Hyaluronic acid modified co-loaded all-trans retinoic acid and arginine liposomes, and preparation method and application thereof

Abstract

The application discloses a hyaluronic acid modified co-loaded all-trans retinoic acid and arginine liposome and a preparation method and application thereof, and belongs to the field of pharmaceutical preparations.The liposome components comprise phospholipid, cholesterol, hyaluronic acid, all-trans retinoic acid and arginine.The prepared liposome can generate nitric oxide in response to a large amount of active oxygen existing in a liver fibrosis microenvironment, the generated nitric oxide is further oxidized into peroxynitrite by ROS, matrix metalloproteinase is activated, an extracellular matrix barrier is reduced, and nanoparticles are promoted to penetrate into a perisinusoidal space of the liver.The liver stellate cells are targeted through receptor-ligand specific combination, and the uptake efficiency of the liver stellate cells is improved.The released all-trans retinoic acid in cells is converted into a resting state by inducing activated liver stellate cells, and the regression of liver fibrosis diseases is promoted.The liposome penetrates through an extracellular matrix to promote nanoparticle penetration, enhances drug delivery, and provides a new way and strategy for efficient delivery of therapeutic drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulations, specifically relating to hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine, their preparation methods, and applications. Background Technology

[0002] Liver fibrosis is a reversible pathological state characterized by the activation of hepatic stellate cells (HSCs) and the abnormal accumulation of extracellular matrix (ECM), caused by chronic liver injury. Excessive ECM deposition forms fibrous scars, further damaging liver structure and progressing to cirrhosis and liver failure. In a normal liver, HSCs located in the perisinusoidal space are in a quiescent state and are the main storage site for vitamin A. When the liver suffers chronic injury, HSCs are activated into myofibroblast-like cells with chemotactic and contractile properties, releasing pro-inflammatory and pro-fibrotic factors and producing large amounts of ECM, thus becoming known as the "executors" of liver fibrosis. Therefore, inhibiting HSC activation is key to anti-fibrotic therapy. In addition to inducing HSC apoptosis and senescence, inducing activated HSCs to revert to a quiescent phenotype can treat liver fibrosis without causing inflammation or toxic side effects. All-trans retinoic acid (RA) is a promising anti-fibrotic drug. It has been reported that hepatic stellate cells (RA) can induce the transformation of activated pancreatic stellate cells to a quiescent state by activating retinoic acid-R-β and inhibiting the transcription and expression of myosin light chain (MLC-2). Due to the high functional similarity between hepatic and pancreatic stellate cells, studies have shown that RA can promote the inactivation of hepatic stellate cells (HSCs), demonstrating potential for anti-hepatic fibrosis therapy.

[0003] Many studies have focused on treating liver fibrosis by targeting HSCs, but have overlooked the barrier of deposited ECM encountered during drug delivery to activated HSCs. This barrier can impair anti-fibrotic efficacy due to low drug delivery efficiency. Therefore, eliminating ECM deposition barriers and targeting HSCs is a key step in improving anti-liver fibrosis efficacy. In normal liver, matrix metalloproteinases (MMPs) and tissue metalloproteinase inhibitors (TIMPs) regulate the dynamic balance between ECM synthesis and degradation. MMPs are a family of zinc-dependent endogenous peptidases, typically produced and secreted by HSCs and Kupffer cells in liver tissue. MMPs can degrade almost all components of the ECM, and their activity is influenced by: 1) gene transcription levels; 2) the conversion of pro-matrix metalloproteinases (pro-MMPs) into active MMPs; and 3) the inhibition of MMP activity by TIMPs. Currently, much research focuses on using collagenase-modified nanoparticles to degrade deposited ECM or delivering gene drugs to block ECM biosynthesis, thereby promoting nanoparticle penetration into HSCs. However, a major drawback of these methods is the tendency for collagenases or gene drugs to become inactive during delivery. Therefore, enhancing the activity of MMPs to break down the ECM barrier under pathological conditions is a promising new direction to explore.

