Disulfide bond-containing lipid molecule for mRNA delivery as well as preparation method and application of disulfide bond-containing lipid molecule

Abstract

The invention discloses a disulfide bond-containing lipid molecule for mRNA delivery and a preparation method and application thereof.The synthesis method of the lipid molecule comprises the steps that 2, 2-dithiodiethanol diacrylate is used as a raw material and further reacts with selected hydrophilic amine to generate an intermediate, and then the intermediate is subjected to a reaction to obtain the disulfide bond-containing lipid molecule for mRNA delivery. Further reacting the intermediate with hydrophobic amine to obtain a lipid molecule finally used for delivering mRNA, wherein the hydrophilic amine raw material is one of 4A1, 4A2 and 4A3; the raw material of the # imgabs0 # hydrophobic amine is one of C18, 2C8 and 2C10; the lipid molecule prepared by the invention contains a head group capable of generating positive ions through ionization, and nucleic acid carries negative charges, so that the lipid molecule and the nucleic acid can be efficiently combined, and the load rate of the nucleic acid is remarkably improved. And the carbon chain length and the branch number of the hydrophobic alkane chain have a crucial influence on the stability of the formed nanoparticles, so that the lipid molecule has a very good prospect in the application of delivering mRNA (messenger Ribonucleic Acid) molecules.

Description

Technical Field

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

[0002] Messenger RNA (mRNA)-based drug technology has attracted widespread attention in the past few years. Due to its superiority, this technology has been widely used in protein replacement therapy, cancer immunotherapy and gene editing. At present, the research on the use of mRNA for disease treatment is developing rapidly. Many related methods are entering clinical trials. Therefore, the clinical application of mRNA-based therapeutics has great potential and can completely change the treatment prospects of some diseases.

[0003] Compared with plasmid DNA (pDNA) or small interfering RNA-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 work efficiently in non-dividing cells or cells that divide very slowly, such as dendritic cells; 2) mRNA does not need to be integrated into the chromosomes of the cell, 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 off-target problems similar to siRNA. In addition, compared with the corresponding recombinant protein, high-charge-density, flexible mRNA has a higher loading efficiency in the carrier; mRNA preparation is relatively simple and has a low biosafety risk.

[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 the 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 direct use in the human body. Since mRNA cannot remain active 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 mRNA non-viral delivery carrier material at present, cationic lipids / lipidoid molecules and cationic polymers can be self-assembled into carriers by mixing and incubating with cholesterol, PEG lipids, and auxiliary lipid molecules (such as DOPE), and then loaded with mRNA to form lipid nanoparticles (LNP). It has broad development space and the best effect in the well-known mRNA delivery system. According to other literature, the efficiency of ionizable cationic lipid molecules in delivering mRNA is determined by the hydrophilic-hydrophobic 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. The present invention discloses a lipid molecule that can effectively bind to mRNA and significantly improve its stability and in vitro and in vivo expression efficiency.

[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) 2-Hydroxyethyl disulfide, triethylamine, and acryloyl chloride are dissolved in a reaction solvent as raw materials, and 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 salt water system and a dichloromethane-water system, and the product is dissolved in dichloromethane. Heat and concentrate, and the concentrated 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 used as raw materials, dissolved in a reaction solvent, and a polymerization inhibitor BHT is added to the system, and the reaction temperature is maintained between 53-60° C. for continuous reaction for 90-100 hours, and finally a crude intermediate product is obtained, which has the physical characteristic of being a yellow oily substance. The yellow oily substance is extracted using an ethyl acetate-water system, and the crude intermediate product is extracted into an ethyl acetate phase, and the intermediate is purified by silica gel column chromatography;

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

[0019]

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

[0021]

[0022] Hydrophobic Amine The structural formula is one of C18, 2C8, and 2C10;

[0023]

[0024] Those with one alkane chain and 18 carbons are called C18. Those with two alkane chains and each chain has 8 carbons are called 2C8. Those with two alkane chains and each chain has 10 carbons are called 2C10. 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 R2 and R3 in 2C10 are straight-chain alkyl groups of C10.

