Preparation method of liver-targeted drug-loaded liposome

Abstract

The invention provides a preparation method of liver-targeted drug-loaded liposome, and belongs to the field of biological medicine. The hepatic targeting carbohydrate chain is constructed through a multi-step enzymatic reaction, and the accuracy of a modification site is ensured. The synthesized galactose ligand is connected to the surface of the liposome through a covalent bond, so that the targeting property and the stability are enhanced. The synthesis of the galactose ligand provides targeting functional groups with clear structures for the liposome, and the groups are integrated to the surface of the liposome through physical entrapment and chemical coupling in the preparation of the liposome, so that active targeting delivery is finally realized. The collaborative design of the two is based on an ASGPR-mediated liver targeting mechanism, and the delivery efficiency is optimized through enzymatic synthesis and a nanotechnology. In in vivo experiments, plasma and liver distributions of liposomes are determined. In an in-vitro experiment, the uptake efficiency of the lipidosome in liver cancer cells is determined. The experiments aim at verifying that the advantages of the lipidosome in liver cancer cell delivery are improved by optimizing the PEG chain length and the exposure of residues.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for preparing liver-targeted drug-loaded liposomes. Background Art

[0002] Liposome delivery systems have garnered significant attention among various delivery systems. Compared to free chemical drugs, liposomes can prolong drug bioavailability in the circulation. As drug carriers, nanoliposomes can encapsulate hydrophilic or lipophilic anticancer drugs, leveraging the enhanced permeability and retention (EPR) effect of solid tumor tissue to enhance drug efficacy, increase tumor accumulation, and reduce drug toxicity.

[0003] In the field of targeted drug delivery (TDD) systems, recent research focuses on achieving tumor-specific active targeting by attaching specific ligands to the outer surface of the phospholipid bilayer. Receptor-mediated endocytosis is an effective method for precisely delivering drugs to specific cell types. This approach achieves high drug concentrations within the cell while minimizing concentrations at non-target sites, achieving a perfect combination of high efficacy and low toxicity.

[0004] The asialoglycoprotein receptor (ASGPR), a member of the C-type lectin family, is one of the most attractive targets for hepatocyte TDD drugs. There is ample evidence that ASGPR is highly expressed only in well-differentiated hepatocytes in the form of hepatocellular carcinoma (HCC), with low expression in the extracellular regions of hepatocytes. Therefore, anticancer drugs targeting ASGPR may be an effective approach for the treatment of HCC.

[0005] ASGPR, a C-type lectin, promotes drug internalization through clathrin-mediated endocytosis and specifically recognizes carbohydrates, particularly D-galactose (Gal) or N-acetylgalactosamine (GalNAc). Notably, ASGPR binds to GalNAc with a 10-50-fold higher affinity than to Gal. Studies have shown that surface modification of liposomes with galactose residues can effectively target drugs to hepatocytes via an ASGPR-dependent pathway. Therefore, surface modification of nanoparticles with Gal or GalNAc is expected to further enhance drug delivery to liver cancer cells.

[0006] After systemic administration, liposomes are primarily cleared from the circulation by macrophages in the liver and spleen. This is because these organs are home to numerous macrophages, which efficiently clear liposomes. Studies have shown that plasma proteins in the blood adsorb heavily onto the liposome surface, forming a protein corona that directs the liposomes to macrophages and hepatocytes. However, surface modification with polyethylene glycol (PEG) chains creates a "stealth effect," reducing protein adsorption and preventing recognition by the reticuloendothelial system (RES).

[0007] Patent ZL201910537743.5 discloses a method for synthesizing a cholesterol-galactose liver-targeting ligand, but the spacer arm uses a hydrophobic hydrocarbon, which results in the galactose target group being unable to effectively separate from the lipid-water interface, hindering ASGPR recognition and resulting in low liver targeting efficiency.

[0008] Patent ZL202011639307.8 improves the ligand structure disclosed in Patent ZL201910537743.5 by replacing the spacer arm with a PEG chain consisting of two PEG units. However, this still fails to completely separate the galactose target from the lipid-water interface, resulting in limited improvement in liver-targeting efficiency. The patent does not explain or verify whether longer PEG chains can improve ASGPR recognition efficiency. Summary of the Invention

[0009] To address the shortcomings of existing patented technologies, the present invention aims to further optimize the liver-targeting performance of liposomes. Using an enzymatic method, the present invention constructs a series of liposomes with surface-modified galactosyl groups. These glycosyl groups are separated from the lipid-water interface by PEG chains of varying lengths. The present invention aims to utilize the anti-opsonization and spacing effects of the PEG chains to improve the exposure of the galactose target groups, thereby providing an optimal interaction configuration with the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes.

