CN118496296A - Carbohydrate cholesterol structures, liposome nanoparticles, compositions and uses thereof - Google Patents

Abstract

The invention discloses a carbohydrate cholesterol structure, liposome nano-particles, a composition and application thereof, and relates to the technical field of drug delivery. A carbohydrate cholesterol structure having a structure represented by the following formula (I) or formula (II): Wherein: r ¹ is selected from a monosaccharide, disaccharide or polysaccharide structure; r ² is selected from H atoms or carbon chains containing 1 to 3 carbon atoms; m and n are any natural number from 1 to 20. The invention provides a carbohydrate cholesterol structure, which is used as a raw material to prepare lipid nano-particles, and the lipid nano-particles are formed by using carbohydrate cholesterol to replace polyethylene glycol, and the lipid nano-particles are composed of cationic liposome, auxiliary liposome and steroid, so that the lipid nano-particles have the advantages of no toxicity, high-efficiency delivery of active agents (including but not limited to nucleic acid), and strong targeting of liver or spleen in vivo.

Description

Carbohydrate cholesterol structures, liposome nanoparticles, compositions and uses thereof

Technical Field

The invention relates to the technical field of drug delivery, in particular to a carbohydrate cholesterol structure, liposome nano particles, a composition and application thereof.

Background

Nucleic acid vaccines are bang on the list of "ten breakthrough technologies worldwide" in 2021 by their great reform in the medical field.

Nucleic acid drugs have the potential to thoroughly change the fields of vaccines, protein substitution therapies, gene editing therapies, and the like. However, nucleic acid therapeutics still face several challenges, including low cell permeability and high sensitivity to degradation by certain nucleic acid molecules, including RNA. Accordingly, there is a need to develop related methods and compositions that facilitate in vivo delivery of nucleic acid molecules for therapeutic and/or prophylactic purposes.

The lipid nanoparticle (Lipid nanoparticles, LNP) technology has high delivery efficiency and good safety, and is the technology most suitable for delivering nucleic acid therapeutic agents. LNP is typically composed of cationic or ionizable lipids, helper lipids, structural lipids, and polyethylene glycol (PEG) lipids. However, because polyethylene glycol has a certain immunogenicity to cells, antibodies against polyethylene glycol have been produced in some people, which results in rapid clearance of active ingredients by the immune system upon repeated injections of nucleic acid therapeutic agents, not only reducing therapeutic or prophylactic effects, but also causing various side effects. Therefore, there is an urgent need to introduce new lipids instead of PEG lipids to break through this limitation.

Disclosure of Invention

The invention provides a carbohydrate cholesterol structure, liposome nano-particles, a composition and application thereof, and aims to solve the problems in the background technology.

In order to achieve the technical purpose, the invention mainly adopts the following technical scheme:

in a first aspect, the invention discloses a carbohydrate cholesterol structure having a structure represented by the following formula (I) or formula (II):

Wherein:

R ¹ is selected from a monosaccharide, disaccharide or polysaccharide structure;

R ² is selected from H atoms or carbon chains containing 1 to 3 carbon atoms;

m and n are any natural number from 1 to 20.

In a preferred embodiment of the present invention, R ¹ is selected from any one of glucose, galactose, mannose, 2-acetamido glucose, 2-acetamido galactose, sialic acid, 2-glucosamine, 2-aminogalactose, gluconic acid, fucose, xylose, lactose, cellobiose, maltobiose, isomaltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and maltooctaose; r ² is H or CH ₂CH₃; m or n is selected from 1, 2, 3.

Preferably, the compound is a compound GC1, a compound GC2, a compound GC3, a compound GC4, a compound GC5, a compound GC6, a compound GC7, a compound GC8, a compound GC9, a compound GC10, a compound GC11, a compound GC12, a compound GC13, a compound GC14, a compound GC15, a compound GC16, a compound GC17 or a pharmaceutically acceptable salt thereof, which are shown in the following structures;

In a second aspect, the invention discloses a liposome nanoparticle composed of carbohydrate cholesterol, comprising the carbohydrate cholesterol structure and cationic lipid, auxiliary lipid and steroid according to the first aspect, wherein the molar ratio between the cationic lipid, auxiliary lipid, steroid and carbohydrate cholesterol is 30-60:5-25:25-50:0.05-10.

In a preferred embodiment of the present invention, the cationic lipid is selected from the group consisting of compound IL1, compound IL2, compound IL3, compound IL4, or a pharmaceutically acceptable salt thereof, as shown in the following structure;

in a preferred embodiment of the present invention, the auxiliary lipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, carbohydrate cholesterol, and lipid.

In a preferred embodiment of the invention, the steroid comprises at least one of cholesterol, vitamin D, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, alpha tocopherol, corticosteroid.

In a third aspect, the present invention discloses the use of a liposomal nanoparticle according to the second aspect for the in vivo delivery of a small molecule therapeutic agent.

In a fourth aspect, the present invention discloses a composition, which is a composition loaded with an active agent by the liposome nanoparticle of the second aspect, the active agent comprising at least one of a prophylactic agent, a therapeutic agent, selected from a nucleic acid, an immunomodulator, an antigen or a fragment thereof, a vaccine, an anti-inflammatory agent, an anti-tumour agent, an antibiotic, an agent acting on the central nervous system, a protein, a peptide, a polypeptide, a small molecule drug, or a mixture thereof;

The nucleic acid is at least one selected from messenger RNA, ribosomal RNA, micro RNA, transfer RNA, small interfering RNA, small nuclear RNA, antisense oligonucleotide, DNA and plasmid.

In a fifth aspect, the invention discloses the use of a composition according to the fourth aspect for the manufacture of a medicament for the treatment or prophylaxis of infectious diseases, cancer, rare diseases, genetic diseases, liver or spleen diseases.

Compared with the prior art, the invention has the following beneficial effects:

The invention provides a carbohydrate cholesterol structure, which is used as a raw material to prepare lipid nano-particles, and the lipid nano-particles are formed by using carbohydrate cholesterol to replace polyethylene glycol, and the lipid nano-particles are composed of cationic liposome, auxiliary liposome and steroid, so that the lipid nano-particles have the advantages of no toxicity, high-efficiency delivery of active agents (including but not limited to nucleic acid), and strong targeting of liver or spleen in vivo.

