Liver targeting lipid carrier as well as preparation method and application thereof

Abstract

The invention discloses a lipid carrier for targeting liver and a preparation method and application thereof, and relates to the field of drug delivery, cholate and mannose are respectively used for modifying two raw materials, namely DSPE-PEG-CA and DSPE-PEG-MAN, consisting of phospholipid molecules DSPE-PEG2000 and synthetic lipidosome, and then the two raw materials, cholesterol, DSPC, DC-cholesterol and the like are mixed according to a certain proportion to obtain the lipid carrier with liver targeting property. The liposome disclosed by the invention has relatively high cell safety and liver targeting property.

Description

Technical Field

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

[0002] DSPE-PEG 2000 It is an amphiphilic (hydrophilic and lipophilic) molecule that is widely used in nanodrug 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 be inserted into the lipid bilayer. PEG2000 (Polyethylene Glycol2000) is a polyethylene glycol part with a molecular weight of 2000, which is hydrophilic, allowing it to be stably suspended in water. DSPE-PEG 2000 Commonly used in the preparation of liposomes, nanoparticles and other drug delivery systems, PEG chains can prevent the immune system from recognizing and clearing the carrier, thus contributing to the "stealth" effect.

[0003] Sodium cholate is a hydrophilic bile acid that binds to ASBT overexpressed on the surface of intestinal epithelial cells and NTCP receptors on the surface of hepatocytes, exerting receptor-mediated endocytosis through the bile acid-ASBT or bile acid-NTCP pathway, thereby enhancing the ability of nanoliposomes to penetrate the intestine and target the liver. Mannose can specifically bind to the mannose receptors on the surface of liver macrophages, targeting liposomes to macrophages in the liver.

[0004] Although specific targeting ligands for the liver (such as mannose, N-acetylgalactosamine (GalNAc), etc.) have been designed, insufficient targeting may still occur: Nonspecific uptake: Liposomes may be taken up by other cells or organs, such as macrophages in the mononuclear phagocytic system (MPS), especially the spleen and lungs. Heterogeneity: Different cell types in the liver (such as hepatocytes, Kupffer cells, endothelial cells) have different uptake efficiencies for targeting molecules, which may lead to uneven drug effects. In addition, although the targeted design reduces systemic distribution, it fails to completely avoid the accumulation of drugs in other parts, which may cause side effects.

[0005] Therefore, there are existing technologies for improving liposome formulations to obtain liposomes with improved targeting, stability, safety and delivery efficiency, such as using PEGylated liposomes to reduce nonspecific clearance, and designing pH-responsive or enzyme-responsive systems to improve intracellular drug release efficiency. The present invention is aimed at the above-mentioned technical defects and has carried out experimental design and research invention. Summary of the invention

[0006] In order to solve the above technical problems, the present invention selects to modify sodium cholate and mannose on the surface of nanoliposomes to construct an oral delivery system with liver targeting.

[0007] Definition Notes:

[0008] DSPC: refers to 1,2-Distearoyl-sn-glycero-3-phosphocholine, also known as 1,2-distearoyl-sn-glycero-3-phosphocholine in Chinese, or distearoylphosphatidylcholine for short. It is a phospholipid commonly used in the preparation of liposomes and nano-drug delivery systems;

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

[0010] DSPE-PEG-CA: distearoylphosphatidylethanolamine polyethylene glycol-cholic acid. Since the PEG in the synthesis reaction is PEG2000, the DSPE-PEG-CA in the specification of the present invention is DSPE-PEG 2000 -CA;

[0011] DSPE-PEG-MAN: distearoylphosphatidylethanolamine polyethylene glycol mannose. Since the PEG in the synthesis reaction is PEG2000, the DSPE-PEG-MAN in the specification of the present invention 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, thus 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 prepared 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 , and 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, the weight ratio of each component in step (3) 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 preparation 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-MAN, DSPE-PEG 2000 -NH2, DC-cholesterol, mix well and transfer to a round-bottom flask with a micro glass syringe. After pre-mixing, shake well in a constant temperature shaker to make all reagents thermodynamically uniform. At this time, the solution is completely clear;

[0021] 4) High-pressure rotary evaporation to obtain a film with uniform thickness and uniform distribution;

[0022] 5) adding buffer and obtaining a solution by sonication;

[0023] 6) The solution obtained in step 5) is drawn into a micro-glass syringe, and extruded back and forth in a liposome extruder. The extruded liposomes are stored below 4° C. overnight to stabilize the liposome particles.

[0024] Furthermore, the operation of step 3) is that the pre-mixing time is 5 to 15 minutes, the constant temperature is 35 to 40° C., and the shaking time is 5 to 15 minutes;

[0025] Furthermore, the operation of step 4) is to perform rotary evaporation at a pressure of 400-600 mbar, a rotation speed of 80-120 rpm, and a temperature of 45-50° C.;

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

[0027] Furthermore, the method further comprises removing unencapsulated drug to be delivered by ultrafiltration, and then quick-freezing the liposomes with liquid nitrogen;

[0028] Furthermore, the method further comprises quick-freezing the obtained liposomes with liquid nitrogen, and re-dissolving them with PBS before use;

[0029] Further; the preparation of DSPE-PEG 2000 -CA method steps are:

[0030] 1) Activating bile acid: dissolving bile acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide (EDC) and N-hydroxysuccinimide (NHS) in dichloromethane, reacting in an ice bath, stirring at room temperature overnight, filtering and rotary evaporating to obtain activated sodium cholate;

[0031] 2) DSPE-PEG 2000 -NH2 is dissolved in DMSO, and activated sodium cholate and triethylamine are added and reacted after they are completely dissolved;

[0032] 3) After the reaction is completed, the solution is placed in a dialysis bag and dialyzed using pure water as the medium;

[0033] 4) After the dialysis is completed, the solution in the dialysis bag is collected and freeze-dried to obtain DSPE-PEG-CA.

[0034] Preferably, step 1) is reacted in an ice bath for 20 to 40 minutes;

[0035] Preferably, step 2) DSPE-PEG 2000 -NH2 and DMSO are added in a ratio of 450-550 mg:3 mL; activated sodium cholate and triethylamine are added in a ratio of 500-600 mg:1 g; the reaction temperature is 38-45°C for 1-3 hours;

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

[0037] Further, the preparation of DSPE-PEG 2000 -MAN method steps are:

[0038] 1) DSPE-PEG 2000 -NH2 is dissolved in DMSO, mannose and triethylamine are added to completely dissolve, and mannose and triethylamine are reacted;

[0039] 2) After the reaction is completed, the solution is placed in a dialysis bag and dialyzed using pure water as the medium;

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

[0041] Preferably, in step 1) DSPE-PEG 2000 -NH2 and DMSO are added in a ratio of 450-550 mg:3 mL; and mannose and triethylamine are added in a ratio of 500-600 mg:1 g; the reaction temperature is 38-45°C for 1-3 hours;

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

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

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

[0045] Furthermore, 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 the present invention include:

[0047] 1) First synthesis of two raw materials DSPE-PEG for liposome composition 2000 -CA and DSPE-PEG 2000 -MAN, and then with cholesterol, DSPC, and DC-cholesterol in a certain ratio to obtain liver-targeted liposomes;

[0048] 2) The five liposomes synthesized by the present invention have no obvious toxicity to myocardial cells H9c2, intestinal epithelial cells Caco-2, liver macrophages Kupffer and hepatocytes AML-12, and are suitable as oral sodium acetate nanocarriers with high cell safety;

[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, intestinal epithelial barrier penetration and liver targeting;

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

[0051] 5) After mice were gavaged with double-labeled sodium acetate liposomes, liver lipid deposition was significantly improved, and the phosphorylation level of liver AMPKα protein was significantly activated. By inhibiting the transcription of Acc1 and Srebf1, CPT1α expression was promoted, and downstream lipid metabolism genes were regulated, effectively reducing liver lipid deposition. In terms of liver inflammation level and oxidative stress, after NAFLD mice were gavaged with double-labeled sodium acetate liposomes for 10 weeks, NFKB protein phosphorylation was significantly inhibited, the release of downstream inflammatory factors was reduced, and liver inflammation and oxidative stress were effectively relieved. BRIEF DESCRIPTION OF THE DRAWINGS

