DE102023001946A1 - Nanoparticles for the transport of active substances with anionic groups, processes for their preparation and their use - Google Patents

Abstract

Lipid nanoparticles containing
a) at least one cationic lipid,
b) at least one phospholipid, and
c) at least one selected stealth lipid, with the proviso that all lipids contained in the lipid nanoparticle have an HLB value greater than or equal to 3.
These nanoparticles can be loaded with active ingredients containing anionic groups, preferably nucleic acids, and are suitable as highly efficient vehicles for the transfer of nucleic acids into cells.

Description

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

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

Lipid nanoparticles (LNP) 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 small molecule drugs. LNP 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 of pharmaceutical active ingredients have also been described, e.g. in Puri, A.; Loomis, K.; Smith, B.; Lee, J.H.; Yavlovich, A.; Heldman, E.; Blumenthal, R., Lipid-based nanoparticles as pharmaceutical drug carriers: from concepts to clinic. Crit. Rev. Ther. Drug Carrier Syst. 2009, 26, 523-80 .

Gene therapy using siRNA and mRNA has become increasingly important in recent years (cf. Akinc, A.; Maier, M.A.; 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, S.J.; 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, H.M.; Essink, B., et al., Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 2021, 384, 403-416 ).

Different nucleic acids such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA or ASOs) must be delivered, which, due to their high molar mass and negative charge, require a transporter in contrast to classical active substances ( Kulkarni, JA; Witzigmann, D.; Thomson, S.B.; Chen, S.; Leavitt, B.R.; Cullis, P.R.; van der Meel, R., The current landscape of nucleic acid therapeutics. Nat. Nanotechnol. 2021, 16, 630-643 ). 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 areas of application or cell types.

With the outbreak of the COVID-19 pandemic at the beginning of 2020, lipid nanoparticles (LNPs) became established as an important drug form for the transport of genetic material (see: Tenchov, R.; Bird, R.; Curtze, A.E.; 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 (see Kulkarni, JA; Witzigmann, D.; Thomson, S.B.; Chen, S.; Leavitt, B.R.; Cullis, P.R.; van der Meel, R., The current landscape of nucleic acid therapeutics. Nat.Nanotechnol. 2021, 16, 630-643; 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, T.C.; Langer, R.; Wood, MJA, Advances in oligonucleotide drug delivery. Nat. Rev. Drug Discov. 2020, 19, 673-694 ); Mipomersen (Kynamro) is an ASO as an orphan drug for hypercholesterolemia. Patisiran (Onpattro) is the first approved representative of novel first-in-class drugs based on RNA interference in 2018 and is used to treat hereditary transthyretin amyloidosis in patients with stage 1 or 2 polyneuropathy ( Buck, J.; Grossen, P.; Cullis, P.R.; Huwyler, J.; Witzigmann, D., Lipid-based DNA therapeutics: Hallmarks of non-viral gene delivery. ACS Nano 2019, 13, 3754-3782 ). In addition, a plasmid encoding human hepatocyte growth factor has been approved for the treatment of patients with critical limb ischemia.

The approval of novel RNA-based systems has further promoted the development of non-viral delivery systems such as N-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 (see Kulkarni, JA; Cullis, P.R.; van der Meel, R., Lipid nanoparticles enabling gene therapies: From concepts to clinical utility, Nucl. Acid Ther. 2018, 28, 146-157; and Schoenmaker, L.; Witzigmann, D.; Kulkarni, JA, et al., mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 2021, 601, 120586 ).

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

For transport, the conserved negatively charged phosphates in the RNA and DNA backbone can be used for interactions with lipids. The cationic lipids play a key role here, as they are responsible for binding to the negatively charged genetic material. They also influence endosomal uptake (cf. Hald Albertsen, C.; Kulkarni, JA; Witzigmann, D., et al., The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv. Drug Del. Rev. 2022, 188, 114416; and Paloncyova, M.; Cechova, P.; Srejber, M., et al., Role of ionizable lipids in SARS-CoV-2 vaccines as revealed by molecular dynamics simulations: From membrane structure to interaction with mRNA fragments. J. Phys. Chem. Lett. 2021, 12, 11199-11205 ).

The development of ionizable lipids is considered a breakthrough for the broad 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 below 7 were developed. These lipids bind to the genetic material through a pH-dependent formulation that starts at a lower pH to allow binding of the genetic material and slowly increases to the physiological pH. The formulations are neutral upon administration, e.g. intravenously or intramuscularly, and are charged under the acidic environment in the endosome, which offers advantages for endosomal release. Protonation in the endosome provides a good interaction with the lipids of the endosomal membrane ( Han, X.; Zhang, H.; Butowska, K.; Swingle, K.L.; Alameh, M.-G.; Weissman, D.; Mitchell, MJ, 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 head group, a dimethylamino base showed high transfection efficiency ( Mon, 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 ).

There are a variety of different ionizable lipids that differ in their potential for gene transfer into LNP ( Semple, S.C.; Akinc, A.; Chen, 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 (cf. Hald Albertsen, C.; Kulkarni, JA; Witzigmann, D., et al., The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv. Drug Del. Rev. 2022, 188, 114416; Zhang, Y.; Sun, C.; Wang, C.; Jankovic, KE; Dong, Y., Lipids and lipid derivatives for RNA delivery. Chem. Rev. 2021, 121, 12181-12277; and Whitehead, K.A.; Dorkin, J.R.; 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 recognition and elimination by the immune system. The stealth effect is achieved by limiting the adhesion of immune-triggering proteins, such as opsonins and immunoglobulins, to the particle surface, which prevents opsonization and leads to a prolonged half-life in the bloodstream after systemic administration (cf. Friedl, J.D.; 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 ).

Camouflaged nanoparticles affect drug delivery, especially in Cancer therapy. In 1994, Langer et al. (Gref, R.; Minamitake, Y.; Peracchia, MT; Trubetskoy, V.; Torchilin, V.; Langer, R., Biodegradable longcirculating polymeric nanospheres. Science 1994, 263, 1600-1603 .). PEG-grafted polymer nanoparticles that can circulate in the blood for a longer period 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 beneficial for drug delivery to the tumor microenvironment due to the enhanced permeation and retention (EPR) phenomenon ( Suk, J.S.; 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 ).

A number of alternatives to PEGylated compounds are known. In general, these show a prolonged systemic circulation time, sustained drug release kinetics and better tumor accumulation.

Examples of alternative polymers to PEG include poly(glycerol) (PG), poly(oxazoline) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethylacrylamide) (PDMA) and poly(N-acryloylmorpholine) (PAcM) (cf. Hoang Thi, TT; Pilkington, E.H.; Nguyen, D.H.; Lee, J.S.; Park, K.D.; Truong, NP, The Importance of poly(ethylene glycol) alternatives for overcoming PEG immunogenicity in drug delivery and bioconjugation. Polymers 2020, 12, 298 ).

Among the so-called stealth lipids, PEG lipids are currently the most important and widely used. In the LNP formulation, PEG lipids are responsible, among other things, for extending the residence time in the organism and they reduce the organism's immune response to the drug (see. Nosova, AS; Koloskova, OO; Nikonova, A.A.; 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.I.; 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 ). It was also shown that they 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, J. W.; 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, K.J.; Dorkin, J.R.; Yang, J.H.; Heartlein, MW; DeRosa, F.; Me, FF; Fenton, O.S.; 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 (Hald 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 ).

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 ).

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

So it is US 7,341,348 B2 a nucleic acid encapsulated with lipids is known. The patent describes a particle made of a lipid layer that encloses a central zone containing nucleic acid and that contains an aminolipid with an amino group with a pK _a value of 4 to 11 and a PEG-DAG conjugate.

Out of WO 2018/081480 A1 LNP is known to contain 40 to 50 mol% of cationic lipid, neutral lipid, steroid, polymer-conjugated lipid and a therapeutic agent encapsulated therein.

WO 2019/089828 A1 discloses a LNP having a bilayer structure containing at least 40 mol% of a cationic lipid and a nucleic acid encapsulated therein.

In EP 3 556 353 A2 LNPs are described that are made up of a cationic lipid, a PEG lipid and an antigen. These LNPs usually contain other components, such as cholesterol or phospholipids. The lipids are dissolved in ethanol to produce the LNPs.

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

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 ). In the case of the COVID-19 vaccine, the latter technique prevailed for commercial scale. One of the advantages of microfluidics is its good reproducibility in production from batch to batch (cf. Maeki, M.; Uno, S.; Niwa, A.; Okada, Y.; Tokeshi, M., Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery. J. Control. Release 2022, 344, 80-96; and Shepherd, S.J.; Issadore, D.; Mitchell, 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 form. The organic solvent must then be removed from the formulation. In addition to evaporation, dialysis or cross-flow filtration is often used (see. Evers, M. J. W.; Kulkarni, J.A; van der Meel, R.; Cullis, P.R.; Vader, P.; Schifflers, 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, J.J.; 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. Pharm. 2011, 420, 118-121; and Terada, T.; Kulkarni, JA; Huynh, A.; Chen, S.; van der Meel, R.; Tam, YYC; Cullis, PR, Characterization of lipid nanoparticles containing ionizable cationic lipids using design-of-experiments approach. Langmuir 2021, 37, 1120-1128 ). In the case of the described LNP, the solvents are dialyzed against PBS. The formulation is then directly adjusted to the physiological pH value of 7.4.

The use of organic solvents for the formulation represents a significant disadvantage. The genetic material becomes more unstable and prolonged storage can lead to the degradation of the lipids (cf. Evers, M. J. W.; Kulkarni, JA; van der Meel, R.; Cullis, P.R.; Vader, P.; Schifflers, RM, State-of-the-art design and rapid-mixing production techniques of lipid nanoparticles for nucleic acid delivery. Small Methods 2018, 2, 1700375; and Roces, CB; Lou, G.; Jain, N.; Abraham, S.; Thomas, A.; Halbert, G. W.; 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.

The material from which the microfluidic chips are made can also be detrimental. Polydimethylsiloxane, for example, absorbs genetic material and tends to swell due to the solvents (see Kwon, H.J.; 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 ).

The elimination of organic solvents for the production of lipid-based nanoparticles is therefore a good way 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, S.B.; Zaifman, J.; Leung, J.; Wagner, PK; Hill, A.; Tam, YYC; Cullis, P.R.; Petkau, TL; Leavitt, BR, Spontaneous, solvent-free entrapment of siRNA within lipid nanoparticles. Nanoscale 2020, 12, 23959-23966 ). One way to avoid the use of ethanol is to homogenize the lipids in an aqueous solution above the melting point. Alternating cooling and heating leads to the formation of LNP (cf. De, A.; Ko, YT, Single pot organic solvent-free thermocycling technology for siRNAionizable 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.

It has now surprisingly been found that LNP can be produced without the need to use organic solvents. In conventional processes, organic solvents, such as ethanol, had to be used in order to be able to introduce the highly lipophilic portions of the lipids used into the LNP. The process according to the invention allows the production of LNP that can be loaded in high concentrations with active substances (e.g. nucleic acids) containing anionic groups, such as phosphate groups.

