A method for modifying phospholipids and its use in the preparation of liver-targeted liposomes

Abstract

The application discloses a method for modifying phospholipids and application thereof in preparation of liver-targeted liposomes, relates to the field of drug delivery, and first utilizes cholate and mannose to modify phospholipid molecules DSPE-PEG 2000 respectively 2000 Two raw materials DSPE-PEG 2000 -CA and DSPE-PEG 2000 -MAN for synthesizing liposomes are synthesized, and then cholesterols, DSPC and DC-cholesterols are added in a certain proportion to obtain liposomes with liver targeting property, which have high cell safety and liver cell targeting property.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of drug delivery, in particular to a method for modifying phospholipids and its application in preparing liver-targeted liposomes. BACKGROUND

[0002] DSPE-PEG 2000 is an amphiphilic (hydrophilic and lipophilic) molecule widely used in nanomedicine carriers, drug delivery systems and biomedical research. Its full name is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000, in which DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine) is a phospholipid with a hydrophobic fatty chain (stearic acid) part that can insert into the lipid bilayer. PEG2000 (Polyethylene Glycol 2000) is a polyethylene glycol part with a molecular weight of 2000, which is hydrophilic and can be stably suspended in water. DSPE-PEG 2000 is commonly used to prepare liposomes, nanoparticles and other drug delivery systems, and is particularly suitable for prolonging the life of drug carriers in the blood circulation in vivo and improving targeting. The PEG chain can prevent the immune system from recognizing and removing the carrier, thus helping However, in the liposomes used for targeting the liver, DSPE-PEG2000 also has some shortcomings, including: 1) low liver targeting efficiency, PEG modification can prolong the circulation time of liposomes, but at the same time it can also reduce the specific binding of liposomes to liver cells. The "stealth" effect of PEG can reduce the ability of liposomes to be recognized by Kupffer cells or hepatocytes in the liver, thereby reducing the liver targeting efficiency. 2) antibody-dependent immune activation, PEG molecules can induce the production of anti-PEG antibodies (anti-PEG antibodies, APAs), leading to increased clearance of the immune system from liposomes, a phenomenon known as accelerated blood clearance effect (ABC effect), which can further affect the liver targeting performance. 3) hindering of cell uptake by "bridge effect", DSPE-PEG2000 may form a physical barrier on the surface of the liposome due to the steric hindrance of the PEG chain, which can hinder the effective uptake of the liposome by liver cells. 4) protein corona effect in blood, PEGylated liposomes can adsorb specific plasma proteins (such as opsonin) in blood, which can change the biodistribution of the liposome and thus affect the liver targeting. 5) PEG shedding problem, under certain physiological conditions, the PEG chain in DSPE-PEG2000 can be shed, leading to the loss of long-circulating properties of the liposome and the accelerated absorption of the liposome by non-specific tissues.

[0003] And the further modification of DSPE-PEG is a technical means to solve the above-mentioned defects. Sodium cholate is a hydrophilic bile acid, which can target and bind to the ASBT overexpressed on the surface of intestinal epithelial cells and the NTCP receptor on the surface of hepatocytes, and then play a role in receptor-mediated endocytosis through the bile acid-ASBT or bile acid-NTCP pathway, thereby enhancing the penetration of nanoliposomes into the intestinal tract and the targeting ability of the liver. Mannose can specifically bind to the mannose receptor on the surface of liver macrophages, and target the liposomes to the macrophages in the liver.

[0004] The present application will be based on the defects in the prior art to study and explore the targeting of the liposome formed by DSPE-PEG. SUMMARY

[0005] To solve the above technical problems, the present application selects sodium cholate and mannose to modify the surface of nanoliposomes to construct an oral delivery system with liver targeting.

[0006] Definition:

[0007] DSPC: refers to 1,2-Distearoyl-sn-glycero-3-phosphocholine, Chinese name 1,2-Distearoyl-sn-glycero-3-phosphocholine, commonly known as distearoyl phosphatidylcholine. It is a phospholipid commonly used in the preparation of liposomes and nanomedicine delivery systems;

[0008] DSPE-PEG 2000 -NH2: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)-2000]-ammonium, commonly known as distearoyl phosphatidyl ethanolamine-polyethylene glycol-ammonium; It 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 effect.

[0009] DSPE-PEG-CA: distearoyl phosphatidyl ethanolamine polyethylene glycol-cholic acid. Since the PEG in the synthesis reaction is PEG2000, DSPE-PEG-CA appearing in the specification of the present application is DSPE-PEG 2000 -CA;

[0010] DSPE-PEG-MAN: distearoyl phosphatidyl ethanolamine polyethylene glycol mannose. Since the PEG in the synthesis reaction is PEG2000, DSPE-PEG-MAN appearing in the specification of the present application is DSPE-PEG2000 -MAN.

[0011] DC-Cholesterol (DC-Chol, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol) is a cholestrol-based cationic lipid commonly used in gene transfection and delivery systems. Its structural feature is to modify the cholestrol molecule into a derivative with a cationic amine group, thereby endowing it with the ability to bind to negatively charged nucleic acids (such as DNA or RNA).

[0012] Specifically:

[0013] The first aspect of the present application is to provide a method for modifying phospholipids, wherein the method is to modify DSPE-PEG 2000 , characterized in that the method comprises:

[0014] 1) activating cholic acid: dissolving cholic acid, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide methiodide (EDC) and N-hydroxysuccinimide (NHS) in dichloromethane, after ice bath reaction, stirring at room temperature overnight, after filtration and rotary evaporation, obtaining activated sodium cholate;

[0015] 2) dissolving DSPE-PEG 2000 -NH2 in DMSO, adding activated sodium cholate and triethylamine to completely dissolve, and then reacting;

[0016] 3) after the reaction is completed, the solution is placed in a dialysis bag and dialyzed with pure water as the medium;

[0017] 4) after dialysis is completed, the solution in the dialysis bag is collected and freeze-dried to obtain DSPE-PEG-CA.

[0018] Further, the ice bath reaction in step 1) is 20-40 min;

[0019] Further, in step 2), the DSPE-PEG 2000 -NH2 is dissolved in DMSO, and the addition ratio is 450-550 mg: 3 mL;

[0020] Further, the addition ratio of activated sodium cholate and triethylamine is 500-600 mg: 1 g;

[0021] Further, the reaction temperature is 38-45°C, and the reaction time is 1-3 h;

[0022] Further, in step 3), the molecular weight of the dialysis bag is MW = 800-1200 Da, and the dialysis time is 18-28 h.

[0023] The second aspect of the present application provides a modified phospholipid DSPE-PEG-CA, wherein the DSPE-PEG-CA is prepared by the method of the first aspect of the present application.

[0024] The third aspect of the present application provides a method for modifying phospholipid, wherein the method is for modifying DSPE-PEG by mannose 2000 , and the method comprises:

[0025] 1) dissolving DSPE-PEG 2000 -NH2 in DMSO, adding mannose and triethylamine to completely dissolve, and reacting the mannose and triethylamine;

[0026] 2) after the reaction is completed, placing the solution in a dialysis bag and dialyzing with pure water as the medium;

[0027] 3) after the dialysis is completed, freeze-drying the solution in the dialysis bag to obtain DSPE-PEG-MAN.

[0028] Further, in step 1), the DSPE-PEG-NH2 is dissolved in DMSO and the addition ratio of DMSO is 450-550 mg: 3 mL.

[0029] Further, the addition ratio of mannose and triethylamine is 500-600 mg: 1 g.

[0030] Further, the reaction temperature is 38-45℃ and the reaction time is 1-3 h.

[0031] Further, in step 2), the molecular weight of the dialysis bag is MW=800-1200 Da and the dialysis time is 18-28 h.

[0032] The fourth aspect of the present application provides a modified phospholipid DSPE-PEG-MAN, wherein the DSPE-PEG-MAN is prepared by the method of the third aspect of the present application.

[0033] The fifth aspect of the present application is the use of the DSPE-PEG-CA of the second aspect and the DSPE-PEG-MAN of the fourth aspect in the preparation of liver-targeting liposomes, and the use comprises the following steps:

[0034] (1) sequentially adding the following raw materials: DSPC, cholesterol, DSPE-PEG-CA, DSPE-PEG-MAN, DSPE-PEG-NH2, and DC-cholesterol, mixing, and then transferring to a round-bottom flask with a micro glass syringe, mixing in advance, and then uniformly shaking in a constant-temperature shaker to make all reagents thermodynamically distributed uniformly, at which time the solution is completely clear;

[0035] (2) high pressure rotary evaporation to obtain a film with uniform thickness and distribution;

[0036] (3) adding a buffer solution to the film formed in step (3) to obtain a solution by ultrasonic treatment;

[0037] (4) using a micro glass syringe to suck the solution obtained in step (3) and repeatedly extruding in a liposome extruder, and then allowing the liposome to stand overnight to stabilize the liposome particles.

[0038] Further, the weight ratio of each component in step (1) is 22.5% to 44.25% of DSPC, 12.5% to 31.5% of cholesterol, 1% to 10% of DSPE-PEG-CA, 1% to 10% of DSPE-PEG-MAN, and 30% of DC-cholesterol.

