Lipid-hydrophilic polymer and its preparation method and application

Abstract

The application belongs to the field of biological medicine, and particularly relates to a lipid-hydrophilic polymer and a preparation method and application thereof. The lipid-hydrophilic polymer provided by the application has a structure shown in formula (I), wherein R1 is a hydrophilic polymer group with a polymerization degree of 20-50, R2 is a C2-C4 alkyl group, R3 is a C2-C4 alkyl group, R4 is a hydrophobic lipid group with a single tail or a C-C double tail, L1 is an ester bond, and L2 is an amide bond or an ester bond. The application introduces a dithiole-sensitive chemical bond between the hydrophilic polymer and the lipid by molecular structure design, and obtains a breakable lipid-hydrophilic polymer. On this basis, by increasing the mole percentage of the lipid-hydrophilic polymer in the lipid nanoparticle, high-efficiency transfection can be realized while the stability of the lipid nanoparticle is enhanced. 10 ~C 22 single tail or C 10 ~C 22 double tail hydrophobic lipid group, L1 is an ester bond, and L2 is an amide bond or an ester bond. The application introduces a dithiole-sensitive chemical bond between the hydrophilic polymer and the lipid by molecular structure design, and obtains a breakable lipid-hydrophilic polymer. On this basis, by increasing the mole percentage of the lipid-hydrophilic polymer in the lipid nanoparticle, high-efficiency transfection can be realized while the stability of the lipid nanoparticle is enhanced. Formula (I).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a lipid-hydrophilic polymer and a preparation method and application thereof. BACKGROUND

[0002] In the modern field of biological medicine, mRNA therapy as a highly potential innovative treatment method has become a key research object in the academic and industrial fields. The core principle of mRNA therapy is to deliver mRNA molecules encoding specific target proteins into cells through a specific method. Cells will use their own nucleic acid translation mechanism to synthesize target proteins in situ, thereby precisely intervening in the occurrence and development process of diseases and showing great application potential in the treatment of various refractory diseases.

[0003] However, mRNA itself has inherent fragility and faces many severe challenges in in vivo application. On the one hand, the phosphodiester structure of mRNA is easily degraded by nucleases in vivo, resulting in damage to its biological activity and functional integrity; on the other hand, mRNA has high negative charge and is mutually repulsive with the negative charge of the cell membrane, and the lipid bilayer structure of the cell membrane has a natural barrier effect on macromolecular substances, making it difficult for mRNA to effectively penetrate the cell membrane and enter the cell interior. These key problems seriously restrict the application of naked mRNA. Among many mRNA delivery carriers, lipid nanoparticles (LNP) have become the core carrier for mRNA delivery due to their good safety and high transfection efficiency. In vaccine development, LNP as a delivery carrier for mRNA has achieved great success, fully verifying its feasibility and reliability in mRNA therapy.

[0004] However, the existing LNP technology still faces a key technical problem: the lipid-polyethylene glycol (PEG) in the formula can improve the stability and blood circulation time of nanoparticles by forming a hydrophilic protective layer, but when the molar percentage of this component exceeds 1.5%, the dense hydration barrier will significantly hinder the interaction between LNP and the cell membrane, resulting in a significant decrease in in vitro and in vivo transfection efficiency. Although theoretically increasing the PEG ratio can optimize the pharmacokinetic properties, in actual application, the transfection efficiency and PEG content present a contradictory relationship, greatly limiting the optimization space of LNP technology. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a lipid-hydrophilic polymer and a preparation method and application thereof. By molecular structure design, the present application introduces a thioenol-sensitive chemical bond between the hydrophilic polymer and the lipid, obtaining a breakable lipid-hydrophilic polymer. On this basis, by increasing the molar percentage of the lipid-hydrophilic polymer in the lipid nanoparticle, it can enhance the stability of the lipid nanoparticle while achieving high-efficiency transfection.

[0006] The application provides a lipid-hydrophilic polymer with the structure shown in formula (I).

[0007] Formula (I);

[0008] In formula (I), R1 is a hydrophilic polymer group with a polymerization degree of 20-50, R2 is C2-C4 alkyl, R3 is C2-C4 alkyl, R4 is a hydrophobic lipid group with C 10 ~C 22 single tail or C 10 ~C 22 double tail, L1 is an ester bond, and L2 is an amide bond or an ester bond.

[0009] Preferably, R1 is the structure of formula (i) or formula (ii).

[0010] Formula (i); Formula (ii);

[0011] Wherein, n is 20-50, and R5 is methyl or ethyl.

[0012] Preferably, R4 is one of the structures of formula (iii) to formula (vi).

[0013] Formula (iii); Formula (iv);

[0014] Formula (v); Formula (vi);

[0015] Wherein, R6-R 12 are independently selected from C 10 ~C 22 alkyl or C 10 ~C 22 unsaturated hydrocarbon group.

[0016] The application provides a preparation method of the lipid-hydrophilic polymer.

[0017] a) mixing and reacting acetone and a mercapto C2-C4 alkyl carboxylic compound to obtain a binary acid with a thio ketone linkage shown in formula (II);

[0018] Formula (II);

[0019] b) reacting the binary acid with excess acetic anhydride to obtain a cyclic anhydride containing a thio ketone structure;

[0020] c) condensing the cyclic anhydride with a hydroxyl-terminated hydrophilic polymer having a structure shown in formula (III) to obtain a graft product having a structure shown in formula (IV) ;

[0021] Formula (III) ; Formula (IV) ;

[0022] d) condensing the graft product with a hydrophobic lipid having a structure shown in formula (V) to obtain a lipid-hydrophilic polymer having a structure shown in formula (I) ;

[0023] Formula (V) ; Formula (I) ;

[0024] In formula (I) - (V), R1 is a hydrophilic polymer group having a polymerization degree of 20 - 50, R2 is a C2 - C4 alkyl group, R3 is a C2 - C4 alkyl group, R4 is a hydrophobic lipid group having a single tail or a double tail, L1 is an ester bond, L2 is an amide bond or an ester bond, and L2' is a chloroamido group or an amino group. 10 ~C 22 single tail or a C 10 ~C 22 double tail, L1 is an ester bond, L2 is an amide bond or an ester bond, and L2' is a chloroamido group or an amino group.

