Lipid-hydrophilic polymer as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a lipid-hydrophilic polymer as well as a preparation method and application thereof. The lipid-hydrophilic polymer provided by the invention has a structure as shown in a formula (I), in the formula (I), 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 C10-C22 single-tailed or C10-C22 double-tailed hydrophobic lipid group, L1 is an ester bond, and L2 is an amido bond or an ester bond. Through molecular structure design, a thioketal sensitive chemical bond is introduced between a hydrophilic polymer and lipid, and the breakable lipid-hydrophilic polymer is obtained. On the basis, by increasing the mole percentage of the lipid-hydrophilic polymer in the lipid nanoparticles, the stability of the lipid nanoparticles can be enhanced, and meanwhile efficient transfection can be achieved. # imgabs0 # formula (I): (1).

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a lipid-hydrophilic polymer and a preparation method and application thereof. Background Art

[0002] In the field of modern biomedicine, mRNA therapy, as a highly promising innovative treatment, has become a key research target in academia and industry. The core principle of mRNA therapy is to efficiently deliver mRNA molecules encoding specific target proteins into cells in a specific way. Cells will use their own nucleic acid translation mechanism to synthesize target proteins in situ, thereby precisely intervening in the occurrence and development of diseases, showing great application potential in the treatment of a variety of intractable diseases.

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

[0004] However, the existing LNP technology still faces a key technical problem: although the lipid-polyethylene glycol (PEG) in its formula can improve the stability and blood circulation time of nanoparticles by forming a hydrophilic protective layer, when the molar percentage of this component exceeds 1.5%, its dense hydration barrier will significantly hinder the interaction between LNP and cell membranes, 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 applications, the transfection efficiency and PEG content show a contradictory relationship of one increasing while the other decreases, which greatly limits the optimization space of LNP technology. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a lipid-hydrophilic polymer and a preparation method and application thereof. The present invention introduces a thioketone sensitive chemical bond between the hydrophilic polymer and the lipid through molecular structure design to obtain a cleavable lipid-hydrophilic polymer. On this basis, by increasing the molar percentage of the lipid-hydrophilic polymer in the lipid nanoparticles, efficient transfection can be achieved while enhancing the stability of the lipid nanoparticles.

[0006] The present invention provides a lipid-hydrophilic polymer having a structure shown in formula (I):

[0007] Formula (I);

[0008] In formula (I), R1 is a hydrophilic polymer group with a degree of polymerization of 20 to 50, R2 is a C2 to C4 alkyl group, R3 is a C2 to 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 or an ester bond.

[0009] Preferably, R1 is a 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, the R4 is one of the structures of formula (iii) to formula (vi):

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

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

[0015] Among them, R6~R 12 Independently selected from C 10 ~C 22 Alkyl or C 10 ~C 22 Unsaturated hydrocarbon group.

[0016] The present invention provides a method for preparing the lipid-hydrophilic polymer described in the above technical solution, comprising the following steps:

[0017] a) mixing acetone with a mercapto C2-C4 alkyl carboxylic acid compound to react to obtain a dibasic acid having a thioketal connecting bond and a structure represented by formula (II);

[0018] Formula (II);

[0019] b) reacting the dibasic acid with excess acetic anhydride to dehydrate the dibasic acid to obtain a cyclic acid anhydride containing a thioketal structure;

[0020] c) condensing the cyclic acid 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);

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

[0022] d) subjecting the grafted product to a condensation reaction with a hydrophobic lipid having a structure represented by formula (V) to obtain a lipid-hydrophilic polymer having a structure represented by formula (I);

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

[0024] In formula (I) to formula (V), R1 is a hydrophilic polymer group with a degree of polymerization of 20 to 50, R2 is a C2 to C4 alkyl group, R3 is a C2 to C4 alkyl group, and R4 is a C 10 ~C 22 Single tail or C 10 ~C 22 The 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.

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

[0026] The molar ratio of the dibasic acid to 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 grafted product to the hydrophobic lipid is 1:(3-10).

[0029] The present invention provides a lipid nanoparticle, comprising: a lipid nanoparticle matrix and a nucleic acid contained in the lipid nanoparticle matrix;

[0030] The components of the lipid nanoparticle matrix include 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.