[0039] Further, the operation of step (2) is performed under the conditions of a pressure of 400 to 600 mbar, a rotation speed of 80 to 120 rpm, and a temperature of 45 to 50°C.

[0040] The DSPE-PEG-CA obtained by the method of the first aspect and the DSPE-PEG-CA obtained by the method of the third aspect are used as carriers in the preparation of drugs for treating non-alcoholic fatty liver.

[0041] The beneficial effects of the present application include:

[0042] 1) For the first time, two raw materials, DSPE-PEG 2000 -CA and DSPE-PEG 2000 -MAN, are synthesized to obtain liposomes with liver targeting properties in a certain proportion with cholesterol, DSPC, and DC-cholesterol;

[0043] 2) The five kinds of liposomes synthesized in the present application have no obvious toxicity to myocardial cells H9c2, intestinal epithelial cells Caco-2, liver macrophages Kupffer, and hepatocytes AML-12, and are suitable as sodium acetate oral nanocarriers with high cell safety;

[0044] 3) The fluorescence intensity of sodium cholate / mannose double-labeled fluorescent liposomes in the liver is significantly higher than that of single-labeled or unlabeled liposomes, indicating that the sodium cholate / mannose liposomes synthesized in the present experiment are a kind of sodium acetate oral delivery nanomaterials with storage stability, cell safety, penetration of intestinal epithelial barrier, and liver targeting;

[0045] 4) Sodium cholate / mannose double-labeled liposomes containing sodium acetate effectively alleviate the lipid deposition and cell inflammation of NAFLD cells;

[0046] 5) After the mice were given double-labeled sodium acetate liposomes by gavage, the liver lipid deposition was significantly improved, the phosphorylation level of liver AMPKα protein was significantly activated, the transcription of Acc1 and Srebf1 was inhibited, CPT1α expression was promoted, and downstream lipid metabolism genes were regulated, effectively reducing liver lipid deposition. In terms of liver inflammation and oxidative stress, after the NAFLD mice were given double-labeled sodium acetate liposomes by gavage for 10 weeks, the phosphorylation of NFκB protein was significantly inhibited, the release of downstream inflammatory factors was reduced, and liver inflammation and oxidative stress were effectively relieved. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 Schematic diagram of synthesis of sodium acetate liposomes modified by cholic acid and mannose;

[0048] Figure 2 DSPE-PEG-CA synthesis, A is the synthesis route, B is the infrared spectrum;

[0049] Figure 3 DSPE-PEG-MAN synthesis, A is the synthesis route, B is the infrared spectrum;

[0050] Figure 4 Release of sodium acetate from liposomes in different groups in serum, simulated gastric juice and simulated intestinal juice after incubation; A: release of sodium acetate in serum; B: release of sodium acetate in simulated gastric juice; C: release of sodium acetate in simulated intestinal juice; different letters represent significant difference (p<0.05), and the same letter represents no significant difference (p>0.05);

[0051] Figure 5 Liposome entrapment efficiency and drug loading under different concentrations of sodium acetate stock solution, A: entrapment efficiency; B: drug loading;

[0052] Figure 6 TEM images of five kinds of liposomes, the scale is 200 nm;

[0053] Figure 7 Cell viability of four kinds of cells incubated with sodium acetate liposomes and sodium acetate solution at concentrations of 1-1500 μM for 24 h, A: H9c2 cells; B: Caco-2 cells; C: Kupffer cells; D: AML-12 cells;

[0054] Figure 8 Sodium acetate liposomes or sodium acetate relieve NAFLD cell lipid deposition A: effect of different liposomes or sodium acetate on cell TG content; B: effect of different liposomes or sodium acetate on cell TC content; C: oil red O staining results of different groups; D: effect of different liposomes or sodium acetate on the relative area of lipid droplets in NAFLD cells; TG, TC and relative lipid droplet area were subjected to significant analysis, # indicates significant difference between the model group and the control group,## p<0.01; * indicates that the experimental group is significantly different from the modeling group, * p<0.05, ** p<0.01; + indicates that the difference between groups is significant, + p<0.05, ++ p<0.01;

[0055] Figure 9 Effect of sodium acetate liposomes or sodium acetate on NAFLD cell inflammatory response related genes, A: IL-1β mRNA relative expression; B: IL-6 mRNA relative expression; C: TNF-α mRNA relative expression; D: IL-4 mRNA relative expression; # indicates that the difference between the modeling group and the control group is significant, ## p<0.01; * indicates that the experimental group is significantly different from the modeling group, * p<0.05, ** p<0.01; + indicates that the difference between groups is significant, + p<0.05, ++ p<0.01;

[0056] Figure 10 In vivo fluorescence imaging of mice after gavage with CY7 liposomes or CY7;

[0057] Figure 11 In vivo fluorescence intensity of main organs of mice after gavage with CY7 liposomes or CY7, A: fluorescence imaging of each organ; B: fluorescence intensity-time curve of liver of mice in each group; C: AUG of fluorescence intensity-time curve of liver of mice 0-24 ; D: fluorescence intensity-time curve of intestinal tract of mice in each group; E: AUG of fluorescence intensity-time curve of intestinal tract of mice 2-24 ;

[0058] Figure 12 Effect of sodium acetate liposomes on body weight change and body weight gain of NAFLD mice: A: body weight growth curve; B: body weight gain; # indicates that the difference between the modeling group and the control group is significant, ## p<0.01; * indicates that the experimental group is significantly different from the modeling group, * p<0.05, ** p<0.01, + indicates that the difference between groups is significant, + p<0.05;

[0059] Figure 13 Effect of sodium acetate liposomes on glucose tolerance of NAFDL mice

[0060] A: OGTT curve; B: area under the OGTT curve; #Indicates that the model group is significantly different from the control group, ## p<0.01; * Indicates that the experimental group is significantly different from the model group, ** p<0.01, + Indicates that the difference between groups is significant, ++ p<0.01;

[0061] Figure 14 Effect of sodium acetate liposome on blood lipid levels in NAFLD mice, A: triglyceride; B: cholesterol; C: low-density lipoprotein cholesterol; D: high-density lipoprotein cholesterol; # Indicates that the model group is significantly different from the control group, ## p<0.01; * Indicates that the experimental group is significantly different from the model group, * p<0.05, ** p<0.01, + Indicates that the difference between groups is significant, + p<0.05, ++ p<0.01;

[0062] Figure 15 Effect of sodium acetate liposome on fat tissue content in NAFLD mice, A: white fat ratio; B: epididymal fat tissue H&E staining results (400x, scale 100 μm); # Indicates that the model group is significantly different from the control group, ## p<0.01; * Indicates that the experimental group is significantly different from the model group, * p<0.05, ** p<0.01, + Indicates that the difference between groups is significant, ++ p<0.01;

[0063] Figure 16 Effect of sodium acetate liposome on liver pathological morphology in NAFLD mice, A: liver appearance (scale 1 cm); B: liver tissue H&E staining (100x, scale 200 μm); C: relative area ratio of liver lipid droplets based on H&E staining sections (n=8); D: liver tissue oil red O staining (100x, scale 200 μm); E: relative area ratio of liver lipid droplets based on oil red O staining (n=8); # Indicates that the model group is significantly different from the control group, ## p<0.01; * Indicates that the experimental group is significantly different from the model group, ** p<0.01, + Indicates that the difference between groups is significant, + p<0.05, ++ p<0.01;

[0064] Figure 17 Effect of sodium acetate liposome on lipid deposition and oxidative stress in the liver of NAFLD mice, A: liver index (%); B: liver triglyceride content; C: liver cholesterol content; D: liver malondialdehyde content; E: liver superoxide dismutase content; # Significant difference between the model group and the control group, ## p<0.01; * Significant difference between the experimental group and the model group, * p<0.05, ** p<0.01; + Significant difference between groups, + p<0.05, ++ p<0.01;

[0065] Figure 18 Effect of sodium acetate liposome on the expression of liver lipid synthesis and metabolism related genes and AMPK protein phosphorylation in NAFLD mice, A-E: Relative expression of liver lipid synthesis and metabolism related genes (Acc1, Fasn, Slc27a2, Srebf1, CPT1α); F: Western blotting of AMPKα, p-AMPKα; G: Relative expression of p-AMPKα / AMPKα; # Significant difference between the model group and the control group, ## p<0.01; * Significant difference between the experimental group and the model group, ** p<0.01; + Significant difference between groups, + p<0.05, ++ p<0.01

[0066] Figure 19 Effect of sodium acetate liposome on liver function in NAFLD mice, A: serum glutathione transaminase; B: serum glutathione transaminase; C: liver glutathione transaminase; D: liver glutathione transaminase; # Significant difference between the model group and the control group, ## p<0.01; * Significant difference between the experimental group and the model group, * p<0.05, ** p<0.01; + Significant difference between groups, + p<0.05, ++ p<0.01

[0067] Figure 20Effect of sodium acetate liposome on liver macrophage inflammatory infiltration of NAFLD mice, A: mouse liver tissue section F4 / 80 staining (200x, scale 100 μm); B: relative area of F4 / 80 positive site; # Significant difference between the model group and the control group, ## p<0.01; * Significant difference between the experimental group and the model group, ** p<0.01; + Significant difference between groups, + p<0.05, ++ p<0.01

[0068] Figure 21 Effect of sodium acetate liposome on inflammation-related cytokines and NFκB protein phosphorylation in liver of NAFLD mice, A-D: relative expression of inflammation-related cytokines in liver, IL-1β, IL-6, TNF-α, IL-4, respectively; E: Western blotting of p-NFκB, NFκB; F: relative expression of p-NFκB / NFκB; # Significant difference between the model group and the control group, ## p<0.01; * Significant difference between the experimental group and the model group, ** p<0.01; + Significant difference between groups, + p<0.05, ++ p<0.01. DETAILED DESCRIPTION

[0069] The concept and the technical effects of the present application will be further described below in combination with specific examples, so as to fully understand the purposes, features and effects of the present application. The methods are all conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative examples of the present application and the descriptions thereof are used to explain the present application and do not constitute undue limitations on the present application. It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.