[0010] To construct ligands containing both galactose and PEG, the present invention synthesized acetylgalactosamine (GalNAc)-terminated PEG chains containing 2, 3, 4, and 5 PEG units and coupled them to cholesterol to ensure secure anchoring within the liposome bilayer. To compare the effects of different galactose ligand types—GalNAc and lactitol (Lac)—on the ligand's affinity for ASGPR, the present invention synthesized Lac-terminated PEG-cholesterol conjugates. Through extensive in vivo animal tissue distribution experiments and in vitro hepatocyte uptake experiments, the present invention determined that the optimal PEG chain length for exposing the GalNAc residue within the liposome bilayer is 3 PEG units. Molecular dynamics simulations were used to elucidate the structure-activity relationship between PEG chain length and ASGPR binding.

[0011] A method for preparing liver-targeted drug-loaded liposomes, comprising: Synthetic liver-targeting carbohydrate ligands: S1: Palladium acetate catalyzes the vinyl exchange of dicarboxyl PEG with vinyl acetate to form divinyl ester: PEG n -divinyl ester; PEG n Where n=1~20, preferably n=1~6. The general structural formula is shown below:

[0012] S2: Enzymatically catalyze the reaction of cholesterol (CHS) with PEG n The vinyl end group of the -divinyl ester undergoes selective esterification to generate single-end vinyl-modified CHS-PEG n -vinyl ester; PEG n Where n = 1 to 20, preferably n = 1, 3 to 6. The general structural formula is shown below:

[0013] S3: Enzymatically catalyze the reaction of the C-6 hydroxyl group of GalNAc with PEG n The vinyl end group of the -vinyl ester undergoes esterification to generate GalNAc-modified CHS-PEG n -6-GalNAc;PEG n Where n=1~20, preferably n=1, 3~6. The general structural formula is shown below:

[0014] and / or The C-6 hydroxyl group of Lac and CHS-PEG n The vinyl end group of the -vinyl ester undergoes esterification to generate Lac-modified CHS-PEG n -6-Lac;PEG n Where n = 1 to 20, preferably n = 1 to 6. The general structural formula is shown below:

[0015] Preparation of liver-targeted drug-loaded liposomes; S4: Using thin film dispersion method, CHS-PEG n -6-GalNAc and / or CHS-PEG n -6-Lac, phospholipids and cholesterol were mixed in proportion to prepare GalNAc or Lac-modified liposomes.

[0016] The specific components are composed of hydrogenated soybean phosphatidylcholine, cholesterol and liver-targeted sugar ligand; the liver-targeted sugar ligand is CHS-PEG n -X; The CHS is a cholesterol skeleton; The PEG n is a polyethylene glycol chain; When n=3, 4, or 5, X is 6-GalNAc When n=2, 3, 4, or 5, X is 6-Lac.

[0017] The molar ratios of hydrogenated soybean phosphatidylcholine, cholesterol, and liver-targeting sugar ligand are shown in the table below.

[0018]

[0019] Furthermore, the drug delivery system includes:

[0020] Preferably, the drug delivery system comprises:

[0021] In the preferred technical solution of the present invention, the drug is selected from drugs for treating liver diseases such as doxorubicin, paclitaxel, lenvatinib, siRNA, naringenin, and naringin.

[0022] Preferably, the particle size of the liposome is 50-150 nm.

[0023] Preferably, the liposome is prepared by the preparation method according to any one of claims 1 to 4.

[0024] Preferably, the surface charge of the liposome is neutral or negative.

[0025] The present invention also provides the use of the liver-targeted drug-loaded liposome in preparing medicines.

[0026] The drug is a hydrophobic or hydrophilic drug, and the drug is selected from doxorubicin, paclitaxel, lenvatinib, siRNA, naringenin, naringin and other drugs for treating liver diseases. The application is to encapsulate the drug inside the liposome by freeze-drying reconstitution or hydration method using ammonium sulfate gradient method to achieve active drug loading.

[0027] One of the purposes of the present invention is to provide a method for preparing the above-mentioned GalNAc or Lac modified liver-targeting sugar ligand molecule, which is prepared by enzyme-catalyzed synthesis, and the reaction route is Figure 1 shown.

