffers in the pH range of 3 to 8, preferably 4 to 7.5,

[0251] II) Presentation of an aqueous solution or dispersion of an active ingredient f) with anionic groups in a buffer in the pH range of 3 to 8, preferably 4 to 7.5

[0252] III) combination of the aqueous dispersions or solutions from steps I) and II); and

[0253] IV) Treatment of the combined aqueous dispersions or solutions from step III) with ultrasound, microfluidics, dual centrifugation, nanoprecipitation or in a vortex mixer, whereby the drug-loaded LNPs are formed.

[0254] In a preferred embodiment of the first variant of the method according to the invention, this includes the following measures:

[0255] V) Initial charge of the LNP-containing aqueous dispersion having a pH value between 3 and 8, preferably 4 to 7.5 from step iii) prepared according to the above process,

[0256] VI) preparing an aqueous solution or dispersion of a nucleic acid in a buffer in the pH range of 3 to 8, preferably 4 to 7.5,

[0257] VII) Mixing both dispersions or solutions from steps V) and VI) in a selected ratio of nucleic acid and lipid a) so that a desired molar N / P ratio of nitrogen atoms in lipid a) to the phosphate groups in the nucleic acid is obtained, preferably an N / P ratio between 1 and 200,

[0258] VIII) Moving the mixture from step VII), and

[0259] IX) optionally subsequent incubation of the resulting mixture. The aqueous dispersions of lipids a), b), and c) for steps i) or I) of the processes according to the invention preferably contain a buffer, in particular an acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer, or mixtures thereof.

[0260] The aqueous solution or dispersion of a nucleic acid for steps v), II) or VI) of the process according to the invention preferably has a pH of 3 to 8, preferably 4 to 7.5.

[0261] The aqueous solution or dispersion of the nucleic acid for steps v), II) or V) of the process according to the invention preferably contains a buffer, in particular an acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer, HBG, HEPES or TRIS buffer.

[0262] The agitation in steps iii), vii), IV), or VIII) of the process according to the invention is preferably carried out by stirring or vortexing. The treatment time in this step is typically between 1 and 120 seconds, in particular between 2 and 60 seconds.

[0263] The incubation in step IX) of the process according to the invention is usually carried out by simply allowing the resulting mixture to stand, for example, for a period of 5 to 60 minutes, preferably 5 to 20 minutes. The mixture can also be incubated in a refrigerator or heating cabinet, for example, at temperatures between 1°C and 80°C.

[0264] The nanoparticles can be separated from the aqueous phase in various ways. Examples include crossflow filtration, centrifugation, ultrafiltration, or dialysis. However, the nanoparticle dispersion can also be used directly after production without further processing. Filtration can remove particles such as aggregates, as well as excess excipients or impurities, from the dispersion. This can change the particle concentration.

[0265] Dissolved molecules can be separated from the dispersion by purification using dialysis / crossflow filtration. The process is largely independent of the particle size of the dispersed particles.

[0266] Centrifugation can also be used to separate dissolved molecules from the dispersion. However, this process also reduces the concentration of dispersed particles. Furthermore, only dispersions containing nanoparticles with a larger diameter, e.g., more than 150 nm, can be treated, and the particles themselves may be damaged. Furthermore, redispersing the particles obtained in this way can be difficult.

[0267] The drug-loaded BLNPs according to the invention are ideally suited as vehicles for the transport of pharmaceutical and agrochemical active ingredients.

[0268] The active ingredient-loaded BLNPs according to the invention are particularly suitable for gene transfer into cells, i.e., for introducing nucleic acids and their functional release into cells. For this purpose, the LNPs containing nucleic acids are added to individual cells, tissues, or a cell culture and taken up by the cells through endocytosis. Surprisingly, it has been shown that high levels of nucleic acids can be transferred into cells using the BLNPs according to the invention.

[0269] The invention therefore also relates to a method for gene transfer into cells, which comprises the following steps: A) contacting cells, tissues or cell cultures with an aqueous dispersion containing the LNPs containing the nucleic acids described above, and

[0270] B) subsequent incubation.

[0271] Preferably, the invention relates to a method for gene transfer into cells, which comprises the following steps:

[0272] C) Providing a cell culture in a bioreactor or incubator,

[0273] D) Addition of an aqueous dispersion containing the LNPs containing the nucleic acids described above,

[0274] E) Distributing the aqueous dispersion in the cell culture, and

[0275] F) subsequent incubation.

[0276] The gene transfer method according to the invention can be carried out using different cells, for example by using single cells, tissues or cell cultures.

[0277] Thus, the nucleic acid-loaded BLNPs according to the invention can be combined with prokaryotic or eukaryotic cells, with tissues derived from eukaryotic cells, or with cell cultures. These can be plant or, preferably, animal cells, including human cells.