[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)azepinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) and 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester;

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

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

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

[0035] The present invention provides a method for preparing the lipid nanoparticles described in the above technical solution, comprising the following steps:

[0036] The lipid phase is mixed with the aqueous phase to self-assemble to obtain lipid nanoparticles;

[0037] The components of the lipid phase include ionizable cationic lipids, cholesterol, auxiliary phospholipids, lipid-hydrophilic polymers and organic solvents; the aqueous phase contains nucleic acids.

[0038] The present invention provides an application of the lipid nanoparticles described in the above technical solution or the lipid nanoparticles prepared by the preparation method described in the above technical solution in nucleic acid delivery.

[0039] Compared with the prior art, the present invention provides a lipid-hydrophilic polymer and a preparation method and application thereof. The lipid-hydrophilic polymer provided by the present invention has a structure shown in formula (I), wherein R1 is a hydrophilic polymer group with a polymerization degree of 20 to 50, R2 is a C2 to C4 alkyl group, R3 is a C2 to C4 alkyl group, and R4 is a C 10 ~C 22 Single tail or C 10 ~C 22The hydrophobic lipid group of the double tail, L1 is an ester bond, and L2 is an amide bond or an ester bond. The present invention introduces a thioketone sensitive chemical bond between the hydrophilic polymer and the lipid through molecular structure design, thereby obtaining a breakable lipid-hydrophilic polymer. On this basis, by increasing the molar percentage of the lipid-hydrophilic polymer in the lipid nanoparticles, efficient transfection can be achieved while enhancing the stability of the lipid nanoparticles. The experimental results show that the lipid-hydrophilic polymer developed by the present invention can achieve efficient transfection of mRNA by intramuscular injection in the range of 1.5~15.5% by molar percentage; when used for intravenous injection, selective transfection of the mouse spleen can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0041] Figure 1 The lipid-hydrophilic polymer DSPE-POx provided in Example 1 of the present invention tk H NMR spectrum of

[0042] Figure 2 Lipid-hydrophilic polymer DSPE-PEG provided in Example 2 of the present invention tk H NMR spectrum of

[0043] Figure 3 DSPE-POx with different molar ratios provided in Example 5 of the present invention tk - Particle size and polydispersity index plots of LNPs;

[0044] Figure 4 DSPE-PEG of different molar ratios provided in Example 5 of the present invention tk - Particle size and polydispersity index plots of LNPs;

[0045] Figure 5 DSPE-POx with different molar ratios provided in Example 5 of the present invention tk - Agarose gel electrophoresis of LNP to test mRNA encapsulation effect;

[0046] Figure 6 DSPE-POx with different molar ratios provided in Example 5 of the present invention tk -TEM morphology characterization of LNP;

[0047] Figure 7DSPE-POx with different molar ratios provided in Example 6 of the present invention tk - Transfection efficiency of LNP in HEK 293T cell line;

[0048] Figure 8 DSPE-POx with different molar ratios provided in Example 7 of the present invention tk -LNP transfection effect diagram in various organs of C57BL / 6 mice; a) is a photo taken using a live imaging system after injection of DSPE-POx tk -LNP bioluminescence image of the transfection effect of various organs; b) DSPE-POx tk -LNP transfection average fluorescence intensity quantitative diagram of each organ; c) DSPE-POx tk -The average fluorescence intensity ratio of each organ transfected with LNP;

[0049] Fig. 9 DSPE-PEG of different molar ratios provided by Example 7 of the present invention tk -LNP transfection effect diagram in various organs of C57BL / 6 mice; a) is a photo taken using a live imaging system after injection of DSPE-PEG tk -LNP bioluminescence image of the transfection effect of various organs; b) DSPE-PEG tk -LNP transfection average fluorescence intensity quantitative diagram of each organ, c) DSPE-PEG tk -The average fluorescence intensity percentage of each organ transfected with LNP. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] The present invention provides a lipid-hydrophilic polymer, which is composed of a hydrophobic lipid, a thioketal linker 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 degree of polymerization of 20 to 50, R2 is a C2 to C4 alkyl group, R3 is a C2 to C4 alkyl group, and R4 is a C 10 ~C 22 Single tail or C 10 ~C 22The 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 invention, in 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, 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 invention, in formula (I), R2 can specifically be a C2 alkyl group, a C3 alkyl group or a C4 alkyl group.

