A liver-targeted liposome and a preparation method and application thereof

Abstract

The application discloses a liver-targeting lipid carrier as well as a preparation method and application thereof, relates to the field of drug delivery, and utilizes cholate and mannose to respectively modify a phospholipid molecule DSPE-PEG 2000 After two raw materials DSPE-PEG-CA and DSPE-PEG-MAN composed of synthetic liposomes are obtained, the liver-targeting lipid carrier is obtained according to a certain proportion of cholesterol, DSPC, DC-cholesterol and the like, and the liposome has high cell safety and liver targeting.

Description

Technical Field

[0001] This invention relates to the field of drug delivery, specifically to a lipid carrier targeting the liver, its preparation method, and its application. Background Technology

[0002] DSPE-PEG 2000 It 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. DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine) is a phospholipid with a hydrophobic fatty acid chain (stearic acid) moiety that can insert into the lipid bilayer. PEG2000 (Polyethylene Glycol 2000) is the polyethylene glycol moiety with a molecular weight of 2000, possessing hydrophilicity that 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 is particularly suitable for extending the lifespan of drug carriers and improving targeting in the bloodstream. The PEG chain can prevent the immune system from recognizing and clearing the carrier, thus contributing to its "stealth" effect.

[0003] 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. It exerts receptor-mediated endocytosis via 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 hepatic macrophages, targeting liposomes to macrophages in the liver.

[0004] Despite the design of liver-specific ligands (such as mannose and N-acetylgalactosamine (GalNAc), insufficient targeting may still occur: Non-specific uptake: Liposomes may be taken up by other cells or organs, such as macrophages in the mononuclear phagocytic system (MPS), especially in the spleen and lungs. Heterogeneity: Different liver cell types (such as hepatocytes, Kupffer cells, and endothelial cells) have different uptake efficiencies for the target molecule, which may lead to uneven drug action. Furthermore, although targeted design reduces systemic distribution, it cannot completely prevent drug accumulation in other sites, potentially causing side effects.

[0005] Therefore, existing technologies exist to improve liposome formulations to obtain liposomes with improved targeting, stability, safety, and delivery efficiency, such as using PEGylated liposomes to reduce non-specific clearance, and designing pH-responsive or enzyme-responsive systems to improve intracellular drug release efficiency. This invention addresses the aforementioned technical deficiencies through experimental design and research. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention modifies the surface of nanoliposomes with sodium cholate and mannose to construct an oral delivery system with liver targeting.

[0007] Definition Explanation:

[0008] DSPC stands for 1,2-Distearoyl-sn-glycero-3-phosphocholine, also known as 1,2-distearate-sn-glycero-3-phosphocholine. It is a phospholipid commonly used in the preparation of liposomes and nanomedicine delivery systems.

[0009] DSPE-PEG 2000 -NH2: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)-2000]-ammonium, abbreviated as distearylphosphatidylethanolamine-polyethylene glycol-amino, is a modified phospholipid commonly used in drug delivery systems (such as liposomes, nanoparticles, etc.), and widely applied in targeted drug delivery and gene therapy. Its amino group (NH2) can be covalently linked to other molecules or target ligands to improve targeting and therapeutic efficacy.

[0010] DSPE-PEG-CA: Disteazylecithin ethanolamine polyethylene glycol-cholic acid. Since the PEG used in the synthesis reaction is PEG2000, DSPE-PEG-CA appearing in this specification is DSPE-PEG. 2000 -CA;

[0011] DSPE-PEG-MAN: Disteazylecithin ethanolamine polyethylene glycol mannose. Since the PEG used in the synthesis reaction is PEG2000, DSPE-PEG-MAN appearing in this specification is DSPE-PEG. 2000 -MAN.

[0012] 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).

[0013] The present invention first provides a liver-targeted lipid carrier, 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 certain proportion, wherein DSPE-PEG-CA is DSPE-PEG modified with bile salts 2000 , DSPE-PEG-MAN is DSPE-PEG modified with mannose 2000 ; the particle size of the liposome is 96±1.5 nm; 0.22 < PDI < 0.25; the Zeta potential is 33±4 mV.

[0014] Furthermore, the weight ratio of each component is as follows: 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-NH2, and 30% of DC-cholesterol.

[0015] Furthermore, in step (3), the weight ratio of each component is 40.5% DSPC, 13.5% cholesterol, 1% DSPE-PEG-CA, 10% DSPE-PEG-MAN, 5% DSPE-PEG-NH2, and 30% DC-cholesterol.

[0016] The second aspect of the present invention provides the application of the drug liposome of the liver described in the first aspect in the preparation of a preparation for treating liver diseases, and the liver diseases are inflammation, tumor, or non-alcoholic fatty liver.

[0017] The third aspect of the present invention is to provide a method for preparing the liver-targeted liposome containing a medicinal ingredient described in the first aspect, and the method includes the following steps:

[0018] 1) Prepare DSPE-PEG-CA;

[0019] 2) Prepare DSPE-PEG-MAN;

[0020] 3) Add the following raw materials in sequence: DSPC, cholesterol, DSPE-PEG 2000 -CA, DSPE-PEG 2000 It should be noted that there is an unclear "①%" in the original text at line 10. I have translated it as "①%" for now. You may need to check and correct this if it's an error in the original.-MAN,DSPE-PEG 2000 -NH2 and DC-cholesterol, after mixing well, transfer to a round-bottom flask using a micro-glass syringe. After pre-mixing, shake well in a constant temperature shaker to ensure that all reagents are thermodynamically distributed. At this point, the solution is completely clear.

[0021] 4) High-pressure rotary evaporation yields a film of uniform thickness and distribution;

[0022] 5) Add buffer solution and sonicate to obtain a solution;

[0023] 6) Use a micro-volume glass syringe to draw up the solution obtained in step 5) and squeeze it back and forth in a liposome extruder. After the liposomes are squeezed, store them at below 4°C overnight to stabilize the liposome particles.

[0024] Furthermore, in step 3), the pre-mixing time is 5-15 min, the constant temperature is 35-40℃, and the shaking time is 5-15 min.

[0025] Further, step 4) involves rotary evaporation at a pressure of 400–600 mbar, a rotation speed of 80–120 rpm, and a temperature of 45–50°C.

[0026] Furthermore, the buffer solution described in step 5) contains either the drug to be delivered or a fluorescent label;

[0027] Furthermore, the method also includes removing unencapsulated drug to be delivered by ultrafiltration, followed by quick-freezing of liposomes with liquid nitrogen;

[0028] Furthermore, the method also includes flash freezing the obtained liposomes with liquid nitrogen and reconstituted them with PBS before use;

[0029] Furthermore; the preparation of DSPE-PEG 2000 The steps for CA are as follows:

[0030] 1) Activated cholic acid: Cholic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide (EDC) and N-hydroxysuccinimide (NHS) were dissolved in dichloromethane, reacted in an ice bath, stirred overnight at room temperature, filtered and rotary evaporated to obtain activated sodium cholate.

[0031] 2) Add DSPE-PEG 2000 -NH2 is dissolved in DMSO, and activated sodium cholate and triethylamine are added and completely dissolved before the reaction occurs.

[0032] 3) After the reaction is complete, place the solution in a dialysis bag and dialyze it with pure water as the medium;

[0033] 4) After dialysis, collect the solution in the dialysis bag and freeze-dry it to obtain DSPE-PEG-CA.

[0034] Preferably, the ice bath reaction in step 1) is carried out for 20–40 minutes;

[0035] Preferably, step 2) DSPE-PEG 2000 The ratio of -NH2 to DMSO is 450–550 mg: 3 mL; the ratio of activated sodium cholate to triethylamine is 500–600 mg: 1 g; the reaction temperature is 38–45 °C and the reaction time is 1–3 h.

[0036] Preferably, the molecular sieve size of the dialysis bag is MW = 800-1200 Da, more preferably 1000 Da; the dialysis time is 18-28 h.

[0037] Furthermore, the preparation of DSPE-PEG... 2000 The steps for using MAN are as follows:

[0038] 1) Add DSPE-PEG 2000 -NH2 is dissolved in DMSO, mannose and triethylamine are added and completely dissolved, and mannose reacts with triethylamine;

[0039] 2) After the reaction is complete, place the solution in a dialysis bag and dialyze it with pure water as the medium;

[0040] 3) After dialysis, freeze-dry the solution in the dialysis bag to obtain DSPE-PEG-MAN.

[0041] Preferably, step 1) DSPE-PEG 2000 -NH2 and DMSO were added in a ratio of 450-550 mg: 3 mL; and mannose and triethylamine were added in a ratio of 500-600 mg: 1 g; the reaction temperature was 38-45℃ and the reaction time was 1-3 h.

[0042] Preferably, in step 2), the molecular sieve size of the dialysis bag is MW = 800-1200 Da, more preferably 1000 Da; the dialysis time is 18-28 h.

