A liver-targeted drug liposome and its application

Abstract

The present invention discloses a liver-targeted drug liposome and its application, which relates to the field of drug delivery, and uses bile salt and mannose to modify the phospholipid molecule DSPE-PEG respectively. 2000 , two raw materials DSPE-PEG composed of synthetic liposomes 2000 ‑CA and DSPE‑PEG 2000 After ‑MAN, liver-targeted liposomes were obtained with cholesterol, DSPC, DC‑cholesterol and water-soluble therapeutic drugs in a certain proportion. The liposomes have high cell safety and liver targeting, and can significantly improve the therapeutic effect of the corresponding drugs after encapsulating the drugs.

Description

Technical Field

[0001] The invention relates to the field of drug delivery, and in particular to a liver-targeted drug liposome and application thereof. Background Art

[0002] Sodium Acetate (chemical formula CH3COONa) is a white crystal or powder formed by sodium acetate salt, which is easily soluble in water. It is widely used in food, industry and medicine, such as as a preservative, flavoring agent in food, and a component of buffer solution.

[0003] In recent years, studies have found that sodium acetate may have a certain therapeutic effect on non-alcoholic fatty liver disease (NAFLD). The main reasons are as follows: Improve lipid metabolism: Sodium acetate, as a short-chain fatty acid, will be converted into acetic acid after entering the body. Acetic acid can promote lipolysis by activating adenylate-activated protein kinase (AMPK), inhibit the accumulation of liver fat, and reduce the synthesis of liver lipids. Antioxidant effect: Sodium acetate may protect liver cells by inhibiting oxidative stress and inflammatory response, thereby reducing liver cell damage and helping to reduce the progression of fatty liver. Regulate intestinal flora: Sodium acetate can promote the growth of beneficial bacteria, thereby regulating the intestinal microbiome and helping to improve the metabolic state of the liver. The balance of intestinal flora helps to reduce the accumulation of fat in the liver. Although sodium acetate has shown a certain anti-fatty liver effect in cell or animal experiments, its clinical application in the treatment of non-alcoholic fatty liver is still in the early research stage.

[0004] Sodium acetate relieves NAFLD by improving lipid metabolism and inhibiting inflammatory responses. However, the effect of direct oral ingestion of sodium acetate or acetic acid in relieving NAFLD is very limited. The main reason is that after sodium acetate is absorbed by the small intestine, it is transmitted to various tissues and organs throughout the body through the portal vein. The sodium acetate content that finally enters the liver is in the micromolar level, which is difficult to reach an effective therapeutic concentration. In addition, the half-life of oral sodium acetate is 20 minutes, indicating that sodium acetate has a short action time in the liver. Therefore, the effect of oral sodium acetate in relieving NAFLD is relatively limited.

[0005] To overcome the above problems, this study intends to encapsulate sodium acetate in liposomes double-modified with sodium cholate and mannose, and utilize the targeting of sodium cholate and mannose to hepatocytes and Kupffer cells to improve the enrichment efficiency of sodium acetate in the liver. At the same time, it prolongs the half-life of sodium acetate in the blood and significantly prolongs the action time of sodium acetate in the liver, thereby enhancing its effect in alleviating liver lipid accumulation.

[0006] DSPE-PEG 2000It is an amphiphilic (hydrophilic and lipophilic) molecule that is widely used in nano-drug carriers, drug delivery systems, and biomedical research. Its full name is 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000, where DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine) is a phospholipid with a hydrophobic fatty chain (stearic acid) part that can be inserted into the lipid bilayer. PEG2000 (Polyethylene Glycol2000) is a polyethylene glycol part with a molecular weight of 2000, which is hydrophilic and allows it to be stably suspended in water. DSPE-PEG 2000 Commonly used in the preparation of liposomes, nanoparticles and other drug delivery systems, PEG chains can prevent the immune system from recognizing and clearing the carrier, thus contributing to the "stealth" effect.

[0007] Sodium cholate is a hydrophilic bile acid that targets and binds to ASBT overexpressed on the surface of intestinal epithelial cells and NTCP receptors on the surface of hepatocytes, exerting receptor-mediated endocytosis through the bile acid-ASBT or bile acid-NTCP pathway, thereby enhancing the ability of nanoliposomes to penetrate the intestine and target the liver. Mannose can specifically bind to mannose receptors on the surface of liver macrophages, targeting liposomes to macrophages in the liver. Based on the above design, nanoliposomes that are stable in the gastrointestinal fluid environment, have intestinal penetration and liver targeting are prepared, and a high concentration of sodium acetate is loaded in the hydrophilic core of the liposome. After oral ingestion, the experimental purposes of intestinal penetration, liver targeting and high-concentration sodium acetate delivery are achieved. Summary of the invention

[0008] In order to solve the above technical problems, the present invention selects to modify sodium cholate and mannose on the surface of nanoliposomes to construct an oral delivery system with liver targeting.

[0009] Definition Notes:

[0010] DSPC: refers to 1,2-Distearoyl-sn-glycero-3-phosphocholine, also known as 1,2-distearoyl-sn-glycero-3-phosphocholine in Chinese, or distearoylphosphatidylcholine for short. It is a phospholipid commonly used in the preparation of liposomes and nano-drug delivery systems;

[0011] DSPE-PEG 2000-NH2: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)-2000]-ammonium, abbreviated as distearoyl phosphatidylethanolamine-polyethylene glycol-amine; this is a modified phospholipid commonly used in drug delivery systems (such as liposomes, nanoparticles, etc.), widely used in targeted drug delivery and gene therapy. Its amino group (NH2) can be used for covalent connection with other molecules or target ligands to improve targeting and therapeutic effects.

[0012] DSPE-PEG-CA: Distearoyl phosphatidylethanolamine polyethylene glycol-cholic acid. Since the PEG in the synthesis reaction is PEG2000, therefore, DSPE-PEG-CA appearing in the specification of the present invention is DSPE-PEG 2000 -CA;

[0013] DSPE-PEG-MAN: Distearoyl phosphatidylethanolamine polyethylene glycol-mannose. Since the PEG in the synthesis reaction is PEG2000, therefore, DSPE-PEG-MAN appearing in the specification of the present invention is DSPE-PEG 2000 -MAN.

[0014] DC-cholesterol (DC-Chol, 3β-[N-(N′,N′-dimethylaminoethyl)carbamoyl]cholesterol) is a cholesterol-based cationic lipid commonly used in gene transfection and delivery systems. Its structural feature is that the cholesterol molecule is modified into a derivative with a cationic amino group, thereby endowing it with the ability to bind to negatively charged nucleic acids (such as DNA or RNA).

[0015] Specifically:

[0016] The present invention first provides a drug liposome targeting the liver, which is characterized in that the liposome is formulated from DSPC, cholesterol, DSPE-PEG-CA, DSPE-PEG-MAN, DSPE-PEG-NH2, and DC-cholesterol in a ratio, wherein DSPE-PEG-CA is DSPE-PEG2000 modified with cholate, and DSPE-PEG-MAN is DSPE-PEG2000 modified with mannose; the drug liposome encapsulates a water-soluble drug solution for treating liver diseases; the particle size of the liposome is 94 ± 0.5 nm; 0.22 < PDI < 0.25; Zeta potential is 33 ± 4 mV; preferably, the particle size is 94.15 ± 0.23 nm, PDI is 0.23 ± 0.1, and Zeta potential is 33 ± 3.71 mV.

[0017] Furthermore, the weight ratio of each component is: 22.5%~44.25% DSPC, 12.5%~31.5% cholesterol, 1%~10% DSPE-PEG-CA, 1%~10 DSPE-PEG-MAN, 5% DSPE-PEG-NH2 and 30% DC-cholesterol.

[0018] Furthermore, the drug is a drug for treating non-alcoholic fatty liver disease, the liposome has an encapsulation rate of 76.6% to 81.57% for the drug, and a drug loading amount of 33.16% to 37.94%.

[0019] Furthermore, the drug is acetic acid, propionic acid butyric acid or an acceptable salt thereof; the drug embedding efficiency is 76.6±0.47%, and the drug loading is 33.16±0.16%.

[0020] The second aspect of the present invention provides the use of the liver drug liposomes described in the first aspect in the preparation of a preparation for treating liver diseases, wherein the liver disease is non-alcoholic fatty liver disease.

[0021] The third aspect of the present invention is to provide the use of the liposome of the liver-targeting drug described in the first aspect in the preparation of a preparation for inhibiting cellular fat accumulation, characterized in that the use is an in vitro application.

[0022] The fourth aspect of the present invention is to provide a method for preparing the liver-targeted liposome containing a medicinal ingredient as described in the first aspect, wherein the method comprises the following steps:

[0023] 1) Preparation of DSPE-PEG-CA;

[0024] 2) Preparation of DSPE-PEG-MAN;

[0025] 3) Add the following raw materials in sequence: DSPC, cholesterol, DSPE-PEG 2000 -CA, DSPE-PEG 2000 -MAN, DSPE-PEG 2000 -NH2, DC-cholesterol, mix well and transfer to a round-bottom flask with a micro glass syringe. After pre-mixing, shake well in a constant temperature shaker to make all reagents thermodynamically uniform. At this time, the solution is completely clear;

[0026] 4) High-pressure rotary evaporation to obtain a film with uniform thickness and distribution;

[0027] 5) adding a water-soluble drug solution to the film formed in step (4), and obtaining a solution by ultrasonication;

[0028] 6) Use a micro-glass syringe to draw the solution obtained in step 5) and squeeze it back and forth in a liposome extruder. Store the liposomes after extrusion at 4 o C or below overnight to allow the liposome particles to stabilize.