[0278] The nucleic acid-loaded LNPs according to the invention can be administered in vivo, for example, subcutaneously or into the muscle, or ex vivo, for example, with immune cells, as in CAR-T therapy. It can also be administered via an RNA vaccine or another vaccine.

[0279] In the context of this description, "parts" are understood to mean the smallest living units of organisms. These can be cells of unicellular or multicellular organisms, which can originate from prokaryotes or eukaryotes. The cells can be microorganisms or individual cells. Cells can be of prokaryotic, plant, or animal origin, or even from fungi. Eukaryotic cells are preferably used, especially those that were originally isolated from tissue and can be permanently cultured—i.e., those that are immortalized.

[0280] For the purposes of this description, “tissues” are understood to mean collections of differentiated cells including their extracellular matrix.

[0281] For the purposes of this description, "cell cultures" refer to combinations of cells or tissues and cell culture medium, where the cells or tissues are cultivated in the cell culture medium outside the organism. Cell lines are used, i.e., cells of a tissue type that can divide during the course of cultivation. Both immortalized (immortalized) cell lines and primary cells (primary culture) can be cultivated. Primary culture is typically understood to mean a non-immortalized cell culture obtained directly from a tissue.

[0282] The cell cultures used according to the invention can be produced and cultivated using standard methods.

[0283] For example, primary cultures can be created from different tissues, for example from tissues of individual organs such as skin, heart, kidney or liver, or from tumor tissue. The tissue cells can be isolated using conventional methods, e.g., by treatment with a protease, which breaks down the proteins that maintain cell association. It may also be appropriate to specifically stimulate certain cell types to divide by adding growth factors or, in the case of poorly growing cell types, to use feeder cells, basement membrane-like matrices or recombinant components of the extracellular matrix. The cells used according to the invention can also be genetically modified by introducing a plasmid as a vector.

[0284] The cells used according to the invention may have a limited lifespan or be immortal cell lines with the ability to divide indefinitely. These can be generated by random mutation, e.g., in tumor cells, or by targeted modification, for example, by the artificial expression of the telomerase gene.

[0285] The cells used according to the invention can be adherent (on surface) growing cells, such as fibroblasts, endothelial cells or cartilage cells, or they can be suspension cells that grow freely floating in the nutrient medium, such as lymphocytes.

[0286] Culture conditions and cell culture media are selected depending on the individual cells being cultivated. Different cell types prefer different culture media, which are specifically formulated. For example, different pH values ​​are adjusted, and the individual culture media can contain different amino acids and / or other nutrients in different concentrations.

[0287] The cells transfected according to the invention can be used in various fields, for example, biotechnology, research, medicine, or veterinary medicine. These can include the production of (recombinant) proteins, virus and / or virus particle production, the study of metabolism, division, and other cellular processes. Furthermore, the cells transfected according to the invention can be used as test systems, for example, in investigating the effects of substances on cell properties, such as signal transduction or toxicity. Other cells preferably used to produce the cells transfected according to the invention are stem cells. These are known to be somatic cells that can differentiate into various cell types or tissues.

[0288] The invention also relates to the use of the LNPs containing nucleic acids described above for gene transfer into cells, i.e. for introducing nucleic acids and their functional release into cells.

[0289] In the process according to the invention, described in the following examples, the property of cationic lipids to form a homogeneous, dispersed system in acidic aqueous solution (e.g., 20 mM NaOAc, pH 5.5) was utilized. Phospholipids, such as DSPC, behave in the same way. This made it possible to withdraw reproducible volumes from adjusted stock solutions. Stealth lipids, such as PEG lipids or POx lipids, can also be incorporated into the formulation in this way, as they are soluble in the aforementioned buffer. Cholesterol was omitted because this molecule is water-insoluble and not required for the construction of the lipid nanoparticles according to the invention.For this reason, the molar composition of the lipid nanoparticles was adjusted for the following examples (cationic lipid / helper lipid / stealth lipid = 81.3 / 16.3 / 2.4); however, the ratio of the components from the original Moderna formulation (50 / 10 / 1.5) was maintained. Controlled mixing and subsequent shearing of the particles using ultrasonic treatment results in the formation of homogeneous, unloaded lipid nanoparticles. The particles formed by ultrasonic treatment differ significantly in shape and size from lipid nanoparticles produced by simple mixing of the mixture.

[0290] The unloaded lipid nanoparticles can be loaded with the desired active ingredient, e.g., the desired genetic material, in a subsequent process step. Both pDNA and RNA were used in the following experiments. The genetic material is diluted, for example, in 20 mM NaOAc buffer, pH 5.5 (MM). Subsequently, after a defined incubation time, equal amounts of nanoparticle suspension and MM are combined by rapid transfer and vortex mixing. Using in vitro test systems, the functionality of the inventive lipid nanoparticles loaded with genetic material with regard to protein expression was demonstrated. After demonstrating that the inventive lipid nanoparticles are generally suitable for transfection with pDNA, the formulation was further optimized with regard to biocompatibility.For this purpose, various buffer systems were tested for the formation of both unloaded and loaded lipid nanoparticles. The data show that an acidic pH is essential for positive transfections. Additionally, mRNA was used as an alternative genetic material. The experiments demonstrated that the lipid nanoparticles of the invention are also suitable for transfection with mRNA. Furthermore, various stealth polymers can be used as an alternative to the commercially available PEG-DMG.