[0043] Furthermore, the molar ratio of DSPC to cholesterol in the liposomes is 3:1;

[0044] Furthermore, in the liposomes, the proportions of DSPE-PEG-CA and DSPE-PEG-MAN are 0.5% to 1.5% and 5% to 15%, respectively, preferably 1% and 10%.

[0045] Further, the weight 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.

[0046] The beneficial effects of this invention include:

[0047] 1) The first synthesis of liposomes using two raw materials, DSPE-PEG 2000 -CA and DSPE-PEG 2000 After -MAN, liposomes with liver-targeting properties were obtained by mixing them with cholesterol, DSPC, and DC-cholesterol in a certain ratio;

[0048] 2) The five liposomes synthesized in this invention have no obvious toxicity to cardiomyocytes H9c2, intestinal epithelial cells Caco-2, liver macrophages Kupffer and hepatocytes AML-12, and are suitable as oral nanocarriers of sodium acetate with high cellular safety.

[0049] 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, ability to penetrate the intestinal epithelial barrier, and liver targeting.

[0050] 4) Sodium cholate / mannose double-labeled liposomes containing sodium acetate effectively alleviated lipid deposition and cellular inflammation in NAFLD cells;

[0051] 5) After oral administration of double-labeled sodium acetate liposomes to mice, 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 hepatic inflammation and oxidative stress, after 10 weeks of oral administration of double-labeled sodium acetate liposomes to NAFLD mice, NFKB protein phosphorylation was significantly inhibited, the release of downstream inflammatory factors was reduced, and hepatic inflammation and oxidative stress were effectively alleviated. Attached Figure Description

[0052] Figure 1 Schematic diagram of the synthesis of sodium acetate liposomes modified with bile acid and mannose;

[0053] Figure 2Synthesis of DSPE-PEG-CA, A is the synthetic route, B is the infrared spectrum;

[0054] Figure 3 Synthesis of DSPE-PEG-MAN, A is the synthetic route, B is the infrared spectrum;

[0055] Figure 4 Sodium acetate release from different groups of liposomes after incubation in serum, simulated gastric juice, and simulated intestinal juice in an orthogonal experiment; A: Sodium acetate release in serum; B: Sodium acetate release in simulated gastric juice; C: Sodium acetate release in simulated intestinal juice; Different letters indicate significant differences (p<0.05), and the same letter indicates no significant differences (p>0.05);

[0056] Figure 5 Liposome encapsulation efficiency and drug loading at different sodium acetate reservoir concentrations; A: encapsulation efficiency; B: drug loading.

[0057] Figure 6 TEM images of five types of liposomes, with a scale bar of 200 nm;

[0058] Figure 7 Cell viability of four cell types after incubation for 24 hours with sodium acetate liposomes at concentrations of 1–1500 μM and sodium acetate solution: A: H9c2 cells; B: Caco-2 cells; C: Kupffer cells; D: AML-12 cells;

[0059] Figure 8 Sodium acetate liposomes or sodium acetate alleviate lipid deposition in NAFLD cells. A: Effects of different liposomes or sodium acetate on cellular TG content; B: Effects of different liposomes or sodium acetate on cellular 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. Significance analyses were performed on TG, TC, and relative lipid droplet area. # indicates a significant difference between the model group and the control group. ## p<0.01; * indicates a significant difference between the experimental group and the model group. * p<0.05, ** p<0.01; + indicates significant difference between groups. + p<0.05, ++ p<0.01;

[0060] Figure 9 Effects of sodium acetate liposomes or sodium acetate on genes related to inflammation response in NAFLD cells: A: Relative expression level of IL-1β mRNA; B: Relative expression level of IL-6 mRNA; C: Relative expression level of TNF-α mRNA; D: Relative expression level of IL-4 mRNA; # indicates significant difference between the model group and the control group. ##p<0.01; * indicates a significant difference between the experimental group and the model group. * p<0.05, ** p<0.01; + indicates significant difference between groups. + p<0.05, ++ p<0.01;

[0061] Figure 10 In vivo fluorescence imaging of mice after gavage administration of CY7 liposomes or CY7;

[0062] Figure 11 Fluorescence intensities of major organs in mice after gavage administration of CY7 liposomes or CY7. A: Fluorescence imaging of each organ; B: Fluorescence intensity-time curve of liver in each group of mice; C: AUG of the fluorescence intensity-time curve of mouse liver. 0-24 D: Fluorescence intensity-time curves of mouse intestines in each group; E: AUG of mouse intestinal fluorescence intensity-time curves. 2-24 ;

[0063] Figure 12 Effects of sodium acetate liposomes on body weight changes and weight gain in NAFLD mice: A: Body weight gain curve; B: Weight gain; # This indicates a significant difference between the model group and the control group. ## p<0.01; * This indicates a significant difference between the experimental group and the model group. * p<0.05, ** p<0.01, + This indicates a significant difference between groups. + p<0.05;

[0064] Figure 13 Effects of sodium acetate liposomes on glucose tolerance in NAFDL mice

[0065] A: OGTT curve; B: Area under the OGTT curve; # This indicates a significant difference between the model group and the control group. ## p<0.01; * This indicates a significant difference between the experimental group and the model group. ** p<0.01, + This indicates a significant difference between groups. ++ p<0.01;

[0066] Figure 14 Sodium acetate liposomes on blood lipid metabolism in NAFLD mice: A: triglycerides; B: cholesterol; C: low-density lipoprotein cholesterol; D: high-density lipoprotein cholesterol; # This indicates a significant difference between the model group and the control group. ## p<0.01; *This indicates a significant difference between the experimental group and the model group. * p<0.05, ** p<0.01, + This indicates a significant difference between groups. + p<0.05, ++ p<0.01;

[0067] Figure 15 Effects of sodium acetate liposomes on adipose tissue content in NAFLD mice: A: Percentage of white adipose tissue; B: H&E staining results of epididymal adipose tissue (400×, scale bar 100μm). # This indicates a significant difference between the model group and the control group. ## p<0.01; * This indicates a significant difference between the experimental group and the model group. * p<0.05, ** p<0.01, + This indicates a significant difference between groups. ++ p<0.01;

[0068] Figure 16 Effects of sodium acetate liposomes on liver pathological morphology in NAFLD mice: A: Liver appearance (scale bar 1 cm); B: Liver tissue H&E staining (100×, scale bar 200 μm); C: Relative area percentage of liver lipid droplets based on H&E stained sections (n=8); D: Liver tissue Oil Red O staining (100×, scale bar 200 μm); E: Relative area percentage of liver lipid droplets based on Oil Red O staining (n=8). # This indicates a significant difference between the model group and the control group. ## p<0.01; * This indicates a significant difference between the experimental group and the model group. ** p<0.01, + This indicates a significant difference between groups. + p<0.05, ++ p<0.01;

[0069] Figure 17 Effects of sodium acetate liposomes on hepatic lipid deposition and oxidative stress in NAFLD mice: A: Liver index (%); B: Hepatic triglyceride content; C: Hepatic cholesterol content; D: Hepatic malondialdehyde content; E: Hepatic superoxide dismutase content. # This indicates a significant difference between the model group and the control group. ## p<0.01; * This indicates a significant difference between the experimental group and the model group. * p<0.05, ** p<0.01; + This indicates a significant difference between groups. + p<0.05,++ p<0.01;

[0070] Figure 18 Effects of sodium acetate liposomes on the expression of genes related to lipid synthesis and metabolism in the liver of NAFLD mice and phosphorylation of AMPK protein. AE: relative expression of mRNAs of genes related to lipid synthesis and metabolism in the liver (Acc1, Fasn, Slc27a2, Srebf1, CPT1α); F: Western blot images of AMPKα and p-AMPKα; G: relative expression level of p-AMPKα / AMPKα. # This indicates a significant difference between the model group and the control group. ## p<0.01; * This indicates a significant difference between the experimental group and the model group. ** p<0.01; + This indicates a significant difference between groups. + p<0.05, ++ p<0.01

[0071] Figure 19 Effects of sodium acetate liposomes on liver function in NAFLD mice: A: serum alanine aminotransferase (ALT); B: serum aspartate aminotransferase (AST); C: liver alanine aminotransferase (ALT); D: liver AST. # This indicates a significant difference between the model group and the control group. ## p<0.01; * This indicates a significant difference between the experimental group and the model group. * p<0.05, ** p<0.01; + This indicates a significant difference between groups. + p<0.05, ++ p<0.01

[0072] Figure 20 Effects of sodium acetate liposomes on inflammatory infiltration of macrophages in the liver of NAFLD mice: A: F4 / 80 staining of mouse liver tissue sections (200×, scale bar 100μm); B: Relative area of ​​F4 / 80 positive sites. # This indicates a significant difference between the model group and the control group. ## p<0.01; * This indicates a significant difference between the experimental group and the model group. ** p<0.01; + This indicates a significant difference between groups. + p<0.05, ++ p<0.01

