Lipid nanoparticles and uses thereof

Abstract

The invention provides a lipid nanoparticle and application thereof, and particularly provides a cholesterol-free LNP delivery system based on sterol ionizable lipid. According to the system, a sterol group is integrated into an ionizable lipid skeleton as a hydrophobic tail through molecular design to form a novel sterol ionizable lipid, and the novel sterol ionizable lipid is self-assembled with an auxiliary lipid and a PEG-lipid to form a stable three-component LNP. The problem of high liver enrichment caused by the fact that traditional four-component LNP depends on cholesterol is solved, the affinity of core lipid and apolipoprotein E is remarkably reduced, and an ApoE / LDLR-mediated liver cell uptake way is effectively avoided. Under the condition that cholesterol is completely not needed, the LNP achieves high encapsulation efficiency and excellent stability on mRNA at the same time, liver enrichment is greatly reduced, the targeting performance on extrahepatic tissues such as the spleen is improved, high transfection efficiency and good biocompatibility are achieved, and an innovative platform is provided for delivery of extrahepatic disease RNA drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a novel lipid nanoparticle and its applications. Background Technology

[0002] Nucleic acid drugs, compared to traditional small-molecule chemical drugs and antibody drugs, are theoretically not limited by the druggability of the target protein. With the advancement of clinical trials and the maturation of related technologies, they have a wider range of applications.

[0003] As exogenous drugs, nucleic acid drugs must overcome multiple obstacles to exert their effects in the body: instability, immunogenicity, low cellular uptake efficiency, and difficulty in endosome escape. Efficient and safe delivery systems are crucial for nucleic acid drugs to overcome these shortcomings and achieve stable, targeted efficacy.

[0004] Currently, lipid nanoparticles (LNPs) have become one of the mainstream delivery systems for nucleic acid drugs, and have been successfully applied in clinical practice in siRNA drugs (Patisiran) and mRNA COVID-19 vaccines.

[0005] Traditional lithium-ionizing polymerases (LNPs) typically employ a four-component system comprising ionizable lipids, cofactor lipids, PEGylated lipids, and cholesterol to achieve efficient encapsulation and structural stability of nucleic acid drugs. However, this system suffers from the following problems: While cholesterol is indispensable for maintaining the stability and high encapsulation efficiency of LNP nanostructures, its high affinity for apolipoprotein E (ApoE) in vivo leads to efficient uptake by hepatocytes via the ApoE / LDLR pathway, resulting in a significant and inherent hepatic enrichment characteristic of traditional LNPs after systemic administration. This "hepatic tropism" severely limits the targeted delivery and application efficacy of LNPs in non-hepatic tissues or organs such as the spleen. Summary of the Invention

[0006] This invention aims to overcome the aforementioned deficiencies of existing technologies and provide a cholesterol-free three-component LNP delivery system based on novel sterolized ionizable lipids (iChol-lipid). The technical problem this invention seeks to solve is: how to construct an LNP carrier that simultaneously achieves high nucleic acid encapsulation efficiency, excellent stability, and significantly reduces liver-specific enrichment without the need for cholesterol, thereby overcoming the technical bottleneck of traditional LNP delivery in extrahepatic tissues.

[0007] The first aspect of this invention provides lipid nanoparticles composed of the following components:

[0008] (a) Sterolylated ionizable lipids;

[0009] (b) Auxiliary lipids;

[0010] (c) PEG lipids or polymers; wherein the molar ratio of sterolated ionizable lipids, auxiliary lipids, and PEG lipids or polymers is n. (可电离脂质) :n (辅助脂质) :n (PEG脂质或聚合物) =40–60:30–60:1–5.

[0011] A second aspect of the present invention provides a lipid nanoparticle composition comprising lipid nanoparticles, a loaded drug and / or a pharmaceutically acceptable excipient.

[0012] A third aspect of the present invention provides the use of a lipid nanoparticle composition in the preparation of a medicament for the prevention and / or treatment of diseases.

[0013] A fourth aspect of the present invention provides a sterolized ionizable lipid.

[0014] The three-component LNP with low liver enrichment based on sterolated ionizable lipids provided by this invention has the following beneficial effects:

[0015] 1. Molecular Structure and Formulation Innovation: Through computer-aided molecular design, a novel iChol-lipid was created by integrating a structurally stabilizing sterol group as a hydrophobic tail into an ionizable lipid backbone. This design enables a single molecule to possess both pKa regulation and structural stabilization functions, thus successfully constructing a three-component LNP system, achieving simplification of formulation components and functional integration.

[0016] 2. Significantly reduces liver-specific enrichment: Compared with cholesterol, the core component iChol-lipid has a significantly reduced affinity for ApoE, which can effectively circumvent the ApoE / LDLR-mediated hepatocyte uptake pathway. From the molecular mechanism level, it greatly reduces the specific enrichment of LNP in the liver and improves its distribution and targeting potential in extrahepatic tissues such as the spleen.

[0017] 3. Excellent overall delivery performance: The mRNA-LNP prepared from this innovative lipid not only maintains high encapsulation efficiency and excellent physical stability, but also exhibits high transfection efficiency, good biocompatibility and safety, ensuring the effective delivery and expression of nucleic acid drugs in vivo.

[0018] 4. Broad prospects for clinical translation: This invention successfully resolves the contradiction between the severe liver enrichment of traditional LNPs and their reliance on cholesterol for stable structure, providing a more targeted and safer innovative delivery platform for the development of RNA drugs for the treatment of extrahepatic diseases, and has great clinical translation and application value. Attached Figure Description

[0019] Figure 1Design of the iChol-lipid molecular library and analysis of its interaction with ApoE. (A) Schematic diagram of iChol-lipid structure and synthesis method, (B) Schematic diagram of the molecular library of bromoalkane intermediates containing cholesterol groups and diester bonds (named: cholesterol alkyl tail library), (C) Library of secondary amine intermediates with alkane tail chains, degradable linkages and hydrophilic head groups, (D) Heat map of the binding free energy of iChol-lipid with ApoE based on DiffDock-L molecular docking simulation.