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.

The BLNPs according to the invention are produced without the use of highly lipophilic steroids and thus contain a smaller number of lipid components than the LNPs known to date. Conventional LNPs contain large amounts of cholesterol. This easily diffuses back and forth between the LNPs and also serum components and lipids in the biological environment. In addition, cholesterol is not produced entirely synthetically, but rather from natural sources, which means that fluctuating qualities and impurities cannot be ruled out. In addition, a process with more steps and components for approval is more complex and therefore more expensive and less robust.

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

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

A further object of the present invention was to provide a simple process for the production of lipid nanoparticles, which can be carried out without the use of organic solvents.

1. a) mindestens ein kationisches Lipid,
2. b) mindestens ein Phospholipid, und
3. c) mindestens ein Stealth-Lipid ausgewählt aus der Gruppe der Lipide enthaltend ein oder mehrere Poly(alkylenoxid)-ketten (nachstehend „PEG-Lipide“), der Lipide enthaltend ein oder mehreren Poly(oxazolin)-ketten (nachstehend „POx-Lipide“), der Lipide enthaltend ein oder mehrere Poly(glycerin)-ketten (nachstehend „PG-Lipide“), der Lipide enthaltend ein oder mehrere Poly(hydroxyalkyl(meth)-acrylat)-ketten (nachstehend „PHAA-Lipide“), der Lipide enthaltend ein oder mehrere Poly(N-(hydroxyalkyl)(meth)acrylamid)-ketten (nachstehend „PHAAA-Lipide“), der Lipide enthaltend ein oder mehrere Poly(vinylpyrrolidon)-ketten (nachstehend „PVP-Lipide“), der Lipide enthaltend ein oder mehrere Poly(N,N-dialkyl(meth)acrylamid)-ketten (nachstehend „PDMAA-Lipide“), der Lipide enthaltend ein oder mehrere Poly(N-(meth)acryloylmorpholin)-ketten (nachstehend „PAM-Lipide“) oder der Lipide enthaltend ein oder mehrere Poly(aminosäure)-ketten (nachstehend „PAA-Lipide“), mit der Maßgabe, dass alle im Lipid-Nanopartikel enthaltenen Lipide einen HLB-Wert von größer gleich 3 aufweisen

The present invention relates to lipid nanoparticles containing

1. a) at least one cationic lipid,
2. b) at least one phospholipid, and
3. c) 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(N-(hydroxyalkyl)(meth)acrylamide) chains (hereinafter "PHAAA lipids"), lipids containing one or more poly(vinylpyrrolidone) chains (hereinafter "PVP lipids"), lipids containing one or more poly(N,N-dialkyl(meth)acrylamide) chains (hereinafter "PDMAA lipids"), lipids containing a or more poly(N-(meth)acryloylmorpholine) chains (hereinafter referred to as "PAM lipids") or the lipids containing one or more poly(amino acid) chains (hereinafter referred to as "PAA lipids"), with the proviso that all lipids contained in the lipid nanoparticle have an HLB value of greater than or equal to 3

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

In the context of this description, “auxiliaries and additives” are understood to mean substances that are added to a formulation in order to give it certain additional properties and/or to facilitate its processing. Examples of excipients and additives are 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 that is useful for the intended application and is not a pharmaceutical or agrochemical active ingredient or a lipid, but can be formulated together with an active ingredient in an active ingredient-lipid complex in order to influence, in particular to improve, the qualitative properties of the LNP. 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.

wobei M, die Molmasse des lipophilen Anteils eines Moleküls ist und M die Molmasse des gesamten Moleküls darstellt.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 $$\text{HLB}=20*\left(1-{\text{M}}_{\text{I}}/\text{M}\right),$$

where M is the molar mass of the lipophilic portion of a molecule and M is the molar mass of the entire molecule.

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 repeat units.

Highly lipophilic compounds generally have HLB values of 1 to 3. These are hydrophilic (oil-soluble) lipids, such as antifoam agents. Compounds with significant hydrophilic portions are dispersible in water and have HLB values of 3 to 9. These include W/O emulsifiers with HLB values of 3 to 6 and wetting agents with HLB values of 7 to 9. Hydrophilic (water-soluble) lipids have HLB values of 9 to 18. These include O/W emulsifiers with HLB values of 8 to 18, detergents with HLB values of 13 to 15 and solubilizers with HLB values of 15 to 18. Phospholipids generally have HLB values of 4 to 5. Preferably, all lipids in the nanoparticles according to the invention have HLB values of 3 to 20, in particular 3 to 18, particularly preferably 4 to 17.5.

The BLNPs according to the invention can be loaded with active substances that have at least one anionic group. The invention therefore also relates to the LNPs described above that are loaded with active substances that have anionic groups.

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%.

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%.

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%.

The percentages given above refer to the total amount of lipids contained in the BLNP.

If the BLNPs according to the invention contain further lipids d) with HLB values of at least 3 which do not belong to one of the groups a) to c), the weight proportion of these lipids d) is at most 10%, preferably at most 5% and in particular at most 1%.

If the BLNPs according to the invention contain auxiliary substances or additives e), their total weight proportion is not more than 5%, preferably not more than 1% and in particular not more than 0.5%.

Preferably, the BLNPs according to the invention contain no further lipids d) and no excipients or additives e).

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

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.

The term “cationic lipid” as used here refers to lipids that have one or more positive net charge(s) at certain pH values, e.g., acidic pH values.

The cationic lipids within the scope of this description also include ionizable cationic lipids. Ionizable cationic lipids are characterized by the weak basicity of their ionizable groups, which influences the charge of the lipid in a pH-dependent manner. As a result, these lipids are positively charged at acidic pH, but are almost charge-neutral at physiological pH.

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

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

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, wherein this amino group(s) has/have a pKa value of 7 to 9.

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 with six to twenty carbon atoms, which can 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 with 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 as alkanolamino groups, preferably as amino groups.

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

worin

• R¹ ein Rest der Formel R⁴R⁵N-(C_mH_2m)-, CH₃-(C_nH_2n)-O-(C_oH₂₀)-, HO-(C_mH_2m)-, HO-CH₂-CH(OH)-CH₂-, CH₃-(CH₂)_n-O-CO-(C_mH_2m)-, CH₃-(C_nH_2n)-CO-O-(C_mH_am)-, NC-(CoH₂o)-, HO-CH₂-CH((CoH₂o)-CH₃ )-, CH₃-(CoH₂₀)-CH(OH)-(C_pH_2p)-, CH₃-(C_nH_2n)-CO-NH-(C_oH₂,)-, (HO-CH((C_qH_2q)-CH₃)-CH((C_oH_2o)-OH)- oder C₆H₁₀(OH)- ist,
• R² und R³ unabhängig voneinander Alkylreste mit sechs bis zwanzig Kohlenstoffatomen, die gegebenenfalls durch eine Estergruppe -CO-O- oder -O-COunterbrochen sein können, und/oder Alkylenreste mit sechs bis zwanzig Kohlenstoffatomen und ein, zwei oder drei nicht direkt zueinander benachbarten Doppelbindungen sind,
• R⁴ und R⁵ unabhängig voneinander Wasserstoff oder Alkylreste mit ein bis fünf Kohlenstoffatomen sind oder beide Reste R⁴ und R⁵ zusammen mit dem gemeinsamen Stickstoffatom einen Pyrrolidin- oder Piperidinrest bilden,
• m eine ganze Zahl von 2 bis 6 ist,
• n eine ganze Zahl von 0 bis 6 bedeutet,
• o und p unabhängig voneinander ganze Zahlen von 1 bis 6 sind,
• q eine ganze Zahl von 2 bis 16 ist, und
• r 0 oder 1 bedeutet.

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

wherein

• R ¹ is a radical of the formula R ⁴ R ⁵ N-(C _m H _2m )-, CH ₃ -(C _n H _2n )-O-(C _o H ₂₀ )-, HO-(C _m H _2m )-, HO-CH ₂ -CH(OH)-CH ₂ -, CH ₃ -(CH ₂ ) _n -O-CO-(C _m H _2m )-, CH ₃ -(C _n H _2n )-CO-O-(C _m H _am )-, NC-(CoH ₂ o)-, HO-CH ₂ -CH((CoH ₂ o)-CH ₃ )-, CH ₃ -(CoH ₂₀ )-CH(OH)-(C _p H _2p )-, CH ₃ -(C _n H _2n )-CO-NH-(C _o H ₂ ,)-, (HO-CH((C _q H _2q )-CH ₃ )-CH((C _o H _2o )-OH)- or C ₆ H ₁₀ (OH)- is,
• R ² and R ³ are independently 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 are independently hydrogen or alkyl radicals having one to five carbon atoms or both radicals R ⁴ and R ⁵ together with the common nitrogen atom form a pyrrolidine or piperidine radical,
• 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 means 0 or 1.

Very particularly preferred are cationic lipids a) of the formula (I) in which R ¹ is a radical of the formula R ⁴ R ⁵ N-(CH ₂ ) _m -, in particular those in which m is 2.

Extremely preferred are cationic lipids a) of the formula (I), in which R ¹ is a radical of the formula R ⁴ R ⁵ N-(CH ₂ ) ₂ -, and R ⁴ and R ⁵ are independently hydrogen, methyl or ethyl or in which one or both radicals R ⁴ and R ⁵ together with the common nitrogen atom form a pyrrolidine or piperidine radical.