[0073] Figure 21Effects of sodium acetate liposomes on phosphorylation of inflammation-related cytokines and NFKB protein in the liver of NAFLD mice. AD: relative expression levels of inflammation-related cytokines in the liver, namely IL-1β, IL-6, TNF-α, and IL-4; E: Western blot images of p-NFKB and NFKB; F: relative expression level of p-NFKB / NFKB. # This indicates a significant difference between the model group and the control group. ## p<0.01; * This indicates a significant difference between the experimental group and the model group. ** p<0.01; + This indicates a significant difference between groups. + p<0.05, ++ p<0.01. Detailed Implementation

[0074] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0075] Example 1: Preparation of liver-targeting sodium acetate liposomes (NaA@CA / MAN-LPs)

[0076] Preparation process as follows Figure 1 As shown

[0077] 1. DSPE-PEG 2000 -CA synthesis

[0078] Cholic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide (EDC), and N-hydroxysuccinimide (NHS) were dissolved in dichloromethane. The mixture was reacted in an ice bath for 30 min, then stirred overnight at room temperature. The resulting product was filtered and rotary evaporated to obtain activated sodium cholate. 500 mg of DSPE-PEG was added... 2000 -NH₂ was dissolved in 3 mL of DMSO, and activated sodium cholate (550 mg) and triethylamine (1 g) were added and completely dissolved. The mixture was then reacted at 40 °C for 2 h. After the reaction, the solution was placed in a dialysis bag (MW = 1000 Da) and dialyzed with pure water for 24 h. After dialysis, the solution in the dialysis bag was collected and lyophilized to obtain DSPE-PEG. 2000 -CA (For synthesis steps, please refer to [link]) Figure 2 A).

[0079] The infrared spectrum of DSPE-PEG-CA is as follows Figure 2 Results B showed that DSPE-PEG 2000 -NH2, after modification with sodium cholate, at 1551 cm⁻¹ -1 The characteristic peak of the -C=O stretching vibration of the carboxylate group appeared at 3425 cm⁻¹. -1 Corresponding to the hydroxyl group in the sodium cholate molecule, 2850-2917 cm -1 This corresponds to the CH stretching vibration in the stearoyl alkyl chain of DSPE-PEG. The stretching vibration of the P=O bond in the phosphate group occurs at 1245 cm⁻¹, while the CO bond stretching vibration in the PEG chain occurs at 1108 cm⁻¹. -1 The CH bending vibration peak of the alkyl chain appears at 1466 cm⁻¹. -1 The above results indicate that sodium cholate has successfully bound to the DSPE-PEG-NH2 molecule.

[0080] 2. DSPE-PEG 2000 -MAN Synthesis

[0081] The synthesis steps and results of DSPE-PEG-MAN are as follows:

[0082] 500mg DSPE-PEG 2000 -NH₂ was dissolved in 3 mL of DMSO, and 550 mg of mannose and 1 g of triethylamine were added and completely dissolved. The reaction was carried out at 40 °C for 2 h. After the reaction, the solution was placed in a dialysis bag with a molecular weight of 1000 Da and dialyzed with pure water for 24 h. After dialysis, the solution in the dialysis bag was lyophilized to obtain DSPE-PEG-MAN (synthetic route as shown in the figure). Figure 3 (As shown in A).

[0083] The results of FTIR (Full-Time Infrared) spectroscopy detection of DSPE-PEG-MAN are as follows: Figure 3 As shown in Figure B: The infrared spectrum of DSPE-PEG-MAN shows the following characteristic peak: 3428 cm⁻¹ -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. -1 In DSPE-PEG 2000 In the FTIR spectrum of -NH2, the amino (-NH2) group appears at 3434 cm⁻¹. -1 However, after modification with mannose, the peak broadens and appears at 3428 cm⁻¹ due to the presence of hydroxyl groups. -1 The stretching vibration of the p=O bond in the phosphate group occurs at 1251 cm⁻¹.-1 The CO bond stretching vibration of the PEG chain appears at 1109 cm⁻¹. -1 The above results indicate that DSPE-PEG-MAN was successfully synthesized.

[0084] 3. Preparation of liver-targeted sodium acetate liposomes

[0085] High-concentration sodium acetate liposomes (NaA@CA / MAN-LPs) capable of penetrating the intestine and targeting the liver were prepared using a thin-film hydration method.

[0086] All raw materials and reagents were stored at -20°C, and the preparation process was carried out on ice.

[0087] (1) Add the following ingredients in sequence according to the liposome formulation: DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine), cholesterol, and DSPE-PEG. 2000 -CA, DSPE-PEG 2000 -MAN,DSPE-PEG 2000 After mixing DC-cholesterol, transfer the solution to a round-bottom flask using a micro-syringe. Mix well for 10 minutes beforehand, then shake in a 37°C constant temperature shaker for 10 minutes to ensure uniform thermodynamic distribution of all reagents. At this point, the solution will be completely clear.

[0088] (2) Rotary evaporation was carried out under the conditions of pressure of 500 mbar, rotation speed of 100 rpm and temperature of 50℃. After the rotary evaporation was completed, a film with uniform thickness and distribution was obtained.

[0089] (3) Add sodium acetate hydration medium to completely submerge the organic membrane, and sonicate for 2 minutes to make the solution turn milky white.

[0090] (4) Assemble the liposome extruder (Avanti Polar Lipids). Place a wetted carbonate membrane filter holder on each of the two black gaskets. Select a 100nm filter membrane, wet it, and place it between the two filter membrane holders. Fix the assembled liposome extruder on the holder. Finally, place the whole assembly on a heating plate and maintain the temperature at 50℃.

[0091] (5) Use a micro-volume glass syringe to draw 1 mL of liposome solution and squeeze it back and forth 21 times in a liposome extruder. After the liposomes are squeezed, store them at 4°C overnight to stabilize the liposome particles.

[0092] (6) Remove unencapsulated sodium acetate by ultrafiltration, then freeze the liposomes with liquid nitrogen, weigh them, and reconstitute them with PBS before use.

[0093] 7) Identification of sodium acetate liposomes includes: HPLC detection of sodium acetate concentration, calculation of encapsulation efficiency and drug loading; liposome particle size, PDI, and Zeta; TEM electron microscopy observation of liposome morphology.

[0094] 4. Orthogonal experiments were used to optimize the composition of NaA@CA / MAN-LPs.

[0095] (1) The ratio of the components in step 1) of the above method is optimized. The components of the liposomes include: DSPC, cholesterol, and DSPE-PEG. 2000 -CA, DSPE-PEG 2000 -MAN,DSPE-PEG 2000 To obtain liposomes with high encapsulation efficiency and good stability, the concentration of sodium acetate stock solution was fixed at 20 mg / mL. Based on orthogonal experiments, the concentrations of DSPC:cholesterol and DSPE-PEG were compared. 2000 -CA, DSPE-PEG 2000 The proportion of -MAN was optimized. Specific factor levels are shown in Table 1.

[0096] Table 1. Optimization Factors for Sodium Acetate Liposome Synthesis

[0097]

[0098] The orthogonal experimental groupings are shown in Table 2:

[0099] Table 2 Orthogonal Experiment Grouping Table

[0100]

[0101] (2) Composition of optimization evaluation indicators

[0102] a. Sodium acetate encapsulation efficiency of liposomes

[0103] a.1 Establishment of a high-performance liquid chromatography (HPLC) method for determination

[0104] The HPLC conditions were as follows: Thermol Hypersil ODS-2C18 column (4.6 mm * 200 mm, 5 μm) was selected; the mobile phase was 0.01 mol / L ammonium dihydrogen phosphate aqueous solution, and 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 °C.

[0105] a.2 Drawing the Standard Curve

[0106] Sodium acetate was accurately weighed into 10 mL volumetric flasks and diluted with PBS to prepare sodium acetate standard solutions with concentrations of 0.01, 0.025, 0.05, 0.1, 0.2, and 0.5 mg / mL. Each sample was analyzed according to HPLC conditions, and the peak area was recorded. A standard curve of AC linearity was plotted with the peak area (A) as the ordinate and the concentration (C) as the abscissa. Sodium acetate was also dissolved in serum, 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. Peak area-time standard curves of acetic acid in serum, simulated gastric juice, and simulated intestinal juice were detected according to HPLC conditions.

[0107] a.3 Determination of encapsulation efficiency of sodium acetate liposomes by ultrafiltration method

[0108] Take an appropriate amount of sodium acetate liposome solution and place it in an activated ultrafiltration tube (MYCO = 3 kDa, Millipore), centrifuge at 12000 rpm for 10 min. Remove the solution from 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 using the following formula.

