Lipid nanoparticle compositions for topical treatment of skin diseases

Abstract

The present invention provides a lipid nanoparticle (LNP) composition for topical treatment of skin diseases and a method of using the same for treating skin diseases.

Description

Technical Field

[0001] This invention relates to the field of drug delivery technology, and more specifically to a lipid nanoparticle (LNP) composition for the topical treatment of skin diseases and its use in the treatment of skin diseases. Background Technology

[0002] Lipid nanoparticles (LNPs) are typically composed of four types of lipid components, including cationic or ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids (PEG lipids). Due to their ability to effectively encapsulate nucleic acid drugs and prevent their degradation in vivo, LNPs have become an important drug delivery system.

[0003] In recent years, LNPs have made significant progress in the field of nucleic acid drug delivery, such as in vaccines that deliver messenger RNA (mRNA), which are typically administered via intramuscular injection. However, after intramuscular injection, LNPs are not confined to the injection site but are widely distributed throughout the body and significantly accumulate in the liver, potentially causing off-target toxicity. Furthermore, existing literature reports that LNPs can be used for systemic or local delivery of therapeutic drugs to cancer or solid tumor tissues (e.g., WO2009127060A1, WO2011000107A1, etc.).

[0004] Although LNPs have shown good potential in drug delivery to various tissues and organs, their application in the treatment of skin diseases, especially in the local delivery of nucleic acid drugs, remains relatively limited.

[0005] The skin, the largest metabolically active organ in the human body, is susceptible to a variety of pathological changes, including inflammatory, neoplastic, traumatic, hormonal, degenerative, and genetically related changes. Bacterial, fungal, or viral infectious skin diseases are common in clinical practice and can cause a variety of dermatological problems. Furthermore, chronic inflammatory skin diseases, such as atopic dermatitis, allergic contact dermatitis, and psoriasis, are often associated with the infiltration of inflammatory T cells. However, many skin diseases, such as psoriasis, atopic dermatitis, and skin cancer, face unique challenges in drug delivery due to the presence of the skin barrier structure. The outermost layer of the skin, the stratum corneum, acts as a protective barrier, significantly limiting the penetration of drug molecules into deeper skin tissues.

[0006] Conventional treatments for skin diseases often lack specificity, limiting patient compliance, safety, and efficacy, and in some cases, may even cause significant systemic toxicity. Specifically, traditional topical preparations, due to their low skin permeability, typically require frequent administration, leading to poor patient compliance during long-term treatment and thus limiting their overall therapeutic effect. Compared to systemic administration methods such as oral or injectable medications, topical drug delivery systems, through targeted local delivery, can improve the effectiveness of treating skin diseases and reduce the occurrence of adverse reactions to some extent.

[0007] Therefore, there is an urgent need for a drug delivery system for treating skin diseases that can deliver active drugs precisely or preferentially into the skin with high or improved delivery efficiency and target specific skin layers, while avoiding cytotoxicity or changes in skin tissue morphology.

[0008] Invention Summary This invention provides a lipid nanoparticle (LNP) composition for the topical treatment of skin diseases. Through careful selection of the lipid components, this composition can form stable nanoparticles and efficiently encapsulate active pharmaceutical ingredients, thereby achieving precise or preferential release of the active pharmaceutical ingredient at the targeted skin site with high or improved delivery efficiency, while minimizing or eliminating systemic release.

[0009] A first aspect of the present invention provides a lipid nanoparticle composition comprising: a nucleic acid; and lipid nanoparticles encapsulating the nucleic acid, wherein the lipid nanoparticles comprise cationic lipids or ionizable cationic lipids, phospholipids, structural lipids, and PEG lipids, wherein: (a) The cationic lipids or ionizable cationic lipids account for about 10 mol% to about 49 mol% of the total lipids; (b) The molar ratio of the structural lipid to the phospholipid is about 2 to about 4; (c) The molar ratio of the PEG lipid to the phospholipid is about 0.05 to about 0.4.

[0010] A second aspect of the present invention provides a method for locally delivering nucleic acids to a subject in need, the method comprising: administering a lipid nanoparticle composition to the subject via topical, transdermal, subcutaneous, intradermal, or intradermal administration, the composition comprising nucleic acids and lipid nanoparticles encapsulating the nucleic acids; the lipid nanoparticles comprising cationic lipids or ionizable cationic lipids, phospholipids, structural lipids, and PEG lipids, wherein: (a) The cationic lipids or ionizable cationic lipids account for approximately 10 mol% to approximately 49 mol% of the total lipids; (b) The molar ratio of the structural lipid to the phospholipid is about 2 to about 4; (c) The molar ratio of the PEG lipid to the phospholipid is about 0.05 to about 0.4.

[0011] A third aspect of the present invention provides a method for treating a skin disease in a subject, comprising: delivering a lipid nanoparticle composition to the subject via topical, transdermal, subcutaneous, intradermal, or intradermal administration, the composition comprising nucleic acid and lipid nanoparticles encapsulating the nucleic acid; the lipid nanoparticles comprising cationic lipids or ionizable cationic lipids, phospholipids, structural lipids, and PEG lipids, wherein: (a) The cationic lipids or ionizable cationic lipids account for about 10 mol% to about 49 mol% of the total lipids; (b) The molar ratio of the structural lipid to the phospholipid is about 2 to about 4; (c) The molar ratio of the PEG lipid to the phospholipid is about 0.05 to about 0.4.

[0012] A fourth aspect of the present invention provides an use for: using the composition to prepare a medicament, the medicament being used to carry out the method of the second or third aspect. The composition comprises nucleic acids and lipid nanoparticles encapsulating the nucleic acids; the lipid nanoparticles comprise cationic lipids or ionizable cationic lipids, phospholipids, structural lipids, and PEG lipids, wherein: (a) The cationic lipids or ionizable cationic lipids account for about 10 mol% to about 49 mol% of the total lipids; (b) The molar ratio of the structural lipid to the phospholipid is about 2 to about 4; (c) The molar ratio of the PEG lipid to the phospholipid is about 0.05 to about 0.4.

[0013] A fifth aspect of the present invention provides a lipid nanoparticle composition for implementing the methods of the second or third aspect, the composition comprising nucleic acids and lipid nanoparticles encapsulating the nucleic acids; the lipid nanoparticles comprising cationic lipids or ionizable cationic lipids, phospholipids, structural lipids, and PEG lipids, wherein: (a) The cationic lipids or ionizable cationic lipids account for about 10 mol% to about 49 mol% of the total lipids; (b) The molar ratio of the structural lipid to the phospholipid is about 2 to about 4; (c) The molar ratio of the PEG lipid to the phospholipid is about 0.05 to about 0.4.

[0014] In some embodiments of the above five aspects, the cationic lipid or ionizable cationic lipid is an ionizable cationic lipid of formula (I) or a salt thereof. (I) wherein m is an integer from 1 to 6 X is selected from the group consisting of: OR , 3 , 5 ,

[0018] , 1 , 5 ,

[0017] , 10 , 4 ,

[0016] , 6 , 2 ,

[0015] , 5 ,

[0019] , , 18 , 18 , 5 , 5 , , , 4 , 5 , , 2 , 3 , , 10 , 1 , , 10 , , 18 , 5 , 、SR 5 、NR 5 R 6 or COOR 5 ; U and V are each independently an optionally substituted C1-C8 alkylene, C2-C8 alkenylene or C2-C8 alkynylene; T is selected from the group consisting of: C(O)O, OC(O), O, S, N(R 5 ), C(O), C(O)N(R 5 ), N(R 5 ), C(O)N(R 5 ), N(R 5 ), C(O)O, C(O)S, C(S)O, S(O), S(O)(O) or C(S); wherein R 5 and R 6 are each independently hydrogen or an optionally substituted C1-C 10 alkyl, C2-C 10 alkenyl or C2-C 10 alkynyl; and R 1 、R 2 、R 3 and R 4 are each independently hydrogen or an optionally substituted C6-C 18 alkyl, C6-C 18 alkenyl or C6-C 18 alkynyl, provided that at most one of R 1 、R 2 、R 3 and R 4 is hydrogen.

[0015] In some embodiments, m is 2 or 4.

[0016] In some embodiments, X is OH or SH. In some embodiments, X is OH.

[0017] In some embodiments, U and V are each independently a C1-C8 alkylene. In some embodiments, U and V are each independently a C5-C8 alkylene.

[0018] In some embodiments, T is C(O)O or OC(O).

