CN118791544A - An amphiphilic nucleoside lipid compound and its preparation method and application - Google Patents

Abstract

The invention discloses an amphiphilic nucleoside lipid compound and a preparation method and application thereof. The amphiphilic nucleoside lipid compound is a nucleoside lipid compound formed on the basis of phenylboronic acid lipids, has higher loading efficiency, higher transfection efficiency and lower toxicity, thereby producing a highly effective therapeutic effect; the preparation method is simple and suitable for large-scale production and supply; a lipid composition constructed by using the amphiphilic nucleoside lipid compound of the invention can improve the safety problem of the lipid composition caused by ionizable lipids while ensuring the gene transfection efficiency, and has broad application prospects in the field of gene drug delivery.

Description

An amphiphilic nucleoside lipid compound and its preparation method and application

Technical Field

The invention relates to an amphiphilic nucleoside lipid compound and also relates to a preparation method and application of the amphiphilic nucleoside lipid compound.

Background Art

siRNA drugs have unique advantages in disease treatment due to their accuracy, effectiveness and safety, but this type of drug itself has certain application limitations. First, they have poor stability and are easily degraded and inactivated by RNA enzymes in the blood or tissues; second, they have a strong negative charge and a large molecular polarity, making it difficult to pass through the lipophilic cell membrane that also has a negative charge, resulting in low cell uptake efficiency. Nanoformulations can just make up for the shortcomings of siRNA drugs. In nano-delivery systems, lipid nanoparticles (LNPs) are widely used in the delivery of siRNA due to their good nucleic acid encapsulation rate and transfection efficiency, low cytotoxicity and immunogenicity. With the launch of Patisiran and the authorization of the two new crown vaccines BNT162b2 and mRNA-1273, LNP has become the only gene drug carrier currently used in clinical practice.

In LNP, the key component is ionizable lipid, which is the key to LNP loading gene drugs. By structurally designing the ionizable lipid, the pKa can be controlled and made to be electrically neutral under physiological pH conditions. After being taken up by cells and entering the acidic lysosomal environment, it carries a positive charge, which is conducive to lysosomal escape. Although ionizable lipids have good safety, in clinical practice, LNP still relies on the pre-use of glucocorticoids and antihistamines before use to reduce adverse reactions. As a key material that causes LNP immune response and long-term toxicity, ionizable lipids have a lot of room for optimization.

The existing strategies for modifying nucleic acid-loaded lipids mainly include: (1) changing the number and length of lipid chains at the tail of ionizable lipids; (2) changing the unsaturation of lipid chains at the tail of ionizable lipids; (3) adding or changing new groups to the structure of marketed ionizable lipids, mostly adding disulfide bonds, carbonate bonds, and benzene rings. However, most of them currently focus only on the loading efficiency and lysosomal escape effect of ionizable lipids, and research on their safety is particularly scarce.

Summary of the invention

Purpose of the invention: The purpose of the present invention is to provide an amphiphilic nucleoside lipid compound, and also to provide a method for preparing the above-mentioned amphiphilic nucleoside lipid compound and its application in preparing lipid compositions.

Technical solution: The amphiphilic nucleoside lipid compound of the present invention has the following structural formula: ;

Wherein, _R1 or _R2 is a saturated or unsaturated/oxygen-containing or oxygen-free alkyl chain with 1-18 carbon atoms; X is adenine A, guanine G, cytosine C, thymine T, uracil U, and one of the rare bases and their derivatives produced by methylation, acetylation, hydrogenation, fluorination or sulfidation of the above bases.

Among them, X is , , , or and one of its derivatives.

Wherein, in R ₁ or R ₂ , the oxygen-containing alkyl chain contains 1 to 3 ester bonds.

Wherein, _R1 or _R2 is , , , , or and its derivatives.

Wherein, the amphiphilic nucleoside lipid compound comprises the following structure: , , , , , , , , , .