[0004] The oxidative stress environment during liver fibrosis is often accompanied by excessive ROS production, further promoting and aggravating the development of fibrosis. Therefore, combined regulation of anti-inflammatory and anti-fibrotic effects is beneficial to accelerating the regression of fibrosis. Nitric oxide (NO) is an endogenous free radical with various biological activities, such as vasodilation, inhibition of platelet aggregation, anti-inflammatory effects, and immune defense. Under ROS conditions, NO reacts with superoxide anion (O2... - NO rapidly forms peroxynitrite (ONOO-), which activates pro-MMPs via cysteine ​​oxidation. Although previous studies have reported significant therapeutic potential in liver fibrosis, few studies have explored the regulation of MMP activity by NO and its impact on ECM collagen degradation, which will drive progress in antifibrotic therapy.

[0005] Inspired by the aforementioned research, this invention aims to enhance the penetration and accumulation of nanoparticles by depleting the collagen deposition barrier, thereby achieving effective anti-hepatic fibrosis treatment. Liposomes, with their natural advantage of targeting the liver, were chosen as carriers for the co-delivery of hydrophobic (RA) and hydrophilic (L-arg) drugs. To improve the uptake efficiency of HSCs, we modified liposomes with hyaluronic acid (HA) to achieve targeted delivery by binding HA to the CD44 receptor overexpressed on activated HSCs. Specifically, L-arg, as an endogenous NO donor, can be catalyzed by endothelial nitric oxide synthase or oxidized by ROS in the inflammatory microenvironment to generate NO and ONOO-. The generated ONOO- can activate pro-MMPs and degrade ECM deposited in the perisinusoidal cavity, providing an opportunity for HSC uptake of nanoparticles. Then, HA-modified liposomes precisely target and deliver RA to HSCs by binding to the CD44 receptor overexpressed on activated HSCs. RA significantly inhibits MLC-2 expression, thereby inhibiting HSC activation, reversing activated HSCs to a quiescent state, and further reducing collagen secretion and deposition. The HA-targeting liposome nanoplatform constructed in this invention can penetrate the ECM barrier in the perisinusoidal cavity of the liver, induce activated HSC cells to become quiescent, reduce collagen production, and achieve deep delivery, precise targeting, and effective treatment of liver fibrosis. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention constructs hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine. The main purpose is to enhance the penetration and accumulation of nano-preparations by consuming the extracellular collagen deposition barrier, thereby achieving highly efficient anti-liver fibrosis treatment.

[0007] The hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine proposed in this invention contain phospholipids, cholesterol, hyaluronic acid, all-trans retinoic acid and arginine.

[0008] The phospholipids mentioned include phospholipids that are directly synthesized into liposomes and phospholipids that are bound to hyaluronic acid;

[0009] The phospholipids used in the direct synthesis of liposomes include egg yolk lecithin (EPC), soybean phosphorycholine (SPC), myristoyl phosphorycholine (DMPC), dilauryl phosphorycholine (DLPC), arachidoyl phosphorycholine (DAPC), dioleoyl phosphorycholine (DOPC), dipalmitoyl phosphorycholine (DPPC), 1-palmitoyl-2-oleoyl phosphorycholine (POPC), and hydrogenated soybean phosphorycholine (HSPC).

[0010] The phospholipids bound to hyaluronic acid include dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearateoylphosphatidylethanolamine (DSPE), disqualoylphosphatidylethanolamine (DEPE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), and 1,2-dilauroylphosphatidylethanolamine (DLPE).

[0011] The hyaluronic acid mentioned is one type of hyaluronic acid with a different molecular weight;

[0012] The drugs mentioned for the treatment of liver fibrosis include all-trans retinoic acid (RA), relaxin, pirfenidone, sorafenib, nintedanib, imatinib, glycyrrhetinic acid, artesunate, glycyrrhizin, glycyrrhizic acid diamine, ferulic acid, silymarin, ursodeoxycholic acid, polyene phosphatidylcholine, anisole, bicyclol, and biphenyl diester.

[0013] The present invention provides a method for preparing hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine, comprising the following steps:

[0014] Step 1: Synthesis of RAL (Relative Acid and Arginine Liposomes)

[0015] Egg yolk lecithin, cholesterol, and all-trans retinoic acid were dissolved in an organic solvent. After sonication, the organic solvent was removed by rotary evaporation to form a uniform and transparent film. The film was dried in a vacuum drying oven, and a 5% glucose solution containing arginine was added to hydrate it. The solution was collected and sonicated under ice bath conditions to obtain a liposome solution (RAL) co-loaded with all-trans retinoic acid and arginine.