[0028] In the preferred technical solution of the present invention, the reaction solvent of step 1 is selected from one or more of tetrahydrofuran, acetone, tert-butanol, benzene, toluene, pyridine, dimethylformamide, acetonitrile and their combinations; the substrate molar ratio is 1:10-1:60 (COOH-PEG n -COOH: vinyl acetate), preferably 1:20-1:40. The catalyst is a co-catalytic system of palladium acetate and 1,10-phenanthroline, with a molar ratio of 1:1-1:5, preferably 1:1-1:1.5. The amount of catalyst (calculated as palladium acetate) is COOH-PEG n The amount of -COOH is 5 mol%, and the reaction temperature is 40-100°C, preferably 30-60°C.

[0029] In the preferred technical solution of the present invention, the reaction solvent of step 2 is selected from one or more of tetrahydrofuran, tert-butanol, acetone, acetonitrile, benzene, toluene, pyridine and their combinations; the substrate molar ratio is 1: 1-1: 10 (cholesterol: PEG n -divinyl ester), preferably at a ratio of 1:1-1:4. The enzyme is selected from one or a combination of two or more of Novozyme 435 (immobilized lipase from Candida antarctica B), PS IM (immobilized lipase from Burkholderia cepacia), Candida rugosa lipase (from Candida rugosa), TL IM (immobilized lipase from Candida rugosa), and RM IM (from Rhizomucor miehei), preferably Novozyme 435. The reaction temperature is 10-100°C, preferably 30-60°C.

[0030] In the preferred technical solution of the present invention, the reaction solvent of step 3 is selected from one or more of tetrahydrofuran, acetone, tert-butanol, acetonitrile, benzene, toluene, pyridine and their combinations; the substrate molar ratio is 1: 1-1: 10 (GalNAc: CHS-PEG n -vinyl ester), preferably at a ratio of 1:2-1:8. The biological enzyme is selected from one or a combination of two or more of Novozyme 435 (immobilized lipase from Candida antarctica B), PS IM (immobilized lipase from Burkholderia cepacia), Candida rugosa lipase (from Candida rugosa), TL IM (immobilized lipase from Candida rugosa), and RM IM (from Rhizomucor miehei). The reaction temperature is 10-100°C, preferably 30-60°C.

[0031] In the preferred technical solution of the present invention, the reaction solvent of step 4 is selected from one or more of tetrahydrofuran, acetone, tert-butanol, acetonitrile, benzene, toluene, pyridine and their combinations; the substrate molar ratio is 1:1-1:99 (Lac: CHS-PEG n-vinyl ester), preferably at a ratio of 1:2-1:8. The biological enzyme is selected from one or a combination of two or more of Novozyme 435 (immobilized lipase from Candida antarctica B), PS IM (immobilized lipase from Burkholderia cepacia), Candida rugosa lipase (from Candida rugosa), TL IM (immobilized lipase from Candida rugosa), and RM IM (from Rhizomucor miehei). The reaction temperature is 10-100°C, preferably 30-60°C.

[0032] The enzymatic ligand synthesis process adopted in the present invention has significant advantages: fewer synthesis steps, the required raw materials are cheap and easily available, the enzymatic reaction conditions are mild, the regional selectivity is high, the reaction efficiency is high, the total yield is about 50% or more (calculated from the first step of the reaction), it is green and environmentally friendly, the production cost is low, and it has great industrial prospects.

[0033] The beneficial technical effects of the present invention are at least as follows: The synthesis of galactose ligands provides liposomes with well-defined targeting functional groups, and liposome preparation integrates these groups into the liposome surface through physical encapsulation and chemical coupling, ultimately achieving active targeted delivery. The collaborative design of the two is based on the ASGPR-mediated liver targeting mechanism, and the delivery efficiency is optimized through enzymatic synthesis and nanotechnology. The present invention measured the plasma half-life, liver uptake, and in vitro hepatocyte uptake of galactose ligand-modified liposomes. These experiments were intended to validate the present invention's design strategy, which optimizes PEG chain length and residue exposure to enhance the advantages of drug-loaded liposomes in hepatocyte delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention is further described with reference to the accompanying drawings. However, the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative effort.

[0035] Figure 1 A synthetic roadmap for liver-targeting glycoligands.

[0036] Figure 2 、 Figure 3 These are the effects of different PEG chain lengths in the linker arm on the affinity of galactose ligand to ASGPR.

[0037] Figure 4 The initial spatial positions of the three sugar ligand molecules in the bilayer membrane, A is the top view, and B is the side view.

[0038] Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 PEG3-divinyl ester 13 C NMR, 1 H NMR, MS and synthetic routes.

[0039] Figure 9 、 Figure 10 、 Figure 11 CHS-PEG3-vinyl ester 13 C NMR, 1 H NMR and synthetic routes.

[0040] Figure 12 、 Figure 13 、 Figure 37 、 Figure 14 CHS-PEG3-6-GalNAc 13 C NMR, 1 H NMR, MS and synthetic routes.