[0291] The key advantage of the lipid nanoparticles according to the invention is that the method of the invention enables the production of lipid nanoparticles for gene transfer without the use of organic solvents. This leads to a reduction in production costs, as rapid removal of the solvents, for example, by dialysis, is eliminated. Furthermore, the genetic material is preserved.

[0292] The examples and figures described below illustrate the invention without limiting it.

[0293] Figures 1A to 1D show the structures of the lipids used in the LNPs used by BioNTech and Moderna. Figure 1A shows the structure of the phospholipid DSPC. Figure 1B shows the structures of the ionizable cationic lipids ALC-0315 and SM-102. Figure 1C shows the structures of the ethoxylated lipids PEG-DMG and ALC-0159. Figure 1D shows the structure of the lipid cholesterol. SM-102 has the molecular formula C44H87NO5 and a molecular weight of 710.18.

[0294] The HLB value calculated according to Griffin is 5.70.

[0295] DSPC has the molecular formula C44H 88 NO8P and has a molecular weight of 790.16. The HLB value calculated according to Griffin is 20*(1-(617.28 / 790.16)=4.38

[0296] PEG-DMG has the molecular formula C124H246O51 and a molecular weight of 2553.28. The Griffin-calculated HLB value is 17.13.

[0297] Cholesterol has the molecular formula C27H 46 O and has a molecular weight of 386.66. The HLB value calculated according to Griffin is 20*(1 -((386.66-17) / 386.66)=0.88

[0298] The composition of the LNP used by BioNTech corresponds to 46.3 mol% ALC-0315, 9.4 mol% DSPC, 42.7 mol% cholesterol and 1.6 mol% ALC-0159.

[0299] The composition of the LNP used by Moderna corresponds to 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% PEG-DMG (see Schoenmaker, L.; Witzigmann, D.; Kulkarni, JA; Verbeke, R.; Kersten, G.; Jiskoot, W.; Crommelin, DJA, mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 2021 , 601, 120586).

[0300] Other stealth lipids used in the experiments described below have the following structures:

[0301] Figure 2 shows the schematic design of a microfluidic device (cf. Maeki, M.; Uno, S.; Niwa, A.; Okada, Y.; Tokeshi, M., Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery. J. Control. Release 2022, 344, 80-96). This is an example of a standard microfluidic device for the production of conventional lipid nanoparticles.

[0302] Figure 3 shows the schematic structure of a commonly used LNP composed of known lipids in known proportions.

[0303] Figure 4 shows a schematic of the process according to the invention. An initial preparation approach was based on a simple mixing process (vortexing), in which various ionizable lipids were mixed with genetic material and tested for transfection. The cationic lipid SM-102 proved to provide the best results, but these were rather subpar. To improve transfection, the helper lipid DSPC and a stealth lipid (here, Chol-POx) were added to the formulation. Step 1) Lipid suspensions are prepared, 2) Dilution of the genetic material, 5 min vortexing, 3) 10 min incubation at room temperature, combining lipids and genetic material, 4) rapid vortexing. Chol-POx is the polymer shown below.

[0304] Chol-POx-52 (n=52) has the molecular formula C240H416N52O56 and has a

[0305] Molar mass of 4926.28. R stands for methyl. The HLB value calculated according to Griffin is 15.27.

[0306] Figure 5 shows the schematic structure of an LNP according to the invention composed of cationic lipid, helper lipid and stealth lipid, which contains genetic material as an active ingredient.

[0307] Figure 6 shows the DLS measurement results of the LNPs produced by vortexing. The production was carried out according to the procedure outlined in Figure 4. The influence of the individual components of the formulation on the particle size was investigated. The combinations were produced from

[0308] F1. Cationic lipid + genetic material,

[0309] F2. Cationic lipid + helper lipid + genetic material,

[0310] F3. Cationic lipid + stealth lipid + genetic material, and F4. the BLNP according to the invention consisting of all four components.

[0311] These particles are plotted from left to right and were each produced at three different N / P ratios (3, 6, 9, vInr). The cationic lipid alone forms measurable lipoplexes (complexes of lipid and genetic material) with the genetic material. It can be seen that the particle size changes depending on the N / P ratio. However, the addition of helper lipid (DSPC) and stealth lipids (Chol-POx) results in the formation of small, homogeneous particles, as indicated by the lower PDI value for these LNPs.