[0025] Preferably, the molar ratio of the acetone to the mercapto C2 - C4 alkyl carboxylic compound is (3 - 5) : 1.

[0026] The molar ratio of the dibasic acid to the acetic anhydride is 1 : (3 - 10).

[0027] The molar ratio of the cyclic anhydride to the hydroxyl-terminated hydrophilic polymer is (3 - 10) : 1.

[0028] The molar ratio of the graft product to the hydrophobic lipid is 1 : (3 - 10).

[0029] The present application provides a lipid nanoparticle, comprising: a lipid nanoparticle matrix and a nucleic acid encapsulated in the lipid nanoparticle matrix.

[0030] The components of the lipid nanoparticle matrix include an ionizable cationic lipid, an auxiliary phospholipid, cholesterol and a lipid-hydrophilic polymer; the lipid-hydrophilic polymer is the lipid-hydrophilic polymer described in the above technical solution or the lipid-hydrophilic polymer prepared by the preparation method described in the above technical solution.

[0031] Preferably, the ionizable cationic lipid is one or more of heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate, ((4-hydroxybutyl)azanediyl)bis(hexan-6,1-diyl)bis(2-hexyldecanoate), and 4-(N,N-dimethylamino)butanoic acid(dilinoleyl) methyl ester;

[0032] The auxiliary phospholipid is one or more of distearoylphosphatidylcholine, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine, 2-(dioctylamino)ethyl nonylphosphate hydrochloride, 2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid, and 1,2-dioleoyl-SN-glycero-3-phosphate-RAC-glycerol sodium salt;

[0033] The nucleic acid is mRNA and / or DNA.

[0034] Preferably, the molar ratio of the ionizable cationic lipid, the auxiliary phospholipid, the cholesterol, and the lipid-hydrophilic polymer is 50:(2~20):(20~50):(1~20).

[0035] The application provides a preparation method of the lipid nanoparticle.

[0036] The lipid phase and the aqueous phase are mixed to obtain the lipid nanoparticle through self-assembly.

[0037] The components of the lipid phase include the ionizable cationic lipid, the cholesterol, the auxiliary phospholipid, the lipid-hydrophilic polymer, and the organic solvent; and the aqueous phase contains the nucleic acid.

[0038] The application provides an application of the lipid nanoparticle or the lipid nanoparticle prepared by the preparation method in nucleic acid delivery.

[0039] Compared with the prior art, the application provides a lipid-hydrophilic polymer, a preparation method and an application thereof. 10 ~C 22 single-tailed or C 10 ~C 22The hydrophobic lipid group of the double-tailed hydrophobic lipid group is L1, and L2 is an ester bond or an amide bond. The application introduces a thioen sensitive chemical bond between the hydrophilic polymer and the lipid by molecular structure design, and obtains a breakable lipid-hydrophilic polymer. On this basis, by increasing the mole percentage of the lipid-hydrophilic polymer in the lipid nanoparticle, efficient transfection can be realized while enhancing the stability of the lipid nanoparticle. Experimental results show that the lipid-hydrophilic polymer developed in the application realizes efficient mRNA transfection by intramuscular injection in the range of 1.5-15.5% in mole percentage; when used for intravenous injection, selective transfection of the mouse spleen can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0041] Figure 1 The lipid-hydrophilic polymer DSPE-POx provided for the embodiment 1 of the present application tk NMR spectrum of hydrogen;

[0042] Figure 2 The lipid-hydrophilic polymer DSPE-PEG provided for the embodiment 2 of the present application tk NMR spectrum of hydrogen;

[0043] Figure 3 The DSPE-POx with different mole ratios provided for the embodiment 5 of the present application tk Particle size and polydispersity index of DSPE-POx-LNP;

[0044] Figure 4 The DSPE-PEG with different mole ratios provided for the embodiment 5 of the present application tk Particle size and polydispersity index of DSPE-POx-LNP;

[0045] Figure 5 The DSPE-POx with different mole ratios provided for the embodiment 5 of the present application tk Agarose gel electrophoresis test of mRNA encapsulation effect of DSPE-POx-LNP;

[0046] Figure 6 The DSPE-POx with different mole ratios provided for the embodiment 5 of the present application tk TEM morphology characterization of DSPE-POx-LNP;

[0047] Figure 7Different molar ratios of DSPE-POx provided for the present embodiment 6 tk Figure of transfection efficiency of LNP in HEK 293T cell line;

[0048] Figure 8 Different molar ratios of DSPE-POx provided for the present embodiment 7 tk Figure of transfection efficiency of LNP in C57BL / 6 mouse organs; wherein, a) is the injection of DSPE-POx tk Figure of bioluminescence of each organ after LNP transfection; b) is DSPE-POx tk Figure of quantification of average fluorescence intensity of each organ after LNP transfection; c) is DSPE-POx tk Figure of average fluorescence intensity ratio in each organ after LNP transfection;

[0049] Figure 9 Different molar ratios of DSPE-PEG provided for the present embodiment 7 tk Figure of transfection efficiency of LNP in C57BL / 6 mouse organs; wherein, a) is the injection of DSPE-PEG tk Figure of bioluminescence of each organ after LNP transfection; b) is DSPE-PEG tk Figure of quantification of average fluorescence intensity of each organ after LNP transfection; c) is DSPE-PEG tk Figure of average fluorescence intensity ratio in each organ after LNP transfection. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0051] The present application provides a lipid-hydrophilic polymer, which is composed of a hydrophobic lipid, a thioether linkage and a hydrophilic polymer, and has a structure shown in formula (I):

[0052] Formula (I);

[0053] In formula (I), R1 is a hydrophilic polymer group with a polymerization degree of 20-50, R2 is C2-C4 alkyl, R3 is C2-C4 alkyl, and R4 is C 10 ~C 22 single-tailed or C 10 ~C 22The hydrophobic lipid group is a double-tailed hydrophobic lipid group, L1 is an ester bond, and L2 is an amide bond or an ester bond.

[0054] In the lipid-hydrophilic polymer provided by the present application, in the formula (I), the R1 is preferably a structure of formula (i) or formula (ii):

[0055] Formula (i); Formula (ii);

[0056] wherein n is 20-50, and can be specifically 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50; and R5 is methyl or ethyl.