The method for preparing the above-mentioned amphiphilic nucleoside lipid compound comprises the following steps:

(1) Halogenated phenylboronic acid reacts with carboimide to prepare intermediate compound 1: ;

(2) The intermediate compound 1 undergoes esterification reaction with nucleoside to obtain the product.

The above-mentioned amphiphilic nucleoside lipid compounds can also be used in the preparation of lipid compositions.

The lipid composition is a liposome, a lipid nanoparticle or a micelle, and is composed of an amphiphilic nucleoside lipid, a lipid material and a loaded drug; the mass ratio of the amphiphilic nucleoside lipid to the lipid material is 1:20 to 10:1.

Wherein, the loaded drug includes one or more of gene drugs, small molecule compounds, polypeptides or proteins; the gene drugs are one or more of siRNA, miRNA, mRNA, ASO, cricRNA, ssRNA, shRNA or ssDNA; the nitrogen-phosphorus ratio (N/P) of the lipid composition to the gene drug is 2-128; the small molecule compound is a compound with preventive/therapeutic effect extracted from animals, plants, minerals, fungal fermentation products or artificially synthesized.

Wherein, the lipid material is dioleoyl phosphatidylcholine, hydrogenated soybean phosphatidylglycerol, egg phosphatidylglycerol, egg phosphatidylinositol, hydrogenated soybean phosphatidylethanolamine, phosphatidylethanolamine, soybean phosphatidylcholine, soybean phosphatidylglycerol, soybean phosphatidylserine, soybean phosphatidylinositol, hydrogenated soybean phosphatidylserine, soybean phosphatidylethanolamine, soybean phosphatidic acid, hydrogenated egg phosphatidylcholine, hydrogenated egg phosphatidylglycerol, egg phosphatidylserine, hydrogenated egg phosphatidylinositol, hydrogenated egg phosphatidylserine, hydrogenated phosphatidylethanolamine, hydrogenated Phosphatidic acid, hydrogenated soybean phosphatidylcholine, hydrogenated soybean phosphatidylinositol, hydrogenated soybean phosphatidic acid, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylinositol, egg phosphatidylcholine, dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylcholine, distearoyl phosphatidylglycerol, phosphatidic acid, dioleyl phosphatidyl-ethanolamine, palmitoyl stearoyl phosphatidylcholine, dipalmitoyl phosphatidic acid, palmitoyl stearoyl phosphatidylglycerol, monooleoyl-phosphatidylethanolamine, tocopherol, fat Ammonium salts of acids, ammonium salts of phospholipids, ammonium salts of glycerides, dilauroylethylphosphocholine, distearoylphosphatidylserine, dimyristoylethylphosphocholine, dipalmitoylethylphosphocholine and distearoylethylphosphocholine, N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride, 1,2-bis(oleoyloxy)-3-(trimethylammonium)propane, distearoylphosphatidylglycerol, dimyristoylphosphatidic acid, distearoylphosphatidic acid, dimyristoylphosphatidylcholine One or more of alcohol, dipalmitoylphosphatidyl inositol, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, dimyristoyl polyethylene glycol, myristoyl lysolecithin, palmitoyl lysolecithin, stearoyl lysolecithin, distearoyl phosphatidylethanolamine-polyethylene glycol, polyethylene glycol distearoylphosphatidylethanolamine-folic acid, phosphatidylcholine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylcholine-polyethylene glycol, cholesterol, lanosterol, sitosterol, stigmasterol, and ergosterol.

Among them, the composition adopts the preparation method including liposome extrusion method, thin film hydration method, nanoprecipitation method, microfluidics, impact jet mixing method, thin film dispersion method, reverse phase evaporation method, multiple emulsion method and solvent injection method, hot melt method, freeze drying method, spray drying method, supercritical reverse phase evaporation method, dialysis method, solvent volatilization method or freeze drying method.