Drawings

FIG. 1 is a nuclear magnetic resonance spectrum and a mass spectrum of a compound GC1 synthesized in example 1 of the present invention;

FIG. 2 is a nuclear magnetic resonance spectrum and a mass spectrum of the compound GC4 synthesized in example 4 of the present invention;

FIG. 3 is a nuclear magnetic resonance spectrum and a mass spectrum of the compound GC5 synthesized in example 5 of the present invention;

FIG. 4 is a nuclear magnetic resonance spectrum and a mass spectrum of the compound GC7 synthesized in example 7 of the present invention;

FIG. 5 is a graph showing the properties of the carbohydrate cholesterol LNP produced in example 18 of the invention;

FIG. 6 is the luminous intensity of images of mice and animals administered with the saccharide cholesterol LNP tail vein injection in example 19 of the present invention;

FIG. 7 shows the intensity of luminescence imaged by mice and animals when carbohydrate cholesterol LNP is administered intramuscularly in example 20 of the present invention.

Detailed Description

The present invention will be described in further detail below in order to make the objects, technical solutions and effects of the present invention more clear and distinct.

In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods. In some instances, related operations of the present application have not been shown or described in the specification in order to avoid obscuring the core portions of the present application, and may be unnecessary to persons skilled in the art from a detailed description of the related operations, which may be presented in the description and general knowledge of one skilled in the art.

Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, the steps or acts in the method descriptions may be sequentially transposed or modified in a manner apparent to those skilled in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.

The ordinal numbers themselves, such as "first", "second", etc., are used herein only to distinguish the described objects and do not have any sequential or technical meaning.

Currently standard lipid nanoparticles are generally composed of cationic lipids, helper lipids, steroids and polyethylene glycol modified lipids. However, polyethylene glycol has certain immunogenicity, and can cause anaphylactic reaction, so that the application range of the existing liposome nano-particles is limited.

The invention uses sugar cholesterol to replace polyethylene glycol, and forms liposome nano particles with cationic liposome, auxiliary liposome and steroid. The carbohydrate cholesterol lipid nanoparticle of the invention is non-toxic, can deliver active agents (including but not limited to nucleic acids) with high efficiency, and has strong spleen or liver targeting in vivo.

In particular, cholesterol is a class of lipid molecules that are important components of the cell membrane of an organism. It is used to maintain the strength and fluidity of the cell membrane. In addition, cholesterol is also a precursor molecule for biosynthesis of steroid hormones and bile acids. Carbohydrates are also one of the most important molecules in life, being hydrophilic, and playing an important role in cell-cell interactions and in recognition of various molecules that promote cell adhesion.

Therefore, the present invention contemplates that sugar and cholesterol are linked by chemical bonds to form sugar cholesterol, which is characterized by amphipathy and can be embedded into a biological membrane structure.

In view of the various functions of carbohydrate cholesterol described above, the present embodiment introduces carbohydrate cholesterol into a formulation of Lipid Nanoparticles (LNPs) to more effectively deliver nucleic acids to cells. The present embodiment uses the carbohydrate cholesterol to replace polyethylene glycol lipid in the common liposome nanoparticle, and the new lipid nanoparticle formulation has many advantages such as high delivery efficiency and specific organ targeting based on the performance of the carbohydrate cholesterol, and can effectively perfect the existing lipid nanoparticle delivery system.

The following is a description of specific examples.

Example 1: synthesis of Compound GC1

To a 500mL single neck round bottom flask was added cholesterol (20 g,51.8 mmol), bromopropyne (1.5 eq,6.7mL,77.6 mmol), tetrabutylammonium iodide (0.1 eq,1.9g,5.2 mmol), dry tetrahydrofuran (200 mL), sodium hydride (3 eq,3.7g,155.3 mmol) was added under ice bath, the ice bath was removed after 30 minutes, and stirring was continued overnight. After the reaction is completed, adding 10mL of water for quenching, then carrying out reduced pressure distillation and concentration on the reaction solution, dissolving the residual solid in ethyl acetate, extracting and washing three times by using a saturated sodium chloride solution, carrying out organic phase drying and concentration, and separating and purifying by a silica gel column (eluent petroleum ether: ethyl acetate=100:1-60:1) to obtain a white solid compound CHO-B (17.9 g, yield 82％).¹H NMR(400MHz,CDCl₃)δ5.36(d,J＝5.0Hz,1H),4.19(d,J＝2.3Hz,2H),3.39(d,J＝4.5Hz,1H),2.47-2.32(m,2H),2.22(s,1H),2.08-1.74(m,5H),1.68-1.40(m,7H),1.42-1.20(m,9H),1.21-1.03(m,7H),1.03-0.79(m,16H),0.67(s,3H).¹³C NMR(100MHz,CDCl₃)δ 140.71,122.04,80.57,78.32,73.91,56.90,56.28,55.23,50.28,42.45,39.91,39.66,38.86,37.26,36.96,36.33,35.93,32.08,32.01,29.85,28.57,28.21,28.16,24.43,23.96,22.97,22.71,21.20,19.48,18.86,12.00.