[0070] Example 1 Preparation of liver-targeting sodium acetate liposome (NaA@CA / MAN-LPs)

[0071] The preparation process is shown in Figure 1

[0072] 1. Synthesis of DSPE-PEG 2000 -CA

[0073] ​Sodium cholate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDC) and N-hydroxysuccinimide (NHS) were dissolved in dichloromethane. After reaction in ice bath for 30 min and stirring at room temperature overnight, the activated sodium cholate was obtained by filtration and rotary evaporation. 500 mg of DSPE-PEG2000-NH2 was dissolved in 3 mL of DMSO, and the activated sodium cholate (550 mg) and triethylamine (1 g) were added and completely dissolved. After reaction at 40 °C for 2 h, the solution was dialyzed in a dialysis bag (MW = 1000 Da) with pure water as the medium for 24 h. After dialysis, the solution in the dialysis bag was collected and lyophilized to obtain DSPE-PEG 2000 -CA (see synthesis steps in Figure 2 A).

[0074] The infrared spectrum of DSPE-PEG-CA is shown in Fig. 1. Figure 2 B shows that, after modification of DSPE-PEG 2000 -NH2 with sodium cholate, a characteristic peak of the C=O stretching vibration of the carboxylate group appeared at 1551 cm -1 . In addition, 3425 cm -1 corresponds to the hydroxyl group in the sodium cholate molecule, and 2850-2917 cm -1 corresponds to the C-H stretching vibration in the stearoyl alkyl chain of DSPE-PEG. The stretching vibration of the P=O bond of the phosphate group appears at 1245 cm-1, and the C-O bond stretching vibration of the PEG chain appears at 1108 cm -1 . The C-H bending vibration peak of the alkyl chain appears at 1466 cm -1 . The above results show that sodium cholate has been successfully conjugated to the DSPE-PEG-NH2 molecule.

[0075] 2. Synthesis of DSPE-PEG 2000 -MAN

[0076] The synthesis steps and results of DSPE-PEG-MAN are as follows:

[0077] 500 mg of DSPE-PEG2000-NH2 was dissolved in 3 mL of DMSO, and 550 mg of mannose and 1 g of triethylamine were added and completely dissolved. After reaction at 40 °C for 2 h, the solution was dialyzed in a dialysis bag (MW = 1000 Da) with pure water as the medium for 24 h. After dialysis, the solution in the dialysis bag was lyophilized to obtain DSPE-PEG-MAN (synthesis route as shown in Figure 3 A).

[0078] The results of FTIR detection of DSPE-PEG-MAN are shown in Fig. 2. Figure 3B shows: 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 C-H bond of the stearoyl alkyl chain in DSPE, 1739 cm -1 corresponding to the aldehyde C=O group. The C-H 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 , and after mannose modification, the peak becomes wide and appears at 3428 cm -1 . The P=O bond of the phosphate group appears at 1251 cm -1 , the C-O bond of the PEG chain appears at 1109 cm -1 , and the above results show that DSPE-PEG-MAN is successfully synthesized.

[0079] 3. Preparation of liver-targeted sodium acetate liposomes

[0080] Intestinal penetration and liver-targeted high-concentration sodium acetate liposomes (NaA@CA / MAN-LPs) were prepared by the thin film hydration method.

[0081] All raw materials and reagents were stored at -20°C, and the preparation process was carried out on ice.

[0082] (1) According to the ratio of liposomes, the following raw materials were added in order: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, DSPE-PEG 2000 -CA, DSPE-PEG 2000 -MAN, DSPE-PEG 2000 , DC-cholesterol, mix well, then transfer to a round-bottom flask with a micro glass syringe, mix well for 10 min, then shake well at 37°C constant temperature shaker for 10 min, so that all reagents are evenly distributed thermodynamically, and the solution is completely clear.

[0083] (2) Perform spin evaporation under the conditions of pressure 500 mbar, rotation speed 100 rpm, and temperature 50°C. After spin evaporation, a film with uniform thickness and uniform distribution is obtained.

[0084] (3) Add sodium acetate hydration medium to completely cover the organic film, and ultrasonic for 2 min, so that the solution becomes milky white.

[0085] (4) Assemble the Liposome Extruder (Avanti Polar Lipids), place a piece of wetted carbon filter membrane support on both black gaskets, select 100 nm filter, wetted and placed between the two filter membrane supports, fix the assembled Liposome Extruder on the support, and finally place the whole on the heating plate, keeping the temperature at 50°C.

[0086] (5) Take 1 mL of the Liposome solution with a micro glass syringe, and extrude it back and forth in the Liposome Extruder for 21 times, and store the extruded Liposome at 4°C overnight to stabilize the Liposome particles.

[0087] (6) Remove the un-wrapped sodium acetate with ultrafiltration, then freeze the Liposome with liquid nitrogen, weigh, and add PBS before use.

[0088] 7) The identification of the sodium acetate Liposome includes: HPLC detection of the sodium acetate concentration, calculation of the encapsulation efficiency and drug loading; particle size, PDI, Zeta of the Liposome; TEM observation of the Liposome morphology.

[0089] 4. Optimization of the composition of NaA@CA / MAN-LPs by orthogonal experiment

[0090] (1) Optimization of the ratio of step 1) in the above method, the composition of the Liposome includes: DSPC, cholesterol, DSPE-PEG 2000 -CA, DSPE-PEG 2000 -MAN, and DSPE-PEG 2000 -CA, DSPE-PEG 2000 -MAN. To obtain a Liposome with high encapsulation efficiency and good stability, the concentration of the sodium acetate stock solution is fixed at 20 mg / mL, and the ratio of DSPC: cholesterol, DSPE-PEG 2000 -CA, DSPE-PEG

[0091] Table 1. Optimization factor level table for the synthesis of sodium acetate Liposome

[0092]

[0093] The grouping of the orthogonal experiment is shown in Table 2:

[0094] Table 2. Orthogonal experiment grouping table

[0095]

[0096] (2) Composition optimization index

[0097] a. Sodium acetate encapsulation efficiency of the Liposome

[0098] a.1Establishment of high performance liquid chromatography (HPLC) determination method

[0099] The HPLC conditions are as follows: the chromatographic column is selected as Thermol Hypersil ODS-2 C18 column (4.6 mm*200 mm, 5 um), the mobile phase is 0.01 mol / L ammonium dihydrogen phosphate aqueous solution, the pH is adjusted to 3.0 by 1 mol / L phosphoric acid solution, the flow rate is 1.0 mL / min, the injection volume is 10 μL, the detection wavelength is 215 nm, and the column temperature is 30 °C.

[0100] a.2Preparation of standard curve

[0101] Precisely weigh sodium acetate into a 10 mL volumetric flask, dilute with PBS, and prepare sodium acetate standard solutions with concentrations of 0.01, 0.025, 0.05, 0.1, 0.2, and 0.5 mg / mL, respectively. Determine each sample according to the HPLC conditions, and record the peak area. Take the main peak area (A) as the vertical coordinate and the concentration (C) as the horizontal coordinate to draw the standard curve of the linear relationship of A-C. Dissolve sodium acetate in serum solution, simulated gastric juice, and simulated intestinal juice to prepare sodium acetate solutions with concentrations of 0.01, 0.025, 0.05, 0.1, 0.2, and 0.5 mg / mL. Detect the peak area-time standard curve of acetic acid in serum, simulated gastric juice, and simulated intestinal juice according to the HPLC conditions.

[0102] a.3Determination of the encapsulation efficiency of sodium acetate liposomes by ultrafiltration method

[0103] Take an appropriate amount of sodium acetate liposome solution and place it in an activated ultrafiltration tube (MYCO = 3K Da, Millipore). Centrifuge at 12000 rpm for 10 min. Take out the solution in the ultrafiltration tube and detect the free sodium acetate content according to the HPLC method in "2.3.5.1". Calculate the encapsulation efficiency of sodium acetate in liposomes according to the following formula.

[0104]

[0105] b.Particle size, PDI, Zeta-potential, and drug loading of NaA@CA / MAN-LPs

[0106] 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 the drug loading of liposomes DL% of NaA@CA / MAN-LPs were detected by ultrafiltration method and high performance liquid chromatography. The calculation formula of the drug loading of liposomes is as follows:

[0107]

[0108] c.Stability of NaA@CA / MAN-LPs in simulated body fluids

[0109] The sterile fetal bovine serum was diluted 10 times with PBS (pH 7.4) to prepare a simulated serum solution. The artificial simulated gastric juice was prepared by adding 0.5 g pepsin to 10 mL pure water, dissolving and then adding 0.82 mL of 1 mol / L hydrochloric acid solution, mixing and then diluting to 50 mL, pH = 1.2. The artificial simulated intestinal juice was prepared by adding 0.34 g potassium phosphate dibasic to 25 mL water, adjusting the pH to 6.8 with 0.4% NaOH; 0.5 g trypsin was dissolved in water, and the two solutions were mixed and diluted to 50 mL.

