Lipid nanoparticle system for delivering nucleic acid in cosmetics and cosmetic composition

Abstract

The invention discloses a lipid nanoparticle system for delivering nucleic acid in cosmetics and a cosmetic composition. Skin care is enhanced by delivery of nucleic acids using lipid nanoparticles (LNPs). According to the present invention, the traditional pharmaceutical lipid delivery system is adopted, such that the transdermal problem of cosmetics in skin cells is fundamentally solved, and the overexpression of effective components in the skin cells is achieved so as to improve the beneficial skin cytokine activity and inhibit the harmful skin cytokine activity.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetic chemistry and biotechnology, and relates to a lipid nanoparticle system and a cosmetic composition for delivering nucleic acids in cosmetics; specifically, the delivery mechanism of the lipid nanoparticle system is used in cosmetic applications, and nanotechnology is used to deliver nucleic acid cosmetic active substances to solve the problem of cosmetic transdermal properties in skin cells and to achieve the inhibition or overexpression of active ingredients in skin cells. Background Art

[0002] The cosmetics industry has traditionally focused on developing products designed to improve the appearance and health of the skin using a variety of active ingredients, including vitamins, antioxidants, peptides, and growth factors. However, the effectiveness of these ingredients is often limited by their stability in the formulation and their ability to penetrate the skin barrier to reach the target cells. To overcome the limitations of skin penetration, the industry has explored various transdermal drug delivery systems, including liposomes, microemulsions, and solid lipid nanoparticles, which improve the delivery of small molecules and peptides. These systems are not effective for larger, more complex molecules such as nucleic acids, which have the potential to regulate gene expression and thus have a more profound impact on skin health.

[0003] Nucleic acids, especially RNA and DNA fragments, have emerged as potent molecules that regulate skin function at the genetic level. They affect cell growth, differentiation, pigmentation, and the aging process by regulating gene expression. Their use in cosmetics aims to achieve effects such as improved skin hydration, reduced pigmentation, and anti-aging. Despite their potential, nucleic acids still face some challenges in their effective use in cosmetic formulations, such as stability and permeability. Nucleic acids are easily degraded by nucleases in the skin and the environment, which rapidly reduces their efficacy. At the same time, nucleic acids have difficulty penetrating the outermost layer of the skin, the stratum corneum, due to their large size and hydrophilicity. Therefore, a delivery system is needed to protect these active substances and help them enter skin cells.

[0004] In the pharmaceutical industry, lipid nanoparticles (LNPs) have revolutionized the delivery of nucleic acids, as evidenced by their successful use in mRNA vaccines and gene therapy. LNPs can encapsulate nucleic acids, protect them from degradation, and promote cell-specific targeting and uptake. This technology has been optimized for stability, biocompatibility, and controlled release, but until now, it has not been widely used in cosmetics.

[0005] Most current cosmetic products fail to fundamentally alter the biological processes of aging and skin damage. While some products may contain ingredients such as epidermal growth factor (EGF) that have genetic influences, these ingredients are often applied in a way that does not ensure their stability or proper delivery to target cells in the skin. As a result, the effects are often superficial and temporary. As consumer awareness and demand for scientifically-backed, more effective skin care products continues to increase, the cosmetics industry is at a crossroads, with a clear market need for advanced delivery systems that can reliably deliver complex bioactive molecules such as nucleic acids into skin cells. Such systems would represent a major leap forward in cosmetic technology, providing products that not only improve the appearance of the skin, but also improve the underlying health and function of the skin.

[0006] Cosmetic technology has made great strides in ingredient formulation and skin penetration, but has yet to fully exploit the potential of nucleic acid-based interventions. The pharmaceutical industry's success with LNPs provides an opportunity to fill this gap. The new methods for applying LNP technology to the cosmetic field are intended to address these challenges by providing an innovative system that can effectively deliver nucleic acids to skin cells, thereby providing a new dimension for skin care products that operate at the genetic level. Summary of the invention

[0007] To solve the above problems, the present invention aims to provide a lipid nanoparticle system for delivering nucleic acids in cosmetics, which uses lipid nanoparticles (LNPs) to encapsulate nucleic acids and deliver them directly to skin cells through cosmetic formulations. This technology takes advantage of the advances in nanoparticle delivery systems originally developed for pharmaceutical applications (such as mRNA vaccines and gene therapy) and applies them to cosmetics; its main purpose is to target specific gene pathways related to skin health, aging and repair, and improve the efficacy of cosmetic treatments.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] The present invention relates to a cosmetic composition comprising

[0010] Lipid nanoparticles (LNPs);

[0011] Nucleic acids encapsulated in the LNPs;

[0012] Wherein, the nucleic acid is used to regulate the expression of genes related to skin conditions.

[0013] As one embodiment of the present invention, the nucleic acid is used to treat skin aging, pigmentation or inflammation, or in other words, to regulate the expression of genes related to skin aging, pigmentation or inflammation.

[0014] As an embodiment of the present invention, the composition is formulated into any one of a cream, a gel, a serum or an emulsion.

[0015] As an embodiment of the present invention, the average diameter of the lipid nanoparticles LNPs is 50-200 nm.

[0016] As one embodiment of the present invention, lipid nanoparticles LNPs are used in the cosmetic composition to deliver nucleic acids to improve the appearance of the skin.

[0017] As an embodiment of the present invention, the cosmetic composition delivers nucleic acid to skin cells, wherein the delivery promotes overexpression of gene protein. In some implementation examples, after the cosmetic composition is applied to the skin, the nucleic acid is delivered to the skin cells to promote overexpression of gene protein.

[0018] As an embodiment of the present invention, the cosmetic composition delivers nucleic acid to skin cells, mainly acting on epidermal and dermal cells of human skin, including fibroblasts and keratinocytes.

[0019] As one embodiment of the present invention, the cosmetic composition is prepared by a method comprising the following steps:

[0020] S1, forming lipid nanoparticles LNPs by combining lipids in a solvent;

[0021] S2, encapsulating nucleic acids in lipid nanoparticles LNPs;

[0022] S3. Adding LNPs encapsulated with nucleic acid into the cosmetic matrix.

[0023] As one embodiment of the present invention, the step of purifying the lipid nanoparticles to remove unencapsulated nucleic acids is also included before S3.

[0024] As an embodiment of the present invention, the nucleic acid-based chemically active substances in S2 are at least partially encapsulated inside the lipid nanoparticles LNPs.

[0025] As an embodiment of the present invention, the encapsulation step in S2 includes using a pipette method, a vortex method and a microfluidic instrument method to ensure that the nucleic acid is evenly distributed in the lipid nanoparticles.

[0026] As an embodiment of the present invention, S3 further includes adjusting the pH value of the cosmetic composition to improve the stability of the lipid nanoparticles.

[0027] As one embodiment of the present invention, the composition of LNPs includes cationic lipids, auxiliary lipids, neutral lipids and cholesterol.

[0028] As an embodiment of the present invention, the cationic lipid is a compound as shown in Formula I:

[0029]

[0030] in:

[0031] Side chain part -W1-L1-(R1) n1 and-W2-L2-(R2) n2 The main chain (i.e., the longest chain) of each independently has 1-40 carbons; preferably, it has 6-24 carbons, and more preferably, it has 12-22 carbons;

[0032] W1 and W2 are each independently C1-C14 alkylene, C2-C14 alkenylene or C2-C14 alkynylene. Preferably, W1 and W2 are each independently C3-C8 alkyl, C3-C8 alkenyl or C3-C8 alkynyl;

[0033] L1 and L2 are each independently selected from the following structures:

[0034]

[0035] Preferably, L1 and L2 are each independently selected from the following structures:

[0036]

[0037] R1 and R2 are each independently H, C3-C22 alkyl, C3-C22 alkenyl, C3-C22 alkynyl, C5-C8 cycloalkyl, C5-C8 cycloalkenyl. Preferably, R1 and R2 are each independently H, C6-C22 alkyl, C6-C22 alkenyl, C6-C22 alkynyl, more preferably C6-C14 alkyl, C6-C14 alkenyl, C6-C14 alkynyl. n1 and n2 are each independently 0, 1 or 2.

[0038] As an embodiment of the present invention, in the compound shown in formula I, R3 and R4 are each independently C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, and the alkyl, alkenyl or alkynyl is arbitrarily substituted by one or more OH, C1-C4 alkoxy, or R3 and R4 are combined with each other to form a 5-8 membered heterocyclic ring, and in addition to the N atom connected to M, the heterocyclic ring optionally includes one or more heteroatoms of N, O, and S. Preferably, R3 and R4 are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 2-hydroxyethyl, 3-hydroxypropyl, or R3 and R4 are combined with each other to form one of the following structures:

[0039]

[0040] Wherein, R is selected from one of C(Ra)2, NRb, O, and S;

[0041] Ra are each independently H, OH, C1-12 alkyl;

[0042] Rb are each independently H, C1-12 alkyl, C(O)C1-12 alkyl;

[0043] a1 is 1, 2, 3, 4, 5, 6, 7 or 8; a2 is 1, 2, 3, 4, 5 or 6; a3 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0044] Among them, R3 and R4 are combined with each other to form tetrahydropyrrol-1-yl, piperidin-1-yl, piperazin-1-yl, 4-methylpiperazin-1-yl.

[0045] As an embodiment of the present invention, in the compound represented by formula I, M is C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene. Preferably, M is -(CH2) s -, s is 1, 2, 3, 4, 5 or 6.

