Composition for improving skin condition

Abstract

The present invention provides a composition for improving skin condition. Compared with the traditional direct filling of collagen by in vitro purification technology, the present invention involves delivering polynucleotides encoding collagen, rather than collagen itself, directly into the skin, which not only preserves the natural spatial structure and function of the protein, resulting in high protein activity, but also causes fewer side effects due to homology. Moreover, this enables long-term endogenous collagen supplementation, thereby maintaining the strength, firmness, and elasticity of the skin, and making the regeneration of skin tissue more natural.

Description

Compositions that improve skin condition Technical Field

[0001] This invention relates to a polynucleotide complex comprising a skin condition-improving compound, a composition comprising the above-mentioned polynucleotide complex, or a method of using the above-mentioned polynucleotide complex; and articles thereof. Background Technology

[0002] Like all organs in the human body, skin undergoes continuous and often cumulative changes over time. Skin aging occurs due to numerous factors, including inherent changes within the skin, the effects of gravity and the action of facial muscles on the skin, loss or displacement of soft tissue, and loss of tissue elasticity. Of concern, the “aging” phenotype of the skin can be accelerated by environmental factors, most notably long-term exposure to ultraviolet radiation (e.g., from the sun). Clinically, the aging phenotype of the skin can be described as wrinkling, sagging, and / or, compared to its younger counterpart, typically exhibiting less elasticity and resilience; however, the changes within this phenotype exist between natural chronological aging and photoaging.

[0003] Numerous skincare products have been developed to improve the appearance of human skin. Wrinkles and skin folds are typically treated with cosmetic facial fillers injected into the dermis and subdermis. Dermal atrophy caused by irreversible collagen loss is a significant characteristic of skin aging. There remains a need in this field to improve skin condition (e.g., to improve skin aging) by replenishing the skin with collagen. Summary of the Invention

[0004] Compared to traditional methods of directly filling collagen through in vitro purification, using in vivo mRNA delivery technology (especially LNP) to replenish collagen in the skin has several advantages. This technology delivers collagen mRNA, rather than collagen itself, directly into the skin, promoting the skin cells' own production of collagen lost due to aging. This preserves the protein's natural spatial structure and function, resulting in high protein activity. Because it is a homologous substance, it has fewer side effects and allows for long-term endogenous collagen replenishment to maintain skin strength, firmness, and elasticity, leading to more natural skin tissue regeneration.

[0005] Invention Details

[0006] The first aspect of the present invention provides a polynucleotide complex comprising a polynucleotide and a polynucleotide protector, wherein the polynucleotide is an mRNA sequence encoding collagen COL17A1.

[0007] In some embodiments, the polynucleotide is encapsulated by the polynucleotide protector.

[0008] In some embodiments, the sequence of the collagen COL17A1 is the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1;

[0009] In some embodiments, the mRNA sequence comprises a nucleotide sequence as shown in SEQ ID NO: 2, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with the nucleotide sequence shown in SEQ ID NO: 2;

[0010] In some embodiments, the mRNA sequence of the present invention further comprises a 5'-UTR, a 3'-UTR, and / or a poly-A tail operably linked to a nucleotide sequence as shown in SEQ ID NO: 6-8.

[0011] In some embodiments, the 5'-UTR comprises a nucleotide sequence as shown in SEQ ID NO:6, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:6;

[0012] In some embodiments, the 3'-UTR comprises a nucleotide sequence as shown in SEQ ID NO:7, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:7;

[0013] In some embodiments, the poly-A tail comprises a polynucleotide sequence as shown in SEQ ID NO:8, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:8.

[0014] In some embodiments, the mRNA sequence comprises a nucleotide sequence as shown in SEQ ID NO: 5, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with the nucleotide sequence shown in SEQ ID NO: 5.

[0015] In some embodiments, the mRNA contains at least one chemically modified nucleoside;

[0016] Optionally, the chemically modified nucleoside is selected from chemically modified uridine;

[0017] Optionally, some or all of the uridine in the mRNA is chemically modified uridine; preferably, all of the uridine in the mRNA is chemically modified uridine.

[0018] Optionally, the chemically modified uridine is selected from any one or more of pseudouridine, N1-methyl-pseudouridine, 2-thiouridine, 4-thiouridine, 2-thio-1-methyl-1-diaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-azauridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 5-methyluridine, and 2-methoxy-uridine. Preferably, the chemically modified uridine is pseudouridine or N1-methyl-pseudouridine.

[0019] In some implementations, the mRNA also includes a 5' cap structure.

[0020] In some embodiments, the 5' cap of the mRNA is selected from any one of m7G(5')ppp(5')(2'-OMeA)pG, m7(3'OMeG)(5')ppp(5')m6(2'OMeA)pG, m7(3'AcmG)(5')ppp(5')(2'OMeA)pG and m7G(5')vppp(5')(2'OMeA)pG, preferably m7G(5')ppp(5')(2'-OMeA)pG.

[0021] Preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeN)pN, where N is a natural or modified nucleoside; preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

[0022] In some embodiments, the polynucleotide protector is selected from liposomes, liposome nanoparticles (LNPs), sol-gels, and nanogels.

[0023] In some preferred embodiments, the polynucleotide protector is a lipid nanoparticle (LNP) comprising ionizable lipids, neutral lipids, structural lipids, and PEG lipids.

[0024] In some embodiments, the ionizable lipid is selected from, but not limited to, one or more of, the following: compound 5 or its stereoisomers, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2 -Dilinyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinyl-4-N-methylpepiazino-[1 [1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1, 2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleothio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), and 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP).Cl), 1,2-dioleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dioleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dioleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylamino Propane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1 2-Dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-carboxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-formylamino)ethyl]-N,N-dimethyl-1-propanetrimonium trifluoroacetate (DOSPA), bis(octadecylaminoglycyl)spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA) (-3-β-oxy)-3'-oxaproloxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), MC3, SM-102, ALC-0315.

[0025] Preferably, the ionizable lipid is selected from compound 5 and its stereoisomers:

[0026] In some embodiments, the neutral lipid is a phospholipid. Preferably, the phospholipid is selected from, but is not limited to, one or more of, the following: dilauroyl lecithin (DLPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DUPC), palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-di-O-octadecyl-sn-glycerol-3-phosphate choline (18:0Diether PC), 1-oleoyl-2-cholestyldimethylsuccinate-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16Lyso PC), 1,2-divinyl-sn-glycerol-3-phosphate choline, 1,2-diarylyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarylyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt (DOPG) or sphingomyelin.

[0027] In some preferred embodiments, the neutral lipid is DSPC. In other preferred embodiments, the neutral lipid is DOPE. In still other preferred embodiments, the neutral lipid is both DSPC and DOPE.