[0016] Step 2: Synthesize the HA-DOPE complex

[0017] Hyaluronic acid was weighed and dissolved in formamide. DOPE, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N,N-dimethyl-4-pyridinium (DMAP) were added and completely dissolved. The mixture was reacted under water bath conditions, poured into ice-cold ether to precipitate, washed, filtered to collect the product, and vacuum dried to obtain the HA-DOPE complex.

[0018] Step 3: Synthesis of Hyaluronic Acid-Modified Co-loaded All-Trans Retinoic Acid and Arginine Liposomes (HRAL)

[0019] The complex of RAL from step 1 and HA-DOPE from step 2 was co-incubated; hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine (HRAL) were collected.

[0020] The above-mentioned method for preparing hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine includes:

[0021] The purity of the egg yolk lecithin mentioned in step 1 is not less than 98%; the purity of the cholesterol mentioned is not less than 99%.

[0022] In step 1, the mass ratio of egg yolk lecithin to all-trans retinoic acid is (5-40):1; the mass ratio of egg yolk lecithin to cholesterol is (1-10):1; and the mass ratio of all-trans retinoic acid to arginine is 1:(1-20).

[0023] The rotary evaporation temperature in step 1 is 30–60°C, and the rotary evaporation time is 0.2–1 h;

[0024] The vacuum drying time mentioned in step 1 is 4–24 hours;

[0025] The hydration temperature in step 1 is 30–60°C, and the hydration time is 0.2–1 h.

[0026] In step 1, the power of the probe ultrasound is 200W or 400W; the total ultrasound duration is 3 to 30 minutes.

[0027] In step 2, the mass ratio of HA, DOPE, EDC, and DMAP is (1-20):(1-10):(1-10):(0.01-1); the ratio of HA to formamide is (1-100) mg / ml.

[0028] The water bath temperature in step 2 is 25–60°C, and the reaction time is 2–20 h.

[0029] In step 2, ethanol is used for washing, and the number of washing cycles is 1 to 5.

[0030] The temperature of the vacuum drying oven in step 2 is 30-60℃, and the drying time is 1-3 days;

[0031] The mass concentration of HA-DOPE in step 3 is (1-1000) mg / ml; the ratio of HA-DOPE to RAL is (1-30):(1-10) mg / ml;

[0032] The incubation temperature in step 3 is 40–65°C; the incubation time is 0.1–2 hours.

[0033] The particle size of the HRAL obtained in step 3 is 60-250 nm.

[0034] Extracellular matrix barriers deposited in the hepatic sinusoidal cavity lead to insufficient drug delivery, severely impacting antifibrotic efficacy. Furthermore, excessive reactive oxygen species (ROS) in the liver microenvironment are a key factor contributing to the progression of liver fibrosis. To address these issues, this invention constructs HA-modified liposomes that co-deliver the hydrophobic drug all-trans retinoic acid (RA) and the hydrophilic drug L-arginine (L-arg). Specifically, excessive ROS in liver fibrosis promotes the conversion of loaded L-arg to NO. Furthermore, NO is further oxidized to ONOO-, which activates inactive pro-MMPs into active MMPs, degrades the ECM, and promotes the penetration and accumulation of more nanoparticles around HSCs. HA modification enhances the targeting effect on HSCs, and the released RA can reduce excessive collagen production by inducing activated HSCs to a quiescent state, resulting in a stronger antifibrotic effect.

[0035] This invention uses normal hepatocytes (L02) and hepatic stellate cells (HSC-T6) as cell models to investigate the cytotoxicity of HRAL. Cytotoxicity assays showed that, at the same concentration, liposomes loaded with all-trans retinoic acid (RL) exhibited weaker cytotoxicity than the RA solution group, indicating that the liposome formulation has a toxicity-reducing effect. Notably, hyaluronic acid-modified liposomes loaded with all-trans retinoic acid (HRL) showed significantly higher cytotoxicity to HSC-T6 cells than RL, indicating that HA has good targeting properties for HSCs. Cell uptake assays showed little difference in uptake intensity between HRL and RL in L02 cells. In contrast, HSC cells in the HRAL and HRL treatment groups showed stronger uptake than RL, indicating that HA-modified liposomes have a stronger affinity for HSC-T6 cells. Pharmacodynamic assays showed that HRAL significantly reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and liver tissue hydroxyproline (Hyp) levels in mice with liver fibrosis, indicating that HRAL can significantly improve liver fibrosis.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] (1) The HA provided by the present invention can specifically bind to the overexpressed CD44 receptor on the surface of activated HSCs, enhance the uptake of nanoparticles by activated HSCs, and improve drug delivery efficiency.