[0057] In the lipid-hydrophilic polymer provided by the present application, in the formula (I), the R2 can be specifically a C2 alkyl group, a C3 alkyl group, or a C4 alkyl group.

[0058] In the lipid-hydrophilic polymer provided by the present application, in the formula (I), the R3 can be specifically a C2 alkyl group, a C3 alkyl group, or a C4 alkyl group.

[0059] In the lipid-hydrophilic polymer provided by the present application, in the formula (I), the R4 is preferably one of the structures of formula (iii) to formula (vi):

[0060] Formula (iii); Formula (iv);

[0061] Formula (v); Formula (vi);

[0062] wherein R6-R 12 are independently selected from C 10 -C 22 alkyl or C 10 -C 22 unsaturated hydrocarbon groups; the carbon atom number of the C 10 -C 22 alkyl group can be specifically C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , or C 22.

[0063] In the lipid-hydrophilic polymer provided by the present application, the lipid-hydrophilic polymer can be specifically a polymer having the structure shown in formula (I-1) or formula (I-2):

[0064] Formula (I-1);

[0065] Formula (I-2).

[0066] The present application also provides a preparation method of the lipid-hydrophilic polymer described in the above technical solution, comprising the following steps:

[0067] a) mixing and reacting acetone with a mercapto C2-C4 alkyl carboxylic acid compound to obtain a binary acid having a thioacetone connecting bond, which has the structure shown in formula (II);

[0068] Formula (II);

[0069] b) dehydrating the binary acid by reacting with excess acetic anhydride to obtain a cyclic anhydride containing a thioacetone structure;

[0070] c) condensing the cyclic anhydride with a hydroxyl-terminated hydrophilic polymer having the structure shown in formula (III) to obtain a grafting product having the structure shown in formula (IV);

[0071] Formula (III); Formula (IV);

[0072] d) condensing the grafting product with a hydrophobic lipid having the structure shown in formula (V) to obtain a lipid-hydrophilic polymer having the structure shown in formula (I);

[0073] Formula (V); Formula (I);

[0074] In formula (I) to formula (V), R1 is a hydrophilic polymer group with a polymerization degree of 20-50, R2 is C2-C4 alkyl, R3 is C2-C4 alkyl, R4 is a hydrophobic lipid group containing C 10 ~C 22 single-tailed or C 10 ~C 22 double-tailed hydrophobic lipid group, L1 is an ester bond, L2 is an amide bond or an ester bond, and L2' is an acyl chloride group or an amino group.

[0075] In the preparation method provided by the present application, in step a), the mercapto C2-C4 alkyl carboxylic acid compound is preferably mercapto acetic acid or mercapto propionic acid.

[0076] In the preparation method provided by the application, in step a), the molar ratio of the acetone to the mercapto C2-C4 alkyl carboxylic acid compound is preferably (3-5):1, and can be specifically 3:1, 3.2:1, 3.5:1, 3.7:1, 4:1, 4.2:1, 4.5:1, 4.7:1 or 5:1.

[0077] In the preparation method provided by the application, in step a), the mixing reaction is preferably carried out under acid catalysis, and the acid catalyst is preferably trifluoroacetic acid.

[0078] In the preparation method provided by the application, in step a), the temperature of the mixing reaction is preferably 10-40℃, and can be specifically 10℃, 15℃, 20℃, 25℃, 30℃, 35℃ or 40℃; and the time of the mixing reaction is not particularly limited, and is preferably 3-12h.

[0079] In the preparation method provided by the application, in step a), after the mixing reaction is completed, post-treatment is carried out; and the process of the post-treatment preferably comprises: low-temperature precipitation of a solid product, then alternating washing and purification using n-hexane and water, and then drying to obtain the target product.

[0080] In the preparation method provided by the application, in step b), the molar ratio of the dibasic acid to the acetic anhydride is preferably 1:(3-10), and can be specifically 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0081] In the preparation method provided by the application, in step b), the reaction is carried out under anhydrous and anaerobic conditions.

[0082] In the preparation method provided by the application, in step b), the temperature of the reaction is preferably 10-40℃, and can be specifically 10℃, 15℃, 20℃, 25℃, 30℃, 35℃ or 40℃; and the time of the reaction is not particularly limited, and is preferably 6-24h.

[0083] In the preparation method provided by the application, in step b), after the mixing reaction is completed, post-treatment is carried out; and the process of the post-treatment preferably comprises: removal of residual acetic anhydride and dibasic acid by evaporation under reduced pressure.

[0084] In the preparation method provided by the present invention, in step c), the hydroxyl-terminated hydrophilic polymer is preferably poly(2-oxazoline) or polyethylene glycol, and the poly(2-oxazoline) is preferably poly(2-methyl-2-oxazoline) or poly(2-ethyl-2-oxazoline); the degree of polymerization of the hydroxyl-terminated hydrophilic polymer is preferably 20 to 50, specifically 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50.

[0085] In the preparation method provided by the present invention, in step c), the molar ratio of the cyclic anhydride to the hydroxyl-terminated hydrophilic polymer is preferably (3-10):1, specifically 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0086] In the preparation method provided by the present invention, in step c), the condensation reaction is carried out in an organic solvent; the organic solvent is preferably dichloromethane and / or chloroform (chloroform).

[0087] In the preparation method provided by the present invention, in step c), the temperature of the condensation reaction is preferably 20-40°C, specifically 20°C, 25°C, 30°C, 35°C, or 40°C; and the time of the condensation reaction is preferably 12-48 hours.

[0088] In the preparation method provided by the present invention, in step c), after the condensation reaction is completed, post-treatment is performed; the post-treatment process preferably includes: ether precipitation, ultrapure water dialysis purification and freeze-drying in sequence.

[0089] In the preparation method provided by the present invention, in step d), the tail structure of the hydrophobic lipid (i.e., R4 in formula (V)) is preferably one of the structures of formula (iii) to formula (vi):

[0090] Formula (iii); Formula (iv);

[0091] Formula (v); Formula (vi);

[0092] Among them, R6~R 12 Independently selected from C 10 ~C 22 Alkyl or C 10 ~C 22 Unsaturated hydrocarbon group; said C 10 ~C 22 The number of carbon atoms can be specifically C10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 or C 22 .

[0093] In the preparation method provided by the application, in step d), the hydrophobic lipid can be specifically di-stearoyl phosphatidyl ethanolamine (DSPE).