[0020] Figure 2 Physicochemical characterization of Tc-LNPs prepared by example iChol-lipid. (a) Particle size and polydispersity index (PDI), (b) Potential, (c) Encapsulation efficiency.

[0021] Figure 3 Expression results of luciferase mRNA delivered by Tc-LNP constructed based on iChol-lipid in HEK293 cells.

[0022] Figure 4 The expression and analysis of luciferase mRNA delivered by Tc-LNP and control LNP constructed based on iChol-lipid in vivo. (a) In vivo luciferase expression after intramuscular injection of 10 Tc-LNPs and control LNPs, (b) Quantitative analysis of fluorescence intensity, optimized formulation ratio of Tc-LNP (c) and its in vivo luciferase expression (d) and quantitative analysis of fluorescence intensity (e), in vivo biodistribution of 10 Tc-LNPs and control LNP after intravenous injection (f, g) and quantitative analysis of luciferase expression mediated by them in different organs (h, i).

[0023] Figure 5 Exemplary Tc-LNP cytotoxicity at different mRNA concentrations; dashed lines represent 100% cell viability.

[0024] Figure 6 The changes in particle size, PDI (a), and encapsulation efficiency (b) of exemplary Tc-LNP after storage at 4°C for 30 days.

[0025] Figure 7 Example: Changes in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and blood urea nitrogen (BUN) levels in mice after intravenous injection of Tc-LNP.

[0026] Figure 8 H&E staining of major organs in mice after intravenous administration of exemplary Tc-LNP.

[0027] Figure 9Comparison of particle size, PDI, and mRNA encapsulation efficiency of ALC-0315LNP under cholesterol-free and cholesterol-free conditions.

[0028] Figure 10 ALC-0315LNP, loaded with luciferase mRNA under cholesterol-free conditions, showed luciferase expression in major organs of mice after intravenous administration. Detailed Implementation

[0029] To address the shortcomings of existing technologies, this invention provides a cholesterol-free LNP delivery system based on sterolized ionizable lipids. This system integrates sterol groups as hydrophobic tails into the ionizable lipid backbone through molecular design, forming novel sterolized ionizable lipids. These lipids then self-assemble with helper lipids and PEG lipids to form a stable three-component LNP. This LNP achieves high encapsulation efficiency and excellent stability of mRNA without the need for cholesterol, significantly reducing liver accumulation and improving targeting to extrahepatic tissues such as the spleen. It also exhibits high transfection efficiency and good biocompatibility, making it suitable for a wider range of clinical applications.

[0030] To achieve the above objectives, the present invention adopts the following technical solution:

[0031] This invention provides lipid nanoparticles composed of the following components:

[0032] (a) Sterolylated ionizable lipids;

[0033] (b) Auxiliary lipids;

[0034] (c) PEG lipids or polymers;

[0035] The molar ratio of sterolated ionizable lipids, auxiliary lipids, and PEG lipids or polymers is n. (可电离脂质) :n (辅助脂质) :n (PEG脂质或聚合物) =40–60:30–60:1–5.

[0036] In some specific embodiments, the molar ratio of sterolated ionizable lipids, cofactor lipids, and PEG lipids is n. (可电离脂质) :n (辅助脂质) :n (PEG脂质) =50–60:40–50:1–2.

[0037] In some specific embodiments, the sterolated ionizable lipid structural features include:

[0038] (a) The structure contains a sterol or a derivative thereof or an analogue thereof;

[0039] (b) The structure comprises a head structure, two tail structures, and a central functional group that connects the head structure and the tail structure together.

[0040] (c) The central functional group is a carbon atom or a nitrogen atom;

[0041] (d) The head structure is a hydrophilic head, including but not limited to amino, hydroxyl, and carboxyl groups; the tail structure is a hydrophobic tail structure, including but not limited to straight / branched hydrocarbon chains; the hydrocarbon chains include biodegradable groups; sterols or their derivatives or analogues are located at the tail end of an alkane chain and are connected to the hydrocarbon chain by ester bonds formed by hydroxyl or carboxyl groups.

[0042] (e) Biodegradable groups include, but are not limited to, -(C=O)O-, -O(C=O)-, -S(C=O)-, -S(C=O)-, -NH(C=O)O-, -O(C=O)NH-, -NH(C=O)-, -(C=O)NH-, and -O(C=O)O-.

[0043] In some specific embodiments, the sterolated ionizable lipid structure is as follows:

[0044]

[0045] Where n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0046] G1 is a C1-C10 alkylene group; or (CH2). a -O-(CH2) b Where a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10;

[0047] G2 and G3 are each independently C1-C10 alkylene groups;

[0048] L1, L2, and L3 are each independently selected from -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, -O(C=O)O-, Where n is 0, 1, ...;

[0049] R1 is a fragment of a sterol or its derivative or analogue; such as Wherein, R is a C1-C10 alkyl group, preferably a C5-C10 alkyl group;

[0050] R2 is

[0051] R3 and R4 are each independently H, a straight-chain or branched alkane group of C1-C20, or a straight-chain or branched olefin group of C2-C20.

[0052] In some specific embodiments, the sterolized ionizable lipid R1 is a sterol fragment, preferably with the following structure.

[0053] In some specific embodiments, the sterolated ionizable lipid is selected from one or more combinations of the group consisting of, stereoisomers of, tautomers of, or pharmaceutically acceptable salts thereof.