[0038] (2) The L-arg provided by the present invention can react with ROS in the liver fibrosis microenvironment to generate NO and ONOO, activate MMPs, break through the extracellular matrix barrier, and promote the penetration of nano-preparations.

[0039] (3) The present invention prepares HA-modified RA and L-arg liposomes that deplete collagen in response to the fibrotic microenvironment, which is expected to achieve efficient delivery of therapeutic drugs and provides new ideas and approaches for the treatment of liver fibrosis. Attached Figure Description

[0040] Figure 1 Synthetic route of HA-DOPE;

[0041] Figure 2 The NMR spectrum for HA-DOPE;

[0042] Figure 3 Particle size diagrams of blank liposome BL, liposome RL loaded with all-trans retinoic acid, and hyaluronic acid-modified liposomes HRL and HRAL loaded with all-trans retinoic acid.

[0043] Figure 4 Potential diagrams for BL, RL, HRL, and HRAL;

[0044] Figure 5 This is a diagram showing drug release in vitro.

[0045] Figure 6 The cytotoxicity of HRAL against HSCs(A) and L02(B);

[0046] Figure 7 Serum ALT levels in each treatment group of fibrotic mice;

[0047] Figure 8 AST levels in different treatment groups of fibrotic mice;

[0048] Figure 9 The content of Hyp in tissues of mice in each treatment group with fibrosis. Detailed Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0050] Example 1

[0051] Preparation of RAL

[0052] Egg yolk lecithin, cholesterol, and all-trans retinoic acid were dissolved in chloroform solution at a mass ratio of 20:5:1. After sonication, the solution was placed in a rotary evaporator and evaporated at 40°C in a water bath to remove the organic solvent, resulting in a uniform, transparent film. This film was then dried in a vacuum oven at 40°C for 12 hours. A 5% glucose solution containing 15 mg of arginine was added, and the film was hydrated at 50°C for 20 minutes. The solution was collected and sonicated under ice bath conditions (200W for 2 minutes, 400W for 4 minutes) to obtain RAL (reactive alpha-linolenic acid).

[0053] Example 2

[0054] Other phospholipids were selected to prepare liposomes co-loaded with all-trans retinoic acid and arginine.

[0055] SPC: Soybean lecithin, cholesterol and all-trans retinoic acid were dissolved in chloroform solution at a mass ratio of 20:5:1. The remaining operations were the same as in Example 1. The resulting liposomes had a particle size of approximately 163.7 nm.

[0056] HSPC: Hydrogenated soybean lecithin, cholesterol and all-trans retinoic acid were dissolved in chloroform solution at a mass ratio of 20:5:1. The remaining operations were the same as in Example 1. The resulting liposomes had a particle size of approximately 186.3 nm.

[0057] Example 3

[0058] Synthesis of HA-DOPE

[0059] Weigh 10 mg of hyaluronic acid and dissolve it in 4 ml of formamide. Add DOPE (3 mg), EDC (1 mg), and DMAP (0.05 mg) and dissolve completely. React at 40 °C for 8 h. Pour the solution into a large amount of ice-cold ether to precipitate the product. Wash the product three times with ethanol, filter and collect the product. Dry the product under vacuum to obtain the HA-DOPE complex.

[0060] The specific synthesis route is as follows: Figure 1 As shown;

[0061] The prepared HA-DOPE complex, through 1 H-NMR (D2O, 300MHz) characterization, corresponding 1 The H-NMR spectrum is attached. Figure 2 As shown.

[0062] On the NMR spectrum of HA, the chemical shift of H on the acetyl-OCCH3 group of hyaluronic acid is 1.98 ppm, and the characteristic peak of the methylene group on the hyaluronic acid sugar ring is observed in the range of 3.25–4.0 ppm. In the NMR spectrum of HA-DOPE, in addition to the above peaks, a characteristic absorption peak of the methylene group on DOPE is also observed in the range of 1.2–1.3 ppm. This preliminarily proves that HA-DOPE was successfully synthesized. Using the acetyl group on the hyaluronic acid side chain (1.98 ppm) as a reference for integration, the integral of the characteristic methylene group on DOPE (1.2–1.3 ppm) was obtained. The grafting rate of DOPE onto HA was found to be 7.8% by the integration ratio.