[0094] In the preparation method provided by the application, in step d), the molar ratio of the graft product to the hydrophobic lipid is preferably 1:(3-10), and can be specifically 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0095] In the preparation method provided by the application, in step d), the condensation reaction is preferably carried out in the presence of a condensing agent; the condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and / or N-hydroxysuccinimide (NHS); and the molar ratio of the graft product, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is preferably 1:(2-5):(2-5), and can be specifically 1:3:3.

[0096] In the preparation method provided by the application, in step d), the graft product is preferably mixed with the condensing agent and an appropriate amount of solvent to activate the terminal carboxyl group of the graft product, and then mixed with the hydrophobic lipid of formula (V) to carry out the condensation reaction. The activation temperature is preferably 10-40℃, and can be specifically 10℃, 15℃, 20℃, 25℃ (room temperature), 30℃, 35℃ or 40℃; and the activation time is preferably 4-8h.

[0097] In the preparation method provided by the application, in step d), the condensation reaction temperature is preferably 20-40℃, and can be specifically 20℃, 25℃, 30℃, 35℃ or 40℃; and the condensation reaction time is preferably 48-72h.

[0098] In the preparation method provided by the application, in step d), after the condensation reaction is completed, post-treatment is carried out; and the post-treatment process preferably includes: sequentially performing ether precipitation, ultrapure water dialysis purification and freeze-drying.

[0099] The application further provides a lipid nanoparticle, comprising: a lipid nanoparticle matrix and a nucleic acid encapsulated in the lipid nanoparticle matrix.

[0100] The components of the lipid nanoparticle matrix comprise ionizable cationic lipids, auxiliary phospholipids, cholesterol and lipid-hydrophilic polymers; the lipid-hydrophilic polymers are the lipid-hydrophilic polymers described in the above technical solution or the lipid-hydrophilic polymers prepared by the preparation method described in the above technical solution.

[0101] In the lipid nanoparticle provided by the application, the ionizable cationic lipids in the lipid nanoparticle matrix are preferably one or more of heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate) (SM-102), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) and 4-(N,N-dimethylamino)butanoic acid (dilinoleyl) methyl ester (DLin-MC3-DMA).

[0102] In the lipid nanoparticle provided by the application, the auxiliary phospholipids in the lipid nanoparticle matrix are one or more of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 2-(dioctylamino)ethyl nonylphosphonate (9A1P9), 2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid (DOPS) and 1,2-dioleoyl-SN-glycero-3-phosphate-RAC-glycerol sodium salt (DOPG).

[0103] In the lipid nanoparticle provided in the present application, the molar ratio of the ionizable cationic lipid, the auxiliary phospholipid, the cholesterol and the lipid-hydrophilic polymer in the lipid nanoparticle matrix is preferably 50:(2~20):(20~50):(1~20); wherein the molar ratio of the ionizable cationic lipid to the auxiliary phospholipid can be specifically 50:2, 50:3, 50:4, 50:5, 50:6, 50:7, 50:8, 50:9, 50:10, 50:11, 50:12, 50:13, 50:14, 50:15, 50:16, 50:17, 50:18, 50:19 or 50:20; the molar ratio of the ionizable cationic lipid to the cholesterol can be specifically 50:20, 50:21, 50:22, 50:23, 50:24, 50:25, 50:26, 50:27, 50:28, 50:29, 50:30, 50:31, 50:32, 50:33, 50:34, 50:35, 50:36, 50:37, 50:38, 50:38.5, 50:39, 50:40, 50:41, 50:42, 50:43, 50:44, 50:45, 50:46, 50:47, 50:48, 50:49 or 50:50; and the molar ratio of the ionizable cationic lipid to the lipid-hydrophilic polymer can be specifically 50:1, 50:1.5, 50:2, 50:2.5, 50:3, 50:3.5, 50:4, 50:4.5, 50:5, 50:5.5, 50:6, 50:6.5, 50:7, 50:7.5, 50:8, 50:8.5, 50:9, 50:9.5, 50:10, 50:10.5, 50:11, 50:11.5, 50:12, 50:12.5, 50:13, 50:13.5, 50:14, 50:14.5, 50:15, 50:15.5, 50:16, 50:16.5, 50:17, 50:17.5, 50:18, 50:18.5, 50:19, 50:19.5 or 50:20.

[0104] In the lipid nanoparticle provided in the present application, the nucleic acid is preferably mRNA and / or DNA.

[0105] In the lipid nanoparticle provided in the present application, the mass ratio of the nucleic acid to the lipid nanoparticle base is preferably 1:(20-50), and can be specifically 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49 or 1:50.

[0106] The present application also provides a preparation method of the lipid nanoparticle as described in the above technical solution, comprising the following steps:

[0107] mixing the lipid phase and the water phase to obtain the lipid nanoparticle by self-assembly;

[0108] The components of the lipid phase include ionizable cationic lipids, cholesterol, auxiliary phospholipids, lipid-hydrophilic polymers and organic solvents; and the water phase contains nucleic acids.

[0109] In the preparation method of the lipid nanoparticle provided in the present application, the ionizable cationic lipids, cholesterol, auxiliary phospholipids and lipid-hydrophilic polymers in the lipid phase have been introduced above, and will not be repeated here; and the organic solvent in the lipid phase is preferably anhydrous ethanol.

[0110] In the preparation method of the lipid nanoparticle provided in the present application, the components of the water phase preferably include nucleic acids and a buffer. The nucleic acids have been introduced above, and will not be repeated here; the buffer is preferably a citric acid-citrate buffer; and the pH value of the buffer is preferably 3.5-4.5, and more preferably 4.

[0111] In the preparation method of the lipid nanoparticle provided in the present application, the mass ratio of the ionizable cationic lipids, cholesterol, auxiliary phospholipids and lipid-hydrophilic polymers in the lipid phase to the nucleic acids in the water phase is preferably (20-50):1, and can be specifically 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1 or 50:1.

[0112] In the preparation method of the lipid nanoparticle provided in the present application, the amount of the buffer is preferably 3-4 times the volume of the organic solvent in the lipid phase.

[0113] The application further provides application of the lipid nanoparticle or the lipid nanoparticle prepared by the preparation method in nucleic acid delivery.

