Disulfide-containing lipid molecules for mRNA delivery and methods of making and uses thereof

Abstract

The application discloses a disulfide-containing lipid molecule for mRNA delivery and a preparation method and application thereof, and a synthesis method of the lipid molecule is as follows: 2,2-dithiodiethanol diacrylate is used as a raw material, and is further reacted with selected hydrophilic amines to generate an intermediate, and the intermediate is further reacted with hydrophobic amines to obtain a final lipid molecule for delivering mRNA, wherein the hydrophilic amine raw material is one of 4A1, 4A2 and 4A3; the hydrophobic amine raw material is one of C18, 2C8 and 2C10; the prepared lipid molecule contains a head group generating a cation by ionization, and nucleic acids carry a negative charge, so that the two can be efficiently combined, thereby significantly improving the loading rate of nucleic acids. And the carbon chain length and the number of branches of the hydrophobic alkane chain have a crucial influence on the stability of the formed nanoparticles, so that the lipid molecule has a good prospect in the application of delivering mRNA molecules.

Description

Technical Field

[0001] The present invention relates to a disulfide bond-containing lipid molecule for mRNA delivery, a preparation method thereof, and uses thereof. Background Art

[0002] Messenger RNA (mRNA)-based drug technologies have garnered significant attention over the past few years. Due to their superior properties, this technology has been widely applied in areas such as protein replacement therapy, cancer immunotherapy, and gene editing. Currently, research into the use of mRNA for disease treatment is developing rapidly, with many approaches entering clinical trials. Therefore, the clinical application of mRNA-based therapeutics holds enormous potential and could revolutionize the treatment of several diseases.

[0003] Compared with plasmid DNA (pDNA) or small interfering RNA (siRNA)-based therapies, mRNA-based therapies have many potential advantages: 1) mRNA directly expresses functional proteins in the cytoplasm and does not need to enter the nucleus like pDNA. Therefore, mRNA therapy eliminates the barrier of crossing the nuclear membrane and can also be highly effective in non-dividing cells or slowly dividing cells, such as dendritic cells; 2) mRNA does not need to be integrated into the cell's chromosomes, which can avoid the risk of insertional gene mutations; 3) Compared with RNA interference, the process of mRNA translation of target proteins is simpler and more direct, without the off-target issues similar to siRNA. In addition, compared with the corresponding recombinant protein, the high charge density and flexibility of mRNA are more efficient in loading into the carrier; the preparation of mRNA is relatively simple, and the biosafety risk is low.

[0004] Due to the immunogenicity of lipid carriers and exogenous mRNA molecules, their therapeutic application is hindered. Although lipid carriers interact with immune cells outside the target cells, mRNA is mainly exposed to intracellular components of the immune system, which will produce cytokine and interferon inflammatory responses, preventing mRNA translation. Therefore, a major problem facing the clinical application of mRNA drugs is to find an effective and safe vector. Viral and non-viral vectors are well known as two widely used gene delivery vectors. Although viral vectors have obvious advantages in delivery efficiency, their safety concerns (such as insertional gene mutations, carcinogenesis or immune responses) greatly reduce the possibility of their direct use in humans. Since mRNA cannot maintain activity in the blood, and there are a series of problems such as high non-specific protein adsorption rate and clearance and recognition by immune-related cells, it requires a suitable non-viral vector delivery vehicle.

[0005] As the main non-viral mRNA delivery carrier material, cationic lipids / lipidoid molecules and cationic polymers are mixed with cholesterol, PEG lipids, and auxiliary lipid molecules (such as DOPE) and incubated to self-assemble into carriers, which are then loaded with mRNA to form lipid nanoparticles (LNPs). Among the well-known mRNA delivery systems, they have broad development potential and the best effect. According to other literature, the efficiency of mRNA delivery by ionizable cationic lipid molecules is determined by the hydrophobic-hydrophilic balance, the type of amine, and the type of auxiliary lipid. Summary of the Invention

[0006] The purpose of the present invention is to provide a disulfide bond-containing lipid molecule for mRNA delivery, a preparation method and use thereof, and the present invention discloses a lipid molecule that can effectively bind to mRNA and significantly improve its stability and expression efficiency in vivo and in vitro.

[0007] The disulfide bond-containing lipid molecule for mRNA delivery has a structure selected from the following:

[0008] (I)

[0009] (II)

[0010] (III)

[0011] (IV)

[0012]

[0013] (v)

[0014]

[0015] The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery comprises the following steps:

[0016] 1) Dissolve 2-hydroxyethyl disulfide, triethylamine, and acryloyl chloride in a reaction solvent and react in an ice-water bath under nitrogen. After completion of the reaction, a crude yellow liquid product is obtained. Filter the product to remove white crystals. Extract the product sequentially with dichloromethane-saturated saline and then dichloromethane-water. The product is dissolved in dichloromethane. Heat and concentrate the product. The residue is purified by silica gel column chromatography to obtain the raw material, 2,2-dithiodiethanol diacrylate.