[0046] As an embodiment of the present invention, in the compound shown in Formula I, the -MN(R3)(R4) has one of the following structures:

[0047]

[0048] As an embodiment of the present invention, in the compound represented by formula I,

[0049] The side chain part -W1-L1-(R1) n1 and-W2-L2-(R2) n2 Each is independently a structure preferably selected from the following group:

[0050]

[0051] R3 and R4 are each independently a C1-C6 alkyl group, the alkyl group is arbitrarily substituted by one or more OH groups, or R3 and R4 are combined with each other to form a 5- to 8-membered heterocyclic ring, which optionally includes one or more heteroatoms selected from N, O, and S in addition to the N atom connected to M;

[0052] M is a C1-C6 alkylene group.

[0053] As an embodiment of the present invention, among the compounds shown in Formula I, the compound is selected from the following group:

[0054]

[0055]

[0056]

[0057] As an embodiment of the present invention, the general synthesis method of the cationic lipid compound is:

[0058]

[0059] The cationic lipids of the present invention can be synthesized by the Ugi four-component reaction "one-pot method", specifically, aldehyde (A), amine (B), carboxylic acid (C) and isonitrile (D) compounds are reacted in a molar ratio of 1:1:1:1. Among them, aldehyde compounds can be purchased from commercial companies or can be obtained by commercial or simple method synthesis of primary alcohols by oxidation of pyridinium chlorochromate (PCC); isonitrile compounds can be purchased from commercial companies or can be obtained by commercial or simple method synthesis of primary amines and ethyl formate to obtain formamide compounds, and the formamide compounds are then dehydrated to obtain; amine and carboxylic acid compounds can be purchased from commercial companies:

[0060]

[0061] As an embodiment of the present invention, in the composition of LNPs, the amount of cationic lipids accounts for 20-55 mol%.

[0062] As one embodiment of the present invention, the auxiliary lipids of LNPs include auxiliary phospholipids; the auxiliary phospholipids include: DSPC, DMPE or DOPE.

[0063] As an embodiment of the present invention, the composition of LNPs further includes auxiliary phospholipids with a substance amount accounting for 8.5-33%.

[0064] As an embodiment of the present invention, the cholesterol includes cholesterol or its derivatives. In the composition of LNPs, the amount of cholesterol accounts for 20-66%.

[0065] As an embodiment of the present invention, the neutral lipid comprises PEG lipid. The PEG lipid comprises: DWG-PEG2000, ALC0159, DSPE-PEG2000 or C14-PEG2000.

[0066] As an embodiment of the present invention, in the composition of LNPs, the amount of neutral lipids accounts for 0.9%-4.1%.

[0067] As an embodiment of the present invention, the nucleic acid comprises any one of mRNA, siRNA, shRNA, DNA, and ds RNA. Preferably, the nucleic acid cosmetic active is mRNA.

[0068] As one embodiment of the present invention, the mRNA comprises at least one of non-replicating linear mRNA, self-replicating linear mRNA, and circular mRNA.

[0069] As an embodiment of the present invention, the mRNA species comprises one of COL1A1 mRNA, COL2A1 mRNA, COL3A1 mRNA, and COL17A1 mRNA. The preferred mRNA species is COL1A1 mRNA.

[0070] The method of applying the aforementioned cosmetic composition also falls within the scope of protection of the present invention. That is, a method of treating a skin condition of a subject in need thereof, comprising applying a therapeutically effective amount of the cosmetic composition to the subject's skin. For example, applying a therapeutically effective amount of the cosmetic composition to the subject's skin.

[0071] As one embodiment of the present invention, the skin condition treated is selected from the group consisting of wrinkles, loss of skin elasticity, and uneven skin tone.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] 1) By screening the results and proportions of each component of LNP, a good skin transdermal effect was achieved.

[0074] 2) By screening the results and proportions of each component of LNP, efficient delivery of collagen nucleic acid was achieved.

[0075] 3) By screening the results and proportions of each component of LNP, overexpression of collagen in human fibroblasts and keratinocytes was achieved.

[0076] 4) Through the protective effect of lipid nanoparticles, the degradation and inactivation of nucleic acid molecules are reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0078] Figure 1 Protein expression of COL1A1 mRNA encapsulated by LNPs constructed with different RD series cationic lipids at a ratio of 10:1 in human keratinocytes.

[0079] Figure 2 The delivery and protein expression effects of COL1A1 mRNA encapsulated by LNPs of different compositions in human keratinocytes at a fixed ratio;

[0080] Figure 3The delivery and protein expression effects of COL3A1 mRNA encapsulated by LNPs of different compositions in human keratinocytes at a fixed ratio;

[0081] Figure 4 The delivery and protein expression effects of COL17A1 mRNA encapsulated by LNPs of different compositions in human keratinocytes at a fixed ratio;

[0082] Figure 5 The delivery and protein expression effects of COL1A1 mRNA encapsulated by LNPs of different compositions in human fibroblasts at a fixed ratio;

[0083] Figure 6 The delivery and protein expression effects of LNP and COL1A1 mRNA in different ratios in human keratinocytes;

[0084] Figure 7 The delivery and protein expression effects of LNP and COL1A1 mRNA in different ratios in human fibroblasts;

[0085] Figure 8 To study the delivery and protein expression effects of LNPs made from different ionizable lipids in a fixed ratio and then encapsulated with COL1A1 mRNA in the same ratio in human keratinocytes.

[0086] Fig. 9 To study the delivery and protein expression effects of LNPs made from different ionizable lipids in a fixed ratio and then encapsulated with COL1A1 mRNA in the same ratio in human fibroblasts.

[0087] Fig.10 Samples of the preferred LNPs encapsulating CY5-labeled COL1A1 mRNA and common LNPs on the market

[0088] The transdermal test effect of samples encapsulating CY5-labeled COL1A1 mRNA. DETAILED DESCRIPTION

[0089] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0090] Example 1. Synthesis of ionizable lipid compounds

[0091] 1.1 Synthesis and characterization of compound 1

[0092]

[0093] 1) Synthesis of Compound 1A

[0094]

[0095] Octadecanol (1A-1, 1.0 g, 3.69 mmol) was dissolved in 30 mL of dichloromethane, and PCC (1.2 g, 5.55 mmol) was added, and the mixture was reacted at 25° C. for 3 hours. After the reaction, the solvent was removed by concentration, and the mixture was purified by silica gel column chromatography, with the eluent being petroleum ether and ethyl acetate in a ratio of 50 / 1 to 10 / 1 (v / v), to obtain product 1A (0.60 g, 95.0%).

[0096] 2) Synthesis of Compound 1D

[0097]

[0098] The compound octadecylamine (1D-1, 1.0 g, 3.71 mmol) was dissolved in 10 mL of chloroform, and ethyl formate (2.76 g, 37.1 mmol) was added, and the mixture was reacted at 60°C for 16 hours. After the reaction, the excess ethyl formate was removed by concentration, and the mixture was purified by silica gel column chromatography, with the eluent being petroleum ether and ethyl acetate in a ratio of 10 / 1 to 0 / 1 (v / v), to obtain the product 1D-2 (0.78 g, 70.0%);

[0099] Compound 1D-2 (0.78 g, 2.62 mmol) was dissolved in 25 mL of chloroform, pyridine (0.63 g, 7.87 mmol) was added, p-toluenesulfonyl chloride (0.68 g, 3.93 mmol) was added at 0°C, and the reaction was continued at 0°C with stirring for 1 hour. After the reaction was completed, a 5% sodium sulfate aqueous solution was added, stirred for 30 minutes, extracted with dichloromethane, concentrated, and the crude product was purified by silica gel column chromatography, the eluent was petroleum ether to ethyl acetate 0 / 1-20 / 1 (v / v), and the product 1D (0.42 g, 57.3%) was obtained.

[0100] 3) Synthesis of Compound 1

[0101]

[0102] Compound 1A (1.0 mmol) and compound 1B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 1D (1.0 mmol) were then added, and the mixture was heated to 40°C for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane: methanol 50 / 1-10:1 (v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 1. LCMS: [M+1]+ =839.6; 1 H NMR (400 MHz, CDCl3) δ = 6.64 (br s, 1H), 4.76-4.65 (m, 1H), 3.64-3.52 (m, 1H), 3.43-3.32 (m, 2H), 3.09 (s, 3H), 2.52-2.33 (m, 4H), 2.29-2.18 (m, 7H), 2.08-1.85 (m, 4H), 1.66-1.59 (m, 2H), 1.55-1.40 (m, 5H), 1.26 (br s, 62H), 0.89 (br t, J = 6.8 Hz, 6H), pale yellow solid.

[0103] 1.2 Synthesis and characterization of compound 2

[0104]

[0105] 1) Synthesis of Compound 2A

[0106]

[0107] Compound 2A-1 (1.0 g, 3.72 mmol) was dissolved in 30 mL of dichloromethane, and PCC (1.2 g, 5.59 mmol) was added, and the mixture was reacted at 25° C. for 3 hours. After the reaction, the solvent was removed by concentration, and the mixture was purified by column chromatography on silica gel with the eluent being petroleum ether to ethyl acetate 50 / 1-10 / 1 (v / v) to obtain product 2A (0.970 g, 98.0%).

[0108] 2) Synthesis of Compound 2D

[0109]

[0110] Compound oleylamine (2D-1, 1.0 g, 3.74 mmol) was dissolved in 10 mL of chloroform, ethyl formate (2.77 g, 37.4 mmol) was added, and the mixture was reacted at 60°C for 16 hours. After the reaction, the excess ethyl formate was removed by concentration, and the mixture was purified by column chromatography on silica gel, with the eluent being petroleum ether and ethyl acetate in a ratio of 10 / 1 to 0 / 1 (v / v), to obtain product 2D-2 (0.78 g, 71.0%).