[0029] Furthermore, the operation of step 3) is that the pre-mixing time is 5 to 15 minutes, the constant temperature is 35 to 40° C., and the shaking time is 5 to 15 minutes;

[0030] Furthermore, the operation of step 4) is to perform rotary evaporation at a pressure of 400-600 mbar, a rotation speed of 80-120 rpm, and a temperature of 45-50°C;

[0031] Furthermore, the buffer solution in step 5) contains a drug to be delivered or a fluorescent marker;

[0032] Furthermore, the method further comprises removing unencapsulated drug to be delivered by ultrafiltration, and then quick-freezing the liposomes with liquid nitrogen;

[0033] Furthermore, the method further comprises quick-freezing the obtained liposomes with liquid nitrogen, and re-dissolving them with PBS before use;

[0034] Further; the preparation of DSPE-PEG 2000 -CA method steps are:

[0035] 1) Activated bile acid: dissolve bile acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide (EDC) and N-hydroxysuccinimide (NHS) in dichloromethane, react in an ice bath, stir at room temperature overnight, filter and rotary evaporate to obtain activated sodium cholate;

[0036] 2) DSPE-PEG 2000 -NH2 is dissolved in DMSO, and activated sodium cholate and triethylamine are added and reacted after they are completely dissolved;

[0037] 3) After the reaction is completed, the solution is placed in a dialysis bag and dialyzed using pure water as the medium;

[0038] 4) After dialysis, the solution in the dialysis bag was collected and freeze-dried to obtain DSPE-PEG-CA.

[0039] Preferably, step 1) the ice bath reaction is for 20 to 40 minutes;

[0040] Preferably, step 2) DSPE-PEG 2000 -NH2 and DMSO are added in a ratio of 450~550mg:3mL; activated sodium cholate and triethylamine are added in a ratio of 500~600mg:1g; the reaction temperature is 38~45℃ for 1~3h;

[0041] Preferably, the molecular sieve size of the dialysis bag is MW=800~1200 Da, more preferably 1000 Da; the dialysis time is 18~28h.

[0042] Further, the preparation of DSPE-PEG 2000 -MAN method steps are:

[0043] 1) DSPE-PEG 2000 -NH2 is dissolved in DMSO, mannose and triethylamine are added to completely dissolve, and mannose and triethylamine are reacted;

[0044] 2) After the reaction is completed, the solution is placed in a dialysis bag and dialyzed using pure water as the medium;

[0045] 3) After dialysis, the solution in the dialysis bag is freeze-dried to obtain DSPE-PEG-MAN.

[0046] Preferably, in step 1) DSPE-PEG 2000 -NH2 and DMSO are added in a ratio of 450~550mg:3mL; and mannose and triethylamine are added in a ratio of 500~600mg:1g; the reaction temperature is 38~45℃ for 1~3h;

[0047] Preferably, in step 2), the molecular sieve size of the dialysis bag is MW=800~1200 Da, more preferably 1000 Da; and the dialysis time is 18~28h.

[0048] Furthermore, the molar ratio of DSPC:cholesterol in the liposome is 3:1;

[0049] Further, in the liposomes, DSPE-PEG-CA and DSPE-PEG-MAN account for 0.5% to 1.5% and 5 to 15%, preferably, 1% and 10%;

[0050] Furthermore, the ratio of each component in the liposome is: 22.5% to 44.25% DSPC, 12.5% ​​to 31.5% cholesterol, 1% to 10% DSPE-PEG-CA, 1% to 10% DSPE-PEG-MAN and 30% DC-cholesterol; the preferred ratio is 40.5% DSPC, 13.5% cholesterol, 1% DSPE-PEG-CA, 10% DSPE-PEG-MAN, 5% DSPE-PEG and 30% DC-cholesterol.

[0051] Furthermore, the drug to be delivered is a water-soluble drug for treating liver-related diseases, preferably the drug to be delivered is a small molecule chemical drug; preferably, the drug is a small molecule chemical drug for treating non-alcoholic fatty liver disease; more preferably, the drug is sodium acetate; more preferably, the drug is sodium acetate, and the concentration of its storage solution is 20~60mg / mL, and most preferably, the drug to be delivered is sodium acetate, and the concentration of its storage solution is 40mg / mL.

[0052] The beneficial effects of the present invention include:

[0053] 1) First synthesis of two raw materials DSPE-PEG for liposomes 2000 -CA and DSPE-PEG 2000 -MAN, and then with cholesterol, DSPC, and DC-cholesterol in a certain ratio to obtain liver-targeted liposomes;

[0054] 2) The five liposomes synthesized by the present invention have no obvious toxicity to myocardial cells H9c2, intestinal epithelial cells Caco-2, liver macrophages Kupffer and hepatocytes AML-12, and are suitable as oral sodium acetate nanocarriers with high cell safety;

[0055] 3) The fluorescence intensity of sodium cholate / mannose dual-labeled fluorescent liposomes in the liver was significantly higher than that of single-labeled or unlabeled liposomes, indicating that the sodium cholate / mannose liposomes synthesized in this experiment are a sodium acetate oral delivery nanomaterial with storage stability, cell safety, intestinal epithelial barrier penetration and liver targeting;

[0056] 4) Sodium cholate / mannose dual-labeled liposomes containing sodium acetate effectively alleviated lipid deposition and cellular inflammation in NAFLD cells;

[0057] 5) After mice were gavaged with double-labeled sodium acetate liposomes, liver lipid deposition was significantly improved, and the phosphorylation level of liver AMPKα protein was significantly activated. By inhibiting the transcription of Acc1 and Srebf1, CPT1α expression was promoted, thereby regulating downstream lipid metabolism genes and effectively reducing liver lipid deposition. In terms of liver inflammation level and oxidative stress, after NAFLD mice were gavaged with double-labeled sodium acetate liposomes for 10 weeks, NFĸB protein phosphorylation was significantly inhibited, the release of downstream inflammatory factors was reduced, and liver inflammation and oxidative stress were effectively alleviated. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of the synthesis of sodium acetate liposomes modified with bile acid and mannose;

[0059] Figure 2 Synthesis of DSPE-PEG-CA, A is the synthesis route, B is the infrared spectrum;

[0060] Figure 3 Synthesis of DSPE-PEG-MAN, A is the synthesis route, B is the infrared spectrum;

[0061] Figure 4 Sodium acetate release of liposomes in different groups after incubation in serum, simulated gastric fluid, and simulated intestinal fluid in the orthogonal experiment; A: sodium acetate release in serum; B: sodium acetate release in simulated gastric fluid; C: sodium acetate release in simulated intestinal fluid; different letters indicate significant differences ( p <0.05, the same letters indicate no significant difference ( p >0.05);

[0062] Figure 5 Liposome embedding rate and drug loading at different sodium acetate stock solution concentrations, A: embedding rate; B: drug loading;

[0063] Figure 6 TEM images of five liposomes, scale bar is 200 nm;

[0064] Figure 7 Cell viability of four cell types after incubation with 1-1500 μM sodium acetate liposomes and sodium acetate solution for 24 h. A: H9c2 cells; B: Caco-2 cells; C: Kupffer cells; D: AML-12 cells;

[0065] Figure 8 Sodium acetate liposomes or sodium acetate alleviate lipid deposition in NAFLD cells A: Effects of different liposomes or sodium acetate on cell TG content; B: Effects of different liposomes or sodium acetate on cell TC content; C: Oil red O staining results of different groups; D: Effects of different liposomes or sodium acetate on the relative area of ​​lipid droplets in NAFLD cells; TG, TC and relative lipid droplet area were analyzed for significance, # indicates significant difference between the modeling group and the control group, ## p <0.01; * indicates significant difference between the experimental group and the modeling group, * p <0.05, ** p <0.01; + indicates significant difference between groups. + p <0.05, ++ p <0.01;

[0066] Fig. 9Effects of sodium acetate liposomes or sodium acetate on genes related to inflammatory response in NAFLD cells, A: relative expression of IL-1β mRNA; B: relative expression of IL-6 mRNA; C: relative expression of TNF-α mRNA; D: relative expression of IL-4 mRNA; # indicates significant difference between the modeling group and the control group, ## p <0.01; * indicates significant difference between the experimental group and the modeling group, * p <0.05, ** p <0.01; + indicates significant difference between groups. + p <0.05, ++ p <0.01;

[0067] Fig.10 In vivo fluorescence imaging of mice after oral administration of CY7 liposomes or CY7;

[0068] Fig.11 Fluorescence intensity of main organs in mice after oral administration of CY7 liposomes or CY7. A: Fluorescence imaging of each organ; B: Fluorescence intensity-time curve of the liver of mice in each group; C: AUG of the fluorescence intensity-time curve of the liver of mice 0-24 ; D: Fluorescence intensity-time curve of the intestine of each group of mice; E: AUG of the fluorescence intensity-time curve of the intestine of mice 2-24 ;

[0069] Fig.12 Effects of sodium acetate liposomes on body weight change and weight gain in NAFLD mice: A: body weight growth curve; B: body weight gain; # It means that the modeling group and the control group are significantly different. ## p <0.01; * It means that the difference between the experimental group and the modeling group is significant. * p <0.05, ** p <0.01, + Indicates significant differences between groups. + p <0.05;

[0070] Fig.13 Effects of sodium acetate liposomes on glucose tolerance in NAFDL mice