To a 100mL single-neck round bottom flask was added D, maltose (3 g,8.8 mmol), pyridine (25 mL), acetic anhydride (15 eq,12mL,132 mmol), stirred until TLC detection reaction ended, the reaction solution was concentrated by reduced pressure distillation, the remaining solid was dissolved in dichloromethane, and extracted sequentially with 1M hydrochloric acid, saturated sodium bicarbonate, saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate and concentrated, and purified by silica gel column separation (eluent petroleum ether: ethyl acetate=3:1-2:1) to give a white solid compound GC1a (4.64 g, yield 78%); dried GC1a (4.64 g,6.8 mmol) was placed in a 200mL round bottom flask, pumped under vacuum with an oil pump and nitrogen protection, dried dichloromethane 50mL, 3-bromo-1-propanol (5 eq,3.1mL,34.2 mmol) and boron trifluoride diethyl ether complex (6 eq,5.1mL,41 mmol) were added, stirred under reflux, after TLC detection reaction was completed, an appropriate amount of saturated sodium bicarbonate solution was added for extraction, and after organic phase drying concentration, the mixture was separated and purified by a silica gel column (eluent petroleum ether: ethyl acetate=3:1-2:1) to give a white solid compound GC1b (2 g, 39% yield); GC1b (2 g,2.6 mmol) was placed in a 100mL round bottom flask, sodium azide (5 eq,860mg,13.2 mmol), tetrabutyl iodinated amine (0.2 eq,195 mg), dried N, N-dimethylformamide 30mL, stirred in an oil bath at 60℃for 2 hours, the reaction was diluted with ethyl acetate and then washed three times with water, and the organic phase was concentrated by drying to give GC1c as a white solid (1.83 g, 96% yield); GC1c (200 mg), CHO-B (1.2 eq,142 mg), anhydrous copper sulfate (2 eq,139 mg), sodium ascorbate (3 eq,165 mg) were placed in a 50mL round bottom flask, evacuated with an oil pump for 1 hour, purged with nitrogen, and methanol and water 2 were added: 1, stirring at room temperature, concentrating the reaction liquid by reduced pressure distillation after TLC detection reaction is finished, and separating and purifying by a silica gel column (eluent petroleum ether: ethyl acetate=3:1-1:1) to obtain a white solid compound GC1d (235 mg, yield 74%); GC1d (235 mg) was placed in a 50mL round bottom flask and 10mL of methanol was added, the pH of the system was adjusted to 9-11 with methanol/sodium methoxide solution, stirring was carried out at room temperature for 1 hour, the reaction was completed, neutralized to pH=7 with Dowex50WX2-100 (H ⁺) acid resin, filtered, concentrated under reduced pressure, and purified by column chromatography on silica gel (eluent dichloromethane: methanol=10:1-8:1) to give compound GC1 (181 mg, yield 88%) as a white solid.

¹H NMR(400MHz,DMSO)δ 8.03(s,1H),5.51(d,J＝3.0Hz,1H),5.43(d,J＝6.1Hz,1H),5.37(d,J＝3.1Hz,1H),5.29(s,1H),5.17(d,J＝4.9Hz,1H),4.99(d,J＝3.6Hz,1H),4.95-4.82(m,2H),4.61(d,J＝3.5Hz,1H),4.57-4.45(m,3H),4.42(dd,J＝12.6,6.3Hz,2H),4.15(d,J＝7.7Hz,1H),3.82-3.64(m,2H),3.63-3.50(m,2H),3.49-3.36(m,4H),3.31-3.12(m,4H),3.12-2.95(m,2H),2.31(s,1H),2.04(dd,J＝13.4,7.1Hz,2H),1.98-1.66(m,4H),1.59-1.17(m,10H),1.17-1.00(m,5H),0.99-0.68(m,15H),0.62(s,3H).¹³C NMR(100MHz,DMSO)δ144.47,140.43,123.83,121.18,102.74,100.86,79.65,77.70,75.17,73.39,73.00,72.47,69.88,60.69,56.23,55.67,49.64,41.89(m),38.89,38.62,36.73,36.31(m),35.73,35.28,31.44(m),30.0727.90,27.43(m),23.90,23.31,22.68(m),22.41(m),19.08(m),18.57(m),11.69.ESI-HRMS：m/z 850.5429[M+H]⁺(calculated for C₄₅H₇₅N₃O₁₂：850.5419[M+H]⁺).

Example 2: synthesis of Compound GC2

The procedure for the synthesis of GC1 was essentially the same as in example 1, except that D-mannose was used instead of D-maltose in example 1.

¹H NMR(400MHz,DMSO)δ 8.04(s,1H),5.29(s,1H),4.68(dd,J＝4.9,1.9Hz,2H),4.53(dd,J＝17.4,11.5Hz,3H),4.38(dt,J＝13.2,6.3Hz,3H),3.71-3.51(m,2H),3.49-3.12(m,15H),2.31(s,1H),2.04(dd,J＝13.2,7.1Hz,2H),1.98-1.66(m,4H),1.57-1.17(m,9H),1.17-1.00(m,5H),1.00-0.69(m,15H),0.62(s,3H).¹³C NMR(100MHz,DMSO)δ 144.59,140.43,123.63,121.18,99.98,74.09,71.00,70.28,66.97,61.22,36.31,35.28,31.44,27.90,27.43,22.66,22.41,19.07,18.57,11.69.ESI-HRMS：m/z 688.4901[M+H]⁺(calculated for C₃₉H₆₅N₃O₇：688.4887[M+H]⁺).

Example 3: synthesis of Compound GC3

The procedure for the synthesis of GC1 was essentially the same as in example 1, except that D-glucose was used in place of D-maltose in example 1.

¹H NMR(400MHz,DMSO)δ 8.07(s,1H),5.32(s,1H),5.09(d,J＝4.7Hz,1H),4.97(d,J＝4.7Hz,1H),4.93(d,J＝4.7Hz,1H),4.88(d,J＝5.6Hz,1H),4.78(d,J＝4.9Hz,1H),4.74(d,J＝6.9Hz,1H),4.61(d,J＝3.6Hz,1H),4.66-4.35(m,4H),4.11(d,J＝7.8Hz,1H),3.69(ddd,J＝35.6,17.4,11.8Hz,2H),3.42(ddd,J＝17.5,11.8,6.0Hz,2H),3.29-2.91(m,4H),2.35(d,J＝13.2Hz,1H),2.21-2.00(m,2H),2.00-1.71(m,4H),1.48(dt,J＝16.8,8.6Hz,3H),1.43-1.27(m,4H),1.24(t,J＝10.7Hz,2H),1.09(dt,J＝16.9,14.8Hz,5H),1.02-0.92(m,5H),0.89(d,J＝6.3Hz,3H),0.83(dd,J＝6.6,1.4Hz,5H),0.64(s,3H).¹³C NMR(100MHz,DMSO)δ 144.46,140.44,123.94,121.21,102.92,98.72,77.64,76.89,76.61,73.38,70.00,65.28,61.04,60.55,56.21,55.62,49.62,46.38,41.88,36.72,36.31(m),35.71,35.26,31.44(m),30.00,27.91,27.44(m),23.90,23.26,22.70(m),22.43(m),20.65,19.09,18.58,11.71.ESI-HRMS：m/z 688.4901[M+H]⁺(calculated for C₃₉H₆₅N₃O₇：688.4893[M+H]⁺).