[0019] In some embodiments, R1 R 2 R 3 and R 4 Each independently represents an optional substituted C6-C 10 Alkyl, C6-C 10 alkenyl or C6-C 10 Alkyne group. In some embodiments, R 1 R 2 R 3 and R 4 Each is independently an optionally substituted C6-C8 alkyl, C6-C8 alkenyl, or C6-C8 alkynyl group. In some embodiments, R 1 R 2 R 3 and R 4 Each is independently a C6-C8 alkyl group.

[0020] In some implementation schemes, R 1 For optional replaced C 10 -C 18 Alkyl, C 10 -C 18 alkenyl or C 10 -C 18 Alkyne group, and R 2 It is hydrogen; R 3 and R 4 Each is independently an optionally substituted C6-C8 alkyl, C6-C8 alkenyl, or C6-C8 alkynyl group. In some embodiments, R 1 C 10 -C 18 Alkyl, C 10 -C 18 alkenyl or C 10 -C 18 alkynyl group, R 2 For hydrogen, R 3 and R 4 Each is independently C6-C8 alkyl, C6-C8 alkenyl, or C6-C8 alkynyl. In some embodiments, R 1 C 10 -C 18 Alkyl, R 2 For hydrogen, R 3 and R 4 Each is independently a C6-C8 alkyl group.

[0021] In some embodiments of the above five aspects, the cationic lipid or ionizable cationic lipid is selected from one or a combination of the following compounds: ((4-hydroxybutyl)azonidyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); heptadecano-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3); 3-(eicosylamino)-N1,N1,4-tridodecyl-1-piperazine ethylamine (KL10); N1-[2-(eicosylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazine ethylamine Diethylamine (KL22); 14,25-tetraazaoctadecane (KL25); 1,2-dioleoyl-N,N-dimethylaminopropane (DLin-DMA); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleoyloxypropane (DODMA); 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); 3-(N-(N′,N′-dimethylaminoethyl)carbamoyl)cholesterol (DC-Chol); dimethyloctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacyloxy-3-dimethyl... Ammonium propane compounds; 1,2-dialkoxy-3-dimethylammonium propane compounds; octadecyl dimethylammonium chloride (DODAC); 1,2-distearate-N,N-dimethyl-3-aminopropane (DSDMA); 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylammonium (DMRIE); 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMEPC); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); 1,2-dioleoxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); 2,3-dioleoxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanetrifluoroacetate (DOSPA); 1,2-Dilinoleoxy-N,N-dimethylaminopropane (DLinDMA); 1,2-Dilinoleoxy-N,N-dimethylaminopropane (DLenDMA); Octadecylamide glycyl spermine (DOGS); 3-Dimethylamino-2-(cholest-5-en-3β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA); 2-[5′-(cholest-5-en-3β-oxy)-3′-oxopentoxy]-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA); N,N-Dimethyl-3,4-dioleoxybenzylamine (DMOBA);1,2-N,N′-Dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP); 2,3-Dilinoleoyloxy-N,N-dimethylaminopropylamine (DLinDAP); 1,2-N,N′-Dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP); 1,2-Dilinoleoylcarbamoyl-3-dimethylaminopropane (DLinCDAP); 2,2-Dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA); 2,2-Dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA); 2,2-Dilinoleoyl-4-(2-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA); (Aminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanium bromide (DMRIE); (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecyloxy)-1-propanium bromide (GAP-DMORIE); (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanium bromide (GAP-DLRIE); (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanium bromide (GAP-DM) RIE); N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecoxy)-1-propanemonium bromide (bAE-DMRIE); N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-ammonium (DOBAQ); 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dienoxy]prop-1-amine (Octyl-CLinDMA); 1,2-dimyristoyl-3-dimethylammonium propane (DMDAP); 1,2-dipalmitoyl-3-dimethylammonium propane (DPDAP); N1-[2- ((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleoyloxy]benzamide (MVL5); 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOEPC); 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropyl-1-ammonium bromide (DLRIE); N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propyl-1-ammonium bromide (DMORIE); di((Z)-non-2-en-1-yl)8,8′-((2-(dimethylamino)ethylthio)carbonyl-N-diyl)dioctanoate (ATX);N,N-Dimethyl-2,3-bis(dodecyloxy)prop-1-amine (DLDMA); N,N-Dimethyl-2,3-bis(tetradecyloxy)prop-1-amine (DMDMA); Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanoic acid ester (L319); N-dodecyl-3-((2-dodecylcarbamoylethyl)-{2-[(2-dodecylcarbamoylethyl)-2-{(2-dodecylcarbamoylethyl)-[2-(2-dodecylcarbamoylethylamino)-ethyl]-amino}ethylamino})propionamide (lipidoid 98N12-5); 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecane-2-ol (lipidoid) 02-200). In some embodiments, the cationic lipid or ionizable cationic lipid is selected from the group consisting of: ((4-hydroxybutyl)azonidyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3). More specifically, the cationic lipid or ionizable cationic lipid is ((4-hydroxybutyl)azonidyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0022] In some embodiments of the above five aspects, the cationic lipid or ionizable cationic lipid constitutes a molar percentage of about 20 mol% to about 49 mol% in the composition. Specifically, the molar percentage of the cationic lipid or ionizable cationic lipid is about 30 mol% to about 49 mol%. More specifically, the molar percentage of the cationic lipid or ionizable cationic lipid is about 35 mol% to about 49 mol%. More specifically, the molar percentage of the cationic lipid or ionizable cationic lipid can be about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 mol.

[0023] In some embodiments of the above five aspects, the phospholipid is selected from one or more of the following: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-diol 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether) PC); 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC); 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC); 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (1,2-dilinolenoyl-sn-glycero-3-phosphocholine);1,2-Diarachidonoyl-sn-glycero-3-phosphocholine; 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine; 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0) PE); 1,2-distearyl-sn-glycero-3-phosphate ethanolamine; 1,2-dilinoleoyl-sn-glycero-3-phosphate ethanolamine; 1,2-dilinoleoyl-sn-glycero-3-phosphate ethanolamine; 1,2-diarachidonicoyl-sn-glycero-3-phosphate ethanolamine; 1,2-docosahexaenooyl-sn-glycero-3-phosphate ethanolamine; 1,2-dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt Salt (DOPG); Sphingomyelin; Dipalmitoylphosphatidylglycerol (DPPG); Palmitoyloleoyl-phosphatidylethanolamine (POPE); Dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal); Dipalmitoylphosphatidylethanolamine (DPPE); Dimyristoylphosphatidylethanolamine (DMPE); Distearatelphosphatidylethanolamine (DSPE); 16-O-monomethylphosphatidylethanolamine (16-O-monomethyl PE); 16-O-dimethylphosphatidylethanolamine (16-O-dimethyl PE) PE); 18-1-trans phosphatidylethanolamine (18-1-trans PE); 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE);1,2-Ditransoleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE); phosphatidylcholine (PC); phosphatidylethanolamine (PE); phosphatidylserine (PS); phosphatidic acid (PA); phosphatidylglycerol (PG). In some embodiments, the phospholipid is DOPE. In some embodiments, the phospholipid is DSPC.

[0024] In some embodiments of the above five aspects, the phospholipid accounts for a molar percentage of about 11 mol% to about 25 mol% in the composition. Specifically, the molar percentage of the phospholipid may be about 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25 mol%.

[0025] In some embodiments of the above five aspects, the structural lipid is selected from one or more of the following: cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, lycopene, ursolic acid, α-tocopherol, hopane compounds, phytosterols, or steroids. Specifically, the structural lipid is cholesterol.

[0026] In some embodiments of the above five aspects, the structural lipids constitute a molar percentage of about 25 mol% to about 40 mol% in the composition. Specifically, the structural lipids constitute a molar percentage of about 30 mol% to about 40 mol%. More specifically, the molar percentage of the structural lipids can be about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40 mol%.

[0027] In some embodiments of the above five aspects, the PEG lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine; PEG-modified phosphatidic acid; PEG-ceramide conjugates, such as PEG-CerC14 or PEG-CerC20; PEG-modified dialkylamines; PEG-modified 1,2-diacyloxypropan-3-amines; PEG-modified diacylglycerol; PEG-modified dialkylglycerol; PEG-c-DOMG; 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol. PEG-DMG; PEG-modified dilinoleoylphosphatidylethanolamine (PEG-DLPE); PEG-modified dimyristoylphosphatidylethanolamine (PEG-DMPE); PEG-modified dipalmitoylphosphatidylcholine (PEG-DPPC); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE); PEG-disterylglycerol (PEG-disteryl) PEG-glycerol (PEG-DSG); PEG-dipalmitoyl; PEG-dioleoyl; PEG-distearate; PEG-diacylglycamide (PEG-DAG); PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE); PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). Specifically, the molecular weight of the PEG group is approximately 1000, 2000, 5000, 10000, 15000, or 20000 Daltons.More specifically, the PEG lipid is DMG-PEG 2000.