[0063] Example 4

[0064] Preparation of HRAL

[0065] Egg yolk lecithin, cholesterol, and all-trans retinoic acid (mass ratio 20:5:1) were dissolved in chloroform solution and sonicated. The solution was then placed in a rotary evaporator and evaporated at 40°C in a water bath to remove the organic solvent, resulting in a uniform, transparent film. This film was then dried in a vacuum drying oven at 40°C for 12 hours. A 5% glucose solution containing 15 mg of arginine was added, and the film was hydrated at 50°C for 20 minutes. The solution was collected and sonicated under ice bath conditions (200W, 2 minutes; 400W, 4 minutes) to obtain RAL. A 10 mg / mL aqueous solution of HA-DOPE was incubated with the RAL at 55°C for 40 minutes using a post-interpolation method to obtain HRAL.

[0066] Appendix Figure 3 The results showed that the prepared HRAL particles had a diameter of approximately 145 nm. (See attached image.) Figure 4 The results showed that, due to the large number of carboxyl groups on HA, which have good hydrophilicity, the liposomes modified with HA had larger particle size and lower potential.

[0067] Example 5

[0068] In vitro release study of HRAL

[0069] In vitro release studies were conducted on the optimally formulated drug-loaded liposomes. RA and HRAL solutions were precisely transferred into dialysis bags (MWCO: 8000 Da), both ends were tied, and each bag was immersed in 30 ml of pH 7.4 PBS solution containing 1% Tween 80. The bags were then placed in a temperature-controlled shaker (37℃, 110 rpm). At pre-set time points, 5 ml of release medium was collected from the solution, and an equal volume of isothermal 1% Tween 80 pH 7.4 PBS release medium was added. The absorbance of the collected samples was measured at 350 nm using high-performance liquid chromatography (HPLC), and the cumulative drug release was calculated. In vitro release curves were plotted (see attached figure). Figure 5 The results showed that the RA solution group and the L-arg solution group could rapidly release the drug. However, HRAL only released 30% of RA and about 25% of L-arg in the first 6 hours in the pH 7.4 PBS release medium. After 48 hours, the cumulative release of RA by HRAL in the pH 7.4 PBS release medium was less than 60%, and the release of L-arg was less than 50%, indicating that the formulation has a significant sustained-release effect.

[0070] Example 6

[0071] Cytotoxicity experiments

[0072] The cytotoxic effects of free RA, RL, HRL, and HRAL were detected using the MTT assay. HSC cells were seeded in 96-well plates (5000 cells / well) and cultured for 24 h, after which the old culture medium was discarded. Then, the different formulations were added and incubated for 24 h. Next, 20 μL of MTT solution (5 mg / ml) was added to each well and incubated for 4 h. 200 μL of DMSO was added to dissolve the formed formaldehyde crystals. Finally, the absorbance of each well was measured at 570 nm using a microplate reader. Untreated cells were used as a control for comparison, and cell viability was calculated for each group. Results are attached. Figure 6 As shown, in the HSC-T6 cell assay, HRL with target modification was significantly more cytotoxic to HSCs than RL without target modification, which also indicates that HA modification can promote the uptake of the agent by HSCs.

[0073] Example 7

[0074] Cell uptake experiment

[0075] HSC or L02 cells were seeded in 6-well plates and allowed to adhere for 24 h. Coumarin 6 (C6) was used instead of RA, and C6-labeled liposome formulations (RL, HRL, and HRAL) were added to the cells for co-culture (C6 concentration: 0.1 μg / ml). The culture medium was discarded, the cells were washed, fixed (in the dark, 20 min), washed with PBS, and the nuclei were stained with DAPI for 10 min. After washing with PBS, a coverslip was placed on top of the slide, the slide was sealed, and the uptake of C6-labeled liposomes in the cells was observed under a confocal microscope. The confocal results showed that HSC cells showed significantly stronger uptake of HRAL than L02 cells, and HSC cells showed significantly stronger uptake of HRAL and HRL than RL. This indicates that HA modification significantly enhanced the uptake of nano-formulations by HSC cells.