Example 4: synthesis of Compound GC4

The procedure for the synthesis of GC1 was essentially the same as in example 1, except that D-cellobiose was used in place of D-maltose in example 1.

¹H NMR(400MHz,DMSO)δ 8.06(s,1H),5.31(s,5H),4.89-4.33(m,7H),4.22(dd,J＝28.4,7.8Hz,2H),4.09-3.56(m,7H),3.53-3.12(m,16H),3.02(dt,J＝16.9,8.9Hz,4H),2.34(d,J＝13.2Hz,1H),2.19-2.00(m,3H),2.00-1.67(m,6H),1.61-1.19(m,11H),1.19-1.02(m,6H),1.01-0.67(m,16H),0.64(s,3H).¹³C NMR(100MHz,DMSO)δ 144.49,140.44,123.91,121.22,103.22,102.58,80.48,77.69,76.83,76.48,74.91,73.23,70.04,65.44,61.03,60.48,56.22,55.64,49.64,46.38,41.89(m),38.89,38.64,36.31(m),31.44(m),27.91,27.43(m),22.67(m),22.41(m),19.07(m),18.57(m),11.69.ESI-HRMS：m/z 850.5429[M+H]⁺(calculated for C₄₅H₇₅N₃O₁₂：850.5422[M+H]⁺).

Example 5: synthesis of Compound GC5

The procedure for the synthesis of GC1 was essentially the same as in example 1, except that D-lactose was used instead of D-maltose in example 1.

¹H NMR(400MHz,DMSO)δ 8.06(s,1H),5.43-5.16(m,2H),5.12(s,1H),4.77(d,J＝51.6Hz,4H),4.52(dd,J＝38.9,20.4Hz,6H),4.17(d,J＝23.2Hz,3H),3.93(d,J＝41.0Hz,1H),3.88-3.43(m,10H),3.27(d,J＝32.2Hz,7H),3.16(d,J＝4.3Hz,1H),3.03(s,1H),2.35(d,J＝14.1Hz,1H),1.92(dd,J＝64.0,42.9Hz,8H),1.68-0.67(m,16H),0.64(s,3H).¹³C NMR(100MHz,DMSO)δ 143.10,140.91,128.06,126.48,113.92,103.90,102.58,80.66,77.74,75.58,74.91,73.32,73.21,70.66,69.85,69.73,69.10,68.16,65.50,60.42,56.29,55.70,49.70,46.47,41.93,38.89,38.62,36.78,36.37,36.31(m),35.76,35.07,33.75,31.43(m),30.12,29.40(m),29.15,29.04,28.81,27.89,27.83,23.90,23.30,22.74(m),22.47(m),20.66,19.08(m),18.57(m),14.03,13.44,11.75.ESI-HRMS：m/z 850.5429[M+H]⁺(calculated for C₄₅H₇₅N₃O_{1 2}：850.5419[M+H]⁺).

Example 6: synthesis of Compound GC6

The procedure for the synthesis of GC1 was essentially the same as in example 1, except that D-isomaltose was used in place of D-maltose in example 1.

¹H NMR(400MHz,DMSO)δ 8.05(d,J＝11.7Hz,1H),5.31(s,1H),5.06(d,J＝38.1Hz,2H),4.82(d,J＝41.1Hz,2H),4.71-4.58(m,2H),4.45(dd,J＝23.5,16.6Hz,4H),4.13(d,J＝7.7Hz,1H),3.87-3.53(m,4H),3.53-3.39(m,4H),3.28-2.91(m,5H),2.35(d,J＝13.2Hz,1H),2.18-1.67(m,6H),1.62-1.19(m,9H),1.19-0.70(m,19H),0.64(s,3H).¹³C NMR(100MHz,DMSO)δ 144.46,140.44,123.88,121.19,102.89,98.26,77.73,76.68,74.81,73.28,72.43,71.96,70.21,65.37,60.87,60.60,56.23,55.66,49.64,46.43,41.89,38.89,38.62,36.73,36.31(m),35.73,35.27,31.44,30.12,27.90,27.43,23.90,23.30,22.68(m),22.42(m),20.66,19.08(m),18.57(m),11.69.ESI-HRMS：m/z 850.5429[M+H]⁺(calculated for C₄₅H₇₅N₃O₁₂：850.5424[M+H]⁺).

Example 7: synthesis of Compound GC7

The procedure for the synthesis of GC1 was essentially the same as in example 1, except that D-maltotriose was used in place of D-maltose in example 1.

1H NMR(400MHz,DMSO)δ8.05(s,1H),5.74(s,2H),5.53(d,J＝34.3Hz,3H),5.31(s,2H),4.97(t,J＝24.7Hz,3H),4.71-4.30(m,6H),4.17(d,J＝7.3Hz,1H),3.88-2.85(m,30H),2.33(s,1H),2.20-1.64(m,8H),1.60-0.70(m,36H),0.64(s,3H).¹³C NMR(100MHz,DMSO)δ144.47,140.46,123.94,121.21,102.70,100.78,99.58,79.44,77.68,76.29,73.37,69.91,60.67,56.24,55.66,49.65,48.60(m),46.43,41.89,39.00,36.73,36.32(m),35.73,35.27(m),31.45(m),30.12,27.98,27.44(m),23.91,23.28,22.69(m),22.42(m),20.66,19.09(m),18.58(m),11.70.ESI-HRMS：m/z1012.5957[M+H]⁺(calculated for C₅₁H₈₅N₃O₁₇：1012.5964[M+H]⁺).

Example 8: synthesis of Compound GC8

The procedure for the synthesis of GC1 was essentially the same as in example 1, except that D, maltohexaose was used in place of D-maltose in example 1.