[0114] In the application, the nucleic acid can be used for selective transfection of the spleen.

[0115] In the application, the nucleic acid is preferably mRNA, and more preferably Flu mRNA.

[0116] In the application, the specific mode of nucleic acid delivery includes but is not limited to injecting the lipid nanoparticle containing the nucleic acid into a living body by intravenous injection.

[0117] The technical scheme provided by the application has at least the following characteristics and advantages:

[0118] (1) The lipid nanoparticle of the application is based on a lipid-hydrophilic polymer containing a thioether linkage, which can realize selective transfection of the spleen of a living body when used for mRNA delivery by adjusting the inherent properties of the lipid-hydrophilic polymer, without the need for other biomolecular targeting transfection technology.

[0119] (2) The lipid nanoparticle of the application can realize efficient loading of mRNA, prevent mRNA leakage, and has high encapsulation efficiency.

[0120] (3) By optimizing the ratio of the thioether-linked lipid-hydrophilic polymer in the lipid nanoparticle, high in vitro and in vivo transfection efficiency can be maintained while ensuring a high ratio of lipid-hydrophilic polymer.

[0121] (4) The hydrophilic polymer, lipid and linkage used in the application have good biocompatibility and good biological safety.

[0122] (5) The preparation process of the lipid nanoparticle of the application is simple and suitable for large-scale production.

[0123] For a clearer understanding, the following examples and comparative examples are described in detail.

[0124] Example 1

[0125] Preparation method of the lipid-hydrophilic polymer DSPE-POx containing a thioether linkage tk The preparation method is as follows:

[0126] Synthesis of the thioketal intermediate 2,2'-(propane-2,2-diylbis(sulfanediyl))diacetic acid (PSDA): Mercaptoacetic acid and acetone were mixed in a molar ratio of 3:1, and an appropriate amount of trifluoroacetic acid was added. The mixture was reacted at room temperature and then a solid product was precipitated at low temperature. The product was purified by alternating washing with n-hexane and water to obtain white solid PSDA.

[0127] Preparation of cyclic anhydrides: PSDA and acetic anhydride are reacted in a molar ratio of 1:5 in the absence of water and oxygen, and the temperature is controlled at ≤25°C. After the reaction is completed, the residual acetic anhydride and acetic acid are evaporated under reduced pressure to obtain a cyclic anhydride product containing a thioacetal bond.

[0128] Polymer grafting reaction: A hydroxyl-terminated poly(2-methyl-2-oxazoline) (PMeOx-OH) with a degree of polymerization of 45 was subjected to a ring-opening grafting reaction with a cyclic anhydride in a molar ratio of 1:5 in chloroform. After ether precipitation, purification by dialysis with ultrapure water, and freeze-drying, a grafted thioketal structure POx-PSDA product was obtained.

[0129] Phospholipid coupling reaction: First, the prepared POx-PSDA was added to a Schlenk reaction tube along with the condensing agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS). To ensure an anhydrous and oxygen-free environment, the reaction tube was purged with nitrogen three times to maintain a nitrogen atmosphere. Subsequently, an appropriate amount of chloroform was added to the reaction tube via syringe, and the reaction was stirred at room temperature for 6 hours to activate the terminal carboxyl groups of POx-PSDA. After activation, distearoylphosphatidylethanolamine (DSPE) was added to the reaction system, and the reaction temperature was raised to 60°C and continued for 72 hours. The molar ratio of POx-PSDA, EDCI, NHS, and DSPE was 1:3:3:5. After completion of the reaction, the product was purified by ether precipitation and dialysis against ultrapure water, followed by lyophilization to obtain the target product, DSPE-POx. tk (Formula I-1).

[0130] Formula (I-1).

[0131] The obtained lipid-hydrophilic polymer DSPE-POx tk H NMR spectrum Figure 1 As shown, 1 H NMR (CDCl3,ppm): δ 0.89 (3H, -CH2CH2(CH2) 14 CH3), 1.27 (28H, -CH2CH2(CH2) 14CH3), 1.63 (2H, -OCOCH2CH2-), 1.63 (6H, -S-C-(CH3)2-S-), 2.14 (3H, -COCH3), 2.25-2.45 (4H, -OCOCH2-S-C-(CH3)2-S-CH2COO-), 2.26-2.45 (2H, -OCOCH2CH2-), 3.07 (3H, -NCH3), 3.48 (4H, -NCH2CH2-), 3.83-4.4 (4H, -CONHCH2CH2O-), 3.83-4.4 (4H, -OCH2-CH-CH2O-), 5.24 (1H, -OCH2-CH-CH2O-).

[0132] Example 2

[0133] Lipid-hydrophilic polymer DSPE-PEG containing thioacetone linkage tk The preparation method is as follows:

[0134] Synthesis of thioacetone intermediate 2,2'-(propane-2,2-diylbis(sulfanediyl)) diacetic acid (PSDA): mercaptoacetic acid and acetone are mixed at a molar ratio of 3:1, an appropriate amount of trifluoroacetic acid is added, and after reaction at room temperature, the solid product is precipitated at low temperature. The product is purified by alternating washing with n-hexane and water to obtain white solid PSDA.

[0135] Preparation of cyclic anhydride: PSDA is reacted with acetic anhydride at a molar ratio of 1:5 under anhydrous and oxygen-free conditions, and the temperature is controlled at ≤25°C. After the reaction is completed, residual acetic anhydride and acetic acid are removed by evaporation under reduced pressure to obtain the cyclic anhydride product containing thioacetone linkage.

[0136] Polymer grafting reaction: polyethylene glycol monomethyl ether with a degree of polymerization of 45 is reacted with cyclic anhydride at a molar ratio of 1:5 in chloroform to undergo ring-opening grafting reaction. After sedimentation with diethyl ether, purification by ultra-pure water dialysis, and freeze-drying, the PEG-PSDA product with grafted thioacetone structure is obtained.