[0017] 2) 2,2-dithiodiethanol diacrylate (labeled as DSDA) and a hydrophilic amine are dissolved in a reaction solvent, a polymerization inhibitor BHT is added to the system, the reaction temperature is maintained at 53-60°C, and the reaction is continued for 90-100 hours, and finally a yellow oil is obtained as a crude intermediate product, the yellow oil is extracted using an ethyl acetate-water system, the crude intermediate product is extracted into the ethyl acetate phase, and the intermediate is purified by silica gel column chromatography;

[0018] 3) the purified intermediate and a hydrophobic amine are used as raw materials, dissolved in a reaction solvent, and reacted in the presence of a polymerization inhibitor BHT at a reaction temperature of 50-60°C for 80-120 hours, and finally a yellow oil is obtained as a final product, and the final product is purified by silica gel column chromatography to complete the preparation; the reaction formula of the lipid molecule of the present application is as follows:

[0019]

[0020] The structural formula of the hydrophilic amine (R1-NH2) is one of 4A1, 4A2, and 4A3:

[0021]

[0022] The structural formula of the hydrophobic amine is one of C18, 2C8, and 2C10.

[0023]

[0024] C18 has one alkane chain and contains 18 carbon atoms. 2C8 has two alkane chains, each chain containing 8 carbon atoms. 2C10 has two alkane chains, each chain containing 10 carbon atoms. For C18, R2 in the hydrophobic amine is a methyl group and R3 is a straight-chain alkyl group of C18, for 2C8, R2 and R3 in the hydrophobic amine are straight-chain alkyl groups of C8, and for 2C10, R2 and R3 are straight-chain alkyl groups of C10.

[0025] From the above structural formula, the specific structure of the hydrophilic amine and the hydrophobic amine in the lipid molecule can be clearly understood. For example, the raw materials for preparing the lipid molecule 4A1S-2C10 are the hydrophilic amine 4A1 and the hydrophobic amine 2C10.

[0026] ​The method for preparing a disulfide-bonded lipid molecule for mRNA delivery is characterized in that in step 1), the solid-liquid ratio of 2-hydroxyethyl disulfide to acryloyl chloride is 1g:1-3ml, preferably 1g:1.5-2ml, the solid-liquid ratio of 2-hydroxyethyl disulfide to triethylamine is 1g:1.5-4ml, preferably 1g:2-2.5ml, the reaction solvent is THF; the reaction time is 20-30h, and the elution solvent for purification by silica gel column chromatography is a mixture of n-hexane and ethyl acetate with a volume ratio of 20-40:1.

[0027] The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery is characterized in that in step 2), the mass ratio of the hydrophilic amine to 2,2-dithiodiethanol diacrylate is 1:10-30, preferably 1:14-28; the mass ratio of 2,2-dithiodiethanol diacrylate to the polymerization inhibitor BHT is 15-30:1, preferably 20-25:1, and the reaction solvent is DMSO.

[0028] The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery is characterized in that in step 2), the reaction temperature is 55-56°C, the reaction time is 95-96h, and silica gel column chromatography is used as the eluent for purification using an ethyl acetate-methanol mixture with a volume ratio of 40 to 50:1 or a n-hexane-ethyl acetate mixture with a volume ratio of 0.5 to 2:1.

[0029] The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery is characterized in that in step 3), the mass ratio of the purified intermediate and the hydrophobic amine is 1:1.5-3, preferably 1:1.8-2.2, the mass ratio of the intermediate and the polymerization inhibitor BHT is 1:0.005-0.015, preferably 1:0.009-0.01, and the reaction solvent is anhydrous THF.

[0030] The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery is characterized in that in step 3), the reaction temperature is 53-60°C, the reaction time is 96-100 hours, and the eluent for purification by silica gel column chromatography is ethyl acetate or a mixture of n-hexane and ethyl acetate with a volume ratio of 1:10-10:1.

[0031] The lipid molecules provided by the present invention have good applications in delivering mRNA molecules.

[0032] Compared with the prior art, the outstanding features of the lipid molecules involved in the present invention are:

[0033] 1) The lipid molecule material synthesized in this invention includes key amine and hydrophobic units, which research has shown to be essential for efficient mRNA delivery vehicles. The present invention utilizes 4A1, 4A2, and 4A3, respectively, to effectively control the material's hydrophilicity, cationic density, and number of hydrophobic chains. Furthermore, the present invention utilizes C18, 2C8, and 2C10, respectively, to effectively control the material's hydrophobicity.

[0034] 2) The lipid molecule product synthesized by the present invention has multiple protonated N groups in its structure, which can effectively bind negatively charged nucleic acids and improve the nucleic acid loading rate; and has an appropriate number and length of hydrophobic alkane chains, which can form highly stable nanoparticles.