[0028] In some embodiments of the above five aspects, the PEG lipid accounts for a molar percentage of about 0.5 mol% to about 5 mol% in the composition. Specifically, the PEG lipid accounts for a molar percentage of about 0.5 mol% to about 4 mol%. More specifically, the PEG lipid accounts for a molar percentage of about 0.5 mol% to about 3 mol%. More specifically, the molar percentage of the PEG lipid can be about 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, or 3.0 mol.

[0029] In some embodiments of the above five aspects, the molar ratio of the structural lipid to the phospholipid is about 2.4 to about 3.6; specifically, the molar ratio of the structural lipid to the phospholipid is about 3.5. In some embodiments of the above five aspects, the molar ratio of the PEG lipid to the phospholipid is about 0.09 to about 0.4, or about 0.1 to about 0.4, or about 0.1 to about 0.3; specifically, the molar ratio of the PEG lipid to the phospholipid is about 0.1 to about 0.2, or about 0.1 to about 0.15.

[0030] In some embodiments of the above five aspects, the molar ratio of the structural lipid to the phospholipid is about 3.3 to about 3.6; specifically, the molar ratio of the structural lipid to the phospholipid is about 3.5. In some embodiments of the above five aspects, the molar ratio of the PEG lipid to the phospholipid is about 0.1 to about 0.12; specifically, the molar ratio of the PEG lipid to the phospholipid is about 0.1.

[0031] In some embodiments of the above five aspects, the lipid nanoparticles contain: ionizable cationic lipids comprising about 20 mol% to about 49 mol% of the total lipids; a molar ratio of structural lipids to phospholipids of about 3.3 to about 3.6; and a molar ratio of PEG lipids to phospholipids of about 0.08 to about 0.12. Specifically, the cationic lipids or ionizable cationic lipids comprise about 30 mol% to about 49 mol% of the total lipids; a molar ratio of structural lipids to phospholipids of about 3.5; and a molar ratio of PEG lipids to phospholipids of about 0.1.

[0032] In some embodiments of the above five aspects, the lipid nanoparticles include: (a) Ionizable cationic lipids, comprising about 10 mol% to about 49 mol% of total lipids, or about 30 mol% to about 49 mol%; (b) The molar ratio of the structural lipid to the phospholipid is approximately 3.5; (c) The molar ratio of the PEG lipid to the phospholipid is approximately 0.1.

[0033] In some embodiments of the above five aspects, the lipid nanoparticles include: (a) Ionizable cationic lipids, accounting for approximately 49 mol% of total lipids; (b) The molar ratio of the structural lipid to the phospholipid is approximately 3.5; (c) The molar ratio of the PEG lipid to the phospholipid is approximately 0.1.

[0034] In some embodiments of the above five aspects, the lipid nanoparticles include: (a) Cationic lipids or ionizable cationic lipids, accounting for approximately 49 mol% of the total lipids; (b) Phospholipids, accounting for approximately 11 mol% of total lipids; (c) Structural lipids, accounting for approximately 39 mol% of total lipids; (d) PEG lipids, accounting for approximately 1.0 mol of the total lipids.

[0035] In some embodiments of the above five aspects, the lipid nanoparticles include: (a) Cationic lipids or ionizable cationic lipids, accounting for approximately 40 mol% of the total lipids; (b) Phospholipids, accounting for approximately 16 mol% of total lipids; (c) Structural lipids, accounting for approximately 40 mol% of total lipids; (d) PEG lipids, accounting for approximately 4.0 mol of the total lipids.

[0036] In some embodiments of the above five aspects, the encapsulation efficiency of the nucleic acid by the lipid nanoparticles is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the nucleic acid is completely encapsulated in the lipid portion of the nucleic acid-lipid particle, thereby enabling the nucleic acid to resist nuclease degradation in aqueous solution.

[0037] In some embodiments of the above five aspects, the N / P ratio of the lipid nanoparticle composition is about 3 to about 8, for example about 4, 4.5, 5, 5.5, 6 or 6.5.

[0038] In some embodiments of the five aspects described above, the weight ratio of the nucleic acid to the cationic lipid or ionizable cationic lipid is from about 1:1 to about 1:100. Specifically, the weight ratio is from about 1:10 to about 1:40, for example from about 1:15 to about 1:25, or for example about 1:20.

[0039] In some embodiments of the five aspects described above, the nucleic acid can be used to treat skin diseases. In some embodiments, the nucleic acid is selected from one or more of the following: Antisense oligonucleotides target specific genes associated with skin diseases (such as psoriasis or eczema-related genes) and inhibit their expression; Small interfering RNA (siRNA) can be used to silence specific genes that cause skin diseases, such as genes involved in excessive melanin production in skin pigmentation disorders. Aptamers are aptamers that bind to specific targets (such as proteins or receptors) associated with skin diseases and regulate their biological activity. DNAzymes can be engineered to cut specific RNA molecules associated with skin diseases, thus providing a potential treatment. Plasmid DNA, which can be used for gene therapy of skin diseases, wherein the DNA is delivered into skin cells to correct genetic defects or enhance the production of therapeutic proteins; Antiviral RNA, which is designed to target and inhibit the replication of viruses such as herpesvirus or wart-associated virus; Ribozymes, which have catalytic activity, can be engineered to cleave specific RNA molecules associated with skin diseases, thus providing a potential therapeutic strategy. Antimicrobial peptides, derived from nucleic acid sequences, have shown potential for use in treating skin infections and skin diseases caused by bacteria or fungi. mRNAs used for collagen production to reverse skin aging-related phenotypes, such as COL1A1 mRNA and COL3A1 mRNA can express collagen, thereby reducing skin wrinkles and improving skin elasticity, producing a cosmetic effect; mRNAs used in gene editing tools, such as Cas9 mRNA and its corresponding guide RNA, can repair mutation sites in disease-causing genes through non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways; for example, by removing exon skipping strategies. COL1A1 Mutations in the gene site generate truncated but functional type VII collagen (COLVII) to repair the phenotype of patients with epidermolysis bullosa. mRNAs used for protein replacement therapy, such as those for hereditary skin diseases caused by gene mutations, like epidermolysis bullosa, can be delivered via [therapeutic route]. COL7A1 Treatment using mRNA.

[0040] In some embodiments of the third, fourth, and fifth aspects described above, the skin disease is selected from hereditary skin diseases and inflammatory skin diseases. The hereditary skin diseases include, but are not limited to, diseases caused by a single mutation in different genes, such as epidermolysis bullosa and Netherton syndrome; the inflammatory skin diseases include, but are not limited to, psoriasis, vitiligo, and atopic dermatitis caused by immune responses. Specifically, epidermolysis bullosa can be treated by using... COL7A1 Treatment with mRNA; Netherton syndrome can be treated with mRNA. SPINK5 Treatment with mRNA; atopic dermatitis can be treated with filaggrin mRNA.

[0041] In some embodiments of the five aspects described above, the composition is prepared as a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition is prepared for local delivery or topical administration. In some embodiments, the composition is prepared in a unit dose form.

[0042] In addition to the high or improved delivery efficiency of the lipid nanoparticles described in this invention at the target skin site, the application of these lipid nanoparticles also has several advantages, including: providing chemical protection for the encapsulated active ingredients (e.g., nucleic acids); enabling the application of unstable or easily degradable pharmaceutical active substances to the skin; improving the bioavailability of drugs; and achieving more effective drug release by promoting drug penetration and retention in the skin.

[0043] Brief description of the attached figures The following drawings, which form part of this specification, are used to further illustrate several aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of the accompanying drawings in conjunction with the detailed description of specific embodiments described herein.

[0044] Figure 1 The statistical results of in vivo fluorescence imaging are presented after delivering luciferase mRNA to mouse skin using lipid nanoparticles with different formulations.

[0045] Figure 2 The effect of varying the molar ratio of ionizable cationic lipids on delivery efficiency was demonstrated.

[0046] Figure 3 Statistical results are presented regarding the delivery of GFP mRNA to mouse skin using A13B.

[0047] Figure 4 Statistical results are presented regarding the delivery of Cas9 mRNA and sgRNA to mouse skin using A13C.

[0048] Figure 5Statistical results are presented for delivering GFP mRNA to mouse skin using different LNPs.