[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 synthesis route, B is the infrared spectrum;

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

[0055] Figure 4 Sodium acetate release of liposomes in different groups after incubation in serum, simulated gastric fluid, and simulated intestinal fluid in the orthogonal experiment; A: sodium acetate release in serum; B: sodium acetate release in simulated gastric fluid; C: sodium acetate release in simulated intestinal fluid; different letters indicate significant differences (p<0.05), and the same letters indicate insignificant differences (p>0.05);

[0056] Figure 5 Liposome embedding rate and drug loading at different sodium acetate stock solution concentrations, A: embedding rate; B: drug loading;

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

[0058] Figure 7 Cell activity of four types of cells after incubation with 1-1500 μM sodium acetate liposomes and sodium acetate solution for 24 hours, 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 cell TG content; B: Effects of different liposomes or sodium acetate on cell TC content; C: Oil red O staining results of different groups; D: Effects of different liposomes or sodium acetate on the relative area of ​​lipid droplets in NAFLD cells; TG, TC and relative lipid droplet area were analyzed for significance, # indicates significant difference between the modeling group and the control group, ## p<0.01; * indicates significant difference between the experimental group and the modeling group, * p<0.05, ** p<0.01; + indicates significant difference between groups. + p<0.05, ++ p<0.01;

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

[0061] Fig.10 In vivo fluorescence imaging of mice after oral administration of CY7 liposomes or CY7;

[0062] Fig.11 Fluorescence intensity of main organs in mice after oral administration of CY7 liposomes or CY7, A: Fluorescence imaging of each organ; B: Fluorescence intensity-time curve of the liver of mice in each group; C: AUG of the fluorescence intensity-time curve of the liver of mice 0-24 ; D: Fluorescence intensity-time curve of the intestine of each group of mice; E: AUG of the fluorescence intensity-time curve of the intestine of mice 2-24 ;

[0063] Fig.12 Effects of sodium acetate liposomes on body weight change and weight gain in NAFLD mice: A: body weight growth curve; B: body weight gain; # It means that the modeling group and the control group are significantly different. ## p<0.01; * It means that the difference between the experimental group and the modeling group is significant. * p<0.05, ** p<0.01, + Indicates significant differences between groups. + p<0.05;

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

[0065] A: OGTT curve; B: area under the OGTT curve; # It means that the modeling group and the control group are significantly different. ## p<0.01; * It means that the difference between the experimental group and the modeling group is significant. ** p<0.01, + Indicates significant differences between groups. ++ p<0.01;

[0066] Fig.14 Effect of sodium acetate liposomes on blood lipid metabolism in NAFLD mice, A: triglycerides; B: cholesterol; C: low-density lipoprotein cholesterol; D: high-density lipoprotein cholesterol; # It means that the modeling group and the control group are significantly different. ## p<0.01; *It means that the difference between the experimental group and the modeling group is significant. * p<0.05, ** p<0.01, + Indicates significant differences between groups. + p<0.05, ++ p<0.01;

[0067] Fig.15 Effects of sodium acetate liposomes on adipose tissue content in NAFLD mice, A: white fat percentage; B: H&E staining results of epididymal adipose tissue (400×, scale bar 100μm); # It means that the modeling group and the control group are significantly different. ## p<0.01; * It means that the difference between the experimental group and the modeling group is significant. * p<0.05, ** p<0.01, + Indicates significant differences between groups. ++ p<0.01;

[0068] Fig.16 Effects of sodium acetate liposomes on liver pathological morphology in NAFLD mice, A: liver appearance (scale bar 1 cm); B: H&E staining of liver tissue (100×, scale bar 200 μm); C: relative area ratio of liver lipid droplets based on H&E staining sections (n=8); D: Oil red O staining of liver tissue (100×, scale bar 200 μm); E: relative area ratio of liver lipid droplets based on Oil red O staining (n=8); # It means that the modeling group and the control group are significantly different. ## p<0.01; * It means that the difference between the experimental group and the modeling group is significant. ** p<0.01, + Indicates significant differences between groups. + p<0.05, ++ p<0.01;

[0069] Fig.17 Effects of sodium acetate liposomes on liver lipid deposition and oxidative stress in NAFLD mice, A: liver index (%); B: liver triglyceride content; C: liver cholesterol content; D: liver malondialdehyde content; E: liver superoxide dismutase content; # It means that the modeling group and the control group are significantly different. ## p<0.01; * It means that the difference between the experimental group and the modeling group is significant. * p<0.05, ** p<0.01; + Indicates significant differences between groups. + p<0.05,++ p<0.01;

[0070] Fig.18 Effects of sodium acetate liposomes on the expression of genes related to lipid synthesis and metabolism and AMPK protein phosphorylation in the liver of NAFLD mice, AE: Relative mRNA expression of genes related to lipid synthesis and metabolism in the liver (Acc1, Fasn, Slc27a2, Srebf1, CPT1α); F: Western blot of AMPKα and p-AMPKα; G: Relative expression of p-AMPKα / AMPKα; # It means that the modeling group and the control group are significantly different. ## p<0.01; * It means that the difference between the experimental group and the modeling group is significant. ** p<0.01; + Indicates significant differences between groups. + p<0.05, ++ p<0.01

[0071] Fig.19 Effects of sodium acetate liposomes on liver function in NAFLD mice, A: serum alanine aminotransferase; B: serum aspartate aminotransferase; C: liver alanine aminotransferase; D: liver aspartate aminotransferase; # It means that the modeling group and the control group are significantly different. ## p<0.01; * It means that the difference between the experimental group and the modeling group is significant. * p<0.05, ** p<0.01; + Indicates significant differences between groups. + p<0.05, ++ p<0.01

[0072] Fig. 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 100 μm); B: relative area of ​​F4 / 80 positive sites; # It means that the modeling group and the control group are significantly different. ## p<0.01; * It means that the difference between the experimental group and the modeling group is significant. ** p<0.01; + Indicates significant differences between groups. + p<0.05, ++ p<0.01

[0073] Fig.21Effects of sodium acetate liposomes on inflammation-related cytokines and NFKB protein phosphorylation in the liver of NAFLD mice. AD: relative expression levels of inflammation-related cytokines in the liver, which are IL-1β, IL-6, TNF-α, and IL-4; E: protein immunoblot of p-NFKB and NFKB; F: relative expression of p-NFKB / NFKB; # It means that the modeling group and the control group are significantly different. ## p<0.01; * It means that the difference between the experimental group and the modeling group is significant. ** p<0.01; + Indicates significant differences between groups. + p<0.05, ++ p<0.01. DETAILED DESCRIPTION

[0074] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

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

[0076] Preparation process Figure 1 Shown

[0077] 1. DSPE-PEG 2000 Synthesis of -CA

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

[0079] The infrared spectrum of DSPE-PEG-CA is shown in Figure 2 B The results show that DSPE-PEG 2000 -NH2 modified with sodium cholate at 1551cm -1 The characteristic peak of -C=O stretching vibration of carboxylate group appeared at 3425cm -1 Corresponding to the hydroxyl group in the sodium cholate molecule, 2850-2917cm -1 The stretching vibration of the P=O bond of the phosphate group appears at 1245 cm-1, and the stretching vibration of the CO bond of the PEG chain appears at 1108 cm-1. -1 The CH bending vibration peak of the alkyl chain appears at 1466 cm -1 The above results show that sodium cholate has been successfully bound to the DSPE-PEG-NH2 molecule.

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

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

[0082] 500 mg DSPE-PEG 2000 -NH2 was dissolved in 3 mL DMSO, 550 mg mannose and 1 g triethylamine were added to completely dissolve, and the mixture was reacted 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 for 24 h with pure water as the medium. After the dialysis, the solution in the dialysis bag was freeze-dried to obtain DSPE-PEG-MAN (synthetic route as shown in Figure 3 A).