[0028] In some embodiments, the structured lipid is selected from, but not limited to, cholesterol, coprosterol, sitosterol, ergosterol, stigmasterol, corticosteroids, or combinations thereof. In some preferred embodiments, the structured lipid is cholesterol. In other embodiments, the structured lipid is cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof.

[0029] In some embodiments, the PEG lipid is a lipid modified with polyethylene glycol (PEG).

[0030] In some embodiments, the PEG lipid is selected from, but not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some preferred embodiments, the PEG lipid is selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. In some more preferred embodiments, the PEG lipid is DMG-PEG2000.

[0031] In some preferred embodiments, the LNP comprises ionizable lipids, neutral lipids, structural lipids, and PEG lipids. Preferably, the molar ratio of the ionizable lipids, neutral lipids, structural lipids, and PEG lipids is (20-60):(5-25):(25-55):(0.5-5). In some more preferred embodiments, the molar ratio of the ionizable lipids, neutral lipids, structural lipids, and PEG lipids is (40-55):(10-15):(35-45):(0.5-2.5).

[0032] In some preferred embodiments, the LNP comprises ionizable lipids, phospholipids, cholesterol, and PEG lipids. Preferably, the molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG lipids is (20–60):(5–25):(25–55):(0.5–5). In some more preferred embodiments, the molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG lipids is (40–55):(10–15):(35–45):(0.5–2.5).

[0033] In some embodiments of this application, the content of ionizable lipids in the lipid nanoparticles is 35 mol%-65 mol%, for example, it can be 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 mol%, 41.5 mol%, 42 mol%, etc. 42.5mol%, 43mol%, 43.5mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol%, 47.5m ol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7mol%, 48.8mol%, 48. 9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8mol%, 49.9mol%, 50mol%, 50.5mol%, 51mol%, 51.5mol%, 52mol%, 52.5mol%, 53mol%, 53.5mol%, 54mol%, 54.5m ol%, 55mol%, 55.5mol%, 56mol%, 56.5mol%, 57mol%, 57.5mol%, 58mol%, 58.5mol%, 59mol%, 59.5mol%, 6 0 mol%, 60.5 mol%, 61 mol%, 61.5 mol%, 62 mol%, 62.5 mol%, 63 mol%, 63.5 mol%, 64 mol%, 64.5 mol%, 65 mol%.

[0034] In some embodiments of this application, the total content of the phospholipids and the structural lipids in the lipid nanoparticles can be 35 mol%-65 mol%, for example, 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 ... 2mol%, 42.5mol%, 43mol%, 43.5mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol%, 47.5mol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7mol%, 48.8mol% , 48.9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8mo l%, 49.9mol%, 50mol%, 50.5mol%, 51mol%, 51.5mol%, 52mol%, 52.5mol%, 53mol%, 53.5mol%, 54mol%, 54. 5mol%, 55mol%, 55.5mol%, 56mol%, 56.5mol%, 57mol%, 57.5mol%, 58mol%, 58.5mol%, 59mol%, 59.5mol%, 60mol%, 60.5mol%, 61mol%, 61.5mol%, 62mol%, 62.5mol%, 63mol%, 63.5mol%, 64mol%, 64.5mol%, 65mol%.

[0035] In some embodiments of this application, the phospholipid content in the lipid nanoparticles can be 5 mol%-15 mol%, for example, 5 mol%, 5.5 mol%, 6 mol%, 6.5 mol%, 7 mol%, 7.5 mol%, 8 mol%, 8.5 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 11.5 mol%, 12 mol%, 12.5 mol%, 13 mol%, 13.5 mol%, 14 mol%, 14.5 mol%, or 15 mol%; for example, it can be 10 mol%-15 mol%.

[0036] In some embodiments of this application, the content of the structural lipids in the lipid nanoparticles can be 30 mol%-50 mol%, for example, 30 mol%, 30.5 mol%, 31 mol%, 31.5 mol%, 32 mol%, 32.5 mol%, 33 mol%, 33.5 mol%, 34 mol%, 34.5 mol%, 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 mol%, 41.5 mol%, 42 mol%, 42.5 mol%, 43 mol%, 43.5 mol%. mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol%, 47.5mol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7m ol%, 48.8mol%, 48.9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8mol%, 49.9mol%, 50mol%; for example, it can be 35mol%-45mol%.

[0037] In some embodiments of this application, the content of the PEG lipid in the lipid nanoparticles is 0.5 mol%-5 mol%, for example, it can be 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, etc. l%, 2.5mol%, 2.6mol%, 2.7mol%, 2.8mol%, 2.9mol%, 3mol%, 3.1mol%, 3.2mol%, 3.3mol%, 3.4mol%, 3.5mol%, 3.6mol%, 3.7mo 1%, 3.8mol%, 3.9mol%, 4mol%, 4.1mol%, 4.2mol%, 4.3mol%, 4.4mol%, 4.5mol%, 4.6mol%, 4.7mol%, 4.8mol%, 4.9mol%, 5mol%.

[0038] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 35 mol%-65 mol%, the combined content of phospholipids and cholesterol is 35 mol%-65 mol%, and the content of PEG lipids is 0.5 mol%-5 mol%.

[0039] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the component ratio is: 50 mol% ionizable lipids, 48.5 mol% phospholipids and cholesterol, and 1.5 mol% PEG lipids.

[0040] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids. The content of ionizable lipids in the lipid nanoparticles is 40 mol%-50 mol%, the content of phospholipids is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipids is 1.5 mol%-2.5 mol%.

[0041] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids. The content of ionizable lipids in the lipid nanoparticles is 48 mol%-49 mol%, the content of phospholipids is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipids is 1.5 mol%-2.5 mol%.

[0042] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 40 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 42.5 mol%, and the content of PEG lipids is 2.5 mol%.

[0043] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 40 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 43.5 mol%, and the content of PEG lipids is 1.5 mol%.

[0044] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 48.5%, the content of phospholipids is 5 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 1.5 mol%.

[0045] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 44.3 mol%, the content of phospholipids is 9.3 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 1.5 mol%.

[0046] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 50 mol%, the content of phospholipids is 5 mol%, the content of cholesterol is 43 mol%, and the content of PEG lipids is 2.1 mol%.

[0047] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 40 mol%, the content of phospholipids is 12.5 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 2.5 mol%.

[0048] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 46 mol%, the content of phospholipids is 11 mol%, the content of cholesterol is 41 mol%, and the content of PEG lipids is 2 mol%.

[0049] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 47.5 mol%, the content of phospholipids is 5 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 2.5 mol%.

[0050] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 40 mol%, the content of phospholipids is 13 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 2 mol%.

[0051] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 50 mol%, the content of phospholipids is 12.5 mol%, the content of cholesterol is 35 mol%, and the content of PEG lipids is 2.5 mol%.