[0035] 3) Compared with the existing invention, the lipid molecule product synthesized by the present invention replaces the two carbons in the middle carbon chain with two sulfurs, and introduces a redox-responsive group, which can accelerate the dissolution of the material in the cell and better escape from the endosomal structure, thereby improving the delivery efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Indicates 4A1S-2C10 1 H-NMR spectrum.

[0037] Figure 2 Indicates 4A2S-C18 1 H-NMR spectrum.

[0038] Figure 3 Indicates 4A2S-2C8 1 H-NMR spectrum.

[0039] Figure 4 Indicates 4A2S-210 1 H-NMR spectrum.

[0040] Figure 5 Indicates 4A3S-210 1 H-NMR spectrum.

[0041] Figure 6 Shows the in vitro mRNA delivery efficiency test results of different delivery vectors.

[0042] Figure 7 The particle sizes and PDI of different delivery vehicles are shown.

[0043] Figure 8 The potentials of different delivery vehicles are shown.

[0044] Figure 9 The encapsulation efficiency of different delivery vehicles is shown.

[0045] Figure 10It shows the comparison of delivery efficiency between sulfur-sulfur bond materials and carbon-carbon bond materials;

[0046] Figure 11 Figure 5 shows the expression of luciferase in mice 6 hours after tail vein injection of 4A1S-2C10 and mRNA lipid nanoparticles.

[0047] Figure 12 Figure 5 shows the expression of luciferase in mice 6 hours after tail vein injection of 4A2S-C18 and mRNA lipid nanoparticles.

[0048] Figure 13 Figure 5 shows the expression of luciferase in mice 6 hours after tail vein injection of 4A2S-2C8 and mRNA lipid nanoparticles.

[0049] Figure 14 Figure 5 shows the expression of luciferase in mice 6 hours after tail vein injection of 4A2S-2C10 and mRNA lipid nanoparticles.

[0050] Figure 15 Figure 5 shows the expression of luciferase in mice 6 hours after tail vein injection of 4A3S-2C10 and mRNA lipid nanoparticles.

[0051] Figure 16 The figure shows the expression of luciferase in mice 6 hours after tail vein injection of naked mRNA.

[0052] Figure 17 It shows the expression of luciferase in various organs of mice 6 hours after injection of the above materials. DETAILED DESCRIPTION

[0053] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0054] Example 1: Synthesis of 4A1S-2C10

[0055] (1) In a three-necked flask, add 10 g of 2-hydroxyethyl disulfide, 24.58 ml of triethylamine, 16 ml of acryloyl chloride, and 100 ml of tetrahydrofuran. Under nitrogen protection, stir in an ice-water bath and react for 24 hours. After the reaction, a yellow liquid crude product is obtained. First, filter it to remove the white crystals. Then extract it with a dichloromethane-saturated salt water system and a dichloromethane-water system in sequence. The product is dissolved in dichloromethane. Heat and concentrate, and the concentrated residue is purified by silica gel column chromatography. The product is purified using an eluent of n-hexane:ethyl acetate = 30:1 (v / v) as a yellow oil, which is recorded as 2,2-dithiodiethanol diacrylate (DSDA).

[0056] (2) In a 50 mL round-bottom flask, 4A1 (0.3 g), DSDA (8.418 g), inhibitor BHT (0.356 g), and DMSO (40 mL) were added. The reaction temperature was 55°C, and the mixture was stirred for 96 hours. After the reaction, a yellow oily crude product was obtained. The product was extracted with ethyl acetate-water and dissolved in ethyl acetate. The residue was concentrated by heating, and purified by silica gel column chromatography. The product was purified with ethyl acetate as an eluent to obtain a yellow oily substance, designated as 4A1S.

[0057] (3) In a brown sample bottle, the purified 4A1S (0.03 g), 2C10 (0.0636 g), BHT (0.29 mg), and anhydrous THF (4 mL) obtained in (1) were added and reacted at 55°C for 96 hours. After the reaction, a white solid was obtained, which was heated and concentrated. The concentrated residue was purified by silica gel column chromatography. The pure product was eluted with ethyl acetate as the eluent and was recorded as 4A1S-2C10. The 1H-NMR spectrum of 4A1S-2C10 is shown in FIG. Figure 1 As shown, according to Figure 1 The chromatographic characterization results showed that δ = 2.8 ppm was the hydrogen atom on the -CH2 junction of the intermediate 4A1S after the reaction between 4A1 and DSDA, with a peak area of ​​4 hydrogen atoms. δ = 2.75 ppm was the hydrogen atom on the CH2 junction of 4A1S and the hydrophobic amine 2C10, with a peak area of ​​4 hydrogen atoms.