• -(C_sH_2s)-CH₃, -CH₂-CH((C_tH_2t)-CH₃)((C_uH_2u)-CH₃), -CH((C_tH_2t)-CH₃)((C_uH_2u)-CH₃),
• -(C_sH_2s)-O-CO-CH((C_tH_2t)-CH₃)((C_uH_2u)-CH₃),
• -(C_sH_2s)-CO-O-CH((C_tH_2t)-CH₃)((C_uH_2u)-CH₃),
• -(C_sH_2s)-CO-O-(CtH₂t)-CH₃, -(C_sH_2s)-O-CO-(C_tH_2t)-CH₃,
• -(C_sH_2s)-O-CO-CH₂-CH((C_tH_2t)-CH₃)((C_uH_2u)-CH₃),
• -(C_sH_2s)-CO-O-CH₂-CH((C_tH_2t)-CH₃)((C_uH_2u)-CH₃),
• -CH((C_sH_2s)-O-CO-CH₂-CH((C_tH_2t)-CH₃)((C_uH_2u)-CH_a))₂,
• -CH((C_sH_2s)-CO-O-CH₂-CH((C_tH_2t)-CH₃)((C_uH_2u)-CH₃))₂,
• -CH((C_sH_2s)-O-CO-CH((C_tH_2t)-CH₃)((C_uH_2u)-CH₃))₂,
• -CH((C_sH_2s)-CO-O-CH((C_tH_2t)-CH₃)((C_uH_2u)-CH₃))₂,
• -(C_tH_2t)-CH=CH-CH₂-CH=CH-(C_uH_2u)-CH₃,
• -CH((C_tH_2t)-CH=CH-CH₂-CH=CH-(C_uH_2u)-CH₃)₂,
• -(C_tH_2t)-CH=CH-(C_uH_2u)-CH₃ und -CH((C_tH_2t)-CH=CH-(C_uH_2u)-CH₃)₂,
• -(C_tH_2t)-CH=C(CH₃)-CH₂-CH=CH-(C_uH_2u)-CH₃,
• -CH((C_tH_2t)-CH=C(CH₃)-CH₂-CH=CH-(C_uH_2u)-CH₃)₂,
• -(C_tH_2t)-CH=C(CH₃)-(C_uH_2u)-CH₃ und -CH((C_tH_2t)-CH=C(CH₃)-(C_uH_2u)-CH₃)₂,
• -(CtH₂t)-C₆H₁₁ und -C₆H₁₀-C(CH₃)₃,

worin
s eine ganze Zahl von 4 bis 20 ist, und
t und u unabhängig voneinander ganze Zahlen von 1 bis 10 bedeuten.Other particularly preferred cationic lipids a) include those of formula (I) in which R ² and R ³ are independently selected from the residues of the group

• -(C _s H _2s )-CH ₃ , -CH ₂ -CH((C _t H _2t )-CH ₃ )((C _u H _2u )-CH ₃ ), -CH((C _t H _2t )-CH ₃ )((C _u H _2u )-CH ₃ ),
• -(C _s H _2s )-O-CO-CH((C _t H _2t )-CH ₃ )((C _u H _2u )-CH ₃ ),
• -(C _s H _2s )-CO-O-CH((C _t H _2t )-CH ₃ )((C _u H _2u )-CH ₃ ),
• -(C _s H _2s )-CO-O-(CtH ₂ t)-CH ₃ , -(C _s H _2s )-O-CO-(C _t H _2t )-CH ₃ ,
• -(C _s H _2s )-O-CO-CH ₂ -CH((C _t H _2t )-CH ₃ )((C _u H _2u )-CH ₃ ),
• -(C _s H _2s )-CO-O-CH ₂ -CH((C _t H _2t )-CH ₃ )((C _u H _2u )-CH ₃ ),
• -CH((C _s H _2s )-O-CO-CH ₂ -CH((C _t H _2t )-CH ₃ )((C _u H _2u )-CH _a )) ₂ ,
• -CH((C _s H _2s )-CO-O-CH ₂ -CH((C _t H _2t )-CH ₃ )((C _u H _2u )-CH ₃ )) ₂ ,
• -CH((C _s H _2s )-O-CO-CH((C _t H _2t )-CH ₃ )((C _u H _2u )-CH ₃ )) ₂ ,
• -CH((C _s H _2s )-CO-O-CH((C _t H _2t )-CH ₃ )((C _u H _2u )-CH ₃ )) ₂ ,
• -(C _t H _2t )-CH=CH-CH ₂ -CH=CH-(C _u H _2u )-CH ₃ ,
• -CH((C _t H _2t )-CH=CH-CH ₂ -CH=CH-(C _u H _2u )-CH ₃ ) ₂ ,
• -(C _t H _2t )-CH=CH-(C _u H _2u )-CH ₃ and -CH((C _t H _2t )-CH=CH-(C _u H _2u )-CH ₃ ) ₂ ,
• -(C _t H _2t )-CH=C(CH ₃ )-CH ₂ -CH=CH-(C _u H _2u )-CH ₃ ,
• -CH((C _t H _2t )-CH=C(CH ₃ )-CH ₂ -CH=CH-(C _u H _2u )-CH ₃ ) ₂ ,
• -(C _t H _2t )-CH=C(CH ₃ )-(C _u H _2u )-CH ₃ and -CH((C _t H _2t )-CH=C(CH ₃ )-(C _u H _2u )-CH ₃ ) ₂ ,
• -(CtH ₂ t)-C ₆ H ₁₁ and -C ₆ H ₁₀ -C(CH ₃ ) ₃ ,

wherein
s is an integer from 4 to 20, and
t and u independently represent integers from 1 to 10.

• R² und R³ unabhängig voneinander ausgewählt werden aus den Resten der Formel -(CH₂)_v-O-CO-R⁵ oder -(CH₂)_v-CO-O-R⁶,
• v eine ganze Zahl von 1 bis 20, vorzugsweise von 5 bis 12 ist, und
• R⁵ und R⁶ unabhängig voneinander Alkylreste mit 6 bis 20 Kohlenstoffatomen und/oder Alkenylreste mit 6 bis 20 Kohlenstoffatomen und mit ein oder vorzugsweise zwei ethylenisch ungesättigten nicht direkt zueinander benachbarten Bindungen bedeuten, insbesondere Reste ausgewählt aus der Gruppe
• -(C_vH_2v)-CH₃, -(CH₂)_w-C₆H₁₁, -C₆H₁₀-C(CH₃)₃, -CH((CH₂)_x-CH₃)((CH₂)_y,-CH₃),
• -CH₂-CH((CH₂)_×-CH₃)((CH₂)_y-CH₃), -CH((CH₂)_×-C(CH₃)₃)((CH₂)_y-C(CH₃)₃) ,
• -CH₂-CH((CH₂)_x-C(CH₃)₃((CH₂)_y-C(CH₃)₃,
• -(CH₂)_w-CH((CH₂)_xCH₃)((CH₂)_y-CH=C(CH₃)₂),

worin

• v eine ganze Zahl von 7 bis 12 ist
• w eine ganze Zahl von 1 bis 4 ist
• x und y unabhängig voneinander ganze Zahlen von 0 bis 12 bedeuten, und ganz besonders bevorzugt Reste ausgewählt aus der Gruppe
  • -CH(CH₂-CH₃)((CH₂)₃-CH₃), -CH((CH₂)₅-CH₃)((CH₂)₇-CH₃),
  • -CH((CH₂)₅-CH₃)((CH₂)₅-CH₃), -CH((CH₂)y-CH₃)((CH₂)₇-CH₃),
  • -CH((CH₂)₅-CH₃)((CH₂)₃-CH₃), -CH((CH₂)₉-CH₃)((CH₂)₁₁-CH₃),
  • -CH((CH₂)₉-CH₃)((CH₂)₇-CH₃), -CH((CH₂)₂-CH₃)((CH₂)₂-CH₃),
  • -CH((CH₂)₅-CH₃)((CH₂)₇-CH₃), CH((CH₂)₃-CH₃)((CH₂)₃-CH₃),
  • -CH(CH₃)-(CH₂)₉-CH₃, -CH₂-CH((CH₂)₅-CH₃)((CH₂)₅-CH₃),
  • -CH₂-CH((CH₂)₇-CH₃)((CH₂)₇-CH₃), -CH₂-CH((CH₂)₅-CH₃)((CH₂)₃-CH₃),
  • -CH₂-CH((CH₂)₉-CH₃)((CH₂)₁₁-CH₃), -CH₂-CH((CH₂)₉-CH₃)((CH₂)₇-CH₃),
  • -CH₂-CH((CH₂)₂-CH₃)((CH₂)₂-CH₃), -CH₂-CH((CH₂)₅-CH₃)((CH₂)₇-CH₃),
  • -CH₂-CH((CH₂)₃-CH₃)((CH₂)₃-CH₃), -(CH₂)₇-CH₃, -(CH₂)₁₀-CH₃, -(CH₂)₁₁-CH₃
  • -CH₂-CH=CH-(CH₂)₇-CH₃, -(CH₂)₈-CH=CH-CH₂-CH=CH-(CH₂)₄-CH₃,
  • -CH₂-CH=C(CH₃)-(CH₂)₃-CH(CH₃)-(CH₂)₃-CH(CH₃)-(CH₂)₃-CH(CH₃)₂,
  • -(CH₂)₂-C₆H₁₁ und -C₆H₁₀-C(CH₃)₃,

Very particularly preferred are cationic lipids a) of the formula (I), in which R ¹ is a radical of the formula R ⁴ R ⁵ N-(CH ₂ ) ₂ -, and R ⁴ and R ⁵ are independently hydrogen, methyl or ethyl or in which or both radicals R ⁴ and R ⁵ together with the common nitrogen atom form a pyrrolidine or piperidine radical,

• R ² and R ³ are independently selected from the radicals of the formula -(CH ₂ ) _v -O-CO-R ⁵ or -(CH ₂ ) _v -CO-OR ⁶ ,
• v is an integer from 1 to 20, preferably from 5 to 12, and
• R ⁵ and R ⁶ independently of one another represent alkyl radicals having 6 to 20 carbon atoms and/or alkenyl radicals having 6 to 20 carbon atoms and having one or preferably two ethylenically unsaturated bonds which are not directly adjacent to one another, in particular radicals selected from the group
• -(C _v H _2v )-CH ₃ , -(CH ₂ ) _w -C ₆ H ₁₁ , -C ₆ H ₁₀ -C(CH ₃ ) ₃ , -CH((CH ₂ ) _x -CH ₃ )((CH ₂ ) _y ,-CH ₃ ),
• -CH ₂ -CH((CH ₂ ) _× -CH ₃ )((CH ₂ ) _y -CH ₃ ), -CH((CH ₂ ) _× -C(CH ₃ ) ₃ )((CH ₂ ) _y -C(CH ₃ ) ₃ ) ,
• -CH ₂ -CH((CH ₂ ) _x -C(CH ₃ ) ₃ ((CH ₂ ) _y -C(CH ₃ ) ₃ ,
• -(CH ₂ ) _w -CH((CH ₂ ) _x CH ₃ )((CH ₂ ) _y -CH=C(CH ₃ ) ₂ ),