[0052] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 50 mol%, the content of phospholipids is 8 mol%, the content of cholesterol is 39.5 mol%, and the content of PEG lipids is 2.5 mol%.

[0053] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 42.3 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 40.3 mol%, and the content of PEG lipids is 2.5 mol%.

[0054] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 46 mol%, the content of phospholipids is 11 mol%, the content of cholesterol is 41 mol%, and the content of PEG lipids is 2 mol%.

[0055] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 47.5 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 35 mol%, and the content of PEG lipids is 2.5 mol%.

[0056] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 48.5 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 35 mol%, and the content of PEG lipids is 1.5 mol%.

[0057] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 50 mol%, the content of phospholipids is 5 mol%, the content of cholesterol is 43.5 mol%, and the content of PEG lipids is 1.5 mol%.

[0058] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 40 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 43.5 mol%, and the content of PEG lipids is 1.5 mol%.

[0059] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 48 mol%, the content of phospholipids is 10.3 mol%, the content of cholesterol is 39.6 mol%, and the content of PEG lipids is 2.1 mol%.

[0060] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the component ratio in the lipid nanoparticles is: 50 mol% ionizable lipids, 10 mol% phospholipids, 38.5 mol% cholesterol, and 1.5 mol% PEG lipids.

[0061] In some embodiments of this application, the molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids in the lipid nanoparticles is 35-65:35-65:0.5-5.

[0062] In the lipid nanoparticles of this application, the molar ratio of the sum of the ionizable lipids, the phospholipids, and the structural lipids, and the PEG lipids is (35-65):(35-65):(0.5-5), where 35-65 can take any value between 35 and 65, for example, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39. .5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 5 6.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 0.5-5 can take any value between 0.5 and 5, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.

[0063] In one specific embodiment, the molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids in the lipid nanoparticles is (40-50):(35-65):(1-3).

[0064] In one specific embodiment, the molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids in the lipid nanoparticles is (50-65):(35-65):(1-3).

[0065] In one specific embodiment, the molar ratio of the sum of the ionizable lipids, the phospholipids, and the structural lipids, and the PEG lipids is not 50:48.5:1.5.

[0066] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (40-65):(5-15):(30-50):(1-3).

[0067] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (40-65):(10-15):(35-45):(1.5-2.5).

[0068] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (40-50):(5-15):(30-50):(1-3). In the molar ratio (40-50):(5-15):(30-50):(1-3), 40-50 can take any value between 40 and 50, for example, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 5-15 can take any value between 5 and 15, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 30-50 can take any value between 30 and 50, for example, The possible values ​​are 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, and 46. 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 1-3 can be any value between 1 and 3, for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.

[0069] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (40-50):(5-15):(35-45):(1.5-2.5). For the molar ratio (40-50):(5-15):(35-45):(1.5-2.5), 40-50 can take any value between 40 and 50, such as 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, and 50; 5-15 can take any value between 5 and 15, such as 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, and 11. 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 35-45 can be any value between 35 and 45, for example, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45; 1.5-2.5 can be any value between 1.5 and 2.5, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5.

[0070] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (50-65):(5-15):(30-50):(1-3). Wherein, for the molar ratio (50-65):(5-15):(30-50):(1-3), 50-65 can take any value between 50 and 65, for example, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60. 5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 5-15 can be any value between 5 and 15, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 30-50 can... Choose any value between 30 and 50, for example: 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 1-3 can be any value between 1 and 3, for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.

[0071] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is not 50:10:38.5:1.5.

[0072] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (50-65):(5-15):(35-45):(1.5-2.5). For the molar ratio (50-65):(5-15):(35-45):(1.5-2.5), 50-65 can take any value between 50 and 65, for example, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 5-15 can take any value between 5 and 15, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 35-45 can be any value between 35 and 45, for example, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45; 1.5-2.5 can be any value between 1.5 and 2.5, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5.

[0073] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5, the sum of DSPC and cholesterol, and DMG-PEG2000 is (35-65):(35-65):(0.5-5).

[0074] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5, DSPC, cholesterol, and DMG-PEG2000 is (40-49):(5-15):(30-50):(1-3).

[0075] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5, DSPC, cholesterol, and DMG-PEG2000 is (40-49):(10-15):(35-45):(1.5-2.5).

[0076] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(5-15):(30-50):(1-3).

[0077] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(10-15):(35-45):(1.5-2.5).

[0078] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5 is 35 mol%-65 mol%, the sum of DSPC and cholesterol is 35 mol%-65 mol%, and the content of DMG-PEG2000 is 0.5 mol%-5 mol%.

[0079] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5 is 40 mol%-50 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0080] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5 is 48 mol%-49 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0081] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5A, DSPC, and cholesterol, and DMG-PEG2000 is (35-65):(35-65):(0.5-5).

[0082] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (40-49):(5-15):(30-50):(1-3).

[0083] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (40-49):(10-15):(35-45):(1.5-2.5).

[0084] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(5-15):(30-50):(1-3).

[0085] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(10-15):(35-45):(1.5-2.5).

[0086] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5A in the lipid nanoparticles is 35 mol%-65 mol%, the combined content of DSPC and cholesterol is 35 mol%-65 mol%, and the content of DMG-PEG2000 is 0.5 mol%-5 mol%.

[0087] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5A is 40 mol%-50 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0088] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5A is 48 mol%-49 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0089] In some embodiments, the molar ratio of the ionizable lipid to the polynucleotide in the polynucleotide complex is 5.2:1.

[0090] A second aspect of the present invention provides a composition comprising the polynucleotide complex of the first aspect of the present invention, an active moisturizing ingredient, a local anesthetic ingredient, a pH buffering ingredient, an osmotic pressure regulator, and water.

[0091] In some embodiments, the present invention provides a composition comprising a polynucleotide complex of the first aspect of the present invention, an active moisturizing ingredient, a local anesthetic ingredient, a pH buffering ingredient, an osmotic pressure regulator, and water.

[0092] In some embodiments, the composition is used to improve skin condition.

[0093] In some embodiments, the polynucleotide is present in the composition at a concentration of 0.4–200 μg / ml, 1–200 μg / ml, 5–100 μg / ml, or 5–50 μg / ml.

[0094] In some embodiments, the polynucleotide in the composition is present in concentrations of 1, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75. 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197 198, 199, 200μg / ml.

[0095] In some embodiments, the molar ratio of the ionizable lipid to nucleotide in the polynucleotide complex of the composition is 5.2:1.

[0096] In some embodiments, the concentration of the active moisturizing ingredient in the composition is 1-10 mg / ml, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml, preferably 5 mg / ml.

[0097] In some embodiments, the active moisturizing ingredient is selected from hyaluronic acid or its salts, preferably sodium hyaluronate.