[0049] Figure 6 Statistical results are presented for the delivery of GFP mRNA to human skin using A13B.

[0050] Figure 7 The effects of lipid nanoparticles delivered via topical administration were demonstrated.

[0051] Figure 8 This demonstrates the systemic effects of locally delivered lipid nanoparticles, in which Figure 8 In B, the three columns of results for each tissue, from left to right, represent intravenous injection, intradermal injection, and delivery via topical route, respectively.

[0052] Figure 9 Demonstrating the effects of lipid nanoparticle therapy Col7a1 c.6485G>A mut / mut The percentage of mice with epidermal adhesion.

[0053] Figure 10 Demonstration of treatment with lipid nanoparticles Col7a1 c.6485G>A mut / mut The proportion of human type VII collagen (COLVII) expressed in mouse skin.

[0054] Figure 11 Demonstrated the use of LNP A13F for human delivery COL7A1 Representative images of immunofluorescence staining of samples taken at different time points after mRNA treatment.

[0055] Detailed description of the invention definition Unless otherwise defined, all technical and scientific terms used in this invention shall have the meanings commonly understood by one of ordinary skill in the art.

[0056] As used in this invention, the following terms and phrases have the following meanings: Unless otherwise specified, the singular form mentioned in this article also includes the plural form.

[0057] As used in this invention, the terms "comprising" or "including" are used to indicate an open definition, that is, including the said elements, but not excluding the inclusion of other elements.

[0058] As used in this invention, the term "about" refers to a variation relative to the stated value within an acceptable range, such as a variation within ±20%, ±10%, or ±5%. In some embodiments, "about" may represent a variation of ±1%, ±2%, ±3%, ±4%, ±5%, ±10%, or ±20%; in other embodiments, "about" may represent a variation of ±1% or ±2%.

[0059] As used herein, the term "lipid nanoparticle" or "LNP" refers to at least one particle with a size on the nanometer scale (e.g., about 1 nm to about 1000 nm) containing one or more types of lipid molecules. The LNPs of this invention may further contain at least one non-lipid-loaded molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNP contains a non-lipid-loaded molecule partially or completely encapsulated within a lipid shell. In some embodiments, the load molecule is a negatively charged molecule (e.g., mRNA). Without any theoretical limitations, it is considered that the cationic lipids in the LNP can interact with the negatively charged load molecule, thereby promoting the incorporation and / or encapsulation of the load molecule during LNP formation. The LNPs of this invention can be prepared by any conventional method and using any conventional equipment, such as microfluidic mixing systems, T-mixers, ethanol injection methods, etc.

[0060] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used interchangeably and encompass any nucleotide polymer linked by phosphodiester bonds. Exemplary nucleic acids include, but are not limited to, RNA and DNA molecules, such as those comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. Nucleic acid molecules can have any three-dimensional structure and can be double-stranded or single-stranded (e.g., sense or antisense strands). Other non-limiting examples include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, microRNA, tracrRNA, crRNA, guide RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes, and nucleic acid primers. Nucleic acid molecules may also contain non-natural or modified nucleotides.

[0061] As used herein, the term "ionizable cationic lipid" refers to a lipid that is generally electrically neutral under near-physiological pH conditions but can carry a positive charge under lower pH conditions (e.g., pH below about 7). As used herein, the term "cationic lipid" refers to a lipid that is generally positively charged under selected pH conditions (e.g., physiological pH).

[0062] As used herein, the term "phospholipid" refers to lipids comprising fatty acid chains and phosphate groups. Unlike positively charged cationic lipids, phospholipids are typically neutral molecules, i.e., they have no overall charge, or may carry a negative charge. Phospholipids are typically zwitterionic compounds, containing both positively and negatively charged components, but are generally uncharged, and are therefore usually classified as neutral lipids. Without being constrained by any theory, phospholipids are considered to be capable of assembling to form one or more lipid bilayer structures.

[0063] As used in this invention, the term "PEG lipid" refers to lipids modified with polyethylene glycol (PEG).

[0064] As used herein, the term "encapsulation" or "encapsulation effect" refers to complete, substantial, or partial encapsulation, restriction, surrounding, or sealing. In some embodiments, a compound, nucleic acid (e.g., mRNA), or other composition may be completely, partially, or substantially encapsulated.

[0065] As used in this invention, the term "encapsulation efficiency" refers to the ratio of the amount of nucleic acid that becomes part of the LNP during LNP preparation to the total amount of nucleic acid initially used to prepare the LNP. For example, if 97 mg of the initially provided 100 mg of nucleic acid is encapsulated in the LNP, the encapsulation efficiency can be expressed as 97%.

[0066] As used in this invention, the terms "patient" or "subject" refer to a living mammalian individual, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In some embodiments, the patient or subject is a primate. Non-limiting examples of human subjects include adults, adolescents, infants, and fetuses.

[0067] As used herein, the term "topical treatment" refers to a treatment of a skin condition in which an active ingredient or composition is delivered locally rather than systemically. For example, in skin treatments, a drug may be applied to multiple or a few sites on the skin and delivered to the skin and adjacent tissues via skin absorption. In some embodiments, topical treatment is achieved through the local delivery or administration of a LNP. In some embodiments, the local delivery includes, but is not limited to, topical, transdermal, subcutaneous, intradermal, or intradermal administration of the LNP. As used herein, "topical delivery or administration" means delivering or administering a drug to any accessible surface of the body (skin) of a subject or patient. In some embodiments of the invention, "topical" means external application to the skin epithelium. As used herein, transdermal, subcutaneous, intradermal, or intradermal delivery or administration of the LNP may be achieved by injection (e.g., using a needle).

[0068] As used in this invention, the term "alkyl" includes a straight-chain or branched, acyclic or cyclic saturated aliphatic hydrocarbon group containing 1 to 24 carbon atoms, for example 1 to 18, or up to 12, or 1 to 8, or 1 to 6 carbon atoms.

[0069] As used in this invention, the term "alkenyl" refers to a monovalent unsaturated hydrocarbon group containing up to 24 carbon atoms, for example 1 to 18, or 1 to 2, or 1 to 8, or 1 to 6 carbon atoms, and containing at least one, and preferably one to two, double bond unsaturations.

[0070] As used in this invention, the term "alkynyl" refers to a monovalent unsaturated hydrocarbon group containing up to 24 carbon atoms, such as 1 to 18, 1 to 12, 1 to 8, or 1 to 6 carbon atoms, and containing at least one, and preferably one to two, triple bond unsaturations.

[0071] As used in this invention, the term "alkylene" refers to a divalent alkyl group as defined herein.

[0072] As used in this invention, the term "alkenyl" refers to a divalent alkenyl group as defined herein.

[0073] As used in this invention, the term "ethynyl" refers to a divalent ethynyl group as defined herein.

[0074] As used herein, the term "optionally substituted" when used before a list of substituents means that each substituent in the list may optionally be substituted by one to three substituents selected from the group consisting of, for example: halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, acyl, amide, acyloxy, amino, aminocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aryl, aryloxy, arylthio, carboxyl, carboxylic acid ester, cyano, cycloalkyl, cycloalkoxy, cycloalkylthio, cycloalkenyl, cycloalkenyloxy, cycloalkenylthio, halogen, hydroxyl, heteroaryl, heteroaryloxy, heteroarylthio, heterocyclic, heterocyclic oxy, heterocyclic thio, nitro, sulfonic acid (SO3H), sulfonyl, sulfonyloxy, mercapto, and alkylthio.

[0075] Example The following embodiments illustrate preferred embodiments of the present invention. Those skilled in the art should understand that the technical solutions disclosed in the following embodiments represent those discovered and proven by the inventors to function well in implementing the present invention, and therefore can be considered preferred methods for implementing the present invention. However, those skilled in the art, under the guidance of this specification, should understand that various changes or substitutions can be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention, and the same or similar technical effects can still be obtained.

[0076] Example 1: Preparation of lipid nanoparticles (LNP) The lipid nanoparticles of this invention are prepared by the following method: 1) According to the molar percentages shown in Table 1, ionizable cationic lipids, structural lipids, phospholipids and PEG lipids were sequentially dissolved in ethanol and mixed to form a lipid organic phase; 2) Dissolve the active ingredient mRNA in a 100mM sodium acetate solution (pH=4) to form an aqueous phase; 3) Using a microfluidic mixing system, the organic phase containing the dissolved lipid mixture and the aqueous phase containing the dissolved mRNA were mixed at a volume ratio of 1:3, with a total mixing flow rate of 4 mL / min. 4) The obtained lipid nanoparticle solution was diluted with phosphate-buffered saline (PBS) and ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 30 kDa to restore the volume to the original preparation volume.