[0109]

[0110] b. Particle size, PDI, Zeta potential, and drug loading of NaA@CA / MAN-LPs

[0111] The particle size, PDI, and Zeta potential of NaA@CA / MAN-LPs were measured using a laser particle size analyzer. The sodium acetate encapsulation efficiency (EE%) and liposome drug loading (DL%) of NaA@CA / MAN-LPs were determined using ultrafiltration and high-performance liquid chromatography (HPLC). The formula for calculating the liposome drug loading is as follows:

[0112]

[0113] c. Stability of NaA@CA / MAN-LPs in simulated body fluids

[0114] Sterile fetal bovine serum was diluted 10-fold with PBS (pH 7.4) to prepare a simulated serum solution. The artificial gastric juice was prepared as follows: 0.5g of pepsin was added to 10mL of pure water, dissolved, and then added to 0.82mL of 1mol / L hydrochloric acid solution. After mixing, the solution was diluted to 50mL, pH=1.2. The artificial intestinal juice was prepared as follows: 0.34g of dipotassium hydrogen phosphate was added to 25mL of water, and the pH was adjusted to 6.8 with 0.4% NaOH; 0.5g of pancreatin was dissolved in water, and the two solutions were mixed and diluted to 50mL.

[0115] 100 μL of NaA@CA / MAN-LPs was mixed with 900 μL of simulated gastric juice, simulated intestinal juice, and serum solution, respectively, and shaken at 1000 rpm at 37 °C. Samples were collected after 2 hours from the group shaken with simulated gastric juice, and after 6 hours from the groups shaken with simulated intestinal juice and serum solutions. The particle size, PDI, and Zeta potential of the collected sample solutions were determined using a laser particle size analyzer. Simultaneously, the sodium acetate release rate after incubation in different media solutions was detected by HPLC. Changes in liposome particle size and encapsulation efficiency before and after incubation were analyzed to characterize the stability of liposomes in the gastrointestinal tract and serum solution.

[0116] Orthogonal experiment results:

[0117] Table 3 shows the sodium acetate encapsulation efficiency and drug loading of liposomes in different groups during the orthogonal experiment. The results show that different DSPC:cholesterol ratios and DSPE-PEG... 2000 -CA and DSPE-PEG 2000 -MAN concentration has a significant impact on liposome encapsulation efficiency and drug loading. The sodium acetate encapsulation efficiency of the nine orthogonal experimental groups of liposomes ranged from 70.52% to 81.15%, with the order from highest to lowest being: 3>7>1>8>4>2>9>6>5. The drug loading of the nine orthogonal experimental groups of liposomes ranged from 20.86% to 31.58%, with the order from highest to lowest being: 3>7>2>1>4>8>9>6>5. These results indicate that liposomes 3 and liposome 7 had significantly higher encapsulation efficiency and drug loading than the remaining seven groups of liposomes. Further evaluation of the particle stability of the nine groups of liposomes after incubation in serum solution, simulated gastric juice, and simulated intestinal juice will be conducted to determine the optimal composition of the liposomes.

[0118] Table 3. Effects of liposome composition on sodium acetate encapsulation efficiency and drug loading.

[0119] Experimental Groups Sodium acetate encapsulation efficiency (EE%) Liposome drug loading (DL%) 1 <![CDATA[77.71±0.47 b ]]> <![CDATA[27.46±0.16 c ]]> 2 <![CDATA[73.41±0.94 e ]]> <![CDATA[27.81±0.36 c <!-- 8 -->]]> 3 <![CDATA[81.15±0.07 a ]]> <![CDATA[31.58±0.03 a ]]> 4 <![CDATA[76.09±0.80 d ]]> <![CDATA[25.62±0.27 d ]]> 5 <![CDATA[70.52±0.87 f ]]> <![CDATA[20.86±0.26 g ]]> 6 <![CDATA[71.28±0.58 f ]]> <![CDATA[23.22±0.19 f ]]> 7 <![CDATA[78.42±0.30 b ]]> <![CDATA[29.70±0.11 b ]]> 8 <![CDATA[76.89±0.64 cd ]]> <![CDATA[24.64±0.21 e ]]> 9 <![CDATA[73.00±0.36 e ]]> <![CDATA[24.58±0.12 e ]]>

[0120] Note: Significance analysis was performed on the sodium acetate encapsulation rate and drug loading of the nine groups of liposomes. Different letters in the same column indicate significant differences (p<0.05), and the same letter indicates no significant differences (p>0.05).

[0121] As shown in Table 4, the particle size changes of liposomes with different compositions after incubation in simulated body fluids are as follows: 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 each group of liposomes increased to varying degrees. After incubation in serum for 6 hours, the liposome particle sizes of each group, from smallest to largest, were: 7<2<8<6<5<1<4<3<9, and the particle size of liposome 7 (132.41±3.67nm) was significantly lower than that of the other eight groups. After incubation in simulated gastric fluid for 2 hours, the liposome particle sizes of different groups, from smallest to largest, were: 7<9<3<8<1<2<6<4<5, and the particle sizes of liposomes 7 and 9 (107.21±2.40nm and 128.70±1.59nm, respectively) were significantly lower than those of the other seven groups. After incubation in simulated intestinal fluid for 6 hours, the liposome particle sizes of different groups, from smallest to largest, were: 4<8<1<7<6<5<9<2<3. Except for liposomes 2 and liposomes 3, there was no significant difference in particle size among the remaining seven groups of liposomes. Table 5 shows the PDI changes of nine groups of liposomes with different compositions after incubation in simulated body fluids. The initial PDI of the nine groups of liposomes ranged from 0.20 to 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 relatively uniform after incubation in simulated body fluids, with no obvious aggregation. Table 6 shows the potential changes of the nine groups of liposomes after incubation in simulated body fluids. It can be seen that the initial Zeta potential of all liposomes was positive, and the charge of the liposomes remained positive after incubation in serum, simulated gastric juice, and simulated intestinal juice.

[0122] Table 4. Particle size changes of liposomes in different groups in serum, simulated gastric juice, and intestinal juice during orthogonal experiments.

[0123]

[0124] Note: The original particle size of the nine groups of liposomes and the particle size of liposomes after incubation in serum, simulated gastric juice, and simulated intestinal juice were analyzed for significance. Different letters in the same column indicate significant differences (p<0.05), and the same letter indicates no significant differences (p>0.05).

[0125] Table 5. Changes in PDI of different groups of liposomes in serum, simulated gastric juice, and intestinal juice during orthogonal experiments.

[0126]

[0127] 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. Different letters in the same column indicate significant differences (p<0.05), and the same letter indicates no significant differences (p>0.05).

[0128] Table 6. Changes in zeta potential of liposomes in different groups in serum, simulated gastric juice, and intestinal juice during orthogonal experiments.

[0129]

[0130] 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. Different letters in the same column indicate significant differences (p<0.05), and the same letter indicates no significant differences (p>0.05).

[0131] See results Figure 4 After incubation in serum for 6 hours, the sodium acetate release from the nine liposome groups ranged from 3.61% to 5.27%. Liposomes 4 and 7 showed significantly lower sodium acetate release than the other groups, at 3.73% ± 0.19% and 3.61% ± 0.32%, respectively. Figure 4 A). After incubation in simulated gastric juice for 2 hours, the sodium acetate release from the nine liposome groups ranged from 12.57% to 16.51%. Liposomes 2 and 7 showed significantly lower sodium acetate release than the other groups, at 12.57% ± 0.32% and 12.70% ± 0.37%, respectively. Figure 4 B). Similarly, after incubation in simulated intestinal fluid for 6 hours, all nine groups of liposomes released sodium acetate, with release amounts ranging from 5.16% to 7.73%. The release amounts of liposomes 3 and 7 were significantly lower than the other seven groups, at 5.16% ± 0.08% and 5.51% ± 0.06%, respectively. Figure 4 C). The above results indicate that, compared with the other lipid groups, liposomes 4 and 7 are more stable in serum, liposomes 2 and 7 are more stable in simulated gastric juice, and liposomes 3 and 7 are more stable in simulated intestinal juice.

[0132] To optimize DSPC:cholesterol and DSPE-PEG in liposomes 2000 -CA and DSPE-PEG 2000The proportion of -MAN was optimized using orthogonal optimization. The results showed that the sodium acetate encapsulation 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%, also ranking second among the nine groups of liposomes. This indicates that the liposomes prepared with the given composition of liposome 7 had a relatively high sodium acetate content. To study the stability of liposomes in different body fluids, the nine groups of liposomes were incubated in serum, simulated gastric juice, and simulated intestinal juice, respectively. Their particle size, PDI, Zeta potential, and sodium acetate release were then measured. The results showed that after incubation in serum, simulated gastric juice, and simulated intestinal juice, the particle size of liposome 7 did not increase significantly compared to other groups; the particle size distribution was uniform, and the particles still carried a positive charge. Furthermore, compared to other liposome groups, liposome 7 exhibited the lowest sodium acetate release after incubation with serum, simulated gastric juice, and simulated intestinal juice, at only 3.61% ± 0.32%, 12.70% ± 0.37%, and 5.51% ± 0.06%, respectively. In summary, at the given liposome ratio of liposome 7, sodium acetate liposomes exhibited the strongest stability, effectively preventing adsorption and structural damage to the liposomes by enzymes in gastrointestinal juice and proteins in serum. Therefore, the optimal ratio for sodium acetate liposomes is a DSPE:cholesterol ratio of 3:1, with 1% DSPE-PEG. 2000 -CA and 10% DSPE-PEG 2000 -MAN.