[0312] Figure 7 shows the results of the transfection experiments. The nanoparticles used are LNPs according to the invention, produced analogously to the particles shown in Figure 6. These particles were transferred to cells to measure transfection. The particles containing stealth lipid and phospholipid were the most efficient. Considering the material usage, a formulation with N / P 6 proved to be optimal.

[0313] Figure 8 outlines another production method for the LNPs according to the invention. The aim was to reduce the size of the resulting LNPs and further improve their efficiency with regard to gene delivery. Of the conceivable methods, ultrasound treatment proved to be the most suitable. The lipid mixture is treated with ultrasound, resulting in significantly smaller, still unloaded lipid particles, which were then loaded with genetic material. Step 1) Lipid suspensions are prepared, 2) LNP assembly by ultrasound treatment, 3) dilution of the genetic material, 5 min vortexing, 4) 10 min incubation at room temperature, combining lipids and genetic material, 5) rapid vortexing.

[0314] Figures 9 and 10 (Figure 10 is an enlarged view of the results from Figure 9) show particle diameters and PDI values ​​of LNPs. The LNPs investigated contain different lipids or lipid combinations as described for Figure 6. LNPs produced using the procedures outlined in Figures 4 (vortexing) and 8 (ultrasonication) are compared. It turned out that the particles produced using the latter procedure are significantly smaller. All measurement data for the LNPs shown below refer to a molar composition of 82.3 mol% SM-102, 16.5 mol% DSPC and 1.2 mol% stealth lipid or 81.3 mol% SM-102, 16.3 mol% DSPC and 2.4 mol% stealth lipid, respectively. The transfection experiments shown were carried out using HEK293T cells. The incubation period is 24 h at 37 °C and the measured values ​​were collected by flow cytometry.

[0315] Figure 11 shows the raw data from the flow cytometer measurement after transfection. The LNPs according to the invention obtained using the method shown in Figure 4 (vortexing) (AC) are compared with the LNPs according to the invention obtained using the method shown in Figure 8 (ultrasound treatment) (DF). In comparison to Figure 11 B (70.05%), the LNPs produced using ultrasound exhibit a significantly higher transfection efficiency of 88.92% (Figure 11 E).

[0316] Figure 12 shows the relative mean fluorescence intensities compared to the raw data from Figure 11. The horizontally striped bars in this case represent the LNPs produced using the method shown in Figure 8. It can be seen that the pure lipoplex made of cationic lipid with anionic active ingredient shows hardly any fluorescence using either method. The LNPs according to the invention, on the other hand, show significant fluorescence values ​​(400x - 900x more than the negative control). Furthermore, to better classify the results, a polyplex made of polyethyleneimine (PEI) was compared with the LNPs. It can be seen that the LNPs produced using ultrasound are superior to the PEI polyplex. Furthermore, the LNPs produced using the method shown in Figure 8 (ultrasound) are significantly superior to the LNPs produced using the method shown in Figure 4 (vortexing).

[0317] Figure 13 shows results obtained with LNP prepared using different buffers. The results shown in the previous figures were obtained with LNP prepared at pH 4.0 in citrate buffer (50 mM). Under these conditions, 50 mM citrate buffer can only be neutralized to pH 7.4 with considerable effort (e.g., dialysis). To facilitate this process, it is necessary to change the buffer systems used. Therefore, other buffer systems were investigated. TRIS, PBS (pH 7.4), and NaOAc (pH 5.5) were tested as solvents for the lipid suspensions. At the same time, the genetic material was dispersed in various buffers (MM). 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. Thus, in addition to the initial combination of 50 mM citrate buffer with 20 mM HBG, 15 new buffer combinations were obtained.In the absence of citrate buffer, the only positive results were obtained from the combination of 20 mM NaOAc pH 5.5 (LNP) with 20 mM NaOAc pH 5.5 (MM). The LNPs were prepared for this series of experiments by vortexing. The advantage of using 20 mM NaOAc is that it is significantly easier to neutralize than citrate buffer. This solvent combination was used for subsequent experiments.

[0318] Figures 14 and 15 show the particle diameters (columns) and PDI values ​​(dots) of LNPs. Figure 14 further illustrates the advantage of the ultrasonic method, as significantly smaller particle sizes can be achieved. Subsequently, we investigated whether changing the buffer system from citrate buffer to NaOAc produces similar particle sizes. Figure 15 shows that this is the case (similar sizes). The buffer systems were varied, and the nanoparticles were measured in the same way after production using DLS.