Principle of the invention: The amphiphilic nucleoside lipid compound of the present invention retains the tertiary amine structure while forming a core lipid based on phenylboronic acid lipids, making full use of the amphiphilicity and gene delivery ability of the core lipids, interacting with nucleic acids through hydrogen bonds and π-π stacking, thereby enhancing the loading of gene drugs; coupled with its endogenous component composition, the toxicity of the core lipids is further reduced relative to ionizable lipids; at the same time, the delivery material obtained by modification with phenylboronic acid has strong flexibility, the boric acid group can react with cis-vicinal dihydroxy compounds, the reaction is rapid and the conditions are mild, and nucleosides are natural cis-vicinal dihydroxy compounds, and phenylboronic acid is used as a spacer group to connect ionizable lipids and nucleosides, which can simply and efficiently prepare core lipids. The obtained core lipids retain the electrostatic interaction between ionizable lipids and nucleic acids, and have non-electrostatic interactions between nucleosides and nucleic acids, so that the gene loading capacity of the material is improved.

Therefore, the core lipid compound formed on the basis of phenylboronic acid lipids is a simple, low-toxic, flexible carrier material with good gene loading efficiency. LNP constructed with this material is expected to improve the safety issues brought by ionizable lipids to LNP while ensuring the gene transfection efficiency, and is expected to become an ideal system for gene drug delivery.

Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The amphiphilic nucleoside lipid compound of the present invention has higher loading efficiency, higher transfection efficiency and lower toxicity, thereby producing a highly effective therapeutic effect and having broad application prospects in the field of gene drug delivery; (2) The preparation method is simple, the raw materials are easily available, the reaction steps are few, and it is suitable for large-scale production and supply.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a synthetic route of the amphiphilic lipid in Example 1;

FIG. 2 is a synthetic route of the amphiphilic nucleoside lipids in Example 1;

Figure 3 is a hydrogen nuclear magnetic resonance spectrum of the amphiphilic lipid compound in Example 1, wherein A is the nuclear magnetic resonance spectrum of the 4-(bromomethyl)phenylboronic acid raw material, B is the nuclear magnetic resonance spectrum of the dinonylamine raw material, and C is the nuclear magnetic resonance spectrum of the amphiphilic lipid Dna-PBA;

FIG4 is a diagram showing the particle size (A) and potential distribution (B) of lipid nanoparticles in Example 3;

FIG5 is a cryo-scanning electron micrograph of lipid nanoparticles in Example 3;

FIG6 is a statistical diagram of particle size and PDI changes of lipid nanoparticles during storage at 4° C. in Example 2;

FIG7 is a graph showing the stability results of lipid nanoparticles co-incubated with plasma in Example 3;

FIG8 is a graph showing the results of investigating the cytotoxicity of the products and lipid nanoparticles in Examples 1 to 3;

FIG9 is a graph showing the results of investigating the cell transfection effect of lipid nanoparticles in Example 4;

FIG. 10 is the NMR spectrum of the intermediate product of PBA-402 in Example 1.

DETAILED DESCRIPTION

The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

Example 1

The preparation process of an amphiphilic lipid is as follows (the synthetic route is shown in Figure 1): the amphiphilic lipid dinonylamine-phenylboronic acid (Dna-PBA) is synthesized by the substitution reaction of bromomethyl and secondary amine. First, dinonylamine (2 mmol, 539.02 mg) and 4-(bromomethyl)phenylboronic acid (1 mmol, 214.85 mg) are dissolved in 5 mL of anhydrous DMF, and Na ₂ CO ₃ (1 mmol, 105.99 mg) is added as an acid binding agent. The reaction is stirred for 12 h under 70°C oil bath conditions. After the reaction, DMF is removed by negative pressure of an oil pump, the crude product is redissolved with 2 mL of CHCl ₃ , and purified by column chromatography to obtain a slightly yellow semi-solid product (340.44 mg, 84.4%). The structure of the collected final product (Dna-PBA) was confirmed by nuclear magnetic resonance hydrogen spectrum.