[0077] 5) After sterile filtration through a bacterial filtration membrane, the resulting lipid nanoparticle solution is sealed and stored in a glass bottle at low temperature.

[0078] In the above embodiments, the ionizable cationic lipids used were ALC-0315 and SM-102, both purchased from Glpbio; the structural lipid was cholesterol, purchased from Glpbio; the phospholipid was DOPE, purchased from Glpbio; the PEG lipid was DMG-PEG2000 (dimyristicoglycerol-polyethylene glycol 2000), purchased from Avanti; and the nucleic acid was luciferase mRNA, purchased from Novoprotein, with a loading of 1.25 μg.

[0079] Various lipid nanoparticle compositions, including A1 to A13 and S1 to S13, were prepared according to the above method. The molar percentages of each component are listed in Table 1.

[0080] Table 1: Molar percentage of each component in each lipid nanoparticle

[0081] The particle size, particle size distribution index (PDI), and zeta potential of the prepared lipid nanoparticles were determined using a nanoparticle size and zeta potential analyzer (Brookhaven, USA). The degree of RNA encapsulation by the lipid nanoparticles was characterized by encapsulation efficiency and empty shell ratio. Encapsulation efficiency reflects the proportion of RNA encapsulated by the lipid nanoparticles, while the empty shell ratio reflects the proportion of empty-shell lipid nanoparticles (i.e., lipid nanoparticles without RNA encapsulation). Encapsulation efficiency and empty shell ratio were determined using a Flow NanoAnalyzer (NanoFCM). Specifically, all nucleic acids in the lipid nanoparticles were labeled with a transmembrane fluorescent dye, and free nucleic acids, empty-shell lipid nanoparticles, and lipid nanoparticles encapsulating RNA were distinguished and quantified by fluorescence intensity and particle size information. The characterization results of LNP A13 are shown in Table 2.

[0082] Table 2: Physicochemical properties characterization results of LNP A13

[0083] Example 2: Evaluation of intradermal delivery efficacy of LNP in mice The lipid nanoparticles encapsulating luciferase mRNA prepared in Example 1 were administered to mouse skin via intradermal injection to induce local expression of the fluorescent protein in the skin, thereby evaluating the delivery effect of different lipid nanoparticles in mouse skin.

[0084] The experimental animals were SPF-grade female CD1 mice, 6–8 weeks old, weighing 20–30 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals underwent at least 7 days of acclimatization before the experiment. During the experiment, mice had free access to food and water, and the environment was characterized by 12 / 12-hour light / dark cycles, an ambient temperature of 20–26°C, and a relative humidity of 40%–70%. Mice were randomly assigned to groups. Lipid nanoparticles encapsulating Luciferase mRNA, prepared according to the method described in Example 1, were administered to mice via intradermal injection. Twenty-four hours after administration, in vivo bioluminescence imaging of the mice was performed using a small animal in vivo imaging system (IVISLUMINA III, PerkinElmer, USA). The specific operating steps are as follows: a D-fluorescein solution with a concentration of 15 mg / mL was prepared using phosphate buffer; the substrate was administered to each mouse via intraperitoneal injection; 5 minutes after the substrate injection, the mice were anesthetized in an anesthesia box using 2.5% isoflurane; subsequently, the anesthetized mice were placed in an IVIS imaging system for fluorescence imaging, and data from the concentrated fluorescence signal areas were collected and analyzed.

[0085] The in vivo delivery efficiency of lipid nanoparticles was expressed as the average fluorescence intensity of different mice in the same experimental group, where a higher fluorescence intensity value indicated a higher in vivo delivery efficiency of lipid nanoparticles.

[0086] Figure 1 The in vivo fluorescence imaging statistics of different lipid nanoparticles after delivery of Luciferase mRNA in mouse skin are shown, with the statistical time point being 24 hours post-injection. The statistical results are presented in the form of box plots, representing the maximum, upper quartile, median, lower quartile, and minimum values ​​from top to bottom; each bar represents the average fluorescence expression level in the skin of 5 mice (n=5).

[0087] Depend on Figure 1 It can be seen that lipid nanoparticles numbered 13 and 5 (such as A13, A5, D13, D5, S5, S13, etc.) showed excellent fluorescence expression 24 hours after intradermal injection in mice.

[0088] Example 3: Effect of changes in the molar ratio of ionizable cationic lipids on delivery performance This embodiment uses A13 as a basis to study the effect of changes in the molar ratio of ionizable cationic lipids on skin delivery efficacy.

[0089] Lipid nanoparticles numbered A13-2 to A13-16 were prepared according to the method described in Example 1, wherein only the molar ratio of ionizable cationic lipids in the lipid system was changed, while the relative ratios of the other three lipids remained unchanged. Subsequently, the intradermal delivery efficiency of the obtained lipid nanoparticles in mice was evaluated according to the method described in Example 2. The composition and delivery efficiency results of the different lipid nanoparticles are shown in Table 3 and... Figure 2 As shown.

[0090] Table 3: Composition and delivery efficiency of lipid nanoparticles with different molar ratios of ionizable cationic lipids

[0091] Unlike WO2009127060A1 and WO2011000107A1, which require an ionizable cationic lipid molar ratio of approximately 50%–85%, the inventors unexpectedly discovered that when the molar ratio of ionizable cationic lipids in lipid nanoparticles exceeds 50%, their intradermal delivery efficiency decreases significantly, by approximately three orders of magnitude, as shown in the comparison between A13-8 and A13-12. This result indicates that when the molar ratio of ionizable cationic lipids is below 50%, lipid nanoparticles can achieve efficient, precise, or preferential local delivery to the skin.

[0092] Example 4: Efficient delivery of GFP mRNA-encapsulated lipid nanoparticles to mouse skin Lipid nanoparticles A13B were prepared using a method similar to that in Example 1. The lipid composition of A13B was the same as that of A13, except that the 1.25 μg luciferase mRNA in Example 1 was replaced with 10 μg green fluorescent protein (GFP) mRNA. The GFP mRNA was purchased from Novoprotein.

[0093] SPF-grade C57 mice were used as experimental animals. A13B lipid nanoparticles encapsulating 10 μg of GFP mRNA were injected into the dorsal skin of the mice. Forty-eight hours post-injection, samples were collected, skin tissue from the injection area was excised, fixed with 4% paraformaldehyde (PFA), embedded in OCT, and then frozen sectioned. Immunostaining with a laboratory-prepared K14 antibody was used to label epidermal basal cells. After staining, the sections were mounted and imaged using a confocal microscope. Subsequently, the Spots function in Imaris software was used to identify DAPI-labeled cells to obtain the total cell count; then, the Spots function was used again to identify GFP-labeled cells to obtain the number of GFP-positive cells. The proportion of GFP-positive cells was calculated by the ratio of the number of GFP-positive cells to the total cell count.

[0094] The delivery efficiency of A13B in mouse skin for delivering GFP mRNA is expressed as the percentage of GFP-positive cells in the total number of cells, where a higher percentage indicates a higher delivery efficiency of A13B.

[0095] Figure 3 The statistical results of A13B delivery of GFP mRNA in mouse skin are shown, with the statistical time point being 48 hours post-injection; each bar represents the average fluorescence expression level in the skin of 5 mice (n=5). Figure 3 As can be seen, A13B can efficiently deliver GFP mRNA in mouse skin, with an average delivery efficiency of about 30%.

[0096] Example 5: Efficient delivery of Cas9 mRNA and sgRNA-encapsulated lipid nanoparticles in mouse skin Lipid nanoparticles A13C were prepared using a method similar to that in Example 1. The lipid composition of A13C was the same as that of A13, except that 1.25 μg of luciferase mRNA was replaced with 10 μg of Cas9 mRNA and 2.5 μg of sgRNA. The Cas9 mRNA was purchased from Apexbio, and the sgRNA was chemically synthesized by Genewiz.

[0097] SPF-grade Ai14 mice were used as experimental animals. A13C lipid nanoparticles encapsulating 10 μg Cas9 mRNA and 2.5 μg sgRNA were injected into the dorsal skin of mice. Forty-eight hours post-injection, skin tissue from the injection area was collected, fixed with 4% paraformaldehyde, embedded in OCT, and then frozen sectioned. Immunostaining with a laboratory-prepared K14 antibody was performed to label epidermal basal cells. After staining, the sections were mounted and imaged using a confocal microscope. Subsequently, the Spots function in Imaris software was used to identify DAPI-labeled cells to obtain the total cell count; then, the Spots function was used again to identify RFP-labeled cells to obtain the number of RFP-positive cells. The proportion of RFP-positive cells was calculated by the ratio of the number of RFP-positive cells to the total cell count.