[0319] Figure 16 shows the transfection efficiency (relative mean fluorescence intensity) of the LNPs prepared with NaOAc buffer. The buffer was acidified once with an equimolar amount of hydrochloric acid (HCl) and adjusted to pH 4.0 to test whether the specific pH of the formulation influences efficacy. It can be seen that the values ​​are highest for pH 5.5. This eliminates the need for further adjustment of the buffer system, and for further optimization, work continued with 20 mM NaOAc at pH 5.5. For further application, it is imperative that the formulation can be neutralized from pH 5.5 to pH 7.4. Figure 17 shows how the pH 5.5 formulation can be neutralized without compromising transfection efficiency. Before bringing the LNPs into contact with the cell culture medium, the samples were then mixed 1 + 1 with the corresponding buffer shown in the figure.This figure shows that, surprisingly, the transfections of the neutralized samples are even better than those without neutralization (pH 5.5). A preferred preparation of the LNPs involves preparing the nanoparticles at pH 5.5 using 20 mM NaOAc followed by neutralization by adding PBS or TBS.

[0320] Figure 18 shows the results of the toxicity measurements. Unloaded LNPs were prepared using the method shown in Figure 8. Two different stealth polymers were used for this purpose: Chol-POx and PEG-DMG. The experiment was conducted on L929 cells, which were incubated with LNP suspensions at various concentrations up to 1000 pM for 24 hours and then analyzed using the Presto-Blue assay. It can be seen that the LNPs according to the invention exhibit no toxicity in the tested range.

[0321] Figures 19 and 20 show DLS measurements (particle sizes) of the LNPs, prepared analogously to Figure 18. Unloaded particles (BLANK) and particles loaded with mRNA and pDNA were measured. The latter were measured both at pH 5.5 (NaOAc) and after neutralization with PBS (pH 7.4). It can be seen that the choice of genetic material has no influence on the size. Neutralization leads to a slight increase in the sizes.

[0322] Figures 21 and 22 show the PDI values ​​of the BLNPs described in Figures 19 and 20. Figure 23 shows the proportion of EGFP-positive cells in a transfection experiment as a function of the amount of genetic material used. BLNPs with the stealth lipid Chol-POx were prepared as shown in Figure 8 and loaded with mRNA and pDNA. It can be seen that the BLNPs according to the invention are significantly superior to PEI.

[0323] Figure 24 lists possible and already used stealth lipids.

[0324] In the experiments shown in Figures 25 and 27 to 29, BLNPs containing 81.3 mol% ionizable / cationic lipid (SM-102), 16.3 mol% phospholipid (DSPC) and 2.4 mol% stealth lipid were used.

[0325] Figure 25 shows that the polymers CHEMS-PEtOxn, CHEMS-PEGn, CHEMS-PMeOx n are very well suited for use in BLNPs. This was demonstrated by the high transfection efficiencies. BLNPs were prepared by sonication as described above, and the transfection efficiency was measured using mRNA.

[0326] Figure 26 shows that the polymers CHEMS-PEtOx listed above n , CHEMS-PEGn, CHEMS-PMeOx nand DMG-PEG differ significantly in their biocompatibility. The PMeOx and PEtOx variants of the CHEMS stealth lipid are significantly less cytotoxic than the PEG variants. In general, PEG is more cytotoxic than POx. The test performed is based on ISO 10993-5 and measures cytotoxicity as the relative metabolic activity of L929 cells after 24 hours of incubation with the corresponding material at the specified concentration.

[0327] Figure 27 shows another specific application of BLNPs. BLNPs were loaded with a Cas9 mRNA and a GFP-specific gRNA and labeled with GFP. +The nanoparticles were applied to HEK-293T cells. In the model experiment, a nearly 100% knockout (KO) efficiency was demonstrated. The nanoparticles were applied once, with a full medium change after 24 hours. The cells were further incubated for 14 days, and the decrease in GFP was measured using flow cytometry.

[0328] Figure 28 demonstrates that BLNPs are also suitable for applications on primary human immune cells. BLNPs were applied to isolated human primary leukocytes and incubated for 18 hours. The cells were then analyzed using antibody staining via flow cytometry. It was demonstrated that transfection occurred only in CD45 / CD14 positive cells, and thus in monocytes.

[0329] Figure 29 combines the expertise of the experiments from Figures 27 and 28.

[0330] Large peritoneal macrophages from genetically modified mice were harvested and treated ex vivo with the CRISPR-Cas9 BLNPs. Figure 29 shows that the mice stably produce GFP (NC). Furthermore, it was shown that the combination of Cas9 mRNA and anti-GFP gRNA resulted in a knockout. This is expressed by the decrease in the GFP signal (right graph, top and bottom).

Claims

Patent claims 1. Lipid nanoparticles containing a) 51 to 94.9 mol% of at least one cationic / ionizable lipid, b) 5 to 40 mol% of at least one phospholipid, and c) 0.1 to 10 mol% of at least one stealth lipid selected from the group of lipids containing one or more poly(alkylene oxide) chains (PEG-lipid) or containing one or more poly(oxazoline) chains (POx-lipid), or containing one or more polyglycerol chains (PG-lipids), or containing one or more poly(hydroxyalkyl(meth)acrylate) chains (PHAA-lipid), or containing one or more poly(V-(hydroxyalkyl)(meth)acrylamide) chains (PHAAA-lipid), or containing one or more poly(vinylpyrrolidone) chains (PVP-lipid), or containing one or more Poly(V,V-dialkyl(meth)acrylamide) chains (PDMAA lipid), or containing one or more poly(V-(meth)acryloylmorpholine) chains (PAM lipids) or containing one or more poly(amino acid) chains (PAA lipids), with the proviso,that all lipids contained in the lipid nanoparticle have an HLB value greater than or equal to 3 and that the percentages given refer to the total mass of the lipids contained in the lipid nanoparticle.