wherein

• v is an integer from 7 to 12
• w is an integer from 1 to 4
• x and y independently represent integers from 0 to 12, and most preferably radicals selected from the group
  • -CH(CH ₂ -CH ₃ )((CH ₂ ) ₃ -CH ₃ ), -CH((CH ₂ ) ₅ -CH ₃ )((CH ₂ ) ₇ -CH ₃ ),
  • -CH((CH ₂ ) ₅ -CH ₃ )((CH ₂ ) ₅ -CH ₃ ), -CH((CH ₂ )y-CH ₃ )((CH ₂ ) ₇ -CH ₃ ),
  • -CH((CH ₂ ) ₅ -CH ₃ )((CH ₂ ) ₃ -CH ₃ ), -CH((CH ₂ ) ₉ -CH ₃ )((CH ₂ ) ₁₁ -CH ₃ ),
  • -CH((CH ₂ ) ₉ -CH ₃ )((CH ₂ ) ₇ -CH ₃ ), -CH((CH ₂ ) ₂ -CH ₃ )((CH ₂ ) ₂ -CH ₃ ),
  • -CH((CH ₂ ) ₅ -CH ₃ )((CH ₂ ) ₇ -CH ₃ ), CH((CH ₂ ) ₃ -CH ₃ )((CH ₂ ) ₃ -CH ₃ ),
  • -CH(CH ₃ )-(CH ₂ ) ₉ -CH ₃ , -CH ₂ -CH((CH ₂ ) ₅ -CH ₃ )((CH ₂ ) ₅ -CH ₃ ),
  • -CH ₂ -CH((CH ₂ ) ₇ -CH ₃ )((CH ₂ ) ₇ -CH ₃ ), -CH ₂ -CH((CH ₂ ) ₅ -CH ₃ )((CH ₂ ) ₃ -CH ₃ ),
  • -CH ₂ -CH((CH ₂ ) ₉ -CH ₃ )((CH ₂ ) ₁₁ -CH ₃ ), -CH ₂ -CH((CH ₂ ) ₉ -CH ₃ )((CH ₂ ) ₇ -CH ₃ ),
  • -CH ₂ -CH((CH ₂ ) ₂ -CH ₃ )((CH ₂ ) ₂ -CH ₃ ), -CH ₂ -CH((CH ₂ ) ₅ -CH ₃ )((CH ₂ ) ₇ -CH ₃ ),
  • -CH ₂ -CH((CH ₂ ) ₃ -CH ₃ )((CH ₂ ) ₃ -CH ₃ ), -(CH ₂ ) ₇ -CH ₃ , -(CH ₂ ) ₁₀ -CH ₃ , -(CH ₂ ) ₁₁ -CH ₃
  • -CH ₂ -CH=CH-(CH ₂ ) ₇ -CH ₃ , -(CH ₂ ) ₈ -CH=CH-CH ₂ -CH=CH-(CH ₂ ) ₄ -CH ₃ ,
  • -CH ₂ -CH=C(CH ₃ )-(CH ₂ ) ₃ -CH(CH ₃ )-(CH ₂ ) ₃ -CH(CH ₃ )-(CH ₂ ) ₃ -CH(CH ₃ ) ₂ ,
  • -(CH ₂ ) ₂ -C ₆ H ₁₁ and -C ₆ H ₁₀ -C(CH ₃ ) ₃ ,

worin

• R¹, R², R³ und r die oben definierte Bedeutung besitzen,
• j eine ganze Zahl ist welche der Anzahl der Stickstoffatome in der Verbindung der Formel (II) entspricht, vorzugsweise 1 oder 2 ist,
• i eine ganze Zahl von 1 bis 5000 ist, und
• X ein i-wertiges Anion bedeutet.

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

wherein

• 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
• X represents an i-valent anion.

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

Examples of inorganic anions X ^i- 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.

Examples of organic anions X ^i- 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.

These anions can exist in the form of polyanions.

worin

• R^1a, R^2a und R^3a unabhängig voneinander Alkylreste mit ein bis zwanzig Kohlenstoffatomen, die gegebenenfalls durch eine Estergruppe -CO-O- oder -O-COunterbrochen sein können, und/oder Alkylenreste mit zwei bis zwanzig Kohlenstoffatomen und ein, zwei oder drei nicht direkt zueinander benachbarten Doppelbindungen sind,
• R^4a ein Alkylrest mit sechs bis zwanzig Kohlenstoffatomen, der gegebenenfalls durch eine Estergruppe -CO-O- oder -O-CO- unterbrochen sein kann, und/oder einen Alkylenrest mit sechs bis zwanzig Kohlenstoffatomen und ein, zwei oder drei nicht direkt zueinander benachbarten Doppelbindungen ist, oder worin zwei Reste R^1a und R^2a zusammen mit dem gemeinsamen Stickstoffatom einen Pyrrolidin- oder Piperidinrest bilden, und
• X, i und j die weiter oben definierte Bedeutung besitzen.

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

wherein

• R ^1a , R ^2a and R ^3a are independently alkyl radicals having one to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO, and/or alkylene radicals having two to twenty carbon atoms and one, two or three double bonds which are not directly adjacent to one another,
• R ^4a is an alkyl radical having six to twenty carbon atoms, which may optionally be interrupted by an ester group -CO-O- or -O-CO-, and/or an alkylene radical having six to twenty carbon atoms and one, two or three double bonds which are not directly adjacent to one another, or in which two radicals R ^1a and R ^2a together with the common nitrogen atom form a pyrrolidine or piperidine radical, and
• X, i and j have the meaning defined above.

The nanoparticles according to the invention very particularly preferably contain cationic lipids which are selected from the group N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)-cholesterol (DC-Chol), N-(1-(2,3-dioleoyloxy)propyl)N-2-(spermine-carboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), Dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), and 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

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.

• LP ein Rest einer Fettsäure ist,
• BG eine (np+1)-wertige Brückengruppe ist,
• np eine ganze Zahl von 1 bis 5 ist, vorzugsweise 1 oder 2,
• Me Wasserstoff, ein ein- oder zweiwertiges Metallkation oder ein Ammoniumkation ist,
• KG eine Kopfgruppe bedeutet, welche einen aliphatischen Rest enthaltend mindestens eine Hydroxylgruppe darstellt, vorzugsweise einen aliphatischen Rest mit einer Hydroxylgruppe und einer Aminogruppe mit einer Hydroxylgruppe und einer quaternären Ammoniumgruppe oder den Rest eines Kohlehydrats mit fünf bis sechs Hydroxylgruppen, wobei
• Reste LP im Rahmen der gegebenen Definitionen innerhalb eines Moleküls unterschiedliche Bedeutungen annehmen können.

A phospholipid b) generally has a structure of the formula (III) (LP) _np -BG-OP(O)(OMe)-O-KG (III) wherein

• LP is a residue of a fatty acid,
• BG is an (np+1)-valent bridging group,
• np is an integer from 1 to 5, preferably 1 or 2,
• Me is hydrogen, a monovalent or divalent metal cation or an ammonium cation,
• KG represents a head group which is an aliphatic radical containing at least one hydroxyl group, preferably an aliphatic radical having one hydroxyl group and one amino group, one hydroxyl group and one quaternary ammonium group or the radical of a carbohydrate having five to six hydroxyl groups, where
• Residues LP can assume different meanings within a molecule within the given definitions.

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

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

Examples of residues of di- to hexavalent aliphatic or cycloaliphatic alcohols are groups derived from ethylene glycol, propylene glycol, glycerin, 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.

worin

• LP ein gesättigter oder ein- bis dreifach-ethylenisch ungesättigter Alkyl- oder Alkenylrest mit sechs bis zwanzig Kohlenstoffatomen ist, wobei mehrere Doppelbindungen nicht direkt zueinander benachbart sind,
• KG und Me die weiter oben definierten Bedeutungen besitzen, und
• die Reste LP im Rahmen der gegebenen Definitionen innerhalb eines Moleküls unterschiedliche Bedeutungen annehmen können.

Preferred phospholipids b) have a residue derived from glycerol as a bridging group and have the structure of formula (IVa) or (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,
• KG and Me have the meanings defined above, and
• the residues LP can assume different meanings within a molecule within the given definitions.

worin

• LP ein gesättigter oder ein- bis dreifach-ethylenisch ungesättigter Alkyl- oder Alkenylrest mit sechs bis zwanzig Kohlenstoffatomen ist, wobei mehrere Doppelbindungen nicht direkt zueinander benachbart sind,
• KG und Me die weiter oben definierten Bedeutungen besitzen,
• mp eine ganze Zahl von 2 bis 8 insbesondere 6 ist, und
• die Reste LP in der Verbindung der Formel (Vb) im Rahmen der gegebenen Definitionen innerhalb eines Moleküls unterschiedliche Bedeutungen annehmen können.

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

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,
• KG and Me have the meanings defined above,
• mp is an integer from 2 to 8, in particular 6, and
• the residues LP in the compound of formula (Vb) can assume different meanings within one molecule within the framework of the given definitions.

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

• pp eine ganze Zahl von 2 bis 6, vorzugsweise 2 ist,
• R⁶ Wasserstoff oder C₁-C₅-Alkyl bedeutet,
• R⁷ und R⁸ unabhängig voneinander C₁-C₆-Alkyl sind, und
• XP ein ip-wertiges Anion bedeutet, und
• ip eine ganze Zahl von 1 bis 3, vorzugsweise 1 oder 2 ist.

Particularly preferred are phospholipids b) with head groups KG of the formula (VIa), (VIb) or (VIc) -O-(C _pp H _2pp )-NH ₂ (Vla) -O-(C _pp H _2pp )-N ⁺ R ⁶ R ⁷ R ⁸ (Xp ^ip- ) _1/ip (Vlb) -O-(C _pp-1 H _2pp-1 )-CH(NH ₂ )(COOH) (VIc) wherein

• pp is an integer from 2 to 6, preferably 2,
• R ⁶ is hydrogen or C ₁ -C ₅ alkyl,
• R ⁷ and R ⁸ are independently C ₁ -C ₆ alkyl, and
• XP means an ip-valent anion, and
• ip is an integer from 1 to 3, preferably 1 or 2.

Particularly preferred are phospholipids b) with head groups KG, which are selected from the group choline, ethanolamine, serine and inositol.

The nanoparticles according to the invention very particularly preferably contain phospholipids b) which are selected from the group distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-sn-glycero-3-phosphoethanolamine-N-(maleimidomethyl) sodium salt (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, 1-Stearioyl-2-oleoylphosphatidyethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (transDOPE).

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

• LPL ein Alkyl- oder Alkenylrest mit 6-20 Kohlenstoffatomen, ein Rest einer Fettsäure, eines Fettalkohols oder ein Sterinrest ist,
• BGL eine (nl+1)-wertige Brückengruppe ist,
• nl eine ganze Zahl von 1 bis 5 ist, vorzugsweise 1 oder 2,
• ml 0 oder 1, vorzugsweise 1 bedeutet,
• ol eine ganze Zahl von 5 bis 500 ist, vorzugsweise 10 bis 200, und R⁹ Wasserstoff, Alkyl mit ein bis sechs Kohlenstoffatomen oder einen Rest
• LPL darstellt, vorzugsweise Wasserstoff, Methyl Ethyl oder ein Sterinrest, wobei
• die Reste LPL im Rahmen der gegebenen Definitionen innerhalb eines Moleküls unterschiedliche Bedeutungen annehmen können.

Preferred stealth lipids c) are PEG lipids. These are in particular lipids with the structure of formula (VII) (LPL) _nl -(BGL) _ml -(O-CH ₂ -CH ₂ ) _ol -OR ⁹ (VII) wherein

• LPL is an alkyl or alkenyl residue with 6-20 carbon atoms, a residue of a fatty acid, a fatty alcohol or a sterol residue,
• BGL is a (nl+1)-valent bridging group,
• nl is an integer from 1 to 5, preferably 1 or 2,
• ml is 0 or 1, preferably 1,
• ol is an integer from 5 to 500, preferably 10 to 200, and R ⁹ is hydrogen, alkyl having one to six carbon atoms or a radical
• LPL, preferably hydrogen, methyl ethyl or a sterol residue, where
• the LPL residues can assume different meanings within a molecule within the given definitions.

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 with six to twenty carbon atoms, and/or mono- to triethylenically unsaturated alkenyl residues with six to twenty carbon atoms, and/or seeded 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, where several double bonds in an alkenyl residue are not directly adjacent to one another.

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.

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

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.

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.