[0041] Figure 15 、 Figure 16 、 Figure 17 CHS-PEG3-6-Lac 13 C NMR, 1 H NMR and synthetic routes.

[0042] Figure 18 、 Figure 19 、 Figure 20 PEG4-divinyl ester 13 C NMR, 1 H NMR and synthetic routes.

[0043] Figure 21 、 Figure 22 、 Figure 23 CHS-PEG4-vinyl ester 13 C NMR, 1 H NMR and synthetic routes.

[0044] Figure 24 、 Figure 25 、 Figure 38 、 Figure 26 CHS-PEG4-6-GalNAc 13 C NMR, 1 H NMR, MS and synthetic routes.

[0045] Figure 27 、 Figure 28 、 Figure 29 PEG5-divinyl ester 13 C NMR, 1 H NMR and synthetic routes.

[0046] Figure 30 、 Figure 31 、 Figure 32 CHS-PEG5-vinyl ester 13 C NMR, 1 H NMR and synthetic routes.

[0047] Figure 33 、 Figure 34 、 Figure 35 CHS-PEG5-6-GalNAc 13 C NMR, 1 H NMR and synthetic routes.

[0048] Figure 36 Unmod-LP@DOX and LP-PEG were intravenously injected at different time points. n -6-GalNAc@DOX (dosage 5 mL·kg -1 (A) DOX concentration in blood and (B) DOX accumulation in liver of mice after 4-8 h of 4-8 h. Data are shown as mean ± SD (n = 3). **: P <0.01. DETAILED DESCRIPTION

[0049] In order to better understand the present invention, the present invention is further described below in conjunction with specific serial numbers, wherein the terms used in the serial numbers are for describing specific embodiments and do not constitute a limitation on the scope of protection of the present invention.

[0050] 1. Experimental Materials Some of the materials used in this invention come from: RM IM (derived from Rhizomucor miehei, immobilized on macroporous anion exchange resin) was purchased from Novozymes; PS IM (derived from Burkholderia cepacia, immobilized on diatomaceous earth) and lipase AYS (derived from Candida rugosa) were purchased from Amano; molecular sieve 4Å was purchased from Aladdin Biochemical Technology Co., Ltd.

[0051] 1,1′-Dioctyl-3,3,3′,3′-tetramethylindoltricarbocyanine iodide (DiR) was purchased from Sigma-Aldrich Shanghai Trading Co., Ltd. (Shanghai, China).

[0052] Candida antarctica lipase B (Novozyme 435, 10000 U∙g⁻¹) and Mucor thermophilus lipase (TL IM, 330 U∙g⁻¹) were purchased from Novozymes Biotechnology Co., Ltd. (Beijing, China).

[0053] Cholesterol (CHS), mass fraction > 98.5%, production batch number A90719, Japan Seika Co., Ltd.; hydrogenated soy phosphatide (HSPC), 1-palmitoyl-2-stearoyl phosphatidylcholine mass fraction > 98.0%, production batch number B40932, Japan Kewpie Co., Ltd. Acetylgalactosamine (GalNAc), lactol (Lac), doxorubicin (DOX), and molecular sieves (pore size 4 Å) were purchased from Aladdin Industries (Shanghai, China).

[0054] Track-etched polycarbonate membranes were purchased from Whatman (Clifton, NJ, USA).

[0055] Dialysis membranes with a molecular weight cutoff of 10 kDa were purchased from Spectrum Laboratories (California, USA).

[0056] The mobile phase used in high performance liquid chromatography (HPLC) analysis was HPLC / spectroscopy grade reagents.

[0057] Other reagents were of analytical grade and used directly without further purification.

[0058] 2 Experimental methods 2.1 Synthesis of liver-targeting carbohydrate ligands Step 1: PEG n -divinyl ester synthesis. Weigh COOH-PEG n 0.1 mol of -COOH, 2 mol of vinylacetate, 0.005 mol of palladium acetate, and 0.0075 mol of 1,10-phenanthroline were added to a 1000 mL three-necked flask. The flask was heated in a 60°C water bath with stirring for 48 hours. After the reaction, the organic layer was washed with aqueous sodium carbonate and dried over magnesium sulfate. The vinyl acetate was removed by rotary evaporation under reduced pressure, followed by distillation at 30 Pa. The 140°C fraction was collected to yield the pure product as a pale yellow oil.