[0098] The delivery efficiency of A13C in mouse skin for Cas9 mRNA and sgRNA is expressed as the percentage of RFP-positive cells in the total number of cells, with a higher percentage indicating higher A13C delivery efficiency.

[0099] Figure 4 The statistical results of A13C delivery of Cas9 mRNA and sgRNA in mouse skin are shown, with the statistical time point being 48 hours post-injection; each bar represents the average percentage of RFP-positive cells in the skin of 5 mice (n=5). Figure 4 It is evident that A13C can efficiently deliver Cas9 mRNA and sgRNA in mouse skin, with an average delivery efficiency of approximately 10%.

[0100] Example 6: Comparison of A13B with commercially available lipid nanoparticles This example compares the delivery of GFP mRNA in mouse skin using A13B and commercially available lipid nanoparticles. A13B is the lipid nanoparticle described in Example 4; the lipid nanoparticles from BioNTech, Moderna, and Alnylam are their respective commercially available lipid nanoparticles, all of which encapsulate GFP mRNA.

[0101] Table 4: Specific Composition of Different Lipid Nanoparticles

[0102] SPF-grade C57 mice were used as experimental animals. Different lipid nanoparticles encapsulating 10 μg of GFP mRNA were injected into the dorsal skin of the mice. Forty-eight hours after injection, samples were collected, skin tissue from the injection area was excised, fixed with 4% paraformaldehyde, embedded in OCT, and then frozen sectioned. Immunostaining with a laboratory-prepared K14 antibody was performed to label the basal layer cells of the epidermis. After staining, the sections were mounted and imaged using a confocal microscope. Subsequently, the Spots function in Imaris software was used to identify DAPI-labeled cells to obtain the total cell count; then, the Spots function was used again to identify GFP-labeled cells to obtain the number of GFP-positive cells. The proportion of GFP-positive cells was calculated by the ratio of the number of GFP-positive cells to the total cell count.

[0103] The delivery efficiency of different lipid nanoparticles in mouse skin for delivering GFP mRNA is expressed as the percentage of GFP-positive cells in the total number of cells, where a higher percentage indicates a higher delivery efficiency of the lipid nanoparticles.

[0104] Figure 5 Table 4 shows the statistical results of GFP mRNA delivery in mouse skin by different lipid nanoparticles, with the statistical time point being 48 hours post-injection; each bar represents the average proportion of GFP-positive cells in the skin of three mice (n=3). Figure 5 As shown in Table 4, A13B delivers GFP mRNA in mouse skin with significantly higher efficiency than other commercially available lipid nanoparticle formulations.

[0105] Example 7: Intradermal delivery in human skin This embodiment evaluates the delivery efficiency of lipid nanoparticles A13B encapsulating GFP mRNA in human skin. The human skin used in the experiment was discarded skin tissue obtained on the day of surgery. After removing subcutaneous adipose tissue using a scalpel and forceps, the skin was cut into small pieces with an area of ​​approximately 1 cm². The skin was washed with phosphate buffer containing 1% penicillin / streptomycin and then placed derm-side down in a Milli-cell suspension culture insert (PET 0.4 μm, 6-well plate) for culture. The culture medium was DMEM + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin, and the culture conditions were 37°C and 5% CO2. After 2–4 hours of culture, lipid nanoparticles A13B encapsulating 10 μg of GFP mRNA were delivered into the human skin via intradermal injection. Forty-eight hours after injection, samples were taken, skin tissue from the injection area was excised, fixed with 4% paraformaldehyde, embedded in OCT, and frozen sectioned. Immunostaining with a laboratory-prepared K14 antibody was used to label epidermal basal cells. After staining, the slides were mounted and imaged using a confocal microscope. Subsequently, the Spots function in Imaris software was used to identify DAPI-labeled cells to obtain the total cell count, and then GFP-labeled cells were identified to obtain the number of GFP-positive cells. The ratio of the two was used to calculate the proportion of GFP-positive cells.

[0106] The delivery efficiency of A13B in human skin for delivering GFP mRNA is expressed as the percentage of GFP-positive cells in the total number of cells, where a higher percentage indicates a higher delivery efficiency of A13B.

[0107] Figure 6 The statistical results of A13B delivery of GFP mRNA in human skin are shown, with the statistical time point being 48 hours post-injection; each bar represents the average fluorescence expression level in three human dermal tissue samples (n=3). Figure 6 It is evident that A13B can efficiently deliver GFP mRNA in human skin, with an average delivery efficiency of approximately 40%.

[0108] Example 8: External delivery of lipid nanoparticles Lipid nanoparticles A13D and A13E were prepared using a method similar to that in Example 1, except that A13D encapsulated 5 μg of luciferase mRNA and A13E encapsulated 10 μg of luciferase mRNA.

[0109] Similar to Example 2, lipid nanoparticles A13 (1.25 μg luciferase mRNA), A13D (5 μg luciferase mRNA), and A13E (10 μg luciferase mRNA) were administered to mice to evaluate the intradermal delivery effect of the lipid nanoparticles, except that the lipid nanoparticles were administered via topical route.

[0110] The data acquisition method is the same as in Example 2, and the results are as follows: Figure 7 As shown. Figure 7 The results show that the lipid nanoparticles of the present invention can effectively deliver mRNA to mouse skin via topical application, and within a certain range, the delivery efficiency increases with the increase of the amount of mRNA encapsulated in the lipid nanoparticles.

[0111] Example 9: Systemic effects of local delivery Following the method in Example 2, lipid nanoparticles A13E encapsulating 10 μg of luciferase mRNA were delivered to mice via intradermal injection and topical application, respectively.

[0112] The data acquisition method is the same as in Example 2, and the results are as follows: Figure 8 As shown, each column represents the fluorescence distribution in the bodies of 3 mice (n=3).

[0113] Figure 8 The results showed that after intradermal injection and topical administration, the fluorescence signal was mainly confined to the skin and did not spread to other organs in the body; while after tail vein injection, the fluorescence signal was mainly concentrated in the liver and spleen and was basically not seen in the skin.

[0114] Example 10 Lipid nanoparticles A13F were prepared using a method similar to that in Example 1, except that 1.25 μg of luciferase mRNA was replaced with 30 μg of human-derived luciferase mRNA. COL7A1 mRNA.

[0115] Skin from Col7a1c.6485G>Amut / mut mice was transplanted onto the backs of Nu / Nu nude mice, and experiments were conducted 20 days after transplantation wound healing. Encapsulated 30 μg of human-derived material was delivered into the transplanted skin. COL7A1 Lipid nanoparticles containing mRNA (A13F) were used. Samples were taken and analyzed on days 5 and 15 after the last administration. Skin tissue from the administration area was excised, embedded in OCT, and frozen sectioned. Immunostaining was performed using human ColVII antibody (Abcam) and Nidogen antibody (Santa Cruz). After staining, images were obtained using confocal microscopy. The degree of phenotypic repair was determined by analyzing the degree of connectivity between the epidermis and dermis and the expression of human ColVII. Higher connectivity between the epidermis and dermis and higher expression of human ColVII indicated better phenotypic repair. Statistical results are presented as box plots, showing the maximum, upper quartile, median, lower quartile, and minimum values ​​from top to bottom; each bar represents the repair results of 3 mice (n=3).

[0116] Figure 9The results showed that on day 5 after drug administration, the lipid nanoparticles treated... Col7a1 c.6485G>A mut / mut The adhesion rate of mouse epidermis was 80%, while that of the PBS control group was only 35%. On day 15 after administration, the epidermis of the PBS control group had completely separated, while the epidermis of the lipid nanoparticle treatment group still retained partial adhesion.

[0117] Figure 10 The results showed that on day 5 after drug administration, the lipid nanoparticles treated... Col7a1 c.6485G>A mut / mut Human ColVII was expressed in approximately 60% of the skin of mice, while no human ColVII expression was detected in the PBS control group; on day 15 after administration, human ColVII was still expressed in approximately 10% of the skin in the lipid nanoparticle treatment group.

[0118] Figure 11 The A13F delivery source was shown. COL7A1 mRNA therapy Col7a1 c.6485G>A mut / mut Representative immunofluorescence staining images of samples taken at different time points after mouse occupancy, with a scale bar of 50 μm.