[0133] Example 2: Sodium acetate stock solution concentration ramp-up experiment

[0134] After orthogonal optimization to determine the proportions of each component in the liposomes, the concentration of the sodium acetate stock solution was optimized to maximize the sodium acetate drug loading of the liposomes. 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 composition. Unencapsulated sodium acetate was removed by ultrafiltration, and the sodium acetate concentration was detected by HPLC to calculate the sodium acetate encapsulation efficiency in the liposomes. The liposomes were lyophilized using liquid nitrogen and weighed; the sodium acetate drug loading of the liposomes was calculated based on their mass.

[0135] The optimal formulation of sodium acetate liposomes was determined to be 40.5% DSPC, 13.5% cholesterol, 1% DSPE-PEG-CA, 10% DSPE-PEG-MAN, and 5% DSPE-PEG. 2000 And 30% DC-cholesterol. A gradient of sodium acetate stock solution concentrations (20, 30, 40, 50, 60 mg / mL) was established to obtain the optimal sodium acetate encapsulation efficiency and loading. The results are shown in [Figure number missing]. Figure 5As the sodium acetate stock solution concentration increased from 20 mg / mL to 60 mg / mL, the encapsulation efficiency of liposomes also increased from 71.09% to 79.36%. When the sodium acetate stock solution concentration exceeded 40 mg / mL, the increase in liposome encapsulation efficiency tended to level off. Figure 5 A), and at this point the liposome loading was at its highest, at 33.07% ( Figure 5 Therefore, when preparing liposomes with high embedding efficiency and loading capacity, the concentration of sodium acetate stock solution selected is 40 mg / mL.

[0136] Example 3: Preparation and characterization of liposomes with different modifications

[0137] After optimization through orthogonal experiments and sodium acetate reservoir ramp-up experiments, the optimal composition ratio of sodium acetate liposomes modified with sodium cholate and mannose, as well as the optimal sodium acetate reservoir concentration, were determined. Based on this, the following 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 different modified liposomes are shown in Table 7.

[0138] Table 7. Proportions of Sodium Acetate Liposomes with Different Modification Types

[0139]

[0140] The particle size, PDI, and Zeta potential of five types of liposomes were measured using dynamic light scattering. The sodium acetate encapsulation efficiency and drug loading of the liposomes were detected by HPLC, and the morphology of the liposomes was observed by transmission electron microscopy (TEM). The specific TEM procedure was as follows: liposomes were diluted and dropped onto a copper grid, allowed to stand for 2 minutes, the liquid on the surface of the copper grid was blotted dry with filter paper, 2% phosphotungstic acid was added for negative staining for 5 minutes, the copper grid was dried, and the morphology of different types of liposomes was observed using a JEM-1400 Flash transmission electron microscope.

[0141] After orthogonal experiments and sodium acetate reservoir ramp-up experiments, the optimal composition ratio for sodium acetate liposomes with high encapsulation efficiency and drug loading was determined. Based on this, five modified sodium acetate liposomes were synthesized: 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); and ⑤ sodium acetate and mannose liposomes without sodium acetate (CA / MAN-LPs). Particle size distribution and encapsulation efficiency / drug loading of the five liposomes were measured using a particle size analyzer, and the results are shown in Table 8. Figure 6 The five liposomes all had a particle size of approximately 100 nm, a PDI < 0.25, and a positive Zeta potential. Except for CA / MAN-LPs, the encapsulation efficiency of NaA@CA / MAN-LPs, NaA@CA-LPs, NaA@MAN-LPs, and NaA@LPs ranged from 76.6% to 81.57%, and the drug loading ranged from 33.16% to 37.94%. This indicates that the five synthesized liposomes are all approximately 100 nm in size, uniformly distributed, and positively charged nanoliposomes with high sodium acetate encapsulation efficiency and drug loading capacity. The appearance of the five liposomes was observed and photographed using transmission electron microscopy. The results are shown in the figure below. Figure 6 The five liposomes were spherical in appearance, with a particle size of about 100 nm, consistent with the particle size measurement results. The particles were evenly distributed and did not aggregate or clump together.

[0142] Table 8 Characterization of different types of sodium acetate liposomes

[0143]

[0144] Example 4: Biosafety of Sodium Acetate Liposomes

[0145] 1. Cell Culture

[0146] 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, the other experimental procedures were the same.

[0147] 2. Liposome safety

[0148] 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.

[0149]

[0150] 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.

[0151] Example 5: Sodium acetate liposomes reduce lipid deposition in liver cell lines and inhibit inflammation.

[0152] 1. Cell Culture

[0153] Hepatocytes AML-12 were revived and cultured using DMEM / F12 complete medium; liver macrophages Kupffer were revived and passaged using RPMI-1640 complete medium.

[0154] 2. Cell modeling

[0155] AML-12 cells in good logarithmic growth phase were digested with 0.25% trypsin (containing EDTA), collected by centrifugation, and counted. The cells were then stored at a rate of 1×10⁻⁶. 6Cells were inoculated into 6-well plates at 2 mL per well and incubated at 37°C in a 5% CO2 incubator. When the cells reached 60% confluence, the culture medium was aspirated, and the cells were washed twice with sterile PBS. Then, serum-free DMEM / F12 medium was added for starvation treatment for 12 h. After the culture was completed, the cells were washed twice with sterile PBS, 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 of the 6-well plate to induce the NAFLD cell model. The control group cells were treated with complete control medium containing BSA.

[0156] 3. Sodium acetate liposomes alleviate lipid deposition in NAFLD cells.

[0157] AML-12 cells were selected and plated according to the "4.3.2 Construction of NAFLD Cell Model" procedure for cells in good logarithmic growth phase. Cells were cultured and starved for 12 hours. Before lipid induction, the cells were divided into six groups: Group 1 received an equal volume of PBS as a blank control; Group 2 received lipid induction medium as the NAFLD model group; Group 3 received sodium cholate / mannose double-labeled sodium acetate liposomes (NaA@CA / MAN-LPs) in lipid induction medium; Group 4 received sodium acetate solution (NaA) in lipid induction medium; Group 5 received unlabeled sodium acetate liposomes (NaA-LPs) in lipid induction medium; and Group 6 received blank double-labeled liposomes (CA / MAN-LPs) without sodium acetate in lipid induction medium. The concentration of sodium acetate solution was 1 mM. After 24 hours of culture, TG, TC, BSA, Oil Red O levels, total RNA extraction, and RT-qPCR detection of lipid metabolism-related genes were performed on all groups of cells.

[0158] The results are as follows Figure 8As shown in the AD diagram, compared with the blank control group, the relative areas of TG, TC, and Oil Red O stained lipid droplets in the NAFLD model group cells were significantly increased, indicating that the NAFLD cell model was successfully constructed after AML-12 cells were induced and cultured with sodium oleate and sodium palmitate for 24 h. After treating NAFLD cells with 1 mM NaA@CA / MAN-LPs and NaA solution alone for 24 h, the relative areas of TG, TC, and Oil Red O stained lipid droplets in the cells were significantly reduced. Similarly, compared with the NAFLD model group, after incubating NAFLD cells with NaA@LPs for 24 h, the intracellular TG and TC contents were significantly reduced, but the relative area of ​​lipid droplets stained with Oil Red O was not significantly improved. After incubating NAFLD cells with blank liposomes (CA / MAN-LPs) without sodium acetate for 24 h, the relative areas of TG, TC, and Oil Red O lipid droplets in the cells were slightly reduced, but there was no significant difference. Analysis of variance within groups showed that, compared with NaA treatment, treatment of NAFLD cells with NaA@CA / MAN-LPs had a more significant effect on alleviating intracellular TG and TC accumulation. The results indicate that incubation of NAFLD cells with sodium acetate / mannose dual-labeled liposomes at a concentration of 1 mM significantly alleviated lipid deposition in NAFLD cells after 24 h, and the alleviating effect of dual-labeled sodium acetate liposomes was significantly better than that of sodium acetate solution alone.

[0159] 4. Sodium acetate liposomes alleviate inflammation of liver macrophages.