[0059] step2: CHS-PEG n -vinyl ester synthesis. Weigh 0.03 mol PEGn 1-divinyl ester and 0.01 mol of cholesterol were added to 10 ml of anhydrous tetrahydrofuran, placed in a stoppered Erlenmeyer flask, and shaken on a thermostatic shaker at 45°C for 30 minutes. Then, 500 mg of Novozyme 435 was added and allowed to react for 24 hours. After the reaction, Novozyme 435 was removed by filtration, and the filtrate was distilled under reduced pressure to obtain a viscous liquid. This was then ultrasonically dissolved in an appropriate amount of methanol, allowed to stand at -20°C for 24 hours to crystallize, and vacuum filtered at low temperature to obtain a white powder.

[0060] Step 3: CHS-PEG n -6-GalNAc synthesis. Weigh 0.4 mmol of GalNAc, CHS-PEG n 0.8 mmol of 2-vinyl ester, 100 mg of TL IM, and 50 mL of anhydrous acetone were placed in a stoppered Erlenmeyer flask and allowed to react in an air bath shaker at 45°C and 250 rpm for 24 h. After the reaction, TL IM was removed by filtration, and the filtrate was distilled under reduced pressure to obtain a white solid. This solid was then dissolved in an appropriate amount of diethyl ether and recrystallized at -20°C. The crystals were washed with diethyl ether and dried to obtain a white solid powder.

[0061] step4: CHS-PEG n -6-Lac synthesis. Weigh 0.4 mmol of Lac, CHS-PEG n -vinyl ester 0.8mmol, Novozyme 435 100mg, anhydrous pyridine 20ml, anhydrous acetone 40ml, placed in a stoppered conical flask, placed in an air bath shaker at 45℃, 250r / min for 24h. After the reaction, Novozyme 435 was removed by filtration, and the filtrate was distilled under reduced pressure to obtain a white solid, which was then dissolved in an appropriate amount of ether and recrystallized at -20℃. The crystals were washed with ether and dried to obtain a white solid powder. Figure 1 shown.

[0062] 2.2 Liposome preparation and characterization 2.2.1 Liposome preparation Doxorubicin (DOX) liposomes were prepared using an ammonium sulfate gradient method. The compositions of different liposome formulations are shown in Table 1. Each component was dissolved in chloroform and placed in a rotary evaporator, heated in a 55°C water bath to evaporate the chloroform until a thin lipid film formed on the inner wall of the round-bottom flask. The film was vacuum dried for 1 h. Next, 10 mL of ammonium sulfate solution (300 mmol·L⁻¹) was added to hydrate the lipid film, which was then incubated at 55°C for 1 h. Blank liposomes were obtained by extrusion through 0.2, 0.1, and 0.05 μm track-etched polycarbonate membranes ten times using a high-pressure extruder (ATS Engineering Co., Ltd., Suzhou, China) in a 65°C water bath under inert nitrogen. The resulting liposomes were passed through a dextran G-50 medium to remove any unencapsulated ammonium sulfate. Then, 200 mL of DOX aqueous solution (10 mg·mL⁻¹) was added and incubated with the blank liposomes at 65°C for 1 h to obtain DOX-loaded liposomes.

[0063] DiR-labeled liposomes were prepared using a thin film dispersion method. The added DiR accounted for 1 mol% of the total liposome volume. The individual liposome components (see Table 1) and DiR were dissolved in chloroform and placed on a rotary evaporator in a 55°C water bath to evaporate the chloroform until a thin lipid film formed on the inner wall of the evaporator. The film was vacuum dried for 1 hour. Next, 10 mL of distilled water was added, followed by incubation at 55°C for 1 hour. DiR-labeled liposomes were extruded through 0.2, 0.1, and 0.05 μm track-etched polycarbonate membranes using a high-pressure extruder at 65°C in a water bath under inert nitrogen atmosphere, performing 10 extrusions each.

[0064]

[0065]

[0066] The data shown here represent the mean ± standard deviation (sample size n=3) 2.2.2 Liposome particle size and zeta potential measurement In the present invention, 100 μL of liposome solution was diluted into 2 mL of normal saline and mixed thoroughly. The particle size distribution, zeta potential, and polydispersity index (PDI) of the liposome particles were measured using a laser scattering particle size analyzer (Malvern Zetasizer Nano ZS90, Worcestershire, UK).

[0067] 2.2.3 Morphological characterization of liposomes The overall morphology of the liposomes was observed using a negative staining transmission electron microscope (TEM, H-7650, Hitachi, Tokyo, Japan). The sample was aspirated and dropwise applied to a 200-mesh carbon-coated copper grid (Electron Microscopy Sciences, Hatfield, Pennsylvania, USA). After 2 minutes, excess liquid was removed from the edge of the grid with filter paper. Phosphotungstic acid (3%, pH 7.0) was added dropwise to the grid. After 2 minutes, excess dye was removed from the edge of the grid with filter paper. Pure water was added dropwise to the grid. Excess water was removed from the edge of the grid with filter paper. All liquid was removed by air drying before TEM observation.