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] The aforementioned "sterol or its derivative or analogue fragment" mainly comprises the skeletal structure of sterol or its derivative or analogue, such as Wherein, R is a C1-C10 alkyl group, preferably a C5-C10 alkyl group;

[0060] Sterols or their derivatives or analogues include, but are not limited to, one or more combinations of cholesterol, sitosterol, ergosterol, cholesterol ketones, cholesterol campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, codostrum, α-tocopherol, or corticosteroids. A preferred sterol is cholesterol or its derivatives; non-limiting examples of cholesterol derivatives include: polar analogues such as 5α-cholesterol, 5α-codostrum, cholesterolyl-(2'-hydroxy)ethyl ether, cholesterolyl-(4'-hydroxy)butyl ether, and 6-ketocholesterol; non-polar analogues such as 5α-cholesterol, cholesterol ketones, 5α-cholesterolone, and decanoic acid cholesterol esters; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analogue such as cholesterolyl-(4'-hydroxy)butyl ether. In some specific implementations, exemplary sterols or their derivatives or analogues include hydrocortisone, prednisone, dexamethasone, betamethasone, triamcinolone, budesonide, testosterone, estrogen, progesterone (such as progesterone) and related derivatives, cholecalciferol (vitamin D3), alfacalcidol, calcitriol, cardiac glycosides (such as digoxin), bile acids (such as ursodeoxycholic acid), and phytosterols (such as β-sitosterol).

[0061] The term "stereoisomer" refers to isomers that have the same atomic connection order but different spatial arrangements of atoms.

[0062] The term "tautomer" refers to the phenomenon where the structure of a compound undergoes an equilibrium interconversion between two functional group isomers, and the corresponding isomers are called tautomers.

[0063] The term "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt. All compounds of the present invention existing as free bases or free acids can be converted into pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids according to methods known to those skilled in the art. Salts of the compounds of the present invention can be converted into their free base or acid forms using standard techniques.

[0064] Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by other methods used in the art, such as ion exchange, and containing an amino group. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, citrate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptate, glyceryl phosphate, gluconate, hemisulfate, heptaate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, and ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Other pharmaceutically acceptable salts include those formed by the quaternization of amines using suitable electrophilic agents (e.g., alkyl halides) to form quaternized alkylated amino salts.

[0065] There is no limitation on the type of "auxiliary lipids", but phospholipid lipids are preferred, including but not limited to: one or more combinations of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and myristoyl phosphatidylglycerol.

[0066] In some specific embodiments, the auxiliary lipid may be selected from: 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC). ), 1,2-Coctodecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 diether PC), dimyristoyl phosphate ethanolamine (DMPE), distearate-phosphatidyl-ethanolamine (DSPE), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), 1- Oleoyl-2-cholestyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-O-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinanoyl-sn-glycerol-3-phosphate choline, 1,2-distearate-sn-glycerol-3-phosphate choline, 1,2-docohexanoyl-sn-glycerol-3-phosphate choline, 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinyl Acyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docohexanoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), diacetyl-phosphatidylethanolamine (DEPE), stearoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, and sphingomyelin are one or more combinations thereof.

[0067] In some specific implementations, phosphatidylcholine is one or a combination of DSPC, DPPC, DMPC, DOPC, and POPC.

[0068] In some specific implementations, the auxiliary lipid is phosphatidylcholine, specifically DSPC.

[0069] In some specific implementations, the auxiliary lipid is phosphatidylcholine, specifically DPPC.

[0070] In some specific embodiments, the auxiliary lipid is phosphatidylcholine, specifically a combination of DSPC and DPPC.

[0071] In some specific embodiments, the auxiliary lipid is phosphatidylethanolamine, specifically DOPE.

[0072] In some specific embodiments, the auxiliary lipid is selected from one or more combinations of DOTAP ((1,2-dioleopropyl)trimethylammonium chloride), DODAP (1,2-dioleoyl-3-dimethylammonium-propane), 18:1PA (1,2-DI(cis-9-octadecenoyl)-SN-glycerol-3-phosphate sodium salt), HS15 (polyethylene glycol (15)-hydroxystearate), and GL67 (N4-arginine cholesterol carbonyl amide).

[0073] The "PEG lipids" mentioned in this invention generally refer to conjugates formed by linking PEG (polyethylene glycol) and lipid molecules through chemical bonds. This includes, but is not limited to, PEG-modified phospholipids and derived lipids, exemplified by one or more combinations of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxy polyethylene glycol bis(tetradecyl)acetamide.

[0074] In some specific embodiments, the PEG lipids include, but are not limited to, PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearate phosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, and polyethylene glycol. Dimethicone (PEG-DMA), PEG distearate glycerol, PEG dipalmitoyl, PEG dioleyl, PEG distearate, PEG diacyl glycamide, PEG dipalmitoyl phosphatidylethanolamine, PEG phosphatidylethanol, PEG phosphatidyl ethylenedimyristyloxypropyl-3-amine, PEG oxypropyl alcoholamine, 1,2-distearate oxypropyl-3-amine-N[methoxy(polyethylene glycol)] (PEG-DSA), methoxypolyethylene glycol lauric acid, methoxypolyethylene glycol bis(tetradecyl acetamide) (ALC0159) or a combination thereof.

[0075] In some specific implementations, the PEG-lipid is PEG-DMG.

[0076] There is no limitation on the type of "polymer," which may include, but is not limited to, amphiphilic block copolymers. Amphiphilic block copolymers are block copolymers composed of hydrophobic polymers and hydrophilic compounds, including but not limited to polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA), glycolide-lactide copolymer (PLCG), polycaprolactone (PCL), polyorthoester, polyanhydride (PAH), polyphosphazene, polyβ-polyurethane (PBAE), poly(α-hydroxy acid), and lactide / glycolic acid copolymer (PLGA or PLG). (These include lactide / glycolic acid copolymers, D-lactide / glycolic acid copolymers, L-lactide / glycolic acid copolymers, and D,L-lactide / glycolic acid copolymers), polyglycolic acid (PGA), polyorthoester (POE), linear or branched polyethylene glycol (PEG), poly(α-hydroxy acid) couplings, polyaspirin, polyphosphazenes, D-lactide, D,L-lactide-caprolactone, D,L-lactide-glycolic acid-caprolactone, dextran, and vinylpyrrole. Alkyl ketones, polyvinyl alcohol (PVA), methacrylates, poly-N-isopropylenamide, SAIB (sucrose acetate isocyanate) hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, carboxymethyl cellulose or its salts, polycarboxyethylene, poly(hydroxyethyl methacrylate), poly(methoxyethyl methacrylate), poly(methoxyethoxy-ethyl methacrylate), polymethyl methacrylate (PMMA), methyl isobutyl acrylate (MMA), PVA-g-PLGA, PEGT-PBT copolymer, PEO-PPO-PEO (pluronics), PEO-PPO-PAA copolymer, PLGA-PEO-PLGA, PEG-PLGA, PLA-PLGA, PEG-PLA, PEG-PCL, poloxamer 407, PEG-PLGA-PEG triblock copolymer, PEG-PLA-PEG triblock copolymer, PEG-PCL-PEG triblock copolymer or their block copolymers with polyethylene glycol (PEG), or one or more combinations of the above polymers or copolymers.