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

[0059] In the lipid-hydrophilic polymer provided by the present invention, in 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] Among them, R6~R 12 Independently selected from C 10 ~C 22 Alkyl or C 10 ~C 22 Unsaturated hydrocarbon group; 10 ~C 22 The number of carbon atoms 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 invention, the lipid-hydrophilic polymer may specifically be a polymer having a structure represented by formula (I-1) or formula (I-2):

[0064] Formula (I-1);

[0065] Formula (I-2).

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

[0067] a) mixing acetone with a mercapto C2-C4 alkyl carboxylic acid compound to react to obtain a dibasic acid having a thioketal connecting bond and a structure represented by formula (II);

[0068] Formula (II);

[0069] b) reacting the dibasic acid with excess acetic anhydride to dehydrate the dibasic acid to obtain a cyclic acid anhydride containing a thioketal structure;

[0070] c) condensing the cyclic acid 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);

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

[0072] d) subjecting the grafted product to a condensation reaction with a hydrophobic lipid having a structure represented by formula (V) to obtain a lipid-hydrophilic polymer having a structure represented by formula (I);

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

[0074] In formula (I) to formula (V), R1 is a hydrophilic polymer group with a degree of polymerization of 20 to 50, R2 is a C2 to C4 alkyl group, R3 is a C2 to C4 alkyl group, and R4 is a C 10 ~C 22 Single tail or C 10 ~C 22 The 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 invention, in step a), the mercapto C2~C4 alkyl carboxylic acid compound is preferably mercaptoacetic acid or mercaptopropionic acid.

[0076] In the preparation method provided by the present invention, in step a), the molar ratio of acetone to the mercapto C2~C4 alkyl carboxylic acid compound is preferably (3~5):1, 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 present invention, in step a), the mixing reaction is preferably carried out under acid catalysis; the acid catalyst is preferably trifluoroacetic acid.

[0078] In the preparation method provided by the present invention, in step a), the temperature of the mixed reaction is preferably 10-40°C, specifically 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C; the time of the mixed reaction is not particularly limited, and is preferably 3-12h.

[0079] In the preparation method provided by the present invention, in step a), after the mixing reaction is completed, post-treatment is performed; the post-treatment process preferably includes: precipitating a solid product at low temperature, then washing and purifying it with n-hexane and water alternately, and then drying to obtain the target product.

[0080] In the preparation method provided by the present invention, in step b), the molar ratio of the dibasic acid to acetic anhydride is preferably 1:(3-10), 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 present invention, in step b), the reaction is carried out under anhydrous and oxygen-free conditions.

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

[0083] In the preparation method provided by the present invention, in step b), after the mixing reaction is completed, post-treatment is performed; the post-treatment process preferably includes: removing 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 20-50, 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 acid 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, 40°C; the time of the condensation reaction is preferably 12-48h.

[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 comprises: 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 (ie, 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; 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 present invention, in step d), the hydrophobic lipid may specifically be distearoylphosphatidylethanolamine (DSPE).

[0094] In the preparation method provided by the present invention, in step d), the molar ratio of the grafted product to the hydrophobic lipid is preferably 1:(3-10), 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 present invention, 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); the molar ratio of the grafted product, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is preferably 1:(2~5):(2~5), specifically 1:3:3.

[0096] In the preparation method provided by the present invention, in step d), the grafted product is preferably first mixed with the condensing agent and an appropriate amount of solvent to complete the activation of the terminal carboxyl group of the grafted product, and then mixed with the hydrophobic lipid of the structure shown in formula (V) for condensation reaction. The activation temperature is preferably 10-40°C, specifically 10°C, 15°C, 20°C, 25°C (room temperature), 30°C, 35°C or 40°C; the activation time is preferably 4-8h.

[0097] In the preparation method provided by the present invention, in step d), the temperature of the condensation reaction is preferably 20-40°C, specifically 20°C, 25°C, 30°C, 35°C or 40°C; the time of the condensation reaction is preferably 48-72h.

[0098] In the preparation method provided by the present invention, in step d), after the condensation reaction is completed, post-treatment is performed; the post-treatment process preferably comprises: ether precipitation, ultrapure water dialysis purification and freeze-drying in sequence.

[0099] The present invention also provides a lipid nanoparticle, comprising: a lipid nanoparticle matrix and a nucleic acid contained in the lipid nanoparticle matrix;

[0100] The components of the lipid nanoparticle matrix include 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 nanoparticles provided by the present invention, the ionizable cationic lipid in the lipid nanoparticle matrix is ​​preferably one or more of heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate) (SM-102), ((4-hydroxybutyl)azepinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) and 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA).