[0137] Phospholipid conjugation reaction: first, the prepared PEG-PSDA and condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were added to a Schlenk reaction tube to ensure the anhydrous and anaerobic environment of the reaction system, the reaction tube was replaced with nitrogen three times, and the reaction tube was placed in a nitrogen protective atmosphere. Subsequently, an appropriate amount of solvent chloroform was added to the reaction tube using a syringe, and the reaction was stirred at room temperature for 6 h to complete the activation process of the terminal carboxyl group of PEG-PSDA. After the activation of the terminal carboxyl group was completed, distearoyl phosphatidyl ethanolamine (DSPE) was added to the reaction system, the reaction temperature was increased to 60°C, and the reaction was continued for 72 h; wherein the molar ratio of POx-PSDA, EDCI, NHS and DSPE was 1:3:3:5. After the reaction was completed, the target product DSPE-PEG tk (Formula I-2).

[0138] (Formula I-2).

[0139] The obtained lipid-hydrophilic polymer DSPE-PEG tk The nuclear magnetic hydrogen spectrum is shown in Figure 2 , 1 H NMR (CDCl3, ppm): δ 0.81 (3H, -CH2CH2(CH2) 14 CH3), 1.18 (28H, -CH2CH2(CH2) 14 CH3), 1.41-1.72(2H, -OCOCH2CH2-), 1.41-1.72 (6H, -S-C-(CH3)2-S-), 2.22 (2H, -OCOCH2CH2-), 3.3(4H, -OCOCH2-S-C-(CH3)2-S-CH2COO-), 3.32 (3H, -OCH3), 3.58 (4H, -OCH2CH2-),3.75-4.31 (4H, -CONHCH2CH2O-), 3.75-4.31 (4H, -OCH2-CH-CH2O-), 5.15 (1H, -OCH2-CH-CH2O-).

[0140] Example 3

[0141] Lipid-hydrophilic polymer DSPE-POx connected by thioketone tk The method for constructing lipid nanoparticles is as follows:

[0142] The lipid-hydrophilic polymer DSPE-POx prepared in Example 1 tk, ionizable cationic lipid SM-102, helper phospholipid DSPC and cholesterol were dissolved in anhydrous ethanol to form a lipid premix solution, and then were mixed in the following molar ratios: ionizable cationic lipid / helper phospholipid / cholesterol / lipid-hydrophilic polymer = 50 / 10 / 38.5 / 1.5, 50 / 10 / 38.5 / 3.5, 50 / 10 / 38.5 / 5.5, 50 / 10 / 38.5 / 7.5, 50 / 10 / 38.5 / 9.5, 50 / 10 / 38.5 / 11.5, 50 / 10 / 38.5 / 13.5, 50 / 10 / 38.5 / 15.5, to obtain a lipid phase containing four-component lipids.

[0143] The mRNA stock solution was added to the citric acid-sodium citrate buffer solution with pH = 4 to form an aqueous phase.

[0144] According to the ratio of the total mass of ionizable cationic lipid, helper phospholipid, cholesterol and lipid-hydrophilic polymer to the mass of mRNA being 40:1, the lipid phase was added to the aqueous phase, and the amount of buffer solution in the aqueous phase was 3 times the volume of organic solvent (anhydrous ethanol) in the lipid phase; after mixing uniformly, eight kinds of DSPE-POx tk lipid nanoparticles (DSPE-POx tk -LNP) with different molar ratios were self-assembled, respectively named as DSPE-POx tk 1.5, DSPE-POx tk 3.5, DSPE-POx tk 5.5, DSPE-POx tk 7.5, DSPE-POx tk 9.5, DSPE-POx tk 11.5, DSPE-POx tk 13.5, DSPE-POx tk 15.5.

[0145] Example 4

[0146] The lipid-hydrophilic polymer DSPE-PEG tk connected by thioketals was prepared according to the following steps:

[0147] The lipid-hydrophilic polymer DSPE-PEG tk , ionizable cationic lipid SM-102, helper phospholipid DSPC and cholesterol were dissolved in anhydrous ethanol to form a lipid premix solution, and then were mixed in the following molar ratios: ionizable cationic lipid / helper phospholipid / cholesterol / lipid-hydrophilic polymer = 50 / 10 / 38.5 / 1.5, 50 / 10 / 38.5 / 11.5, to obtain a lipid phase containing four-component lipids.

[0148] The mRNA stock solution was added to the citric acid-sodium citrate buffer solution with pH = 4 to form the water phase.

[0149] According to the ratio of the total mass of the ionizable cationic lipid, the auxiliary phospholipid, the cholesterol and the lipid-hydrophilic polymer to the mass of the mRNA being 40:1, the lipid phase was added to the water phase, and the amount of the buffer solution in the water phase was 3 times the volume of the organic solvent (absolute ethanol) in the lipid phase; after uniform mixing, the DSPE-PEG tk lipid nanoparticles (DSPE-PEG tk -LNP) with a molar ratio of two were obtained by self-assembly, and were named DSPE-PEG tk 1.5, respectively. tk 11.5.

[0150] Comparative Example 1

[0151] The method for constructing the lipid nanoparticles from DMG-PEG was as follows:

[0152] The purchased control group lipid-hydrophilic polymer DMG-PEG (1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, abbreviated as: DMG-PEG 2000, manufacturer: Shanghai McLean Biochemical Technology Co., Ltd.), ionizable cationic lipid SM-102, auxiliary phospholipid DSPC and cholesterol were dissolved in absolute ethanol to form a lipid premix solution, and then were blended in the following molar ratios: ionizable cationic lipid / auxiliary phospholipid / cholesterol / lipid-hydrophilic polymer = 50 / 10 / 38.5 / 1.5, to obtain a lipid phase containing four components of lipids.

[0153] The mRNA stock solution was added to the citric acid-sodium citrate buffer solution with pH = 4 to form the water phase.

[0154] According to the ratio of the total mass of the ionizable cationic lipid, the auxiliary phospholipid, the cholesterol and the lipid-hydrophilic polymer to the mass of the mRNA being 40:1, the lipid phase was added to the water phase, and the amount of the buffer solution in the water phase was 3 times the volume of the organic solvent (absolute ethanol) in the lipid phase; after uniform mixing, the DMG-PEG lipid nanoparticles (DMG-PEG-LNP) were obtained by self-assembly, and were named DMG-PEG 1.5.