[0160] Select Kupffer cells in good logarithmic growth phase, at 5 × 10⁶ cells / year. 6 Cells were densely seeded into plates at 37°C and incubated in a 5% CO2 incubator. When the cells reached 60% confluence, the culture medium was aspirated, and the cells were washed twice with sterile PBS. Then, the cells were starved for 12 hours in serum-free RPMI-1640 medium, washed twice with sterile PBS, and then divided into groups. Except for the blank control group, the remaining five groups of cells were treated with 20 ng / mL LPS to induce cellular inflammation six hours before the end of treatment. The specific treatment conditions for each group were as follows: the blank control group was treated with an equal volume of PBS solution without LPS; the model group was treated with LPS to induce cellular inflammation 6 hours before the end of incubation; the double-labeled liposome group was treated with 1 mM sodium cholate / mannose double-labeled sodium acetate liposomes (NaA@CA / MAN-LPs); the sodium acetate group was treated with 1 mM sodium acetate solution; the unlabeled liposome group was treated with 1 mM unlabeled sodium acetate liposomes (NaA-LPs); and the blank liposome group was treated with blank double-labeled liposomes (CA / MAN-LPs) without sodium acetate. After processing, total RNA was extracted from cells for RT-qPCR detection of inflammation-related genes.

[0161] The results are as follows Figure 9As shown in the AD diagram, compared with the control group, LPS-induced Kupffer cells exhibited significantly upregulated expression of pro-inflammatory factors IL-1β, IL-6, and TNF-α mRNA, and significantly downregulated expression of anti-inflammatory factor IL-4 mRNA, indicating that the model group of Kupffer cells produced a significant inflammatory response. Compared with the model group, treatment with sodium acetate-containing sodium cholate / mannose double-labeled liposomes (NaA@CA / MAN-LPs), unlabeled liposomes (NaA@LPs), and sodium acetate solution alone (NaA) significantly reduced the expression of IL-1β, IL-6, and TNF-α mRNA in Kupffer cells, while significantly increasing the expression of IL-4 mRNA. This indicates that sodium acetate and sodium acetate liposomes effectively inhibited the activation of inflammatory pathways in Kupffer cells. The unlabeled double-labeled blank liposomes (CA / MAN-LPs) had no significant effect on the high expression of pro-inflammatory factors and the low expression of anti-inflammatory factors in Kupffer cells, indicating that liposome materials had neither alleviating nor promoting effects on Kupffer cells in an inflammatory state. Intra-group differential analysis of the four treatment groups showed that, compared with unlabeled sodium acetate liposomes and sodium acetate alone, treatment with sodium acetate-labeled liposomes significantly alleviated the high expression of IL-1β, IL-6, and TNF-α mRNA and the low expression of IL-4 mRNA in Kupffer cells. These results indicate that sodium acetate-labeled liposomes effectively alleviate the inflammatory response in Kupffer cells, and their intervention effect is significantly better than that of sodium acetate alone or unlabeled sodium acetate liposomes.

[0162] Example 6: In vivo targeted localization of liposomes

[0163] Fifteen male C57 mice were used, divided into five groups of three. 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. In vivo fluorescence imaging was performed on the anesthetized mice at 0.5, 1, 1.5, 2, and 2.5 hours after gavage. The results are as follows: Figure 10 As 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.

[0164] 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 mouse liver reached its peak at 14 h, with values ​​of 0.303, 0.208, 0.147, 0.084, and 0.038 [(p / sec / cm2 / sr) / μM / cm2], respectively. The fluorescence intensity-time curves of the five fluorescent liposomes were integrated, AUG was calculated, and significance was analyzed. The results are as follows: Figure 11 C. The fluorescence intensity of the five liposomes in the liver, according to AUG0-24, from highest to lowest, was: 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 single fluorescent dyes, unmodified fluorescent liposomes, mannose-labeled fluorescent liposomes, or sodium cholate-labeled fluorescent liposomes. 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 AUG2-24 over 2-24 hours was calculated, and the results are as follows: Figure 11 As shown in E, the fluorescence intensity accumulated by CY7@CA / MAN-LPs in the intestine from 2 to 24 hours was significantly higher than that of CY7@MAN-LPs, CY7@LPs and CY7, but there was no significant difference compared with fluorescent liposomes labeled with sodium cholate alone. This indicates that the sodium cholate ligand modified on the surface of liposomes is beneficial to promoting the intestinal absorption of liposomes, and its mechanism may be related to the ASBT receptor in the mouse ileum.

[0165] The results above demonstrate that, compared with fluorescent liposomes labeled with sodium cholate or mannose alone, unlabeled fluorescent liposomes, or single fluorescent dyes, fluorescent liposomes labeled with sodium cholate / mannose are more effective at penetrating the mouse intestine, targeting liver tissue, and exerting their effects for a longer period of time.

[0166] Example 7: Sodium acetate liposomes alleviate NAFLD in mice

[0167] 1. Animal models

[0168] The animals used in this experiment were 6-week-old male C57BL / 6J mice without specific pathogens (Specific pathogen Free, SPF), weighing 18 ± 1.0 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (certificate number SYXK (Beijing) 2021-0001). The experimental design passed the review of the Laboratory Animal Welfare and Ethics Committee of Zhongyan Zichuang (Beijing) Biotechnology Co., Ltd. (batch number ZYZC202402006S), and all animal handling and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council

[0169] 2. Model establishment and intervention

[0170] Animal grouping and intervention treatment

[0171] Before the formal experiment began, all mice were adaptively fed for one week. During the feeding process, the mice had free access to sterile water. After one week, the mice were randomly divided into six groups of 8 mice each (4 mice per cage), and then a sodium acetate liposome gavage experiment was carried out, with gavage once every other day for 10 weeks. The experimental design plan for animals, the specific grouping and intervention methods are as follows:

[0172] (1) Normal control group: Fed with basal maintenance diet, and gavaged with PBS every other day from week 0 to week 10

[0173] (2) NAFLD model group: Fed with HFD diet, and gavaged with PBS every other day from week 0 to week 10

[0174] (3) Dual-labeled liposome intervention group (NaA@CA / MAN-LPs): Fed with HFD diet, and gavaged with sodium cholate / mannose dual-labeled sodium acetate liposomes (200 μL, the concentration of sodium acetate in the dual-labeled liposomes was 15 mg / mL) every other day from week 0 to week 10

[0175] (4) Sodium acetate intervention group (NaA): Fed with HFD diet, and gavaged with sodium acetate solution (200 μL, the concentration of the sodium acetate solution was 15 mg / mL) every other day from week 0 to week 10

[0176] (5) Unlabeled liposome intervention group (NaA@LPs): Fed with HFD diet, and gavaged with unlabeled sodium acetate liposomes (200 μL, the concentration of sodium acetate in the unlabeled liposomes was 15 mg / mL) every other day from week 0 to week 10

[0177] (6) Blank liposome intervention group (CA / MAN-LPs): Fed with HFD diet, and gavaged with blank liposomes (200 μL, the concentration of the blank liposomes was the same as that of the liposomes in the other groups, which was 46 mg / mL) every other day from week 0 to week 10

[0178] Evaluation of the improvement of HFD-induced NAFLD function in mice using sodium acetate liposomes using methods commonly used in the art.

[0179] Body weight change trend, organ index, oral glucose tolerance test (OGTT), blood biochemical index measurement, tissue hematoxylin and eosin (H&E) staining and Oil Red O staining, liver tissue F4 / 80 immunohistochemical staining, detection of changes in mouse liver lipid content, detection of mouse liver function index, detection of mouse liver oxidative damage (MDA and SOD monitoring), extraction of total RNA from liver tissue and real-time quantitative PCR determination (genes Acc1, Fasn, Srebf1, CPT1α, IL-1β, IL-4, IL-6, TNF-α, GAPDH); Western blotting.

[0180] 1) Weight results as follows Figure 12 As shown in Figures 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 alone (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 all three groups was significantly lower than that of the NAFLD model group, indicating that sodium acetate, double-labeled sodium acetate liposomes, and unlabeled liposomes all effectively alleviated the weight gain in NAFLD mice. After 10 weeks of intervention with blank double-labeled liposomes (without sodium acetate), the mice lost 1.76g of body weight, 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 weight gain. Analysis of the body weight changes in the four intervention groups 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 effect of double-labeled liposomes in alleviating the weight gain in NAFLD mice was significantly higher than that of sodium acetate treatment alone. Figure 12 A, B);

[0181] 2) Glucose tolerance results as follows Figure 13As shown: Compared with the NAFLD model group, the area under the OGTT curve of sodium acetate liposomes (sodium cholate / mannose dual-labeled), sodium acetate solution alone, and unlabeled sodium acetate liposomes was significantly reduced (p < 0.01), indicating that glucose tolerance was significantly restored after treatment in these three groups. After 10 weeks of intervention with blank liposomes without sodium acetate, the area under the OGTT curve of NAFLD mice decreased, but there was no significant difference compared with the NAFLD group. Analysis of the AUG (autogenous glucose tolerance) of the four treatment groups showed that the AUG of OGTT in the dual-labeled sodium acetate liposome group after 10 weeks of intervention was significantly lower than that in mice treated with sodium acetate solution alone and unlabeled sodium acetate liposomes. These results indicate that oral administration of sodium acetate, unlabeled sodium acetate liposomes, and dual-labeled sodium acetate liposomes effectively improved glucose tolerance in NAFLD mice, and the sodium acetate / mannose dual-labeled sodium acetate liposomes showed the most significant effect in improving glucose tolerance. Figure 13 AB).