[0098] In some embodiments, the local anesthetic component is present in a concentration of 1-10 mg / ml in the composition, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml, preferably 3 mg / ml.

[0099] In some embodiments, the local anesthetic component is selected from lidocaine, rucaine, tetracaine, bupivacaine, ropivacaine or salts thereof, preferably lidocaine hydrochloride.

[0100] In some embodiments, the pH buffering component is selected from sodium dihydrogen phosphate or its hydrate and / or disodium hydrogen phosphate or its hydrate. Preferably, the pH buffering component is selected from sodium dihydrogen phosphate monohydrate and / or disodium hydrogen phosphate dodecahydrate.

[0101] In some embodiments, the composition has a pH of 7.0-7.6.

[0102] In some embodiments, the osmotic pressure regulator is sodium chloride.

[0103] In some embodiments, the composition has an osmotic pressure of 270 mOsmol / kg to 350 mOsmol / kg.

[0104] In some implementations, the water is water for injection.

[0105] In some embodiments, the composition is formulated with (polynucleotides 0.4–200 μg / ml).

[0106] In some embodiments, the composition is formulated with (polynucleotides 0.4–200 μg / ml).

[0107] In some embodiments, the composition is formulated with (polynucleotides 0.4–200 μg / ml).

[0108] In some embodiments, the composition is formulated with a polynucleotide concentration of 5–50 μg / ml.

[0109] In some embodiments, the composition is formulated with a polynucleotide concentration of 5–50 μg / ml.

[0110] In some embodiments, the composition is formulated with a polynucleotide concentration of 5–50 μg / ml.

[0111] In some embodiments, the composition is formulated as (polynucleotide 1.7 μg / ml).

[0112] In some embodiments, the composition is formulated as (polynucleotide 1.7 μg / ml).

[0113] In some embodiments, the composition is formulated as (polynucleotide 1.7 μg / ml).

[0114] In some embodiments, the composition is formulated as (5 μg / ml of polynucleotides).

[0115] In some embodiments, the composition is formulated as (5 μg / ml of polynucleotides).

[0116] In some embodiments, the composition is formulated as (5 μg / ml of polynucleotides).

[0117] In some embodiments, the composition is formulated as (15 μg / ml of polynucleotides).

[0118] In some embodiments, the composition is formulated as (15 μg / ml of polynucleotides).

[0119] In some embodiments, the composition is formulated as (15 μg / ml of polynucleotides).

[0120] The third aspect of the present invention provides:

[0121] (1) A method of supplementing collagen in a subject in need, comprising administering to the subject a polynucleotide complex as described in the first aspect of this application or a composition as described in the second aspect of this application.

[0122] (2) A method of improving skin aging in subjects in need, comprising administering to the subject a polynucleotide complex as described in the first aspect of this application or a composition as described in the second aspect of this application.

[0123] (3) A method for increasing and / or improving at least one of the texture, smoothness, elasticity, or tension of the skin of a subject in need, said method comprising administering to the subject a polynucleotide complex as described in the first aspect of this application or a composition as described in the second aspect of this application.

[0124] (4) A method for reducing the occurrence of one or more superficial pits in the skin in a subject in need, the method comprising administering to the subject a polynucleotide complex as described in the first aspect of this application or a composition as described in the second aspect of this application.

[0125] In some embodiments of this application, skin aging is selected from skin aging caused by increasing age or skin aging caused by ultraviolet light or sunlight exposure.

[0126] In some embodiments of this application, one or more superficial depressions in the skin are selected from the group consisting of: nasolabial folds, crow's feet, frown lines, worry lines, scars, frown lines, drooping eyebrows, tear troughs, nasolabial folds, bunny lines, drooping cheeks / midfaces, marionette lines, poppy dimpling, smile lines, laugh lines, chin wrinkles, neck wrinkles, platysma bands, and any combination thereof.

[0127] In some implementations, the increase and / or improvement in skin texture, smoothness, elasticity, or tension is selected from: (a) treatment, reduction, and / or prevention of fine lines and / or wrinkles compared to before application; (b) reduction in skin pore size; (c) improvement in skin thickness, fullness, and / or firmness; (d) improvement in skin smoothness, suppleness, and / or softness; (e) improvement in skin color, radiance, and / or translucency; (f) improvement in procollagen and / or collagen production; and (g) improvement and / or retexturization of skin texture. (h) Improved appearance of skin contour; (i) Restoration of skin luster and / or brightness; (j) Improvement of skin appearance diminished due to aging and / or menopause; (k) Improved skin hydration; (l) Increased skin elasticity and / or resilience; (m) Treatment, reduction and / or prevention of skin sagging; (n) Improved skin firmness; (o) Reduction of pigmentation spots, freckled skin and / or scars; (p) Improvement of skin optical properties in terms of light diffraction or reflection; or (q) Any combination thereof.

[0128] In some embodiments of this application, the subject is a human being.

[0129] In some embodiments of this application, the administration is performed percutaneously, subcutaneously, and / or intradermally to the subject or via superficial injection.

[0130] In some embodiments, the dosage form of the product or drug includes, but is not limited to, topical dosage forms, gels, lotions, creams, ointments, injectable dosage forms, microneedles, etc., preferably injectable solutions.

[0131] In some implementations, the dosage form is a transdermal dosage form, a subcutaneous dosage form, and / or an intradermal dosage form.

[0132] In some implementations, the product or drug is administered via superficial injection.

[0133] In some implementations, the product or drug is administered via dermal injection.

[0134] definition

[0135] To facilitate understanding of this invention, certain terms are defined below. Further definitions of the following terms and other terms are set forth throughout the specification.

[0136] As used herein, the term “messenger RNA (mRNA)” or “mRNA” refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. Typically, mRNA consists of ribonucleotides. mRNA may contain one or more coding and noncoding regions. mRNA may be purified from natural sources, or generated using recombinant expression systems and optionally purified, chemically synthesized, etc. Unless otherwise stated, mRNA sequences in this document are presented in a 5' to 3' orientation.

[0137] An open reading frame (ORF) is a continuous segment of DNA or RNA that encodes a protein or polypeptide. Typically, an ORF contains a translation start signal or start codon such as ATG or AUG, and a stop codon.

[0138] The untranslated region (UTR) refers to the non-translated nucleic acid at the 5' end (5'-UTR) and / or 3' end (3'-UTR) of the open reading frame, meaning it will be transcribed but not translated into an amino acid sequence. The 5'-UTR begins at the transcription start site (+1 position) and continues to the start codon (but does not include the start codon). Typically, the 5'-UTR often has characteristics like the Kozak sequence. The 3'-UTR begins immediately after the stop codon and continues until the transcription termination signal. Exemplary 3'- and 5'-UTRs include α- and β-globin, albumin, HSD17B4, and eukaryotic elongation factor 1α. Additionally, viral 5' and 3' UTRs can also be used, including orthopoxvirus and cytomegalovirus UTR sequences.