[0102] In the lipid nanoparticles provided by the present invention, 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)ethylnonyl hydrogen phosphate (9A1P9), 2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropionic acid (DOPS) and 1,2-dioleoyl-SN-glycero-3-phospho-RAC-glycerol sodium salt (DOPG).

[0103] In the lipid nanoparticles provided by the present invention, the molar ratio of the ionizable cationic lipid, auxiliary phospholipid, cholesterol and 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 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, 50:20, 50:21, 50:22, 50:23, 50:24, 50:25 :14, 50:15, 50:16, 50:17, 50:18, 50:19 or 50:20; the molar ratio of the ionizable cationic lipid to cholesterol can be 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, 50:39, 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; the molar ratio of the ionizable cationic lipid to the lipid-hydrophilic polymer can be 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: or 50:20.

[0104] In the lipid nanoparticles provided by the present invention, the nucleic acid is preferably mRNA and / or DNA.

[0105] In the lipid nanoparticles provided by the present invention, the mass ratio of the nucleic acid to the lipid nanoparticle matrix is ​​preferably 1:(20-50), 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 invention also provides a method for preparing the lipid nanoparticles described in the above technical solution, comprising the following steps:

[0107] The lipid phase is mixed with the aqueous phase to self-assemble to obtain lipid nanoparticles;

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

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

[0110] In the method for preparing lipid nanoparticles provided by the present invention, the components of the aqueous phase preferably include nucleic acids and buffer. The nucleic acids have been introduced above and will not be described again here; the buffer is preferably a citric acid-sodium citrate buffer; and the pH value of the buffer is preferably 3.5-4.5, more preferably 4.

[0111] In the method for preparing lipid nanoparticles provided by the present invention, the mass ratio of the total mass of ionizable cationic lipids, cholesterol, auxiliary phospholipids and lipid-hydrophilic polymers in the lipid phase to the mass ratio of nucleic acids in the aqueous phase is preferably (20-50):1, 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 method for preparing lipid nanoparticles provided by the present invention, the amount of the buffer is preferably 3 to 4 times the volume of the organic solvent in the lipid phase.

[0113] The present invention also provides an application of the lipid nanoparticles described in the above technical solution or the lipid nanoparticles prepared by the preparation method described in the above technical solution in nucleic acid delivery.

[0114] In the application provided by the present invention, it is used for delivering nucleic acid to achieve spleen selective transfection.

[0115] In the application provided by the present invention, the nucleic acid is preferably mRNA, more preferably Flu mRNA.

[0116] In the application provided by the present invention, the specific method of delivering the nucleic acid includes but is not limited to injecting the lipid nanoparticles containing the nucleic acid into the organism by intravenous injection.

[0117] The technical solution provided by the present invention has at least the following characteristics and advantages:

[0118] (1) The lipid nanoparticles of the present invention are based on lipid-hydrophilic polymers containing thioacetal linkages. When used to deliver mRNA, the inherent properties of the lipid-hydrophilic polymers themselves are adjusted to achieve selective transfection of mRNA into the spleen of the test organism without the need to use other biomolecule targeted transfection technologies.

[0119] (2) When loading mRNA, the lipid nanoparticles of the present invention can achieve efficient encapsulation, prevent mRNA leakage, and have high encapsulation efficiency.

[0120] (3) By optimizing the ratio of thioketone-linked lipids to hydrophilic polymers in lipid nanoparticles, high in vitro and in vivo transfection efficiencies can be maintained while ensuring a high lipid-hydrophilic polymer addition ratio.

[0121] (4) The hydrophilic polymer, lipid and connecting bond used in the present invention have good biocompatibility and good biosafety.

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

[0123] For the purpose of greater clarity, the present invention is described in detail with reference to the following examples and comparative examples.

[0124] Example 1

[0125] Lipid-hydrophilic polymer DSPE-POx containing thioketal linker tk The preparation method comprises the following steps:

[0126] Synthesis of thioacetal intermediate 2,2'-(propane-2,2-diylbis(sulfanediyl))diacetic acid (PSDA): Mix thioacetic acid and acetone in a molar ratio of 3:1, add an appropriate amount of trifluoroacetic acid, react at room temperature, and precipitate a solid product at low temperature. The product is purified by alternating washing with n-hexane and water to obtain a white solid PSDA.