[0111] Compound 2D-2 (0.78 g, 2.66 mmol) was dissolved in 25 mL of chloroform, pyridine (0.63 g, 7.97 mmol) was added, p-toluenesulfonyl chloride (0.76 g, 3.98 mmol) was added at 0°C, and the reaction was continued at 0°C with stirring for 1 hour. After the reaction was completed, a 5% sodium sulfate aqueous solution was added, stirred for 30 minutes, extracted with dichloromethane, concentrated, and the crude product was purified by column chromatography on silica gel, the eluent being petroleum ether to ethyl acetate 50 / 1-20 / 1 (v / v), to obtain product 2D (0.44 g, 60.0%).

[0112] 3) Synthesis of Compound 2

[0113]

[0114] Compound 2A (1.0 mmol) and compound 1B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 2D (1.0 mmol) were then added, and the mixture was heated to 40°C for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (20:1, v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 2. LCMS: [M+1] + =834.6; 1 H NMR (500 MHz, CDCl3) δ 6.64 (br s, J = 6.0 Hz, 1H), 5.45-5.24 (m, 4H), 4.71 (br t, J = 8.4 Hz, 1H), 3.67-3.53 (m, 1H), 3.43-3.27 (m, 2H), 3.22-3.07 (m, 4H), 2.97 -2.62 (m, 1H), 2.54-2.10 (m, 11H), 2.06-1.86 (m, 9H), 1.79-1.59 (m, 6H), 1.55-1.40 (m, 4H), 1.37-1.14 (m, 45H), 0.88 (t, J = 6.8 Hz, 6H), yellow oil.

[0115] 1.3 Synthesis and characterization of compound 3

[0116]

[0117] 1) Synthesis of compound 3A

[0118]

[0119] Compound 3A-1 (1.0 g, 3.30 mmol) and 5-hexen-1-ol (0.36 g, 3.60 mmol) were dissolved in 30 mL of dichloromethane, 4-dimethylaminopyridine (DMAP, 0.60 g, 4.96 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.95 g, 4.96 mmol) were added, and the mixture was stirred at 25°C for 1 hour. After the reaction was completed, the solvent was removed by concentration, and the mixture was purified by column chromatography on silica gel, with the eluent being petroleum ether to ethyl acetate 50 / 1-10 / 1 (v / v), to obtain compound 3A-2 (1.03 g, 81.0%);

[0120] Compound 3A-2 (1.03 g, 2.68 mmol) was dissolved in 20 mL of tetrahydrofuran, and tetrabutylammonium fluoride solution (4 mL, 4.0 mmol) was added, and the mixture was reacted at 25°C for 2 hours. After the reaction, the solvent was removed by concentration, and the mixture was purified by column chromatography on silica gel, with the eluent being petroleum ether and ethyl acetate in a ratio of 50 / 1 to 10 / 1 (v / v), to obtain product 3A-3 (0.69 g, 95.0%).

[0121] Compound 3A-3 (0.69 g, 2.54 mmol) was dissolved in 20 mL of dichloromethane, and PCC (0.83 g, 3.82 mmol) was added, and the mixture was reacted at 25° C. for 3 hours. After the reaction, the solvent was removed by concentration, and the mixture was purified by column chromatography on silica gel with the eluent being petroleum ether to ethyl acetate 50 / 1-10 / 1 (v / v) to obtain product 3A (0.53 g, 77.0%).

[0122] 2) Synthesis of Compound 3

[0123]

[0124] Compound 3A (1.0 mmol) and compound 1B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 1D (1.0 mmol) were then added, and the mixture was heated to 40°C for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (20:1, v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 3. LCMS: [M+1] + =839.5; 1H NMR(500MHz, CDCl3)δ6.62(br s,1H),5.78(ddt,J=16.9,10.2,6.7Hz,1H),5.05-4.91(m,2H),4.68(br s,1H),4.05(t,J=6.7Hz,2H),3.56(ddt,J=9.5,6.2,3.1Hz,1H),3.38-3.23(m,2H),3.21-3.06(m,4H), 2.49-2.33(m,3H),2.31-2.15(m,10H),2.09-2.05(m,2H),1.97-1.86(m,2H),1.76-1.40(m,17H),1.35 -1.13 (m, 40H), 0.87 (t, J=6.9 Hz, 3H), yellow oil.

[0125] 1.4 Synthesis and characterization of compound 4

[0126]

[0127] 1) Synthesis of compound 4A

[0128]

[0129] Compound 4A-1 (1.0 g, 3.30 mmol) and 7-tridecanol (0.72 g, 3.60 mmol) were dissolved in 30 mL of dichloromethane, 4-dimethylaminopyridine (DMAP, 0.60 g, 4.96 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.95 g, 4.96 mmol) were added, and the mixture was stirred at 25°C for 3 hours. After the reaction was completed, the solvent was removed by concentration, and the mixture was purified by silica gel column chromatography, with the eluent being petroleum ether to ethyl acetate 50 / 1-10 / 1 (v / v), to obtain compound 4A-2 (1.21 g, 76.0%);

[0130] Compound 4A-2 (1.21 g, 2.51 mmol) was dissolved in 20 mL of tetrahydrofuran, and tetrabutylammonium fluoride solution (3.76 mL, 3.76 mmol) was added, and the mixture was reacted at 25°C for 2 hours. After the reaction, the solvent was removed by concentration, and the mixture was purified by silica gel column chromatography, with the eluent being petroleum ether and ethyl acetate 50 / 1-10 / 1 (v / v), to obtain product 4A-3 (0.86 g, 92.1%);

[0131] Compound 4A-3 (0.86 g, 2.30 mmol) was dissolved in 20 mL of dichloromethane, and PCC (0.75 g, 3.46 mmol) was added, and the mixture was reacted at 25° C. for 3 hours. After the reaction, the solvent was removed by concentration, and the mixture was purified by silica gel column chromatography, with the eluent being petroleum ether and ethyl acetate 50 / 1-10 / 1 (v / v), to obtain product 4A (0.60 g, 70.0%).

[0132] 2) Synthesis of compound 4

[0133]

[0134] Compound 4A (1.0 mmol) and compound 1B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 1D (1.0 mmol) were then added, and the mixture was heated to 40°C for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (20:1, v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 4. LCMS: [M+1] + =939.6; 1 H NMR (500 MHz, CDCl3) δ 6.62 (br s, 1H), 4.85 (t, J = 6.3 Hz, 1H), 4.69 (br t, J = 7.8 Hz, 1H), 3.60-3.54 (m, 1H), 3.39-3.22 (m, 2H), 3.21-3.08 (m, 4H), 2.49-2.33 (m, 3H), 2.28-2.20 (m, 10H), 2.01 -1.84 (m, 2H), 1.81-1.54 (m, 9H), 1.55 -1.36 (m, 8H), 1.33 -1.13 (m, 56H), 0.88-0.85 (m, 9H), yellow oil.

[0135] 1.5 Synthesis and characterization of compound 5

[0136]

[0137] 1) Synthesis of compound 5D

[0138]

[0139] Tert-butyloxycarbonyl-11-aminoundecanoic acid (5D-1, 5.0 g, 16.6 mmol) and 7-tridecanol (3.66 g, 18.2 mmol) were dissolved in 80 mL of dichloromethane, 4-dimethylaminopyridine (DMAP, 3.04 g, 24.9 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 3.82 g, 19.9 mmol) were added, and the mixture was stirred at 25°C for 3 hours. After the reaction was completed, the solvent was removed by concentration, and the mixture was purified by column chromatography on silica gel, with the eluent being petroleum ether to ethyl acetate 50 / 1-10 / 1 (v / v), to obtain the product 5D-2 (7.9 g, 98.4%);

[0140] Compound 2B (7.9 g, 16.3 mmol) was dissolved in 100 mL of dichloromethane, trifluoroacetic acid (TFA, 77.0 g, 675 mmol) was added at 0°C, and the reaction was continued at 0°C for 30 minutes. After the reaction, the solvent was concentrated and the pH was adjusted to about 10 with saturated sodium carbonate aqueous solution, and the dichloromethane was extracted. The organic phases were combined and concentrated to obtain a crude product 5D-3 (5.9 g) which was directly used in the next step;

[0141] Compound 5D-3 (5.9 g, 15.4 mmol) was dissolved in 10 mL of chloroform, and ethyl formate (11.4 g, 154 mmol) was added, and the mixture was reacted at 60°C for 16 hours. After the reaction, the excess ethyl formate was removed by concentration, and the mixture was purified by column chromatography on silica gel, with the eluent being petroleum ether and ethyl acetate in a ratio of 10 / 1 to 0 / 1 (v / v), to obtain product 5D-4 (5.0 g, 78.9%);

[0142] Compound 5D-4 (2.5 g, 6.07 mmol) was dissolved in 25 mL of chloroform, pyridine (1.44 g, 18.2 mmol) was added, p-toluenesulfonyl chloride (1.74 g, 9.11 mmol) was added at 0°C, and the reaction was continued at 0°C with stirring for 1 hour. After the reaction was completed, a 5% sodium sulfate aqueous solution was added, stirred for 30 minutes, extracted with dichloromethane, concentrated, and the crude product was purified by column chromatography on silica gel, the eluent being petroleum ether to ethyl acetate 50 / 1-20 / 1 (v / v), to obtain product 5D (1.3 g, 54.4%).