The amphiphilic nucleoside lipid compound of the present invention is prepared as follows (the synthetic route is shown in FIG2 ):

Take Dna-PBA (806.72 mg, 2 mmol) in Example 1 and dissolve it in 10 mL of a mixed solution of tetrahydrofuran and methanol (1:1), and place it in a 37°C water bath with stirring; dissolve adenosine (Ado, 2.5 mmol, 668.10 mg) in 10 mL of a dilute hydrochloric acid solution with a pH of 2.0, and add it dropwise to the Dna-PBA solution, continue the reaction for 1 hour, remove the organic solvent by rotary evaporation, and remove the remaining water by freeze drying to obtain the product Dna-PBA-Ado (1142.41 mg, 90.0%).

Preparation of PBA-301: Dna-PBA (806.72 mg, 2 mmol) in Example 1 was dissolved in 10 mL of a mixed solution of tetrahydrofuran and methanol (1:1), and stirred in a 37°C water bath; Guanosine (2.5 mmol, 708.10 mg) was dissolved in 10 mL of a pH 2.0 dilute hydrochloric acid solution, and added dropwise to the Dna-PBA solution, and the reaction was continued for 1 h. The organic solvent was removed by rotary evaporation, and the remaining water was removed by freeze drying to obtain the product PBA-301 (1107.44 mg, 85.1%);

.

Preparation of PBA-302: Dna-PBA (806.72 mg, 2 mmol) in Example 1 was dissolved in 10 mL of a mixed solution of tetrahydrofuran and methanol (1:1), and stirred in a 37°C water bath; uridine (2.5 mmol, 610.50 mg) was dissolved in 10 mL of a pH 2.0 dilute hydrochloric acid solution, and added dropwise to the Dna-PBA solution, and the reaction was continued for 1 h. The organic solvent was removed by rotary evaporation, and the remaining water was removed by freeze drying to obtain the product PBA-302 (982.28 mg, 80.3%);

.

Preparation of PBA-303: Dna-PBA (806.72 mg, 2 mmol) in Example 1 was dissolved in 10 mL of a mixed solution of tetrahydrofuran and methanol (1:1), and stirred in a 37°C water bath; cytosine nucleoside (Citicoline, 2.5 mmol, 608.05 mg) was dissolved in 10 mL of a pH 2.0 dilute hydrochloric acid solution, and added dropwise to the Dna-PBA solution, and the reaction was continued for 1 h. The organic solvent was removed by rotary evaporation, and the remaining water was removed by freeze drying to obtain the product PBA-303 (1014.90 mg, 83.1%);

.

Preparation of PBA-304: Dna-PBA (806.72 mg, 2 mmol) in Example 1 was dissolved in 10 mL of a mixed solution of tetrahydrofuran and methanol (1:1), and stirred in a 37°C water bath; Pseudouricdine (2.5 mmol, 610.50 mg) was dissolved in 10 mL of a pH 2.0 dilute hydrochloric acid solution, and added dropwise to the Dna-PBA solution, and the reaction was continued for 1 h. The organic solvent was removed by rotary evaporation, and the remaining water was removed by freeze drying to obtain the product PBA-304 (1081.36 mg, 88.4%).

.

Preparation of PBA-401: First, dissolve dioctadecylamine (2 mmol, 1043.98 mg) and 4-(bromomethyl)phenylboronic acid (1 mmol, 214.85 mg) in 5 mL of anhydrous DMF, add Na ₂ CO ₃ (1 mmol, 105.99 mg) as an acid-binding agent, and stir the reaction in an oil bath at 70°C for 24 h. After the reaction, remove DMF with negative pressure from an oil pump, redissolve the crude product with 2 mL of CHCl ₃ , and purify it by column chromatography to obtain a slightly yellow semisolid product (428.33 mg, 65.3%). The above-mentioned synthetic product (2 mmol, 1311.88 mg) was dissolved in 10 mL of a mixed solution of tetrahydrofuran and methanol (1:1), and stirred in a 37°C water bath; adenosine (Ado, 2.5 mmol, 668.10 mg) was dissolved in 10 mL of a pH 2.0 dilute hydrochloric acid solution, and added dropwise to the solution of the synthetic product, and the reaction was continued for 1 h. The organic solvent was removed by rotary evaporation, and the remaining water was removed by freeze drying to obtain the product PBA-401 (1298.80 mg, 73.2%);

.