[0110] 100 μL of NaA@CA / MAN-LPs were taken and mixed with 900 μL of simulated gastric juice, simulated intestinal juice and serum solution, respectively, and incubated at 37°C with constant shaking at 1000 rpm. The sample solution was collected after 2 h for the group incubated with simulated gastric juice, and after 6 h for the groups incubated with simulated intestinal juice and serum solution. The particle size, PDI and Zeta-potential of the collected sample solution were determined by laser particle size analyzer, and the release rate of sodium acetate after incubation in different medium solutions was detected by HPLC method to analyze the changes in particle size and embedding rate of the liposomes before and after incubation, and to characterize the stability of the liposomes in the gastrointestinal tract and serum solution.

[0111] Orthogonal experiment results:

[0112] Table 3 shows the sodium acetate embedding rate and drug loading of the liposomes in different groups in the orthogonal experiment. The results show that different DSPC: cholesterol ratios, as well as DSPE-PEG 2000 -CA and DSPE-PEG 2000 The concentration of MAN has an important influence on the embedding rate and drug loading of the liposomes. The sodium acetate embedding rate of the nine groups of liposomes in the orthogonal experiment was between 70.52% and 81.15%, and the order from high to low was: 3 > 7 > 1 > 8 > 4 > 2 > 9 > 6 > 5. The drug loading of the nine groups of liposomes in the orthogonal experiment was between 20.86% and 31.58%, and the order from high to low was: 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, and the subsequent evaluation of the particle stability of the nine groups of liposomes after incubation in serum solution, simulated gastric juice and simulated intestinal juice will determine the optimal ratio of the composition of the liposomes.

[0113] Table 3 shows the sodium acetate embedding rate and drug loading of the liposomes in different groups in the orthogonal experiment. The results show that different DSPC: cholesterol ratios, as well as DSPE-PEG

[0114]

[0115]

[0116] Note: The sodium acetate embedding rate and drug loading of nine groups of liposomes were analyzed for significance, respectively. Different letters in the same column indicate significant difference (p <0.05), and the same letter indicates no significant difference (p >0.05).

[0117] From the results of the particle size changes of liposomes with different compositions in the simulated body fluid after incubation, the initial particle size of the nine groups of liposomes was about 100 nm. After incubation in serum, simulated gastric juice and simulated intestinal juice for 6 h or 2 h, the particle size of the liposomes increased to varying degrees. After incubation in serum for 6 h, the particle size of the liposomes from small to large was 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 juice for 2 h, the particle size of the different groups of liposomes from small to large was 7<9<3<8<1<2<6<4<5, and the particle size of liposome 7 and liposome 9 (107.21±2.40 nm, 128.70±1.59 nm) was significantly lower than that of the remaining seven groups. After incubation in simulated intestinal juice for 6 h, the particle size of the different groups of liposomes from small to large was 4<8<1<7<6<5<9<2<3, and the particle size of the remaining seven groups of liposomes had no significant difference except for liposome 2 and liposome 3. Table 5 shows the PDI changes of the nine groups of liposomes after incubation in simulated body fluid. The initial PDI of the nine groups of liposomes was between 0.20 and 0.22. After incubation in serum, simulated gastric juice and simulated intestinal juice, the PDI of all liposomes was <0.4, indicating that the particle size distribution of all liposomes was uniform after incubation in simulated body fluid, and there was no obvious aggregation. From the potential changes of the nine groups of liposomes in the simulated body fluid after incubation in Table 6, the initial Zeta potential of the liposomes was positive charge. After incubation in serum, simulated gastric juice and simulated intestinal juice, the charge of the liposomes remained positive.

[0118] Table 4 Particle size changes of different groups of liposomes in serum, simulated gastric juice and intestinal juice in orthogonal experiment

[0119]

[0120] Note: The original particle size of the nine groups of liposomes and the particle size of the liposomes after incubation in serum, simulated gastric juice and simulated intestinal juice were analyzed for significance, respectively. Different letters in the same column indicate significant difference (p <0.05), and the same letter indicates no significant difference (p >0.05).

[0121] Table 5 PDI changes of different groups of liposomes in serum, simulated gastric juice and intestinal juice in orthogonal experiment

[0122]

[0123] Note: The original particle size of the nine groups of liposomes and the PDI results after incubation in serum, simulated gastric juice, and simulated intestinal juice were analyzed for significance, respectively. Different letters in the same column indicate significant differences (p < 0.05), and the same letter indicates no significant difference (p > 0.05).

[0124] Table 6 Zeta-potential changes of different groups of liposomes in serum, simulated gastric juice, and intestinal juice in orthogonal experiments

[0125]

[0126] Note: The original particle size of the nine groups of liposomes and the Zeta potential after incubation in serum, simulated gastric juice, and simulated intestinal juice were analyzed for significance, respectively. Different letters in the same column indicate significant differences (p < 0.05), and the same letter indicates no significant difference (p > 0.05).

[0127] Results are shown in Figure 4 After 6h incubation in serum, the sodium acetate release of the nine groups of liposomes was between 3.61% and 5.27%, among which the sodium acetate release of liposome 4 and liposome 7 was significantly lower than that of the other groups, with release amounts of 3.73% ± 0.19% and 3.61% ± 0.32% (A), respectively. Figure 4 After 2h incubation in simulated gastric juice, the sodium acetate release of the nine groups of liposomes was between 12.57% and 16.51%, among which the sodium acetate release of liposome 2 and liposome 7 was significantly lower than that of the other groups, with release amounts of 12.57% ± 0.32% and 12.70% ± 0.37% (B), respectively. Figure 4 Similarly, after 6h incubation in simulated intestinal juice, the sodium acetate release of the nine groups of liposomes was between 5.16% and 7.73%, among which the release of liposome 3 and liposome 7 was significantly lower than that of the other seven groups, with release amounts of 5.16% ± 0.08% and 5.51% ± 0.06% (C), respectively. Figure 4 The above results show that compared with the other groups of liposomes, liposome 4 and 7 are more stable in serum, liposome 2 and 7 are more stable in simulated gastric juice, and liposome 3 and 7 are more stable in simulated intestinal juice.

[0128] To optimize the DSPC: cholesterol and DSPE-PEG 2000 -CA and DSPE-PEG 2000The proportion of MAN was optimized by orthogonal optimization method. According to the above results, the sodium acetate embedding rate of liposome 7 was 78.42% ± 0.30%, ranking second among the nine groups of liposomes, and the drug loading was 29.70% ± 0.11%, ranking second among the nine groups of liposomes, indicating that under the composition ratio of liposome 7, the prepared sodium acetate liposome had a higher sodium acetate content. To study the stability of liposomes in different body fluids, the particle size, PDI, Zeta potential and sodium acetate release of the nine groups of liposomes were detected after incubation in serum, simulated gastric juice and simulated intestinal juice. According to the above results, compared with other groups, the particle size of liposome 7 increased insignificantly after incubation in serum, simulated gastric juice and simulated intestinal juice, the particle size distribution was uniform and still had a positive charge. In addition, compared with other groups of liposomes, the sodium acetate release in liposome 7 was the lowest after incubation in serum, simulated gastric juice and simulated intestinal juice, which was only 3.61% ± 0.32%, 12.70% ± 0.37% and 5.51% ± 0.06%. In summary, under the liposome ratio of liposome 7, sodium acetate liposome has the strongest stability, effectively preventing the adsorption of enzymes in gastrointestinal juice and proteins in serum on the liposome and the destruction of the structure of the liposome, so the most suitable ratio of sodium acetate liposome is DSPE: cholesterol ratio of 3:1, and 1% of DSPE-PEG 2000 -CA and 10% of DSPE-PEG 2000 -MAN.

[0129] Example 2 Sodium acetate stock solution concentration ramping experiment

[0130] After orthogonal experiment optimization, the ratio of each component of the liposome was determined, and the concentration of the sodium acetate stock solution was optimized to maximize the sodium acetate drug loading of the liposome. 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 method to calculate the sodium acetate encapsulation efficiency of the liposome. The liposome was freeze-dried by liquid nitrogen rapid freezing method, and the sodium acetate drug loading of the liposome was calculated according to the mass of the liposome.

[0131] The optimal ratio of sodium acetate liposome 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, and the results are shown in Table 3. Figure 5With the increase of sodium acetate stock solution concentration from 20 mg / mL to 60 mg / mL, the entrapment efficiency of liposomes also increased from 71.09% to 79.36%, and when the concentration of sodium acetate stock solution exceeded 40 mg / mL, the increase of entrapment efficiency of liposomes tended to be flat (A), and at this time the loading capacity of liposomes was the highest, 33.07% (B), so when preparing liposomes with high entrapment efficiency and loading capacity, the concentration of sodium acetate stock solution selected was 40 mg / mL. Figure 5 A), and at this time the loading capacity of liposomes was the highest, 33.07% (B), so when preparing liposomes with high entrapment efficiency and loading capacity, the concentration of sodium acetate stock solution selected was 40 mg / mL. Figure 5 B).