[0143] 3) Synthesis of Compound 5

[0144]

[0145] Compound 2A (1.0 mmol) and compound 1B (1.0 mmol) were placed in a 10 mL reaction bottle, 5.0 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 5D (1.0 mmol) were then added, and the mixture was heated to 40° C. and reacted for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation using a silica gel chromatography plate.

[0146] Methanol (20:1, v / v) was used as the eluent and the product was purified by silica gel column chromatography to obtain the final product 5. LCMS: [M+1] + =991.8;

[0147] 1 H NMR (500MHz, CDCl3) δ6.66 (br s, 1H), 5.45-5.18 (m, 2H), 4.87 (p, J=6.

[0148] 3Hz,1H),4.69(br t,J=7.8Hz,1H),3.70-3.53(m,1H),3.40-3.23(m,2H),3.2

[0149] 0-3.08(m,4H),2.50-2.36(m,6H),2.29-2.25(m,4H),2.11 -1.85(m,6H),1.81-1.

[0150] 54 (m, 12H), 1.54-1.38 (m, 9H), 1.37-1.17 (m, 53H), 0.89-0.86 (m, 9H), yellow oil.

[0151] 1.6 Synthesis and characterization of compound 6

[0152]

[0153] 1) Synthesis of Compound 6A

[0154]

[0155] Compound 6A-1 (1.0 g, 3.75 mmol) was dissolved in 30 mL of dichloromethane, and PCC (1.2 g, 5.63 mmol) was added, and the mixture was reacted at 25° C. for 3 hours. After the reaction, the solvent was removed by concentration, and the mixture was purified by column chromatography on silica gel with the eluent being petroleum ether to ethyl acetate 50 / 1-10 / 1 (v / v) to obtain product 6A (0.94 g, 95.1%).

[0156] 2) Synthesis of Compound 6

[0157]

[0158] Compound 6A (1.0 mmol) and compound 1B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 h. Compound 1C (1.0 mmol) and compound 5D (1.0 mmol) were then added, and the temperature was raised to 40°C for 10 h. After the reaction was completed by TLC monitoring, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (20:1, v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 6. LCMS: [M+1] + =949.7; 1 H NMR (500 MHz, CDCl3) δ 6.64 (br s, 1H), 5.47-5.27 (m, 4H), 4.94-4.83 (m, 1H), 4.70 (br s, 1H), 3.65-3.54 (m, 1H), 3.44-3.22 (m, 2H), 3.21-3.08 (m, 4H), 2.82-2.66 (m, 2H), 2.54-2.36 (m, 4H), 2.34-2.20 (m, 8H), 2.08-1.87 (m, 6H), 1.78-1.58 (m, 7H), 1.56-1.41 (m, 8H), 1.39 -1.19 (m, 45H), 0.99-0.80 (m, 9H), yellow oil.

[0159] 1.7 Synthesis and characterization of compound 7

[0160]

[0161] 1) Synthesis of compound 7

[0162]

[0163] Compound 4A (1.0 mmol) and compound 1B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 5D (1.0 mmol) were then added, and the mixture was heated to 40° C. and reacted for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation using a silica gel chromatography plate.

[0164] Methanol (20:1, v / v) was used as the eluent and the product was purified by silica gel column chromatography to obtain the final product 7. LCMS [M+1] + =1053.

[0165] 8; 1 H NMR (400MHz, CDCl3) δ = 6.72-6.56 (m, 1H), 4.87 (quin, J = 6.0Hz, 2

[0166] H),4.79-4.63(m,1H),3.64-3.55(m,1H),3.43-3.28(m,2H),3.27-3.08(m,

[0167] 4H),2.52-2.34(m,8H),2.33-2.25(m,6H),1.99-1.90(m,10H),1.76-1.66(m,6H),1.51(br d,J=6.0Hz,10H),1.27(br d,J=6.8Hz,54H),0.88(br t,J=6.

[0168] 8Hz, 12H), yellow oil.

[0169] 1.8 Synthesis and characterization of compound 8

[0170]

[0171] 1) Synthesis of compound 8

[0172]

[0173] Compound 4A (1.0 mmol) and compound 8B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 5D (1.0 mmol) were then added, and the mixture was heated to 40°C for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (20:1, v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 8. All other compounds can be synthesized by the same method. LC-MS: [M+1] + =1081.9; 1 H NMR (400 MHz, CDCl3) δ = 6.62 (br s, 1H), 4.87 (t, J = 6.4 Hz, 2H), 4.71 (br t, J = 6.8 Hz, 1H), 3.41-3.23 (m, 2H), 3.21-3.09 (m, 4H), 2.61-2.38 (m, 8H), 2.28 (dt, J = 2.8, 7.6 Hz, 5H), 2.00-1.84 (m, 8H), 1.79-1.64 (m, 8H), 1.51 (br d, J = 6.0 Hz, 10H), 1.35-1.22 (m, 54H), 1.04 (br t, J = 6.8 Hz, 6H), 0.93-0.81 (m, 12H), yellow oil.

[0174] 1.9 Synthesis and characterization of compound 9

[0175]

[0176] 1) Synthesis of compound 9

[0177]

[0178] Compound 4A (1.0 mmol) and compound 5B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 5D (1.0 mmol) were then added, and the mixture was heated to 40°C for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (20:1, v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 9. LCMS: [M+1] + =1094.0; 1 H NMR (400MHz,CDCl3)δ=6.58(br s,1H),4.86-4.73(m,2H),4.61(br t,J=7.2Hz,1H),3.57-3.46(m,1H),3.34-3.15(m,2H),3.13-3.00(m,3H),2.29(br d,J=6.4Hz,6H),2.26-2.16(m,6H),1.92-1.78(m,3H),1.68(br s,4H),1.57-1.48(m,10H),1.47-1.33(m,16H),1.28-1.15(m,54H),0.89-0.76(m,12H),yellow oil.

[0179] 1.10 Synthesis and characterization of compound 10

[0180]

[0181] 1) Synthesis of compound 10

[0182]

[0183] Compound 4A (1.0 mmol) and compound 10B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 h. Compound 1C (1.0 mmol) and compound 5D (1.0 mmol) were then added, and the mixture was heated to 40 °C for 10 h. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (2

[0184] 0:1, v / v) as the eluent, and purified by silica gel column chromatography to obtain the final product 10. LCMS: [M+1] + =1108.7; 1 H

[0185] NMR (400MHz, CDCl3) δ = 6.57 (br s, 1H), 4.79 (quin, J = 6.4Hz, 2H), 4.63 (br

[0186] t,J=6.4Hz,1H),3.56-3.45(m,2H),3.35-3.17(m,3H),3.16-3.02(m,5H),2.45-2.35(m, 6H),2.30-2.24(m,8H),2.23-2.17(m,6H),1.91-1.80(m,4H),1.68-1.61(m,6H),1.43(br d,J=6.0Hz,12H),1.27-1.14(m,54H),0.81(t,J

[0187] =6.8Hz,12H), yellow oil.

[0188] 1.11 Synthesis and characterization of compound 11

[0189]

[0190] 1) Synthesis of compound 11

[0191]

[0192] Compound 4A (1.0 mmol) and compound 11B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 5D (1.0 mmol) were then added, and the mixture was heated to 40°C for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (20:1, v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 11. LCMS: [M+1] + =1109.3; 1H NMR (400 MHz, CDCl3) δ = 6.60 (br s, 1H), 4.87 (quin, J = 6.0 Hz, 2H), 4.71 (br s, 1H), 3.63-3.56 (m, 1H), 3.32-3.09 (m, 6H), 2.60 (br s, 2H), 2.56-2.24 (m, 10H), 2.00-1.86 (m, 2H), 1.73-1.57 (m, 14H), 1.56-1.45 (m, 14H), 1.27 (br d, J = 7.3 Hz, 54H), 01.09 (br s, 6H), 0.92-0.84 (m, 12H), yellow oil.

[0193] 1.12 Synthesis and characterization of compound 12

[0194]

[0195] 1) Synthesis of compound 12

[0196]

[0197] Compound 4A (1.0 mmol) and compound 12B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 5D (1.0 mmol) were then added, and the mixture was heated to 40°C for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (20:1, v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 12. LCMS: [M+1] + =1113.8; 1 H NMR (400MHz, CDCl3) δ = 6.66 (br s, 1H), 4.91-4.83 (m, 2H), 4.82-4.73 (m, 1H), 3.83-3.65 (m, 4H), 3.50 (br d, J = 4.0 Hz, 2H), 3.47-3.32 (m, 2H), 3.30-3.09 (m, 5H), 2.96-2.89 (m, 1H), 2.81-2.74 (m, 2H), 2.74-2.61 (m, 4H), 2.53-2.42 (m, 4H), 2.42-2.33 (m, 4H), 2.28 (dt, J = 4.0, 7.6 Hz, 5H), 1.99-1.87 (m, 3H), 1.77-1.70 (m, 5H), 1.51 (br d, J = 5.6 Hz, 10H), 1.27 (br s, 54H), 0.92-0.84 (m, 12H)., yellow oil.

[0198] 1.13 Synthesis and characterization of compound 13

[0199]

[0200] 1) Synthesis of compound 13

[0201]

[0202] Compound 2A (1.0 mmol) and compound 8B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 2D (1.0 mmol) were then added, and the mixture was heated to 40°C for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (20:1, v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 13. LCMS: [M+1] + =863.8; 1 H NMR (500 MHz, CDCl3) δ 6.63 (br s, 1H), 5.46-5.23 (m, 4H), 4.71 (br s, 1H), 3.69-3.52 (m, 1H), 3.43-3.23 (m, 2H), 3.22-3.08 (m, 4H), 2.92-2.68 (m, 1H), 2.60-2.31 (m, 9H), 2.07-1.85 (m, 9H), 1.79-1.55 (m, 6H), 1.52-1.39 (m, 4H), 1.38-1.16 (m, 45H), 1.02 (t, J = 7.1 Hz, 6H), 0.88 (t, J = 6.8 Hz, 6H), yellow oil.