[0119] The above results indicate that human-derived lipid nanoparticles A13F can be delivered... COL7A1 mRNA can effectively repair Col7a1 c.6485G>A mut / mut Disease phenotypes in mice.

[0120] Patients with recessive dystrophic epidermolysis bullosa (RDEB) carry a mutation in the Col7a1 gene, which encodes type VII collagen (ColVII). This mutation leads to abnormal function of the ColVII protein, preventing it from fulfilling its role in connecting the epidermis and dermis. This results in the epidermis and dermis easily separating, forming blisters and skin lesions. A model was constructed based on the mutation in RDEB patients. Col7a1 c.6485G>A mut / mut The mouse model exhibited a similar phenotype and typically died shortly after birth. This embodiment further validates that the lipid nanoparticles described in this invention can be used to treat skin diseases.

[0121] It should be understood that any prior art documents mentioned in this article do not constitute an acknowledgment that such documents belong to the general technical common sense of any country.

[0122] All publications, patents, patent applications, and published patent applications mentioned in this document by way of identifiable reference are incorporated herein by reference in their entirety. Although the invention has been described in considerable detail for the foregoing through examples and illustrations, those skilled in the art can make various minor modifications and alterations without departing from the spirit and scope of the invention. Therefore, the foregoing descriptions and embodiments should not be construed as limiting the scope of protection of this invention.

Claims

1. A lipid nanoparticle (LNP) composition, the composition comprising: nucleic acid; and lipid nanoparticles, the lipid nanoparticles encapsulating the nucleic acid and comprising cationic lipids or ionizable cationic lipids, phospholipids, structural lipids, and PEG lipids, wherein: (a) The cationic lipid or ionizable cationic lipid accounts for about 10 mol% to about 49 mol% of the total lipids present in the composition; (b) The molar ratio of the structural lipid to the phospholipid is about 2 to about 4; and (c) The molar ratio of the PEG lipid to the phospholipid is about 0.05 to about 0.

4.

2. A method for locally delivering nucleic acid to a subject in need, the method comprising administering an LNP composition to the subject locally, percutaneously, subcutaneously, epidermally, or intradermally, the composition comprising: nucleic acid; and lipid nanoparticles, the lipid nanoparticles encapsulating the nucleic acid and comprising cationic lipids or ionizable cationic lipids, phospholipids, structural lipids, and PEG lipids, wherein: (a) The cationic lipid or ionizable cationic lipid accounts for about 10 mol% to about 49 mol% of the total lipids present in the composition; (b) The molar ratio of the structural lipid to the phospholipid is about 2 to about 4; and (c) The molar ratio of the PEG lipid to the phospholipid is about 0.05 to about 0.

4.

3. A method for treating a skin disease in a subject, the method comprising delivering an LNP composition to the subject topically, percutaneously, subcutaneously, epidermally, or intradermally, the composition comprising: nucleic acid; and lipid nanoparticles, the lipid nanoparticles encapsulating the nucleic acid and comprising cationic lipids or ionizable cationic lipids, phospholipids, structural lipids, and PEG lipids, wherein: (a) The cationic lipid or ionizable cationic lipid accounts for about 10 mol% to about 49 mol% of the total lipids present in the composition; (b) The molar ratio of the structural lipid to the phospholipid is about 2 to about 4; and (c) The molar ratio of the PEG lipid to the phospholipid is about 0.05 to about 0.

4.

4. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the cationic lipid or ionizable cationic lipid is an ionizable cationic lipid of formula (I) or a salt thereof. (I) in m is an integer from 1 to 6; X can be selected from the following groups: OR 5 SR 5 NR 5 R 6 or COOR 5 ; U and V are each independently optional substituted C1-C8 alkylene, C2-C8 alkenylene, or C2-C8 ynylene; T can be selected from the following groups: C(O)O, OC(O), O, S, N(R) 5 ), C(O), C(O)N(R) 5 ), N(R 5 )C(O), OC(O)N(R 5 ), N(R 5 )C(O)O, C(O)S, C(S)O, S(O), S(O)(O) or C(S); Where R 5 and R 6 Each is independently hydrogen or optionally substituted C1-C 10 Alkyl, C2-C 10 alkenyl or C2-C 10 acetylinyl group; and R 1 R 2 R 3 and R 4 Each is independently hydrogen or optionally substituted C6-C 18 Alkyl, C6-C 18 alkenyl or C6-C 18 Alkyne group, provided that R 1 R 2 R 3 and R 4 At most one of them is hydrogen.

5. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the cationic lipid or ionizable cationic lipid is selected from the following compounds or salts thereof: ((4-hydroxybutyl)azonidyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3); 3-(dococosylamino)-N1,N1,4-tristidodecyl-1-piperazineethylamine (KL10); N1-[2-(dococosylamino)ethyl]- N1,N4,N4-Tridecyl-1,4-piperazine diethylamine (KL22); 14,25-Toctanediyl-15,18,21,24-tetraazaoctadecane (KL25); 1,2-Dilinoleoxy-N,N-dimethylaminopropane (DLin-DMA); 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP); N,N-Dimethyl-2,3-dioleoyloxypropane (DODMA); 1,2-Di-O-octadecenyl-3-trimethylammonium propane (DOTMA); 3-(N-(N′,N′-dimethylaminoethyl)carbamoyl)cholesterol (DC-Chol); Dimethyloctadecylammonium (DDAB); 1,2-Dioleoyl-3-dimethylammonium propane Alkane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane compounds; 1,2-dialkoxy-3-dimethylammonium propane compounds; octadecyl dimethylammonium chloride (DODAC); 1,2-distearate-N,N-dimethyl-3-aminopropane (DSDMA); 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylammonium (DMRIE); 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMEPC); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); 1,2-dioleoylpropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); 2,3-dioleoyl-N-[2-(spermine carboxyl) [Amide)ethyl]-N,N-dimethyl-1-propanetrifluoroacetate (DOSPA); 1,2-dilinoleoxy-N,N-dimethylaminopropane (DLinDMA); 1,2-dilinoleoxy-N,N-dimethylaminopropane (DLenDMA); Octadecylamide glycyl spermine (DOGS); 3-Dimethylamino-2-(cholest-5-en-3β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA); 2-[5′-(cholest-5-en-3β-oxy)-3′-oxopentoxy]-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA);N,N-Dimethyl-3,4-dioleoylbenzylamine (DMOBA); 1,2-N,N′-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP); 2,3-Dilinoleoyloxy-N,N-dimethylaminopropane (DLinDAP); 1,2-N,N′-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP); 1,2-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLinCDAP); 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA); 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin -K-XTC2-DMA); 2,2-Dilinyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanenium bromide (DMRIE); (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecyloxy)-1-propanenium bromide (GAP-DMORIE); (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanenium bromide (GAP-DLRIE); (±)-N-(3-aminopropyl)-N,N- Dimethyl-2,3-bis(tetradecyloxy)-1-propanediamine bromide (GAP-DMRIE); N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanediamine bromide (bAE-DMRIE); N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-ammonium (DOBAQ); 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dienoxy]propane-1-amine (Octyl-CLinDMA); 1,2-dimyristoyl-3-dimethylammonium propane (DMDA) P); 1,2-Dipalmitoyl-3-dimethylammonium propane (DPDAP); N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleoyloxy]benzamide (MVL5); 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOEPC); 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylprop-1-ammonium bromide (DLRIE); N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)prop-1-ammonium bromide (DMORIE); di((Z)-non-2-en-1-yl) 8,8′-((2-(dimethylamino)ethylthio)carbonyl-N-diyl)dioctanoate (ATX);N,N-Dimethyl-2,3-bis(dodecyloxy)prop-1-amine (DLDMA); N,N-Dimethyl-2,3-bis(tetradecyloxy)prop-1-amine (DMDMA); Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanoic acid ester (L319); N-dodecyl-3-((2-dodecylcarbamoylethyl)-{2-[(2-dodecylcarbamoylethyl)-2-{(2-dodecylcarbamoylethyl)-[2-(2-dodecylcarbamoylethylamino)-ethyl]-amino}ethylamino})propionamide (lipidoid 98N12-5); or 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecane-2-ol (lipidoid) 02-200).

6. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the cationic lipid or ionizable cationic lipid is selected from the group consisting of: ((4-hydroxybutyl)azonyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3).

7. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the cationic lipid or ionizable cationic lipid accounts for about 30 mol% to about 49 mol% of the total lipids present in the composition.

8. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the cationic lipid or ionizable cationic lipid accounts for about 35 mol% to about 49 mol% of the total lipids present in the composition.

9. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the cationic lipid or ionizable cationic lipid accounts for about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, or about 49 mol% of the total lipids present in the composition.

10. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the phospholipid is selected from the group consisting of: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DMPC). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-Diundecanoyl-sn-glycero-3-phosphocholine (DUPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0) Diether PC); 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC); 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC); 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (1,2-dilinolenoyl-sn-glycero-3-phosphocholine).1,2-Diarachidonoyl-sn-glycero-3-phosphocholine; 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine; 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0) PE); 1,2-distearyl-sn-glycero-3-phosphate ethanolamine; 1,2-dilinoleoyl-sn-glycero-3-phosphate ethanolamine; 1,2-dilinoleoyl-sn-glycero-3-phosphate ethanolamine; 1,2-diarachidonicoyl-sn-glycero-3-phosphate ethanolamine; 1,2-docosahexaenooyl-sn-glycero-3-phosphate ethanolamine; 1,2-dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt Salt (DOPG); Sphingomyelin; Dipalmitoylphosphatidylglycerol (DPPG); Palmitoyloleoyl-phosphatidylethanolamine (POPE); Dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal); Dipalmitoylphosphatidylethanolamine (DPPE); Dimyristoylphosphatidylethanolamine (DMPE); Distearatelphosphatidylethanolamine (DSPE); 16-O-monomethylphosphatidylethanolamine (16-O-monomethyl PE); 16-O-dimethylphosphatidylethanolamine (16-O-dimethyl PE) PE); 18-1-trans phosphatidylethanolamine (18-1-trans PE); 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE);1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE); phosphatidylcholine (PC); phosphatidylethanolamine (PE); phosphatidylserine (PS); phosphatidic acid (PA); phosphatidylglycerol (PG).

11. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

12. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the structural lipid is selected from the group consisting of: cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, lycopene, ursolic acid, α-tocopherol, hopane compounds, phytosterols or steroids.

13. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the structural lipid is cholesterol.

14. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the structural lipid accounts for about 25 mol% to about 40 mol% of the total lipids present in the composition.

15. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the structural lipids comprise about 30 mol% to about 40 mol% of the total lipids present in the composition.

16. The LNP composition according to claim 1, or the method according to claim 2 or 3, wherein the PEG lipid is selected from the following compounds: PEG-modified phosphatidylethanolamine; PEG-modified phosphatidic acid; PEG-ceramide conjugates, such as PEG-CerC14 or PEG-CerC20; PEG-modified dialkylamines; PEG-modified 1,2-diacyloxypropan-3-amines; PEG-modified diacylglycerol; PEG-modified dialkylglycerol; PEG-c-DOMG; 1,2-dimyristoyl-sn-glycerolmethoxypolyethylene glycol. PEG-DMG; PEG-modified dilinoleoylphosphatidylethanolamine (PEG-DLPE); PEG-modified dimyristoylphosphatidylethanolamine (PEG-DMPE); PEG-modified dipalmitoylphosphatidylcholine (PEG-DPPC); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE); PEG-disterylglycerol (PEG-DSG); PEG-dipalmitoyl; PEG-dioleoyl; PEG-distearoyl; PEG-diacylglycamide (PEG-DAG); PEG-dipalmitoylphosphatidylethanolamine (PEG-dipalmitoyl); phosphatidylethanolamine (PEG-DPPE); PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA).

17. The LNP composition of claim 1 or the method of claim 2 or 3, wherein the molecular weight of the PEG group is about 1000, 2000, 5000, 10,000, 15,000 or 20,000 Daltons.

18. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the PEG lipid is DMG-PEG2000.

19. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the molar ratio of the structural lipid to the phospholipid is about 2.4 to about 3.

6.

20. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the molar ratio of the structural lipid to the phospholipid is about 3.

5.

21. The LNP composition of claim 1 or the method of claim 2 or 3, wherein the molar ratio of the PEG lipid to the phospholipid is about 0.09 to about 0.4, or about 0.1 to about 0.4, or about 0.1 to about 0.3, or about 0.1 to about 0.2, or about 0.1 to about 0.

15.

22. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the molar ratio of the structural lipid to the phospholipid is about 3.3 to about 3.6, or about 3.

5.

23. The LNP composition according to claim 1, or the method according to claim 2 or 3, wherein the molar ratio of the PEG lipid to the phospholipid is about 0.1 to about 0.12, or about 0.

1.

24. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein: The ionizable cationic lipid comprises about 20 mol% to about 49 mol% of the total lipids present in the composition, the molar ratio of the structural lipid to the phospholipid is about 3.3 to 3.6; and the molar ratio of the PEG lipid to the phospholipid is about 0.08 to 0.12; or The cationic lipid or ionizable cationic lipid comprises about 30 mol% to about 49 mol% of the total lipids present in the composition, the molar ratio of the structural lipid to the phospholipid is about 3.5, and the molar ratio of the PEG lipid to the phospholipid is about 0.1; or The ionizable cationic lipid comprises about 20 mol% to about 49 mol% of the total lipids present in the composition, the molar ratio of the structural lipid to the phospholipid is about 3.3 to 3.6; and the molar ratio of the PEG lipid to the phospholipid is about 0.08 to 0.12; or The cationic lipid or ionizable cationic lipid accounts for about 30 mol% to about 49 mol% of the total lipids present in the composition, the molar ratio of the structural lipid to the phospholipid is about 3.5, and the molar ratio of the PEG lipid to the phospholipid is about 0.

1.

25. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the lipid nanoparticles comprise: (a) About 20 mol% to about 49 mol% of the total lipids present in the composition, or about 30 mol% to about 49 mol% of ionizable cationic lipids; (b) The molar ratio of the structural lipid to the phospholipid is approximately 3.5; and (c) The molar ratio of the PEG lipid to the phospholipid is approximately 0.

1.

26. The LNP composition according to claim 1, or the method according to claim 2 or 3, wherein the lipid nanoparticles comprise: (a) Approximately 49 mol% of the total lipids present in the composition; (b) The molar ratio of the structural lipid to the phospholipid is approximately 3.5; and (c) The molar ratio of the PEG lipid to the phospholipid is approximately 0.

1.

27. The LNP composition according to claim 1, or the method according to claim 2 or 3, wherein the lipid nanoparticles comprise: (a) Cationic lipids or ionizable cationic lipids comprising approximately 49 mol% of the total lipids present in the composition; (b) Phospholipids comprising approximately 11 mol% of the total lipids present in the composition; (c) Structural lipids comprising approximately 39 mol% of the total lipids present in the composition; and (d) PEG lipids comprising about 1.0 mol% of the total lipids present in the composition.

28. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the lipid nanoparticles comprise: (a) Cationic lipids or ionizable cationic lipids comprising about 40 mol% of the total lipids present in the composition; (b) Phospholipids comprising approximately 16 mol% of the total lipids present in the composition; (c) Structural lipids comprising approximately 40 mol% of the total lipids present in the composition; and (d) PEG lipids comprising approximately 4.0 mol% of the total lipids present in the composition.

29. The LNP composition according to claim 1, or the method according to claim 2 or 3, wherein the nucleic acid encapsulation percentage of the LNP is at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

30. The LNP according to claim 1 or the method according to claim 2 or 3, wherein the N / P ratio of the LNP composition is 3 to 8.

31. The LNP composition according to claim 1 or the method according to claim 2 or 3, wherein the composition comprises the nucleic acid to the cationic lipid or ionizable cationic lipid in a weight ratio of about 1:1 to about 1:

100.

32. The LNP composition according to claim 1, or the method according to claim 2 or 3, wherein the nucleic acid is effective in treating skin diseases.

33. The LNP composition according to claim 1, or the method according to claim 2 or 3, wherein the nucleic acid is selected from the group consisting of: antisense oligonucleotides, siRNA, aptamers, DNAzymes, plasmid DNA, antiviral RNA, ribozymes, or antimicrobial peptides.

34. The method according to claim 2 or 3, wherein the skin disease is selected from: hereditary skin diseases caused by functional protein defects, congenital ichthyosis, psoriasis, melanoma, or cosmetic-related skin conditions.

35. The LNP composition according to claim 1, or the method according to claim 2 or 3, wherein the composition is formulated as a pharmaceutical composition and further comprises an excipient.

36. The LNP composition of claim 1, or the method of claim 2 or 3, wherein the composition is formulated for local delivery or local application.

37. The LNP composition according to claim 1, or the method according to claim 2 or 3, wherein the composition is formulated in unit doses.

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