Preparation of PBA-402: First, ditetradecylamine (2 mmol, 819.54 mg) and 4-(bromomethyl)phenylboronic acid (1 mmol, 214.85 mg) were dissolved in 5 mL of anhydrous DMF, and Na ₂ CO ₃ (1 mmol, 105.99 mg) was added as an acid-binding agent. The mixture was stirred in an oil bath at 70°C for 18 h. After the reaction, DMF was removed by negative pressure of an oil pump, and the crude product was redissolved with 2 mL of CHCl ₃ and purified by column chromatography to obtain a slightly yellow semisolid product (427.37 mg, 78.6%). The above-mentioned synthetic product, namely the intermediate product PBA-402 (2 mmol, 1087.46 mg), was dissolved in 10 mL of a mixed solution of tetrahydrofuran and methanol (1:1), and stirred in a 37°C water bath; adenosine (Ado, 2.5 mmol, 668.10 mg) was dissolved in 10 mL of a pH 2.0 dilute hydrochloric acid solution, and added dropwise to the solution of the synthetic product, and the reaction was continued for 1 h. The organic solvent was removed by rotary evaporation, and the remaining water was removed by freeze drying to obtain the product PBA-402 (1258.50 mg, 81.2%);

.

Preparation of PBA-403: First, dissolve didodecylamine (2 mmol, 707.34 mg) and 4-(bromomethyl)phenylboronic acid (1 mmol, 214.85 mg) in 5 mL of anhydrous DMF, add Na ₂ CO ₃ (1 mmol, 105.99 mg) as an acid-binding agent, and stir the reaction in an oil bath at 70°C for 18 h. After the reaction, remove DMF with negative pressure from an oil pump, redissolve the crude product with 2 mL of CHCl ₃ , and purify it by column chromatography to obtain a slightly yellow semisolid product (381.81 mg, 78.3%). The above-mentioned synthetic product (2 mmol, 975.24 mg) was dissolved in 10 mL of a mixed solution of tetrahydrofuran and methanol (1:1), and stirred in a 37°C water bath; adenosine (Ado, 2.5 mmol, 668.10 mg) was dissolved in 10 mL of a dilute hydrochloric acid solution of pH 2.0, and added dropwise to the solution of the synthetic product, and the reaction was continued for 1 h. The organic solvent was removed by rotary evaporation, and the remaining water was removed by freeze drying to obtain the product PBA-403 (1213.40 mg, 84.4%);

.

Preparation of PBA-502: First, 3,3'-iminodipropionic acid (2 mmol, 322.32 mg) and 4-(bromomethyl)phenylboronic acid (1 mmol, 214.85 mg) were dissolved in 5 mL of anhydrous DMF, triethylamine (1 mmol, 101.19 mg) was added, and the mixture was stirred in an oil bath at 50°C for 24 h. After the reaction, the mixture was purified by column chromatography and re-dissolved in 5 mL of DMF. 5-Hydroxyvaleric acid (2 mmol, 236.26 mg), EDCI (2 mmol, 383.40 mg) and DMAP (2 mmol, 244.34 mg) were added. The mixture was reacted in a water bath at 40°C for 24 h. After the reaction, the mixture was purified by column chromatography and re-dissolved in 5 mL of DMF. Excess n-butanol, EDCI (2 mmol, 383.4 mg) and DMAP (2 mmol, 244.34 mg) were added. The oil pump was dried and then purified by CHCl _3. Redissolve and purify by column chromatography to obtain an oily product (342.04 mg, 56.3%). Take the above synthetic product (2 mmol, 1215.10 mg) and dissolve it in 10 mL of a mixed solution of tetrahydrofuran and methanol (1:1), and place it in a 37°C water bath for stirring; dissolve adenosine (Adenosine, Ado, 2.5 mmol, 668.10 mg) in 10 mL of a pH 2.0 dilute hydrochloric acid solution, and add it dropwise to the solution of the synthetic product, continue the reaction for 1 hour, remove the organic solvent by rotary evaporation, and remove the remaining water by freeze drying to obtain the product PBA-502 (1296.72 mg, 77.3%);

.