[0071] A: OGTT curve; B: area under the OGTT curve; # It means that the modeling group and the control group are significantly different. ## p<0.01; * It means that the difference between the experimental group and the modeling group is significant. ** p <0.01, + Indicates significant differences between groups. ++ p <0.01;

[0072] Fig.14 Effect of sodium acetate liposomes on blood lipid metabolism in NAFLD mice, A: triglycerides; B: cholesterol; C: low-density lipoprotein cholesterol; D: high-density lipoprotein cholesterol; # It means that the modeling group and the control group are significantly different. ## p <0.01; * It means that the difference between the experimental group and the modeling group is significant. * p <0.05, ** p <0.01, + Indicates significant differences between groups. + p <0.05, ++ p <0.01;

[0073] Fig.15 Effects of sodium acetate liposomes on adipose tissue content in NAFLD mice, A: white fat percentage; B: H&E staining results of epididymal adipose tissue (400×, scale bar 100 μm); # It means that the modeling group and the control group are significantly different. ## p <0.01; * It means that the difference between the experimental group and the modeling group is significant. * p <0.05, ** p <0.01, + Indicates significant differences between groups. ++ p <0.01;

[0074] Fig.16 Effects of sodium acetate liposomes on liver pathological morphology in NAFLD mice, A: liver appearance (scale bar 1 cm); B: H&E staining of liver tissue (100×, scale bar 200 μm); C: relative area ratio of liver lipid droplets based on H&E staining sections (n=8); D: Oil red O staining of liver tissue (100×, scale bar 200 μm); E: relative area ratio of liver lipid droplets based on Oil red O staining (n=8); # It means that the modeling group and the control group are significantly different. ## p<0.01; * It means that the difference between the experimental group and the modeling group is significant. ** p <0.01, + Indicates significant differences between groups. + p <0.05, ++ p <0.01;

[0075] Fig.17 Effects of sodium acetate liposomes on liver lipid deposition and oxidative stress in NAFLD mice, A: liver index (%); B: liver triglyceride content; C: liver cholesterol content; D: liver malondialdehyde content; E: liver superoxide dismutase content; # It means that the modeling group and the control group are significantly different. ## p <0.01; * It means that the difference between the experimental group and the modeling group is significant. * p <0.05, ** p <0.01; + Indicates significant differences between groups. + p <0.05, ++ p <0.01;

[0076] Fig.18 Effects of sodium acetate liposomes on the expression of genes related to lipid synthesis and metabolism and AMPK protein phosphorylation in the liver of NAFLD mice,AE: Relative mRNA expression of genes related to lipid synthesis and metabolism in the liver (Acc1, Fasn, Slc27a2, Srebf1, CPT1α);F: Western blot of AMPKα and p-AMPKα;G: Relative expression of p-AMPKα / AMPKα; # It means that the modeling group and the control group are significantly different. ## p <0.01; * It means that the difference between the experimental group and the modeling group is significant. ** p <0.01; + Indicates significant differences between groups. + p <0.05, ++ p <0.01;

[0077] Fig.19Effects of sodium acetate liposomes on liver function in NAFLD mice, A: serum alanine aminotransferase; B: serum aspartate aminotransferase; C: liver alanine aminotransferase; D: liver aspartate aminotransferase; # It means that the modeling group and the control group are significantly different. ## p <0.01; * It means that the difference between the experimental group and the modeling group is significant. * p <0.05, ** p <0.01; + Indicates significant differences between groups. + p <0.05, ++ p <0.01;

[0078] Fig. 20 Effects of sodium acetate liposomes on inflammatory infiltration of liver macrophages in NAFLD mice, A: F4 / 80 staining of mouse liver tissue sections (200×, scale bar 100 μm); B: relative area of ​​F4 / 80 positive sites; # It means that the modeling group and the control group are significantly different. ## p <0.01; * It means that the difference between the experimental group and the modeling group is significant. ** p <0.01; + Indicates significant differences between groups. + p <0.05, ++ p <0.01;

[0079] Fig.21 Effects of sodium acetate liposomes on inflammatory cytokines and NFĸB protein phosphorylation in the liver of NAFLD mice. AD: Relative expression of inflammatory cytokines in the liver, which are IL-1β, IL-6, TNF-α, and IL-4; E: p - Western blotting of NFĸB and NFĸB; F: p -Relative expression of NFĸB / NFĸB; # It means that the modeling group and the control group are significantly different. ## p <0.01; * It means that the difference between the experimental group and the modeling group is significant. ** p <0.01; + Indicates significant differences between groups. + p <0.05, ++p <0.01. DETAILED DESCRIPTION

[0080] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0081] Example 1 Preparation of liver-targeted sodium acetate liposomes (NaA@CA / MAN-LPs)

[0082] Preparation process Figure 1 Shown

[0083] 1. DSPE-PEG 2000 Synthesis of -CA

[0084] Cholic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide (EDC) and N-hydroxysuccinimide (NHS) were dissolved in dichloromethane, reacted in an ice bath for 30 min, stirred at room temperature overnight, filtered and evaporated to obtain activated sodium cholate. 500 mg DSPE-PEG2000-NH2 was dissolved in 3 mL DMSO, activated sodium cholate (550 mg) and triethylamine (1 g) were added and completely dissolved, and then reacted at 40 ° C for 2 h. After the reaction was completed, the solution was placed in a dialysis bag (MW = 1000 Da) and dialyzed with pure water as the medium for 24 h. After the dialysis was completed, the solution in the dialysis bag was collected and freeze-dried to obtain DSPE-PEG 2000 -CA (Synthesis steps see Figure 2 A).

[0085] The infrared spectrum of DSPE-PEG-CA is shown in Figure 2 B The results show that DSPE-PEG 2000 After -NH2 was modified with sodium cholate, the -1 The characteristic peak of -C=O stretching vibration of carboxylate group appeared at 3425 cm -1 Corresponding to the hydroxyl group in the sodium cholate molecule, 2850-2917 cm -1 Corresponding to the CH stretching vibration in the stearyl alkyl chain in DSPE-PEG. The stretching vibration of the P=O bond of the phosphate group appears at 1245 cm-1, and the stretching vibration of the CO bond of the PEG chain appears at 1108 cm -1The CH bending vibration peak of the alkyl chain appears at 1466 cm -1 The above results show that sodium cholate has been successfully bound to the DSPE-PEG-NH2 molecule.

[0086] 2. DSPE-PEG 2000 -Synthesis of MAN

[0087] The synthesis steps and results of DSPE-PEG-MAN are as follows:

[0088] Dissolve 500 mg DSPE-PEG2000-NH2 in 3 mL DMSO, add 550 mg mannose and 1 g triethylamine to completely dissolve, and react at 40 °C for 2 h. After the reaction, place the solution in a dialysis bag with a molecular weight of 1000 Da and dialyze it with pure water for 24 h. After the dialysis, freeze-dry the solution in the dialysis bag to obtain DSPE-PEG-MAN (synthetic route as shown in Figure 3 A).

[0089] The results of infrared spectroscopy (FTIR) detection of DSPE-PEG-MAN are as follows Figure 3 As shown in B: The infrared spectrum of DSPE-PEG-MAN shows the following characteristic peaks: 3428 cm -1 Corresponding to the hydroxyl group in the mannose molecule, 2917 cm -1 Corresponding to the CH bond of the stearoyl alkyl chain in DSPE, 1739 cm -1 The corresponding aldehyde C=O group. The CH bending vibration peak of the alkyl chain appears at 1460 cm -1 In the FTIR spectrum of DSPE-PEG2000-NH2, the amino (-NH2) group appears at 3434 cm -1 After modification with mannose, the peak becomes broader and appears at 3428 cm due to the presence of hydroxyl groups. -1 The stretching vibration of the P=O bond of the phosphate group appears at 1251 cm -1 The CO bond stretching vibration of the PEG chain appears at 1109 cm -1 The above results show that DSPE-PEG-MAN was successfully synthesized.

[0090] 3. Preparation of liver-targeted sodium acetate liposomes

[0091] Intestinal penetrating and liver-targeting high-concentration sodium acetate liposomes (NaA@CA / MAN-LPs) were prepared by thin film hydration method.

[0092] All raw materials and reagents were stored at -20 o The preparation process was kept on ice at C.

[0093] (1) Add the following raw materials in order according to the liposome ratio: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, Distearoylphosphatidylcholine), cholesterol, DSPE-PEG 2000 -CA, DSPE-PEG 2000 -MAN, DSPE-PEG 2000 , DC-cholesterol, mix well and transfer to a round-bottom flask with a micro-glass syringe. Pre-mix for 10 min and incubate at 37 o C constant temperature shaker for 10 min to ensure that all reagents are thermodynamically evenly distributed and the solution is completely clear.

[0094] (2) At a pressure of 500 mbar, a speed of 100 rpm, and a temperature of 50 o C conditions, and at the end of the rotary evaporation, a film with uniform thickness and uniform distribution is obtained.

[0095] (3) Add sodium acetate as hydration medium to completely cover the organic membrane and sonicate for 2 min until the solution turns milky white.

[0096] (4) Assemble the liposome extruder (Avanti Polar Lipids). Place a wet carbonate membrane filter holder on each of the two black washers. Select a 100 nm filter membrane, wet it and place it between the two filter holders. Fix the assembled liposome extruder on the holder and finally place the whole thing on a heating plate at 50 °C. o C.

[0097] (5) Use a micro-glass syringe to draw 1 mL of liposome solution and squeeze it back and forth 21 times in the liposome extruder. Store the liposomes after extrusion at 4 o C overnight to stabilize the liposome particles.