Examples of amino alcohol residues are groups derived from residues of the formula >N-R-O-, in which R is a divalent organic residue, preferably an alkylene residue, the PEG residue is bonded to the oxygen atom and one or two LPL residues are bonded to the nitrogen atom. Other amino alcohol residues can have several amino groups and/or oxygen atoms, for example aminophenols with two hydroxy groups and/or amino groups.

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.

worin

• LPL ein gesättigter oder ein- bis dreifach-ethylenisch ungesättigter Alkyl- oder Alkenylrest mit sechs bis zwanzig Kohlenstoffatomen ist, wobei mehrere Doppelbindungen nicht direkt zueinander benachbart sind, oder ein Sterinrest, R⁹ und ol die weiter oben definierten Bedeutungen besitzen, und die Reste LPL im Rahmen der gegebenen Definitionen innerhalb eines Moleküls unterschiedliche Bedeutungen annehmen können.

Preferred stealth lipids c) have a glycerol-derived residue as a bridging group and have the structure of formula (VIIIa) or (VIIIb)

wherein

• LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, or a sterol radical, R ⁹ and ol have the meanings defined above, and the LPL radicals can assume different meanings within a molecule within the framework of the given definitions.

worin

• LPL ein gesättigter oder ein- bis dreifach-ethylenisch ungesättigter Alkyl- oder Alkenylrest mit sechs bis zwanzig Kohlenstoffatomen ist, wobei mehrere Doppelbindungen nicht direkt zueinander benachbart sind, R⁹ und ol die weiter oben definierten Bedeutungen besitzen, und die Reste LPL in der Verbindung der Formel (IXb) im Rahmen der gegebenen Definitionen innerhalb eines Moleküls unterschiedliche Bedeutungen annehmen können.

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

wherein

• LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, R ⁹ and ol have the meanings defined above, and the LPL radicals in the compound of formula (IXb) can assume different meanings within one molecule within the framework of the given definitions.

• LPL ein gesättigter oder ein- bis dreifach-ethylenisch ungesättigter Alkyl- oder Alkenylrest mit sechs bis zwanzig Kohlenstoffatomen ist, wobei mehrere Doppelbindungen nicht direkt zueinander benachbart sind, und R⁹ und ol die weiter oben definierten Bedeutungen besitzen.

Further preferred stealth lipids c) have a residue derived from succinic acid as a bridging group and have the structure of formula (X) LPL-O-OC-CH ₂ -CH ₂ -CO-O-CH ₂ -CH ₂ -(O-CH ₂ -CH ₂ ) _ol-1 -OR ⁹ (X) wherein

• LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, and R ⁹ and ol have the meanings defined above.

The PEG lipids c) preferably used according to the invention include the substance listed below, wherein n is a number between 15 and 200, preferably between 18 and 70.

The nanoparticles according to the invention very particularly preferably contain PEG lipids which are selected from the group 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-(ω-methoxy-(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-Di(tetradecanoxy)propyl-N-(ω-methoxy-(polyethoxy)ethyl)carbamate.

Other particularly preferred stealth lipids c) are POx lipids. These are generally polymers which, in addition to at least one lipid residue, have a polyoxazoline residue connected to it. sen, the latter being produced by the polymerization of oxazoline. The connection of both residues can be achieved by a covalent bond or via a bridging group BGL.

worin R Wasserstoff oder ein einwertiger organischer Rest ist.Polymers produced by the polymerization of oxazoline monomers have the recurring structural element of formula (XI)

where R is hydrogen or a monovalent organic radical.

worin

• LPL ein Alkyl- oder Alkenylrest mit 6-20 Kohlenstoffatomen, ein Rest einer Fettsäure, eines Fettalkohols oder ein Sterinrest ist,
• BGL eine (nl+1)-wertige Brückengruppe ist,
• nl eine ganze Zahl von 1 bis 5 ist, vorzugsweise 1 oder 2,
• ml 0 oder 1, vorzugsweise 1 bedeutet,
• ol eine ganze Zahl von 5 bis 500 ist, vorzugsweise 10 bis 200,
• R¹⁰ Wasserstoff, Alkyl mit ein bis sechs Kohlenstoffatomen oder ein Rest
• LPL darstellt, vorzugsweise Wasserstoff, Methyl Ethyl oder ein Sterinrest, und
• R¹¹ Wasserstoff oder C₁-C₄-Alkyl bedeutet, wobei
• Reste LPL, und R¹¹ im Rahmen der gegebenen Definitionen innerhalb eines Moleküls unterschiedliche Bedeutungen annehmen können.

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

wherein

• LPL is an alkyl or alkenyl residue with 6-20 carbon atoms, a residue of a fatty acid, a fatty alcohol or a sterol residue,
• BGL is a (nl+1)-valent bridging group,
• nl is an integer from 1 to 5, preferably 1 or 2,
• ml is 0 or 1, preferably 1,
• ol is an integer from 5 to 500, preferably 10 to 200,
• R ¹⁰ is hydrogen, alkyl having one to six carbon atoms or a radical
• LPL, preferably hydrogen, methyl ethyl or a sterol residue, and
• R ¹¹ is hydrogen or C ₁ -C ₄ alkyl, where
• Residues LPL and R ¹¹ can have different meanings within a molecule within the given definitions.

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 with six to twenty carbon atoms, and/or mono- to tri-ethylenically unsaturated alkenyl residues with six to twenty carbon atoms, and/or saturated or mono- to tri-ethylenically unsaturated fatty acid residues with six to twenty carbon atoms, and/or saturated or mono- to tri-ethylenically unsaturated fatty alcohol residues with six to twenty carbon atoms, and/or sterol residues, wherein several double bonds in an alkenyl residue are not directly adjacent to one another.

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, where the bridging group BGL is the residue of a di- to hexavalent aliphatic or cycloaliphatic alcohol, or the residue of a di- to hexavalent carboxylic acid, or a carbamate residue, or the residue of an amino alcohol.

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.

worin

• LPL ein gesättigter oder ein- bis dreifach-ethylenisch ungesättigter Alkyl- oder Alkenylrest mit sechs bis zwanzig Kohlenstoffatomen ist, wobei mehrere Doppelbindungen nicht direkt zueinander benachbart sind, R¹⁰, R¹¹ und ol die weiter oben definierten Bedeutungen besitzen, und
• die Reste LPL im Rahmen der gegebenen Definitionen innerhalb eines Moleküls unterschiedliche Bedeutungen annehmen können.

Preferred POx lipids have a glycerol-derived residue as a bridging group and have the structure of formula (XIIIa) or (XIIIb)

wherein

• LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, R ¹⁰ , R ¹¹ and ol have the meanings defined above, and
• the LPL residues can assume different meanings within a molecule within the given definitions.

worin

• LPL ein gesättigter oder ein- bis dreifach-ethylenisch ungesättigter Alkyl- oder Alkenylrest mit sechs bis zwanzig Kohlenstoffatomen ist, wobei mehrere Doppelbindungen nicht direkt zueinander benachbart sind, und R¹⁰, R¹¹ und ol die weiter oben definierten Bedeutungen besitzen.

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

wherein

• LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, and R ¹⁰ , R ¹¹ and ol have the meanings defined above.

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

• LPL ein Alkyl- oder Alkenylrest mit 6-20 Kohlenstoffatomen, ein Rest einer Fettsäure, eines Fettalkohols oder ein Sterinrest ist,
• BGL eine (nl+1)-wertige Brückengruppe ist,
• nl eine ganze Zahl von 1 bis 5 ist, vorzugsweise 1 oder 2,
• ml 0 oder 1, vorzugsweise 1 bedeutet, und
• POLY ein Rest der Formeln (XVa), (XVIb), (XVIc), (XVId), (XVle), (XVIf) oder (XVIg) ist
  
  
  
  
  worin R¹² Wasserstoff oder LPL bedeutet,
  • R¹³ Wasserstoff oder ein einwertiger organischer Rest, wie Alkyl, Cycloalkyl, Aryl oder Aralkyl bedeutet,
  • R¹⁴ Wasserstoff oder Alkyl mit ein bis sechs Kohlenstoffatomen ist, insbesondere Wasserstoff oder Methyl,
  • R¹⁵ und R¹⁶ unabhängig voneinander Wasserstoff oder Alkyl mit ein bis sechs Kohlenstoffatomen bedeuten, insbesondere Alkyl mit ein bis vier Kohlenstoffatomen, R¹⁷ Wasserstoff, Alkyl mit ein bis sechs Kohlenstoffatomen, das gegebenenfalls mit einem Hydroxyl-, Amin-, Phenyl-, Hydroxyphenyl-, Carboxyl- oder Amidrest substituiert ist,
  • NMORPH einen Morphonylrest bedeutet, der über das Ringstickstoffatom mit der Carbonylgruppe verbunden ist,
  • r, s, t und u Zahlen größer gleich 1, bevorzugt zwischen 1 und 5000 bedeuten, wobei die
  • Reste LPL im Rahmen der gegebenen Definitionen innerhalb eines Moleküls unterschiedliche Bedeutungen annehmen können.

Other preferred stealth lipids c) have a structure of the formula (XV) (LPL) _nl -(BGL) _ml -POLY (XV) wherein

• LPL is an alkyl or alkenyl residue with 6-20 carbon atoms, a residue of a fatty acid, a fatty alcohol or a sterol residue,
• BGL is a (nl+1)-valent bridging group,
• nl is an integer from 1 to 5, preferably 1 or 2,
• ml is 0 or 1, preferably 1, and
• POLY is a radical of the formulas (XVa), (XVIb), (XVIc), (XVId), (XVle), (XVIf) or (XVIg)
  
  
  
  
  where R ¹² is hydrogen or LPL,
  • R ¹³ is hydrogen or a monovalent organic radical such as alkyl, cycloalkyl, aryl or aralkyl,
  • R ¹⁴ is hydrogen or alkyl having one to six carbon atoms, in particular hydrogen or methyl,
  • R ¹⁵ and R ¹⁶ independently of one another are hydrogen or alkyl having one to six carbon atoms, in particular alkyl having one to four carbon atoms, R ¹⁷ is hydrogen, alkyl having one to six carbon atoms, which is optionally substituted by a hydroxyl, amine, phenyl, hydroxyphenyl, carboxyl or amide radical,
  • NMORPH means a morphonyl residue 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
  • LPL residues can assume different meanings within a molecule within the given definitions.

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

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

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

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

Particularly preferred stealth lipids c) of the formula (XV) are those in which POLY is a radical of the formula (XVIf), in which R ¹⁴ is methyl and R ¹⁷ is hydrogen.

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

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

Preferred active ingredients f) are nucleic acids. In the context of the present description, this refers to naturally occurring nucleic acids including modified derivatives thereof. Modified nucleic acids can contain modified nucleotides or be modified in other ways, for example by introducing chemical modifications. Nucleic acids form complexes with the cationic lipids a) via the phosphate ester residues present therein.