[0203] 1.14 Synthesis and characterization of compound 14

[0204]

[0205] Chemical formula: C 52 H 97 N3O2S2

[0206] Molecular weight:859.70

[0207] 1) Synthesis of compound 14

[0208]

[0209] Compound 6A (1.0 mmol) and compound 8B (1.0 mmol) were placed in a 10 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 2D (1.0 mmol) were then added, and the mixture was heated to 40°C for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (20:1, v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 15. LCMS: [M+1] + =861.5; 1 H NMR (500 MHz, CDCl3) δ 6.62 (br s, 1H), 5.48 -5.21 (m, 6H), 4.72 (br s, 1H), 3.65-3.53 (m, 1H), 3.43-3.23 (m, 2H), 3.23 -3.03 (m, 4H), 2.92 -2.61 (m, 1H), 2.58-2.31 (m, 9H), 2.16-1.87 (m, 9H), 1.78 -1.57 (m, 6H), 1.53-1.39 (m, 4H), 1.38-1.20 (m, 41H), 1.02 (t, J = 7.1 Hz, 6H), 0.88 (t, J = 6.8 Hz, 6H), yellow oil.

[0210] 1.15 Synthesis and characterization of compound 15

[0211]

[0212] Chemical formula: C 56 H 105 N3O6S2

[0213] Molecular weight:979.74

[0214] 1) Synthesis of compound 15A

[0215]

[0216] N-tert-Butyloxycarbonyl-8-aminooctanoic acid (15A-1, 1.0 g, 3.86 mmol) and cis-2-nonen-1-ol (0.6 g, 4.24 mmol) were dissolved in 20 mL of dichloromethane, 4-dimethylaminopyridine (DMAP, 0.71 g, 5.78 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.11 g, 5.78 mmol) were added, and the mixture was stirred at 25°C for 3 hours. After the reaction was completed, the solvent was removed by concentration, and the mixture was purified by column chromatography on silica gel, with the eluent being petroleum ether to ethyl acetate 50 / 1-10 / 1 (v / v), to obtain the product 15A-2 (1.42 g, 96.0%);

[0217] Compound 15A-2 (1.42 g, 16.3 mmol) was dissolved in 100 mL of dichloromethane, trifluoroacetic acid (TFA, 17.7 g, 155 mmol) was added at 0°C, and the reaction was continued at 0°C for 30 minutes. After the reaction was completed, the solvent was concentrated and removed, the pH was adjusted to about 10 with saturated sodium carbonate aqueous solution, and extracted with dichloromethane. The organic phases were combined and concentrated to obtain a crude product 15A-3 (1.0 g) which was directly used in the next step;

[0218] Compound 15A-3 (1.0 g, 3.70 mmol) was dissolved in 10 mL of chloroform, and ethyl formate (2.74 g, 37.0 mmol) was added, and the mixture was reacted at 60°C for 16 hours. After the reaction, the excess ethyl formate was removed by concentration, and the mixture was purified by column chromatography on silica gel, with the eluent being petroleum ether and ethyl acetate in a ratio of 10 / 1 to 0 / 1 (v / v), to obtain product 15A-4 (1.0 g, 86.9%).

[0219] Compound 15A-4 (1.0 g, 3.21 mmol) was dissolved in 25 mL of chloroform, pyridine (0.76 g, 9.63 mmol) was added, p-toluenesulfonyl chloride (0.92 g, 4.82 mmol) was added at 0°C, and the reaction was continued at 0°C with stirring for 1 hour. After the reaction was completed, a 5% aqueous sodium sulfate solution was added, stirred for 30 minutes, extracted with dichloromethane, concentrated, and the crude product was purified by column chromatography on silica gel, the eluent being petroleum ether to ethyl acetate 50 / 1-20 / 1 (v / v), to obtain product 15A (0.53 g, 52.7%).

[0220] 2) Synthesis of compound 15

[0221] Compound 4A (1.0 mmol) and compound 8B (1.0 mmol) were placed in a 10.0 mL reaction bottle, 5 mL of anhydrous methanol was added, and the mixture was stirred at room temperature for 0.5 hours. Compound 1C (1.0 mmol) and compound 15D (1.0 mmol) were then added, and the mixture was heated to 40°C for 10 hours. After the reaction was completed, the reaction solvent was removed by rotary evaporation, and dichloromethane:methanol (20:1, v / v) was used as the eluent. The mixture was purified by silica gel column chromatography to obtain the final product 15. LCMS: [M+1] + =980.6; 1H NMR (400MHz, CDCl3) δ = 6.64 (br s,1H),5.70-5.62(m,1H),5.58-5.51(m,1H),4.88(quin,J=6.4Hz,1H),4. 75-4.67(m,1H),4.64(d,J=6.8Hz,2H),3.64-3.57(m,1H),3.48-2.87(m,6H ),2.64-2.21(m,12H),2.12(q,J=7.2Hz,2H),2.02-1.86(m,2H),1.81-1.66 (m,6H),1.65-1.57(m,8H),1.56-1.46(m,8H),1.39-1.24(m,40H),1.04(br d, J = 6.5 Hz, 4H), 0.93-0.87 (m, 9H), yellow oil.

[0222] Example 2: Delivery and protein expression effects of COL1A1 mRNA encapsulated by LNPs constructed with different RD series cationic lipids at a fixed ratio in human keratinocytes

[0223] 2.1 Lipid Nanoparticle (LNP) Formulation

[0224] First, all lipids (ionizable lipids, cholesterol, auxiliary phospholipids, PEGylated lipids) were dissolved in anhydrous ethanol to prepare a solution with a concentration of 10 mg / mL for use. Then, from the prepared lipid solution, RD series lipids, DOPE, cholesterol and DMG-PEG2000 were added to form an ethanol solution (organic phase) at a molar ratio of 60:34:11:0.5. According to the dose of 2 μg COL1A1 mRNA per well, mRNA was dissolved in 16 μL of RNase-free citric acid-sodium citrate buffer (pH=3-5). Then, according to the ratio of LNP-mRNA=10:1, the ethanol phase (organic phase) and the buffer phase (aqueous phase) were quickly mixed by a pipette at a volume ratio of 1 / 3, and incubated for 15 minutes to obtain LNPs encapsulating COL1A1 mRNA. The prepared LNPs encapsulating COL1A1-mRNA were stored at 4°C or -80°C until use.

[0225] The above RD series lipids are shown in Table 1:

[0226] Table 1

[0227]

[0228]

[0229] 2.2 Culture of human keratinocytes

[0230] Shake the cryotube containing 1mL of cell suspension quickly in a 37℃ water bath to thaw, add 5mL of DMEM (high glucose) complete medium and mix well. Centrifuge at 1000RPM for 5 minutes, discard the supernatant, add 8mL of DMEM (high glucose) complete medium and blow evenly. Then add all the cell suspension to the culture bottle and culture overnight (or add the cell suspension to a 6cm dish), and culture overnight. Change the medium and check the cell density on the second day. If the cell density reaches 80%, you can start to plate, and plant 60,000 cells per well in a 6-well cell culture plate, and culture overnight until the cells adhere to the wall for cell transfection.

[0231] 2.3 Transfection of human keratinocytes

[0232] According to the ratio of LNP-mRNA=10:1, the ethanol phase (organic phase) and the buffer phase (aqueous phase) shown in Table 1 were quickly mixed by a pipette at a volume ratio of 1 / 3 and incubated for 15 minutes to obtain LNPs encapsulating COL1A1-mRNA.

[0233] At the same time, the culture medium was replaced with fresh DMEM (high glucose) medium. After the incubation, the prepared LNPs encapsulating COL1A1-mRNA were diluted with 1x PBS to an ethanol concentration of less than 5%, and added to the cell culture plate. After 6 hours, the culture medium was replaced with DMEM (high glucose) complete medium and cultured for 24 hours. Western Boltting was used to verify mRNA expression. Each group of experiments was repeated three times, and the data were averaged.

[0234] 2.4 Protein expression determination method - Western Boltting to verify mRNA expression

[0235] Collect cells 24 hours after transfection, lyse the cells with cell lysis buffer, generally lyse for 20-30 minutes, centrifuge, take out the supernatant to determine the protein concentration, prepare Bicinchoninic Acid (BCA) working solution according to the amount of the sample, add 200μL of BCA working solution and 20μL of human keratinocyte protein extract to each well of the 96-well plate, and incubate in a cell culture incubator for 30 minutes. After incubation, place it in an ELISA reader to measure the absorption peak at 562nm, and then calculate the protein concentration according to the conversion formula of absorbance and protein concentration (the calculated value is in μg / μL).

[0236] After calculating the protein concentration, the protein concentration of each sample needs to be balanced. Use tissue lysis buffer to dilute the protein sample to 1μg / μL. At this time, it is necessary to determine the volume of the protein solution in each sample tube (suck it out one by one with a gun and calculate the volume each time), and then use the formula n1V1=n2(V1+V) to add tissue lysis buffer for dilution. Then add 5× loading buffer, and the volume of loading buffer added is the volume of the diluted protein solution divided by 4. Then place it on a metal bath at 100℃ for 10 minutes, and then store it at -20℃.