[0098] (6) Remove unencapsulated sodium acetate by ultrafiltration, then quickly freeze the liposomes in liquid nitrogen, weigh them, and reconstitute them in PBS before use.

[0099] 7) Identification of sodium acetate liposomes includes: HPLC detection of sodium acetate concentration, calculation of encapsulation efficiency and drug loading; liposome particle size, PDI, Zeta; TEM electron microscopy observation of liposome morphology.

[0100] 4. Orthogonal experiment to optimize the composition of NaA@CA / MAN-LPs

[0101] (1) The ratio of step 1) in the above method is optimized, and the components of the liposome include: DSPC, cholesterol, DSPE-PEG 2000 -CA, DSPE-PEG2000 -MAN, DSPE-PEG 2000 In order to obtain liposomes with high embedding efficiency and good stability, the concentration of sodium acetate stock solution was fixed at 20 mg / mL. According to the orthogonal test, DSPC: cholesterol, DSPE-PEG 2000 -CA, DSPE-PEG 2000 -MAN ratio is optimized. The specific factor levels are shown in Table 1.

[0102] Table 1 Optimization factor level table of sodium acetate liposome synthesis

[0103]

[0104] The orthogonal experiment grouping is shown in Table 2:

[0105] Table 2 Orthogonal experiment grouping table

[0106]

[0107] (2) Composition optimization evaluation indicators

[0108] a. Sodium acetate encapsulation efficiency of liposomes

[0109] a.1 Establishment of high performance liquid chromatography (HPLC) assay method

[0110] The HPLC conditions were as follows: Thermol Hypersil ODS-2 C18 column (4.6 mm * 200 mm, 5 um) was used as the chromatographic column, the mobile phase was 0.01 mol / L ammonium dihydrogen phosphate aqueous solution, the pH was adjusted to 3.0 with 1 mol / L phosphoric acid solution, the flow rate was 1.0 mL / min, the injection volume was 10 μL, the detection wavelength was 215 nm, and the column temperature was 30 o C.

[0111] a.2 Drawing of standard curve

[0112] Sodium acetate was accurately weighed in a 10 mL volumetric flask, and PBS was added to dilute it to prepare sodium acetate standard solutions with concentrations of 0.01, 0.025, 0.05, 0.1, 0.2, and 0.5 mg / mL, respectively. Each sample was measured according to the conditions of HPLC, and the peak area was recorded. The standard curve of AC linear relationship was drawn with the main peak area (A) as the ordinate and the concentration (C) as the abscissa. Sodium acetate was dissolved in serum solution, simulated gastric fluid, and simulated intestinal fluid to prepare sodium acetate solutions with concentrations of 0.01, 0.025, 0.05, 0.1, 0.2, and 0.5 mg / mL, respectively. The peak area-time standard curve of acetic acid in serum, simulated gastric fluid, and simulated intestinal fluid was detected according to the conditions of HPLC.

[0113] a.3 Determination of encapsulation efficiency of sodium acetate liposomes by ultrafiltration

[0114] Take an appropriate amount of sodium acetate liposome solution, place it in an activated ultrafiltration tube (MYCO=3K Da, Millipore), and centrifuge at 12000 rpm for 10 min. Take out the solution in the ultrafiltration tube, detect the free sodium acetate content according to the HPLC method in "2.3.5.1", and calculate the encapsulation rate of sodium acetate in liposomes according to the following formula.

[0115]

[0116] b. Particle size, PDI, Zeta-potential and drug loading of NaA@CA / MAN-LPs

[0117] The particle size, PDI, and Zeta potential of NaA@CA / MAN-LPs were measured by laser particle size analyzer. The sodium acetate encapsulation efficiency EE% and liposome drug loading DL% of NaA@CA / MAN-LPs were detected by ultrafiltration and high performance liquid chromatography. The calculation formula of liposome drug loading is as follows:

[0118]

[0119] c. Stability of NaA@CA / MAN-LPs in simulated body fluids

[0120] Sterile fetal bovine serum was diluted 10 times with PBS (pH 7.4) to prepare a simulated serum solution. The method for preparing artificial simulated gastric fluid was as follows: 0.5 g pepsin was added to 10 mL pure water, dissolved and added to 0.82 mL 1 mol / L hydrochloric acid solution, mixed and diluted to 50 mL, pH = 1.2. The method for preparing artificial simulated intestinal fluid was as follows: 0.34 g potassium dihydrogen phosphate was added to 25 mL water, and the pH was adjusted to 6.8 with 0.4% NaOH; 0.5 g pancreatin was dissolved in water, and the two solutions were mixed and diluted to 50 mL.

[0121] 100 μL of NaA@CA / MAN-LPs was mixed with 900 μL of simulated gastric fluid, simulated intestinal fluid and serum solution respectively and incubated at 37 o C, 1000 rpm constant temperature shaking. The sample solution was collected after 2 hours in the group shaken with simulated gastric fluid, and after 6 hours in the group shaken with simulated intestinal fluid and serum solution. The collected sample solution was measured by laser particle size analyzer for particle size, PDI and Zeta-potential of the sample, and the sodium acetate release rate after incubation with different medium solutions was detected by HPLC, and the changes in particle size and embedding rate of liposomes before and after incubation were analyzed to characterize the stability of liposomes in gastrointestinal tract and serum solution.

[0122] Orthogonal experiment results:

[0123] Table 3 shows the sodium acetate embedding rate and drug loading of liposomes in different groups in the orthogonal experiment. The results show that different DSPC: cholesterol ratios and DSPE-PEG 2000 -CA and DSPE-PEG 2000 -MAN concentration has an important effect on the liposome embedding rate and drug loading. The sodium acetate embedding rate of the nine orthogonal experimental group liposomes is between 70.52% and 81.15%, and the order from large to small is: 3>7>1>8>4>2>9>6>5. The drug loading of the nine orthogonal experimental group liposomes is between 20.86% and 31.58%, and the order from large to small is: 3>7>2>1>4>8>9>6>5. The above results show that the embedding rate and drug loading of liposome 3 and liposome 7 are significantly higher than those of the remaining seven groups of liposomes.

[0124] Table 3 Effect of liposome composition on sodium acetate embedding efficiency and drug loading

[0125]

[0126] Note: The sodium acetate embedding rate and drug loading of the nine groups of liposomes were analyzed for significance. Different letters in the same column of data indicate significant differences ( p <0.05, the same letters indicate no significant difference ( p >0.05).

[0127] From the results of particle size changes of liposomes of different compositions after incubation in simulated body fluids in Table 4, it can be seen that the initial particle size of the nine groups of liposomes is about 100 nm. After each group of liposomes is incubated in serum, simulated gastric fluid and simulated intestinal fluid for 6 h or 2 h, the particle size of the liposomes increases to varying degrees. After incubation in serum for 6 h, the particle sizes of liposomes in each group were 7<2<8<6<5<1<4<3<9, and the particle size of liposome 7 (132.41±3.67 nm) was significantly lower than that of the other eight groups. After incubation in simulated gastric fluid for 2 h, the particle sizes of liposomes in different groups were 7<9<3<8<1<2<6<4<5, and the particle sizes of liposome 7 and liposome 9 (107.21±2.40 nm, 128.70±1.59 nm) were significantly lower than those of the other seven groups. After incubation in simulated intestinal fluid for 6 h, the particle sizes of liposomes in different groups were 4<8<1<7<6<5<9<2<3, and there was no significant difference in the particle sizes of liposomes in the other seven groups except liposome 2 and liposome 3. Table 5 shows the changes in PDI of nine groups of liposomes with different compositions after incubation in simulated body fluids. The initial PDI of the nine groups of liposomes is between 0.20 and 0.22. After incubation in serum, simulated gastric fluid and simulated intestinal fluid, the PDI of all liposomes is <0.4, indicating that after incubation in simulated body fluids, the particle size distribution of all liposomes is relatively uniform, without obvious aggregation. From the changes in the potential of the nine groups of liposomes after incubation in simulated body fluids in Table 6, it can be seen that the initial Zeta potential of the liposomes is positive, and after incubation in serum, simulated gastric fluid and simulated intestinal fluid, the charge of the liposomes remains positive.

[0128] Table 4 Changes in particle size of liposomes in different groups in serum, simulated gastric fluid and intestinal fluid in orthogonal experiments

[0129]

[0130] Note: The original particle sizes of the nine groups of liposomes and the particle sizes of the liposomes after incubation in serum, simulated gastric fluid, and simulated intestinal fluid were analyzed for significance. Different letters in the same column of data indicate significant differences ( p <0.05, the same letters indicate no significant difference ( p >0.05).

[0131] Table 5 PDI changes of liposomes in different groups in serum, simulated gastric fluid and intestinal fluid in orthogonal experiment

[0132]

[0133] Note: The original particle sizes of the nine groups of liposomes and the PDI results after incubation in serum, simulated gastric fluid, and simulated intestinal fluid were analyzed for significance. Different letters in the same column of data indicate significant differences ( p<0.05, the same letters indicate no significant difference ( p >0.05).

[0134] Table 6 Zeta-potential changes of liposomes in different groups in serum, simulated gastric fluid and intestinal fluid in orthogonal experiment

[0135]

[0136] Note: The original particle size of the nine groups of liposomes and the Zeta potential after incubation in serum, simulated gastric fluid, and simulated intestinal fluid were analyzed for significance. Different letters in the same column of data indicate significant differences ( p <0.05, the same letters indicate no significant difference ( p >0.05).