¹H NMR(400MHz,DMSO)δ 8.06(s,1H),5.45(d,J＝112.6Hz,9H),5.01(s,5H),4.48(d,J＝33.3Hz,7H),4.18(d,J＝6.7Hz,1H),3.95-3.44(m,17H),3.27-2.77(m,6H),2.34(d,J＝11.6Hz,1H),1.91(dd,J＝66.9,42.9Hz,6H),1.58-0.70(m,28H),0.64(s,3H).¹³C NMR(100MHz,DMSO)δ 144.50,140.48,123.94,121.24,102.73,100.87,100.61-100.23(m),79.90,79.67(m),77.65,76.24,73.17(m),72.61,72.12(m),71.76(m),69.96,60.85,60.56(m),60.30(m),60.43,56.25(m),55.65(m),49.66,41.91,38.90,38.67,36.40(m),31.46(m),31.20,28.00,27.86,27.45(m),23.93,23.27,22.72(m),22.45(m),19.11(m),18.61(m),11.73.ESI-HRMS：m/z 1498.7542[M+H]⁺(calculated for C₆₉H₁₁₅N₃O₃₂：1498.7537[M+H]⁺).

Example 9: synthesis of Compound GC9

The procedure for the synthesis of GC1 was essentially the same as in example 1, except that D-maltoheptaose was used in place of D-maltose in example 1.

¹H NMR(400MHz,DMSO)δ 8.06(s,1H),5.51(d,J＝38.8Hz,8H),5.31(s,2H),5.11-4.96(m,4H),4.88(s,1H),4.67-4.29(m,7H),4.18(d,J＝7.6Hz,1H),3.87-3.40(m,20H),3.15(dd,J＝77.7,5.9Hz,5H),2.35(d,J＝15.0Hz,1H),1.98(ddd,J＝73.0,37.9,24.6Hz,7H),1.62-1.02(m,17H),1.02-0.69(m,14H),0.64(s,3H).¹³C NMR(100MHz,DMSO)δ 144.50,140.47,129.66,123.91,121.23,102.73,100.83(m),100.46(m),100.25,79.81,79.41,77.66,76.20,75.12,74.01-72.79(m),72.57,72.09(m),71.12(m),60.85,60.57(m),60.33(m),56.24,55.65,49.65,46.47,41.90,38.76,38.45,36.33(m),35.74,35.28(m),31.46(m),30.11,28.87,27.93,27.44(m),22.71(m),22.44(m),19.10(m),18.59(m),11.72.ESI-HRMS：m/z 1660.8070[M+H]⁺(calculated for C₆₉H₁₁₅N₃O₃₂：1660.8090[M+H]⁺).

Example 10: synthesis of Compound GC10

The procedure for the synthesis of GC1 was essentially the same as in example 1, except that D-maltooctaose was used in place of D-maltose in example 1.

¹H NMR(400MHz,DMSO)δ 8.06(s,1H),5.97-5.37(m,8H),5.27(d,J＝34.7Hz,1H),5.13-4.76(m,5H),4.74-4.34(m,6H),4.18(d,J＝7.6Hz,1H),3.94-3.43(m,16H),3.26-2.83(m,3H),2.34(s,1H),2.21-1.65(m,5H),1.63-0.72(m,21H),0.64(s,3H).¹³C NMR(100MHz,DMSO)δ 144.51,140.48,123.93,121.25,102.73,100.84-99.58(m),79.82-79.07(m),73.51,73.35,73.16(m),72.57,72.04(m),71.69(m),60.71,60.34(m),56.24,49.65,46.46,41.90,39.01,38.67,36.73,36.41(m),36.33,35.27,31.47(m),28.71(m),27.93(m),27.44(m),23.92,23.27,22.72(m),22.45(m),19.11(m),18.60(m),11.73.ESI-HRMS：m/z 1822.8598[M+H]⁺(calculated for C₈₁H₁₃₅N₃O₄₂：1822.8609[M+H]⁺).

Example 11: synthesis of Compound GC11

The procedure for the synthesis of GC1 was essentially the same as in example 1, except that D-mannose was used instead of D-maltose in example 1.

¹H NMR(400MHz,DMSO)δ 8.07(s,1H),5.31(s,1H),4.76(d,J＝39.7Hz,4H),4.60(d,J＝11.2Hz,2H),4.52(s,2H),4.48-4.29(m,3H),3.80-3.53(m,5H),3.51-3.28(m,12H),3.23(s,1H),2.33(s,1H),2.17-1.67(m,7H),1.60-1.19(m,10H),1.18-0.70(m,20H),0.64(s,3H).¹³C NMR(100MHz,DMSO)δ 144.60,140.44,123.69,121.22,102.74,100.87,77.70,73.62,71.99,71.06,70.31,66.92,66.68,65.76,63.34,61.19,60.59,56.23,55.64,49.64,46.74,41.90(m),39.00,38.62,36.73,36.33(m),35.73,35.28,31.45(m),29.91,27.91,27.45(m),23.92,23.28,22.71(m),22.44(m),20.67,19.10(m),18.59(m),11.72(m).ESI-HRMS：m/z 850.5429[M+H]⁺(calculated for C₄₅H₇₅N₃O₁₂：850.5414[M+H]⁺).

Example 12: synthesis of Compound GC12

GC12a (30 mg), CHO-B (1.5 eq,26 mg), anhydrous copper sulfate (2 eq,20 mg), sodium ascorbate (3 eq,24 mg) were placed in a 25mL round bottom flask, evacuated with an oil pump for 1 hour, purged with nitrogen, and methanol and water 2 were added: 1, stirring at room temperature, concentrating the reaction solution by distillation under reduced pressure after TLC detection reaction is finished, and separating and purifying by a C18 reverse phase chromatographic column (eluent deionized water: methanol=1:0-0:1) to obtain a white solid compound GC12 (28 mg, yield 60%).