The physicochemical properties of nucleic acids form the basis for interaction with carrier materials, and small changes in the sequence can lead to different biological effects and allow for rapid adaptation to different indications without having to adapt the entire formulation process. Compared to conventional drugs, nucleic acids are biopolymers with a higher molar mass (about 333 Da per nucleotide) and a strong negative charge, which leads to good water solubility. In addition, their stability in the presence of degradation enzymes is low and they can trigger an immune response based on evolutionarily optimized mechanisms that protect the organism from viral gene manipulation. Despite these hurdles, nucleic acids enable modulation of gene expression, whereas classical drugs often have no causal effect. Some successful, approved systems for the application of nucleic acids are in DNA is usually present as a double-stranded variant (dsDNA), in which two single-stranded DNA chains (ssDNA) are linked together by hydrogen bonds and hydrophobic interactions between the complementary base sequences, resulting in the familiar double helix conformation. dsDNA is a semi-flexible polymer with a high negative charge density. DNA is often encoded in plasmids (pDNA) containing a few thousand base pairs (bp) for therapeutic applications. When pDNA enters the cell nuclei, it can affect gene expression. By repairing defective genes, whether congenital or acquired, gene therapy currently offers highly specific and potentially even curative therapies for diseases with no treatment or cure options. In addition to DNA, Short interfering RNA (siRNA) is a double-stranded RNA that is usually 19-25 bp long. Due to the different sugar backbone, they have a higher linear charge density and stiffness than DNA. Therapeutic siRNA temporarily switches off genes (knock-down) because the translation of the target mRNA is hindered. In contrast, single-stranded antisense oligonucleotides (ASO) can switch off translation by binding to the corresponding mRNA. Both types of RNA are already in clinical use, as in 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 or siRNA and ssDNA. Since the bases are accessible, mRNAs have a stronger amphiphilic character, which allows hydrophobic interactions with potential transport molecules. Unmodified mRNA is more labile to nucleases due to its single-stranded character and has a 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 and thus bypass the nuclear membrane barrier.

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

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.

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

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

Combinations of DNA and RNA can also be used in the BLNPs according to the invention.

Modified nucleic acids, also called xenonucleic acids (XNA), offer a number of 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. XNA can exhibit increased biostability and, in addition, can increasingly be developed in vitro, accelerating lead discovery (Duffy, K.; Arangundy-Franklin, S.; Holliger, P., Modified nucleic acids: replication, evolution, and next-generation therapeutics. BMC Biol. 2020,18, 112).

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

The nanoparticles according to the invention can be characterized by their particle diameter. Typical particle diameters (for example z-average) are in the range of less than or equal to 900 nm, preferably less than or equal to 500 nm, particularly preferably between 30 and 500 nm, very particularly preferably between 40 and 250 nm and in particular between 50 and 200 nm. 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 (IS013321, ISO22412). A refractive index of 1.33 for ultrapure water was assumed for size determination.

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.

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

The BLNPs according to the invention can also be characterized by their polydispersity index (or PDI). The PDI indicates the width of the distribution of the particle sizes of particles. Values between 0 (monodisperse) and 1 (polydisperse) can be assumed. For the purposes of the present 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 by means of cumulant analysis of the correlation function.

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

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.

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

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

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 proportion is 10% or less, in particular less than 5%.

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.

The BLNPs of the invention may be in solid form as a powder or they may form a dispersion and be dispersed in aqueous solvents, the particles being in solid form in the dispersing medium.

In a preferred embodiment, the BLNPs according to the invention form a disperse phase in water or in an aqueous buffer solution.

The BLNPs according to the invention can be produced by assembly. 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 set to 3 to 8, preferably 4 to 7.5, e.g. by using an acetate buffer or another suitable buffer such as citrate buffer, lactate buffer, phosphate buffer and phosphate-citrate buffer. 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 set to a value between 3 and 8, particularly preferably to a value between 4 and 7.5. A buffer solution containing acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer, HEPES, TRIS, or just salts is particularly suitable for this purpose. The aqueous dispersions of the lipids and the active ingredient solution or dispersion are combined with each other, whereby the amounts of active ingredient and cationic lipid a) are selected so 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 by vortexing and/or by sonication with ultrasound. The resulting nanoparticles are preferably left to stand for some time before further use, for example between 5 and 20 minutes, in order to enable a bond between lipid a) and active ingredient e) (hereinafter referred to as "incubation"). After production 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).

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

The aqueous dispersion of the BLNP and the active ingredient solution or dispersion are then combined with one another, with the amounts being chosen so that a desired active ingredient/lipid ratio, e.g. a desired N/P ratio, is achieved. 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 by sonicating with ultrasound. Here too, the resulting active ingredient-loaded nanoparticles are left to stand for some time before further use, for example between 5 and 20 minutes, to enable a bond between lipid a) and active ingredient e) (hereinafter referred to as "incubation"). After production in 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 dispersion medium or remain in the dispersion medium.

In addition to the cationic lipid a), the other lipids b) and c) and the active ingredient f), one or more auxiliary and additive substances e) may be present in the dispersion medium during their nanoprecipitation. Alternatively, these auxiliary and additive substances e) can be added after the nucleic acid copolymer complex has been dispersed in the aqueous phase.

Water is used as a dispersing medium. Buffer substances, salts, sugars or acids and bases can be added to this in order to adjust the desired pH value or osmolarity.

The processes according to the invention are characterized by the fact that the use of organic solvents such as ethanol can be dispensed with during the production of the lipid nanoparticles. A subsequent separation of the solvent can therefore be omitted.

• i) Vorlage von wässrigen Dispersionen der Lipide a), b) und c) in Puffern im pH-Bereich von 3 bis 8, bevorzugt 4 bis 7,5
• ii) Kombination der wässrigen Dispersionen aus Schritt i); und
• iii) Behandlung der kombinierten wässrigen Dispersionen aus Schritt ii) mit einem Mischverfahren ausgewählt aus der Gruppe bestehend aus Ultraschall, Dualzentrifugation, Nanofällung, Mikrofluidik oder im Vortexmischer, wobei sich die Nanopartikel ausbilden.

The invention also relates to a process for producing the BLNP described above comprising the following measures:

• 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
• ii) combining 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.

• iv) Vorlage der LNP enthaltenden wässrigen Dispersion aus Schritt iii) gemäß dem vorstehenden Verfahren,
• v) Vorlage einer wässrigen Lösung oder Dispersion eines Wirkstoffs f) mit anionischen Gruppen in einem Puffer im pH-Bereich von 3 bis 8, bevorzugt 4 bis 7,5
• vi) Kombination der wässrigen Dispersionen oder Lösungen aus Schritten iv) und v), und
• vii) Behandlung der kombinierten wässrigen Dispersionen oder Lösungen aus Schritt vi) mit Ultraschall, Mikrofluidik, Dualzentrifugation, Ultraschall, Nanofällung oder im Vortexmischer, wobei sich die mit Wirkstoff beladenen LNP ausbilden.

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

• iv) introducing the LNP-containing aqueous dispersion from step iii) according to the above process,
• v) 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
• vi) combining the aqueous dispersions or solutions from steps iv) and v), and
• vii) treating the combined aqueous dispersions or solutions from step vi) with ultrasound, microfluidics, dual centrifugation, ultrasound, nanoprecipitation or in a vortex mixer to form the drug-loaded LNPs.

• I) Vorlage von wässrigen Dispersionen der Lipide a), b) und c) in Puffern im pH-Bereich von 3 bis 8, bevorzugt 4 bis 7,5,
• II) Vorlage einer wässrigen Lösung oder Dispersion eines Wirkstoffs f) mit anionischen Gruppen in einem Puffer im pH-Bereich von 3 bis 8, bevorzugt 4 bis 7,5
• III) Kombination der wässrigen Dispersionen oder Lösungen aus Schritten I) und II); und
• IV) Behandlung der kombinierten wässrigen Dispersionen oder Lösungen aus Schritt III) mit Ultraschall, Mikrofluidik, Dualzentrifugation, Nanofällung oder im Vortexmischer, wobei sich die mit Wirkstoff beladenen LNP ausbilden.

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:

• 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,
• II) Preparation 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
• 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 LNPs are formed.

• V) Vorlage der LNP enthaltenden wässrigen Dispersion mit einem pH-Wert zwischen 3 bis 8, bevorzugt 4 bis 7,5 aus Schritt iii) hergestellt gemäß dem vorstehenden Verfahren,
• VI) Herstellen einer wässrigen Lösung oder Dispersion einer Nukleinsäure in einem Puffer im pH-Bereich von 3 bis 8, bevorzugt 4 bis 7,5,
• VII) Vermischen beider Dispersionen oder Lösungen aus Schritten V) und VI) in einem ausgewählten Mengenverhältnis von Nukleinsäure und Lipid a), so dass ein gewünschtes molares N/P-Verhältnis von Stickstoffatomen im Lipid a) zu den Phosphatgruppen in der Nukleinsäure erhalten wird, vorzugsweise ein N/P-Verhältnis zwischen 1 und 200,
• VIII) Bewegen der Mischung aus Schritt VII), und
• IX) gegebenenfalls nachfolgende Inkubation der erhaltenen Mischung.

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

• V) introducing the LNP-containing aqueous dispersion having a pH value between 3 to 8, preferably 4 to 7.5 from step iii) prepared according to the above process,
• 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 200,
• VIII) agitating the mixture from step VII), and
• IX) optionally subsequent incubation of the resulting mixture.

The aqueous dispersions of the lipids a), b) and c) for steps i) or l) of the processes according to the invention preferably contain a buffer, in particular an acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer or mixtures thereof.

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

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

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

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.

The nanoparticles can be separated from the aqueous phase in different ways. Examples are crossflow filtration, centrifugation, ultrafiltration or dialysis. The dispersion of the nanoparticles can also be used preferably directly after production without further processing.

By cleaning by filtration, particles such as aggregates, but also excess auxiliary materials or impurities can be separated from the dispersion. The particle concentration can change in the process.

Dissolved molecules can be separated from the dispersion by cleaning using dialysis/crossflow filtration. The process is largely independent of the particle size with regard to the dispersed particles.

Dissolved molecules can also be separated from the dispersion by cleaning using centrifugation. However, this process also reduces the concentration of the dispersed particles. In addition, only dispersions with nanoparticles of larger diameter, e.g. more than 150 nm, can be treated and the particles can be damaged. Furthermore, redispersing the particles obtained in this way can be difficult.

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

In particular, the BLNPs loaded with active substances according to the invention are suitable for gene transfer into cells, i.e. for introducing nucleic acids and 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.

1. A) Inkontaktbringen von Zellen, Geweben oder Zellkulturen mit einer wässrigen Dispersion enthaltend die oben beschriebenen Nukleinsäuren enthaltenden LNP, und
2. B) anschließendes Inkubieren.

The invention therefore also relates to a method for gene transfer into cells, which comprises the following steps:

1. A) contacting cells, tissues or cell cultures with an aqueous dispersion containing the LNPs containing the nucleic acids described above, and
2. B) subsequent incubation.