[0132] Example 3 Preparation and characterization of liposomes with different modifications

[0133] After optimization by orthogonal experiment and sodium acetate stock solution climbing experiment, the optimal ratio of sodium cholate, mannose modified sodium acetate liposome composition and the concentration of sodium acetate stock solution were determined. On this basis, the following different modified liposomes were prepared: ① sodium cholate, mannose modified sodium acetate liposomes (NaA@CA / MAN-LPs); ② sodium cholate modified sodium acetate liposomes alone (NaA@CA-LPs); ③ mannose modified sodium acetate liposomes alone (NaA@MAN-LPs); ④ unmodified sodium acetate liposomes (NaA@LPs); ⑤ sodium cholate, mannose liposomes without sodium acetate (CA / MAN-LPs). The specific composition of different modified liposomes is shown in Table 7.

[0134] Table 7 Composition of sodium acetate liposomes with different modifications

[0135]

[0136] The particle size, PDI and Zeta potential of the five kinds of liposomes were measured by dynamic light scattering method, the entrapment efficiency and drug loading capacity of the liposomes were detected by HPLC, and the morphology of the liposomes was observed by transmission electron microscopy (TEM). The specific operation steps of TEM are as follows: take the diluted liposomes, drop onto the copper mesh, stand for 2 min, use filter paper to absorb the liquid on the surface of the copper mesh, add 2% phosphotungstic acid negative staining for 5 min, then bake the copper mesh, and observe the morphology of different types of liposomes by JEM-1400Flash transmission electron microscope.

[0137] After orthogonal experiment and sodium acetate stock solution ramping experiment, the composition ratio of sodium acetate liposome with high embedding rate and loading capacity was determined. On this basis, five kinds of different modified liposomes were synthesized, which were ① sodium cholate and mannose modified sodium acetate liposome (NaA@CA / MAN-LPs); ② sodium cholate modified sodium acetate liposome (NaA@CA-LPs) alone; ③ mannose modified sodium acetate liposome (NaA@MAN-LPs) alone; ④ unmodified sodium acetate liposome (NaA@LPs); ⑤ sodium cholate and mannose liposome without sodium acetate (CA / MAN-LPs). The particle size instrument and embedding rate and drug loading were detected for the five kinds of liposomes, and the results were shown in Table 8. Figure 6 The particle size of the five kinds of liposomes was about 100 nm, PDI was less than 0.25, and Zeta potential was positive. Except for CA / MAN-LPs, the embedding rate of NaA@CA / MAN-LPs, NaA@CA-LPs, NaA@MAN-LPs and NaA@LPs was between 76.6% and 81.57%, and the drug loading was between 33.16% and 37.94%, which indicated that the five kinds of liposomes synthesized were about 100 nm, the particles were uniformly distributed and the nanoliposomes were positively charged, and had high sodium acetate embedding rate and liposome drug loading. Transmission electron microscopy was used to observe and take pictures of the appearance of the five kinds of liposomes, and the results were shown in Figure 6 The appearance of the five kinds of liposomes was spherical, the particle size was about 100 nm, which was consistent with the particle size measurement results, and the particles were uniformly distributed without aggregation or clumping.

[0138] Table 8 Characterization of different types of sodium acetate liposomes

[0139]

[0140] Example 4 Biological safety of sodium acetate liposome

[0141] 1. Cell culture

[0142] Caco-2 was resuscitated and subcultured using DMEM complete medium, AML-12 and H9c2 were resuscitated and subcultured using DMEM / F12 complete medium, and Kupffer was resuscitated and subcultured using RPMI 1640 complete medium. Except for the different types of culture medium, the other experimental operation steps were the same.

[0143] 2. Liposome safety

[0144] 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 were detected using the CCK-8 assay. Cell suspensions in good growth condition were used to adjust the cell density of H9c2, Caco-2, Kupffer, and AML-12 cells to 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a rate of 100 μL per well into 96-well cell culture plates. After the cells reached approximately 60% confluence, the culture medium was aspirated, and cell culture medium containing one of five different liposomes or sodium acetate solutions (1, 10, 100, 200, 500, 1000, and 1500 μM) was added to each well. The cells were then cultured for 24 h. After the culture was completed, the culture medium was aspirated, and 100 μL of the corresponding complete culture medium and 10 μL of CCK-8 solution were added to each well. The plates were then incubated at 37°C for 30 min. The absorbance of each well was measured at 450 nm using a multi-mode microplate reader, and cell viability was calculated according to the formula.

[0145]

[0146] Cell safety results are shown in Figure 7 The results showed that within the sodium acetate concentration range of 1–1500 μM, the four cell types ( Figure 7 The cell viability after incubation in five types of liposomes and sodium acetate solution for 24 hours was greater than 85%, indicating that the prepared double-labeled sodium acetate liposomes, single-labeled sodium acetate liposomes, unmodified sodium acetate liposomes and sodium acetate-free liposomes had good safety for cardiomyocytes, intestinal epithelial cells, liver macrophages and hepatocytes, and are suitable as nanocarriers for oral delivery of sodium acetate.

[0147] Example 5: Sodium acetate liposomes reduce lipid deposition in liver cell lines and inhibit inflammation.

[0148] 1. Cell Culture

[0149] Hepatocytes AML-12 were revived and cultured using DMEM / F12 complete medium; liver macrophages Kupffer were revived and passaged using RPMI-1640 complete medium.

[0150] 2. Cell modeling

[0151] AML-12 cells in good logarithmic growth phase were collected by centrifugation and counted. The cells were then divided into cells at a concentration of 1×10⁻⁶. 6 Cells were inoculated into 6-well plates, and 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. Control cells were treated with complete control medium containing BSA.

[0152] 3. Sodium acetate liposomes alleviate NAFLD cell lipid deposition

[0153] Select AML-12 cells, take the logarithmic growth period of good cell plating, culture and starvation treatment for 12h, when the lipid induction, the cells are divided into six groups, the first group is added with equal volume of PBS as a blank control group; the second group is added with lipid induction medium as a NAFLD modeling group; the third group is added with sodium cholate / mannose double-labeled sodium acetate liposomes (NaA@CA / MAN-LPs) in the lipid induction medium; the fourth group is added with sodium acetate solution (NaA) in the lipid induction medium; the fifth group is added with unmarked sodium acetate liposomes (NaA-LPs) in the lipid induction medium; the sixth group is added with blank double-labeled liposomes (CA / MAN-LPs) without sodium acetate in the lipid induction medium, wherein the concentration of sodium acetate solution is 1mM. After 24h of culture, TG, TC, BSA, oil red O detection, total RNA extraction and RT-qPCR detection of lipid metabolism related genes of cells in all groups are performed.

[0154] The results are shown in A-D of Figure 8 As shown in A-D of Figure 1, after 24h of treatment of NAFLD cells with 1mM concentration of NaA@CA / MAN-LPs and NaA solution alone, the intracellular TG, TC and oil red O stained lipid droplet relative area were significantly reduced; similarly compared with the NAFLD modeling group, after 24h of incubation of NAFLD cells with NaA@LPs, the intracellular TG and TC content was significantly reduced, but the relative area of lipid droplet after oil red O staining was not significantly improved, and after 24h of incubation of NAFLD cells with blank liposomes (CA / MAN-LPs) without sodium acetate, the cell TG, TC and oil red O lipid droplet relative area was slightly reduced, but there was no significant difference. Intra-group variance analysis showed that compared with NaA treatment, NaA@CA / MAN-LPs treatment of NAFLD cells had more significant alleviating effect on intracellular TG and TC accumulation, and the results showed that after 24h of incubation of NAFLD cells with 1mM concentration of sodium cholate / mannose double-labeled liposomes, the lipid deposition in NAFLD cells was significantly alleviated, and the alleviating effect of double-labeled sodium acetate liposomes was significantly better than that of sodium acetate solution alone.

[0155] 4. Sodium acetate liposomes alleviate liver macrophage inflammation

[0156] Kupffer cells in good state of logarithmic growth phase were selected, and except for the blank control group, the remaining five groups of cells were induced to inflammation by adding LPS at a concentration of 20 ng / mL at six hours before the end of treatment. The specific treatment conditions of the cells in each group were as follows: the cells in the blank control group were added with PBS solution of the same volume without LPS treatment; the cells in the modeling group were added with LPS to induce cell inflammation at 6 h before the end of incubation; the cells in the double-labeled liposome group were added with 1 mM sodium cholate / mannose double-labeled sodium acetate liposome (NaA@CA / MAN-LPs); the cells in the sodium acetate group were added with 1 mM sodium acetate solution; the cells in the non-labeled liposome group were added with 1 mM non-labeled sodium acetate liposome (NaA-LPs); and the cells in the blank liposome group were added with blank double-labeled liposome (CA / MAN-LPs) without sodium acetate. After the treatment, total RNA of the cells was extracted for RT-qPCR detection of inflammation-related genes.