¹H NMR(400MHz,DMSO)δ 8.32(s,1H),7.89(s,1H),7.78(d,J＝8.7Hz,1H),5.33(s,1H),5.06(s,1H),4.89-4.11(m,11H),4.10-3.88(m,2H),3.87-3.46(m,9H),3.27-2.83(m,5H),2.74(d,J＝8.3Hz,1H),2.37(d,J＝11.0Hz,1H),2.08(ddd,J＝23.4,8.7,2.8Hz,1H),2.00-1.59(m,10H),1.59-0.70(m,34H),0.65(s,3H).¹³C NMR(100MHz,DMSO)δ 171.15,169.48,148.85,142.81123.15,121.69,104.37,102.63,100.89,81.30,78.18,76.74-75.32(m),74.40-71.04(m),69.04,67.09,63.76,60.94,56.05,54.11,49.93,42.33(m),42.20,36.77(m),36.43,31.89(m),28.26(m),27.87,25.55,23.14(m),22.87(m),21.85,21.09(m),19.28(m),12.15(m).ESI-HRMS：m/z1168.6492[M+H]⁺(calculated for C₅₇H₉₃N₅O₂₀：1168.6489[M+H]⁺).

Example 13: synthesis of Compound GC13

To a 100mL single neck round bottom flask was added β -sitosterol (Sitosterol) (3 g,7.2 mmol), bromopropyne (1.5 eq,0.95mL,10.9 mmol), tetrabutylammonium iodide (0.1 eq,535mg,1.4 mmol), dry tetrahydrofuran (30 mL), sodium hydride (3 eq,521mg,21.7 mmol) was added under ice bath, the ice bath was removed after 30 minutes and stirring continued overnight. After the reaction is completed, adding 10mL of water for quenching, then decompressing, distilling and concentrating the reaction liquid, dissolving the residual solid in ethyl acetate, extracting and washing with saturated sodium chloride solution for three times, concentrating by organic phase drying, and separating and purifying by a silica gel column (eluent petroleum ether: ethyl acetate=100:1-60:1) to obtain a white solid compound St-B (1.9 g, yield 59%); GC1c (200 mg), st-B (1.2 eq,151 mg), anhydrous copper sulfate (2 eq,139 mg), sodium ascorbate (3 eq,165 mg) were placed in a 50mL round bottom flask, evacuated with an oil pump for 1 hour, purged with nitrogen, and methanol and water 2 were added: 1, stirring at room temperature, concentrating the reaction liquid by reduced pressure distillation after TLC detection reaction is finished, and separating and purifying by a silica gel column (eluent petroleum ether: ethyl acetate=3:1-1:1) to obtain a white solid compound GC13a (267 mg, yield 82%); GC13a (267 mg) was placed in a 50mL round bottom flask and 10mL of methanol was added, the pH of the system was adjusted to 9-11 with methanol/sodium methoxide solution, stirring was performed at room temperature for 1 hour, the reaction was completed, neutralized to pH=7 with Dowex50WX2-100 (H ⁺) acid resin, filtered, concentrated under reduced pressure, and purified by column chromatography on silica gel (eluent dichloromethane: methanol=10:1-8:1) to give GC13 (172 mg, 86% yield) as a white solid.

¹H NMR(400MHz,DMSO)δ 8.06(d,J＝2.7Hz,1H),5.52(d,J＝25.5Hz,2H),5.26(d,J＝39.1Hz,2H),4.99(dd,J＝17.6,14.2Hz,3H),4.69--4.34(m,5H),4.17(d,J＝7.7Hz,1H),3.88-3.52(m,4H),3.45(ddd,J＝20.4,12.8,8.5Hz,5H),3.24(ddd,J＝29.0,20.4,11.3Hz,4H),3.14-2.97(m,2H),2.34(d,J＝10.9Hz,1H),2.20-1.70(m,6H),1.69-1.03(m,14H),1.03-0.69(m,17H),0.63(d,J＝11.7Hz,3H).¹³C NMR(100MHz,DMSO)δ 144.47,140.42,123.86,121.17,102.74,100.87,79.66,77.73,75.17,73.39,73.00,72.48,60.70,55.53,45.19(m),41.88(m),40.04,38.89,36.72,36.42(m),35.59(m),33.41,31.44(m),28.74(m),27.91,22.65(m),19.69(m),19.07(m),18.64(m),11.78,11.67.ESI-HRMS：m/z 878.5742[M+H]⁺(calculated for C₄₅H₇₅N₃O₁₂：878.5710[M+H]⁺).

Example 14: synthesis of Compound GC14

D-maltose (3 g,8.8 mmol), pyridine (25 mL) and acetic anhydride (15 eq,12mL,132 mmol) are added into a 100mL single-neck round bottom flask, the mixture is stirred until the TLC detection reaction is finished, the reaction liquid is distilled and concentrated under reduced pressure, the residual solid is dissolved in dichloromethane, 1M hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride solution are sequentially used for extraction, an organic phase is dried by anhydrous sodium sulfate and then concentrated, and the organic phase is separated and purified by a silica gel column (eluent petroleum ether: ethyl acetate=3:1-2:1) to obtain a white solid compound GC1a (4.64 g, yield 78%); to a 100mL single neck round bottom flask was added GC1a (2.42 g,3.6 mmol), tetrahydrofuran (20 mL), benzylamine (1.2 eq,468 μl,4.3 mmol), stirred until TLC detection was completed, the reaction was concentrated by distillation under reduced pressure, the remaining solid was dissolved in dichloromethane, and extracted sequentially with 1M hydrochloric acid, saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate and concentrated, and purified by column chromatography over silica gel (eluent petroleum ether: ethyl acetate=3:1 to 1:1) to give GC14a (1.83 g, 81% yield) as a white solid; dried GC14a (1.83 g,2.9 mmol) was placed in a 100mL round bottom flask, pumped under vacuum for 1 hour, nitrogen was turned over, dried dichloromethane 30mL, trichloroacetonitrile (5 eq,1.44mL,14.4 mmol), 1, 8-diazabicyclo [5.4.0] undec-7-ene (0.2 eq, 80. Mu.L) were added, stirred under reflux, after the TLC detection reaction ended, directly concentrated and purified by silica gel column separation (eluent petroleum ether: ethyl acetate=5:1-3:1) to give GC14b as a white solid (1.6 g, 73% yield); placing 200mg of cholesterol and 14b (1.3 eq,672 mg) of cholesterol in a 50mL round bottom flask, vacuumizing an oil pump for 1 hour, transferring nitrogen protection, adding 10mL of dried dichloromethane and trimethyl silicone triflate (0.2 eq,18 μl) under ice bath conditions, transferring to room temperature, continuously stirring for 30 minutes, concentrating the reaction liquid by reduced pressure distillation after TLC detection is finished, and separating and purifying by a silica gel column (eluent petroleum ether: ethyl acetate=6:1-3:1) to obtain a white solid compound GC14c (263 mg, yield 53%); GC14c (263 m, g) was placed in a 50mL round bottom flask and 10mL of methanol was added, the pH of the system was adjusted to 9-11 with methanol/sodium methoxide solution, stirring was carried out at room temperature for 1 hour, the reaction was completed, neutralized to pH=7 with Dowex50WX2-100 (H ⁺) acid resin, filtered, concentrated under reduced pressure, and purified by column on silica gel (eluent dichloromethane: methanol=12:1-9:1) to give GC14 (161 mg, yield 83%) as a white solid.