[0068] 2.2.4 Liposome encapsulation efficiency measurement The drug loading of doxorubicin (DOX) was determined by high-performance liquid chromatography (HPLC). An XBridge peptideBEH C18 column (250 mm × 4.6 mm, 5 μm, Waters Corporation, Milford, MA, USA) was used as the column. The mobile phase consisted of methanol, acetonitrile, and phosphate buffer, pH 3.0, in a 20:30:50 ratio. The HPLC flow rate was 1.0 mL min⁻¹. The excitation and emission wavelengths of the fluorescence detector were 470 nm and 580 nm, respectively. Daunorubicin was used as the internal standard. The sample injection volume was 10 μL. Peak areas were measured, and the DOX loading in the sample was calculated using the internal standard method.

[0069] The encapsulation efficiency of DOX in liposomes was determined by dialysis. The encapsulation efficiency was calculated using the following formula: EE = (Wdialysis / Wtotal) × 100% (Formula 1) Wherein, EE is the encapsulation efficiency of DOX, Wdialysis is the total mass of DOX in the dialysis bag after 24 h of dialysis, and Wtotal is the total mass of DOX in the initial sample before dialysis.

[0070] 2.3 Uptake of liposomes modified with galactose ligands containing different PEG units by liver cancer cells The cellular uptake of DiR-labeled liposomes modified with galactose ligands containing different PEG units mediated by ASGPR was evaluated in HepG2 cells, which are known to overexpress ASGPR. HepG2 cells were plated at 1×10 5 Cells were seeded at a density of 100 cells / well in a 24-well plate, 1 mL of growth medium was added to each well, and cultured overnight at 37°C to allow them to adhere. Afterwards, the growth medium was discarded and the cells were incubated with 0.099 mM (0.2 mg × mL −1Ligand-modified DiR-labeled liposomes were incubated at 37°C for 1 hour. For inhibition experiments, HepG2 cells were pre-incubated with an excess of 20 mM acetylgalactosamine (GalNAc) for 30 minutes. Subsequently, ligand-modified DiR-labeled liposomes were added to the HepG2 cells. After 1 hour, the solution was removed, and the cells were washed three times with cold PBS. The cells were then trypsinized and harvested to obtain a cell suspension for analysis by flow cytometry (Beckman Coulter, Inc., Pennsylvania, USA). Data from 10,000 gated events were collected and analyzed using FlowJo software (version 10.6.2).

[0071] 2.4 Experimental uptake of liposomes modified with different sugar ligands by liver cancer cells The cellular uptake of ASGPR-mediated DiR-labeled liposomes modified with different sugar ligands was evaluated in HepG2 cells, which are known to overexpress ASGPR. HepG2 cells were plated at 1×10 5 Cells were seeded at a density of 100 cells / well in a 24-well plate, 1 mL of growth medium was added to each well, and cultured at 37°C overnight to allow them to adhere. Afterwards, the growth medium was discarded, and the cells were incubated with ligand-modified DiR-labeled liposomes at a concentration of 0.099 mM (0.2 mg×mL−1 DiR) at 37°C for 1 h. Subsequently, ligand-modified DiR-labeled liposomes were added to HepG2 cells. After 1 h, the solution was removed and the cells were washed three times with ice-cold PBS. The cells were then trypsinized and collected to obtain a cell suspension, which was analyzed by flow cytometry (BeckmanCoulter, Inc., Pennsylvania, USA). Data of 10,000 gated events were collected and analyzed using FlowJo software (version 10.6.2). 2.5 Mouse liver uptake and plasma clearance experiment Male Balb / c mice (weighing 18-22 g) were provided by the Guangdong Medical Laboratory Animal Center and acclimated for one week under standard experimental conditions (temperature 25 ± 2°C; humidity 60 ± 5%; 12-h dark / light cycle) prior to the experiment. All animals were specific pathogen-free (SPF) and had free access to food and water. Experimental procedures adhered to the Regulations on Laboratory Animal Care, and the study protocol was approved by the Medical Ethics Committee of Jiaying University (approval number: JYDWLL2024-09).