[0137] Results Figure 4 After incubation in serum for 6 h, the sodium acetate release of the nine groups of liposomes ranged from 3.61% to 5.27%, among which the sodium acetate release of liposome 4 and liposome 7 was significantly lower than that of the other groups, with the release amounts being 3.73%±0.19% and 3.61%±0.32%, respectively ( Figure 4 A). After incubation in simulated gastric fluid for 2 h, the sodium acetate release of the nine groups of liposomes ranged from 12.57% to 16.51%, among which the sodium acetate release of liposome 2 and liposome 7 was significantly lower than that of the other groups of liposomes, which were 12.57%±0.32% and 12.70%±0.37%, respectively ( Figure 4 B). Similarly, after incubation in simulated intestinal fluid for 6 h, sodium acetate was released to a certain extent in all nine groups of liposomes, and the release amount was between 5.16% and 7.73%. The release amount of liposome 3 and liposome 7 was significantly lower than that of the other seven groups of liposomes, and their release amounts were 5.16%±0.08% and 5.51%±0.06%, respectively ( Figure 4 C). The above results show that, compared with the other groups of lipids, liposomes 4 and 7 are more stable in serum, liposomes 2 and 7 are more stable in simulated gastric fluid, and liposomes 3 and 7 are more stable in simulated intestinal fluid.

[0138] To optimize DSPC:cholesterol and DSPE-PEG in liposomes 2000 -CA and DSPE-PEG 2000-MAN ratio, the above three levels were optimized by orthogonal optimization method. From the above results, it can be seen that the sodium acetate embedding rate of liposome 7 is 78.42%±0.30%, ranking second among the nine groups of liposomes, and the drug loading is 29.70%±0.11%, ranking second among the nine groups of liposomes, indicating that the sodium acetate content of the prepared liposomes is high under the composition ratio of liposome 7. In order to study the stability of liposomes in different body fluids, the nine groups of liposomes were incubated in serum, simulated gastric fluid and simulated intestinal fluid, and their particle size, PDI, Zeta potential and sodium acetate release were detected. According to the above results, after incubation in serum, simulated gastric fluid and simulated intestinal fluid, the particle size of liposome 7 did not increase significantly compared with other groups, and the particle size distribution was uniform and still positively charged. In addition, compared with other groups of liposomes, liposome 7 has the lowest release of sodium acetate in serum, simulated gastric fluid and simulated intestinal fluid, which is only 3.61%±0.32%, 12.70%±0.37% and 5.51%±0.06%. Therefore, the most suitable ratio of sodium acetate liposomes is DSPE: cholesterol ratio of 3:1 and 1% DSPE-PEG 2000 -CA and 10% DSPE-PEG 2000 -MAN.

[0139] Example 2 Sodium acetate stock solution concentration ramping experiment

[0140] Sodium acetate stock solutions with concentrations of 20, 30, 40, 50, and 60 mg / mL were prepared, and NaA@CA / MAN-LPs were synthesized according to the optimal liposome ratio. Unencapsulated sodium acetate was removed by ultrafiltration, and the sodium acetate concentration was detected by HPLC to calculate the sodium acetate encapsulation rate in the liposomes. The liposomes were freeze-dried by liquid nitrogen rapid freezing and weighed, and the sodium acetate drug loading of the liposomes was calculated based on the liposome mass.

[0141] The optimal ratio of sodium acetate liposomes was determined to be 40.5% DSPC, 13.5% cholesterol, 1% DSPE-PEG-CA, 10% DSPE-PEG-MAN, 5% DSPE-PEG2000 and 30% DC-cholesterol. The concentration of the gradient sodium acetate stock solution (20, 30, 40, 50, 60 mg / mL) was set to obtain the optimal sodium acetate embedding rate and loading capacity. The results are shown in Figure 5 As the sodium acetate stock solution concentration increased from 20 mg / mL to 60 mg / mL, the liposome encapsulation efficiency also increased from 71.09% to 79.36%. When the sodium acetate stock solution concentration exceeded 40 mg / mL, the increase in the liposome encapsulation efficiency tended to be flat ( Figure 5 A), and the liposome loading capacity was the highest at this time, which was 33.07% ( Figure 5B), therefore, when preparing liposomes with high encapsulation efficiency and loading capacity, the selected sodium acetate stock concentration was 40 mg / mL.

[0142] Example 3 Preparation and characterization of liposomes with different modifications

[0143] The following differently modified liposomes were prepared: ① sodium acetate liposomes modified with sodium cholate and mannose (NaA@CA / MAN-LPs); ② sodium acetate liposomes modified with sodium cholate alone (NaA@CA-LPs); ③ sodium acetate liposomes modified with mannose alone (NaA@MAN-LPs); ④ unmodified sodium acetate liposomes (NaA@LPs); ⑤ sodium cholate and mannose liposomes without sodium acetate (CA / MAN-LPs). The specific compositions of the differently modified liposomes are shown in Table 7.

[0144] Table 7 Proportions of sodium acetate liposomes with different modification types

[0145]

[0146] Dynamic light scattering was used to measure the particle size, PDI and Zeta potential of the five liposomes, HPLC was used to detect the sodium acetate embedding rate and drug loading of the liposomes, and transmission electron microscopy (TEM) was used to observe the morphology of the liposomes. The specific operation steps of TEM were as follows: the liposomes were diluted and dropped onto a copper mesh, left to stand for 2 min, the liquid on the surface of the copper mesh was dried with filter paper, 2% phosphotungstic acid was added for negative staining for 5 min, the copper mesh was dried, and the morphology of different types of liposomes was observed using a JEM-1400Flash transmission electron microscope.

[0147] Five liposomes with different modifications were synthesized, namely ① sodium acetate liposomes modified with sodium cholate and mannose (NaA@CA / MAN-LPs); ② sodium acetate liposomes modified with sodium cholate alone (NaA@CA-LPs); ③ sodium acetate liposomes modified with mannose alone (NaA@MAN-LPs); ④ sodium acetate liposomes without modification (NaA@LPs); ⑤ sodium cholate and mannose liposomes without sodium acetate (CA / MAN-LPs). The five liposomes were tested by particle size analyzer and drug loading capacity. The results are shown in Table 8. Figure 6 The particle size of the five liposomes was about 100 nm, PDI < 0.25, and Zeta potential was positive. Except for CA / MAN-LPs, the embedding rates of NaA@CA / MAN-LPs, NaA@CA-LPs, NaA@MAN-LPs, and NaA@LPs were between 76.6% and 81.57%, and the drug loading was between 33.16% and 37.94%, indicating that the five synthesized liposomes were all about 100 nm, with uniform particle distribution and positively charged nanoliposomes, and had high sodium acetate embedding rate and liposome drug loading. Figure 6 The five liposomes shown are spherical in appearance, with a particle size of about 100 nm, which is consistent with the particle size measurement results. The particles are evenly distributed without aggregation or clumping.

[0148] Table 8 Characterization of different types of sodium acetate liposomes

[0149]

[0150] Example 4 Biological safety of sodium acetate liposomes

[0151] 1. Cell culture

[0152] Caco-2 was revived and passaged using DMEM complete medium, AML-12 and H9c2 were revived and passaged using DMEM / F12 complete medium, and Kupffer was revived and passaged using RPMI 1640 complete medium. Except for the different types of medium, other experimental operation steps were the same.

[0153] 2. Liposome safety

[0154] The CCK-8 method was used to detect the effects of five liposomes (NaA@CA / MAN-NPs, NaA@CA-NPs, NaA@ MAN-NPs, NaA@NPs, CA / MAN-NPs) and sodium acetate solution on cell viability.

[0155] Cell safety results are shown in Figure 7 The results showed that in the range of 1~1500 μM sodium acetate concentration, four types of cells ( Figure 7 The cell viability of AD cells after incubation in five liposomes and sodium acetate solution for 24 h was greater than 85%, indicating that the prepared double-labeled modified sodium acetate had good safety for cardiomyocytes, intestinal epithelial cells, liver macrophages and hepatocytes, and was suitable as a nanocarrier for oral delivery of sodium acetate.

[0156] Example 5 Sodium acetate liposomes reduce lipid deposition in liver cell lines and inhibit inflammation

[0157] 1. Cell culture

[0158] The hepatocyte AML-12 was revived and cultured using DMEM / F12 complete medium; the liver macrophage Kupffer was revived and passaged using RPMI-1640 complete medium.

[0159] 2. Cell modeling

[0160] AML-12 cells in good logarithmic growth phase were obtained, digested with trypsin and then inoculated into 6-well plates for culture. 2 mL of cell lipid induction medium (containing 0.5 mM sodium oleate and 0.25 mM sodium palmitate) was added to each well to induce the NAFLD cell model. The control group cells used control complete medium containing BSA.

[0161] 3. Sodium acetate liposomes alleviate lipid deposition in NAFLD cells

[0162] AML-12 cells were selected, and cells in good logarithmic growth phase were plated, cultured and starved for 12 hours. When lipid induction was performed, the cells were divided into six groups. The first group was added with an equal volume of PBS as a blank control group; the second group was added with lipid induction medium as a NAFLD model group; the third group was added with sodium cholate / mannose double-labeled sodium acetate liposomes (NaA@CA / MAN-LPs) in the lipid induction medium; the fourth group was added with sodium acetate solution (NaA) in the lipid induction medium; the fifth group was added with unlabeled sodium acetate liposomes (NaA-LPs) in the lipid induction medium; the sixth group was added with blank double-labeled liposomes (CA / MAN-LPs) without sodium acetate in the lipid induction medium, where the concentration of sodium acetate solution was 1 mM. After 24 hours of culture, TG, TC, BSA, Oil Red O detection, total cell RNA extraction and RT-qPCR detection of lipid metabolism-related genes were performed on all grouped cells.