2. Lipid nanoparticles according to claim 1, characterized in that they are loaded with active substances containing anionic groups.

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

4. Lipid nanoparticles according to at least one of claims 1 to 3, characterized in that they contain no further lipids d) and no auxiliary substances or additives e) in addition to the lipids a), b) and c).

5. Lipid nanoparticles according to at least one of claims 1 to 4, characterized in that the cationic lipid a) contains no phosphate groups and at least one amino group.

6. Lipid nanoparticles according to claim 5, characterized in that the cationic lipid a) is an ionizable lipid which forms a positive charge on the nitrogen atom via protonation in the acidic pH range from 4 to 7 and which is almost neutral in the basic pH range above 7.

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

8. 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) R 3 R 1 -N-(CO) r -R 2 (I), wherein R 1 a residue of the formula R 4 R5 N-(C m H 2m )-, CH3-(CnH2n)-O-(CoH2o)-, HO-(C m H 2m )-, HO-CH2-CH(OH)-CH2-, CH3-(CH2)nO-CO-(C m H2m)-, CH3-(C n H2n)-CO-O- (C m H 2m )-, NC-(C O H2O)-, HO-CH2-CH((C O H2O)-CH3)-, CH3-(C O H2O)-CH(OH)- (C p H2p)-, CH3-(CnH2n)-CO-NH-(CoH2o)-, (HO-CH((C q H 2q )-CH3)-CH((C o H2o)-OH)- or CßH-ic / OH)-, R 2 and R 3 independently of one another alkyl radicals having six to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO-, and / or alkylene radicals having six to twenty carbon atoms and one, two or three double bonds not directly adjacent to each other, R 4 and R 5 are independently hydrogen or alkyl radicals having one to five carbon atoms or both radicals R 4 and R5 together with the common nitrogen atom form a pyrrolidine or piperidine residue, m is an integer from 2 to 6, n is an integer from 0 to 6, o and p are independently integers from 1 to 6, q is an integer from 2 to 16, and r is 0 or 1.

9. Lipid nanoparticles according to at least one of claims 1 to 8, characterized in that the phospholipid b) has the structure of formula (IVa) or (IVb) OP(O)(OMe)-O-KG I LP-CO-O-CH2-CH-CH2-O-CO-LP (IVa) O-CO-LP I LP-CO-O-CH2-CH-CH2-OP(O)(OMe)-O-KG (IVb) wherein LP is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to each other, Me is hydrogen, a monovalent or divalent metal cation or an ammonium cation, KG represents a head group which is an aliphatic radical containing at least one hydroxyl group, preferably an aliphatic residue with one hydroxyl group and one amino group with one hydroxyl group and one quaternary ammonium group or the residue of a carbohydrate with five to six hydroxyl groups, and the residues LP can take on different meanings within a molecule within the given definitions.

10. Lipid nanoparticles according to at least one of claims 1 to 8, characterized in that the phospholipid b) has the structure of formula (Va) or (Vb) NH-CO-LP I CH3-(CH2-CH2)m P -CH=CH-CH(OH)-CH-OP(O)(OMe)-O-KG (Va) LP-N-CO-LP I CH3-(CH2-CH2) mp -CH=CH-CH(OH)-CH-OP(O)(OMe)-O-KG (Vb) where LP is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to each other, Me is hydrogen, a monovalent or divalent metal cation or an ammonium cation, KG represents a head group which represents an aliphatic radical containing at least one hydroxyl group, preferably an aliphatic radical having a hydroxyl group and an amino group, a hydroxyl group and a quaternary ammonium group, or the radical of a carbohydrate having five to six hydroxyl groups, and mp is an integer from 2 to 8, in particular 6, and the LP residues in the compound of formula (Vb) can assume different meanings within one molecule within the framework of the given definitions.

11. Lipid nanoparticles according to at least one of claims 1 to 10, characterized in that the stealth lipid c) has the structure of formula (V11a) or (V11b) O-CH2-CH2-(O-CH2-CH2)OI-I-OR 9 I LPL-CO-O-CH2-CH-CH2-O-CO-LPL (Villa) O-CO-LPL I LPL-CO-O-CH2-CH-CH2-O-CH2-CH2-(O-CH2-CH2)0I-I-OR 9 (Vlllb) wherein LPL is a saturated or mono- to tri-ethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, wherein several double bonds are not directly adjacent to each other, or a sterol radical, ol is an integer from 5 to 500, preferably 10 to 200, R 9represents hydrogen, alkyl having one to six carbon atoms or a radical -LPL, preferably hydrogen, methyl ethyl or a sterol radical, and the radicals LPL can assume different meanings within a molecule within the framework of the given definitions.