[0083] The results of infrared spectroscopy (FTIR) detection of DSPE-PEG-MAN are as follows Figure 3 As shown in B: The infrared spectrum of DSPE-PEG-MAN shows the following characteristic peaks: 3428cm -1 Corresponding to the hydroxyl group in the mannose molecule, 2917 cm -1 Corresponding to the CH bond of the stearoyl alkyl chain in DSPE, 1739 cm -1 The corresponding aldehyde C=O group. The CH bending vibration peak of the alkyl chain appears at 1460 cm -1 In DSPE-PEG 2000 In the FTIR spectrum of -NH2, the amino group (-NH2) appears at 3434 cm -1 After modification with mannose, the peak becomes broader and appears at 3428 cm due to the presence of hydroxyl groups. -1 The stretching vibration of the P=O bond of the phosphate group appears at 1251 cm-1 The CO bond stretching vibration of the PEG chain appears at 1109 cm -1 The above results show that DSPE-PEG-MAN was successfully synthesized.

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

[0085] Intestinal penetrating and liver-targeting high-concentration sodium acetate liposomes (NaA@CA / MAN-LPs) were prepared by thin film hydration method.

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

[0087] (1) Add the following raw materials in order according to the liposome ratio: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, Distearoylphosphatidylcholine), cholesterol, DSPE-PEG 2000 -CA, DSPE-PEG 2000 -MAN, DSPE-PEG 2000 , DC-cholesterol, mix well and transfer to a round-bottom flask with a micro glass syringe. Pre-mix for 10 minutes and shake in a 37°C constant temperature shaker for 10 minutes to ensure that all reagents are thermodynamically evenly distributed. At this time, the solution is completely clear.

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

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

[0090] (4) Assemble a liposome extruder (Avanti Polar Lipids). Place a wet carbonate membrane filter holder on each of the two black washers. Select a 100 nm filter membrane, wet it and place it between the two filter holders. Fix the assembled liposome extruder on the holder and finally place the whole on a heating plate at 50°C.

[0091] (5) 1 mL of liposome solution was drawn into a micro-glass syringe and extruded 21 times in a liposome extruder. The extruded liposomes were stored at 4°C overnight to stabilize the liposome particles.

[0092] (6) Unencapsulated sodium acetate was removed by ultrafiltration, and the liposomes were then quickly frozen with liquid nitrogen, weighed, and reconstituted with PBS before use.

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

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

[0095] (1) The ratio of step 1) in the above method is optimized, and the components of the liposome include: DSPC, cholesterol, DSPE-PEG 2000 -CA, DSPE-PEG 2000 -MAN, DSPE-PEG 2000 In order to obtain liposomes with high embedding efficiency and good stability, the concentration of sodium acetate stock solution was fixed at 20 mg / mL. According to the orthogonal test, DSPC: cholesterol, DSPE-PEG 2000 -CA, DSPE-PEG 2000 -MAN ratio is optimized. The specific factor levels are shown in Table 1.

[0096] Table 1 Optimization factor level table of sodium acetate liposome synthesis

[0097]

[0098] The orthogonal experiment grouping is shown in Table 2:

[0099] Table 2 Orthogonal experiment grouping table

[0100]

[0101] (2) Composition optimization evaluation index

[0102] a. Sodium acetate encapsulation efficiency of liposomes

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

[0104] The HPLC conditions were as follows: the chromatographic column was Thermol Hypersil ODS-2C18 column (4.6 mm*200 mm, 5 um), the mobile phase was 0.01 mol / L aqueous ammonium dihydrogen phosphate solution, the pH was adjusted to 3.0 with 1 mol / L phosphoric acid solution, the flow rate was 1.0 mL / min, the injection volume was 10 μL, the detection wavelength was 215 nm, and the column temperature was 30 °C.

[0105] A.2 Drawing of standard curve

[0106] Sodium acetate was accurately weighed in a 10 mL volumetric flask, and PBS was added for dilution to prepare sodium acetate standard solutions with concentrations of 0.01, 0.025, 0.05, 0.1, 0.2, and 0.5 mg / mL, respectively. Each sample was measured under HPLC conditions, and the peak area was recorded. The standard curve of AC linear relationship was drawn with the main peak area (A) as the ordinate and the concentration (C) as the abscissa. Sodium acetate was dissolved in serum solution, simulated gastric fluid, and simulated intestinal fluid to prepare sodium acetate solutions with concentrations of 0.01, 0.025, 0.05, 0.1, 0.2, and 0.5 mg / mL, respectively. The peak area-time standard curve of acetic acid in serum, simulated gastric fluid, and simulated intestinal fluid was detected under HPLC conditions.

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

[0108] Take an appropriate amount of sodium acetate liposome solution, place it in an activated ultrafiltration tube (MYCO=3K Da, Millipore), and centrifuge at 12000rpm for 10min. Take out the solution in the ultrafiltration tube, detect the free sodium acetate content according to the HPLC method in "2.3.5.1", and calculate the encapsulation rate of sodium acetate in liposomes according to the following formula.

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

[0112]

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

[0114] Sterile fetal bovine serum was diluted 10 times with PBS (pH 7.4) to prepare a simulated serum solution. The method for preparing artificial simulated gastric fluid was as follows: 0.5g pepsin was added to 10mL pure water, dissolved and added to 0.82mL 1mol / L hydrochloric acid solution, mixed and diluted to 50mL, pH = 1.2. The method for preparing artificial simulated intestinal fluid was as follows: 0.34g potassium dihydrogen phosphate was added to 25mL water, and the pH was adjusted to 6.8 with 0.4% NaOH; 0.5g pancreatin was dissolved in water, 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 fluid, simulated intestinal fluid and serum solution, respectively, and then oscillated at 1000 rpm at 37°C. The sample solution was collected after 2 hours in the group oscillating with simulated gastric fluid, and after 6 hours in the group oscillating with simulated intestinal fluid and serum solution. The collected sample solution was measured for particle size, PDI and Zeta potential by laser particle size analyzer, and the sodium acetate release rate after incubation with different medium solutions was detected by HPLC. The changes in particle size and encapsulation rate of liposomes before and after incubation were analyzed to characterize the stability of liposomes in gastrointestinal tract and serum solution.

[0116] Orthogonal experiment results:

[0117] Table 3 shows the sodium acetate embedding rate and drug loading of liposomes in different groups in the orthogonal experiment. The results show that different DSPC: cholesterol ratios and DSPE-PEG 2000 -CA and DSPE-PEG 2000 -MAN concentration has an important influence on the liposome embedding rate and drug loading. The sodium acetate embedding rate of the nine orthogonal experimental group liposomes is between 70.52% and 81.15%, and the order from large to small is: 3>7>1>8>4>2>9>6>5. The drug loading of the nine orthogonal experimental group liposomes is between 20.86% and 31.58%, and the order from large to small is: 3>7>2>1>4>8>9>6>5. The above results show that the embedding rate and drug loading of liposome 3 and liposome 7 are significantly higher than those of the remaining seven groups of liposomes. The particle stability of the nine groups of liposomes after incubation in serum solution, simulated gastric fluid and simulated intestinal fluid will be further evaluated to determine the optimal ratio of liposome composition.

[0118] Table 3 Effect of liposome composition on sodium acetate embedding efficiency and drug loading

[0119] Experimental Grouping Sodium acetate embedding rate (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: The sodium acetate embedding rate and drug loading capacity of the nine groups of liposomes were analyzed for significance. Different letters in the same column of data indicate significant differences (p<0.05), and the same letters indicate insignificant differences (p>0.05).

[0121] From the results of particle size changes of liposomes of different compositions after incubation in simulated body fluids in Table 4, it can be seen that the initial particle size of the nine groups of liposomes is about 100 nm. After each group of liposomes is incubated in serum, simulated gastric fluid and simulated intestinal fluid for 6h or 2h, the particle size of the liposomes increases to varying degrees. After incubation in serum for 6 hours, the particle sizes of liposomes in each group were 7<2<8<6<5<1<4<3<9 from small to large, 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 particle sizes of liposomes in different groups were 7<9<3<8<1<2<6<4<5 from small to large, and the particle sizes of liposome 7 and liposome 9 (107.21±2.40nm, 128.70±1.59nm) were significantly lower than those of the other seven groups of liposomes; after incubation in simulated intestinal fluid for 6 hours, the particle sizes of liposomes in different groups were 4<8<1<7<6<5<9<2<3 from small to large, and except for liposome 2 and liposome 3, there was no significant difference in the particle sizes of the remaining seven groups of liposomes. Table 5 shows the changes in PDI of nine groups of liposomes with different compositions after incubation in simulated body fluids. The initial PDI of the nine groups of liposomes was between 0.20 and 0.22. After incubation in serum, simulated gastric fluid and simulated intestinal fluid, the PDI of all liposomes was <0.4, indicating that after incubation in simulated body fluids, the particle size distribution of all liposomes was relatively uniform and there was no obvious aggregation. From the changes in the potential of the nine groups of liposomes after incubation in simulated body fluids in Table 6, it can be seen that the initial Zeta potential of the liposomes was positive. After incubation in serum, simulated gastric fluid and simulated intestinal fluid, the charge of the liposomes remained positive.