[0163] The results are as follows Figure 8 As shown in the AD, the relative areas of lipid droplets after TG, TC and Oil Red O staining in the NAFLD model group cells increased significantly, and the NAFLD cell model was constructed. After 24 h of treatment of NAFLD cells with 1 mM concentration of NaA@CA / MAN-LPs and NaA solution alone, the relative areas of intracellular TG, TC and Oil Red O stained lipid droplets were significantly reduced; similarly, compared with the NAFLD model group, after 24 h of incubation of NAFLD cells with NaA@LPs, the intracellular TG and TC contents were significantly reduced, but the relative area of ​​lipid droplets after Oil Red O staining did not improve significantly. After 24 h of incubation of NAFLD cells with blank liposomes (CA / MAN-LPs) without sodium acetate, the relative areas of cell TG, TC and Oil Red O lipid droplets decreased slightly, but there was no significant difference. Intra-group analysis of variance showed that compared with NaA treatment, NaA@CA / MAN-LPs treatment of NAFLD cells had a more significant alleviating effect on intracellular TG and TC accumulation. The results showed that incubation of NAFLD cells with sodium cholate / mannose double-labeled liposomes with a sodium acetate concentration of 1 mM for 24 h significantly alleviated lipid deposition in NAFLD cells, and the alleviating effect of double-labeled sodium acetate liposomes was significantly better than that of sodium acetate solution alone.

[0164] 4. Sodium acetate liposomes relieve liver macrophage inflammation

[0165] Kupffer cells in good logarithmic growth phase were selected as experimental objects and treated in groups. Except for the blank control group, the remaining five groups of cells were treated with LPS at a concentration of 20 ng / mL six hours before the end of treatment to induce cell inflammation. The specific treatment conditions of each group of cells were as follows: the cells in the blank control group were added with an equal volume of PBS solution and no LPS treatment; the cells in the modeling group were added with LPS 6 h before the end of incubation to induce cell inflammation; 1 mM sodium cholate / mannose double-labeled sodium acetate liposomes (NaA@CA / MAN-LPs) were added to the double-labeled liposome group; 1 mM sodium acetate solution was added to the cells in the sodium acetate group; 1 mM unlabeled sodium acetate liposomes (NaA-LPs) were added to the cells in the unlabeled liposome group; blank double-labeled liposomes (CA / MAN-LPs) without sodium acetate were added to the cells in the blank liposome group. After the treatment, total RNA of the cells was extracted for RT-qPCR detection of inflammation-related genes.

[0166] The results are as follows Fig. 9 As shown in AD, the expression of proinflammatory factors IL-1β, IL-6 and TNF-α mRNA in Kupffer cells induced by LPS was significantly upregulated, and the expression of anti-inflammatory factor IL-4 mRNA was significantly downregulated, indicating that the Kupffer cells in the model group produced a significant inflammatory response. Compared with the model group, after treatment with sodium acetate-containing sodium cholate / mannose double-labeled liposomes (NaA@CA / MAN-LPs), unlabeled liposomes (NaA@LPs) and sodium acetate solution (NaA), the expression of IL-1β, IL-6 and TNF-α mRNA in Kupffer cells was significantly reduced, and the expression of IL-4 mRNA was significantly increased, indicating that sodium acetate and sodium acetate liposomes effectively inhibited the activation of Kupffer cell inflammatory pathways. Double-labeled blank liposomes (CA / MAN-LPs) without sodium acetate had no significant effect on the high expression of proinflammatory factors and the low expression of anti-inflammatory factors in Kupffer cells, indicating that the liposome material has neither ameliorative nor promoting effect on Kupffer under inflammatory state.

[0167] Example 6 Liposome Targeting in vivo

[0168] Fifteen C57 male mice were selected, 3 mice in each group, for a total of 5 groups, and CY7@CA / MAN-LPs, CY7@CA-LPs, CY7@MAN-LPs, CY7@LPs and CY7 with the same fluorescence intensity were gavaged orally. The mice were anesthetized and subjected to in vivo fluorescence imaging at 0.5, 1, 1.5, 2, and 2.5 h after gavage. The results are shown in the figure. Fig.10As shown. The fluorescence intensity of mice directly gavaged with CY7 fluorescent dye and unlabeled CY7-liposomes was generally low within 0-2.5 h. After gavage with sodium cholate or mannose-modified liposomes encapsulating CY7, the fluorescent liposomes accumulated in the abdomen, and the fluorescence intensity showed a trend of first increasing and then decreasing within 0-2.5 h. The fluorescence intensity at 2.5 h was still clearly visible. The accumulation of the three sodium cholate or mannose-labeled fluorescent liposomes in the abdomen was from high to low: sodium cholate / mannose dual-labeled fluorescent liposomes> sodium cholate single-labeled fluorescent liposomes> mannose single-labeled fluorescent liposomes. The above results show that sodium cholate / mannose dual-labeled liposomes have stronger intestinal penetration and liver targeting capabilities, and deliver more fluorescent dyes to the liver.

[0169] C57 mice were randomly divided into 5 groups and gavaged with CY7@CA / MAN-LPs, CY7@CA-LPs, CY7@MAN-LPs, CY7@LPs and CY7 of the same fluorescence intensity. At 2 h, 6 h, 10 h, 14 h, 18 h and 24 h, the mice were killed after anesthesia, and the main organs (heart, liver, spleen, lung, kidney and gastrointestinal tract) were removed. The fluorescence intensity of each organ was immediately detected in a fluorescence imager. The results are shown in Figure 5. Fig.11 A; The liver fluorescence intensity change curves of five groups of fluorescent liposomes were drawn with liver fluorescence intensity as the ordinate and time as the abscissa ( Fig.11 B) The five groups of curves showed an overall trend of increasing first and then decreasing. The fluorescence intensity in the mouse liver reached a peak at 14 h, which were 0.303, 0.208, 0.147, 0.084, 0.038 [(p / sec / cm 2 / sr) / μM / cm 2 ]; The fluorescence intensity-time curves of the five groups of fluorescent liposomes were integrated, AUG was calculated and the significance was analyzed. The results are as follows Fig.11 C, Fluorescence intensity of five liposomes in liver AUG 0-24 From high to low, the order is: CY7@CA / MAN-LPs>CY7@CA-LPs>CY7@MAN-LPs>CY7@LPs>CY7, and the accumulation content of sodium cholate / mannose modified fluorescent liposomes in the liver is significantly higher than that of single fluorescent dye, unmodified fluorescent liposomes, single mannose labeled fluorescent liposomes or single sodium cholate labeled fluorescent liposomes. Similarly, the fluorescence intensity in the intestines of the five groups of mice was quantified, such as Fig.11 As shown in D, the fluorescence intensity in the mouse intestine gradually decreased within 2-24 h. The fluorescence intensity AUG of the mouse intestine within 2-24 h was calculated. 2-24 , the results are as follows Fig.11As shown in E, the fluorescence intensity of CY7@CA / MAN-LPs accumulated in the intestine for 2-24 h was significantly higher than that of CY7@MAN-LPs, CY7@LPs and CY7. The above results indicate that sodium cholate / mannose dual-labeled fluorescent liposomes more effectively penetrate the mouse intestine, target the liver tissue and exert their effects for a long time.

[0170] Example 7 Sodium acetate liposomes alleviate NAFLD in mice

[0171] 1. Animal Model

[0172] The animals used in this experiment were 6-week-old specific pathogen free (SPF) C57BL / 6J male mice, weighing 18±1.0 g, purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd. (certificate SYXK (Beijing) 2021-0001). The experimental design was reviewed by the Laboratory Animal Welfare Ethics Committee of Zhongyanzi Chuang (Beijing) Biotechnology Co., Ltd. (approval number ZYZC202402006S), and all animal handling and experimental procedures were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.

[0173] 2. Modeling and intervention

[0174] The mice were randomly divided into six groups, with 8 mice in each group (4 mice / cage), and then the sodium acetate liposome gavage experiment was performed once every other day for 10 weeks. The animal experiment design, specific grouping and intervention methods are as follows:

[0175] (1) Normal control group: Feeding with basic diet, and gavage with PBS every other day from week 0 to week 10

[0176] (2) NAFLD model group: HFD feeding, from week 0 to week 10, intragastric administration of PBS every other day

[0177] (3) Dual-labeled liposome intervention group (NaA@CA / MAN-LPs): HFD diet, oral administration of sodium cholate / mannose dual-labeled sodium acetate liposomes (200 μL, sodium acetate concentration in dual-labeled liposomes was 15 mg / mL) every other day from week 0 to week 10

[0178] (4) Sodium acetate intervention group (NaA): HFD diet was used, and sodium acetate solution (200 μL, the concentration of sodium acetate solution was 15 mg / mL) was gavage every other day from week 0 to week 10.

[0179] (5) Unlabeled liposome intervention group (NaA@LPs): HFD diet was used, and unlabeled sodium acetate liposomes (200 μL, sodium acetate concentration in unlabeled liposomes was 15 mg / mL) were gavaged every other day from week 0 to week 10.

[0180] (6) Blank liposome intervention group (CA / MAN-LPs): The mice were fed with HFD and gavaged with blank liposomes (200 μL) every other day from week 0 to week 10. The concentration of blank liposomes was consistent with that of liposomes in other groups, which was 46 mg / mL.