[0058] Example 2: Synthesis of 4A2S-C18

[0059] (1) In a three-necked flask, add 10 g of 2-hydroxyethyl disulfide, 24.58 ml of triethylamine, 16 ml of acryloyl chloride, and 100 ml of tetrahydrofuran. Under nitrogen protection, stir in an ice-water bath and react for 24 hours. After the reaction, a yellow liquid crude product is obtained. First, filter it to remove the white crystals. Then extract it with a dichloromethane-saturated salt water system and a dichloromethane-water system in sequence. The product is dissolved in dichloromethane. Heat and concentrate, and the concentrated residue is purified by silica gel column chromatography. The product is purified using an eluent of n-hexane:ethyl acetate = 30:1 (v / v) as a yellow oil, which is recorded as 2,2-dithiodiethanol diacrylate (DSDA).

[0060] (2) 4A2 (0.30 g), DSDA (4.323 g), inhibitor BHT (0.181 g), and DMSO (40 mL) were added to a 50 mL round-bottom flask and stirred at 55°C for 96 hours. After the reaction, a yellow oily crude product was obtained, which was extracted with ethyl acetate-water and dissolved in ethyl acetate. The residue was concentrated by heating, and purified by silica gel column chromatography using an eluent of ethyl acetate:methanol = 50:1 (v / v) to obtain the product as a yellow oily intermediate, designated as 4A2S.

[0061] (3) In a brown sample bottle, the purified 4A2S (0.03 g), C18 (0.0569 g), BHT (0.27 mg), and anhydrous THF (4 mL) obtained in step (1) were added and reacted at 55° C. for 96 hours. After the reaction, a yellow oil was obtained, which was concentrated by heating. The concentrated residue was purified by silica gel column chromatography. The pure product was eluted with an eluent of n-hexane:ethyl acetate = 1:10 (v / v), which was recorded as 4A2S-C18. 1 H-NMR spectrum Figure 2 As shown, according to Figure 2 Spectral characterization results show that δ = 2.8 ppm represents the hydrogen atom on the -CH2 junction of the intermediate 4A2S after the reaction between 4A2 and DSDA, with a peak area of ​​4 hydrogen atoms. δ = 2.75 ppm represents the hydrogen atom on the CH2 junction of 4A2S and the hydrophobic amine C18, with a peak area of ​​4 hydrogen atoms.

[0062] Example 3: Synthesis of 4A2S-2C8

[0063] (1) In a three-necked flask, add 10 g of 2-hydroxyethyl disulfide, 24.58 ml of triethylamine, 16 ml of acryloyl chloride, and 100 ml of tetrahydrofuran. Under nitrogen protection, stir in an ice-water bath and react for 24 hours. After the reaction, a yellow liquid crude product is obtained. First, filter it to remove the white crystals. Then extract it with a dichloromethane-saturated salt water system and a dichloromethane-water system in sequence. The product is dissolved in dichloromethane. Heat and concentrate, and the concentrated residue is purified by silica gel column chromatography. The product is purified using an eluent of n-hexane:ethyl acetate = 30:1 (v / v) as a yellow oil, which is recorded as 2,2-dithiodiethanol diacrylate (DSDA).

[0064] (2) 4A2 (0.30 g), DSDA (4.323 g), inhibitor BHT (0.181 g), and DMSO (40 mL) were added to a 50 mL round-bottom flask and stirred at 55°C for 96 hours. After the reaction, a yellow oily crude product was obtained, which was extracted with ethyl acetate-water and dissolved in ethyl acetate. The residue was concentrated by heating, and purified by silica gel column chromatography using an eluent of ethyl acetate:methanol = 50:1 (v / v) to obtain the product as a yellow oily intermediate, designated as 4A2S.

[0065] (3) In a brown sample bottle, the purified 4A2S (0.03 g), 2C10 (0.0597 g), BHT (0.27 mg), and anhydrous THF (4 mL) obtained in step (1) were added and reacted at 55° C. for 96 hours. After the reaction, a yellow oil was obtained, which was heated and concentrated. The concentrated residue was purified by silica gel column chromatography. The pure product was eluted with an eluent of n-hexane:ethyl acetate = 10:1 (v / v), which was recorded as 4A2S-2C8. 1 H-NMR spectrum Figure 3 As shown, according to Figure 3 Spectral characterization results show that δ = 2.8 ppm represents the hydrogen atom on the -CH2 junction of the intermediate 4A2S after the reaction between 4A2 and DSDA, with a peak area of ​​4 hydrogen atoms. δ = 2.75 ppm represents the hydrogen atom on the CH2 junction of 4A2S and the hydrophobic amine 2C8, with a peak area of ​​4 hydrogen atoms.

[0066] Example 4: Synthesis of 4A2S-2C10

[0067] (1) In a three-necked flask, add 10 g of 2-hydroxyethyl disulfide, 24.58 ml of triethylamine, 16 ml of acryloyl chloride, and 100 ml of tetrahydrofuran. Under nitrogen protection, stir in an ice-water bath and react for 24 hours. After the reaction, a yellow liquid crude product is obtained. First, filter it to remove the white crystals. Then extract it with a dichloromethane-saturated salt water system and a dichloromethane-water system in sequence. The product is dissolved in dichloromethane. Heat and concentrate, and the concentrated residue is purified by silica gel column chromatography. The product is purified using an eluent of n-hexane:ethyl acetate = 30:1 (v / v) as a yellow oil, which is recorded as 2,2-dithiodiethanol diacrylate (DSDA).