[0157] The results are shown as A-D in Figure 9 Compared with the modeling group, the mRNA expressions of IL-1β, IL-6 and TNF-α in the Kupffer cells were significantly reduced, and the mRNA expression of IL-4 was significantly increased after treatment with sodium cholate / mannose double-labeled liposome (NaA@CA / MAN-LPs) containing sodium acetate, non-labeled liposome (NaA@LPs) and sodium acetate solution (NaA) alone, indicating that sodium acetate and sodium acetate liposome effectively inhibited the activation of the Kupffer cell inflammation pathway. The intragroup difference analysis results of the four treatment groups showed that compared with non-labeled sodium acetate liposome and sodium acetate alone, the sodium acetate-containing double-labeled liposome treatment had a more significant effect on the alleviation of high expression of IL-1β, IL-6 and TNF-α mRNA and low expression of IL-4 mRNA in the Kupffer cells. The above results show that the sodium acetate double-labeled liposome effectively alleviates the inflammatory response of the Kupffer cells, and its intervention effect is significantly better than that of sodium acetate alone or non-labeled sodium acetate liposome intervention.

[0158] Example 6 Liposome in vivo targeting positioning

[0159] Fifteen C57 male mice were taken, and each group of 3 mice, a total of 5 groups, were respectively given CY7@CA / MAN-LPs, CY7@CA-LPs, CY7@MAN-LPs, CY7@LPs and CY7 with the same fluorescence intensity. After the administration, the mice in anesthetized state were subjected to in vivo fluorescence imaging at 0.5, 1, 1.5, 2 and 2.5 h, and the results are shown in Figure 10As shown in the figure, mice directly administered CY7 fluorescent dye and unlabeled CY7-liposomes generally showed low fluorescence intensity within 0-2.5 h. However, after gavage administration of CY7-encapsulated sodium cholate or mannose-modified liposomes, 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, with the fluorescence intensity still clearly visible at 2.5 h. The accumulation amount of the three sodium cholate or mannose-labeled fluorescent liposomes in the abdomen, from highest to lowest, was: sodium cholate / mannose dual-labeled fluorescent liposomes > sodium cholate single-labeled fluorescent liposomes > mannose single-labeled fluorescent liposomes. These results indicate that sodium cholate / mannose dual-labeled liposomes have stronger intestinal penetration and liver targeting capabilities, delivering more fluorescent dye to the liver.

[0160] To more directly detect the amount of liposomes with different modifications that cross the intestine and enter the liver, C57 mice were randomly divided into 5 groups. Each group was administered CY7@CA / MAN-LPs, CY7@CA-LPs, CY7@MAN-LPs, CY7@LPs, and CY7 with the same fluorescence intensity via gavage. At 2h, 6h, 10h, 14h, 18h, and 24h, the mice were anesthetized and sacrificed. Major organs (heart, liver, spleen, lungs, kidneys, and gastrointestinal tract) were harvested, and the fluorescence intensity of each organ was immediately detected using a fluorescence imaging system. The results are shown below. Figure 11 A; Plot the liver fluorescence intensity change curves of the five groups of fluorescent liposomes with liver fluorescence intensity as the ordinate and time as the abscissa. Figure 11 B) The five curves generally showed a trend of first increasing and then decreasing. The fluorescence intensity in the mouse liver reached its peak at 14 h, with values ​​of 0.303, 0.208, 0.147, 0.084, and 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 significance was analyzed. The results are as follows: Figure 11 C, fluorescence intensity of five liposomes in the liver (AUG) 0-24 The fluorescence intensity was ranked from highest to lowest as follows: CY7@CA / MAN-LPs > CY7@CA-LPs > CY7@MAN-LPs > CY7@LPs > CY7. Furthermore, the accumulation of sodium cholate / mannose-modified fluorescent liposomes in the liver was significantly higher than that of fluorescent liposomes with single fluorescent dyes, unmodified fluorescent liposomes, fluorescent liposomes labeled with mannose alone, or fluorescent liposomes labeled with sodium cholate alone. Similarly, the fluorescence intensity in the intestines of the five groups of mice was quantified. Figure 11 As shown in D, the fluorescence intensity in the mouse intestine gradually decreased over 2-24 hours. The intestinal fluorescence intensity AUG of the mouse during 2-24 hours was calculated. 2-24 The result is as follows Figure 11As shown in FIG. 8, the fluorescence intensity of CY7@CA / MAN-LPs accumulated in the intestinal tract for 2-24 h was significantly higher than that of CY7@MAN-LPs, CY7@LPs and CY7, but was not significantly different from that of the fluorescent liposome labeled with sodium cholate alone, indicating that the sodium cholate ligand on the surface of the liposome is beneficial to promote the intestinal absorption of the liposome, and the mechanism may be related to the ASBT receptor of the mouse ileum.

[0161] The above results show that the fluorescent liposome labeled with sodium cholate / mannose is more effective in penetrating the mouse intestinal tract, targeting the liver tissue and playing a long-term role compared with the fluorescent liposome labeled with sodium cholate or mannose alone, unlabeled fluorescent liposome or fluorescent dye alone.

[0162] Example 7 Sodium acetate liposome relieves NAFLD in mice

[0163] 1. Animal model

[0164] 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 Vital River Laboratory Animal Technology Co., Ltd. (License No. SYXK(Jing)2021-0001). The experimental design was reviewed by the Experimental Animal Welfare Ethics Committee of Zhizuo (Beijing) Biological Technology Co., Ltd. (Batch No. ZYZC202402006S), and all animal handling and experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Research Council

[0165] 2. Modeling and intervention

[0166] Animal grouping and intervention treatment

[0167] Before the formal experiment began, all mice were adaptively fed for one week, and during the feeding process, the mice freely drank sterile water. After one week, the mice were randomly divided into six groups, 8 in each group (4 / cage), and then the sodium acetate liposome gavage experiment was performed, once every other day, for 10 weeks. The animal experiment design scheme, specific grouping and intervention methods are as follows:

[0168] (1) Normal control group: basic maintenance feed, gavage with PBS every other day from week 0 to week 10

[0169] (2) NAFLD model group: HFD feed, gavage with PBS every other day from week 0 to week 10

[0170] (3) Double-labeled liposome intervention group (NaA@CA / MAN-LPs): HFD feed, gavage with sodium cholate / mannose double-labeled sodium acetate liposome (200 μL, the concentration of sodium acetate in the double-labeled liposome is 15 mg / mL) every other day from week 0 to week 10

[0171] (4) Sodium acetate intervention group (NaA): HFD feeding, sodium acetate solution (200 μL, the concentration of sodium acetate solution is 15 mg / mL) was gavaged every other day from the 0th week to the 10th week.

[0172] (5) Non-labeled liposome intervention group (NaA@LPs): HFD feeding, non-labeled sodium acetate liposome (200 μL, the concentration of sodium acetate in non-labeled liposome is 15 mg / mL) was gavaged every other day from the 0th week to the 10th week.

[0173] (6) Blank liposome intervention group (CA / MAN-LPs): HFD feeding, blank liposome (200 μL, the concentration of blank liposome is 46 mg / mL, which is consistent with the concentration of liposome in the rest groups) was gavaged every other day from the 0th week to the 10th week.

[0174] Evaluation of sodium acetate liposome on improving the function of HFD-induced NAFLD mice by using the methods common in the art

[0175] Body weight trend, organ index, oral glucose tolerance test (OGTT), blood biochemical index determination, hematoxylin and eosin (H&E) staining and oil red O staining, liver tissue F4 / 80 immunohistochemical staining, detection of changes in liver lipid content in mice, detection of liver function indicators in mice, detection of oxidative damage in mouse liver (MDA and SOD monitoring), total RNA extraction from liver tissue and real-time quantitative PCR determination (genes Acc1, Fasn, Srebf1, CPT1α, IL-1β, IL-4, IL-6, TNF-α, GAPDH); Western Blot.

[0176] 1) Body weight results are as follows Figure 12A-B) showed that the body weight of NAFLD mice decreased by 18.08%, 13.88% and 6.45% after 10 weeks of intervention of double-labeled sodium acetate liposomes (NaA@CA / MAN-LPs), unlabeled sodium acetate liposomes (NaA@LPs) and sodium acetate solution (NaA) respectively. At the end of the tenth week of intervention, the body weight of the three groups of mice increased significantly lower than that of the NAFLD model 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 of blank double-labeled liposomes without sodium acetate, the body weight decreased by 1.76g, which was not significantly lower than that of the NAFLD model group, indicating that the blank liposome material had no significant effect on the body weight of NAFLD mice and did not cause the weight gain of mice. Analysis of the body weight changes of the four groups of intervention-treated mice showed that the body weight of the double-labeled liposome group was significantly lower than that of the blank liposome group and the sodium acetate group, indicating that the double-labeled liposome had a significantly higher effect on alleviating the weight gain of NAFLD mice than the sodium acetate treatment alone Figure 12 A, B) ;

[0177] 2) The results of glucose tolerance are shown in Figure 13 A-B) showed that the body weight of NAFLD mice decreased by 18.08%, 13.88% and 6.45% after 10 weeks of intervention of double-labeled sodium acetate liposomes (NaA@CA / MAN-LPs), unlabeled sodium acetate liposomes (NaA@LPs) and sodium acetate solution (NaA) respectively. At the end of the tenth week of intervention, the body weight of the three groups of mice increased significantly lower than that of the NAFLD model 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 of blank double-labeled liposomes without sodium acetate, the body weight decreased by 1.76g, which was not significantly lower than that of the NAFLD model group, indicating that the blank liposome material had no significant effect on the body weight of NAFLD mice and did not cause the weight gain of mice. Analysis of the body weight changes of the four groups of intervention-treated mice showed that the body weight of the double-labeled liposome group was significantly lower than that of the blank liposome group and the sodium acetate group, indicating that the double-labeled liposome had a significantly higher effect on alleviating the weight gain of NAFLD mice than the sodium acetate treatment alone Figure 13 A-B).