[0077] In some embodiments of the present invention, the weight-average molecular weight of PEG in the PEG lipid is 1000 to 10000, for example 1000 to 2000, 2000 to 4000, 4000 to 6000, 6000 to 8000, 8000 to 10000, preferably 2000.

[0078] A second aspect of the present invention provides a lipid nanoparticle composition comprising lipid nanoparticles, a loaded drug and / or a pharmaceutically acceptable excipient.

[0079] The "drugs" described in this invention include, but are not limited to, any one or a combination of multiple of nucleic acids, small molecules, and proteins.

[0080] The "nucleic acid" described in this invention can be a nucleotide polymer of any length. This includes, but is not limited to, single-stranded DNA, double-stranded DNA, plasmid DNA, short isoforms, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), micro-non-coding RNA (miRNA and siRNA), telomerase RNA, small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense oligonucleotides (ASO), ribozymes, asymmetric interfering RNA (aiRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), guide RNA (gRNA), small guide RNA (sgRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholine antisense oligonucleotides, morpholine-substituted oligonucleotides, or bio-customized oligonucleotides, or one or more combinations thereof.

[0081] In some embodiments of the present invention, the nucleic acid is mRNA. mRNA is a single-stranded ribonucleic acid transcribed from one strand of DNA as a template, carrying genetic information and directing protein synthesis. mRNA can be monocistronic or polycistronic.

[0082] The term "small molecule" as used in this invention refers to a compound that is not a protein or nucleic acid molecule. Small molecules can be small molecules of therapeutic and / or preventative agents, such as antibiotics, anti-inflammatory drugs, anticancer drugs, antiviral drugs, immunosuppressants, analgesics, antifungal drugs, antiparasitic drugs, anticonvulsants, antidepressants, anti-anxiety drugs, antipsychotics, etc.

[0083] The term "protein" as used in this invention refers to a molecule or complex comprising one or more polypeptides having secondary, tertiary, and / or quaternary structures. The secondary, tertiary, and / or quaternary structures of proteins are typically stabilized using non-covalent bonds such as ionic bonds, hydrogen bonds, hydrophobic interactions, and / or van der Waals interactions. Additionally, or alternatively, proteins may include disulfide bonds, for example, between thiol groups of cysteine ​​residues. Exemplary proteins include, but are not limited to, antibodies, antigens or fragments thereof, fusion proteins, recombinant proteins, polypeptides, short peptides, enzymes, etc.

[0084] The pharmaceutical compositions of this invention also comprise pharmaceutically acceptable excipients. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 4-8, preferably about 5-7, although the pH value may vary depending on the nature of the formulated substances and the condition to be treated. The formulated pharmaceutical compositions can be administered via conventional routes, including but not limited to: intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, intracavitary injection, inhalation, implantation, etc.

[0085] The term "pharmaceutical acceptable" as used in this invention means that when the drug is properly administered to animals or humans, it does not produce adverse, allergic, or other adverse reactions.

[0086] "Pharmaceutical-acceptable excipients" should be compatible with the active ingredient, meaning they can be mixed with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can be used as pharmaceutically acceptable excipients include sugars such as glucose, mannitol, sucrose, lactose, trehalose, and maltose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; alcohols such as ethanol, propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; surfactants; lyophilization protectants; colorants; flavoring agents; tableting agents; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; buffer solutions, and combinations thereof. These substances are used as needed to improve the stability of the formulation or to help improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0087] The pharmaceutical compositions of the present invention can be formulated into inhaled nebulized formulations (such as dry powder formulations, aerosol formulations, inhaled droplet formulations, etc.), implantable gel formulations, microneedle formulations, and can also be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions should preferably be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, approximately 10 micrograms / kg body weight to approximately 50 milligrams / kg body weight per day.

[0088] In some embodiments of the present invention, the pharmaceutical composition comprises LNPs, the LNPs having an average particle size of 60–120 nm, which may be 60–70 nm, 70–80 nm, 80–90 nm, 90–100 nm, 100–110 nm, 110–120 nm, or 60–80 nm, 80–100 nm, 100–120 nm.

[0089] In some embodiments of the present invention, the pharmaceutical composition comprises nucleic acid lipid nanoparticles, wherein the nucleic acid is encapsulated in the nanolipid nanoparticles at a rate greater than 80%, and may be 80%–85%, 85%–90%, 90%–95%, 95%–97%, 97%–99%, or more than 99%.

[0090] This invention provides the use of lipid nanoparticle compositions in the preparation of medicaments for the prevention and / or treatment of diseases.

[0091] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0092] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, this invention can be implemented using any prior art methods, apparatus, and materials similar to or equivalent to those described in the embodiments of this invention, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention.

[0093] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. All materials and equipment used in this invention are commercially available products.

[0094] Example 1: Construction of a lipid library and its interaction with ApoE

[0095] A bromoalkane intermediate containing a cholesterol structural fragment and a diester bond (cholesterol alkyl tail) and a secondary amine intermediate with an alkane tail chain, degradable linkage, and a hydrophilic head group were constructed. Sterol-derived ionizable lipids (iChol-lipid) were then synthesized via nucleophilic substitution reactions. (See appendix) Figure 1 A). The cholesterol alkyl tail intermediate library is composed of carbon chain-conjugated cholesterol (see appendix). Figure 1 B, Chol6–Chol18); 15 structurally diverse secondary amine intermediates (see appendix) Figure 1 C), encompassing head groups of varying polarity (e.g., ethanol, butanol, diethylene glycol), alkane tail chains (straight-chain C6–C9, branched-chain C10–C18), and various degradable linkages (e.g., ester bonds, carbamates, acetals, etc.). An iChol-lipid molecular library was constructed using combinatorial chemistry strategies. The interaction between iChol-lipid and ApoE was then demonstrated using a DiffDock-L molecular docking model.