[0072] Male Balb / c mice fasted for 12 hours were divided into five groups: Group I (Unmod-LP@DOX), Group II (LP-PEG2-6-GalNAc@DOX), Group III (LP-PEG3-6-GalNAc@DOX), Group IV (LP-PEG4-6-GalNAc@DOX), and Group V (LP-PEG5-6-GalNAc@DOX). Each group received a single injection of 5 mL kg⁻¹ (based on body weight) of liposomal DOX formulation (phospholipid dosage 20 mg kg⁻¹) via the tail vein. Three mice were included in each group at each time point. Blood samples were collected from the orbital venous plexus at various time points (5, 15, 30, 45, and 60 minutes). The mice were then euthanized by cervical dislocation. After perfusion of the left ventricle with normal saline to remove residual blood, the liver, lung, spleen, kidney, and heart tissues were immediately removed, rinsed with normal saline, blotted dry with filter paper, and weighed. Whole blood samples were centrifuged at 500 × g for 15 min to separate plasma. Plasma and tissue samples were stored at -20°C until testing.

[0073] DOX concentrations in plasma and tissues were determined by HPLC: 0.8 mL of acidic ethanol (0.3 M HCl) and 0.1 mL of epirubicin (40 μg mL⁻¹, internal standard) were added to 0.1 mL of plasma. Tissue samples were homogenized in saline at a 1:2 (mass / volume ratio), and 0.2 mL of the homogenate was processed in the same manner. The mixed sample was incubated in the dark at 4°C for 30 min, centrifuged at 10,000 rpm for 5 min, and 10 μL of the supernatant was injected for analysis. 2.5 Molecular Dynamics Simulation 2.5.1 System Setup In order to simulate and understand the dynamic conformation of the ligand in the bilayer membrane, we used cholesterol-galactose ligands (CHS-PEG n -6-GalNAc, n=2, 3, 4, 5), was incorporated into a bilayer membrane composed of distearoylphosphatidylcholine (DSPC) and CHS in a 2:1 ratio (33% cholesterol) (see Table 2-1 for system composition). As a starting configuration, CHS-PEG n -6-GalNAc was placed in the center of the box, CHS-PEG n The cholesterol residues in the -6-GalNAc are aligned with the coordinates of the cholesterol molecules in the bilayer membrane along the z-axis, ensuring that the GalNAc residues are fully exposed to the solvent in the initial state.

[0074] In order to simulate and understand the dynamic clustering of multiple GalNAc residues on the bilayer membrane surface, we used three CHS-PEG nThe 6-GalNAc molecules were incorporated into the bilayer membrane composed of DSPC and CHS in a ratio of 2:1 with an initial spacing of 25 Å (close to the average value of the optimal binding site of ASGPR) (see Table 2). 2.5.2 Molecular model parameterization and molecular dynamics simulation parameters The specific description is as follows: lipid molecules and ligand molecules are generated based on the CHARMM36 lipid force field and CGenFF. Each system uses the TIP3P water model and is neutralized by adding 150 mM NaCl. All simulations are performed using GROMACS2021.2, and the input files are generated by CHARMM-GUI.

[0075] The system was equilibrated using the default six-step equilibration protocol of the CHARMM-GUI membrane builder. NVT (number, volume, and temperature constant) dynamics were first applied with a time step of 1 fs for 250 ps. Subsequently, the NPT (number, pressure, and temperature constant) ensemble was applied with time steps of 1 fs for 125 ps and 2 fs for 1.5 ns. During equilibration, positional and dihedral restraints were applied to the lipid and water molecules, and their force constants were gradually reduced. Finally, a production simulation was performed on all systems with a time step of 2 fs. The LINCS algorithm was used to maintain the covalent bond lengths involving hydrogen atoms. Van der Waals interactions were cut off at 12 Å, and a force transfer function between 10 and 12 Å was used. Electrostatic interactions were calculated using the particle-mesh Ewald method. Temperature (310 K) and pressure (1 bar) were controlled using the Nosé–Hoover and Parrinello–Rahman methods, respectively. The membrane and water were coupled to independent heat baths, and the system was thermalized with a time constant of 1.0 ps. The pressure was maintained semi-isotropic with a time constant of 5.0 ps.

[0076]

[0077] DSPC stands for Dipalmitoylphosphatidylcholine CHS stands for Cholesterol Sulfate Water refers to the total number of water molecules in the simulation system 3 Results and Discussion Cholesterol, one of the most important components of cell membranes, is widely available, inexpensive, safe, and non-toxic to humans. It is commonly used to graft various functional groups to modify liposomes. Therefore, the present invention set out to develop an alternative cholesterol derivatization method involving the conjugation of active ingredients (drugs or ligands) to cholesterol residues via a polyethylene glycol (PEG) spacer. Specifically, the present invention describes the synthesis of cholesterol-anchored galactose derivatives using medium-length PEG chains as spacers. PEG chains of this size have been reported to enhance the plasma circulation time of attached particles, potentially by reducing adsorption of opsonized plasma proteins and, consequently, reducing delivery to cells by the mononuclear phagocyte system. Similar to the design of immunoliposomes carrying antibodies distal to the bilayer-anchored PEG chains, the present invention hypothesizes that this structure would combine the "macrophage evasion" properties of PEG chains with the specific targeting properties of galactose residues.