[0178] 3) The results of blood lipid metabolism are shown in Figure 14A-D shows that compared with the NAFLD modeling group, after the mice were gavaged with sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate solution alone and unmarked sodium acetate liposomes, the serum TG, TC and LDL-C levels of the mice were significantly reduced, and the HDL-C level was significantly increased (p<0.01), indicating that unmarked liposomes containing sodium acetate, double-labeled liposomes and sodium acetate can significantly alleviate the blood lipid levels of NAFLD. After gavaging the mice with double-labeled liposomes without sodium acetate, the serum TG, TC and LDL-C levels of the mice showed no significant change, and the HDL-C level was significantly increased (p<0.05), indicating that the blank liposome material does not aggravate the serum lipid metabolism level of NAFLD mice and increases the HDL-C content to a certain extent. The serum lipid metabolism parameters of the mice in the double-labeled liposome group, the sodium acetate group, the unmarked liposome group and the blank liposome group were analyzed for intragroup significance, and the results showed that compared with gavaging sodium acetate solution alone, gavaging sodium cholate / mannose double-labeled sodium acetate liposomes significantly reduced the serum TG content of the mice and significantly increased the HDL-C content, indicating that double-labeled sodium acetate liposomes are more effective than gavaging sodium acetate alone in alleviating NAFLD dyslipidemia.

[0179] 4) Sodium acetate liposomes improve the fat tissue content of HFD-induced NAFLD mice as shown in Figure 15 Figure 15 A: Compared with the NAFLD modeling group, gavaging sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate solution alone, unmarked sodium acetate liposomes and blank liposome material significantly alleviated the proportion of white adipose tissue in NAFLD mice, which was reduced by 3.56%, 2.20%, 2.05% and 0.54%, respectively. Intragroup difference analysis of the total white adipose tissue content of the mice in the four intervention groups showed that the proportion of white adipose tissue after sodium cholate / mannose double-labeled sodium acetate liposome intervention was significantly lower than that after gavaging sodium acetate solution alone or unmarked liposomes containing sodium acetate. The epididymal fat tissue of the mice was stained with H&E, and the staining results are shown in Figure 15 B: Compared with the NAFLD modeling group, the epididymal fat vacuole area of the mice gavaged with sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate solution alone and unmarked sodium acetate liposomes was significantly reduced, the epididymal fat cell section area was significantly reduced, the number of fat cells under the same field of view was significantly increased, and the changes in the double-labeled liposome group were the most obvious. In summary, unmarked liposomes, double-labeled liposomes and sodium acetate effectively reduce the white fat content and the volume of epididymal fat cells in NAFLD mice, and sodium cholate / mannose double-labeled sodium acetate liposomes have the most significant effect on improving the fat tissue content of NAFLD mice.

[0180] 5) Sodium acetate liposomes improve the liver pathological morphology of NAFLD mice as shown in Figure 16 Figure 16 ​​As shown in A, after gavage with blank liposome without sodium acetate, the appearance of the liver was similar to that of the NAFLD model group, and no obvious change was observed. However, after gavage with sodium cholate / mannose double-labeled sodium acetate liposome, sodium acetate solution alone or unlabeled sodium acetate liposome for 10 weeks, the color of the liver of the NAFLD mice returned to reddish brown, and the smooth surface feeling was restored, indicating that sodium acetate or sodium acetate-containing two kinds of liposomes had the function of relieving the deposition of NAFLD liver. Figure 16 B is the H&E staining result, the liver lobule structure of the NAFLD model group of mice was destroyed, and the hepatocytes were ballooned, showing that the hepatocytes were enlarged and contained large lipid droplets, and part of the hepatocytes appeared mixed type of large vacuolar and microvesicular fatty degeneration, accompanied by inflammatory cell infiltration; After gavage with sodium cholate / mannose double-labeled sodium acetate liposome, sodium acetate solution alone or unlabeled sodium acetate liposome, the number and area of vacuoles in the liver of mice were significantly reduced, indicating that the liver fatty degeneration was significantly improved. After gavage with blank liposome without sodium acetate, the area of vacuoles in the liver of mice was reduced, but the number was still more, indicating that the liposome alone had very limited effect on the relief of liver lipid deposition in NAFLD mice. Further quantitative analysis of the lipid droplet area in the H&E staining section of the liver, sodium cholate / mannose double-labeled sodium acetate liposome, sodium acetate solution and unlabeled sodium acetate liposome significantly reduced the liver lipid droplet area of NAFLD mice (p<0.01), the above three groups of sodium acetate intervention for 10 weeks, reduced the liver lipid droplet area of NAFLD mice by 46.39%, 40.97% and 39.33% respectively. Figure 16 C) Oil red O staining results are shown in Figure 16 D, blank liposome without sodium acetate has no effect on the relief of liver lipid deposition in NAFLD mice. Sodium cholate / mannose double-labeled sodium acetate liposome, sodium acetate alone or unlabeled sodium acetate liposome significantly reduced the liver lipid deposition of NAFLD mice, and the double-labeled sodium acetate liposome had the most obvious effect, and the oil red O staining section at the end of intervention was closer to that of the normal group of mice Figure 16 D) The proportion of red lipid droplet area to total section area was quantitatively analyzed by Image J software, and the relative lipid droplet area of each group was calculated based on the control group as the standard. The H&E staining results were consistent. Compared with the control group, the liver lipid droplets of NAFLD model group and blank liposome group of mice increased by 14.37 times and 13.67 times respectively, sodium cholate / mannose double-labeled sodium acetate liposome, sodium acetate alone or unlabeled sodium acetate liposome significantly reduced the liver lipid droplet area of NAFLD mice after 10 weeks of intervention, compared with the NAFLD model group, the liver lipid droplet area at the end of intervention was reduced by 88.27%, 73.45% and 70.40% respectively Figure 16 E).

[0181] 6) The results of sodium acetate liposome improving liver lipid accumulation and oxidative stress in NAFLD mice are shown inFigure 17 The results showed that the liver index of the NAFLD mice treated with the two kinds of liposomes containing sodium acetate (NaA@CA / MAN-LPs and NaA@LPs) for 10 weeks was significantly lower than that of the mice treated with sodium acetate solution alone, indicating that the liposomes containing sodium acetate were superior to sodium acetate solution alone in improving the increase in liver mass of the NAFLD mice. Figure 17 A) The contents of TG and TC in the livers of the mice were detected by using a kit, and the results are shown in Figure 17 B, C, after 10 weeks of intragastric administration of the two kinds of liposomes containing sodium acetate or sodium acetate solution, the TG and TC contents in the livers of the NAFLD mice were significantly reduced, and the lipid deposition in the livers of the mice was significantly improved. The results of the intragroup difference analysis showed that the TG and TC contents in the livers of the mice treated with the double-labeled sodium acetate liposomes were significantly lower than those in the sodium acetate group. The levels of MDA and SOD in the livers of the mice were detected by using a kit, and the results are shown in Figure 17 D, E, after 10 weeks of intragastric administration of the two kinds of liposomes containing sodium acetate or sodium acetate solution, the TG and TC contents in the livers of the NAFLD mice were significantly reduced, and the lipid deposition in the livers of the mice was significantly improved. The results of the intragroup difference analysis showed that the TG and TC contents in the livers of the mice treated with the double-labeled sodium acetate liposomes were significantly lower than those in the sodium acetate group. The levels of MDA and SOD in the livers of the mice were detected by using a kit, and the results are shown in

[0182] 7) The expression of the genes related to lipid metabolism in the livers of the NAFLD mice, and the results are shown in Figure 18 A-E, compared with the NAFLD model group, the blank liposomes without sodium acetate had no significant effect on the expression of the genes related to lipid synthesis and transport in the livers of the NAFLD mice. After 10 weeks of intervention with the double-labeled sodium acetate liposomes, sodium acetate solution, or unlabeled sodium acetate liposomes, the mRNA expression levels of Acaca, Fasn, Slc27a2, and Srebf1 in the livers of the NAFLD mice were significantly reduced, and the mRNA expression level of CPT1a was significantly up-regulated. The above results indicated that sodium acetate or liposomes containing sodium acetate effectively alleviated the up-regulation of the expression of the genes related to lipogenesis in the livers of the NAFLD mice and promoted the expression of the fatty acid transporter CPT1a. The phosphorylation of the AMPKa protein in the livers of the NAFLD mice treated with sodium acetate or sodium acetate liposomes for 10 weeks was analyzed by Western blot, and the results are shown in Figure 18 F, after 10 weeks of intragastric administration of the double-labeled sodium acetate liposomes, sodium acetate solution, or unlabeled sodium acetate liposomes, the phosphorylation expression level of the AMPKa protein in the livers of the NAFLD mice was significantly increasedFigure 18 F, G). After gavage with blank liposome, double-labeled sodium cholate / mannose liposome, and sodium acetate alone, the expression of Acc1 and Srebf1c mRNA in the liver was significantly down-regulated, and the expression of CPT1a was significantly up-regulated. The above results show that sodium acetate or double-labeled sodium cholate / mannose liposome can inhibit the transcription of Acc1 and Srebf1c, promote the expression of CPT1a by activating the AMPK pathway, and further regulate downstream lipid metabolism genes to reduce liver lipid deposition.