[0096] The ApoE protein sequence (P08226) was obtained from the UniProt database, and its three-dimensional structure was predicted using AlphaFold. Lipid-protein interaction simulations were performed using the DiffDock-L service on the Neurosnap platform. Each lipid underwent 100 independent simulations, and the docking conformation with the highest score was selected after analysis. Molecular docking is shown in Figure D. DiffDock-L molecular docking simulations showed that over 80% of iChol-lipids had significantly higher binding free energies to ApoE than cholesterol (the predicted binding free energy of cholesterol to ApoE using DiffDock-L was -2.02 kJ / mol), indicating a generally weakened ApoE binding affinity. Figure 1 D).

[0097] Example 2: Sterolylation of Ionizable Lipids

[0098]

[0099] Synthesis of Compound 1: N,N-dicyclohexylcarbodiimide (DCC, 3.20 g, 15.52 mmol), 4-dimethylaminopyridine (DMAP, 1.58 g, 12.93 mmol), and succinic acid (3.05 g, 25.86 mmol) were dissolved in dichloromethane (DCM, 50 mL). The solution was stirred at 0 °C for 30 min under nitrogen protection. Cholesterol (5.00 g, 12.93 mmol) was then added. The reaction mixture was heated to 25 °C and stirred for 12 h. Thin-layer chromatography (TLC) analysis indicated that the reaction was complete. The reaction mixture was filtered, the filtrate was collected, washed with saturated NaHCO3 solution (50 mL × 3), and concentrated to obtain the crude product. The crude product was purified by column chromatography (silica gel, DCM) to obtain the pure product (3.18 g, yield 50.5%).

[0100] Synthesis of Chol6: DCC (1.02 g, 4.93 mmol), DMAP (0.15 g, 1.23 mmol), and Compound 1 (2.00 g, 4.11 mmol) were dissolved in dichloromethane (DCM, 20 mL). The solution was stirred at 0 °C for 30 min under nitrogen protection. Then, 4-bromobutanol (0.62 g, 4.11 mmol) was added. The reaction mixture was heated to 25 °C and stirred for 12 h. TLC analysis indicated that the reaction was complete. The reaction mixture was filtered, and the filtrate was collected and concentrated to obtain the crude product. The crude product was purified by column chromatography (silica gel, 2.0% EA / heptane) to give the pure product (1.86 g, yield 72.8%).

[0101] Chol10, Chol12, Chol13, Chol14, Chol15, and Chol18 were synthesized using the methods described above.

[0102]

[0103] Synthesis of Compound 5: DCC (4.53 g, 21.96 mmol), DMAP (0.73 g, 5.99 mmol), and 2-hexyldecanoic acid (5.12 g, 19.97 mmol) were dissolved in dichloromethane (DCM, 60 mL). The solution was stirred at 0 °C for 30 min under nitrogen protection. Then, 6-bromo-1-hexanol (3.62 g, 19.97 mmol) was slowly added dropwise. The reaction mixture was heated to 25 °C and stirred for 12 h. Thin-layer chromatography (TLC) analysis indicated that the reaction was complete. The reaction mixture was filtered, and the filtrate was collected and concentrated to obtain the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate / n-heptane = 1 / 50) to give a pure product as a colorless oil (7.41 g, yield 88.5%).

[0104] Synthesis of compound C4A1: Compound 5 (2.48 g, 5.91 mmol), 4-amino-1-butanol (2.63 g, 29.56 mmol), and ethanol (5 mL) were added to a round-bottom flask. The solution was stirred at 80 °C for 12 hours under nitrogen protection. TLC analysis indicated that the reaction was complete. The reaction mixture was diluted with 100 mL of ethyl acetate (EA) and then washed successively with water (100 mL), saturated NaHCO3 solution (100 mL), and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and filtered. The filtrate was collected and concentrated to give the crude product. The crude product was purified by column chromatography (silica gel, methanol / dichloromethane = 1 / 10) to give a pure product as a colorless oil (2.03 g, yield 80.3%).

[0105] C2A1, C2A2, C2A3, C2A4, C4A2, C4A3, C4A4, C4OA1, C4OA2, C4OA3, and C4OA4 were synthesized using the methods described above.

[0106]

[0107] Synthesis of Compound 9: 9-Heptadecanol (4.00 g, 15.60 mmol), N,N'-disuccinimidyl carbonate (1.83 g, 15.60 mmol), and DMAP (2.29 g, 18.72 mmol) were dissolved in N,N-dimethylformamide (DMF, 60 mL). The solution was stirred at 70 °C for 12 h under nitrogen protection. The reaction mixture was cooled to 25 °C, and then 6-aminohexane-1-ol (1.83 g, 15.60 mmol) was added. The mixture was stirred at 70 °C for another 12 h. Thin-layer chromatography (TLC) analysis indicated that the reaction was complete. The mixture was concentrated, diluted with 100 mL of ethyl acetate (EA), and then washed successively with water (100 mL), saturated NaHCO3 solution (100 mL), and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and filtered. The filtrate was collected and concentrated to give the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate / n-heptane = 1 / 20) to obtain a pure product as a colorless oil (2.03 g, yield 80.3%).