[0068] (2) 4A2 (0.30 g), DSDA (4.323 g), inhibitor BHT (0.181 g), and DMSO (40 mL) were added to a 50 mL round-bottom flask and stirred at 55°C for 96 hours. After the reaction, a yellow oily crude product was obtained, which was extracted with ethyl acetate-water and dissolved in ethyl acetate. The residue was concentrated by heating, and purified by silica gel column chromatography using an eluent of ethyl acetate:methanol = 50:1 (v / v) to obtain the product as a yellow oily intermediate, designated as 4A2S.

[0069] (3) In a brown sample bottle, the purified 4A2S (0.03 g), 2C10 (0.0597 g), BHT (0.27 mg), and anhydrous THF (4 mL) obtained in step (1) were added and reacted at 55° C. for 96 hours. After the reaction, a white solid was obtained, which was heated and concentrated. The concentrated residue was purified by silica gel column chromatography and eluted with an eluent of n-hexane:ethyl acetate = 10:1 (v / v). The pure product was recorded as 4A2S-2C10. 1 H-NMR spectrum Figure 4 As shown, according to Figure 4 Spectral characterization results show that δ = 2.8 ppm represents the hydrogen atom on the -CH2 junction of the intermediate 4A2S after the reaction between 4A2 and DSDA, with a peak area of ​​4 hydrogen atoms. δ = 2.75 ppm represents the hydrogen atom on the CH2 junction of 4A2S and the hydrophobic amine 2C10, with a peak area of ​​4 hydrogen atoms.

[0070] Example 5: Synthesis of 4A3S-2C10

[0071] (1) In a three-necked flask, add 10 g of 2-hydroxyethyl disulfide, 24.58 ml of triethylamine, 16 ml of acryloyl chloride, and 100 ml of tetrahydrofuran. Under nitrogen protection, stir in an ice-water bath and react for 24 hours. After the reaction, a yellow liquid crude product is obtained. First, filter it to remove the white crystals. Then extract it with a dichloromethane-saturated salt water system and a dichloromethane-water system in sequence. The product is dissolved in dichloromethane. Heat and concentrate, and the concentrated residue is purified by silica gel column chromatography. The product is purified using an eluent of n-hexane:ethyl acetate = 30:1 (v / v) as a yellow oil, which is recorded as 2,2-dithiodiethanol diacrylate (DSDA).

[0072] (2) 4A3 (0.30 g), DSDA (4.239 g), inhibitor BHT (0.17 g), and DMSO (40 mL) were added to a 50 mL round-bottom flask and stirred at 55°C for 96 hours. After the reaction, a yellow oily crude product was obtained, which was extracted with ethyl acetate-water and dissolved in ethyl acetate. The residue was concentrated by heating, and purified by silica gel column chromatography using an eluent of ethyl acetate:n-hexane = 2:1 (v / v) to obtain the product as a yellow oily intermediate, designated as 4A3S.

[0073] (3) In a brown sample bottle, the purified 4A3S (0.03 g), 2C10 (0.0596 g), BHT (0.27 mg), and anhydrous THF (4 mL) obtained in step (1) were added and reacted at 55° C. for 96 hours. After the reaction, a white solid was obtained, which was heated and concentrated. The concentrated residue was purified by silica gel column chromatography and eluted with an eluent of n-hexane:ethyl acetate = 1:10 (v / v). The pure product was recorded as 4A3S-2C10. 1 H-NMR spectrum Figure 5 As shown, according to Figure 5 Spectral characterization results show that δ = 2.8 ppm represents the hydrogen atom on the -CH2 junction of the intermediate 4A3S after the reaction between 4A3 and DSDA, with a peak area of ​​4 hydrogen atoms. δ = 2.75 ppm represents the hydrogen atom on the CH2 junction of 4A3S and the hydrophobic amine 2C10, with a peak area of ​​4 hydrogen atoms.

[0074] 1. The structure of the lipid molecules of the present invention is confirmed by nuclear magnetic spectrum, and the nuclear magnetic spectrum is as follows Figure 1-Figure 5 shown.

[0075] Compared with the previous patent, the innovation of this patent lies in replacing the carbon-carbon bond with a sulfur-sulfur bond in the connecting part. In order to demonstrate the advantages of the compound of the present invention, the following two control compounds are set up:

[0076] The preparation of the control compound 4A1C-2C10 was carried out by repeating the steps of Example 1, except that "in step (2), DSDA was replaced with an equal mass of 1,6-hexanediol diacrylate (DAHE), and the product finally obtained in step (2) was denoted as 4A1C. In step (3), 4A1S was replaced with an equal mass of 4A1C, and the product finally obtained in step (3) was denoted as 4A1C-2C10." The other conditions remained unchanged.