[0181] Body weight change trend, organ index, oral glucose tolerance (OGTT), blood biochemical indexes, tissue hematoxylin and eosin (H&E) staining and Oil Red O staining, liver tissue F4 / 80 immunohistochemical staining, mouse liver lipid content change detection, mouse liver function index detection, mouse liver oxidative damage detection (MDA and SOD monitoring), liver tissue total RNA extraction and real-time quantitative PCR determination (genes Acc1, Fasn, Srebf1, CPT1α, IL-1β, IL-4, IL-6, TNF-α, GAPDH); protein immunoblotting (Western Blot).

[0182] 1) Weight results are as follows Fig.12 As shown in AB: After 10 weeks of intervention with double-labeled sodium acetate liposomes (NaA@CA / MAN-LPs), unlabeled sodium acetate liposomes (NaA@ LPs) and sodium acetate solution (NaA), the body weight of NAFLD mice decreased by 18.08%, 13.88% and 6.45%, respectively. At the end of the tenth week of intervention, the weight gain of the three groups of mice was significantly lower than that of the NAFLD modeling group, indicating that sodium acetate, sodium acetate double-labeled liposomes and unlabeled liposomes can effectively alleviate the weight gain of NAFLD mice. After 10 weeks of intervention with blank double-labeled liposomes without sodium acetate, the body weight of mice decreased by 1.76 g, which was not significantly reduced compared with the NAFLD modeling group, indicating that the blank liposome material had no significant effect on the body weight of NAFLD mice and did not cause weight gain in mice.

[0183] 2) Glucose tolerance results such as Fig.13 As shown: Compared with the NAFLD model group mice, the area under the OGTT curve of sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution alone and unlabeled sodium acetate liposomes was significantly reduced ( p <0.01), indicating that the glucose tolerance of mice was significantly restored after the above three treatments; the AUG of mice in the four treatment groups was analyzed for inter-group differences, and the results showed that the AUG of OGTT of mice in the double-labeled sodium acetate liposome group after 10 weeks of intervention was significantly lower than that of mice gavaged with sodium acetate solution alone and unlabeled sodium acetate liposomes. The above results indicate that sodium cholate / mannose double-labeled sodium acetate liposomes have the most significant effect in improving glucose tolerance ( Fig.13 AB).

[0184] 3) Blood lipid metabolism results such as Fig.14 As shown in AD, compared with the NAFLD model group, after mice were gavaged with sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution alone, and unlabeled sodium acetate liposomes, the levels of serum TG, TC, and LDL-C were significantly reduced, and the level of HDL-C was significantly increased ( p <0.01), indicating that unlabeled liposomes, double-labeled liposomes and sodium acetate containing sodium acetate can significantly alleviate the blood lipid level of NAFLD. The intra-group significance analysis of serum lipid metabolism parameters of mice in the double-labeled liposome group, sodium acetate group, unlabeled liposome group and blank liposome group showed that the effect of double-labeled sodium acetate liposomes in alleviating dyslipidemia in NAFLD was better than that of sodium acetate alone.

[0185] 4) Sodium acetate liposomes improve adipose tissue content in HFD-induced NAFLD mice Fig.15 As shown, Fig.15 A: Compared with the NAFLD model group mice, oral administration of sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution alone, unlabeled sodium acetate liposomes, and blank liposome materials significantly alleviated the proportion of white adipose tissue in NAFLD mice, with each group decreasing by 3.56%, 2.20%, 2.05%, and 0.54%, respectively. Intra-group difference analysis of the total content of white adipose tissue of mice in the four intervention groups was performed, and the results showed that the proportion of white adipose tissue after intervention with sodium cholate / mannose dual-labeled sodium acetate liposomes was significantly lower than that after oral administration of sodium acetate solution alone or unlabeled liposomes containing sodium acetate. H&E staining was performed on the epididymal adipose tissue of mice, and the staining results are shown as follows. Fig.15 As shown in Figure B, compared with the NAFLD model group, the epididymal fat vacuoles area of ​​sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution alone, and unlabeled sodium acetate liposomes were significantly reduced, the epididymal fat cell section area was significantly reduced, and the number of fat cells in the same field of view was significantly increased, and the changes in the dual-labeled liposome group were the most obvious. In summary, sodium cholate / mannose dual-labeled sodium acetate liposomes have the most significant effect in improving the adipose tissue content of NAFLD mice.

[0186] 5) The results of sodium acetate liposomes improving liver pathological morphology in NAFLD mice are as follows Fig.16 shown. Fig.16As shown in A, the livers of the control group mice fed with ordinary feed were smooth, reddish brown, and had a certain degree of tissue elasticity. Compared with the control group, the livers of the NAFLD model mice constructed after 10 weeks of HFD diet became lighter white overall, and obvious "fat droplet white spots" could be visually seen. After 10 weeks of gavage with sodium acetate liposomes doubly labeled with sodium bile / mannose, sodium acetate solution alone, or unlabeled sodium acetate liposomes, the color of the livers of NAFLD mice returned to reddish brown, and the surface smoothness was restored, indicating that sodium acetate or two liposomes containing sodium acetate have the function of alleviating NAFLD liver deposition. The H&E results of the livers of the NAFLD modeling group mice showed ( Fig.16 B) The structure of mouse liver lobules was destroyed, and hepatocytes underwent ballooning, which was manifested by enlarged hepatocytes containing large lipid droplets. Some hepatocytes showed mixed macrovesicular and microvesicular fatty changes, accompanied by inflammatory cell infiltration. After intragastric administration of sodium acetate liposomes double-labeled with sodium bile acid / mannose, sodium acetate solution alone, or unlabeled sodium acetate liposomes, the number and area of ​​vacuoles in the mouse liver were significantly reduced, indicating that hepatic fatty degeneration was significantly improved. After intragastric administration of blank liposomes without sodium acetate, the area of ​​vacuoles in the mouse liver was reduced, but the number was still large, indicating that the effect of liposomes alone on the alleviation of lipid deposition in the liver of NAFLD mice was very limited. Further quantitative analysis of the lipid droplet area in the liver H&E stained sections showed that the liver lipid droplet area of ​​the NAFLD model group mice was 1.93 times that of the healthy control group mice. Sodium acetate liposomes double-labeled with sodium cholate / mannose, sodium acetate solution and unlabeled sodium acetate liposomes significantly reduced the liver lipid droplet area of ​​NAFLD mice (p<0.01). After 10 weeks of sodium acetate intervention, the liver lipid droplet area of ​​NAFLD mice was reduced by 46.39%, 40.97% and 39.33%. ( Fig.16 C). Oil Red O staining results are shown in Fig.16 D, The number of red lipid droplets in the liver of mice in the NAFLD group and the blank liposome group increased significantly, the size became significantly larger, and the oil red color was bright, indicating that a large amount of fat was deposited in the liver of mice in the NAFLD group. The double-labeled liposome material without sodium acetate had no effect on alleviating lipid deposition in the liver of NAFLD mice. Sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate alone, or sodium acetate-free liposomes significantly reduced liver lipid deposition in NAFLD mice, and the alleviating effect of double-labeled sodium acetate liposomes was the most obvious. The Oil Red O-stained sections at the end of the intervention were closer to those of normal mice ( Fig.16D). The liver lipid droplets of mice in the NAFLD model group and the blank liposome group increased significantly, which were 14.37 times and 13.67 times that of the control group, respectively. After 10 weeks of intervention with sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate alone, and unlabeled sodium acetate liposomes, the liver lipid droplet area of ​​NAFLD mice was significantly reduced. Compared with the NAFLD model group, the liver lipid droplet area at the end of the intervention was reduced by 88.27%, 73.45%, and 70.40%, respectively ( Fig.16 E).

[0187] 6) Sodium acetate liposomes improve hepatic lipid accumulation and oxidative stress in NAFLD mice Fig.17 After 10 weeks of intervention with two sodium acetate-containing liposomes (NaA@CA / MAN-LPs and NaA@LPs) in NAFLD mice, the liver index was significantly lower than that of mice given sodium acetate solution alone ( Fig.17 A). The TG and TC contents in mouse liver were tested. The results are as follows Fig.17 B, C. The levels of TG and TC in the liver of mice in the NAFLD model group increased significantly ( p <0.01). After 10 weeks of oral administration of blank liposomes without sodium acetate, there was no significant improvement in the TG and TC content in the liver of mice. After 10 weeks of oral administration of two liposomes containing sodium acetate or sodium acetate solution, the TG and TC content in the liver of NAFLD mice was significantly reduced, and lipid deposition in the liver of mice was significantly improved. The results of intra-group difference analysis showed that the TG and TC content in the liver of mice gavaged with double-labeled sodium acetate liposomes was significantly lower than that in the sodium acetate group. The levels of lipid-promoting oxidants MDA and antioxidant SOD in the liver of mice were detected by the kit method. The results are as follows Fig.17 As shown in D and E. After oral administration of sodium acetate liposomes, sodium acetate solution or unlabeled sodium acetate liposomes with sodium bile acid / mannose dual labeling, the MDA level in the liver of NAFLD mice was significantly reduced, and the SOD level was significantly increased ( p <0.01). The results of intragroup difference analysis showed that double-labeled sodium acetate liposomes significantly alleviated the increase of MDA and the decrease of SOD levels in the liver of NAFLD mice, which was significantly better than intragastric administration of sodium acetate solution.