[0108] Synthesis of Compound 10: Compound 9 (5.00 g, 12.51 mmol), triethylamine (3.92 g, 38.78 mmol), and DMAP (0.15 g, 1.25 mmol) were dissolved in dichloromethane (DCM, 100 mL). The solution was stirred at 0 °C for 10 min under nitrogen protection. Then, methanesulfonic anhydride (3.27 g, 18.77 mmol, dissolved in DCM to prepare a 0.2 g / mL solution) was slowly added dropwise. The reaction mixture was heated to 25 °C and stirred for 2 h. TLC analysis indicated that the reaction was complete. The reaction was quenched by adding 3.25 mL of water, and the mixture was washed successively with water (100 mL), 10.0% citric acid (w / w) solution (100 mL), and saturated NaHCO3 solution (100 mL). The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and filtered. The filtrate was collected and concentrated to give the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate / n-heptane = 1 / 50) to obtain a pure product (5.56 g, yield 93.0%).

[0109] Synthesis of C4AC: Compound 10 (5.00 g, 10.47 mmol), 4-amino-1-butanol (4.66 g, 52.33 mmol), and acetonitrile (MeCN, 25 mL) were added to a round-bottom flask. The solution was stirred at 70 °C for 12 h under nitrogen protection. TLC analysis indicated that the reaction was complete. The reaction mixture was concentrated and diluted with 100 mL of ethyl acetate (EA), followed by washing successively with water (100 mL), saturated NaHCO3 solution (100 mL), and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and filtered. The filtrate was collected and concentrated to give the crude product. The crude product was purified by column chromatography (silica gel, methanol / dichloromethane = 1 / 10) to give the pure product (4.16 g, yield 84.4%).

[0110]

[0111] Synthesis of Compound 11: Compound 10 (3.00 g, 6.28 mmol) was dissolved in tetrahydrofuran (THF, 30 mL). The solution was stirred at 0 °C for 10 min under nitrogen protection. Sodium hydride (0.50 g, 12.56 mmol, 60.0%, w / w) and methyl iodide (1.34 g, 9.42 mmol) were then added sequentially. The reaction mixture was heated to 25 °C and stirred for 48 h. Thin-layer chromatography (TLC) analysis indicated that the reaction was complete. The mixture was concentrated, diluted with 50 mL of ethyl acetate (EA), and then washed sequentially with water (50 mL), saturated NaHCO3 solution (50 mL), and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and filtered. The filtrate was collected and concentrated to give the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate / n-heptane = 1 / 50) to obtain a pure product as a colorless oil (2.41 g, yield 78.0%).

[0112] C4ACM was synthesized following the same steps as C4AC.

[0113]

[0114] Synthesis of C4AAc: Compound 12 (2.00 g, 3.94 mmol, synthesized according to the method described in patent and international publication number WO 2022 / 140252A1), 4-amino-1-butanol (0.70 g, 7.88 mmol), and acetonitrile (MeCN, 10 mL) were added to a round-bottom flask. The solution was stirred at 50 °C for 12 hours under nitrogen protection. Thin-layer chromatography (TLC) analysis showed that the reaction was complete. The reaction mixture was diluted with 50 mL of ethyl acetate (EA), and then washed successively with water (100 mL), saturated NaHCO3 solution (100 mL), and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and filtered. The filtrate was collected and concentrated to give the crude product. The crude product was purified by column chromatography (silica gel, silica gel, methanol / dichloromethane = 1 / 10) to give a pure product as a colorless oil (1.04 g, yield 51.2%).

[0115]

[0116] Synthesis of iChol15-C4A2: Chol15 (200.0 mg, 0.27 mmol), C4A1 (114.4 mg, 0.27 mmol), K2CO3 (55.5 mg, 0.41 mmol), and KI (4.4 mg, 0.027 mmol) were added to a thick-walled pressure reaction tube (10 mL), followed by 2 mL of a tetrahydrofuran / acetonitrile (THF / MeCN = 1 / 1, v / v) mixed solvent. The reaction tube was tightly sealed under nitrogen protection and stirred at 80 °C for 24 hours. After the reaction was complete, the mixture was diluted with n-heptane (20 mL) and filtered through a 0.22 μm filter membrane. The filtrate was collected and concentrated to obtain the crude product. The crude product was purified by column chromatography (silica gel, 0-8.0% ethanol / dichloromethane gradient elution) to obtain the pure product (203.1 mg, yield 69.4%).

[0117] Other target lipids were synthesized using a similar method.

[0118] The proton NMR spectral data of some compounds are shown in the table below:

[0119]

[0120]

[0121] Example 3: Preparation of three-component LNP (Tc-LNP)

[0122] Tc-LNP was prepared using the following experimental method.

[0123] Step 1: Dissolve iChol-lipid, DSPC, and PEG-lipid in ethanol according to the designed formulation ratio (iChol-lipid / DSPC / DMG-PEG molar ratio of 50 / 50 / 1.5) to prepare a lipid ethanol solution (Lipid concentration 10 mg / mL). Dissolve the LNP prepared from the commercially available comparative sample ALC0315 (ALC0315 / DSPC / Cholesterol / DMG-PEG molar percentage of 46.3 / 9.4 / 42.7 / 1.6) in ethanol, and dissolve the LNP prepared from the commercially available comparative sample MC3 (MC3 / DSPC / Cholesterol / DMG-PEG molar percentage of 50 / 10 / 38.5 / 1.5) in ethanol to obtain a lipid ethanol solution (Lipid concentration 10 mg / mL).

[0124] Step 2: Prepare mRNA with a lipid nanoparticle (LNP) to mRNA mass ratio of 10:1 to 30:1, and dilute the mRNA to 0.2 mg / mL using citrate or sodium acetate buffer (pH=3 or 5).

[0125] Step 3: Thoroughly mix the lipid ethanol solution obtained in Step 1 with the mRNA solution at a volume ratio of 1:5 to 1:1. The obtained nanoparticles are purified by ultrafiltration and dialysis. After sterilization by filtration, the particle size and PDI of the mRNA-LNPs (lipid nanoparticles encapsulating mRNA) are characterized using a Malvern Zetasizer Nano ZS analyzer. The encapsulation efficiency of the mRNA is then determined using a Ribogreen RNA quantification kit (Thermo Fisher). Representative Tc-LNPs are shown in the attached figure. Figure 2 As shown, the lipid nanoparticles of the present invention can form stable nanostructures with narrow size distribution (PDI<0.3). The size varies with the structure of different lipid nanoparticles. In the range of 80-100 nm, the zeta potential is -1 mV to -3 mV, and the encapsulation efficiency is greater than 80%.