[0183] 8) Sodium acetate liposome improves liver function of NAFLD mice: The serum AST and ALT levels were detected, and the results are shown in Figure 19 A, B. After gavage with blank liposome without sodium acetate, the serum ALT and AST levels of NAFLD mice did not decrease significantly, but after gavage with double-labeled sodium cholate / mannose liposome containing sodium acetate, sodium acetate solution, or unlabeled sodium acetate liposome, the serum AST and ALT levels of mice decreased significantly. The liver ALT and AST levels were detected, and the results are shown in Figure 19 C, D. Compared with the control group, the liver ALT and AST levels of NAFLD model mice increased significantly (p<0.01). Consistent with the results of serum ALT and AST, the liver ALT and AST levels of mice in the blank liposome group without sodium acetate did not improve significantly. Gavage with double-labeled liposome containing sodium acetate, unlabeled liposome, or sodium acetate solution alone effectively improved the liver ALT and AST levels of NAFLD mice. Intragroup difference analysis showed that the effect of double-labeled sodium cholate / mannose liposome containing sodium acetate on reducing the increase of ALT and AST content was significantly better than that of gavage with sodium acetate alone

[0184] 9) Sodium acetate liposome improves the liver inflammation level and oxidative stress of NAFLD mice. The results are shown in Figure 20 Compared with the liver of NAFLD model mice, the liver macrophage infiltration of mice in the blank liposome group did not improve significantly, and the liver macrophage infiltration of mice in the double-labeled sodium acetate liposome group, sodium acetate solution group, or unlabeled sodium acetate liposome group improved significantly Figure 20 A). Image J software was used to quantitatively analyze the positive area of immunohistochemical staining. The statistical results showed that the F4 / 80 positive area of the liver of NAFLD model mice was significantly higher than that of the control group (p<0.01), and after gavage with blank liposome for 10 weeks, the relative positive area of liver macrophages of NAFLD mice decreased but did not improve significantly. After gavage with double-labeled sodium acetate liposome, sodium acetate solution alone, or unlabeled sodium acetate liposome for 10 weeks, the relative positive area of F4 / 80 in the liver of mice decreased by 62.46%, 40.99%, and 50.16%, respectively Figure 20B). The analysis of differences in the area of ​​F4 / 80 positive cells in the liver of mice in the four intervention groups showed that sodium cholate / mannose double-labeled sodium acetate liposomes significantly improved macrophage infiltration in the liver of NAFLD mice than sodium acetate solution alone or unlabeled sodium acetate liposomes (p<0.01).

[0185] 10) Further analysis was conducted to detect the expression of liver inflammation and oxidative stress-related cytokines in mice, such as... Figure 21 As shown in AD. Compared with the NAFLD model group, after 10 weeks of gavage administration of blank liposomes without sodium acetate, the expression of pro-inflammatory factors in the liver did not decrease significantly, and the level of anti-inflammatory factors did not increase significantly. After 10 weeks of gavage administration of sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate solution alone, or unlabeled sodium acetate liposomes, the expression levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α mRNA in the liver of NAFLD mice were significantly reduced, while the expression level of anti-inflammatory factor IL-4 mRNA was significantly increased. This indicates that sodium acetate or sodium acetate-containing double-labeled and unlabeled liposomes have a significant alleviating effect on liver inflammation in NAFLD mice. Compared with the NAFLD model group, after gavage administration of sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate solution, or unlabeled sodium acetate liposomes, the phosphorylation level of NFκB in the liver of mice was significantly downregulated, indicating a reduction in HFD-induced liver inflammatory response. The intragroup differential analysis results showed that the alleviating effect of double-labeled or unlabeled sodium acetate liposomes on the increased phosphorylation level of inflammatory proteins was significantly better than that of sodium acetate intervention alone. Figure 21 E, F).

[0186] In summary, after 10 weeks of gavage administration of sodium cholate / mannose-labeled sodium acetate liposomes to HFD-induced NAFLD mice, the weight gain of the mice was significantly reduced, glucose tolerance was effectively improved, and serum lipid markers showed that the double-labeled sodium acetate liposomes effectively alleviated the increase of TG, TC, and LDL-C and the decrease of HDL-C in the liver of NAFLD mice. Histopathological results of liver tissue sections from NAFLD mice showed that after gavage administration of double-labeled sodium acetate liposomes, hepatic lipid deposition was significantly improved, and the phosphorylation level of hepatic 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 hepatic lipid deposition. Regarding liver inflammation and oxidative stress, after 10 weeks of gavage administration of double-labeled sodium acetate liposomes to NAFLD mice, NFκB protein phosphorylation was significantly inhibited, the release of downstream inflammatory factors was reduced, and liver inflammation and oxidative stress were effectively alleviated. The dual-labeled sodium acetate liposomes were superior to the sodium acetate solution of the same concentration administered by gavage alone in alleviating liver lipid deposition and oxidative stress in NAFLD mice. This indicates that the sodium cholate / mannose dual-labeled liposomes successfully targeted and delivered sodium acetate to the liver, causing sodium acetate to accumulate in the liver and more effectively alleviating liver lipid accumulation and inhibiting inflammatory response.

Claims

1. Use of DSPE-PEG-CA and DSPE-PEG-MAN in the preparation of a liver-targeted liposome, the use comprising the steps of: (1) The following raw materials are added in turn: DSPC, cholesterol, DSPE-PEG-CA, DSPE-PEG-MAN, DSPE-PEG 2000 -NH2, DC-cholesterol, and mixed, then transferred to a round-bottom flask with a micro glass syringe, pre-mixed, and shaken in a constant temperature shaker to make all reagents thermodynamically distributed uniformly, at which time the solution is completely clear; (2) high-pressure rotary evaporation to obtain a film with uniform thickness and distribution; (3) adding water or buffer to the film formed in step (2) and obtaining a solution by ultrasonic treatment; (4) using a micro glass syringe to suck the solution obtained in step (3), and extruding back and forth in a liposome extruder, and extruding the liposome overnight to stabilize the liposome particles; wherein the weight ratio of each component in step (1) is: 40.5% DSPC, 13.5% cholesterols, 1% DSPE-PEG-CA, 10% DSPE-PEG 2000 -NH2 and 30% DC-cholesterol; wherein The operation of step (2) is carried out under the conditions of pressure 400-600 mbar, rotation speed 80-120 rpm, and temperature 45-50 ℃. Wherein, DSPE-PEG-CA is cholic acid salt modified DSPE-PEG 2000 , DSPE-PEG-MAN is mannose modified DSPE-PEG 2000 .

2. Use according to claim 1, characterized in that, The preparation method of the DSPE-PEG-CA is as follows: 1) activation of cholic acid: dissolve sodium cholate, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide methiodide (EDC) and N-hydroxysuccinimide (NHS) in dichloromethane, carry out ice bath reaction, stir overnight at room temperature, filter and rotary evaporate to obtain activated sodium cholate; 2) DSPE-PEG 2000 -NH2 was dissolved in DMSO, and activated sodium cholate and triethylamine were added and completely dissolved, and then the reaction was performed; 3) after the reaction is completed, place the solution in a dialysis bag and dialyze with pure water as the medium; 4) After dialysis, the solution in the dialysis bag is collected and freeze-dried to obtain DSPE-PEG-CA, wherein the ice bath reaction in step 1) is 20-40 min; the molecular sieve size of the dialysis bag in step 2) is MW=800-1200 Da, and the dialysis time is 18-28 h. 2000 The addition ratio of -NH2 and DMSO is 450-550 mg:3 mL; the addition ratio of activated sodium cholate and triethylamine is 500-600 mg:1 g; the reaction temperature is 38-45 DEG C, and the reaction time is 1-3 h; wherein the molecular sieve size of the dialysis bag in step 3) is MW=800-1200 Da, and the dialysis time is 18-28 h.

3. Use according to claim 1, characterized in that, The preparation method of the DSPE-PEG-MAN is as follows: 1) Dissolve DSPE-PEG 2000 -NH2 in DMSO, add mannose and triethylamine to complete dissolution, and mannose with triethylamine, reaction; 2) after the reaction is completed, place the solution in a dialysis bag and dialyze with pure water as the medium; 3) After dialysis, the solution in the dialysis bag is freeze-dried to obtain DSPE-PEG-MAN; wherein step 1) DSPE-PEG 2000 The addition ratio of -NH2 and DMSO is 450-550 mg: 3 mL; The addition ratio of mannose and triethylamine is 500-600 mg: 1 g, the reaction temperature is 38-45 ℃, and the reaction time is 1-3 h; the molecular weight of the dialysis bag in step 2) is MW=800-1200 Da, and the dialysis time is 18-28 h.

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