[0127] Preparation of cyclic anhydride: PSDA and acetic anhydride are reacted at a molar ratio of 1:5 under anhydrous and oxygen-free conditions, and the temperature is controlled to be ≤25°C. After the reaction is completed, the residual acetic anhydride and acetic acid are removed by reduced pressure evaporation to obtain a cyclic anhydride product containing a thioacetal bond.

[0128] Polymer grafting reaction: Poly(2-methyl-2-oxazoline) (PMeOx-OH) with a degree of polymerization of 45 and a hydroxyl-terminated poly(2-methyl-2-oxazoline) (PMeOx-OH) 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 and the condensation agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were added to the Schlenk reaction tube. To ensure the anhydrous and oxygen-free environment of the reaction system, the reaction tube was replaced with nitrogen three times to keep it 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 hours to complete the activation process of the terminal carboxyl group of POx-PSDA. After the activation of the terminal carboxyl group was completed, distearoylphosphatidylethanolamine (DSPE) was added to the reaction system, the reaction temperature was raised to 60°C and the reaction was continued for 72 hours; 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-POx was finally obtained by ether precipitation and ultrapure water dialysis purification and freeze-drying. 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, -SC-(CH3)2-S-), 2.14 (3H, -COCH3), 2.25-2.45 (4H, -OCOCH2-SC-(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 thioketal linker tk The preparation method comprises the following steps:

[0134] Synthesis of thioacetal intermediate 2,2'-(propane-2,2-diylbis(sulfanediyl))diacetic acid (PSDA): Mix thioacetic acid and acetone in a molar ratio of 3:1, add an appropriate amount of trifluoroacetic acid, react at room temperature, and precipitate a solid product at low temperature. The product is purified by alternating washing with n-hexane and water to obtain a white solid PSDA.

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

[0136] Polymer grafting reaction: Polyethylene glycol monomethyl ether with a degree of polymerization of 45 and cyclic acid anhydride are subjected to a ring-opening grafting reaction in chloroform at a molar ratio of 1:5. After ether precipitation, ultrapure water dialysis purification and freeze-drying, a PEG-PSDA product with a grafted thioketone structure is obtained.

[0137] Phospholipid coupling reaction: First, the prepared PEG-PSDA and the condensation agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were added to the Schlenk reaction tube. To ensure the anhydrous and oxygen-free environment of the reaction system, the reaction tube was replaced with nitrogen three times to keep it 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 hours to complete the activation process of the terminal carboxyl group of PEG-PSDA. After the activation of the terminal carboxyl group was completed, distearoylphosphatidylethanolamine (DSPE) was added to the reaction system, the reaction temperature was raised to 60°C and the reaction was continued for 72 hours; 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 was finally obtained by ether precipitation and ultrapure water dialysis purification and freeze-drying. tk (Formula I-2).

[0138] Formula (I-2).

[0139] The obtained lipid-hydrophilic polymer DSPE-PEG tk H NMR spectrum Figure 2 As shown, 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, -SC-(CH3)2-S-), 2.22 (2H, -OCOCH2CH2-), 3.3(4H, -OCOCH2-SC-(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 linked by thioketal tk The method for constructing lipid nanoparticles comprises the following steps:

[0142] The lipid-hydrophilic polymer DSPE-POx prepared in Example 1 tk, ionizable cationic lipid SM-102, auxiliary phospholipid DSPC and cholesterol were dissolved in anhydrous ethanol 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 / 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 components of lipids.

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

[0144] According to the total mass ratio of ionizable cationic lipids, auxiliary phospholipids, cholesterol and lipid-hydrophilic polymer to mRNA of 40:1, the lipid phase was added to the aqueous phase, and the amount of buffer in the aqueous phase was 3 times the volume of the organic solvent (anhydrous ethanol) in the lipid phase; after mixing evenly, 8 DSPE-POx with different molar ratios were self-assembled. tk Lipid nanoparticles (DSPE-POx tk -LNP), 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] Lipid-hydrophilic polymer DSPE-PEG linked by thioketal tk The method for constructing lipid nanoparticles comprises the following steps:

[0147] The lipid-hydrophilic polymer DSPE-PEG prepared in Example 2 tk , ionizable cationic lipid SM-102, auxiliary phospholipid DSPC and cholesterol were dissolved in anhydrous ethanol 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, to obtain a lipid phase containing four components of lipids.