12. Lipid nanoparticles according to at least one of claims 1 to 10, characterized in that the stealth lipid c) has the structure of formula (IXa) or (IXb) LPL-NH-CO-O-CH2-CH2-(O-CH2-CH2)OI-I-OR 9 (IXa) LPL I LPL-N-CO-O-CH2-CH2-(O-CH2-CH2)OI-I-OR 9 (IXb) wherein LPL is a saturated or mono- to tri-ethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, wherein several double bonds are not directly adjacent to one another, or a sterol radical, ol is an integer from 5 to 500, preferably 10 to 200, R 9represents hydrogen, alkyl having one to six carbon atoms or a radical -LPL, preferably hydrogen, methyl ethyl or a sterol radical, and the radicals LPL in the compound of formula (IXb) can assume different meanings within one molecule within the framework of the given definitions.

13. Lipid nanoparticles according to at least one of claims 1 to 11, characterized in that the stealth lipid c) is a POx lipid.

14. Lipid nanoparticles according to claim 13, characterized in that the POx lipid has a structure of formula (XI Ia) or (XIIIb) COR 11 I O-(CH2-CH2-N)oi-OR 10 I LPL-CO-O-CH2-CH-CH2-O-CO-LPL (Xllla) O-CO-LPL COR 11 II LRL-CO-0-CH2-CH-CH2-0-(CH2-CH2-N)OI-OR 10(XIIIb) wherein LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, wherein several double bonds are not directly adjacent to one another, or a sterol radical, ol is an integer from 5 to 500, preferably 10 to 200, R 10 represents hydrogen, alkyl having one to six carbon atoms or a radical -LPL, preferably hydrogen, methyl ethyl or a sterol radical, and R 11 hydrogen or Ci-C4-alkyl, where the LPL radicals can take on different meanings within a molecule within the given definitions.

15. Lipid nanoparticles according to at least one of claims 1 to 14, characterized in that their lipids are only cationic lipids a), phospholipids b) and stealth lipids c), and that the cationic lipids a) are selected from the group A / ,A / -dioleyl-A / , / V-dimethylammonium chloride (DODAC); A / -(2,3-dioleyloxy)propyl)-A / , / V, / V-trimethylammonium chloride (DOTMA); N,N-distear \-N,N-dimethylammonium bromide (DDAB); A / -(2,3-dioleyloxy)propyl)-A / , / V,A / -trimethylammonium chloride (DOTAP); 3-(A / -( / V,A / '-dimethylaminoethane)-carbamoyl)-cholesterol (DC-Chol), A / -(1-(2,3-dioleoyloxy)propyl)-A / -2-(spermine-carboxamido)ethyl)-A / ,A / -dimethylammonium trifluoroacetate (DOSPA), 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), dioctadecylamidoglycyl carboxysperm in (DOGS), 1 ,2-Dioleoyl-3-dimethylammoniumpropan (DODAP), A / ,A / -Dimethyl-2,3-dioleoyloxy)propylamin (DODMA), N-(1 ,2-Dimyristyloxyprop-3- yl)-A / , / V-dimethyl- / V-hydroxyethylammoniumbromid (DMRIE), 1 ,2-Dilinoleyloxy- A / ,A / -dimethylaminopropan (DLinDMA), oder 1 ,2-Dilinolenyloxy-A / , / V-dimethyl- aminopropan (DLenDMA), dass die Phospholipide b) ausgewählt sind aus der Gruppe Distearoylphosphatidyl- cholin (DSPC), Dioleoylphosphatidylcholin (DOPC), Dipalmitoylphosphatidyl- cholin (DPPC), dioleoylphosphatidylglycerin (DOPG), Dipalmitoylphosphatidyl- glycerin (DPPG), Dioleoylphosphatidylethanol-amin (DOPE), Palmitoyloleoyl- phosphatidylcholin (POPO), Palmitoyloleoyl-phosphatidylethanolamin (POPE), Dioleoyl-sn-glycero-3-phosphoethanolamin-N-(maleimidomethyl) Natriumsalz (DOPE-mal), Dipalmitoyl-phosphatidylethanol-amin (DPPE), Dimyristoyl- phosphoethanolamin (DMPE), Distearoylphosphatidyl-ethanolamin (DSPE), 16- O-Monomethyl PE, 16-0-Dimethyl PE, 18-1 -Trans PE,1-Stearioyl-2-oleoylphosphatidylethanolamine (SOPE), or 1,2-dielaidoyl-sn-glycero-3-phosphatidylethanolamine (transDOPE), and that the stealth lipids c) are selected from the group pegylated diacylglycerol (PEG-DAG), in particular 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), PEG-succinate-diacylglycerol (PEG-S-DAG), in particular 4-O-(2',3'-di(tetradecanoyloxy)-propyl-1-O-(w-methoxy-(polyethoxy)ethyl)butanedioate (PEG-S-DMG), pegylated ceramide (PEG-cer), or PEG-dialkoxypropylcarbamate, in particular cü-Methoxy(polyethoxy)ethyl-A / -(2,3-di(tetradecanoxy)propyl)-carbamate or 2,3-di(tetradecanoxy)propyl- / V-(w-methoxy-(polyethoxy)ethyl)-carbamate., 16. Lipid nanoparticles according to claim 2, characterized in that the active ingredient is a nucleic acid, preferably a DNA and / or RNA, in particular a nucleic acid selected from the group consisting of A-DNA, B-DNA, Z-DNA, mtDNA, bacterial DNA, antisense DNA, viral DNA, Plasmids, hnRNA, mRNA, tRNA, rRNA, mtRNA, snRNA, snoRNA, scRNA, siRNAa, miRNA, antisense RNA, bacterial RNA and viral RNA.