Preparation of PBA-504: First, 3,3'-iminodipropionic acid (2 mmol, 322.32 mg) and 4-(bromomethyl)phenylboronic acid (1 mmol, 214.85 mg) were dissolved in 5 mL of anhydrous DMF, triethylamine (1 mmol, 101.19 mg) was added, and the reaction was stirred in an oil bath at 50°C for 24 h. After the reaction, column chromatography was used for purification and 5 mL of DMF was used for redissolution; n-decanol (2 mmol, 316.56 mg), EDCI (2 mmol, 383.4 mg) and DMAP (2 mmol, 244.34 mg) were added. After the reaction, column chromatography was used for purification and the oily product (409.27 mg, 71.1%) was obtained after the oil pump was used for drying. The above-mentioned synthetic product (2 mmol, 1151.28 mg) was dissolved in 10 mL of a mixed solution of tetrahydrofuran and methanol (1:1), and placed in a 37°C water bath with stirring; adenosine (Ado, 2.5 mmol, 668.10 mg) was dissolved in 10 mL of a pH 2.0 dilute hydrochloric acid solution, and added dropwise to the solution of the synthetic product, and the reaction was continued for 1 h. The organic solvent was removed by rotary evaporation, and the remaining water was removed by freeze drying to obtain the product PBA-504 (1126.36 mg, 69.8%);

.

Example 2

Prescription composition:

;

Preparation process: LNP was prepared by nanoprecipitation method. Accurately weigh 5 times the amount of Dna-PBA, DOPC, cholesterol, and DMG-PEG, add 0.5 mL of tetrahydrofuran (THF), and dissolve in water bath ultrasound as the organic phase. Take about 30 μg of siRNA, add 60 μL of DEPC water to dissolve, and then add 840 μL of pH 6.8 HEPES buffer as the aqueous phase. Under vigorous magnetic stirring, add 0.1 mL of organic phase dropwise into the aqueous phase. After stirring evenly, remove the residual THF by rotary evaporation to obtain PBA@LNP-siRNA.

Example 3

Prescription composition:

;

Preparation process: LNP was prepared by microfluidic process. Accurately weigh the prescribed amount of Dna-PBA-Ado, DOPC, cholesterol, and DMG-PEG, add 300 μL of tetrahydrofuran: methanol (1:1) and sonicate until completely dissolved. Take about 100 μg of siRNA, add 60 μL of DEPC water to dissolve, and then add 840 μL of pH 6.8 HEPES buffer as the aqueous phase. Add the corresponding solutions to the two-phase injection port of the microfluidic control, mix the aqueous phase and the organic phase evenly through the program setting, dilute the preparation obtained at the collection port 40 times with PBS, remove the organic phase by ultrafiltration, and obtain Ado@LNP-siRNA.

Example 4

Prescription composition:

;

Preparation process: LNP was prepared by nanoprecipitation method. Five times the amount of Dna-PBA-Ado, DOPC, cholesterol, and DMG-PEG were accurately weighed, and 0.5 ml of methanol: tetrahydrofuran (1:1) was added and ultrasonicated until completely dissolved as the organic phase; 0.9 mL of pH 6.8 HEPES Buffer was added to a 10 mL flask as the aqueous phase, 0.03 mg of siRNA was added under stirring and continued to stir for 1 min, and then 100 μL of organic phase was added and continued to stir for 5 min. Then, AMD3100 methanol solution was added and continued to stir for 3 min. Finally, the organic solvent was removed by vacuum rotary evaporation to obtain Ado/AMD@LNP-siRNA.

The preparation prepared in Example 3 was taken, and the particle size and potential were detected using a Malven Zetasizer. As shown in Figure 4, nearly spherical lipid nanoparticles with a small particle size and uniform PDI were prepared, with a particle size of about 130 nm and a PDI of less than 0.3. Figure 5 is a cryo-scanning electron micrograph of the prepared preparation.