[0122] Table 4 Changes in particle size of liposomes in different groups in serum, simulated gastric fluid and intestinal fluid in orthogonal experiments

[0123]

[0124] Note: The significance analysis was performed on the original particle size of the nine groups of liposomes and the particle size of the liposomes after incubation in serum, simulated gastric fluid, and simulated intestinal fluid. Different letters in the same column of data indicate significant differences (p<0.05), and the same letters indicate insignificant differences (p>0.05).

[0125] Table 5 PDI changes of liposomes in different groups in serum, simulated gastric fluid and intestinal fluid in orthogonal experiment

[0126]

[0127] Note: The original particle sizes of the nine groups of liposomes and the PDI results after incubation in serum, simulated gastric fluid, and simulated intestinal fluid were analyzed for significance. Different letters in the same column of data indicate significant differences (p<0.05), and the same letters indicate insignificant differences (p>0.05).

[0128] Table 6 Zeta-potential changes of liposomes in different groups in serum, simulated gastric fluid and intestinal fluid in orthogonal experiment

[0129]

[0130] Note: The original particle size of the nine groups of liposomes and the Zeta potential after incubation in serum, simulated gastric fluid and simulated intestinal fluid were analyzed for significance. Different letters in the same column of data indicate significant differences (p<0.05), and the same letters indicate insignificant differences (p>0.05).

[0131] Results Figure 4 After incubation in serum for 6 hours, the sodium acetate release of the nine groups of liposomes ranged from 3.61% to 5.27%, among which the sodium acetate release of liposome 4 and liposome 7 was significantly lower than that of the other groups, with the release amounts being 3.73%±0.19% and 3.61%±0.32% ( Figure 4 A). After incubation in simulated gastric fluid for 2 h, the sodium acetate release of the nine groups of liposomes ranged from 12.57% to 16.51%, among which the sodium acetate release of liposome 2 and liposome 7 was significantly lower than that of the other groups of liposomes, which were 12.57%±0.32% and 12.70%±0.37%, respectively ( Figure 4 B). After incubation in simulated intestinal fluid for 6 hours, all nine groups of liposomes released sodium acetate to a certain extent, and the release amount was between 5.16% and 7.73%. The release amount of liposome 3 and liposome 7 was significantly lower than that of the other seven groups of liposomes, and their release amounts were 5.16%±0.08% and 5.51%±0.06% respectively ( Figure 4 C). The above results show that, compared with the other groups of lipids, liposomes 4 and 7 are more stable in serum, liposomes 2 and 7 are more stable in simulated gastric fluid, and liposomes 3 and 7 are more stable in simulated intestinal fluid.

[0132] To optimize DSPC:cholesterol and DSPE-PEG in liposomes 2000 -CA and DSPE-PEG 2000-MAN ratio, the above three levels were optimized by orthogonal optimization method. From the above results, it can be seen that the sodium acetate embedding rate of liposome 7 is 78.42%±0.30%, ranking second among the nine groups of liposomes, and the drug loading is 29.70%±0.11%, ranking second among the nine groups of liposomes, indicating that the sodium acetate content of the prepared liposomes is high under the composition ratio of liposome 7. In order to study the stability of liposomes in different body fluids, the nine groups of liposomes were incubated in serum, simulated gastric fluid and simulated intestinal fluid, and their particle size, PDI, Zeta potential and sodium acetate release were detected. According to the above results, after incubation in serum, simulated gastric fluid and simulated intestinal fluid, the particle size of liposome 7 did not increase significantly compared with other groups, and the particle size distribution was uniform and still positively charged. In addition, compared with other groups of liposomes, liposome 7 has the lowest release of sodium acetate in liposomes after incubation with serum, simulated gastric fluid and simulated intestinal fluid, which is 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 liposomes have the strongest stability, effectively preventing the adsorption and structural destruction of enzymes in gastrointestinal fluid and proteins in serum. Therefore, the most effective ratio of sodium acetate liposomes is DSPE: cholesterol ratio of 3:1, and 1% DSPE-PEG 2000 -CA and 10% DSPE-PEG 2000 -MAN.

[0133] Example 2 Sodium acetate storage solution concentration climbing experiment

[0134] 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. The 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. The unencapsulated sodium acetate was removed by ultrafiltration, and the sodium acetate concentration was detected by HPLC to calculate the sodium acetate encapsulation rate in the liposome. The liposomes were freeze-dried by liquid nitrogen rapid freezing and weighed, and the sodium acetate drug loading of the liposomes was calculated based on the liposome mass.

[0135] The optimal ratio of sodium acetate liposomes was determined to be 40.5% DSPC, 13.5% cholesterol, 1% DSPE-PEG-CA, 10% DSPE-PEG-MAN, 5% DSPE-PEG 2000 and 30% DC-cholesterol. The concentration of the gradient sodium acetate stock solution (20, 30, 40, 50, 60 mg / mL) was set to obtain the optimal sodium acetate embedding rate and loading capacity. The results are shown in Figure 5As the sodium acetate stock concentration increased from 20 mg / mL to 60 mg / mL, the liposome embedding rate also increased from 71.09% to 79.36%. When the sodium acetate stock concentration exceeded 40 mg / mL, the increase in the liposome embedding rate tended to be flat ( Figure 5 A), and the liposome loading was the highest at this time, which was 33.07% ( Figure 5 B), therefore, when preparing liposomes with high encapsulation efficiency and loading capacity, the selected sodium acetate stock concentration was 40 mg / mL.

[0136] Example 3 Preparation and characterization of differently modified liposomes

[0137] After optimization by orthogonal experiment and sodium acetate stock solution climbing experiment, the optimal ratio of sodium cholate and mannose modified sodium acetate liposomes and sodium acetate stock solution concentration were determined. On this basis, the following differently modified liposomes were prepared: ① sodium cholate and mannose modified sodium acetate liposomes (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 composition of the differently modified liposomes is 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 liposomes were measured by dynamic light scattering, the sodium acetate embedding rate and drug loading of liposomes were detected by HPLC, and the morphology of liposomes was observed by transmission electron microscopy (TEM). The specific operation steps of TEM are as follows: after diluting the liposomes, drop them onto a copper mesh, let them stand for 2 minutes, use filter paper to absorb the liquid on the surface of the copper mesh, add 2% phosphotungstic acid for negative staining for 5 minutes, dry the copper mesh, and use a JEM-1400Flash transmission electron microscope to observe the morphology of different types of liposomes.

[0141] After orthogonal experiments and sodium acetate storage ramp experiments, the composition ratio of sodium acetate liposomes with high encapsulation rate and loading amount was determined. On this basis, five liposomes with different modifications were synthesized, namely ① sodium acetate liposomes modified with sodium cholate and mannose (NaA@CA / MAN-LPs); ② sodium acetate liposomes modified with sodium cholate alone (NaA@CA-LPs); ③ sodium acetate liposomes modified with mannose alone (NaA@MAN-LPs); ④ sodium acetate liposomes without modification (NaA@LPs); ⑤ sodium cholate and mannose liposomes without sodium acetate (CA / MAN-LPs). The five liposomes were tested by particle size analyzer and encapsulation rate and drug loading, and the results are shown in Table 8. Figure 6 . The particle size of the five liposomes is about 100nm, PDI < 0.25, and the Zeta potential is positive. Except for CA / MAN-LPs, the embedding rates of NaA@CA / MAN-LPs, NaA@CA-LPs, NaA@MAN-LPs, and NaA@LPs are between 76.6% and 81.57%, and the drug loading is between 33.16% and 37.94%, indicating that the five synthesized liposomes are all about 100nm, with uniform particle distribution and positively charged nanoliposomes, and have high sodium acetate embedding rate and liposome drug loading. The appearance of the five liposomes was observed and photographed using a transmission electron microscope. The results are shown in Figure 2. Figure 6 The appearance of the five liposomes was spherical, with a particle size of about 100 nm, which was consistent with the particle size measurement results. The particles were evenly distributed without aggregation or clumping.