[0237] Take out the extracted protein and marker and thaw them. Put the plate into the electrophoresis box (with the short plate facing inwards), fill it with electrophoresis solution, and then load the sample. First run at 80V for 30 minutes to let the protein run through the concentrated gel, then run at 120V for 1.5 hours to let the protein run on the separation gel. The time for electrophoresis on the separation gel can be adjusted appropriately according to the molecular weight of the protein.

[0238] After the electrophoresis is completed, the gel is taken out, the target protein band is cut off according to the marker, and the internal reference band is cut off at the same time, and placed in the electrotransfer device (black sponge at the bottom, white sponge at the top). After the gel strips are laid, the cut PMSF membrane (after cutting, it is activated with methanol for 10 seconds, and the upper left corner of the membrane is cut off) is laid on the strips, covered with the electrotransfer plate and placed in the electrotransfer device, two small ice bags are placed in the electrotransfer device, the entire device is placed on ice, and the voltage is 59V for 2 hours.

[0239] After electroporation, the membrane was removed and blocked with 5% skim milk powder (1x TBST) at room temperature for 1 hour. After blocking, the membrane was removed and rinsed once with TBST, and then the membrane was incubated with primary antibodies at 4°C overnight.

[0240] After the primary antibody incubation, the membrane was removed and washed three times with TBST for 10 minutes each time, then the secondary antibody was added and incubated at room temperature for 1.5 hours. After the secondary antibody incubation, the membrane can be developed with a 1:1 developer solution. The mRNA expression can be determined based on the development results.

[0241] 2.5 Protein expression results of COL1A1 mRNA encapsulated by LNPs constructed with different RD series cationic lipids at a ratio of 10:1 in human keratinocytes

[0242] Figure 1 Protein expression of COL1A1 mRNA encapsulated by LNPs constructed with different RD series cationic lipids at a ratio of 10:1 in human keratinocytes; Figure 1 As shown, it can be clearly found that through the optimization of the LNP formula, the delivery effect of RD007 cationic lipid is better than that of other RD series cationic lipids. Therefore, RD007 was selected as the optimal solution for the RD series lipids for subsequent experiments.

[0243] Example 3: Delivery and protein expression effects of LNPs with different compositions encapsulating COL1A1 mRNA at a fixed ratio in human keratinocytes

[0244] 3.1 Lipid Nanoparticle (LNP) Formulation

[0245] First, all lipids (ionizable lipids, cholesterol, auxiliary phospholipids, PEGylated lipids) were dissolved in anhydrous ethanol to prepare solutions with a concentration of 10 mg / mL for use, and then RD007 (Compound 7 in Example 1), DOPE, cholesterol and DMG-PEG2000 were mixed in different molar ratios from the prepared lipid solution to form an ethanol solution (organic phase), and the mRNA was dissolved in 16 μL of RNase-free citric acid-sodium citrate buffer (pH=3-5) at a dose of 2 μg COL1A1 mRNA per well, and then the ethanol phase (organic phase) and the buffer phase (aqueous phase) were quickly mixed by a pipette at a volume ratio of 1 / 3 according to the ratio of LNP-mRNA=10:1, and incubated for 15 minutes to obtain LNPs encapsulating COL1A1 mRNA. The prepared LNPs encapsulating COL1A1-mRNA were stored at 4°C or -80°C until use.

[0246] The molar ratios of the above different proportions are shown in Table 2:

[0247] Table 2

[0248]

[0249]

[0250] 3.2 Culture of human keratinocytes

[0251] Shake the cryotube containing 1mL of cell suspension quickly in a 37℃ water bath to thaw, add 5mL of DMEM (high glucose) complete medium and mix well. Centrifuge at 1000RPM for 5 minutes, discard the supernatant, add 8mL of DMEM (high glucose) complete medium and blow evenly. Then add all the cell suspension to the culture bottle and culture overnight (or add the cell suspension to a 6cm dish), and culture overnight. Change the medium and check the cell density on the second day. If the cell density reaches 80%, you can start to plate, and plant 60,000 cells per well in a 6-well cell culture plate, and culture overnight until the cells adhere to the wall for cell transfection.

[0252] 3.3 Transfection of human keratinocytes

[0253] According to the ratio of LNP-mRNA=10:1, the ethanol phase (organic phase) and the buffer phase (aqueous phase) shown in Table 1 were quickly mixed by a pipette at a volume ratio of 1 / 3 and incubated for 15 minutes to obtain LNPs encapsulating COL1A1-mRNA.

[0254] At the same time, the culture medium was replaced with fresh DMEM (high glucose) medium. After the incubation, the prepared LNPs encapsulating COL1A1-mRNA were diluted with 1x PBS to an ethanol concentration of less than 5%, and added to the cell culture plate. After 6 hours, the culture medium was replaced with DMEM (high glucose) complete medium and cultured for 24 hours. Western Boltting was used to verify mRNA expression. Each group of experiments was repeated three times, and the data were averaged.

[0255] 3.4 Protein expression determination method - Western Boltting to verify mRNA expression

[0256] Collect cells 24 hours after transfection, lyse the cells with cell lysis buffer, generally lyse for 20-30 minutes, centrifuge, take out the supernatant to determine the protein concentration, prepare Bicinchoninic Acid (BCA) working solution according to the amount of the sample, add 200μL of BCA working solution and 20μL of human keratinocyte protein extract to each well of the 96-well plate, and incubate in a cell culture incubator for 30 minutes. After incubation, place it in an ELISA reader to measure the absorption peak at 562nm, and then calculate the protein concentration according to the conversion formula of absorbance and protein concentration (the calculated value is in μg / μL).

[0257] After calculating the protein concentration, the protein concentration of each sample needs to be balanced. Use tissue lysis buffer to dilute the protein sample to 1μg / μL. At this time, it is necessary to determine the volume of the protein solution in each sample tube (suck it out one by one with a gun and calculate the volume each time), and then use the formula n1V1=n2(V1+V) to add tissue lysis buffer for dilution. Then add 5× loading buffer, and the volume of loading buffer added is the volume of the diluted protein solution divided by 4. Then place it on a metal bath at 100℃ for 10 minutes, and then store it at -20℃.

[0258] Take out the extracted protein and marker and thaw them. Put the plate into the electrophoresis box (with the short plate facing inwards), fill it with electrophoresis solution, and then load the sample. First run at 80V for 30 minutes to let the protein run through the concentrated gel, then run at 120V for 1.5 hours to let the protein run on the separation gel. The time for electrophoresis on the separation gel can be adjusted appropriately according to the molecular weight of the protein.

[0259] After the electrophoresis is completed, the gel is taken out, the target protein band is cut off according to the marker, and the internal reference band is cut off at the same time, and placed in the electrotransfer device (black sponge at the bottom, white sponge at the top). After the gel strips are laid, the cut PMSF membrane (after cutting, it is activated with methanol for 10 seconds, and the upper left corner of the membrane is cut off) is laid on the strips, covered with the electrotransfer plate and placed in the electrotransfer device, two small ice bags are placed in the electrotransfer device, the entire device is placed on ice, and the voltage is 59V for 2 hours.

[0260] After electroporation, the membrane was removed and blocked with 5% skim milk powder (1x TBST) at room temperature for 1 hour. After blocking, the membrane was removed and rinsed once with TBST, and then the membrane was incubated with primary antibodies at 4°C overnight.

[0261] After the primary antibody incubation, the membrane was removed and washed three times with TBST for 10 minutes each time, then the secondary antibody was added and incubated at room temperature for 1.5 hours. After the secondary antibody incubation, the membrane can be developed with a 1:1 developer solution. The mRNA expression can be determined based on the development results.

[0262] 3.5 Protein expression results of COL1A1 mRNA encapsulated by LNPs of different compositions at a ratio of 10:1 in human keratinocytes

[0263] Figure 2 The protein expression of COL1A1 mRNA encapsulated by LNPs of different compositions in human keratinocytes at a ratio of 10:1; Figure 2 As shown, it can be clearly found that the delivery effect of the A14 formula has been significantly increased by optimizing the LNP formula, and its expression level has increased by 30% compared with the original formula (A0).

[0264] Example 4: Delivery and protein expression effects of LNPs with different compositions encapsulating COL3A1 mRNA at a fixed ratio in human keratinocytes

[0265] 4.1 Lipid Nanoparticle (LNP) Formulation

[0266] First, all lipids (ionizable lipids, cholesterol, auxiliary phospholipids, PEGylated lipids) were dissolved in anhydrous ethanol to prepare solutions with a concentration of 10 mg / mL for use. Then, RD007, DOPE, cholesterol and DMG-PEG2000 were mixed in different molar ratios (same as Table 1) from the prepared lipid solution to form an ethanol solution (organic phase). According to the dose of 2 μg COL3A1 mRNA per well, mRNA was dissolved in 16 μL RNase-free citric acid-sodium citrate buffer (pH=3-5). Then, according to the ratio of LNP-mRNA=10:1, the ethanol phase (organic phase) and the buffer phase (aqueous phase) were quickly mixed by pipette at a volume ratio of 1 / 3, and incubated for 15 minutes to obtain LNPs encapsulating COL3A1 mRNA. The prepared LNPs encapsulating COL3A1-mRNA were stored at 4°C or -80°C until use.

[0267] 4.2 Culture of human keratinocytes

[0268] Same as 3.2 in Example 3.