[0025] From the above structural formula, we can clearly understand the specific structures of the hydrophilic amine and the hydrophobic amine in the lipid molecule. For example, the raw material hydrophilic amine and the hydrophobic amine used to prepare the lipid molecule 4A1S-2C10 are 4A1 and 2C10 respectively.

[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 mixed solution of n-hexane-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 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 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 an eluent to purify the product 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 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 uses 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 molecular material synthesized by the present invention includes key amines and hydrophobic units, which are proved to be essential groups for efficient mRNA delivery carriers. In the selection of hydrophilic amines, the present invention respectively adopts 4A1, 4A2, and 4A3 to effectively regulate the hydrophilicity, cationic density, and number of hydrophobic chains of the material. In terms of hydrophobic amines, the present invention respectively adopts hydrophobic amines C18, 2C8, and 2C10 to effectively regulate the hydrophobicity of the material.

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

[0035] 3) Compared with the existing invention, the two carbons in the middle carbon chain part of the lipid molecule product synthesized by the present invention are replaced by two sulfurs, and a redox-responsive group is introduced, 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 Represents the in vitro mRNA delivery efficiency test results of different delivery vectors.

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

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

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

[0045] Fig.10It represents the comparison of delivery efficiency between sulfur-sulfur bond materials and carbon-carbon bond materials;

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

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

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

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

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

[0051] Fig.16 It shows the expression of luciferase in mice injected with naked mRNA by tail vein for 6 hours.

[0052] Fig.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 in conjunction with 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 h. After the reaction, a yellow liquid crude product is obtained. First, filter out the white crystals, and extract 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), DMSO (40 mL) were added, the reaction temperature was 55°C, stirred, and the reaction was carried out for 96 hours. After the reaction was completed, a yellow oily crude product was obtained, which was extracted with ethyl acetate-water and dissolved in ethyl acetate. The concentrated residue was purified by silica gel column chromatography. The product was purified by ethyl acetate as an eluent as a yellow oily substance, which was recorded as 4A1S.

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

[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 h. After the reaction, a yellow liquid crude product is obtained. First, filter out the white crystals, and extract 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) In a 50 mL round-bottom flask, 4A2 (0.30 g), DSDA (4.323 g), inhibitor BHT (0.181 g), DMSO (40 mL) were added, stirred and dissolved at 55°C, and reacted 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 concentrated residue was purified by silica gel column chromatography using an eluent of ethyl acetate: methanol = 50:1 (v / v) to purify the product, which was a yellow oily intermediate, recorded as 4A2S.

[0061] (3) In a brown sample bottle, add the purified 4A2S (0.03 g) obtained in step (1), C18 (0.0569 g), BHT (0.27 mg), and anhydrous THF (4 mL), and react 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 = 1:10 (v / v), which was recorded as 4A2S-C18. 1 H-NMR spectrum Figure 2 As shown, according to Figure 2 The spectrum characterization results show that δ=2.8ppm is the hydrogen atom on -CH2 at the junction of 4A2S, the intermediate after the reaction of 4A2 and DSDA, and its peak area is 4 hydrogens. δ=2.75ppm is the hydrogen atom on CH2 at the junction of 4A2S and hydrophobic amine C18, and its peak area is 4 hydrogens.

[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 h. After the reaction, a yellow liquid crude product is obtained. First, filter out the white crystals, and extract 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) In a 50 mL round-bottom flask, 4A2 (0.30 g), DSDA (4.323 g), inhibitor BHT (0.181 g), DMSO (40 mL) were added, stirred and dissolved at 55°C, and reacted 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 concentrated residue was purified by silica gel column chromatography using an eluent of ethyl acetate: methanol = 50:1 (v / v) to purify the product, which was a yellow oily intermediate, recorded as 4A2S.

[0065] (3) In a brown sample bottle, add the purified 4A2S (0.03 g) obtained in step (1), 2C10 (0.0597 g), BHT (0.27 mg), and anhydrous THF (4 mL), and react at 55° C. for 96 hours. After the reaction, a yellow oil was obtained, which was concentrated by heating, and 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 The spectrum characterization results show that δ=2.8ppm is the hydrogen atom on -CH2 at the junction of 4A2S, the intermediate after the reaction of 4A2 and DSDA, and its peak area is 4 hydrogens. δ=2.75ppm is the hydrogen atom on CH2 at the junction of 4A2S and hydrophobic amine 2C8, and its peak area is 4 hydrogens.