[0126] Example 4: Transfection efficiency verification experiment

[0127] HEK293 cells were planted at a density of 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 mcg / well in 96-well plates and cultured for 24 hours. Tc-LNP cells loaded with luciferase mRNA were added to each well at a dose of 25 ng of luciferase mRNA. Bio-Lumi was used to... TM II. Firefly luciferase assay kit to assess luciferase expression. Experimental results (see attached) Figure 3As shown in the figure, most lipid molecules exhibited good biological activity (luciferase expression). Ten lipids with the best biological activity at the cellular level were selected for further experiments.

[0128] Ten selected lipid-based Tc-LNPs were used in animal experiments along with ALC0315 LNP and MC3 LNP. BALB / c mice were intramuscularly injected with LNPs (5 μg / mouse, n=3), and luciferase expression levels were quantitatively detected. Six hours post-injection, anesthetized mice were intraperitoneally injected with D-luciferin (200 μL, 15 mg / mL), and bioluminescent signals were detected using an IVIS Spectrum imaging system. The results showed that the transfection efficiency of the seven iChol-lipid-based Tc-LNPs at the injection site was comparable to or higher than that of commercial vectors (including ALC-0315 and MC3 LNPs). In particular, the iChol15-C4A2 and iChol15-C4ACM-based Tc-LNPs (iChol15-C4A2 Tc-LNPs and iChol15-C4ACM Tc-LNPs) exhibited the best mRNA delivery performance in vivo, as shown in the attached figure. Figure 4 As shown in a and 4b. Furthermore, compared to ALC-0315LNP, which exhibits strong hepatic luciferase activity, Tc-LNPs showed significantly reduced hepatic luciferase expression (see appendix). Figure 4 a).

[0129] The Tc-LNP formulation was optimized using the following composition ranges: iChol-lipid (40-60 mol), DSPC (30-60 mol), and DMG-PEG2000 (1-5 mol), as shown in the attached formula. Figure 4 As shown in Figure c. Tc-LNP was prepared using the ethanol dilution method and administered intramuscularly to Balb / c mice (1 μg / mouse, n=3). Six hours post-injection, luciferase expression at the injection site was assessed using an IVIS system. The results showed that formulation 8 (iChol-lipid / DSPC / DMG-PEG2000 molar percentages of 53.8 / 44.8 / 1.4) exhibited the best transfection efficiency (see attached figure). Figure 4 (as shown in d and 4e) is the preferred formulation.

[0130] In the intravenous mRNA delivery study, Tc-LNPs were prepared at a molar ratio of 60 / 50 / 1.5 (iChol-lipid / DSPC / DMG-PEG2000). Tc-LNPs, MC3, and ALC-0315 LNPs encapsulating luciferase mRNA were prepared according to the established protocol. Mice were administered mRNA intravenously at a dose of 0.1 mg / kg, and bioluminescent signals in major organs were quantitatively analyzed using an IVIS imaging system 6 hours post-injection. The results showed that compared to LNPs prepared from MC3 and ALC-0315, Tc-LNPs exhibited significantly lower mRNA expression levels in the liver and were more abundant in the spleen (see attached image). Figure 4 f-4i). iChol15-C4A2Tc-LNPs and iChol15-C4ACM Tc-LNPs showed the highest transfection efficiency of spleen mRNA mediated in vivo (see appendix). Figure 4 The luminescence intensity ratios of iChol15-C4A2 and iChol15-C4ACM Tc-LNPs in the spleen and liver were 4.78 and 5.20, respectively, which were 20 to 50 times higher than those of LNPs prepared by MC3 (0.24) and ALC-0315 (0.12). (See attached image) Figure 4 h).

[0131] Example 5: Isolation and Proteomics Analysis of LNP Protein Crown

[0132] Whole blood from mice was collected into EDTA anticoagulant tubes and centrifuged (1000g, 4℃) to obtain plasma. The plasma was centrifuged twice (15300g, 4℃) to remove protein aggregates. LNP was mixed with plasma at a 1:1 volume ratio and incubated at 37℃ for 1 hour. After incubation, the mixture was centrifuged (15300g, 4℃), and the precipitate was washed three times with pre-chilled PBS (pH 7.4) to remove unadsorbed proteins. The precipitate was resuspended in an appropriate amount of 2% SDS solution, and protein concentration was determined by the BCA method. The sample was heat-denatured (100℃, 15 min) and then stored at -20℃ for later use.

[0133] The isolated protein corona samples were subjected to SDS-PAGE electrophoresis at 90V until the protein bands migrated to approximately 1 cm within the gel. After Coomassie brilliant blue staining, the target protein bands were aseptically excised and stored at 4°C for subsequent LC-MS analysis. Quantitative analysis of proteins adsorbed on the surfaces of the three LNPs showed that Tc-LNPs exhibited significantly reduced apolipoprotein adsorption (4.7% for Tc-LNPs and 12.1% for ALC-0315LNPs). Furthermore, Tc-LNPs showed extremely low ApoE adsorption, with only 0.4% of the protein corona composed of ApoE. This proportion was reduced by 90% compared to ALC-0315LNPs (6.2%). The significantly reduced ApoE adsorption by Tc-LNPs directly led to decreased hepatocyte uptake and transfection efficiency.

[0134] Example 6: Cell viability experiment:

[0135] Cell viability was detected using the CCK-8 assay kit: HEK293 cells (1×10⁻⁶) were cultured using the assay kit. 4 Cells / mL, 100 μL / well were seeded in 96-well plates and cultured for 24 hours. Afterward, the medium was replaced with Tc-LNP medium containing the specified concentration of mRNA and cultured for another 24 hours. Then, the medium was replaced with fresh medium containing 10 μL of CCK-8 working solution and incubated at 37°C for 2 hours. The absorbance at 450 nm was measured using a microplate reader, with untreated cells serving as a control. The experimental results are shown in (see attached). Figure 5 Tc-LNP prepared from the exemplary lipids iChol15-C4A2 and iChol15-C4ACM showed no significant cytotoxicity in the mRNA concentration range of 0.25–4 μg / mL.