[0269] 4.3 Transfection of human keratinocytes

[0270] According to the ratio of LNP-mRNA=10:1, the ethanol phase (organic phase) and the buffer phase (aqueous phase) shown in Table 1 were quickly mixed by a pipette at a volume ratio of 1 / 3 and incubated for 15 minutes to obtain LNPs encapsulating COL3A1-mRNA.

[0271] At the same time, the culture medium was replaced with fresh DMEM (high glucose) medium. After the incubation, the prepared LNPs were diluted with 1xPBS to an ethanol concentration of less than 5%, and added to the cell culture plate. After 6 hours, the culture medium was replaced with DMEM (high glucose) complete medium and cultured for 24 hours. Western Boltting was used to verify mRNA expression. Each group of experiments was repeated three times, and the data were averaged.

[0272] 4.4 Protein expression determination method - Western Boltting to verify mRNA expression

[0273] Same as 3.4 in Example 3.

[0274] 4.5 Protein expression of COL3A1 mRNA encapsulated by LNPs of different compositions at a fixed ratio in human keratinocytes

[0275] Figure 3 The protein expression of COL3A1 mRNA encapsulated by LNPs of different compositions in human keratinocytes at a fixed ratio (10:1); Figure 3As shown, it can be clearly found that the delivery effect of the A14 formula has been significantly increased by optimizing the LNP formula, and its expression level has increased by 13% compared with the original formula.

[0276] Example 5. Delivery and protein expression effects of LNPs with different compositions encapsulating COL17A1 mRNA at a fixed ratio in human keratinocytes

[0277] 5.1 Lipid Nanoparticle (LNP) Formulation

[0278] First, all lipids (ionizable lipids, cholesterol, auxiliary phospholipids, PEGylated lipids) were dissolved in anhydrous ethanol to prepare solutions with a concentration of 10 mg / mL for use. Then, RD007, DOPE, cholesterol and DMG-PEG2000 were mixed in different molar ratios (same as Table 1) from the prepared lipid solution to form an ethanol solution (organic phase). According to the dose of 2 μg COL17A1 mRNA per well, mRNA was dissolved in 16 μL RNase-free citric acid-sodium citrate buffer (pH=3-5). Then, according to the ratio of LNP-mRNA=10:1, the ethanol phase (organic phase) and the buffer phase (aqueous phase) were quickly mixed by a pipette at a volume ratio of 1 / 3, and incubated for 15 minutes to obtain LNPs encapsulating COL17A1 mRNA. The prepared LNPs encapsulating COL17A1-mRNA were stored at 4°C or -80°C until use.

[0279] 5.2 Culture of human keratinocytes

[0280] Same as 3.2 in Example 3.

[0281] 5.3 Transfection of human keratinocytes

[0282] According to the ratio of LNP-mRNA=10:1, the ethanol phase (organic phase) and the buffer phase (aqueous phase) shown in Table 1 were quickly mixed by a pipette at a volume ratio of 1 / 3 and incubated for 15 minutes to obtain LNPs encapsulating COL17A1-mRNA.

[0283] At the same time, the culture medium was replaced with fresh DMEM (high glucose) medium. After the incubation, the prepared LNPs were diluted with 1xPBS to an ethanol concentration of less than 5%, and added to the cell culture plate. After 6 hours, the culture medium was replaced with DMEM (high glucose) complete medium and cultured for 24 hours. Western Boltting was used to verify mRNA expression. Each group of experiments was repeated three times, and the data were averaged.

[0284] 5.4 Protein expression determination method - Western Boltting to verify mRNA expression

[0285] Same as 3.4 in Example 3.

[0286] 5.5 Protein expression of COL17A1 mRNA encapsulated by LNPs of different compositions at a fixed ratio in human keratinocytes

[0287] Figure 4 The protein expression of COL17A1 mRNA encapsulated by LNPs of different compositions in human keratinocytes at a fixed ratio (10:1); Figure 4 As shown, it can be clearly found that the delivery effect of the A14 formula has been significantly increased by optimizing the LNP formula, and its expression level has increased by 19% compared with the original formula.

[0288] Example 6. Delivery and protein expression effects of LNPs with different compositions encapsulating COL1A1 mRNA at a fixed ratio in human fibroblasts

[0289] 6.1 Lipid Nanoparticle (LNP) Formulation

[0290] Same as 3.1 in Example 3.

[0291] 6.2 Culture of human fibroblasts

[0292] Same as 3.2 in Example 3.

[0293] 6.3 Transfection of human fibroblasts

[0294] Same as 3.3 in Example 3.

[0295] 6.4 Protein expression determination method - Western Boltting to verify mRNA expression

[0296] Same as 3.4 in Example 3.

[0297] 6.5 Protein expression of COL1A1 mRNA encapsulated by LNPs of different compositions at fixed ratios in human fibroblasts

[0298] Figure 5 Protein expression of COL1A1 mRNA encapsulated by LNPs of different compositions in human fibroblasts at a fixed ratio (10:1); Figure 5 As shown, it can be clearly found that the delivery effect of the A14 formula has been significantly increased by optimizing the LNP formula, and its expression level has increased by 11% compared with the original formula.

[0299] Example 7: Delivery and protein expression effects of LNP and COL1A1 mRNA in human keratinocytes at different ratios

[0300] 7.1 Lipid Nanoparticle (LNP) Formulation

[0301] First, all lipids (ionizable lipids, cholesterol, auxiliary phospholipids, PEGylated lipids) were dissolved in anhydrous ethanol to prepare a solution with a concentration of 10 mg / mL for use, and then RD007, DOPE, cholesterol and DMG-PEG2000 were mixed in a molar ratio of 60:34:11:0.5 from the prepared lipid solution to form an ethanol solution (organic phase), and the mRNA was dissolved in 16 μL of RNase-free citric acid-sodium citrate buffer (pH=3-5) at a dose of 2 μg COL1A1 mRNA per well, and then the ethanol phase (organic phase) and the buffer phase (aqueous phase) were quickly mixed by a pipette at a volume ratio of 1 / 3 according to different LNP-mRNA ratios, and incubated for 15 minutes to obtain LNPs encapsulating COL1A1 mRNA. The prepared LNPs were stored at 4°C or -80°C until use.

[0302] Among them, different LNP-mRNA ratios are shown in Table 3 below:

[0303] Table 3

[0304]

[0305] 7.2 Culture of human keratinocytes

[0306] Basically the same as 3.2 in Example 3.

[0307] 7.3 Transfection of human keratinocytes

[0308] Basically the same as 3.3 in Example 3.

[0309] 7.4 Protein expression determination method - Western Boltting to verify mRNA expression

[0310] Basically the same as 3.4 in Example 3.

[0311] Protein expression of 7.5LNP and COL1A1 mRNA in human keratinocytes at different ratios

[0312] Figure 6 The protein expression of LNP and COL1A1 mRNA in human keratinocytes at different ratios; Figure 6 As shown in the figure, with the increase of the LNP-mRNA ratio, the transfection efficiency and protein expression level showed a trend of first increasing and then decreasing. When the ratio was 10:1 and 15:1, the expression level was not much different, with a difference of only 0.4%. However, considering the cost issue, LNP-mRNA = 10:1 is preferred.

[0313] Example 8: Delivery and protein expression effects of LNP and COL1A1 mRNA in human fibroblasts at different ratios

[0314] 8.1 Lipid Nanoparticle (LNP) Formulation

[0315] Same as 7.1 in Example 7.

[0316] 8.2 Culture of human fibroblasts

[0317] Same as 6.2 in Example 6.

[0318] 8.3 Transfection of human fibroblasts

[0319] Same as 6.3 in Example 6.

[0320] 8.4 Protein expression determination method - Western Boltting to verify mRNA expression

[0321] Same as 3.4 in Example 3.

[0322] Protein expression of 8.5LNP and COL1A1 mRNA in human fibroblasts at different ratios

[0323] Figure 7 The protein expression of LNP and COL1A1 mRNA in human fibroblasts at different ratios; Figure 7 As shown in the figure, with the increase of the LNP-mRNA ratio, the transfection efficiency and protein expression level showed a trend of first increasing and then decreasing. The delivery efficiency and protein expression level reached the highest when LNP-mRNA = 10:1, so the preferred ratio is 10:1.

[0324] Example 9: Delivery and protein expression effects of different ionizable lipids prepared into LNPs at a fixed ratio and then encapsulated with COL1A1 mRNA at the same ratio in human keratinocytes

[0325] 9.1 Lipid Nanoparticle (LNP) Formulation

[0326] First, all lipids (ionizable lipids, cholesterol, auxiliary phospholipids, PEGylated lipids) were dissolved in anhydrous ethanol to prepare a solution with a concentration of 10 mg / mL for use, and then different cationic lipids, DOPE, cholesterol and DMG-PEG2000 were mixed at a molar ratio of 60:34:11:0.5 from the prepared lipid solution to form an ethanol solution (organic phase), and the mRNA was dissolved in 16 μL of RNase-free citric acid-sodium citrate buffer (pH=3-5) at a dose of 2 μg COL1A1 mRNA per well, and then the ethanol phase (organic phase) and the buffer phase (aqueous phase) were quickly mixed by a pipette at a volume ratio of 1 / 3 according to the ratio of LNP-mRNA=10:1, and incubated for 15 minutes to obtain LNPs encapsulating COL1A1 mRNA. The prepared LNPs encapsulating COL1A1 mRNA were stored at 4°C or -80°C until use.

[0327] Among them, different cationic lipids are shown in Table 4 below:

[0328] Table 4

[0329]

[0330] 9.2 Culture of human keratinocytes

[0331] Same as 3.2 in Example 3.