[0139] The poly-A tail refers to polyadenine nucleotides, which are usually located at the 3' end of polynucleotides (e.g., mRNA) and can increase the stability of polynucleotide molecules.

[0140] A 5' cap structure refers to the structure formed by modifying the 5' end of eukaryotic mRNA. In some embodiments, a suitable cap is 7-methylguanosine monophosphate (“m7G”), which is linked to the 5' end of the first nucleotide via a triphosphate bridge, resulting in m7G(5')ppp(5')N, where N represents the first transcribed nucleotide. Depending on the degree of methylation, three types of caps can be formed: CAP 0, CAP I, and CAP II. Guanosine is linked to the 5' end of the mRNA via a 5'-5' pyrophosphate bond. When the 7th nitrogen atom in guanosine is methylated to form m7G(5')ppp(5')N, the cap is called "CAP 0". If the 2'-O position of the first nucleotide of the mRNA is also methylated, forming m7GpppNm (also represented as m7G(5')ppp(5')(2'-OMeN)pN), it is called "CAP I". If the 2'-O positions of both the first and second nucleotides of the mRNA are methylated, becoming m7G-pppNmNm (also represented as m7G(5')ppp(5')(2'-OMeN)p(2'-OMeN)), it is called "CAP II". For example, a cap for mRNA produced by in vitro transcription is m7G(5')ppp(5')G, which has been used as a dinucleotide cap in transcription using T7 or SP6 RNA polymerase in vitro to obtain mRNA with a cap structure at its 5' end. Alternatively, a pre-formed dinucleotide form, m7G(5')ppp(5')G (“m7GpppG”), can be used as a transcription initiator for the in vitro synthesis of capped mRNA. A pre-formed trinucleotide form, m7G(5')ppp(5')(2'-OMeA)pG, can also be used as a transcription initiator. Other cap analogs can be found, for example, in Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs with superi or translational properties”, RNA, 9:1108-1122 (2003).

[0141] As used herein, “chemically modified nucleosides” include modifications to nucleosides that differ from those chemical substances found in naturally occurring mRNA. For example, chemically modified nucleosides include, but are not limited to, any one or more of pseudouridine, N1-methyl-pseuuridine, 2-thiouridine, 4-thiouridine, 2-thio-1-methyl-1-diaza-pseuuridine, 2-thio-1-methyl-pseuuridine, 2-thio-5-azauridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-pseuuridine, 5-azauridine, dihydropseuuridine, 5-methoxyuridine, 5-methyluridine, and 2-methoxyuridine. Preferred chemically modified nucleosides are pseudouridine, which can enhance mRNA stability and translational ability, as well as reduce immunogenicity in vivo. See, for example, Molecular Therapy 16(11): 1833-1840 (2008). The methods and techniques for modifying nucleotides are well known to those skilled in the art.

[0142] Sequence identity refers to the overall correlation between polymer molecules, such as the overall correlation between oligonucleotide molecules (e.g., DNA and / or RNA molecules) and / or polypeptide molecules. The percentage of identity between two polynucleotide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and identical sequences can be ignored for non-comparison purposes). Molecules are identical at that position when a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of shared identical positions, taking into account the number of gaps and the length of each gap, which need to be introduced to achieve optimal alignment of the two sequences. Sequence comparison and determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. Non-limiting examples of algorithms suitable for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Nuc. Acids Res., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol., 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). Another example is global alignment algorithms for determining the percentage of sequence identity, such as the Needleman-Wunsch algorithm for aligning protein or nucleotide (e.g., RNA) sequences. Attached Figure Description

[0143] Figure 1 shows the stability study of DNA plasmids containing different polyA molecules.

[0144] Figure 2 shows the expression level of COL17A1 protein in cells after transfection with mRNAs encoding different sequences of collagen.

[0145] Figure 3 shows the HE and Masson staining results of a mouse model of skin photoaging.

[0146] Figure 4 shows the effect of mRNA encoding collagen on dermal collagen fiber content in a mouse model of skin photoaging.

[0147] Figure 5 shows the effect of mRNA encoding collagen on dermal thickness in a mouse model of skin photoaging. Specific Implementation

[0148] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0149] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the nucleotide sequences described in the instructions are written from the 5' to the 3' end, and the amino acid sequences are written from the amino terminus to the carboxyl terminus. In case of discrepancies between the sequences in the instructions and the sequence listing, the sequences described in the instructions shall prevail.

[0150] Example 1: Preparation of DNA plasmid encoding collagen mRNA and the mRNA

[0151] 1.1 Synthesis of collagen sequences and construction of recombinant vectors

[0152] Design and synthesize DNA fragments containing the ORF sequence encoding collagen for transcription to obtain the corresponding mRNA. In the examples of this application, DNA fragments for mRNA transcription were constructed and prepared. The fragments sequentially included, from the 5' end to the 3' end: XbaI restriction site sequence (TCTAGA), T7 promoter sequence, transcription start site sequence (AGG), 5' UTR sequence (SEQ ID NO: 6), ORF sequence encoding collagen (SEQ ID NO: 2), 3' UTR sequence (SEQ ID NO: 7), sequence encoding poly A tail, SapI restriction site sequence (CGAAGAGC), and NotI restriction site sequence (GCGGCCGC).

[0153] The above DNA fragment was digested with XbaI and NotI, and ligated with the pUC57-GW-Kan (GenScript) vector backbone fragment digested with XbaI and NotI to construct recombinant plasmids respectively containing each COL17A1 collagen coding sequence.

[0154] 1.2 Screening of poly A elements of mRNA encoding collagen COL17A1

[0155] During the previous research, the inventors found that using the known 120A tail (120 consecutive As) in the mRNA encoding collagen would cause defects such as base deletion during DNA plasmid replication, resulting in instability. Therefore, the inventors developed a poly A element, RG2, which can significantly improve the replication stability of DNA plasmids.

[0156] After the constructed vector plasmid was confirmed to be correct by sequencing, it was transferred into Escherichia coli DH5α. The transformed plate was grown at 30°C, and plasmid extraction and sequencing were completed. After sequencing, the stability and base deletion of different poly(A) variants were analyzed and calculated according to the sequencing results; the replication stability was represented by the proportion of clones with no base changes, and the higher the proportion, the higher the replication stability of the plasmid in Escherichia coli. Finally, chi-square test was used for data statistics. The results showed that the replication stability of COL17A1-RG2 was significantly higher than that of COL17A1-A120 (89.0% V.S. 70.8%, p<0.01, χ2 test).