The preparation prepared in Example 2 was placed in an environment at 4° C., and the changes in the particle size and PDI of the preparation were observed.

Figure 6 shows the changes in particle size and PDI of LNP. It can be seen that lipid nanoparticles have high stability at 4°C and have a strong protective effect on the loaded nucleic acid drugs and chemical drugs.

The preparation prepared in Example 3 was mixed with plasma and placed in a 37°C air bath. After incubation with plasma for 0 h, 0.5 h, 1 h, 2 h, 4 h, and 6 h, SDS solution was added, mixed, and placed in a 60°C water bath for 5 min to inactivate the enzyme in the plasma. Then, heparin sodium solution was added, mixed evenly, and incubated at room temperature for 10 min to extract siRNA from the preparation, and then the samples were subjected to nucleic acid electrophoresis.

FIG7 shows that the brightness of the free siRNA band displaced from the preparation is close to that at 0 h, indicating that the preparation can effectively isolate the loaded siRNA from the enzymes in the plasma within 6 h and maintain the stability of the nucleic acid drug.

Dna-PBA, PBA@LNP-siRNA, and Ado@LNP-siRNA were taken for cytotoxicity detection, and the cytotoxicity was determined by MTT method. The AML-12 cells with good growth status were digested, centrifuged, counted, and resuspended with fresh culture medium. They were inoculated in 96-well cell culture plates at a cell density of 5×10 ³ /well. Later, 180 μL of culture medium containing different concentrations of Dna-PBA, PBA@LNP-siRNA, and Ado@LNP-siRNA were added, and a blank solvent group was set as a control. Five replicate wells were set for each sample and placed in a cell culture incubator for incubation for 48 h. 20 μL of 5 mg/mL MTT solution was added to each well. After continuing to incubate in the incubator for 4 h, the culture medium in the well was discarded, 200 μL of DMSO was added, and the well was placed on a shaker for 10 min. After shaking and dissolving, the absorbance at 570 nm was measured using a microplate detection system. The cell survival rate was calculated according to the following formula to evaluate the cytotoxicity of the preparation to normal stem cells;

.

Figure 8 shows that with increasing doses, Ado@LNP-siRNA has lower toxicity compared with Dna-PBA or PBA@LNP-siRNA.

Preparation of fluorescently labeled Ado/AMD@LNP-FAM-siRNA: After scraping RAW264.7 cells in the logarithmic growth phase with a cell scraper, the cells were collected, counted, and inoculated in a confocal dish at a density of 1×10 ⁶ / dish. After the cells adhered to the wall, Ado/AMD@LNP-FAM-siRNA encapsulated with fluorescently labeled siRNA was administered, and the final concentration of siRNA was 50nM. After incubation for 1h or 4h, the drug-containing medium was aspirated, the cells were washed twice with PBS, and 1mL of blank medium solution containing 50nM Lyso-Tracker Red was added to each well, and incubated at 37℃ for 35min. The cells were washed twice with PBS, and 0.5mL of 4% paraformaldehyde solution was added to fix the cells at room temperature for 20min. The paraformaldehyde solution was aspirated, the cells were washed twice with PBS, and then 100μL of DAPI staining solution was added to each sample for staining for 10min, and then the cells were washed twice with PBS. Add 200 μL PBS to the confocal dish and place it under CLSM to collect multi-channel images.

Figure 9 shows that the prepared LNPs have good transfection efficiency and good lysosomal escape performance.

Claims (10)

1. An amphiphilic nucleoside lipid compound characterized by the following structural formula:；

Wherein R ₁ or R ₂ is a saturated or unsaturated/oxygen-containing or oxygen-free alkyl chain having 1 to 18 carbon atoms;

x is one of rare bases and derivatives thereof generated by methylation, acetylation, hydrogenation, fluorination or sulfuration of adenine A, guanine G, cytosine C, thymine T and uracil U.