¹H NMR(400MHz,DMSO)δ 5.76-5.21(m,3H),5.16-4.73(m,3H),4.73-4.21(m,2H),3.95-3.38(m,8H),3.25-2.89(m,3H),2.56(d,J＝13.2Hz,2H),2.42-2.05(m,2H),1.88(ddd,J＝32.6,20.3,10.8Hz,4H),1.65-0.71(m,30H),0.65(s,3H).¹³C NMR(100MHz,DMSO)δ 140.62,121.16,100.91,96.91,73.60-72.74(m),72.58,71.26,69.90,60.79,60.45,56.21,55.61,41.88,35.69,35.23,31.44(m),29.13,28.94-28.74(m),27.81,27.43(m),23.89,23.23,22.69(m),22.41(m),20.62,19.10(m),18.57(m),11.69(m).

Example 15: synthesis of Compound GC15

The procedure for the synthesis of GC14 was essentially the same as in example 14, except that D-glucose was used in place of D-maltose in example 14.

¹H NMR(400MHz,DMSO)δ 5.32(s,1H),4.87(s,2H),4.54-4.06(m,2H),3.64(d,J＝11.1Hz,1H),2.99(dd,J＝72.6,19.5Hz,3H),2.34(s,1H),2.12(s,1H),1.90(dd,J＝43.7,25.1Hz,4H),1.57-0.70(m,26H),0.64(s,3H).¹³C NMR(100MHz,DMSO)δ140.50,121.24,100.87,79.21,77.02,76.80,73.50,70.12,61.13,56.24,55.65,41.91,36.89,36.26,35.73,35.26,31.47(m),29.31,27.84,27.45(m),23.92,23.26,22.71(m),22.44(m),20.66,19.14(m),18.60(m),11.72(m).

Example 16: synthesis of Compound GC16

The procedure for the synthesis of GC14 was essentially the same as in example 14, except that D-cellobiose was used in place of D-maltose in example 14.

¹H NMR(400MHz,DMSO)δ 5.75(s,2H),5.28(d,J＝33.0Hz,1H),5.02(s,2H),4.79-4.41(m,2H),4.27(d,J＝6.9Hz,1H),3.64(d,J＝44.9Hz,3H),3.21-2.72(m,4H),2.35(s,1H),2.19-1.68(m,4H),1.62-0.71(m,22H),0.64(s,3H).¹³C NMR(100MHz,DMSO)δ 140.41,121.26,103.55,100.59,77.22,76.80,76.47,75.05,70.03,61.03,56.21,55.62,54.92(m),49.63,41.88(m),36.85,36.40(m),36.22,35.69,31.43(m),27.43(m),22.69(m),22.42(m),20.64,19.11(m),18.57(m),11.70(m).

Example 17: synthesis of Compound GC17

The procedure for the synthesis of GC14 was essentially the same as in example 14, except that D-maltotriose was used in place of D, maltose in example 14.

¹H NMR(400MHz,DMSO)δ 5.54(s,2H),5.32(s,1H),5.23-4.75(m,3H),4.55(s,2H),4.27(d,J＝7.5Hz,1H),3.84-3.49(m,6H),3.27-2.72(m,4H),2.36(d,J＝13.2Hz,1H),2.19-1.65(m,4H),1.65-0.71(m,23H),0.65(s,3H).¹³C NMR(100MHz,DMSO)δ 140.85,121.69,101.28,101.02,77.63,75.49,73.83,61.24,50.06,42.64(m),38.78,37.42,36.66(m),36.15,35.67,31.89(m),29.71(m),27.87(m),24.33(m),22.99(m),22.35(m),21.08,19.55(m),19.01(m),12.13(m).

Example 18: preparation and characterization of carbohydrate cholesterol lipid nanoparticles

Cationic lipid IL1 was mixed with DSPC, cholesterol, carbohydrate cholesterol at 50:10:38.5:1.5 in absolute ethanol at a total lipid concentration of 10mM. Luciferase mRNA (Fluc mRNA) was dissolved in aqueous sodium acetate (10 mm, ph=4.6) and mRNA concentration was 0.1mg/mL. Using a microfluidic instrument to control the flow rate ratio of the ethanol solution to the sodium acetate aqueous solution to be 1: and 3, preparing a solution of lipid nano particles in a microfluidic chip, diluting and centrifuging with PBS at 4 ℃ to remove ethanol, diluting and centrifuging again for three times, and diluting the obtained concentrated solution with PBS for later use. The size and PDI of the lipid nanoparticles were determined by dynamic light scattering in a 173 ° backscatter detection mode using Malvern Zetasizer Nano ZS. The encapsulation efficiency of the lipid nanoparticle was determined using Quant it Ribogreen RNA quantitative assay kit (ThermoFisher Scientific). The final lipid nanoparticle particle size and encapsulation efficiency are shown in table 1 and fig. 5.

TABLE 1 characterization of carbohydrate cholesterol LNP

As can be seen from Table 1 and FIG. 5, the carbohydrate cholesterol can completely replace PEG to prepare LNP for coating nucleic acid molecules, the particle size of the obtained mRNA-LNP is 80-150 nm, the uniformity is good, and the encapsulation efficiency is high.