• C) Bereitstellen einer Zellkultur in einem Bioreaktor oder Inkubator,
• D) Zugabe einer wässrigen Dispersion enthaltend die oben beschriebenen Nukleinsäuren enthaltenden LNP,
• E) Verteilen der wässrigen Dispersion in der Zellkultur, und
• F) anschließendes inkubieren.

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

• C) Providing a cell culture in a bioreactor or incubator,
• D) Addition of an aqueous dispersion containing the LNP containing nucleic acids described above,
• E) distributing the aqueous dispersion in the cell culture, and
• F) subsequent incubation.

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.

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

The application of the LNP loaded with nucleic acids according to the invention can be carried out in vivo, for example under the skin or in the muscle, or the application can also be carried out ex vivo, for example with immune cells, as in CAR-T therapy. It can also be an RNA vaccination or another vaccination.

In the context of this description, "cells" are understood to mean the smallest living units of organisms. These can be cells of single- or multi-celled 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 can originate from fungi. Eukaryotic cells are preferably used, especially those that were originally isolated from tissue and can be permanently cultivated, i.e. that are immortalized.

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

In the context of this description, "cell cultures" refers to combinations of cells or tissues and cell culture medium, whereby the cells or tissues are cultivated in the cell culture medium outside the organism. Cell lines are used, i.e. cells of a tissue type that can divide during the course of cultivation. Both immortalized cell lines and primary cells (primary culture) can be cultivated. Primary culture is usually understood to mean a non-immortalized cell culture that was obtained directly from a tissue.

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

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 methods known per se, e.g. by treatment with a protease, which breaks down the proteins that maintain the cell association. It may also be appropriate to 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.

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

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

Culture conditions and cell culture media are selected depending on the individual cells being cultured. The different cell types prefer different nutrient media, which are specifically composed. For example, different pH values are set and the individual nutrient media can contain different amino acids and/or other nutrients in different concentrations.

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

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

In the process according to the invention, described by the following examples, the property of the cationic lipids to form a homogeneous, disperse system in acidic aqueous solution (e.g. 20 mM NaOAc, pH 5.5) was used. The phospholipid, e.g. DSPC, behaves in the same way. This made it possible to take reproducible volumes from adjusted stock solutions. The stealth lipids, such as PEG lipids or POx lipids, can also be transferred to the formulation in this way, since they are soluble in the buffer mentioned above. Cholesterol was omitted, since this molecule is insoluble in water and is not required for the construction of the lipid nanoparticles according to the invention. For this reason, the molar composition of the lipid nanoparticles was adapted for the following examples (cationic Lipid/helper lipid/stealth lipid = 81.3/16.3/2.4); however, the ratio of the components to each other from the original Moderna formulation (50/10/1.5) was retained. Controlled mixing and subsequent shearing of the particles using ultrasound treatment results in the formation of homogeneous, unloaded lipid nanoparticles. The particles formed by ultrasound treatment differ significantly in shape and size from lipid nanoparticles that are produced by simply mixing the mixture.

The unloaded lipid nanoparticles can be loaded with the desired active ingredient, e.g. the desired genetic material, in a subsequent process step. 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). Then, after a defined incubation time, equal amounts of nanoparticle suspension and MM are combined by quickly transferring them into one another and mixing them using a vortexer. Using in vitro test systems, the functionality of the lipid nanoparticles loaded with genetic material according to the invention could be demonstrated with regard to protein expression. After it was shown that the lipid nanoparticles according to the invention are basically suitable for transfections with pDNA, the formulation was further optimized with regard to biocompatibility. For this purpose, various buffer systems were tested for the formation of the unloaded lipid nanoparticles and the loaded lipid nanoparticles. The data show that an acidic pH is essential for positive transfections. In addition, mRNA was used as an alternative genetic material. The experiments showed that the lipid nanoparticles according to the invention are also suitable for transfection with mRNA. In addition, various stealth polymers can be used as an alternative to the commercial PEG-DMG.

The main advantage of the lipid nanoparticles according to the invention is that the method according to the invention enables the production of lipid nanoparticles for gene transfer without the use of organic solvents. This leads to a reduction in production costs, since the rapid removal of the solvents, for example by dialysis, is no longer necessary. In addition, the genetic material is protected.

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

show the structures of the lipids used in the LNPs used by BioNTech and Moderna. shows the structure of the phospholipid DSPC. shows the structures of the ionizable cationic lipids ALC-0315 and SM-102. shows the structures of the ethoxylated lipids PEG-DMG and ALC-0159. shows the structure of the lipid cholesterol.

SM-102 has the molecular formula C ₄₄ H ₈₇ NO ₅ and a molecular weight of 710.18. The HLB value calculated according to Griffin is 5.70.

DSPC has the molecular formula C ₄₄ H ₈₈ NO ₈ P and a molecular weight of 790.16. The HLB value calculated according to Griffin is 20*(1-(617.28/790.16)=4.38

PEG-DMG has the molecular formula C ₁₂₄ H ₂₄₆ O ₅₁ and a molecular weight of 2553.28. The HLB value calculated according to Griffin is 17.13.

Cholesterol has the molecular formula C ₂₇ H ₄₆ O and a molecular mass of 386.66. The HLB value calculated according to Griffin is 20*(1-((386.66-17)/386.66)=0.88

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. 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 each 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 ).

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

shows the schematic structure of a conventionally used LNP composed of the known lipids in known proportions.

shows a scheme of the method according to the invention. A first approach to production was based on a simple mixing process (vortexing), in which various ionizable lipids were mixed with genetic material and tested for transfection. It turned out that the cationic lipid SM-102 gave the best results, but these were rather below average. To improve the transfections, 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, vortexing for 5 sec, 3) 10 min incubation at room temperature, combining lipids and genetic material, 4) rapid vortexing.

Chol-POx is the polymer shown below

Chol-POx-52 (n=52) has the molecular formula C _24o H ₄₁₆ N ₅₂ O ₅₆ and a molecular weight of 4926.28. R stands for methyl. The HLB value calculated according to Griffin is 15.27.

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.

1. 1. Kationischem Lipid + genetisches Material (Legende: -),
2. 2. Kationisches Lipid + Helferlipid + genetisches Material (Legende: DSPC),
3. 3. Kationisches Lipid + Stealth Lipid + genetisches Material (Legende: Chol-POx),
4. 4. der erfindungsgemäße BLNP aus allen 4 Komponenten (Legende: DSPC & Chol-POx).

shows the measurement results obtained by DLS of the LNP produced by vortexing. The production was carried out according to the method described in The aim was to investigate the influence of the individual components of the formulation on the particle size. The combinations were produced from

1. 1. Cationic lipid + genetic material (legend: -),
2. 2. Cationic lipid + helper lipid + genetic material (legend: DSPC),
3. 3. Cationic lipid + stealth lipid + genetic material (legend: Chol-POx),
4. 4. the BLNP according to the invention from all 4 components (legend: DSPC & Chol-POx).

These particles are shown from left to right and were each produced at 3 different N/P ratios (3, 6, 9). 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, by adding helper lipid (DSPC) and stealth lipids (Chol-POx), small, homogeneous particles are formed, as can be seen from the lower PDI value for these LNPs.

In Results of the transfection experiments are shown. The nanoparticles used are LNPs according to the invention, which are analogous to the particles from were produced. The particles in the figure were supplemented with two additional amounts of Chol-POx (1.2% and 3.6%). These particles were transferred to cells to measure the transfection. The particles with stealth lipid and phospholipid are the most efficient. After weighing up the material usage, a formulation with N/P 6 turned out to be optimal.

outlines another method for producing the LNP according to the invention. The aim was to reduce the size of the resulting LNP and to further improve the efficiency of gene transport. improve. Of the possible methods, treatment with ultrasound turned out to be the most suitable. The lipid mixture is treated with ultrasound and significantly smaller, still unloaded lipid particles are created, which are then loaded with genetic material. Step 1) Lipid suspensions are prepared, 2) LNP assembly by ultrasound treatment, 3) dilution of the genetic material, vortexing for 5 seconds, 4) 10 min incubation at room temperature, combining lipids and genetic material, 5) rapid vortexing.

( provides an enlarged view of the results show particle diameters and PDI values of LNP. The LNPs investigated contain different lipids or lipid combinations as described for . LNPs are compared with those in (vortexing) and 8 (ultrasonic treatment). It turned out that the particles produced with the latter method are significantly smaller.

All measurement data for the LNP shown below refer to a molar composition of 82.3 mol% SM-102, 16.5 mol% DSPC and 1.2 mol% Stealth Lipid or 81.3 mol% SM-102, 16.3 mol% DSPC and 2.4 mol% Stealth Lipid. The transfection experiments shown were carried out using HEK293T cells. The incubation period is 24 h at 37 °C and the measured values were recorded using flow cytometry.

In The raw data from the measurement using a flow cytometer after transfection are shown. The LNP according to the invention are compared with the method according to (Vortexen) (AC) with the LNP according to the invention according to the method (Ultrasound treatment) (DF). In comparison between (70.05%), the LNPs produced by ultrasound show a significantly higher transfection efficiency of 88.92% ( on.

In the relative mean fluorescence intensities are added to the raw data from The horizontal bars in this case represent the LNP, which was calculated using the method according to It can be seen that the pure lipoplex made of cationic lipid with anionic active ingredient shows hardly any fluorescence in both methods. The LNP according to the invention, on the other hand, show clear fluorescence values (400x - 900x more than the negative control). In addition, a polyplex made of polyethyleneimine (PEI) was compared with the LNP to better classify the results. It can be seen that the LNP produced by ultrasound is superior to the PEI polyplex. In addition, the LNPs produced according to the method (ultrasound) the LNP produced using method (Vortexen) clearly superior.

shows results obtained with LNPs prepared using different buffers. The results shown in the previous figures were obtained with LNPs prepared at pH 4.0 in citrate buffer (50 mM). Under these circumstances, 50 mM citrate buffer can only be neutralized to pH 7.4 with great 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 different 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. This resulted in 15 new buffer combinations in addition to the initial combination of 50 mM citrate buffer with 20 mM HBG. In the absence of citrate buffer, the only positive result was obtained from the combination of 20 mM NaOAc pH 5.5 (LNP) with 20 mM NaOAc pH 5.5 (MM). The LNP were prepared for this series of experiments by vortexing. The advantage of using 20 mM NaOAc is that it is much easier to neutralize than citrate buffer. This solvent combination was used for the further experiments.

In the Particle diameters (columns) and PDI values (dots) of LNP are shown. once again illustrates the advantage of the ultrasound method, as significantly smaller particle sizes can be achieved. In the following, it was investigated whether changing the buffer system from citrate buffer to NaOAc produces similar particle sizes. In it can be seen that this is the case (similar sizes). The buffer systems were varied and the nanoparticles were measured in the same way using DLS after production.

shows the transfection efficiency (relative mean fluorescence intensity) of the LNP produced with NaOAc buffer. The buffer was acidified once with an equimolar amount of hydrochloric acid (HCl) and adjusted to pH 4.0 to test whether the specific pH value of the formulation influences the effectiveness. It can be seen that the values for pH 5.5 are the highest. This means that further To adapt the buffer system and for further optimization, work was continued with 20 mM NaOAc at pH 5.5.