[0076] Example 8

[0077] Anti-mouse liver fibrosis experiment

[0078] First, a liver fibrosis model was established using 6-8 week old male C57BL / 6 mice. Mice were randomly assigned to 6 groups: 1) normal group (ipOil for 4 weeks + ivPBS for 3 weeks), 2) liver fibrosis model group (ipCCl4 for 7 weeks), 3) liver fibrosis + RA treatment group (ipCCl4 for 4 weeks + ivRA for 3 weeks), 4) liver fibrosis + RL treatment group (ipCCl4 for 4 weeks + ivRL for 3 weeks), 5) liver fibrosis + HRL treatment group (ipCCl4 for 4 weeks + ivHRL for 3 weeks), and 6) liver fibrosis + HRAL treatment group (ipCCl4 for 4 weeks + ivHRAL for 3 weeks). An olive oil solution containing 20% ​​CCl4 (CCl4: 0.75 μL / g) was prepared and administered via intraperitoneal injection (ip) twice weekly for 4 weeks. Each treatment group received RA at a dose of 2.5 mg / kg via tail vein injection (iv), twice weekly for 3 weeks. Results are attached. Figure 7 , Figure 8 and Figure 9 As shown, compared with the liver fibrosis model group, each treatment group had a certain degree of inhibitory effect on the progression of liver fibrosis in mice. Preferably, HRAL treatment significantly reduced the serum ALT and AST levels in the model group mice and significantly reduced the Hyp content in the liver tissue, exhibiting the strongest anti-fibrotic effect.

[0079] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any improvements and modifications made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine, characterized in that, The hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine include egg yolk lecithin, cholesterol, hyaluronic acid, all-trans retinoic acid, and arginine. The preparation method includes the following steps: Step 1: Synthesis of liposomes co-loaded with all-trans retinoic acid and arginine Egg yolk lecithin, cholesterol, and all-trans retinoic acid were dissolved in an organic solvent at a mass ratio of 20:5:

1. After ultrasonic dissolution, the organic solvent was removed by rotary evaporation to form a uniform and transparent film. The film was then dried in a vacuum drying oven. A 5% glucose solution containing arginine was added to hydrate the film. The solution was collected and ultrasonicated under ice bath conditions to obtain a liposome solution co-loaded with all-trans retinoic acid and arginine, denoted as RAL. The mass ratio of all-trans retinoic acid to arginine was 1:(1~20). Step 2: Synthesize the HA-DOPE complex Hyaluronic acid was weighed and dissolved in formamide. Dioleoylphosphatidylethanolamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N,N-dimethyl-4-pyridinium amine were added and completely dissolved. The mixture was reacted under water bath conditions, and the product was precipitated in ice-cold ether. The product was washed with ethanol, filtered, collected, and dried under vacuum to obtain the HA-DOPE complex. The mass ratio of hyaluronic acid, dioleoylphosphatidylethanolamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N,N-dimethyl-4-pyridinium amine was 10:3:1:0.

05. Step 3: Synthesis of hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine The complex of RAL from step 1 and HA-DOPE from step 2 (10 mg / ml) was co-incubated at 40-65°C for 0.1-2 h, and hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine were collected.

2. The method for preparing hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine according to claim 1, characterized in that, The purity of the egg yolk lecithin mentioned in step 1 is not less than 98%; the purity of the cholesterol is not less than 99%; the rotary evaporation temperature is 30~60℃, and the rotary evaporation time is 0.2~1h; the temperature of the vacuum drying oven is 40℃, and the vacuum drying time is 4~24h; the hydration temperature is 30~60℃, and the hydration time is 0.2~1h; the ultrasonic power of the probe is 200W or 400W; and the total ultrasonic testing time is 3~30min.

3. The method for preparing hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine according to claim 1, characterized in that, The water bath temperature in step 2 is 25~60℃, the reaction time is 2~20h, and the number of ethanol washings is 1~5 times.

4. The method for preparing hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine according to claim 1, characterized in that, The hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine had a particle size of 60-250 nm.

5. The application of the liposomes prepared by the method of claim 1 for preparing hyaluronic acid-modified liposomes co-loaded with all-trans retinoic acid and arginine in the preparation of anti-liver fibrosis drugs.

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