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

[0143]

[0144] Example 4 Sodium acetate liposome biosafety

[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, other experimental operation steps were the same.

[0147] 2. Liposome safety

[0148] The CCK-8 method was used to detect the effects of five liposomes (NaA@CA / MAN-NPs, NaA@CA-NPs, NaA@MAN-NPs, NaA@NPs, CA / MAN-NPs) and sodium acetate solution on cell viability. The cell suspension with good growth status was taken and the cell density of H9c2, Caco-2, Kupffer, and AML-12 was adjusted to 5×10 5 cell / mL, and inoculated into a 96-well cell culture plate at a volume of 100 μL per well. After the cells grew to about 60%, the culture medium was aspirated, and cell culture medium containing five liposomes or sodium acetate solutions of 1, 10, 100, 200, 500, 1000, and 1500 μM sodium acetate was added respectively to continue culturing for 24 hours. 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, and the plates were cultured at 37°C for 30 minutes. The absorbance of each well was measured at a wavelength of 450 nm using a multifunctional microplate reader, and the cell viability was calculated according to the formula.

[0149]

[0150] Cell safety results are shown in Figure 7 The results showed that in the range of 1 to 1500 μM sodium acetate concentration, the four cell types ( Figure 7 The cell viability of AD) after incubation in five liposomes and sodium acetate solution for 24 h 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 have 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] The hepatocyte AML-12 was revived and cultured using DMEM / F12 complete medium; the liver macrophage Kupffer was revived and passaged using RPMI-1640 complete medium.

[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 the cells were counted. 6The cells / well were inoculated into a 6-well plate, 2 mL per well, and incubated in a 37°C 5% CO2 incubator. When the cells grew to 60%, the culture medium was aspirated, and the cells were washed twice with sterile PBS, and then starved for 12 hours with serum-free DMEM / F12 medium. After the culture, 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 used a control complete 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 cells in good logarithmic growth phase in "4.3.2 Construction of NAFLD cell model", cultured and starved for 12 hours. When lipid induction was performed, the cells were divided into six groups. The first group was added with an equal volume of PBS as a blank control group; the second group was added with lipid induction medium as the NAFLD modeling group; the third group was added with sodium cholate / mannose double-labeled sodium acetate liposomes (NaA@CA / MAN-LPs) in the lipid induction medium; the fourth group was added with sodium acetate solution (NaA) in the lipid induction medium; the fifth group was added with unlabeled sodium acetate liposomes (NaA-LPs) in the lipid induction medium; the sixth group was added with blank double-labeled liposomes (CA / MAN-LPs) without sodium acetate in the lipid induction medium, where the concentration of sodium acetate solution was 1mM. After 24 hours of culture, all grouped cells were tested for TG, TC, BSA, Oil Red O, total cell RNA extraction, and RT-qPCR detection of lipid metabolism-related genes.

[0158] The results are as follows Figure 8As shown in the AD, compared with the blank control group, the relative areas of TG, TC and lipid droplets after Oil Red O staining in the NAFLD model group cells increased significantly, 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 hours. After 1mM concentration of NaA@CA / MAN-LPs and NaA solution alone were treated with NAFLD cells for 24 hours, the relative areas of intracellular TG, TC and lipid droplets stained with Oil Red O were significantly reduced; also compared with the NAFLD model group, after NaA@LPs were incubated with NAFLD cells for 24 hours, the intracellular TG and TC contents were significantly reduced, but the relative area of ​​lipid droplets after Oil Red O staining did not improve significantly. After blank liposomes (CA / MAN-LPs) without sodium acetate were incubated with NAFLD cells for 24 hours, the relative areas of cell TG, TC and Oil Red O lipid droplets decreased slightly, but there was no significant difference. Intra-group analysis of variance showed that compared with NaA treatment, NaA@CA / MAN-LPs treatment of NAFLD cells had a more significant effect on alleviating the accumulation of intracellular TG and TC. The results showed that incubation of NAFLD cells with sodium cholate / mannose double-labeled liposomes with a sodium acetate concentration of 1 mM for 24 hours significantly alleviated lipid deposition in NAFLD cells, and the alleviating effect of double-labeled sodium acetate liposomes was significantly better than that of sodium acetate solution alone.

[0159] 4. Sodium acetate liposomes relieve liver macrophage inflammation

[0160] Select Kupffer cells in good logarithmic growth phase and add 5×10 6 The cells were plated at a density of 100 cells / well and incubated in a 37℃ 5% CO2 incubator. When the cells grew to 60%, the culture medium was aspirated, washed twice with sterile PBS, and then starved for 12 hours with serum-free RPMI-1640 medium. After washing twice with sterile PBS, the cells were treated in groups. Except for the blank control group, the remaining five groups of cells were treated with LPS at a concentration of 20 ng / mL six hours before the end of the treatment to induce cell inflammation. The specific treatment conditions of each group of cells are as follows: the cells of the blank control group were treated with an equal volume of PBS solution without LPS treatment; the cells of the modeling group were treated with LPS 6 hours before the end of the incubation to induce cell inflammation; 1 mM sodium cholate / mannose double-labeled sodium acetate liposomes (NaA@CA / MAN-LPs) were added to the double-labeled liposome group; 1 mM sodium acetate solution was added to the cells of the sodium acetate group; 1 mM unlabeled sodium acetate liposomes (NaA-LPs) were added to the cells of the unlabeled liposome group; blank double-labeled liposomes (CA / MAN-LPs) without sodium acetate were added to the cells of the blank liposome group. After the treatment, total cellular RNA was extracted for RT-qPCR detection of inflammation-related genes.

[0161] The results are as follows Fig. 9As shown in AD, compared with the control group cells, the expression of pro-inflammatory factors IL-1β, IL-6 and TNF-α mRNA in Kupffer cells induced by LPS was significantly upregulated, and the expression of anti-inflammatory factor IL-4 mRNA was significantly downregulated, indicating that the Kupffer cells in the model group produced a significant inflammatory response. Compared with the model group, after treatment with sodium acetate-containing sodium cholate / mannose double-labeled liposomes (NaA@CA / MAN-LPs), unlabeled liposomes (NaA@LPs) and sodium acetate solution alone (NaA), the expression of IL-1β, IL-6 and TNF-α mRNA in Kupffer cells was significantly reduced, and the expression of IL-4 mRNA was significantly increased, indicating that sodium acetate and sodium acetate liposomes effectively inhibited the activation of Kupffer cell inflammatory pathways. Double-labeled blank liposomes without sodium acetate (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 the liposome material has neither ameliorative nor promoting effect on Kupffer under inflammatory state. The results of intra-group difference analysis of the four treatment groups showed that compared with unlabeled sodium acetate liposomes and sodium acetate alone, double-labeled liposomes containing sodium acetate significantly alleviated the high expression of IL-1β, IL-6 and TNF-α mRNA and low expression of IL-4 mRNA in Kupffer cells. The above results indicate that sodium acetate double-labeled liposomes effectively alleviate the inflammatory response of Kupffer cells, and its intervention effect is significantly better than that of single sodium acetate or unlabeled sodium acetate liposomes.