[0182] 3) Results of lipid metabolism, such as Figure 14 As shown in the AD diagram, compared with the NAFLD model group, after gavage administration of sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate solution alone, and unlabeled sodium acetate liposomes, the serum TG, TC, and LDL-C levels of mice were significantly reduced, while the HDL-C level was significantly increased (p<0.01). This indicates that unlabeled liposomes containing sodium acetate, double-labeled liposomes, and sodium acetate can all significantly alleviate the lipid levels in NAFLD. However, after gavage administration of double-labeled liposomes without sodium acetate, the serum TG, TC, and LDL-C levels of mice did not change significantly, while the HDL-C level increased significantly (p<0.05). This indicates that the blank liposome material does not aggravate the serum lipid metabolism level in NAFLD mice and increases the HDL-C content to some extent. Intra-group significance analysis was performed on serum lipid metabolism parameters in mice in the double-labeled liposome group, sodium acetate group, unlabeled liposome group, and blank liposome group. The results showed that compared with sodium acetate solution alone, oral administration of sodium acetate liposomes double-labeled with sodium cholate / mannose significantly reduced serum TG levels and significantly increased HDL-C levels, indicating that double-labeled sodium acetate liposomes were more effective than sodium acetate alone in alleviating NAFLD dyslipidemia.

[0183] 4) Sodium acetate liposomes improve adipose tissue content in HFD-induced NAFLD mice, such as... Figure 15 As shown, Figure 15A: Compared with the NAFLD model group mice, gavage administration of sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate solution alone, unlabeled sodium acetate liposomes, and blank liposomes all significantly reduced the proportion of white adipose tissue in NAFLD mice, decreasing by 3.56%, 2.20%, 2.05%, and 0.54%, respectively. Intra-group difference analysis of the total white adipose tissue content in the four intervention groups showed that the proportion of white adipose tissue after intervention with sodium cholate / mannose double-labeled sodium acetate liposomes was significantly lower than that after gavage administration of sodium acetate solution alone or unlabeled liposomes containing sodium acetate. H&E staining of mouse epididymal adipose tissue was performed, and the staining results are as follows... Figure 15 As shown in Figure B, compared with the control group, the epididymal adipocytes of the model group mice were significantly enlarged. Compared with the NAFLD model group, the epididymal fat vacuolar area was significantly reduced and the cross-sectional area of ​​epididymal adipocytes was significantly reduced in the groups treated with sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate solution alone, and unlabeled sodium acetate liposomes. The number of adipocytes in the same field of view was significantly increased, and the changes were most pronounced in the double-labeled liposome group. In conclusion, unlabeled liposomes, double-labeled liposomes, and sodium acetate effectively reduced the white fat content and epididymal adipocyte volume in NAFLD mice, with sodium cholate / mannose double-labeled sodium acetate liposomes showing the most significant effect in improving adipose tissue content in NAFLD mice.

[0184] 5) Results of sodium acetate liposomes improving liver pathological morphology in NAFLD mice: Figure 16 As shown. Figure 16 As shown in Figure A, the livers of control mice fed a normal diet were smooth, reddish-brown, and had some tissue elasticity. Compared to the control group, the livers of NAFLD model mice constructed after 10 weeks of HFD diet became lighter in color, and obvious "lipid droplets" were clearly visible. After gavage administration of blank liposomes without sodium acetate, the liver morphology was similar to that of the NAFLD model group, with no significant changes. However, after gavage administration of sodium acetate liposomes double-labeled with sodium cholate / mannose, sodium acetate solution alone, or unlabeled sodium acetate liposomes for 10 weeks, the liver color of NAFLD mice returned to reddish-brown, and the surface smoothness was restored, indicating that sodium acetate or the two types of liposomes containing sodium acetate have the function of alleviating liver deposition in NAFLD mice. H&E sections of liver tissue from healthy control mice showed normal lobular structure, with hepatocyte cords arranged radially from terminal venules in the central area of ​​the lobules. The hepatocytes were evenly distributed and brownish-red, with clear nuclei and no lipid droplets or inflammatory cell aggregation in the cytoplasm. Figure 16B) Hematologic and epithelial studies (H&E) of the livers of NAFLD model mice showed that the lobular structure of the liver was disrupted, and hepatocytes underwent ballooning degeneration, characterized by enlarged hepatocytes containing large lipid droplets. Some hepatocytes exhibited a mixed type of steatosis, consisting of macrovesicles and microvesicles, accompanied by inflammatory cell infiltration. After gavage administration of sodium acetate liposomes double-labeled with sodium cholate / mannose, sodium acetate solution alone, or unlabeled sodium acetate liposomes, the number and area of ​​vacuoles in the mouse liver were significantly reduced, indicating a significant improvement in hepatic steatosis. After gavage administration of blank liposomes without sodium acetate, the area of ​​vacuoles in the mouse liver decreased, but the number remained relatively high, indicating that liposomes alone had a very limited effect on alleviating lipid deposition in the liver of NAFLD mice. Further quantitative analysis of lipid droplet area in liver H&E stained sections revealed that the lipid droplet area in the liver of NAFLD model group mice was 1.93 times that of healthy control mice. Sodium acetate / mannose double-labeled sodium acetate liposomes, sodium acetate solution, and unlabeled sodium acetate liposomes significantly reduced the lipid droplet area in the liver of NAFLD mice (p<0.01). After 10 weeks of sodium acetate intervention, the lipid droplet area in the liver of NAFLD mice decreased by 46.39%, 40.97%, and 39.33%, respectively. Figure 16 C). In addition to H&E staining, Oil Red O staining was performed on the liver tissues of mice in each group. The results are shown in [Figure 1]. Figure 16 D. In healthy mice fed a normal diet, the number and size of red lipid droplets in the liver were low, while in the NAFLD group and the blank liposome group, the number and size of red lipid droplets in the liver were significantly increased, and the oil red color was bright. This indicates that a large amount of fat was deposited in the liver of NAFLD mice, and the double-labeled liposome material without sodium acetate had no alleviating effect on lipid deposition in the liver of NAFLD mice. Sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate alone, or sodium acetate unlabeled liposomes significantly reduced lipid deposition in the liver of NAFLD mice, and the double-labeled sodium acetate liposomes showed the most significant alleviating effect. The Oil Red O stained sections at the intervention endpoint were closer to those of the normal group mice. Figure 16 D). The proportion of red lipid droplets to the total slice area was quantitatively analyzed using ImageJ software. The relative lipid droplet area of ​​each group was calculated using the control group as the standard. Consistent with H&E staining results, compared to the control group, the number of liver lipid droplets in the NAFLD model group and the blank liposome group was significantly increased, at 14.37 times and 13.67 times that of the control group, respectively. After 10 weeks of intervention with sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate alone, and unlabeled sodium acetate liposomes, the liver lipid droplet area of ​​NAFLD mice was significantly reduced. Compared to the NAFLD model group, the liver lipid droplet area at the intervention endpoint decreased by 88.27%, 73.45%, and 70.40%, respectively. Figure 16 E).

[0185] 6) The results of sodium acetate liposomes in improving hepatic lipid accumulation and oxidative stress in NAFLD mice are as follows: Figure 17As shown in the figure. The results showed that after 10 weeks of intervention with two liposomes containing sodium acetate (NaA@CA / MAN-LPs and NaA@LPs) in NAFLD mice, the liver index was significantly lower than that of sodium acetate solution alone, indicating that liposomes containing sodium acetate were more effective than sodium acetate alone in improving liver quality in NAFLD mice. Figure 17 A). The levels of TG and TC in mouse liver were detected using a kit method, and the results are as follows: Figure 17 Compared with the NAFLD group, mice in groups B and C showed no significant improvement in liver TG and TC levels after 10 weeks of gavage administration of blank liposomes without sodium acetate. However, after 10 weeks of gavage administration of either of the two liposomes containing sodium acetate or sodium acetate solution, the TG and TC levels in the liver of NAFLD mice were significantly reduced, and liver lipid deposition was significantly improved. Intragroup differential analysis showed that the liver TG and TC levels in mice administered double-labeled sodium acetate liposomes were significantly lower than those in the sodium acetate group. The levels of lipid peroxidation oxidase (MDA) and superoxide dismutase (SOD) in the liver of mice were detected using a kit method, and the results are as follows: Figure 17 As shown in D and E, after gavage administration of blank liposomes without sodium acetate to NAFLD mice, the liver MDA and SOD levels were not significantly improved. However, after gavage administration of sodium acetate liposomes double-labeled with sodium cholate / mannose, sodium acetate solution, or unlabeled sodium acetate liposomes, the liver MDA level of NAFLD mice was significantly reduced, and the SOD level was significantly increased (p<0.01). Intragroup differential analysis showed that double-labeled sodium acetate liposomes significantly alleviated the increase in liver MDA and the decrease in SOD levels in NAFLD mice compared to gavage administration of sodium acetate solution. This indicates that encapsulating sodium acetate in sodium cholate / mannose double-labeled liposomes significantly increased the alleviating effect of sodium acetate on liver inflammation and oxidative stress in NAFLD mice.