[0077] The preparation of the control compound 4A3C-2C10 was carried out by repeating the steps of Example 5, except that "in step (2), DSDA was replaced with an equal mass of 1,6-hexanediol diacrylate (DAHE), and the product finally obtained in step (2) was recorded as 4A3C. In step (3), 4A3S was replaced with an equal mass of 4A3C, and the product finally obtained in step (3) was recorded as 4A3C-2C10." The other conditions remained unchanged.

[0078] 2. In vitro mRNA delivery efficiency

[0079] The synthesized lipid carrier was mixed with the auxiliary lipids DOPE, cholesterol, and PEG2000-DMG at a molar ratio of 38.5:30:30:1.5 and dissolved in anhydrous ethanol to produce an ethanol solution. Luciferase mRNA (Fluc mRNA) was dissolved in a sodium citrate solution (10 mM, pH = 4.2) to produce a sodium citrate solution. The ethanol solution was mixed with the sodium citrate solution (10 mM, pH = 4.2) at a ratio of 1:3 (v / v), incubated for 1 hour, and then diluted with PBS to 5 times the original volume to produce the desired nanoparticle solution. Each liter of the composite nanoparticle solution contained 1.67 mg of luciferase mRNA and 33.4 mg of the delivery vector, with a mass ratio of delivery vector to luciferase mRNA (Fluc mRNA) of approximately 20:1.

[0080] After IGrov1 cells were cultured in DMEM medium until adherent, the composite plasmid solution prepared above was added (at a volume of 75 μL / mL of cell solution) and transfected for 48 hours under these conditions. In vitro mRNA delivery efficiency was tested.

[0081] A blank control group was also established. Specifically, IGrov1 cells were cultured in DMEM medium until adherent, and then a small amount of DMEM medium (75 μL / mL cell solution) was added. Transfection was continued under these conditions for 48 hours to test the in vitro mRNA delivery efficiency.

[0082] Through the above operation process, the lipid molecules 4A1S-2C10, 4A2S-C18, 4A2S-2C8, 4A2S-2C10, 4A3S-2C10 synthesized in Examples 1-5 of the present invention and the commercially available positive control reagent MC3 were used as delivery vectors, mixed with auxiliary lipids and mRNA encoding luciferase (luciferase), and prepared nanoparticles. The nanoparticles were added to IGrov1 cells. After 48 hours of transfection, the in vitro delivery efficiency of the mRNA was evaluated by the expression of luciferase in the cells. The in vitro mRNA delivery efficiency test results of different delivery vectors are shown in FIG. Figure 6 . Figure 6 The fluorescence intensity of the different delivery vector groups on the vertical axis is the relative value of the ratio to the fluorescence intensity of the MC3 group, and the cell viability (ie, the number of surviving cells) is the relative value of the ratio to the cell viability of the blank control group. Figure 6 The high or low fluorescence intensity reflects the mRNA delivery efficiency of different carriers. It can be seen that the mRNA delivery efficiency of the lipid carriers synthesized in Examples 1 and 3-5 of the present invention is significantly higher than that of the commercially available positive control reagent MC3.

[0083] In addition, through the above operation process, the sulfur-sulfur bond materials 4A1S-2C10, 4A3S-2C10, and the carbon-carbon bond material control compounds 4A1C-2C10 and the control compound 4A3C-2C10 were used as delivery carriers, mixed with auxiliary lipids and mRNA encoding luciferase to prepare nanoparticles, and added to IGrov1 cells. After 48 hours of transfection, the in vitro delivery efficiency of mRNA was evaluated by the level of luciferase expression in the cells. The comparison of the delivery efficiency of sulfur-sulfur bond materials and carbon-carbon bond materials is shown in Figure 2. Figure 10 . Figure 10 The cell viability (i.e., number of surviving cells) of the different delivery vector groups in the middle vertical axis is the relative value of the ratio of the cell viability of the blank control group. Figure 10 It can be seen that the mRNA delivery efficiency of sulfur-sulfur bond materials is significantly higher than that of carbon-carbon bond materials.

[0084] 3. Characterization of the Physicochemical Properties of Delivery Materials

[0085] The synthesized lipid carrier was mixed with the auxiliary lipids DOPE, cholesterol, and PEG2000-DMG at a molar ratio of 38.5:30:30:1.5 and dissolved in anhydrous ethanol to produce an ethanol solution. Luciferase mRNA (Fluc mRNA) was dissolved in a sodium citrate solution (10 mM, pH = 4.2) to produce a sodium citrate solution. The ethanol solution was mixed with the sodium citrate solution (10 mM, pH = 4.2) at a ratio of 1:3 (v / v), incubated for 1 hour, and then diluted with PBS to 5 times the original volume to produce the desired nanoparticle solution. Each liter of the composite nanoparticle solution contained 1.67 mg of luciferase mRNA and 33.4 mg of the delivery vector, with a mass ratio of delivery vector to luciferase mRNA (Fluc mRNA) of approximately 20:1.