[0162] Example 6 Liposome Targeting in Vivo

[0163] Fifteen C57 male mice were selected, 3 mice in each group, for a total of 5 groups, and CY7@CA / MAN-LPs, CY7@CA-LPs, CY7@MAN-LPs, CY7@LPs and CY7 with the same fluorescence intensity were gavaged respectively. In vivo fluorescence imaging of the mice under anesthesia was performed at 0.5, 1, 1.5, 2, and 2.5 h after gavage. The results are shown in the figure. Fig.10 As shown. The fluorescence intensity of mice directly gavaged with CY7 fluorescent dye and unlabeled CY7-liposomes was generally low within 0-2.5h. After gavage with sodium cholate or mannose-modified liposomes encapsulating CY7, the fluorescent liposomes accumulated in the abdomen, and the fluorescence intensity showed a trend of first increasing and then decreasing within 0-2.5h. The fluorescence intensity at 2.5h was still clearly visible. The accumulation of the three sodium cholate or mannose-labeled fluorescent liposomes in the abdomen was from high to low: sodium cholate / mannose dual-labeled fluorescent liposomes> sodium cholate single-labeled fluorescent liposomes> mannose single-labeled fluorescent liposomes. The above results show that sodium cholate / mannose dual-labeled liposomes have stronger intestinal penetration and liver targeting capabilities, and deliver more fluorescent dyes to the liver.

[0164] In order to more intuitively detect the content of differently modified liposomes passing through the intestine and entering the liver, C57 mice were randomly divided into 5 groups and gavaged with CY7@CA / MAN-LPs, CY7@CA-LPs, CY7@MAN-LPs, CY7@LPs and CY7 of the same fluorescence intensity. At 2h, 6h, 10h, 14h, 18h and 24h, the mice were killed after anesthesia, and the main organs (heart, liver, spleen, lung, kidney and gastrointestinal tract) were removed, and the fluorescence intensity of each organ was immediately detected in a fluorescence imager. The results are shown in Figure 5. Fig.11 A; The liver fluorescence intensity change curves of five groups of fluorescent liposomes were drawn with liver fluorescence intensity as the ordinate and time as the abscissa ( Fig.11 B), the five groups of curves showed an overall trend of increasing first and then decreasing. The fluorescence intensity in the mouse liver reached a peak at 14h, which were: 0.303, 0.208, 0.147, 0.084, 0.038 [(p / sec / cm2 / sr) / μM / cm2]; the fluorescence intensity-time curves of the five groups of fluorescent liposomes were integrated, AUG was calculated and the significance was analyzed. The results are shown in Fig.11 C, the fluorescence intensity of the five liposomes in the liver AUG0-24 from high to low is: CY7@CA / MAN-LPs>CY7@CA-LPs>CY7@MAN-LPs>CY7@LPs>CY7, and the accumulation content of sodium cholate / mannose modified fluorescent liposomes in the liver is significantly higher than that of single fluorescent dye, unmodified fluorescent liposomes, single mannose labeled fluorescent liposomes or single sodium cholate labeled fluorescent liposomes. Similarly, the fluorescence intensity in the intestines of the five groups of mice was quantified, as shown in Fig.11 As shown in D, the fluorescence intensity in the mouse intestine gradually decreased within 2-24 hours. The fluorescence intensity AUG2-24 of the mouse intestine within 2-24 hours was calculated. The results are shown in Fig.11 As shown in E, the fluorescence intensity of CY7@CA / MAN-LPs accumulated in the intestine for 2-24 h was significantly higher than that of CY7@MAN-LPs, CY7@LPs and CY7, but there was no significant difference with the fluorescent liposomes labeled with sodium cholate alone, indicating that the sodium cholate ligand modified on the liposome surface is beneficial to promote the intestinal absorption of liposomes, and its mechanism may be related to the ASBT receptor in the mouse ileum.

[0165] The above results indicate that compared with fluorescent liposomes labeled with sodium cholate or mannose alone, unlabeled fluorescent liposomes or fluorescent dyes alone, sodium cholate / mannose dual-labeled fluorescent liposomes are more effective in penetrating the mouse intestine, targeting the liver tissue and exerting their effects for a long time.

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

[0167] 1. Animal Model

[0168] The animals used in this experiment were 6-week-old C57BL / 6J male mice weighing 18±1.0 g and purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd. (certificate SYXK(Beijing)2021-0001). The experimental design was reviewed by the Laboratory Animal Welfare Ethics Committee of Zhongyanzi Chuang (Beijing) Biotechnology Co., Ltd. (approval number ZYZC202402006S), and all animal handling and experimental procedures were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.

[0169] 2. Modeling and intervention

[0170] Animal grouping and intervention treatment

[0171] Before the formal experiment began, all mice were adaptively fed for one week, and during the feeding process, the mice were free to drink sterile water. One week later, the mice were randomly divided into six groups, with 8 mice in each group (4 mice / cage), and then sodium acetate liposome gavage experiment was performed every other day for 10 weeks. The animal experiment design plan, specific grouping and intervention methods are as follows:

[0172] (1) Normal control group: Feeding with basic diet, and gavage with PBS every other day from week 0 to week 10

[0173] (2) NAFLD model group: HFD diet, from week 0 to week 10, intragastric administration of PBS every other day

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

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

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

[0177] (6) Blank liposome intervention group (CA / MAN-LPs): The mice were fed with HFD and gavaged with blank liposomes (200 μL) every other day from week 0 to week 10. The concentration of blank liposomes was consistent with that of liposomes in other groups, which was 46 mg / mL.

[0178] Evaluation of sodium acetate liposomes in improving the function of HFD-induced NAFLD mice using common methods in the field

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

[0180] 1) Weight results such as Fig.12 As shown in AB: After 10 weeks of intervention with double-labeled sodium acetate liposomes (NaA@CA / MAN-LPs), unlabeled sodium acetate liposomes (NaA@LPs) and sodium acetate solution 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 the three groups of mice was 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 with blank double-labeled liposomes without sodium acetate, the body weight of mice decreased by 1.76g, which was not significantly reduced compared with the NAFLD model group, indicating that the blank liposome material had no significant effect on the body weight of NAFLD mice and would not cause weight gain in mice. The changes in body weight of mice treated with the four interventions were analyzed. The body weight of mice in 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 of NAFLD mice was significantly higher than that of sodium acetate alone ( Fig.12 A, B);

[0181] 2) Glucose tolerance results such as Fig.13As shown: Compared with the NAFLD model group mice, the area under the OGTT curve of sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution alone, and unlabeled sodium acetate liposomes was significantly reduced (p < 0.01), indicating that the glucose tolerance of mice in the above three groups was significantly restored after treatment; 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 with the NAFLD group. The AUG of mice in the four treatment groups was analyzed for inter-group differences, and the results showed that the AUG of OGTT of mice in the dual-labeled sodium acetate liposome group after 10 weeks of intervention was significantly lower than that of mice gavaged with sodium acetate solution alone and unlabeled sodium acetate liposomes. The above results show that sodium acetate, unlabeled sodium acetate liposomes, and dual-labeled sodium acetate liposomes can effectively improve the glucose tolerance of NAFLD mice after gavage, and sodium cholate / mannose dual-labeled sodium acetate liposomes have the most significant effect on improving glucose tolerance ( Fig.13 AB).

[0182] 3) Blood lipid metabolism results such as Fig.14 As shown in AD, compared with the NAFLD modeling group, after mice were gavaged with sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution alone, and unlabeled sodium acetate liposomes, the levels of serum TG, TC, and LDL-C in mice were significantly reduced, and the level of HDL-C was significantly increased (p<0.01), indicating that unlabeled liposomes, dual-labeled liposomes, and sodium acetate containing sodium acetate can significantly alleviate the blood lipid level of NAFLD. After gavage with dual-labeled liposomes without sodium acetate, the levels of serum TG, TC, and LDL-C in mice did not change significantly, and the level of HDL-C increased significantly (p<0.05), indicating that blank liposome materials did not aggravate the serum lipid metabolism level of NAFLD mice and increased the HDL-C content to a certain extent. The serum lipid metabolism parameters of mice in the double-labeled liposome group, sodium acetate group, unlabeled liposome group and blank liposome group were analyzed within the group. The results showed that compared with oral administration of sodium acetate solution alone, the serum TG content of mice was significantly decreased and the HDL-C content was significantly increased after oral administration of sodium cholate / mannose double-labeled sodium acetate liposomes, indicating that the effect of double-labeled sodium acetate liposomes in alleviating dyslipidemia in NAFLD is better than oral administration of sodium acetate alone.