[0186] 7) The expression of lipid metabolism-related genes in the liver of NAFLD mice was inhibited by liposomes, and the results are as follows: Figure 18 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 acetate liposomes double-labeled with sodium cholate / mannose, sodium acetate solution, or unlabeled sodium acetate liposomes, the expression levels of Acaca, Fasn, Slc27a2, and Srebf1 mRNAs in the liver of NAFLD mice were significantly reduced, while the expression level of CPT1α mRNA was significantly upregulated. These results indicate that sodium acetate or sodium acetate-containing liposomes effectively alleviated the upregulation of adipogenesis-related genes in the liver of NAFLD mice and promoted the expression of the fatty acid transporter CPT1α. Western blot analysis was used to analyze the phosphorylation of AMPKα protein in the liver of NAFLD mice after 10 weeks of gavage administration of sodium acetate or sodium acetate liposomes. The results are shown in [Figure 1]. Figure 18F. Compared with control mice, hepatic AMPKα protein phosphorylation was significantly inhibited in HFD-induced NAFLD model mice and in mice treated with blank liposomes without sodium acetate. After gavage administration of sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate solution, or unlabeled sodium acetate liposomes to NAFLD mice, the phosphorylation expression level of hepatic AMPKα protein was significantly increased. Figure 18 (F, G). After gavage administration of unlabeled liposomes containing sodium acetate, double-labeled liposomes, and sodium acetate alone, the expression of Acc1 and Srebf1 cmRNA in the liver was significantly downregulated, while the expression of CPT1α was significantly upregulated. These results indicate that sodium acetate or double-labeled sodium cholate liposomes activate the AMPK pathway, inhibit the transcription of Acc1 and Srebf1c, promote CPT1α expression, and thus regulate downstream lipid metabolism genes, thereby reducing hepatic lipid deposition.

[0187] 8) Sodium acetate liposomes improve liver function in NAFLD mice: Serum AST and ALT levels were measured, and the results are as follows: Figure 19 As shown in A and B, after NAFLD mice were administered blank liposomes without sodium acetate by gavage, serum ALT and AST levels did not decrease significantly. However, after gavage with sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate solution, or unlabeled sodium acetate liposomes, serum AST and ALT levels in mice decreased significantly. Liver ALT and AST levels were measured, and the results are shown in [Figure 1]. Figure 19 C, D. Compared with the control group, the liver ALT and AST levels of mice in the NAFLD model group were significantly increased (p<0.01). Consistent with the serum ALT and AST results, the liver ALT and AST levels of mice in the blank liposome group without sodium acetate were not significantly improved. Gavage administration of double-labeled liposomes containing sodium acetate, unlabeled liposomes, or sodium acetate solution alone effectively improved the liver ALT and AST levels of NAFLD mice. Intra-group differential analysis showed that sodium cholate / mannose double-labeled sodium acetate liposomes were significantly more effective than sodium acetate alone in alleviating the increase in ALT and AST levels.

[0188] 9) The results of sodium acetate liposomes in improving liver inflammation and oxidative stress in NAFLD mice are as follows: Figure 20 As shown. Compared with the liver of mice in the NAFLD model group, the macrophage infiltration in the liver of mice in the blank liposome group was not significantly improved, while the macrophage infiltration in the liver of mice in the double-labeled sodium acetate liposome, sodium acetate solution, or unlabeled sodium acetate liposome groups was significantly improved. Figure 20A). ImageJ software was used to quantitatively analyze the positive area of ​​immunohistochemical staining. Statistical results showed that the F4 / 80 positive area in the liver of NAFLD model group mice was significantly higher than that in the control group (p<0.01). After 10 weeks of gavage administration of blank liposomes, the relative positive area of ​​macrophages in the liver of NAFLD mice decreased but did not significantly improve. After 10 weeks of gavage administration of double-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. Figure 20 B). 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).

[0189] 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 NFKB in the liver of mice was significantly downregulated, indicating a reduction in HFD-induced liver inflammatory response. The results of intragroup differential analysis 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).

[0190] 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, NFKB 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. A liver-targeted lipid vehicle, characterized in that, The lipid carrier described above is prepared by mixing DSPC, cholesterol, DSPE-PEG-CA, DSPE-PEG-MAN, DSPE-PEG-NH2, and DC-cholesterol in a specific ratio, where DSPE-PEG-CA is DSPE-PEG modified with cholate 2000 , and DSPE-PEG-MAN is DSPE-PEG modified with mannose 2000 ; the particle size of the lipid carrier is 96 ± 1.5 nm; 0.22 < PDI < 0.25; Zeta potential is 33 ± 4 mV; the weight ratio of each component is as follows: 40.5% DSPC, 13.5% cholesterol, 1% DSPE-PEG-CA, 10% DSPE-PEG-MAN, 5% DSPE-PEG-NH2, and 30% DC-cholesterol.

2. The liver-targeted lipid vehicle according to claim 1, characterized in that, The lipid carrier is prepared by the following method: (1) DSPE-PEG is modified with cholate salt 2000 DSPE-PEG-CA is obtained; (2) Utilizing mannose-modified DSPE-PEG 2000 resulting in DSPE-PEG-MAN; (3) sequentially adding the following raw materials: DSPC, cholesterol, DSPE-PEG-CA, DSPE-PEG-MAN, DSPE-PEG-NH2, DC-cholesterol, mixing, and then transferring into a round-bottom flask with a micro glass syringe, uniformly mixing in advance, and uniformly shaking in a constant-temperature shaker to make all reagents thermodynamically distributed, at which time the solution is completely clear; (4) high-pressure rotary evaporation to obtain a film with uniform thickness and uniform distribution; (5) adding a buffer solution to the film formed in step (4); (6) using a micro glass syringe to suck the solution obtained in step (5), and repeatedly extruding in a liposome extruder, and overnight to stabilize the liposome particles.

3. The method of producing a liver-targeted liposomal carrier according to claim 1, wherein, including the following steps: (1) DSPE-PEG is modified with cholate salt 2000 DSPE-PEG-CA is obtained; (2) Utilizing mannose-modified DSPE-PEG 2000 resulting in DSPE-PEG-MAN; (3) sequentially adding the following raw materials: DSPC, cholesterol, DSPE-PEG-CA, DSPE-PEG-MAN, DSPE-PEG-NH2, DC-cholesterol, mixing, and then transferring into a round-bottom flask with a micro glass syringe, uniformly mixing in advance, and uniformly shaking in a constant-temperature shaker to make all reagents thermodynamically distributed, at which time the solution is completely clear; (4) high-pressure rotary evaporation to obtain a film with uniform thickness and uniform distribution; (5) adding a buffer solution to the film formed in step (4); (6) using a micro glass syringe to suck the solution obtained in step (5), and repeatedly extruding in a liposome extruder, and overnight to stabilize the liposome particles.

4. The method of claim 3, wherein, The operation of step (1) is: 1) activate cholic acid: dissolve cholic acid, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide methiodide (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; 2) DSPE-PEG 2000 -NH2 is dissolved in DMSO, and activated sodium cholate and triethylamine are added and completely dissolved, and then the reaction is carried out; 3) After the reaction is completed, the solution is placed in a dialysis bag and dialyzed 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.

5. The method of claim 4, wherein, Wherein: Step 1) the ice bath reaction is 20~40min; Step 2) the DSPE-PEG 2000 -NH2 and DMSO and the addition ratio of 450~550mg:3mL; the addition ratio of activated sodium cholate and triethylamine is 500~600mg:1g; the reaction temperature is 38~45℃, and the reaction time is 1~3h; Step 3) the molecular sieve size of the dialysis bag is MW=800~1200 Da, and the dialysis time is 18~28h.

6. The method of claim 3, wherein, The operation of step (2) is: 1) DSPE-PEG 2000 -NH2 was dissolved in DMSO, mannose and triethylamine were added to complete dissolution, and the reaction was carried out; 2) After the reaction is completed, the solution is placed in a dialysis bag and dialyzed with pure water as the medium; 3) After dialysis, the solution in the dialysis bag is freeze-dried to obtain DSPE-PEG-MAN.

7. The method of claim 6, wherein, wherein step 1) DSPE-PEG 2000 The addition ratio of -NH2 to DMSO is 450-550 mg: 3 mL. And the addition ratio of mannose and triethylamine is 500-600 mg: 1 g; the reaction temperature is 38-45°C, 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.

8. The liver-targeting lipid carrier of claim 1 for use in the preparation of a preparation for treating liver diseases.

9. Use according to claim 8, characterized in that, The liver disease is inflammation, cancer, or non-alcoholic fatty liver.

Images