[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 h. After the reaction, a yellow liquid crude product is obtained. First, filter out the white crystals, and extract 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) In a 50 mL round-bottom flask, 4A2 (0.30 g), DSDA (4.323 g), inhibitor BHT (0.181 g), DMSO (40 mL) were added, stirred and dissolved at 55°C, and reacted 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 concentrated residue was purified by silica gel column chromatography using an eluent of ethyl acetate: methanol = 50:1 (v / v) to purify the product, which was a yellow oily intermediate, recorded as 4A2S.

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

[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 h. After the reaction, a yellow liquid crude product is obtained. First, filter out the white crystals, and extract 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) In a 50 mL round-bottom flask, 4A3 (0.30 g), DSDA (4.239 g), inhibitor BHT (0.17 g), DMSO (40 mL) were added, stirred and dissolved at 55°C, and reacted 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 concentrated residue was heated and concentrated, and purified by silica gel column chromatography, and the product was purified with an eluent of ethyl acetate: n-hexane = 2:1 (v / v) to obtain a yellow oily intermediate, which was recorded as 4A3S.

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

[0074] 1. The structure of the lipid molecule of the present invention is confirmed by nuclear magnetic resonance spectroscopy. Figure 1-Figure 5 shown.

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

[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 by 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 by an equal mass of 4A1C, and the product finally obtained in step (3) was denoted as 4A1C-2C10", and the other conditions remained unchanged.

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

[0078] 2. In vitro mRNA delivery efficiency

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

[0080] After IGrov1 cells were cultured in DMEM medium until they were attached, the above-prepared composite plasmid solution was added (the amount added was 75 μL / mL cell solution), and transfection was performed under this condition for 48 hours. The in vitro mRNA delivery efficiency was tested.

[0081] In addition, a blank control group was set up, specifically: after IGrov1 cells were cultured in DMEM medium until they were in an adherent state, a small amount of DMEM medium (the added amount was 75 μL / mL cell solution) was added, and transfection was performed under this condition for 48 hours. The in vitro mRNA delivery efficiency was tested.

[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 to prepare nanoparticles, and added to IGrov1 cells. After 48 hours of transfection, the in vitro delivery efficiency of 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 Figure 6 . Figure 6 The fluorescence intensity of different delivery vector groups in the middle 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 intensity of the fluorescence reflects the efficiency of mRNA delivery by 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 expression of luciferase in the cells. The delivery efficiency of the sulfur-sulfur bond materials and the carbon-carbon bond materials is compared. Fig.10 . Fig.10 The cell viability (i.e., the number of surviving cells) of the different delivery vector groups in the middle vertical axis are all relative values ​​to the cell viability of the blank control group. Fig.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 and dissolved in anhydrous ethanol with auxiliary lipid DOPE, cholesterol, and PEG2000-DMG at a molar ratio of 38.5:30:30:1.5 to obtain an ethanol solution. Luciferase mRNA (Fluc mRNA) was dissolved in a sodium citrate solution (10mM, pH=4.2) to obtain a sodium citrate solution. The ethanol solution and the sodium citrate solution (10mM, pH=4.2) were controlled to be mixed at a ratio of 1:3 (v / v), incubated for 1h after mixing, and then diluted with PBS to 5 times the original volume to obtain the desired nanoparticle solution, wherein each liter of the composite nanoparticle solution contained 1.67mg of luciferase mRNA and 33.4mg of the delivery vector, and the mass ratio of the delivery vector to the luciferase mRNA (Fluc mRNA) was about 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 Fig. 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 sodium citrate solution (10mM, pH=4.2). The ratio of ethanol solution to sodium citrate solution (10mM, pH=4.2) was controlled to be 1:3, and the mass ratio of lipid molecules to luciferase mRNA (Fluc mRNA) was about 20:1. After incubation, it was diluted 5 times with PBS to obtain a composite nanoparticle solution (each liter of composite nanoparticle solution contained 1.67mg luciferase mRNA and 33.4mg lipid molecules).