[0188] 7) Effect of liposomes on the expression of genes related to lipid metabolism in the liver of NAFLD mice. Fig.18 AE. Compared with the control group, the mRNA expression of genes related to lipid synthesis regulation (Acc1, Fasn, Slc27a2, Srebf1) in the liver of NAFLD mice induced by HFD diet was significantly increased ( p <0.01), and the mRNA expression level of fatty acid transport gene CPT1α was significantly decreased ( p<0.01); compared with the NAFLD model group, blank liposomes without sodium acetate had no significant effect on the gene expression of lipid synthesis and transport in the liver of NAFLD mice. After 10 weeks of intervention with sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution or unlabeled sodium acetate liposomes, the expression levels of Acaca, Fasn, Slc27a2, and Srebf1 mRNA in the liver of NAFLD mice were significantly reduced, and the expression level of CPT1α mRNA was significantly upregulated. Western blot technology was used to analyze the phosphorylation of AMPKα protein in the liver of NAFLD mice after 10 weeks of oral administration of sodium acetate or sodium acetate liposomes. The results are shown in Fig.18 F. Compared with the control group, the phosphorylation of AMPKα protein in the liver of the NAFLD model group induced by HFD and the blank liposome intervention group without sodium acetate was significantly inhibited. After NAFLD mice were gavaged with sodium bile / mannose dual-labeled sodium acetate liposomes, sodium acetate solution or unlabeled sodium acetate lipids, the phosphorylation level of AMPKα protein in the liver was significantly increased ( Fig.18 F, G). After oral administration of unlabeled liposomes, double-labeled liposomes, and sodium acetate alone containing sodium acetate, the mRNA expression of liver Acc1 and Srebf1c was significantly downregulated, and the expression of CPT1α was significantly upregulated. The above results indicate that sodium acetate or double-labeled sodium cholate liposomes inhibit the transcription of Acc1 and Srebf1c and promote the expression of CPT1α by activating the AMPK pathway, thereby regulating downstream lipid metabolism genes and reducing liver lipid deposition.

[0189] 8) Sodium acetate liposomes improve liver function in NAFLD mice: Serum AST and ALT levels were measured. The results are as follows Fig.19 As shown in A and B. After NAFLD mice were gavaged with blank liposomes without sodium acetate, the serum ALT and AST levels did not decrease significantly, but after gavage with sodium acetate liposomes double-labeled with sodium cholate / mannose, sodium acetate solution or unlabeled sodium acetate liposomes, the serum AST and ALT levels of mice were significantly reduced. The liver ALT and AST levels are shown in Fig.19 C, D. Intragastric administration of double-labeled liposomes containing sodium acetate, unlabeled liposomes or sodium acetate solution alone effectively improved the levels of ALT and AST in the liver of NAFLD mice. Intra-group difference analysis showed that the effect of sodium cholate / mannose double-labeled sodium acetate liposomes in alleviating the increase in ALT and AST levels was significantly better than that of intragastric administration of sodium acetate alone.

[0190] 9) Sodium acetate liposomes improve liver inflammation and oxidative stress in NAFLD mice Fig. 20 The hepatic macrophage infiltration of mice in the double-labeled sodium acetate liposome, sodium acetate solution or unlabeled sodium acetate liposome groups was significantly improved ( Fig. 20A). Quantitative analysis showed that after 10 weeks of oral administration of dual-labeled sodium acetate liposomes, sodium acetate solution alone or unlabeled sodium acetate liposomes to NAFLD mice, the relative positive area of ​​F4 / 80 in the liver of mice decreased by 62.46%, 40.99% and 50.16%, respectively ( Fig. 20 B). The results of the intergroup differences in the area of ​​F4 / 80-positive cells in the liver of mice in the four intervention groups showed that the effect of sodium cholate / mannose dual-labeled sodium acetate liposomes on improving the infiltration of macrophages in the liver of NAFLD mice was significantly better than that of sodium acetate solution alone or unlabeled sodium acetate liposomes ( p <0.01).

[0191] 10) Further testing of the expression of cytokines related to liver inflammation and oxidative stress in mice, such as Fig.21 As shown in AD. After 10 weeks of oral administration of sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution alone, or unlabeled sodium acetate liposomes, the mRNA expression levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the liver of NAFLD mice were significantly reduced, and the mRNA expression level of anti-inflammatory factor IL-4 was significantly increased, indicating that sodium acetate or dual-labeled and unlabeled liposomes containing sodium acetate have a significant alleviating effect on liver inflammation in NAFLD mice. Compared with the NAFLD model group mice, the phosphorylation level of NFĸB in the mouse liver was significantly downregulated after oral administration of sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution, or unlabeled sodium acetate liposomes, indicating that the HFD-induced liver inflammatory response was reduced. The results of intra-group difference analysis of the treatment groups showed that the alleviating effect of dual-labeled or unlabeled sodium acetate liposomes on the increased phosphorylation level of inflammatory proteins was significantly better than that of sodium acetate intervention alone ( Fig.21 E, F).

[0192] In summary, after 10 weeks of oral administration of sodium cholate / mannose dual-labeled sodium acetate liposomes to NAFLD mice induced by HFD, the weight gain of mice was significantly reduced, and glucose tolerance was effectively improved. The detection of serum lipid indexes showed that dual-labeled sodium acetate liposomes effectively alleviated the increase of TG, TC, and LDL-C in the liver of NAFLD mice and the decrease of HDL-C. The results of liver pathological tissue sections of NAFLD mice showed that after oral administration of dual-labeled sodium acetate liposomes to mice, liver lipid deposition was significantly improved, and the phosphorylation level of liver AMPKα protein was significantly activated. By inhibiting the transcription of Acc1 and Srebf1, CPT1α expression was promoted, and downstream lipid metabolism genes were regulated, effectively reducing liver lipid deposition. In terms of liver inflammation level and oxidative stress, after 10 weeks of oral administration of dual-labeled sodium acetate liposomes to NAFLD mice, NFĸB protein phosphorylation was significantly inhibited, the release of downstream inflammatory factors was reduced, and liver inflammation and oxidative stress were effectively alleviated. The effect of dual-labeled sodium acetate liposomes on alleviating liver lipid deposition and oxidative stress in NAFLD mice was better than that of oral administration of sodium acetate solution of the same concentration alone, indicating that sodium cholate / mannose dual-labeled liposomes successfully targeted the delivery of sodium acetate to the liver, causing sodium acetate to accumulate in the liver, and more effectively alleviating liver lipid accumulation and inhibiting inflammatory responses.

Claims

1. A liver-targeted drug liposome, characterized in that: The liposome described above is prepared from DSPC, cholesterol, DSPE-PEG-CA, DSPE-PEG-MAN, and DSPE-PEG 2000 , and DC-cholesterol in a certain proportion, where DSPE-PEG-CA is DSPE-PEG modified with bile salts 2000 , and DSPE-PEG-MAN is DSPE-PEG modified with mannose 2000 ; the drug liposome encapsulates a water-soluble drug solution for treating liver diseases; the particle size of the liposome is 95 ± 0.5 nm; 0.22 < PDI < 0.25; the Zeta potential is 33 ± 4 mv, and the weight ratio of each component is: 22.5% - 44.25% of DSPC, 12.5% - 31.5% of cholesterol, 1% - 10% of DSPE-PEG-CA, 1% - 10% of DSPE-PEG-MAN, 5% of DSPE-PEG 2000 and 30% of DC-cholesterol; The drug is acetic acid, propionic acid, butyric acid or an acceptable salt thereof; the drug embedding rate is 76.6±0.47%, and the drug loading is 33.16±0.16%.

2. A method for preparing the liver-targeted drug liposome according to claim 1, characterized in that: The method comprises the following steps: (1) Modification of DSPE-PEG with bile salt 2000 Obtain DSPE-PEG-CA; (2) Modification of DSPE-PEG with mannose 2000 Obtain DSPE-PEG-MAN; (3) Add the following raw materials in sequence: DSPC, cholesterol, DSPE-PEG-CA, DSPE-PEG-MAN, DSPE-PEG 2000 , DC-cholesterol, mix well and transfer to a round-bottom flask with a micro-glass syringe. After pre-mixing, shake well in a constant temperature shaker to make all reagents thermodynamically uniform. At this time, the solution is completely clear; (4) High-pressure rotary evaporation to obtain a film with uniform thickness and distribution; (5) adding a water-soluble drug solution to the film formed in step (4), and obtaining a solution by ultrasonication; (6) Use a micro-glass syringe to draw the solution obtained in step (5), extrude it back and forth in a liposome extruder, and leave the extruded liposomes overnight to stabilize the liposome particles.

3. The preparation method according to claim 2, characterized in that: The concentration of the water-soluble drug solution in step (5) is 20-60 mg / mL.

4. The preparation method according to claim 3, characterized in that: The weight ratio of each component in step (3) is: 22.5%~44.25% DSPC, 12.5%~31.5% cholesterol, 1%~10% DSPE-PEG-CA, 1%~10% DSPE-PEG-MAN, 5% DSPE-PEG 2000 and 30% DC-cholesterol; the mother liquor concentration of the drug aqueous solution in step (5) is 40~60mg / mL.

5. The preparation method according to claim 4, characterized in that: The weight ratio of each component in step (3) is 40.5% DSPC, 13.5% cholesterol, 1% DSPE-PEG-CA, 10% DSPE-PEG-MAN, and 5% DSPE-PEG 2000 and 30% DC-cholesterol, the concentration of the drug aqueous solution in step (5) is 40 mg / mL.

6. Use of the liver-targeted drug liposome according to claim 1 in preparing a preparation for treating liver diseases, characterized in that: The liver disease is non-alcoholic fatty liver disease.

7. Use of the liver-targeted drug liposome according to claim 1 in preparing a preparation for inhibiting cell fat accumulation, characterized in that: The use described is an in vitro application.

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