[0148] The mRNA stock solution was added to a citric acid-sodium citrate buffer at pH = 4 to form an aqueous phase.

[0149] According to the total mass ratio of ionizable cationic lipids, auxiliary phospholipids, cholesterol and lipid-hydrophilic polymer to mRNA of 40:1, the lipid phase was added to the aqueous phase, and the amount of buffer in the aqueous phase was 3 times the volume of the organic solvent (anhydrous ethanol) in the lipid phase; after mixing evenly, two molar ratios of DSPE-PEG were self-assembled. tk Lipid nanoparticles (DSPE-PEG tk -LNP), named DSPE-PEG tk 1.5 DSPE-PEG tk 11.5.

[0150] Comparative Example 1

[0151] The method for constructing lipid nanoparticles by DMG-PEG is as follows:

[0152] The purchased control lipid-hydrophilic polymer DMG-PEG (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, abbreviated as: DMG-PEG2000, manufacturer: Shanghai MacLean Biochemical Technology Co., Ltd.), ionizable cationic lipid SM-102, auxiliary phospholipid DSPC and cholesterol were dissolved in anhydrous ethanol 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 to obtain a lipid phase containing four components of lipids.

[0153] The mRNA stock solution was added to a citric acid-sodium citrate buffer at pH = 4 to form an aqueous phase.

[0154] According to the total mass ratio of ionizable cationic lipids, auxiliary phospholipids, cholesterol and lipid-hydrophilic polymer to mRNA of 40:1, the lipid phase was added to the aqueous phase, and 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, DMG-PEG lipid nanoparticles (DMG-PEG-LNP) were self-assembled and named DMG-PEG 1.5.

[0155] Comparative Example 2

[0156] The method for constructing lipid nanoparticles from DSPE-PEG and DMG-PEG is 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, to obtain a lipid phase containing four components of lipids.

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

[0159] According to the total mass ratio of ionizable cationic lipids, auxiliary phospholipids, cholesterol and lipid-hydrophilic polymer to mRNA of 40:1, the lipid phase was added to the aqueous phase, and 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-LNP) 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 was 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 Figure 3~Figure 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 in Example 5 of the present invention tk - Particle size and polydispersity index plot of LNPs.

[0164] pass Figure 3~Figure 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, between 120 and 220 nm, and all samples maintain good dispersion properties, and most of the PDI values ​​measured are less than 0.2.

[0165] (2) Evaluation of DSPE-POx by agarose gel electrophoresis tk The specific operation includes: after the gel mold is cleaned with enzyme-free water, 0.5 g agarose is mixed with 50 mL 1×TAE buffer and heated until completely dissolved, 5 μL Gel-Red nucleic acid dye is added and injection molded; after the colloid solidifies, it is placed in the electrophoresis tank, and the buffer is injected to immerse the gel surface by 1 cm, then the LNP sample is mixed with 2×RNA loading buffer in equal volumes, and 1 μg mRNA is loaded into the well, and 5 μL DNA Ladder is set as a reference; after electrophoresis at a constant voltage of 120 V for 40 min, the electrophoresis spectrum is collected by a 302 nm ultraviolet 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 of LNP to test 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 When the molar ratio is gradually increased in the range of 5.5-11.5, the LNP system shows a regular particle size reduction phenomenon. Although the particle size changes with the adjustment of the component ratio, all samples still maintain a complete spherical self-assembled structure with a smooth surface and no significant agglomerate formation.

[0171] Example 6

[0172] The transfection effect of lipid nanoparticles with different molar ratios on luciferase mRNA in vitro was evaluated as follows:

[0173] Evaluation of DSPE-PEG using an in vitro cell transfection model tk The functional expression efficiency of -LNP was determined by: 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 three replicate 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 of 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 the best 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 by intravenous injection of lipid nanoparticles with different molar ratios was evaluated by the following steps:

[0178] An in vivo transfection evaluation model was established in mice. C57BL / 6 experimental animals of appropriate age (6-8 weeks old, 18-20 g in weight) were randomly divided into groups and injected with LNPs containing 2 μg Fluc mRNA via tail vein injection. 24 h after injection, 15 mg / mL D-luciferin potassium salt solution (200 μL / mouse) was delivered by intraperitoneal injection. After 8 min, the mice were euthanized and the target organs such as heart, liver, spleen, lung, kidney and lymph nodes were collected by dissection. An in vivo imaging system was used to collect bioluminescent signals at the organ level, and the fluorescence intensity distribution characteristics of each organ were quantitatively evaluated by professional image analysis software.