[0136] Example 7: LNP Stability Study

[0137] Tc-LNPs loaded with luciferase mRNA were prepared using iChol-lipid / DSPC / DMG-PEG2000 (molar ratio 60 / 50 / 1.5). Particle size, PDI, and encapsulation efficiency were measured at 0, 1, 3, 7, 14, and 30 days after preparation, under storage conditions at 4°C. Results showed (see attached diagram). Figure 6 (a) and (6b) the exemplary lipid iChol15-C4A2 maintained stable particle size, PDI and encapsulation efficiency over 30 days.

[0138] Example 8: In vivo safety experiment

[0139] iChol15-C4A2 and iChol15-C4ACM Tc-LNP were prepared at a molar ratio of 60 / 50 / 1.5. Balb / c mice were administered Tc-LNP (0.5 mg mRNA / kg) intravenously, with PBS (pH 7.4) as a negative control. Whole blood and major organs were collected 24 hours after injection, and liver function (ALT, AST) and kidney function (BUN, CREA) were assessed using a biochemical analyzer. After tissue sectioning and H&E staining, images of the sections were acquired using a fluorescence microscope. Experimental results are shown in (see attached). Figure 7 and 8 Compared with the PBS control group, Tc-LNPs did not cause significant changes in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), or creatinine (CRE) levels. No pathological abnormalities were observed in major organs, including the heart, liver, spleen, lungs, and kidneys. These results demonstrate the good stability and biocompatibility of Tc-LNPs, highlighting their potential for clinical applications.

[0140] Example 9: Validation Experiment of Cholesterol Removal by ALC-0315LNP

[0141] After removing cholesterol from ALC-0315LNP, the LNP exhibited significant physicochemical instability. Specifically, the LNP particle size increased by 216% (from 76.6 nm to 241.3 nm), the polydispersity index (PDI) increased by 87% (from 0.135 to 0.252), and the encapsulation efficiency decreased by 30% (from 89.7% to 60.1%). (See Appendix) Figure 9 At the functional level, detection of luciferase mRNA expression revealed that the in vitro transfection efficiency of mRNA decreased by approximately one order of magnitude (see appendix). Figure 10 The above data indicate that cholesterol plays a crucial role in maintaining the structural integrity of LNPs, ensuring efficient mRNA encapsulation, and improving delivery efficiency. While the three-component LNP formulation prepared in this invention does not contain cholesterol, it still maintains high stability and transfection efficiency, demonstrating excellent performance.

[0142] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A lipid nanoparticle, comprising the following components: (a) Sterolylated ionizable lipids; (b) Auxiliary lipids; (c) PEG lipids or polymers; The molar ratio of sterolated ionizable lipids, auxiliary lipids, and PEG lipids or polymers is n. (可电离脂质) :n (辅助脂质) :n (PEG脂质或聚合物) =40–60:30–60:1–5.

2. The lipid nanoparticles according to claim 1, characterized in that, The molar ratio of the sterolized ionizable lipid, the auxiliary lipid, and the PEG lipid is n. (可电离脂质) :n (辅助脂质) :n (PEG脂质) =50–60:40–50:1–2.

3. The lipid nanoparticles according to claim 1, characterized in that, The auxiliary lipid is selected from one or more combinations of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, myristoyl phosphatidylglycerol, DOTAP, DODAP, 18:1PA, HS15, and GL67.

4. The lipid nanoparticles according to claim 2, characterized in that, The PEG lipid is selected from one or more combinations of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxy polyethylene glycol bis(tetradecyl)acetamide.

5. The sterolized ionizable lipid structure according to claim 1 is as follows: in, n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; G1 is a C1-C10 alkylene group; or (CH2). a -O-(CH2) b Where a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10; G2 and G3 are each independently C1-C10 alkylene groups; L1, L2, and L3 are each independently selected from -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, -O(C=O)O-, Where n is 0, 1, ...; R1 is a fragment of a sterol or its derivative or analogue; such as Wherein, R is a C1-C10 alkyl group, preferably a C5-C10 alkyl group; R2 is R3 and R4 are each independently H, a straight-chain or branched alkane group of C1-C20, or a straight-chain or branched olefin group of C2-C20.

6. The sterolized ionizable lipid according to claim 5, characterized in that... R1 is a sterol fragment, preferably with the following structure.

7. The sterolized ionizable lipid according to claim 6, characterized in that, The lipids are selected from one or more combinations of the following groups, their stereoisomers, their tautomers, or their pharmaceutically acceptable salts.

8. A composition comprising any of the lipid nanoparticles according to claims 1-7, characterized in that, The lipid nanoparticle composition further comprises a loaded drug and / or pharmaceutically acceptable excipients; preferably, the loaded drug comprises one or more of nucleic acid molecules, small molecule compounds, and proteins.

9. Use of the composition of claim 8 in the preparation of medicaments for the prevention and / or treatment of diseases.

10. A sterolated ionizable lipid, with the following structure: in, n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; G1 is a C1-C10 alkylene group; or (CH2). a -O-(CH2) b Where a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10; G2 and G3 are each independently C1-C10 alkylene groups; L1, L2, and L3 are each independently selected from -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, -O(C=O)O-, Where n is 0, 1, ...; R1 is a fragment of a sterol or its derivative or analogue; such as Wherein, R is a C1-C10 alkyl group, preferably a C5-C10 alkyl group; preferably, R1 is... R2 is R3 and R4 are each independently H, a straight-chain or branched alkane group of C1-C20, or a straight-chain or branched olefin group of C2-C20; Preferably, the lipid is selected from one or more combinations of the following groups, their stereoisomers, their tautomers, or their pharmaceutically acceptable salts:

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