Example 19: in vivo delivery Performance test of carbohydrate cholesterol lipid nanoparticles (tail vein injection)

Female BALB/c mice of 6-8 weeks old are selected, the weight is about 20g, the feeding environment is SPF-grade feeding room, and animal experiments are strictly carried out by referring to national institutes of health guidelines and animal ethics requirements. 3 mice were randomly selected and the lipid nanoparticles encapsulating luciferase (Fluc) mRNA were injected at 0.5mg/kg using tail vein. After 6 hours, 200. Mu.L of 10mg/mL of D-potassium fluorescein salt was intraperitoneally injected into each mouse, and after 10 minutes, the mice were placed under a living imaging system (PERKINELMER IVIS Spectrum), and the total luminous intensity of each mouse was observed and recorded by photographing. After euthanasia of the mice, the mice were dissected, heart, liver, spleen, lung, kidney were obtained, imaged, the luminous intensity of each organ was observed, and photographed. Intravenous administration mode of delivery of Fluc mRNA expression intensities are shown in table 2 and fig. 6.

TABLE 2 expression intensity of carbohydrate cholesterol LNP intravenous administration delivery of Fluc mRNA

The data in Table 2 and FIG. 6 show that the ability of multiple carbohydrate cholesterol LNPs to deliver Fluc mRNA in vivo by tail vein injection is significantly better than PEG-LNP, with DMG-PEG2K as a control, and anatomical experiments show that luminescence is primarily in the liver and spleen, with carbohydrate cholesterol LNP having liver and spleen targeting.

Example 20: in vivo delivery Performance test of carbohydrate cholesterol lipid nanoparticles (intramuscular injection)

Female BALB/c mice of 6-8 weeks old are selected, the weight is about 20g, the feeding environment is SPF-grade feeding room, and animal experiments are strictly carried out by referring to national institutes of health guidelines and animal ethics requirements. 3 mice were randomly selected and lipid nanoparticles encapsulating luciferase mRNA were used at a dose of 0.15mg/kg by intramuscular injection. After 6 hours, 200. Mu.L of 10mg/mL of D-potassium fluorescein salt was intraperitoneally injected into each mouse, and after 10 minutes, the mice were placed under a living imaging system (PERKINELMER IVIS Spectrum), and the total luminous intensity of each mouse was observed and recorded by photographing. Intramuscular administration mode the expression intensity of the delivery of Fluc mRNA is shown in table 3 and fig. 7.

TABLE 3 expression intensity of carbohydrate cholesterol LNP intramuscular administration delivery of Fluc mRNA

LNP formulation	Sugar cholesterol	Total luminous intensity (p/sec)
			1	GC1	6.12E+8
3	GC4	5.46E+8
			5	GC6	1.65E+8
6	GC7	3.07E+8
			15	PEG2K	2.98E+8

The data in Table 3 and FIG. 7 show that the ability of multiple carbohydrate cholesterol LNPs to deliver Fluc mRNA at the intramuscular injection site was significantly better than PEG-LNP when administered by intramuscular injection with DMG-PEG2K as a control.

The present invention is not limited to the above embodiments, and any equivalent embodiments which can be changed or modified by the technical disclosure described above can be applied to other fields, but any simple modification, equivalent changes and modification made to the above embodiments according to the technical matter of the present invention will still fall within the scope of the technical disclosure.

Claims (10)

1. A carbohydrate cholesterol structure characterized by having a structure represented by the following formula (I) or formula (II):

Wherein:

R ¹ is selected from a monosaccharide, disaccharide or polysaccharide structure;

R ² is selected from H atoms or carbon chains containing 1 to 3 carbon atoms;

m and n are any natural number from 1 to 20.

2. The carbohydrate cholesterol structure of claim 1 wherein R ¹ is selected from any of glucose, galactose, mannose, 2-acetamido glucose, 2-acetamido galactose, sialic acid, 2-glucosamine, 2-aminogalactose, gluconic acid, fucose, xylose, lactose, cellobiose, maltobiose, isomalt, mannobiose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and maltooctaose; r ² is H or CH ₂CH₃; m or n is selected from 1, 2, 3.

3. The carbohydrate cholesterol structure of any of claims 1 or 2, which is a compound GC1, a compound GC2, a compound GC3, a compound GC4, a compound GC5, a compound GC6, a compound GC7, a compound GC8, a compound GC9, a compound GC10, a compound GC11, a compound GC12, a compound GC13, a compound GC14, a compound GC15, a compound GC16, a compound GC17, or a pharmaceutically acceptable salt thereof, as shown in the following structure;

4. Liposome nanoparticle composed of carbohydrate cholesterol, characterized by comprising the carbohydrate cholesterol structure according to any one of claims 1-2 and a cationic lipid, an auxiliary lipid and a steroid, wherein the molar ratio between the cationic lipid, the auxiliary lipid, the steroid and the carbohydrate cholesterol is 30-60:5-25:25-50:0.05-10.

5. The liposomal nanoparticle of carbohydrate cholesterol of claim 4, wherein the cationic lipid is selected from the group consisting of compound IL1, compound IL2, compound IL3, compound IL4, and pharmaceutically acceptable salts thereof, as shown in the following structure;

6. The liposome nanoparticle of carbohydrate cholesterol according to claim 4, wherein the auxiliary lipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, carbohydrate cholesterol, and lipid.

7. The liposomal nanoparticle of carbohydrate cholesterol of claim 4, wherein the steroid comprises at least one of cholesterol, vitamin D, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, alpha tocopherol, corticosteroid.

8. Use of the liposome nanoparticle of any one of claims 4-7 for the preparation of an in vivo delivery of a small molecule therapeutic.

9. A composition comprising the liposome nanoparticle of any one of claims 4 to 7 loaded with an active agent comprising at least one of a prophylactic agent, a therapeutic agent, a nucleic acid, an immunomodulator, an antigen or a fragment thereof, a vaccine, an anti-inflammatory agent, an anti-tumour agent, an antibiotic, an agent acting on the central nervous system, a protein, a peptide, a polypeptide, a small molecule drug, or a mixture thereof;

The nucleic acid is at least one selected from messenger RNA, ribosomal RNA, micro RNA, transfer RNA, small interfering RNA, small nuclear RNA, antisense oligonucleotide, DNA and plasmid.

10. Use of a composition according to claim 9 in the manufacture of a medicament for the treatment or prophylaxis of infectious diseases, cancer, rare diseases, genetic diseases, liver or spleen diseases.