[0086] The particle size and surface potential of lipid nanoparticles were determined by dynamic light scattering (DLS). The particle size and PDI test results are shown in Figure 7 , the potential test results are shown in Figure 8 . The Quant-iT RiboGreenAssay kit was used to determine the concentration of free mRNA in the nanoparticles to quantify the efficiency of nanoparticle encapsulation of mRNA. The specific steps are to take 100 μL of nanoparticle solution diluted 8 times with buffer and add it to a 96-well black board, then add 100 μL of RiboGreen reagent diluted 2000 times with TE buffer, and then gently blow it and use a microplate reader (λex=485nm, λem=535nm) to detect the fluorescence intensity to quantify the free mRNA. The loading rate of mRNA in the nanoparticles can be obtained by calculation. The test results are shown in Figure 9 .

[0087] 4. In vivo mRNA delivery effect test

[0088] Lipid molecule experimental group: Lipid molecules were mixed with DOPE, cholesterol, and PEG2000-DMG at a molar ratio of 38.5:30:30:1.5 and dissolved in anhydrous ethanol. Luciferase mRNA (Fluc mRNA) was dissolved in a sodium citrate solution (10 mM, pH = 4.2). The ethanol solution and sodium citrate solution (10 mM, pH = 4.2) were mixed at a ratio of 1:3, resulting in a lipid molecule to luciferase mRNA (Fluc mRNA) mass ratio of approximately 20:1. After incubation, the solution was diluted fivefold with PBS to obtain a composite nanoparticle solution (containing 1.67 mg of luciferase mRNA and 33.4 mg of lipid molecules per liter of composite nanoparticle solution).

[0089] Naked mRNA group: Luciferase mRNA (Fluc mRNA) was dissolved in sodium citrate solution (10 mM, pH =

[0090] 4.2), and then diluted with PBS after incubation to obtain an mRNA nanoparticle solution (containing 1.67 mg luciferase mRNA per liter of nanoparticle solution).

[0091] Nanoparticle solutions prepared from the lipid molecule or naked mRNA groups were used to test the mRNA delivery efficacy in vivo in 5-week-old female BALB / c mice weighing approximately 20g. Each mouse received an injection volume of 0.2mL of the composite nanoparticle solution. The corresponding lipid-mRNA composite nanoparticle solution or mRNA nanoparticle solution was injected into the mice via the tail vein. Six hours after administration, the luciferase expression efficiency in the mice was measured using a small animal imaging device.

[0092] According to the above process, when the lipid molecules were 4A1S-2C10, 4A2S-C18, 4A2S-2C8, 4A2S-2C10, and 4A3S-2C10, the expression results of luciferase in mice were shown in Figure 2. Figure 11-15 As shown. Figure 11-15 It can be seen that 6 hours after the injection of the composite nanoparticle solution formed by the five lipid molecules and mRNA, the mice all had strong fluorescence expression. The results showed that after the injection of the composite nanoparticle solution formed by the lipid molecules and mRNA, the mice had strong luciferase expression in their bodies, especially 4A2S-2C8 and 4A2S-2C10, whose expression levels could reach 10 9 At this order of magnitude, the other three lipid carriers also have relatively excellent mRNA delivery effects in vivo.

[0093] According to the above process, when the injected agent is mRNA nanoparticle solution, the expression results of luciferase in mice are shown in Figure 16 As shown. Figure 16 It can be seen that there is no expression of luciferase after naked mRNA is injected into the tail vein of mice.

[0094] According to the experimental results, the lipid molecules 4A1S-2C10, 4A2S-C18, 4A2S-2C8, 4A2S-2C10, and 4A3S-2C10 synthesized according to Examples 1-5 all have good in vivo mRNA delivery efficiency.

[0095] 5. Organ Distribution of mRNA Lipid Nanoparticles

[0096] The in vivo mRNA delivery effect test of the lipid molecule experimental group and the naked mRNA group was performed 24 hours later. Organs were dissected and imaged (liver, spleen, lung, heart, and kidney). The results of luciferase expression in each organ of the mice are shown in the figure. Figure 17 ,according to Figure 17 The results of in vivo imaging of the lipid molecules synthesized in Examples 1-5 of the present invention were consistent with the in vivo organ distribution results: 4A1S-2C10, 4A2S-2C8, and 4A2S-2C10 had the best delivery effects, while 4A3S-2C10 had a moderate delivery effect. All four materials effectively delivered luc-mRNA to the spleen, resulting in high levels of luciferase expression. However, 4A2S-C18 was almost incapable of delivering luciferase.

[0097] The contents described in this specification are merely an enumeration of implementation forms of the inventive concept, and the protection scope of the present invention should not be considered as being limited to the specific forms described in the embodiments.