[0179] The experimental results are as follows Figure 8~Figure 9 As shown, Figure 8 DSPE-POx with different molar ratios provided in Example 7 of the present invention tk -LNP transfection effect diagram in various organs of C57BL / 6 mice; a) is a photo taken using a live imaging system after injection of DSPE-POx tk -LNP bioluminescence image of the transfection effect of various organs; b) DSPE-POx tk -LNP transfection average fluorescence intensity quantitative diagram of each organ; c) DSPE-POx tk -The average fluorescence intensity percentage of each organ transfected with LNP. Fig. 9 DSPE-PEG of different molar ratios provided by Example 7 of the present invention tk -LNP transfection effect diagram in various organs of C57BL / 6 mice; a) is a photo taken using a live imaging system after injection of DSPE-PEG tk -LNP bioluminescence image of the transfection effect of various organs; b) DSPE-PEG tk -LNP transfection average fluorescence intensity quantitative diagram of each organ, c) DSPE-PEG tk -The average fluorescence intensity percentage of each organ transfected with LNP.

[0180] pass Figure 8~Figure 9 It can be seen that the lipid-hydrophilic polymer modified LNP system showed selective transfection ability in the spleen, and its fluorescence signal intensity accounted for more than 80% of the total signal of each organ; tk and DSPE-PEG tk When the molar ratios in the lipid components reached 11.5 and 1.5, respectively, the system showed optimal spleen transfection efficiency, and the relative fluorescence intensity was increased by nearly 6 times and 2 times compared with the control groups DMG-PEG 1.5 and DSPE-PEG 1.5, respectively, showing a significant spleen-selective delivery advantage.

[0181] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A lipid-hydrophilic polymer, characterized in that The lipid-hydrophilic polymer has a structure shown in formula (I): Formula (I); In formula (I), R1 is a hydrophilic polymer group with a degree of polymerization of 20 to 50, R2 is a C2 to C4 alkyl group, R3 is a C2 to 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 or an ester bond.

2. The lipid-hydrophilic polymer according to claim 1, characterized in that The R1 is a structure of formula (i) or formula (ii): Formula (i); Formula (ii); Wherein, n is 20-50, and R5 is methyl or ethyl.

3. The lipid-hydrophilic polymer 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.

4. A method for preparing a lipid-hydrophilic polymer according to any one of claims 1 to 3, characterized in that: The following steps are involved: a) mixing acetone with a mercapto C2-C4 alkyl carboxylic acid compound to react to obtain a dibasic acid having a thioketal connecting bond 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 acid 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) subjecting the grafted product to a condensation reaction 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 hydrophilic polymer group with a degree of polymerization of 20 to 50, R2 is a C2 to C4 alkyl group, R3 is a C2 to C4 alkyl group, and R4 is a C 10 ~C 22 Single tail or C 10 ~C 22 The 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.

5. The preparation method according to claim 4, 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).

6. A lipid nanoparticle, characterized in that: 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; the lipid-hydrophilic polymers are the lipid-hydrophilic polymers described in any one of claims 1 to 3 or the lipid-hydrophilic polymers prepared by the preparation method described in any one of claims 4 to 5.

7. The lipid nanoparticle according to claim 6, 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-glycero-3-phosphoethanolamine, 2-(dioctylamino)ethylnonyl hydrogen phosphate, 2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropionic acid and 1,2-dioleoyl-SN-glycero-3-phospho-RAC-glycerol sodium salt; The nucleic acid is mRNA and / or DNA.

8. The lipid nanoparticle according to claim 6, characterized in that The molar ratio of the ionizable cationic lipid, auxiliary phospholipid, cholesterol and lipid-hydrophilic polymer is 50:(2-20):(20-50):(1-20).

9. A method for preparing lipid nanoparticles according to any one of claims 6 to 8, characterized in that: The following steps are involved: The lipid phase is mixed with the aqueous phase to self-assemble to obtain lipid nanoparticles; The components of the lipid phase include ionizable cationic lipids, cholesterol, auxiliary phospholipids, lipid-hydrophilic polymers and organic solvents; the aqueous phase contains nucleic acids.

10. Use of the lipid nanoparticles according to any one of claims 6 to 8 or the lipid nanoparticles prepared by the preparation method according to claim 9 in nucleic acid delivery.

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