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

[0184] 5) The results of sodium acetate liposomes improving liver pathological morphology in NAFLD mice are as follows Fig.16 shown. Fig.16 As shown in A, the livers of the control group mice fed with ordinary feed were smooth, reddish brown, and had a certain degree of tissue elasticity. Compared with the control group, the overall color of the liver of the NAFLD model mice constructed after 10 weeks of HFD diet became lighter white, and obvious "fat droplet white spots" could be visually seen. After gavage with blank liposomes without sodium acetate, the appearance of the liver was similar to that of the NAFLD modeling group, with no significant changes. However, after gavage with sodium acetate liposomes doubly labeled with sodium bile / mannose, sodium acetate solution alone, or unlabeled sodium acetate liposomes for 10 weeks, the color of the liver of NAFLD mice returned to reddish brown, and the surface smoothness was restored, indicating that sodium acetate or the two liposomes containing sodium acetate have the function of alleviating NAFLD liver deposition. H&E sections of liver tissue of mice in the healthy control group showed that the liver lobule structure was normal, with hepatocyte cords arranged radially from the terminal venules to the outside in the central area of ​​the liver lobule, the hepatocytes were evenly distributed and brown, the hepatocyte nuclei were clear in color, and there were no fat droplets or inflammatory cell aggregations in the cytoplasm ( Fig.16B); H&E results of the liver of mice in the NAFLD model group showed that the structure of the mouse liver lobule was destroyed, and the liver cells underwent ballooning, which was manifested by enlarged liver cells containing large lipid droplets. Some liver cells showed mixed macrovesicular and microvesicular fatty changes, accompanied by inflammatory cell infiltration; after gavage with sodium acetate liposomes double-labeled with sodium bile / mannose, sodium acetate solution alone, or unlabeled sodium acetate liposomes, the number and area of ​​vacuoles in the mouse liver were significantly reduced, indicating that liver fatty degeneration was significantly improved. After gavage with blank liposomes without sodium acetate, the area of ​​vacuoles in the mouse liver decreased, but the number was still large, indicating that the effect of liposomes alone on the alleviation of lipid deposition in the liver of NAFLD mice was very limited. Further quantitative analysis of the lipid droplet area in the liver H&E stained sections showed that the liver lipid droplet area of ​​the NAFLD model group mice was 1.93 times that of the healthy control group mice. Sodium acetate liposomes double-labeled with sodium cholate / mannose, sodium acetate solution and unlabeled sodium acetate liposomes significantly reduced the liver lipid droplet area of ​​NAFLD mice (p<0.01). After 10 weeks of sodium acetate intervention, the liver lipid droplet area of ​​NAFLD mice was reduced by 46.39%, 40.97% and 39.33% respectively. Fig.16 C). In addition to H&E staining, the liver tissues of mice in each group were stained with Oil Red O. The results are shown in Fig.16 D. The number of red lipid droplets in the liver of healthy mice fed with ordinary diet was small and small in size, while the number of red lipid droplets in the liver of mice in the NAFLD group and blank liposome group increased significantly, the size became significantly larger, and the oil red color was bright, indicating that a large amount of fat was deposited in the liver of mice in the NAFLD group. The double-labeled liposome material without sodium acetate had no effect on alleviating lipid deposition in the liver of NAFLD mice. Sodium cholate / mannose double-labeled sodium acetate liposomes, sodium acetate alone or sodium acetate-free liposomes significantly reduced liver lipid deposition in NAFLD mice, and the alleviating effect of double-labeled sodium acetate liposomes was the most obvious. The Oil Red O-stained sections at the intervention endpoint were closer to those of normal group mice ( Fig.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 with the control group as the standard. The results were consistent with the H&E staining results. Compared with the control group, the liver lipid droplets of mice in the NAFLD model group and the blank liposome group increased significantly, which were 14.37 times and 13.67 times of the control group, respectively. After 10 weeks of intervention with sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate alone, and unlabeled sodium acetate liposomes, the liver lipid droplet area of ​​NAFLD mice was significantly reduced. Compared with the NAFLD model group, the liver lipid droplet area at the end of the intervention was reduced by 88.27%, 73.45%, and 70.40%, respectively ( Fig.16 E).

[0185] 6) Sodium acetate liposomes improve hepatic lipid accumulation and oxidative stress in NAFLD mice Fig.17The results showed that after 10 weeks of intervention with two sodium acetate-containing liposomes (NaA@CA / MAN-LPs and NaA@LPs) in NAFLD mice, the liver index was significantly lower than that of the single oral administration of sodium acetate solution, indicating that the effect of sodium acetate-containing liposomes on improving the increase in liver mass in NAFLD mice was better than that of the single oral administration of sodium acetate ( Fig.17 A). The TG and TC content in mouse liver was detected by the kit method. The results are as follows Fig.17 B, C, compared with the mice in the NAFLD group, there was no significant improvement in the TG and TC content in the liver of mice after 10 weeks of oral gavage with blank liposomes without sodium acetate. After 10 weeks of oral gavage with two liposomes containing sodium acetate or sodium acetate solution, the TG and TC content in the liver of NAFLD mice was significantly reduced, and the lipid deposition in the liver of mice was significantly improved. The results of intra-group difference analysis showed that the TG and TC content in the liver of mice gavaged with double-labeled sodium acetate liposomes was significantly lower than that in the sodium acetate group. The levels of lipid-promoting oxidants MDA and antioxidant SOD in the liver of mice were detected by the kit method, and the results are shown in Fig.17 As shown in D and E, after gavage with blank liposomes without sodium acetate, the liver MDA and SOD levels of NAFLD mice did not significantly improve. After gavage with sodium cholate / mannose double-labeled sodium acetate liposomes, 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). The results of intra-group difference analysis showed that double-labeled sodium acetate liposomes were significantly better than gavage with sodium acetate solution in alleviating the increase of MDA and the decrease of SOD levels in the liver of NAFLD mice, indicating 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) Effect of liposomes on the expression of genes related to lipid metabolism in the liver of NAFLD mice. Fig.18 AE, compared with the NAFLD model group, blank liposomes without sodium acetate had no significant effect on the gene expression of lipid synthesis and transport in the liver of NAFLD mice. After 10 weeks of intervention with sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution or unlabeled sodium acetate liposomes, the mRNA expression levels of Acaca, Fasn, Slc27a2, and Srebf1 in the liver of NAFLD mice were significantly reduced, and the mRNA expression level of CPT1α was significantly upregulated. The above results indicate that sodium acetate or liposomes containing sodium acetate effectively alleviate the upregulation of the expression of genes related to lipogenesis in the liver of NAFLD mice, and promote the expression of fatty acid transporter CPT1α. Western blot technology was used to analyze the phosphorylation of AMPKα protein in the liver of NAFLD mice after 10 weeks of oral administration of sodium acetate or sodium acetate liposomes. The results are shown in Table 1. Fig.18F. Compared with the control group, the phosphorylation of AMPKα protein in the liver of the NAFLD model group induced by HFD and the blank liposome intervention group without sodium acetate was significantly inhibited. After oral administration of sodium bile / mannose dual-labeled sodium acetate liposomes, sodium acetate solution or unlabeled sodium acetate lipids to NAFLD mice, the phosphorylation level of AMPKα protein in the liver was significantly increased ( Fig.18 F, G). After oral administration of unlabeled liposomes, double-labeled liposomes, and sodium acetate alone, the expression of cmRNA in the liver Acc1 and Srebf1 was significantly downregulated, and the expression of CPT1α was significantly upregulated. The above results indicate that sodium acetate or double-labeled sodium cholate liposomes inhibit the transcription of Acc1 and Srebf1c and promote the expression of CPT1α by activating the AMPK pathway, thereby regulating downstream lipid metabolism genes and reducing liver lipid deposition.

[0187] 8) Sodium acetate liposomes improve liver function in NAFLD mice: Serum AST and ALT levels were measured. The results are as follows Fig.19 As shown in A and B, after NAFLD mice were gavaged with blank liposomes without sodium acetate, the serum ALT and AST levels did not decrease significantly, but after gavage with sodium acetate liposomes double-labeled with sodium cholate / mannose, sodium acetate solution or unlabeled sodium acetate liposomes, the serum AST and ALT levels of mice were significantly reduced. The liver ALT and AST levels were detected, and the results are shown in Fig.19 C, D. Compared with the control group, the liver ALT and AST levels of mice in the NAFLD model group increased significantly (p<0.01). Consistent with the results of serum ALT and AST, there was no significant improvement in the liver ALT and AST levels of mice in the blank liposome group without sodium acetate. Oral 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 difference analysis showed that the effect of sodium cholate / mannose double-labeled sodium acetate liposomes in alleviating the increase in ALT and AST levels was significantly better than oral administration of sodium acetate alone.