Claims

1. A disulfide bond-containing lipid molecule for mRNA delivery, wherein the structure is selected from one of the following: 。 2. The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery according to claim 1, characterized in that The following steps are involved: 1) 2,2-dithiodiethanol diacrylate and a hydrophilic amine are dissolved as raw materials in a reaction solvent, and a polymerization inhibitor BHT is added to the system. The reaction temperature is maintained between 53°C and 60°C and the reaction is continued for 90-100 hours to obtain a crude intermediate product characterized by a yellow oil. The yellow oil is extracted with an ethyl acetate-water system. The crude intermediate product is extracted into an ethyl acetate phase and purified by silica gel column chromatography. The hydrophilic amine raw material is one of 4A1, 4A2, and 4A3. ； 2) The intermediate purified in step 1) and a hydrophobic amine are dissolved in a reaction solvent, and BHT is added to react. The reaction temperature is maintained between 50°C and 60°C and the reaction is continued for 80-120 hours. After the reaction, a yellow oil is obtained, which is finally purified by silica gel column chromatography to obtain the final product; wherein the hydrophobic amine raw material is one of C18, 2C8, and 2C10; 。 3. The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery according to claim 2, characterized in that Step 1) The preparation method of 2,2-dithiodiethanol diacrylate comprises the following steps: 2-Hydroxyethyl disulfide, triethylamine, and acryloyl chloride are dissolved as raw materials in a reaction solvent, and the reaction is stirred in an ice-water bath under nitrogen protection. After the reaction, a yellow liquid crude product is obtained, which is first filtered to remove white crystals, and then extracted with a dichloromethane-saturated brine system and a dichloromethane-water system in sequence. The product is dissolved in dichloromethane, heated and concentrated, and the concentrated residue is purified by silica gel column chromatography to obtain 2,2-dithiodiethanol diacrylate.

4. The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery according to claim 3, characterized in that In the preparation method of 2,2-dithiodiethanol diacrylate, the solid-liquid ratio of 2-hydroxyethyl disulfide to acryloyl chloride is 1 g:1-3 ml, the solid-liquid ratio of 2-hydroxyethyl disulfide to triethylamine is 1 g:1.5-4 ml, the reaction solvent is THF, the reaction time is 20-30 hours, and the elution solvent for purification by silica gel column chromatography is a mixture of n-hexane and ethyl acetate with a volume ratio of 20-40:

1.

5. The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery according to claim 4, characterized in that In the preparation method of 2,2-dithiodiethanol diacrylate, the solid-liquid ratio of 2-hydroxyethyl disulfide to acryloyl chloride is 1g:1.5-2ml, and the solid-liquid ratio of 2-hydroxyethyl disulfide to triethylamine is 1g:2-2.5ml.

6. The method for synthesizing a disulfide bond-containing lipid molecule for mRNA delivery according to claim 2, wherein In step 1), the mass ratio of the hydrophilic amine to 2,2-dithiodiethanol diacrylate is 1:10-30; the mass ratio of 2,2-dithiodiethanol diacrylate to the polymerization inhibitor BHT is 15-30:1, and the reaction solvent is DMSO.

7. A method for synthesizing a disulfide bond-containing lipid molecule for mRNA delivery according to claim 6, characterized in that In step 1), the mass ratio of the hydrophilic amine to 2,2-dithiodiethanol diacrylate is 1:14-28; the mass ratio of 2,2-dithiodiethanol diacrylate to the polymerization inhibitor BHT is 20-25:

1.

8. The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery according to claim 2, characterized in that In step 1), the reaction temperature is 55-56° C., the reaction time is 95-96 h, and purification is carried out by silica gel column chromatography using a 40-50:1 volume ratio ethyl acetate-methanol mixture or a 0.5-2:1 volume ratio n-hexane-ethyl acetate mixture or ethyl acetate as the eluent.

9. The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery according to claim 2, characterized in that In step 2), the mass ratio of the purified intermediate to the hydrophobic amine is 1:1.5-3, the mass ratio of the intermediate to the polymerization inhibitor BHT is 1:0.005-0.015, and the reaction solvent is anhydrous THF.

10. The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery according to claim 9, characterized in that In step 2), the mass ratio of the purified intermediate to the hydrophobic amine is 1:1.8-2.2, and the mass ratio of the intermediate to the polymerization inhibitor BHT is 1:0.009-0.

01.

11. The method for preparing a disulfide bond-containing lipid molecule for mRNA delivery according to claim 2, characterized in that In step 2), the reaction temperature is 53-60° C., the reaction time is 96-100 hours, and the eluent for purification by silica gel column chromatography is ethyl acetate or a mixture of n-hexane and ethyl acetate in a volume ratio of 1:10-10:

1.

12. Use of the disulfide bond-containing lipid molecule for mRNA delivery according to claim 1 in delivering mRNA molecules.

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