[0155] Comparative Example 2

[0156] The method for constructing the lipid nanoparticles from DSPE-PEG and DMG-PEG was as follows:

[0157] The purchased control lipid-hydrophilic polymer DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], abbreviated as: DSPE-PEG2000, manufacturer: Shanghai MacLean Biochemical Technology Co., Ltd.) or DMG-PEG (same as comparative example 1) was dissolved in anhydrous ethanol with ionizable cationic lipid SM-102, auxiliary phospholipid DSPC and cholesterol to form a lipid premix, and then blended in the following molar ratios: ionizable cationic lipid / auxiliary phospholipid / cholesterol / lipid-hydrophilic polymer = 50 / 10 / 38.5 / 1.5, 50 / 10 / 38.5 / 11.5, respectively, to obtain a lipid phase containing four components of lipids.

[0158] The mRNA stock solution was added to a citric acid-sodium citrate buffer solution at pH 4 to form an aqueous phase.

[0159] The lipid phase was added to the aqueous phase at a mass ratio of 40:1 between the total mass of ionizable cationic lipids, auxiliary phospholipids, cholesterol, and lipid-hydrophilic polymers and the mass of mRNA. The amount of buffer in the aqueous phase was 3 times the volume of the organic solvent (anhydrous ethanol) in the lipid phase. After uniform mixing, two DSPE-PEG lipid nanoparticles (DSPE-PEG-LNPs) with two molar ratios were self-assembled and named DSPE-PEG 1.5 and DSPE-PEG 11.5, respectively.

[0160] Example 5

[0161] The physicochemical properties of lipid nanoparticles (LNPs) with different molar ratios are characterized as follows:

[0162] (1) Determination of particle size and polydispersity index (PDI): The LNP sample was diluted with phosphate buffered saline (PBS) to a final volume of 800 μL and transferred to a standard quartz cuvette after the system stabilized. The hydrodynamic diameter and polydispersity index (PDI) were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS nanoparticle size analyzer at a constant temperature of 25°C.

[0163] The experimental results are as follows Figures 3-4 As shown, Figure 3 DSPE-POx with different molar ratios provided in Example 5 of the present invention tk - Particle size and polydispersity index graph of LNP, Figure 4 DSPE-PEG of different molar ratios provided by Example 5 of the present invention tk - Particle size and polydispersity index plot of LNPs.

[0164] pass Figures 3-4It can be seen that: with the gradual increase in the molar ratio of the hydrophilic polymer in the lipid component, the average particle size of LNP shows a decreasing trend, ranging from 120 to 220 nm, and all samples maintain good dispersion properties, with most PDI values ​​measured to be less than 0.2.

[0165] (2) Evaluation of DSPE-POx by agarose gel electrophoresis tk The nucleic acid encapsulation efficiency of LNP was investigated. The specific steps included: washing the gel mold with enzyme-free water, mixing 0.5 g agarose with 50 mL 1×TAE buffer, heating until completely dissolved, adding 5 μL Gel-Red nucleic acid dye, and injection molding; after the colloid solidified, placing it in an electrophoresis tank, injecting buffer to immerse 1 cm of the gel surface, then mixing equal volumes of LNP sample and 2× RNA loading buffer, loading the wells with 1 μg mRNA loading amount, and setting 5 μL DNA ladder as a reference; after electrophoresis at a constant voltage of 120 V for 40 min, the electrophoresis pattern was acquired using a 302 nm UV imaging system.

[0166] The experimental results are as follows Figure 5 As shown, Figure 5 DSPE-POx with different molar ratios provided in Example 5 of the present invention tk -Agarose gel electrophoresis test of LNP mRNA encapsulation effect.

[0167] pass Figure 5 It can be seen that when the hydrophilic polymer-lipid molar ratio varies in the range of 1.5 to 7.5, the encapsulated nucleic acid material does not show obvious migration bands under the action of the electric field, confirming that LNP can effectively maintain the integrity of the nucleic acid encapsulation structure under different ratio conditions.

[0168] (3) Transmission electron microscopy (TEM) images were taken by JEOL JEM-1011 (JEOL Ltd.) with a field of view of 100 nm.

[0169] The experimental results are as follows Figure 6 As shown, Figure 6 DSPE-POx with different molar ratios provided in Example 5 of the present invention tk -TEM morphology characterization of LNP.

[0170] pass Figure 6 It can be seen that when DSPE-POx tk The LNP system exhibited a regular particle size reduction as the molar ratio increased from 5.5 to 11.5. Despite the changes in particle size as the component ratios were adjusted, all samples maintained a complete spherical self-assembled structure with a smooth surface and no significant agglomerate formation.

[0171] Example 6

[0172] The transfection efficiency of luciferase mRNA was evaluated in vitro using lipid nanoparticles with different molar ratios. The specific steps are as follows:

[0173] Evaluation of DSPE-PEG using an in vitro cell transfection model tk The functional expression efficiency of LNP was determined by the following procedures: HEK 293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double-antibody in a 37°C, 5% CO2 constant temperature incubator to the logarithmic growth phase; 1.5×10 4 The cells were seeded into 96-well plates at a predetermined density per well. After 24 h of adherence culture, DSPE-PEG loaded with Fluc mRNA was quantitatively added to each well. tk -LNP (0.2 μg mRNA / well), set up triplicate wells; after continuing to culture for 24 h, replace the cell lysis buffer (50 μL / well), add the luciferase substrate reaction system, and use a chemiluminescence detector to quantitatively analyze the luminescence intensity.

[0174] The experimental results are as follows Figure 7 As shown, Figure 7 DSPE-POx with different molar ratios provided in Example 6 of the present invention tk - Graph showing the transfection efficiency of LNPs in HEK 293T cell line.

[0175] pass Figure 7 It can be seen that with the increase of the amount of lipid-hydrophilic polymer added, the transfection ability of LNP also has a certain fluctuation. tk The LNP transfection ability was optimal when the molar ratio was 15.5, which confirmed that the LNP delivery system can achieve efficient intracellular expression of mRNA.

[0176] Example 7

[0177] The transfection effect of luciferase mRNA in mice was evaluated by intravenous injection of lipid nanoparticles with different molar ratios. The specific steps are as follows:

[0178] A mouse in vivo transfection evaluation model was established, and C57BL / 6 strain experimental animals of appropriate age (6-8 weeks old, 18-20 g in weight) were randomly divided into groups, and then were given LNP loaded with 2 μg Fluc mRNA through the tail vein injection route. 24 h after injection, 15 mg / mL D-luciferin potassium salt solution (200 μL / each) was delivered through intraperitoneal injection, and 8 min later, euthanasia was performed and target organs such as heart, liver, spleen, lung, kidney and lymph node were collected. The in vivo imaging system was used to collect the organ level bioluminescence signal, and the professional image analysis software was used to quantitatively evaluate the fluorescence intensity distribution characteristics of each organ.