[0188] 9) Sodium acetate liposomes improve liver inflammation and oxidative stress in NAFLD mice Fig. 20 Compared with the livers of mice in the NAFLD model group, the infiltration of macrophages in the livers of mice in the blank liposome group was not significantly improved, while the infiltration of macrophages in the livers of mice in the double-labeled sodium acetate liposome, sodium acetate solution or unlabeled sodium acetate liposome groups was significantly improved ( Fig. 20A). 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 the NAFLD model group mice was significantly higher than that of the control group (p<0.01). After 10 weeks of oral administration of blank liposomes, the relative positive area of ​​liver macrophages in NAFLD mice decreased but there was no significant improvement. After 10 weeks of oral 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 mouse liver decreased by 62.46%, 40.99% and 50.16%, respectively ( Fig. 20 B). The results of the intergroup difference analysis of the F4 / 80 positive cell area in the liver of mice in the four intervention groups showed that the effect of sodium cholate / mannose dual-labeled sodium acetate liposomes on improving the macrophage infiltration in the liver of NAFLD mice was significantly better than that of sodium acetate solution alone or unlabeled sodium acetate liposomes (p<0.01).

[0189] 10) The expression of cytokines related to inflammation and oxidative stress in mouse liver was further detected, such as Fig.21 As shown in AD. Compared with the NAFLD model group, after 10 weeks of oral gavage with blank liposomes without sodium acetate, there was no significant decrease in the expression of pro-inflammatory factors in the liver, and there was no significant increase in the level of anti-inflammatory factors. After 10 weeks of oral gavage with sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution alone, or unlabeled sodium acetate liposomes, the mRNA expression levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the liver of NAFLD mice were significantly reduced, and the mRNA expression level of anti-inflammatory factor IL-4 was significantly increased, indicating that sodium acetate or dual-labeled and unlabeled liposomes containing sodium acetate have a significant alleviating effect on liver inflammation in NAFLD mice. Compared with the NAFLD model group mice, after oral gavage with sodium cholate / mannose dual-labeled sodium acetate liposomes, sodium acetate solution, or unlabeled sodium acetate liposomes, the phosphorylation level of NFKB in the mouse liver was significantly downregulated, indicating that the HFD-induced liver inflammatory response was reduced. The results of intra-group difference analysis of the treatment groups showed that the alleviating effect of dual-labeled or unlabeled sodium acetate liposomes on the increased phosphorylation level of inflammatory proteins was significantly better than that of sodium acetate intervention alone ( Fig.21 E, F).

[0190] In summary, after 10 weeks of oral administration of sodium cholate / mannose dual-labeled sodium acetate liposomes to HFD-induced NAFLD mice, the weight gain of mice was significantly reduced, and glucose tolerance was effectively improved. The detection of serum lipid indexes showed that dual-labeled sodium acetate liposomes effectively alleviated the increase of TG, TC, and LDL-C in the liver of NAFLD mice and the decrease of HDL-C. The results of liver pathological tissue sections of NAFLD mice showed that after oral administration of dual-labeled sodium acetate liposomes to mice, liver lipid deposition was significantly improved, and the phosphorylation level of liver AMPKα protein was significantly activated. By inhibiting the transcription of Acc1 and Srebf1, CPT1α expression was promoted, and downstream lipid metabolism genes were regulated, effectively reducing liver lipid deposition. In terms of liver inflammation level and oxidative stress, after 10 weeks of oral administration of dual-labeled sodium acetate liposomes to NAFLD mice, NFKB protein phosphorylation was significantly inhibited, the release of downstream inflammatory factors was reduced, and liver inflammation and oxidative stress were effectively alleviated. The effect of dual-labeled sodium acetate liposomes on alleviating liver lipid deposition and oxidative stress in NAFLD mice was better than that of oral administration of sodium acetate solution of the same concentration alone, indicating that sodium cholate / mannose dual-labeled liposomes successfully targeted the delivery of sodium acetate to the liver, causing sodium acetate to accumulate in the liver, and more effectively alleviating liver lipid accumulation and inhibiting inflammatory responses.

Claims

1. A lipid carrier targeting the liver, characterized in that: The lipid carrier described above is prepared from DSPC, cholesterol, DSPE-PEG-CA, DSPE-PEG-MAN, DSPE-PEG-NH2, and DC-cholesterol in a certain proportion, where 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; Zeta potential is 33 ± 4 mV.

2. The liver-targeting lipid carrier according to claim 1, characterized in that The weight ratio of each component 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, 5% of DSPE-PEG-NH2 and 30% of DC-cholesterol.

3. The method for preparing the liver-targeting lipid carrier according to claim 1 or 2, characterized in that: The steps include: (1) Modification of DSPE-PEG with bile salt 2000 Obtain DSPE-PEG-CA; (2) Modification of DSPE-PEG with mannose 2000 Obtain DSPE-PEG-MAN; (3) Add the following raw materials in sequence: DSPC, cholesterol, DSPE-PEG-CA, DSPE-PEG-MAN, DSPE-PEG-NH2, and DC-cholesterol, mix well, and 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 evenly distributed. At this 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 membrane formed in step (4); (6) The solution obtained in step (5) is drawn into a micro-glass syringe, and extruded back and forth in a liposome extruder. The extruded liposomes are left overnight to stabilize the liposome particles.

4. The method according to claim 3, characterized in that: The operation of step (1) is: 1) Activating bile acid: dissolving bile acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide (EDC) and N-hydroxysuccinimide (NHS) in dichloromethane, reacting in an ice bath, stirring at room temperature overnight, filtering and rotary evaporating to obtain activated sodium cholate; 2) DSPE-PEG 2000 -NH2 is dissolved in DMSO, and activated sodium cholate and triethylamine are added and reacted after they are completely dissolved; 3) After the reaction is completed, the solution is placed in a dialysis bag and dialyzed using pure water as the medium; 4) After the dialysis is completed, the solution in the dialysis bag is collected and freeze-dried to obtain DSPE-PEG-CA.

5. The method according to claim 4, characterized in that in: Step 1) ice bath reaction for 20-40 min; Step 2) DSPE-PEG 2000 -NH2 and DMSO are added in a ratio of 450-550 mg:3 mL; activated sodium cholate and triethylamine are added in a ratio of 500-600 mg:1 g; the reaction temperature is 38-45°C for 1-3 hours; the molecular sieve size of the dialysis bag in step 3) is MW=800-1200 Da, and the dialysis time is 18-28 hours.

6. The method according to claim 3, characterized in that The operation of step (2) is: 1) DSPE-PEG 2000 -NH2 is dissolved in DMSO, mannose and triethylamine are added to completely dissolve, and mannose and triethylamine are reacted; 2) After the reaction is completed, the solution is placed in a dialysis bag and dialyzed using pure water as the medium; 3) After the dialysis is completed, the solution in the dialysis bag is freeze-dried to obtain DSPE-PEG-MAN.

7. The method according to claim 6, characterized in that Wherein step 1) DSPE-PEG 2000 -NH2 and DMSO are added in a ratio of 450-550 mg:3 mL; The addition ratio of mannose to triethylamine is 500-600 mg:1 g; the reaction temperature is 38-45° C. for 1-3 hours; step 2) the molecular sieve size of the dialysis bag is MW=800-1200 Da, and the dialysis time is 18-28 hours.

8. The method according to claim 3, characterized in that The weight ratio of the components in step (3) is 40.5% DSPC, 13.5% cholesterol, 1% DSPE-PEG-CA, 10% DSPE-PEG-MAN, 5% DSPE-PEG-NH2 and 30% DC-cholesterol.

9. Use of the liver-targeting lipid carrier according to claim 1 or 2 in the preparation of a preparation for treating liver diseases.

10. The use according to claim 9, characterized in that: The liver diseases are inflammation, cancer, and non-alcoholic fatty liver disease.

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