2. The amphiphilic nucleoside lipid compound according to claim 1, wherein X is、、、Or (b)。

3. The amphiphilic nucleoside lipid compound of claim 1, wherein in R ₁ or R ₂, the oxygen-containing alkyl chain contains 1 to 3 ester linkages.

4. The amphiphilic nucleoside lipid compound of claim 1, wherein R ₁ or R ₂ is、、、、Or (b)。

5. A method for preparing an amphiphilic nucleoside lipid compound according to claim 1, comprising the steps of:

(1) Halogenated phenylboronic acid and carbon chain imine undergo halogenation reaction to prepare an intermediate compound 1: ；

(2) The intermediate compound 1 and nucleoside are subjected to esterification reaction to obtain the compound.

6. Use of an amphiphilic nucleoside lipid compound according to claim 1 for the preparation of a lipid composition.

7. The use according to claim 6, wherein the lipid composition is a liposome, a lipid nanoparticle or a micelle, consisting of an amphiphilic nucleoside lipid, a lipid material and a drug-loaded substance; the mass ratio of the amphiphilic nucleoside lipid to the lipid material is 1:20-10:1.

8. The use of claim 7, wherein the drug-loading comprises one or more of a genetic drug, a small molecule compound, a polypeptide, or a protein, wherein the genetic drug is one or more of siRNA, miRNA, mRNA, ASO, cricRNA, ssRNA, shRNA or ssDNA.

9. The use according to claim 7, wherein, the lipid material is selected from the group consisting of di-oleoyl lecithin, hydrogenated soybean phosphatidylglycerol, phosphatidylinositol, hydrogenated soybean phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, hydrogenated soybean phosphatidylserine, soybean phosphatidylethanolamine, soybean phosphatidic acid, hydrogenated phosphatidylcholine, hydrogenated phosphatidylglycerol, phosphatidylserine, hydrogenated phosphatidylinositol, hydrogenated phosphatidylserine, hydrogenated phosphatidylethanolamine, hydrogenated phosphatidic acid, hydrogenated soybean phosphatidylcholine, hydrogenated soybean phosphatidylinositol, hydrogenated soybean phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylinositol, lecithin, dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylcholine, distearoyl phosphatidylglycerol, phosphatidic acid, dioleyl phosphatidyl-ethanolamine, palmitoyl stearoyl phosphatidylcholine, dipalmitoyl phosphatidic acid, palmitoyl stearoyl phosphatidylglycerol, monooleoyl-phosphatidylethanolamine, tocopherols, ammonium salts of fatty acids, ammonium salts of phospholipids, ammonium salts of glycerides, dilauryl ethyl phosphorylcholine, distearoyl phosphatidylserine, dimyristoyl ethyl phosphorylcholine, dipalmitoyl ethyl phosphorylcholine and distearoyl ethyl phosphorylcholine, N- (2, 3-di- (9- (Z) -octadecenyloxy) -prop-1-yl-N, N, N-trimethyl ammonium chloride, 1, 2-bis (oleoyloxy) -3- (trimethylammonium) propane, distearoyl phosphatidylglycerol, dimyristoyl phosphatidic acid, distearoyl phosphatidic acid, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, dimyristoyl phosphatidylserine, dimyristoyl polyethylene glycol, myristoyl lysolecithin, palmitoyl lysolecithin, stearoyl lysolecithin, distearoyl phosphatidylethanolamine-polyethylene glycol, polyethylene glycol distearoyl phosphatidylethanolamine-folic acid, phosphatidylcholine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol, distearoyl phosphatidylcholine-polyethylene glycol, cholesterol, lanosterol, sitosterol, stigmasterol, ergosterol, or one or more of the following.

10. The use according to claim 7, wherein the composition is prepared by a method comprising liposome extrusion, film hydration, nano precipitation, micro flow control, impact jet mixing, film dispersion, reverse phase evaporation, re-emulsion and solvent injection, hot melt, freeze drying, spray drying, supercritical reverse phase evaporation, dialysis, solvent evaporation or lyophilization.