[0157] Table 1. Evaluation of the replication stability of DNA plasmids encoding collagen by different poly A

[0158] 1.3 Preparation of mRNA

[0159] 1.3.1 Plasmid linearization

[0160] The recombinant plasmids prepared in Example 1.1 were linearized by digestion with the restriction endonuclease SapⅠ at 37°C for 3 hours. The reaction system is shown in Table 2.

[0161] Table 2. Plasmid linearization enzyme digestion system

[0162] After the reaction was completed, 2 μL of the enzyme digestion product was subjected to 1% agarose gel electrophoresis to detect the linearization of the plasmids. Subsequently, each linearized recombinant plasmid was purified using a PCR product recovery kit (Comway Century).

[0163] 1.3.2 In vitro transcription and purification of mRNA products

[0164] Using the linearized recombinant plasmid obtained in Example 1.3.1 as a template, in vitro transcription was performed using a High Yield T7RNA Synthesis Kit (Shanghai Zhaowei Technology Development Co., Ltd., product catalog number ON-040) according to the instructions, with the reaction time being 37°C for 3 hours. The transcription system is shown in Table 3.

[0165] Table 3. In vitro transcription system

[0166] Among them, ΨUTP (100mM) is pseudouridine triphosphate, also represented as N1-Me-pUTP.

[0167] CleanCap AG is m7G(5')ppp(5')(2'-OMeA)pG, used as the transcription initiation primer.

[0168] After transcription, 1 μL of DNase I was added, and the mixture was incubated at 37°C for 15 min. Then, 15 μL of ammonium acetate stop solution was added and mixed well. Next, 1 / 3 volume of 7.5 M Lithium Chloride (LiCl) precipitation solution was added (to a final concentration of 2.5 M), and the mixture was incubated at -20°C for 30 min. The mixture was centrifuged at 12000 g for 15 min, and the RNA precipitate was discarded. 1 mL of 70% ethanol was added to wash the RNA, and the mixture was centrifuged at 12000 g for 5 min, discarding the supernatant. After drying, 50 μL of RNase-free water was added to dissolve the precipitate, and mRNA quantification was performed using a UV spectrophotometer to obtain 5'-capped mRNA encoding collagen.

[0169] 1.4 Optimization of Nucleic Acid Sequences

[0170] The ORF sequence (SEQ ID NO: 2) encoding COL17A1 collagen in Section 1.1 (hereinafter referred to as Optimized Group 1) was obtained through nucleic acid sequence optimization. To verify its performance advantage over other sequences, we optimized another ORF sequence (SEQ ID NO: 4) (hereinafter referred to as Optimized Group 2) based on the wild-type ORF sequence (SEQ ID NO: 3) encoding COL17A1 collagen, and compared the expression levels of the three sequences. The specific process is as follows:

[0171] (1) 293T cells were seeded in 6-well plates, with 5 x 10 cells per well. 5 100 cells. Transfection was performed 24 hours later when the cell mixture reached approximately 70%-80%.

[0172] (2) Mix 2 μg mRNA (the plasmid was constructed according to the method in Section 1.1, the difference being that the corresponding ORF sequence was replaced and the mRNA was prepared according to Section 1.3) with 4 μl of transfection reagent Lipofectamine™ 3000 and let stand at room temperature for 15 min.

[0173] (3) Add mRNA and Lipofectamine™ 3000 to 293T cells;

[0174] (4) Cell lysis was performed 24 hours after transfection: the culture medium was removed, the cells were washed once with pre-cooled PBS, and 200 μl of cell lysis buffer was added to each well.

[0175] (5) Western Blot: Based on the protein concentration determination results, 5 μg of each sample was loaded and separated on a 4-12% gradient gel. After electrophoresis, the protein samples were transferred to a 0.22 μm nitrocellulose membrane by wet transfer, and then co-incubated with COL17A1 specific antibody (primary antibody) and fluorescent secondary antibody in sequence before detection. Imaging was performed on the Bio-Rad gel imaging system ChemiDoc™ MP Imaging System.

[0176] A multi-parameter optimization design approach was employed to improve sequence stability and gene expression efficiency by optimizing key parameters such as Codon usage bias, GC content, and mRNA secondary structure. Furthermore, the optimization process incorporated customized requirements for specific patterns, including optimizing Shine-Dalgarno or Kozak sequences to enhance gene expression efficiency, and modifying RNA instability motifs to regulate expression efficiency. Simultaneously, sequence stability was further ensured by eliminating or inserting restriction enzyme sites and reducing repetitive sequences (direct repeats, inverted repeats, and dimer repeats). The aim was to obtain an optimized nucleic acid sequence that balances RNA stability and expression efficiency through this comprehensive optimization strategy.

[0177] As shown in Figure 2, the expression level of optimized group 1 (corresponding ORF is SEQ ID NO:2) is more than 1.5 times that of the wild-type group (corresponding ORF is SEQ ID NO:3), while the expression level of optimized group 2 (corresponding ORF is SEQ ID NO:4) is only about 20% of that of the wild-type group.

[0178] It is evident that the nucleic acid sequence with ORF sequence SEQ ID NO:2 exhibits better expression performance.

[0179] 1.5 Preparation of polynucleotide complexes

[0180] For example, the full-length mRNA sequence used to translate and generate COL17A1 in this application embodiment is shown in SEQ ID NO: 5 (from the transcription start site sequence AGG to the poly A tail).

[0181] The mRNA stock solution obtained in step 1.3.2 was dispersed separately in 20 mM acetic acid solution (pH 5.0) and adjusted to a final concentration of 200 μg / mL. The mixture was then prepared by mixing ionizable lipids (compound 5A): cholesterol: DSPC: DMG-PEG2000 at a molar ratio of 40–55: 35–45: 10–15: 0.5–2.5 to form a lipid mixture. The flow rates of the aqueous and oil phases were controlled using a T-junction method to mix the mRNA with the lipid mixture separately, with a total flow rate of 12–120 mL / min and a flow rate ratio (aqueous phase: organic phase) of 3:1. The syringe pump was started to mix the mRNA solution with the lipid mixture to form an LNP solution. Subsequently, the solution was diluted 1–5 times with 2 mmol / L acetic acid solution, concentrated by ultrafiltration centrifugation, and subjected to three solution replacements. The pH of the obtained solution was adjusted to 7.0–8.0 with Tris aqueous solution to obtain the LNP-loaded mRNA solution. In addition, empty LNPs without mRNA were prepared using the same steps as a control.

[0182] The concentration of mRNA loaded in LNPs and the particle size of LNPs were determined using a Ribogreen RNA quantification kit (Invitrogen, R11490) and a Malvern ZetaSizer particle size analyzer, respectively. Characterization data of exemplary LNPs are shown in Table 4.