[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] Using the nanoparticle solution prepared in the above lipid molecule experimental group or naked mRNA group, 5-week-old female BALB / c mice weighing about 20g were selected, and the injection volume of each mouse was 0.2mL of the composite nanoparticle solution. Under this condition, the in vivo mRNA delivery effect was tested. The composite nanoparticle solution or mRNA nanoparticle solution formed by the corresponding lipid molecule and mRNA was injected into the mouse through the tail vein, and the expression efficiency of luciferase in the mouse was detected using a small animal imaging instrument 6 hours after administration.

[0092] According to the above process, when the lipid molecules are selected as 4A1S-2C10, 4A2S-C18, 4A2S-2C8, 4A2S-2C10, and 4A3S-2C10, the expression results of luciferase in mice are 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 Fig.16 As shown. Fig.16 It can be seen that there was no expression of luciferase after naked mRNA was 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] In vivo mRNA delivery effect test of lipid molecule experimental group and naked mRNA group. Organs were dissected and imaged 24 hours later (the organs dissected were liver, spleen, lung, heart and kidney). The expression results of luciferase in various organs of mice are shown in the figure. Fig.17 ,according to Fig.17 As a result, the in vivo imaging results 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, and 4A3S-2C10 had a moderate delivery effect. The four materials could effectively deliver luc-mRNA to the spleen and express a large amount of luciferase. However, 4A2S-C18 could hardly complete the delivery.

[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 regarded 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. A method for preparing a disulfide bond-containing lipid molecule for mRNA delivery as claimed in claim 1, characterized in that The following steps are involved: 1) dissolving 2,2-dithiodiethanol diacrylate and a hydrophilic amine as raw materials in a reaction solvent, adding a polymerization inhibitor BHT to the system, maintaining the reaction temperature between 53-60° C. for continuous reaction for 90-100 hours, and finally obtaining a crude intermediate product, which is physically characterized by a yellow oily substance, extracting the yellow oily substance using an ethyl acetate-water system, extracting the crude intermediate product into an ethyl acetate phase, and purifying the intermediate by silica gel column chromatography; wherein the hydrophilic amine raw material is one of 4A1, 4A2, and 4A3; 2) The intermediate purified in step 1) and a hydrophobic amine are used as raw materials, dissolved in a reaction solvent, and then BHT is added to react, the reaction temperature is maintained between 50-60° C. and the reaction is continued for 80-120 hours. After the reaction is completed, a yellow oil is obtained, and 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. A method for preparing a disulfide bond-containing lipid molecule for mRNA delivery as claimed in claim 2, characterized in that Step 1) The method for preparing 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. A method for preparing a disulfide bond-containing lipid molecule for mRNA delivery as claimed in 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 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 mixed solution of n-hexane-ethyl acetate with a volume ratio of 20-40:

1.

5. A method for synthesizing a disulfide bond-containing lipid molecule for mRNA delivery as claimed in claim 2, characterized in that In step 1), the mass ratio of hydrophilic amine to 2,2-dithiodiethanol diacrylate is 1:10-30, preferably 1:14-28; the mass ratio of 2,2-dithiodiethanol diacrylate to polymerization inhibitor BHT is 15-30:1, preferably 20-25:1, and the reaction solvent is DMSO.

6. A method for preparing a disulfide bond-containing lipid molecule for mRNA delivery as claimed in claim 2, characterized in that In step 1), the reaction temperature is 55-56° C., the reaction time is 95-96 h, and the product is purified 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.

7. A method for preparing a disulfide bond-containing lipid molecule for mRNA delivery as claimed in claim 2, characterized in that In step 2), the mass ratio of the purified intermediate to the hydrophobic amine is 1:1.5-3, preferably 1:1.8-2.2, the mass ratio of the intermediate to the inhibitor BHT is 1:0.005-0.015, preferably 1:0.009-0.01, and the reaction solvent is anhydrous THF.

8. A method for preparing a disulfide bond-containing lipid molecule for mRNA delivery as claimed in 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.

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

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