[0179] The experimental results are shown in Figures 8-9 , Figure 8 The different molar ratios of DSPE-POx provided in Example 7 of the present application tk The transfection effect of LNP in each organ of C57BL / 6 mice is shown in Figure 1; wherein a) is the transfection effect of LNP in each organ of C57BL / 6 mice, b) is the bioluminescence image of the transfection effect of each organ after injection of DSPE-POx tk LNP, c) is the quantitative graph of the average fluorescence intensity of each organ transfected by DSPE-POx tk LNP, c) is the quantitative graph of the average fluorescence intensity of each organ transfected by DSPE-POx tk LNP, c) is the quantitative graph of the average fluorescence intensity of each organ transfected by DSPE-POx Figure 9 The different molar ratios of DSPE-PEG provided in Example 7 of the present application tk The transfection effect of LNP in each organ of C57BL / 6 mice is shown in Figure 1; wherein a) is the transfection effect of LNP in each organ of C57BL / 6 mice, b) is the bioluminescence image of the transfection effect of each organ after injection of DSPE-POx tk LNP, c) is the quantitative graph of the average fluorescence intensity of each organ transfected by DSPE-POx tk LNP, c) is the quantitative graph of the average fluorescence intensity of each organ transfected by DSPE-POx tk LNP, c) is the quantitative graph of the average fluorescence intensity of each organ transfected by DSPE-POx

[0180] It can be seen that: Figures 8-9 The lipid-hydrophilic polymer modified LNP system shows selective transfection ability in the spleen, and the fluorescence signal intensity accounts for more than 80% of the total signal amount of each organ; when the molar ratio of DSPE-POx tk and DSPE-PEG tk in the lipid component reaches 11.5 and 1.5 respectively, the system shows the best transfection efficiency in the spleen, and the relative fluorescence intensity is increased by nearly 6 times and 2 times respectively compared with the control groups DMG-PEG 1.5 and DSPE-PEG 1.5, which shows a significant advantage of selective delivery in the spleen.

[0181] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. Application of lipid nanoparticles in nucleic acid delivery; The lipid nanoparticles include: A lipid nanoparticle matrix and a nucleic acid entrapped in the lipid nanoparticle matrix; The components of the lipid nanoparticle matrix include ionizable cationic lipids, auxiliary phospholipids, cholesterol and lipid-hydrophilic polymers, and the lipid-hydrophilic polymer has a structure shown in formula (I): Formula (I); In formula (I), R1 is a structure of formula (i) or formula (ii): Formula (i); Formula (ii); In formula (i) and formula (ii), n is 20 to 50, and R5 is methyl or ethyl; In formula (I), R2 is a C2~C4 alkyl group, R3 is a C2~C4 alkyl group, and R4 is a C 10 ~C 22 Single tail or C 10 ~C 22 A double-tailed hydrophobic lipid group, L1 is an ester bond, and L2 is an amide bond; The molar ratio of the ionizable cationic lipid, auxiliary phospholipid, cholesterol and lipid-hydrophilic polymer is 50:(2-20):(20-50):(1-20); The nucleic acid is mRNA, and the nucleic acid delivery can achieve selective transfection of the spleen.

2. The use according to claim 1, characterized in that The R4 is one of the structures of formula (iii) to formula (vi): Formula (iii); Formula (iv); Formula (v); Formula (vi); Among them, R6~R 12 Independently selected from C 10 ~C 22 Alkyl or C 10 ~C 22 Unsaturated hydrocarbon group.

3. The use according to claim 1, characterized in that The lipid-hydrophilic polymer is prepared according to the following steps: a) mixing acetone with a mercapto C2-C4 alkyl carboxylic acid compound to react to obtain a dibasic acid having a thioketal linkage and a structure represented by formula (II); Formula (II); b) reacting the dibasic acid with excess acetic anhydride to dehydrate the dibasic acid to obtain a cyclic acid anhydride containing a thioketal structure; c) condensing the cyclic anhydride with a hydroxyl-terminated hydrophilic polymer having a structure represented by formula (III) to obtain a grafted product having a structure represented by formula (IV); Formula (III); Formula (IV); d) condensing the grafted product with a hydrophobic lipid having a structure represented by formula (V) to obtain a lipid-hydrophilic polymer having a structure represented by formula (I); Formula (V); Formula (I); In formula (I) to formula (V), R1 is a structure of formula (i) or formula (ii): Formula (i); Formula (ii); In formula (i) and formula (ii), n is 20 to 50, and R5 is methyl or ethyl; In formula (I) to formula (V), R2 is a C2~C4 alkyl group, R3 is a C2~C4 alkyl group, and R4 is a C 10 ~C 22 Single tail or C 10 ~C 22 The double-tailed hydrophobic lipid group has L1 as an ester bond, L2 as an amide bond, and L2' as an amino group.

4. The use according to claim 3, characterized in that The molar ratio of the acetone to the mercapto C2~C4 alkyl carboxylic acid compound is (3~5):1; The molar ratio of the dibasic acid to acetic anhydride is 1:(3-10); The molar ratio of the cyclic anhydride to the hydroxyl-terminated hydrophilic polymer is (3-10):1; The molar ratio of the grafted product to the hydrophobic lipid is 1:(3-10).

5. The use according to claim 1, characterized in that The ionizable cationic lipid is one or more of heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate), ((4-hydroxybutyl)azepinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), and 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester; The auxiliary phospholipid is one or more of distearoylphosphatidylcholine, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, 2-(dioctylamino)ethylnonyl hydrogen phosphate and 1,2-dioleoyl-SN-glycerol-3-phospho-RAC-glycerol sodium salt.

6. The use according to claim 1, characterized in that The lipid nanoparticles are prepared according to the following steps: The lipid phase is mixed with the aqueous phase to self-assemble into lipid nanoparticles; The components of the lipid phase include ionizable cationic lipid, cholesterol, auxiliary phospholipid, lipid-hydrophilic polymer and organic solvent; the aqueous phase contains nucleic acid.

Images