[0332] 9.3 Transfection of human keratinocytes

[0333] Same as 3.3 in Example 3.

[0334] 9.4 Protein expression determination method - Western Boltting to verify mRNA expression

[0335] Same as 3.4 in Example 3.

[0336] 9.5 Protein expression effect of LNP-COL1A1 mRNA prepared by using different ionizable lipids at a fixed ratio in human keratinocytes

[0337] Figure 8 Protein expression of LNP-COL1A1 mRNA in human keratinocytes prepared for different ionizable lipids; e.g. Figure 8As shown, it can be known that mRNA has no way to enter cells to promote the overexpression of type I collagen without a carrier. Therefore, the present invention uses lipid nanoparticles as a delivery carrier to package and deliver mRNA in order to achieve the purpose of mRNA entering cells to overexpress the target protein. At the same time, it can be seen that different LNPs have different expression effects, but most commercial LNPs are not ideal. Relatively speaking, the LNP in Example 1 of the present invention has a high expression effect and can promote the overexpression of type I collagen in cells, with an efficiency increased by 30%.

[0338] Example 10: Delivery and protein expression effects of LNPs prepared with different ionizable lipids in a fixed ratio and then encapsulated with COL1A1 mRNA in the same ratio in human fibroblasts

[0339] 10.1 Lipid Nanoparticle (LNP) Formulation

[0340] Same as 9.1 in Example 9.

[0341] 10.2 Culture of human fibroblasts

[0342] Same as 6.2 in Example 6.

[0343] 10.3 Transfection of human fibroblasts

[0344] Same as 6.3 in Example 6.

[0345] 10.4 Protein expression determination method - Western Boltting to verify mRNA expression

[0346] Same as 3.4 in Example 3.

[0347] 10.5 Protein expression effect of LNP-COL1A1 mRNA prepared by using different ionizable lipids at a fixed ratio in human fibroblasts

[0348] Fig. 9 Protein expression of LNP-COL1A1 mRNA in human fibroblasts prepared with different ionizable lipids; e.g. Fig. 9 As shown, different LNPs have different expression effects, but most commercial LNPs are not ideal. Relatively speaking, our independently developed LNPs have high expression effects and can promote the overexpression of type I collagen in cells, with an efficiency increased by 11%.

[0349] Example 11. Transdermal test results of a sample of a preferred LNP encapsulated CY5-labeled COL1A1 mRNA and a sample of a common LNP encapsulated CY5-labeled COL1A1 mRNA on the market

[0350] According to RD007 / cholesterol / DOPE / DMG-PEG2000=60:34:11:0.5 and SM102 / cholesterol

[0351] / DOPE / DMG-PEG2000=60:34:11:0.5 molar ratio to form an ethanol solution (organic phase), according to the dosage of 2μg CY5-COL1A1 mRNA per well, the mRNA was dissolved in 16μL RNase-free citric acid-sodium citrate buffer (pH=3~5), and then the ethanol phase (organic phase) and the buffer phase (aqueous phase) were quickly mixed by a pipette at a volume ratio of 1 / 3 according to the ratio of LNP-mRNA=10:1, and incubated for 15 minutes to obtain LNPs encapsulating CY5-COL1A1 mRNA. The prepared LNPs encapsulating CY5-COL1A1 mRNA were stored at 4℃ or -80℃ until use.

[0352] The prepared LNP sample encapsulating CY5-COL1A1 mRNA was subjected to a transdermal test on suckling pig skin. The test process is as follows:

[0353] Add receiving solution to the receiving chamber: use a pipette to draw 7.0 mL of receiving solution (physiological saline) into the receiving chamber, and place the matching magnetic stirring bar in the receiving chamber.

[0354] Assembly and fixation of the test model: Fix the piglet skin between the diffusion chamber and the receiving chamber of the Franz cell diffusion tank, with the piglet skin stratum corneum facing the diffusion chamber and the dermis facing the receiving chamber. After fixing the piglet skin, add 0.5mL of receiving solution (normal saline) to the sampling tube with a pipette according to the liquid height of the sampling tube, so that the piglet skin dermis is in close contact with the receiving solution, and the total volume of the receiving solution is 7.5mL.

[0355] The Franz cell diffusion pool was fixed in the transdermal absorption diffusion instrument, and the electromagnetic stirrer was turned on to stir at a speed of 300 rpm, and a constant temperature water bath of (32±1)°C was maintained, and it was ensured that there were no bubbles in the water bath interlayer.

[0356] Loading: After the water bath temperature of the diffusion instrument is constant, the sample is loaded. Use a pipette to draw LNP-CY5-COL1A1 mRNA and add it to the surface of the suckling pig skin, so that it is evenly distributed on the surface of the suckling pig skin and act for 15 minutes (as a blank group), 2 hours, 4 hours, 6 hours, 8 hours, and 12 hours respectively. After the action is over, the supernatant is discarded.

[0357] Quantitative analysis: The expression level of marker protein in suckling pig skin was quantitatively analyzed by fluorescence microscope imaging and analysis software. The experimental results are as follows: Fig.10 As shown. Fig.10As shown, through the transdermal fluorescence quantitative test, it can be seen that the RD007-LNP independently developed by the present invention has more beneficial transdermal properties, and the transdermal efficiency is increased by about 80% compared with the common samples on the market.

[0358] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A cosmetic composition comprising: Lipid nanoparticles LNPs; Nucleic acids encapsulated in the LNPs; Wherein, the nucleic acid is used to regulate the expression of genes related to skin conditions.

2. The cosmetic composition according to claim 1, characterized in that Including at least one of the following technical solutions: A1. The average diameter of the lipid nanoparticles LNPs is 50-200 nm; A2. The cosmetic composition uses lipid nanoparticles LNPs to deliver nucleic acids to improve the appearance of the skin; A3, the cosmetic composition delivers nucleic acid to skin cells, wherein the delivery promotes overexpression of gene protein; A4. The cosmetic composition delivers nucleic acid to skin cells, mainly acting on epidermal and dermal cells of human skin, including fibroblasts and keratinocytes.

3. The cosmetic composition according to claim 1, characterized in that The cosmetic composition is prepared by a method comprising the following steps: S1, forming lipid nanoparticles LNPs by combining lipids in a solvent; S2, encapsulating nucleic acids in lipid nanoparticles LNPs; S3. Adding LNPs encapsulated with nucleic acid into the cosmetic matrix.

4. The cosmetic composition according to claim 3, characterized in that Including at least one of the following technical solutions: Before B1 and S3, there is also a step of purifying the lipid nanoparticles to remove unencapsulated nucleic acids; In B2 and S2, nucleic acid chemically active substances are at least partially encapsulated inside lipid nanoparticles LNPs; The encapsulation steps in B3 and S2 include the use of pipetting, vortexing, and microfluidic instrumentation to ensure uniform distribution of nucleic acids within lipid nanoparticles; B4 and S3 also include adjusting the pH value of the cosmetic composition to improve the stability of the lipid nanoparticles.

5. The cosmetic composition according to claim 1 or 3, characterized in that: The composition of LNPs includes cationic lipids, auxiliary lipids, neutral lipids and cholesterol.

6. The cosmetic composition according to claim 5, characterized in that The cationic lipid is a compound as shown in Formula I: in, Side chain part -W1-L1-(R1) n1 and-W2-L2-(R2) n2 Each independently has 1 to 40 carbon atoms in the main chain; W1 and W2 are each independently C1-C14 alkylene, C2-C14 alkenylene or C2-C14 alkynylene; L1 and L2 are each independently selected from the following structures: R1 and R2 are each independently H, C3-C22 alkyl, C3-C22 alkenyl, C3-C22 alkynyl, C5-C8 cycloalkyl, C5-C8 cycloalkenyl; R3 and R4 are each independently C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, and the alkyl, alkenyl or alkynyl is arbitrarily substituted by one or more OH, C1-C4 alkoxy, or R3 and R4 are combined with each other to form a 5- to 8-membered heterocyclic ring, and in addition to the N atom connected to M, the heterocyclic ring optionally includes one or more heteroatoms of N, O, and S; M is a C1-C6 alkylene group, a C2-C6 alkenylene group or a C2-C6 alkynylene group.

7. The cosmetic composition according to claim 5, characterized in that Include at least one of the following technical features: C1. In the composition of LNPs, the amount of cationic lipids is 20-55 mol%; C2. The auxiliary lipids of LNPs include auxiliary phospholipids; the auxiliary phospholipids include: DSPC, DMPE or DOP E; C3, LNPs also include auxiliary phospholipids with a substance amount of 8.5-33%; C4. The cholesterol includes cholesterol or its derivatives; in the composition of LNPs, the amount of cholesterol accounts for 20-66%; C5. The neutral lipid includes PEG lipid; the PEG lipid includes: DWG-PEG2000, ALC0159, DSPE-PEG2000 or C14-PEG2000; In the composition of C6 and LNPs, the amount of neutral lipids accounts for 0.9%-4.1%.

8. The cosmetic composition according to claim 1, characterized in that The nucleic acid is selected from any one of mRNA, siRNA, shRNA, DNA and dsRNA.

9. The cosmetic composition according to claim 8, characterized in that The mRNA comprises at least one of non-replicating linear mRNA, self-replicating linear mRNA, and circular mRNA; And / or, the mRNA species comprises one of COL1A1 mRNA, COL2A1 mRNA, COL3A1 mRNA, and COL17A1 mRNA.

10. A method for applying the cosmetic composition according to any one of claims 1 to 9, characterized in that: It comprises applying a therapeutically effective amount of the cosmetic composition to the skin of a subject.

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