[0183] Table 4. Characterization data of collagen mRNA-LNP

[0184] 1.6 Preparation of Compositions for Improving Skin Condition

[0185] Preparation of sodium hyaluronate-lidocaine hydrochloride phosphate buffer: Weigh the prescribed amounts of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate, sodium chloride, and lidocaine hydrochloride into a solution preparation bag. Add water and stir to dissolve. Then weigh the prescribed amount of sodium hyaluronate and add it to the solution. Add water to the total volume and stir until the sodium hyaluronate is completely swollen.

[0186] Mixing: The polynucleotide complex was mixed with sodium hyaluronate-lidocaine hydrochloride phosphate buffer. The resulting solution was filtered through two stages of 0.22 μm PES sterile filters and then poured into washed and dried vials. After capping, crimping, and light inspection, the product was considered finished. The composition had an osmotic pressure of 270 mOsmol / kg to 350 mOsmol / kg and a pH of 7.0 to 7.6. Table 5 shows the formulation of the composition.

[0187] Table 5. Product Composition:

[0188] Example 2: In vivo verification of collagen mRNA

[0189] 2.1 Construction of a mouse model of skin photoaging

[0190] The mouse model of skin photoaging was constructed according to the method described in the reference (Yi You et al., Intradermally delivered mRNA encapsulating extracellular vesicles for collagen-replacement therapy. Nat. Biomed. Eng., 2023). Specifically, 10-12 week old female nude mice were subjected to UVB irradiation of their back skin every other day for a total of 8 weeks to establish the mouse model of skin photoaging. Specifically, mice were anesthetized with 1.5% isoflurane, and a UV lamp (Philips; a 311nm UVB lamp) was placed 30 cm away from the back of the mouse for irradiation. The UV irradiation dose was monitored using a UV illuminometer. The specific irradiation doses were: 60 mJ / cm² per irradiation for the first two weeks, 120 mJ / cm² per irradiation for the third week, 180 mJ / cm² per irradiation for the fourth week, and 240 mJ / cm² per irradiation for the fifth to eighth weeks.

[0191] After irradiation, the mouse skin tissue was stained with HE and Masson staining. Based on the staining results, the skin tissue structure and the distribution and morphology of collagen fibers were evaluated and analyzed to verify the successful establishment of the photoaging mouse model. The results are shown in Figure 3. Compared with normal mouse skin that had not undergone UV irradiation, the irradiated mice exhibited typical photoaging tissue structure characteristics: thickened epidermis, smoothed wavy connections between the epidermis and dermis, disappearance of papillae and dermal papillae, and abnormal changes in dermal collagen fibers such as disordered arrangement, uneven distribution, degeneration, and breakage. These results indicate that the photoaging mouse model of skin in this embodiment was successfully established, producing the expected pathological structure, and can be used for in vivo efficacy evaluation.

[0192] 2.2 Evaluation of the anti-aging effect of mRNA encoding collagen

[0193] Fifteen mice that successfully developed the aging model as described in Section 2.1 were randomly divided into three groups of five each. Five normal mice that did not undergo the aging model were selected as negative controls. According to the groupings shown in Table 6, mice were intradermally injected with either LNP prepared in Table 4 of Example 1.5 or the product prepared in Table 5 of Example 1.6 on days 0, 4, 7, 14, and 21. The normal control group received no treatment, while the model control group received intradermal injections of PBS. On day 28, skin samples were collected from the mice and stained with HE and Masson's stain.

[0194] Table 6. Grouping of mice for evaluating anti-aging effects

[0195] Digital images are acquired by scanning tissue sections to observe changes in histopathological structure.

[0196] Masson staining of skin collagen fiber percentage: 10X fields of view were randomly selected, and collagen fibers were selected using ImageJ image analysis software. The percentage of collagen fiber area to the total skin tissue area was calculated. Statistical analysis of the significance between data groups was performed using the two-tailed unpaired t-test in GraphPad (*p<0.05, **p<0.01).

[0197] The analysis results are shown in Figure 4. Compared with normal mice, the proportion of collagen fibers in the model control group was reduced, confirming the successful construction of the mouse model. Compared with the model control group, the dermal collagen fiber content of mice in both the COL17A1 nucleic acid complex group (p<0.01) and the COL17A1 combination group (p<0.05) was significantly increased. These results indicate that COL17A1-mRN can effectively alleviate collagen fiber loss caused by ultraviolet radiation in vivo.

[0198] The analysis results are shown in Figure 5. Compared with normal mice, the dermal thickness of the model control group was significantly reduced (p<0.01), further confirming that the model mice have obvious photoaging pathological characteristics. Compared with the model control group, the dermal thickness of both the COL17A1 nucleic acid complex group (p<0.01) and the COL17A1 combination group (p<0.001) was significantly increased. This result indicates that COL17A1-mRNA has a mitigating effect on the decrease in dermal thickness due to photoaging in the in vivo disease model.

[0199] The results above indicate that mRNA encoding COL17A1 can effectively alleviate symptoms of skin aging.

[0200] The sequences used in the above embodiments of this application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences for the embodiments of this application and are not intended to limit the scope of this application. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences, and when they represent RNA sequences, "T" represents uridine.

[0201] sequence list

Claims

1. A composition for improving skin condition, comprising a polynucleotide complex, an active moisturizing ingredient, a local anesthetic ingredient, a pH buffering ingredient, an osmotic pressure regulator, and water, or thereof comprising a polynucleotide complex, an active moisturizing ingredient, a local anesthetic ingredient, a pH buffering ingredient, an osmotic pressure regulator, and water; The polynucleotide complex comprises a polynucleotide and a polynucleotide protector, wherein the polynucleotide is an mRNA sequence encoding collagen COL17A1, the polynucleotide is encapsulated by the polynucleotide protector, and the mRNA sequence comprises a nucleotide sequence as shown in SEQ ID NO: 2, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with the nucleotide sequence shown in SEQ ID NO:

2.

2. The composition according to claim 1, wherein the mRNA sequence further comprises an operatively linked 5'-UTR, 3'-UTR, and / or poly-A tail.

3. The composition according to any one of claims 1-2, wherein the poly-A tail comprises a polynucleotide sequence as shown in SEQ ID NO:8, or comprises a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:

8.

4. The composition according to any one of claims 1-3, wherein the 5'-UTR comprises a nucleotide sequence as shown in SEQ ID NO:6, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:6, and / or the 3'-UTR comprises a nucleotide sequence as shown in SEQ ID NO:7, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:

7.

5. The composition according to any one of claims 1-4, wherein the mRNA sequence comprises or is a nucleotide sequence as shown in SEQ ID NO: 5, or comprises or is a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with the nucleotide sequence shown in SEQ ID NO:

5.