For further use, it is imperative that the formulation can be neutralized from pH 5.5 to pH 7.4. In shows how the pH 5.5 formulation can be neutralized without affecting the transfection efficiency. Before bringing the LNP into contact with the cell culture medium, the samples were mixed 1 + 1 with the corresponding buffer shown. This figure shows that the transfections of the neutralized samples are surprisingly even better than without neutralization (pH 5.5). A preferred preparation of the LNP therefore involves preparing the nanoparticles at pH 5.5 using 20 mM NaOAc followed by neutralization by adding PBS.

In The results of the toxicity measurements are shown. For this purpose, unloaded LNP were tested according to the method from manufactured. Two different stealth polymers were used for this purpose. One was Chol-POx, the other was PEG-DMG. The test was carried out on L929 cells and these were incubated with LNP suspensions in various concentrations up to 826 µg/mL for 24 hours and then evaluated using the Presto-Blue assay. It can be seen that the LNP according to the invention does not exhibit any toxicity in the tested range.

In DLS measurements (particle sizes) of the LNP are carried out, analogous to described. Unloaded particles were measured (BLANK) as well as particles loaded with mRNA and pDNA. The latter were measured both at pH 5.5 (NaOAc) and after neutralization with PBS (pH 7.4). It can be seen that the choice of genetic material has no influence on the size. Neutralization leads to a slight increase in the sizes.

show the PDI values of the described LNP.

shows the proportion of EGFP positive cells in a transfection experiment depending on the amount of genetic material used. BLNP with the stealth lipid Chol-POx were transfected according to and loaded with mRNA and pDNA. It can be seen that the BLNPs according to the invention are clearly superior to PEI.

lists possible and already used PEG and POx lipids.

QUOTES INCLUDED IN THE DESCRIPTION

This list of documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions.

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• EP 3 556 353 A2 [0023]

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Claims (27)

Lipid nanoparticles containing a) at least one cationic/ionizable lipid, b) at least one phospholipid, and c) 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(N-(hydroxyalkyl)(meth)acrylamide) chains (PHAAA lipid), or containing one or more poly(vinylpyrrolidone) chains (PVP lipid), or containing one or more poly(N,N-dialkyl(meth)acrylamide) chains (PDMAA lipid), or containing one or more Poly(N-(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. Lipid nanoparticles after claim 1 , characterized in that they are loaded with active substances containing anionic groups. Lipid nanoparticles after claim 1 , characterized in that the molar mass fraction of the cationic lipid a) is 51 to 94.9%, the molar mass fraction of the phospholipid b) is 5 to 40%, and the molar mass fraction of the stealth lipid c) is 0.1 to 10%, wherein the percentages stated refer to the total mass of the lipids contained in the LNP. Lipid nanoparticles according to at least one of the Claims 1 until 3 , characterized in that , in addition to the lipids a), b) and c), they contain no further lipids d) and no auxiliary or additive substances e). Lipid nanoparticles according to at least one of the Claims 1 until 4 , characterized in that the cationic lipid a) contains no phosphate groups and at least one amino group. Lipid nanoparticles after 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. Lipid nanoparticles after claim 6 , characterized in that the cationic lipid a) contains one to two amino groups with a pKa value of 7 to 9. Lipid nanoparticles according to at least one of the Claims 1 until 7 , characterized in that the cationic lipid a) has the structure of formula (I)

where R ¹ is a radical of the formula R ⁴ R ⁵ N-(C _m H _2m )-, CH ₃ -(C _n H _2n )-O-(C _o H _2o )-, HO-(C _m H _2m )-, HO-CH ₂ -CH(OH)-CH ₂ -, CH ₃ -(CH ₂ ) _n -O-CO-(C _m H _2m )-, CH ₃ -(C _n H _2n )-CO-O-(C _m H _2m )-, NC-(C _o H _2o )-, HO-CH ₂ -CH((C _o H _2o )-CH ₃ )-, CH ₃ -(C _o H _2o )-CH(OH)-(CpH _2p )-, CH ₃ -(C _n H _2n )-CO-NH-(C _{o H2o} )-, (HO-CH((C _q H2 _q )-CH3)-CH((C _o H _2o )-OH)- or C ₆ H ₁₀ (OH)-, R ² and R ³ are independently 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 which are not directly adjacent to one another, R ⁴ and R ⁵ are independently hydrogen or alkyl radicals having one to five carbon atoms or both radicals R ⁴ and R ⁵ together with the common nitrogen atom form a pyrrolidine or piperidine radical, 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. Lipid nanoparticles according to at least one of the Claims 1 until 8 , characterized in that the phospholipid b) has the structure of formula (IVa) or (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 one another, Me is hydrogen, a mono- or divalent metal cation or an ammonium cation, KG is a head group which represents an aliphatic radical containing at least one hydroxyl group, preferably an aliphatic radical with one hydroxyl group and one amino group with one hydroxyl group and one quaternary ammonium group or the radical of a carbohydrate with five to six hydroxyl groups, and the radicals LP can assume different meanings within a molecule within the framework of the given definitions. Lipid nanoparticles according to at least one of the Claims 1 until 8 , characterized in that the phospholipid b) has the structure of formula (Va) or (Vb)

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 one another, Me is hydrogen, a mono- or divalent metal cation or an ammonium cation, KG is a head group which represents an aliphatic radical containing at least one hydroxyl group, preferably an aliphatic radical with one hydroxyl group and one amino group with one hydroxyl group and one quaternary ammonium group or the radical of a carbohydrate with five to six hydroxyl groups, and mp is an integer from 2 to 8, in particular 6, and the residues LP in the compound of formula (Vb) can assume different meanings within one molecule within the framework of the given definitions. Lipid nanoparticles according to at least one of the Claims 1 until 10 , characterized in that the stealth lipid c) has the structure of formula (VIIIa) or (VIIIb)

wherein LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, or a sterol radical, ol is an integer from 5 to 500, preferably 10 to 200, R ⁹ represents hydrogen, alkyl having one to six carbon atoms or a radical -LPL, preferably hydrogen, methyl ethyl or a sterol radical, and the radicals LPL can assume different meanings within a molecule within the framework of the given definitions. Lipid nanoparticles according to at least one of the Claims 1 until 10 , characterized in that the stealth lipid c) has the structure of formula (IXa) or (IXb) LPL-NH-CO-O-CH ₂ -CH ₂ -(O-CH ₂ -CH ₂ ) _ol-1 -OR ⁹ (IXa)

wherein LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, or is a sterol radical, ol is an integer from 5 to 500, preferably 10 to 200, R ⁹ is 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 the formula (IXb) can assume different meanings within a molecule within the framework of the given definitions. Lipid nanoparticles according to at least one of the Claims 1 until 11 , characterized in that the stealth lipid c) is a POx lipid. Lipid nanoparticles after claim 13 , characterized in that the POx lipid has a structure of formula (XIIIa) or (XIIIb)

wherein LPL is a saturated or mono- to triethylenically unsaturated alkyl or alkenyl radical having six to twenty carbon atoms, where several double bonds are not directly adjacent to one another, or is a sterol radical, ol is an integer from 5 to 500, preferably 10 to 200, R ¹⁰ is hydrogen, alkyl having one to six carbon atoms or a radical -LPL, preferably hydrogen, methyl ethyl or a sterol radical, and R ¹¹ is hydrogen or C ₁ -C ₄ alkyl, where the LPL radicals can assume different meanings within a molecule within the framework of the given definitions. Lipid nanoparticles according to at least one of the Claims 1 until 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 N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)-cholesterol (DC-Chol), N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), Dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), or 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), that the phospholipids b) are selected from the group distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), Dioleoylphosphatidylethanolamine (DOPE), Palmitoyloleoylphosphatidylcholine (POPC), Palmitoyloleoyl-phosphatidylethanolamine (POPE), Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(maleimidomethyl) sodium salt (DOPE-mal), Dipalmitoyl-phosphatidylethanolamine (DPPE), Dimyristoylphosphoethanolamine (DMPE), Distearoylphosphatidylethanolamine (DSPE), 16-0-Monomethyl PE, 16-0-Dimethyl PE, 18-1-Trans PE, 1-Stearioyl-2-oleoylphosphatidyethanolamine (SOPE), or 1,2-Dielaidoyl-sn-glycero-3-phosphoethanolamine (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 phosphatidylethanola min (PEG-PE), PEG-succinate-diacylglycerol (PEG-S-DAG), in particular 4-O-(2',3'-di(tetradecanoyloxy)-propyl-1-O-(ω-methoxy-(polyethoxy)ethyl)butanedioate (PEG-S-DMG), pegylated ceramide (PEG-cer), or PEG-dialkoxypropylcarbamate, in particular ω-Methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)-carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy-(polyethoxy)ethyl)-carbamate. Lipid nanoparticles after 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. Lipid nanoparticles according to at least one of the Claims 1 until 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, the particle diameter being determined by dynamic light scattering (DLS). Lipid nanoparticles according to at least one of the Claims 1 until 17 , characterized in that they have 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). Lipid nanoparticles after 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 preferably between 3 and 15. Lipid nanoparticles after 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. Process for the preparation of lipid nanoparticles according to one of the Claims 1 , 3 until 15 or 17 until 18 comprising the following measures: i) presentation of aqueous dispersions of 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) 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. Process for the preparation of lipid nanoparticles according to one of the Claims 2 , 16 or 19 until 20 comprising the following measures: iv) introducing the aqueous dispersion containing lipid nanoparticles from step iii) of the process according to claim 21 , v) introducing an aqueous solution or dispersion of an active ingredient with anionic groups into a buffer in the pH range from 3 to 8, preferably 4 to 7.5 vi) combining the aqueous dispersions or solutions from steps iv) and v), and vii) treating the combined aqueous dispersions or solutions from step vi) with ultrasound, microfluidics, dual centrifugation, nanoprecipitation or in a vortex mixer, whereby the lipid nanoparticles loaded with the active ingredient are formed. Process for the preparation of lipid nanoparticles according to one of the Claims 2 , 16 or 19 until 20 comprising the following measures: I) presentation of aqueous dispersions of lipids a), b) and c) after claim 1 in buffers in the pH range from 3 to 8, preferably 4 to 7.5, II) introducing 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 III) combining the aqueous dispersions or solutions from steps I) and II); and IV) treating 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. procedure 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) agitating the mixture from step VII), and IX) optionally subsequent incubation of the resulting mixture. A method for gene transfer into cells, which comprises the following steps: A) contacting cells, tissues or cell cultures with an aqueous dispersion containing lipid nanoparticles containing nucleic acids according to claim 16 , and B) subsequent incubation. procedure according to claim 25 which comprises the following steps: C) providing a cell culture in a bioreactor or incubator, D) adding an aqueous dispersion containing lipid nanoparticles containing nucleic acids after claim 16 , E) distributing the aqueous dispersion in the cell culture, and F) subsequent incubation. Use of nanoparticles according to claim 2 , 16 or 19 until 20 for gene transfer into cells.

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