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

18. Lipid nanoparticles according to at least one of claims 1 to 17, characterized in that they contain a PDI TG-value of the particle size distribution in the range between 0.01 and 0.4, preferably between 0.02 and 0.3 and particularly preferably between 0.05 and 0.2, wherein the polydispersity index is determined by dynamic light scattering (DLS).

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

20. Lipid nanoparticles according to claim 19, characterized in that they have a diameter determined by means of DLS (z-average) between 40 and 250 nm, in particular between 50 and 200 nm, a polydispersity index of the particle diameters between 0.05 and 0.2 and an N / P ratio between 3 and 15.

21. A process for the preparation of lipid nanoparticles according to any one of claims 1, 3 to 15 or 17 to 18, comprising the following measures: i) initial charging of aqueous dispersions of the lipids a), b) and c) according to claim 1 in buffers in the pH range from 3 to 8, preferably 4 to 7.5, ii) combination of the aqueous dispersions from step i); and iii) treating the combined aqueous dispersions from step ii) with a mixing process selected from the group consisting of ultrasound, dual centrifugation, nanoprecipitation, microfluidics or in a vortex mixer, whereby the nanoparticles are formed.

22. A process for producing lipid nanoparticles according to any one of claims 2, 16 or 19 to 20, comprising the following measures: iv) initial charge of the lipid nanoparticle-containing aqueous dispersion from step iii) of the process according to claim 21, v) initial charge of an aqueous solution or dispersion of an active ingredient with anionic groups in a buffer in the pH range from 3 to 8, preferably 4 to 7.5 vi) combination of the aqueous dispersions or solutions from steps iv) and v), and vii) treatment of the combined aqueous dispersions or solutions from step vi) with ultrasound, microfluidics, dual centrifugation, nanoprecipitation or in a vortex mixer, whereby the active ingredient-loaded lipid nanoparticles are formed.

23. A process for producing lipid nanoparticles according to any one of claims 2, 16 or 19 to 20 comprising the following measures: I) introducing aqueous dispersions of the lipids a), b) and c) according to claim 1 in buffers in the pH range from 3 to 8, preferably 4 to 7.5, II) Introduction of an aqueous solution or dispersion of an active ingredient with anionic groups in a buffer in the pH range of 3 to 8, preferably 4 to 7.5 III) combination of the aqueous dispersions or solutions from steps I) and II); and IV) Treatment of the combined aqueous dispersions or solutions from step III) with ultrasound, microfluidics, dual centrifugation, Nanoprecipitation or in a vortex mixer, whereby the drug-loaded lipid nanoparticles are formed.

24. Method according to claim 22 comprising the following measures: V) introducing the aqueous dispersion containing lipid nanoparticles with a pH value between 3 and 8 from step iii) of the process according to claim 21, VI) preparing an aqueous solution or dispersion of a nucleic acid in a buffer in the pH range of 3 to 8, preferably 4 to 7.5, VII) Mixing both dispersions or solutions from steps V) and VI) in a selected ratio of nucleic acid and lipid a) so that a desired molar N / P ratio of nitrogen atoms in lipid a) to the phosphate groups in the nucleic acid is obtained, preferably an N / P ratio between 1 and 100, VIII) Moving the mixture from step VII), and IX) if necessary, subsequent incubation of the resulting mixture.

25. A method for gene transfer into cells, 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 incubation.

26. The method according to claim 25, which includes the following steps: C) Providing a cell culture in a bioreactor or incubator, D) Addition of an aqueous dispersion containing nucleic acid-containing lipid nanoparticles according to claim 16, E) Distributing the aqueous dispersion in the cell culture, and F) subsequent incubation.

27. Use of the nanoparticles according to claim 2, 16 or 19 to 20 for gene transfer into cells.

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