6. The composition according to any one of claims 1-5, wherein the mRNA comprises at least one chemically modified nucleoside; optionally, the chemically modified nucleoside is selected from chemically modified uridine; optionally, part or all of the uridine in the mRNA is chemically modified uridine; preferably, all the uridine in the mRNA is chemically modified uridine.

7. The composition according to claim 6, wherein the chemically modified uridine is selected from any one or more of pseudouridine, N1-methyl-pseudouridine, 2-thiouridine, 4-thiouridine, 2-thio-1-methyl-1-diaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-azauridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 5-methyluridine, and 2-methoxy-uridine, preferably, the chemically modified uridine is pseudouridine or N1-methyl-pseudouridine.

8. The composition according to any one of claims 1-7, wherein the mRNA further comprises a 5' cap structure; preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

9. The composition according to any one of claims 1-8, wherein the polynucleotide protector is selected from liposomes, liposome nanoparticles (LNPs), sol-gels and nanogels; preferably, the polynucleotide protector is a lipid nanoparticle (LNP), the lipid nanoparticle comprising ionizable lipids, neutral lipids, structural lipids, and PEG lipids.

10. The composition according to any one of claims 1-9, wherein the ionizable lipid is selected from compound 5 or its stereoisomer.

11. The composition according to any one of claims 1-10, wherein the neutral lipid is a phospholipid; preferably, the phospholipid is selected from, but not limited to, one or more of, the following: dilauroyl lecithin (DLPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DUPC), palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-di-O-octadecyl-sn-glycerol-3-phosphate choline (18:0Diether PC), 1-oleoyl-2-cholestyl dimethylsuccinate-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-divinyl-sn-glycerol-3-phosphate choline, 1,2-diaryl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diaryl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt (DOPG) or sphingomyelin, preferably, the neutral lipid is DSPC and / or DOPE.

12. The composition according to any one of claims 1-11, wherein the structural lipid is selected from cholesterol, coccosterol, sitosterol, ergosterol, stigmasterol, corticosteroids or combinations thereof; preferably, the structural lipid is cholesterol.

13. The composition according to any one of claims 1-12, wherein the PEG lipid is a lipid modified with polyethylene glycol (PEG); preferably, the PEG lipid is selected from, but not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof; more preferably, the PEG lipid is selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid; even more preferably, the PEG lipid is DMG-PEG2000.

14. The composition according to any one of claims 1-13, wherein the molar ratio of the ionizable lipid, the neutral lipid, the structural lipid, and the PEG lipid in the polynucleotide protector is preferably (20-60):(5-25):(25-55):(0.5-5); preferably, the molar ratio of the ionizable lipid, the neutral lipid, the structural lipid, and the PEG lipid is (40-55):(10-15):(35-45):(0.5-2.5).

15. The composition according to any one of claims 1-14, wherein the molar ratio of the ionizable lipid to the polynucleotide in the polynucleotide complex is 5.2:

1.

16. The composition according to any one of claims 1-15, wherein the polynucleotide is present in a concentration of 0.4-200 μg / ml, 1-200 μg / ml, 5-100 μg / ml, or 5-50 μg / ml; preferably 15 μg / ml.

17. The composition according to any one of claims 1-16, wherein the concentration of the active moisturizing ingredient in the composition is 1-10 mg / ml, preferably 5 mg / ml; preferably the active moisturizing ingredient is selected from hyaluronic acid or its salt, and more preferably sodium hyaluronate.

18. The composition according to any one of claims 1-17, wherein the concentration of the local anesthetic component in the composition is 1-10 mg / ml, preferably 3 mg / ml; preferably the local anesthetic component is selected from lidocaine, lucaine, tetracaine, bupivacaine, ropivacaine or salts thereof, and more preferably lidocaine hydrochloride.

19. The composition according to any one of claims 1-18, wherein the pH buffering component is selected from sodium dihydrogen phosphate or its hydrate and / or disodium hydrogen phosphate or its hydrate; preferably, the pH buffering component is selected from sodium dihydrogen phosphate monohydrate and / or disodium hydrogen phosphate dodecahydrate.

20. The composition according to any one of claims 1-19, wherein the pH of the composition is 7.0-7.

6.

21. The composition according to any one of claims 1-20, wherein the osmotic pressure regulator is sodium chloride.

22. The composition according to any one of claims 1-21, wherein the osmotic pressure of the composition is 270 mOsmol / kg to 350 mOsmol / kg.

23. The composition according to any one of claims 1-22, wherein the water is water for injection.

24. A method of supplementing collagen in a subject in need, the method comprising administering to the subject the composition of any one of claims 1-23.

25. A method for improving skin aging in a subject in need, the method comprising administering to the subject the composition of any one of claims 1-23.

26. A method for increasing and / or improving at least one of the texture, smoothness, elasticity, or tension of the skin of a subject in need, said method comprising administering to the subject the composition of any one of claims 1-23.

27. A method for reducing the occurrence of one or more superficial pits in the skin in a subject in need, the method comprising administering to the subject the composition of any one of claims 1-23.

28. The method of claim 25, wherein, The skin aging referred to is skin aging caused by increasing age or skin aging caused by ultraviolet light or sunlight exposure.

29. The method of claim 26, wherein, The increase or improvement in skin texture, smoothness, elasticity and / or tension is selected from: (a) fine lines and / or wrinkles are treated, reduced and / or prevented compared with before application; (b) skin pore size is reduced; (c) Improved skin thickness, fullness, and / or firmness; (d) Improved skin smoothness, suppleness, and / or softness; (e) Improved skin tone, radiance, and / or translucency; (f) Improved procollagen and / or collagen production; (g) Improved skin texture and / or enhanced retexturation; (h) Improved appearance of skin contour. (i) Skin luster and / or radiance are restored; (j) Improvement of skin appearance due to aging and / or menopause; (k) Improvement of skin hydration; (l) Increased skin elasticity and / or resilience; (m) Treatment, reduction and / or prevention of skin sagging; (n) Improvement of skin firmness; (o) Reduction of pigmentation spots, freckled skin and / or scars; (p) Improvements in the skin's optical properties in terms of light diffraction or reflection; or (q) any combination thereof.

30. The method of claim 27, wherein, One or more superficial depressions in the skin are selected from the group consisting of: nasolabial folds, crow's feet, frown lines, worry lines, scars, frown lines, drooping eyebrows, tear troughs, nasolabial folds, bunny lines, drooping cheeks / midface, marionette lines, poppy dimpling, smile lines, laugh lines, chin wrinkles, neck wrinkles, platysma bands, and any combination thereof.

31. The method according to any one of claims 24-30, wherein, The subjects were human.

32. The method according to any one of claims 24-30, wherein, The application method is selected from percutaneous, subcutaneous, intradermal, and / or superficial injection methods.

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