[0078] Since galactose residues are relatively small and PEG chains are highly flexible, the present invention speculates that after exceeding a certain PEG chain length, the formation of random coils may hinder the formation of galactose clusters required for optimal interaction with ASGPR. Therefore, the present invention selects a shorter PEG chain, that is, a spacer length that meets the required separation of galactose groups from the lipid-water interface, to ensure that the PEG chain has a certain rigidity to eliminate the coil effect. The experimental results are shown in Figure 2. Figure 2 As shown, the affinity of the galactose ligand for ASGPR initially increases and then decreases with the increase in the number of PEG units in the linker. The liposome containing three PEG units (DiR-LP-PEG3-6-GalNAc) exhibits the strongest binding, with an uptake rate 5.28 times that of conventional liposomes (DiR-Unmod-LP) and 1.97 times that of DiR-LP-PEG2-6-GalNAc. The increasing length of the galactose ligand linker with the addition of PEG units reduces steric hindrance to ASGPR binding, leading to enhanced binding. As the linker length increases, the increased flexibility of the PEG linker may result in a less favorable conformation for ASGPR binding on the liposome surface, leading to a weakening of the liposome-ASGPR binding affinity. The precise mechanism will be further explored in subsequent experiments.

[0079] To confirm whether the high uptake rate of HepG2 cells for galactose ligand-modified liposomes is due to active endocytosis mediated by ASGPR, GalNAc was added to the cell culture medium in advance to competitively inhibit ASGPR's recognition of galactose ligands on the liposome surface. The experimental results are shown in Figure 2. Figure 3After the addition of GalNAc, the mean fluorescence intensity of the galactose ligand-modified liposomes decreased significantly, indicating that the high uptake rate of galactose ligand-modified liposomes by HepG2 cells may be due to the recognition of the galactose ligand-modified liposomes by the cell membrane surface ASGPR, which then activates the active endocytosis process mediated by clathrin.

[0080]

[0081] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0082] Subsequently, the present invention studied the effect of different glycosyl-terminated cholesterol conjugates containing 3 PEG units on the affinity of ASGPR. Figure 4 As shown, there was no significant difference in the uptake efficiency of HepG2 cells for liposomes modified with ligands with Lac (DiR-LP-PEG3-6-Lac) or GalNAc (DiR-LP-PEG3-6-GalNAc) at the end, but it was significantly higher than DiR-LP-PEG2-6-GalNAc, indicating that there was no significant difference in the affinity of GalNAc and Lac for ASGPR, but the amount of PEG contained in the ligand structure had a significant effect on the affinity of ASGPR.

[0083]

[0084] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0085] To evaluate the CHS-PEG with different PEG chain lengths n -6-GalNAc modified liposomes in mice blood clearance and liver distribution characteristics, doxorubicin loaded ordinary liposomes (Unmod-LP@DOX) and CHS-PEG n -6-GalNAc modified liposomes (LP-PEG n -6-GalNAc@DOX, n=2, 3, 4, 5) were injected into mice via the tail vein, and the DOX concentrations in various organs were analyzed at different time points (see Figure 35 Unmod-LP@DOX has a lower affinity for the reticuloendothelial system, showing lower liver uptake (60 min after injection, the uptake was 6.42 ± 0.45% of the dose) and higher serum residual ratio (72.95 ± 4.83%). n-6-GalNAc@DOX exhibited rapid blood clearance, with blood concentrations decreasing to 14.99 ± 2.05% (n = 2), 11.11 ± 2.93% (n = 3), 16.43 ± 1.31% (n = 4), and 16.77 ± 0.99% (n = 5) of the initial dose within 60 min. This improvement in serum clearance was primarily attributed to enhanced hepatic uptake, which increased to 48.39 ± 1.61% (n = 2), 68.77 ± 4.00% (n = 3), 53.00 ± 4.44% (n = 4), and 50.07 ± 2.98% (n = 5) of the initial dose within 60 min, respectively.

[0086] In vivo experimental data showed that the liver uptake rate increased first and then decreased with the length of the PEG chain, among which LP-PEG3-6-GalNAc@DOX had the highest liver uptake efficiency ( P <0.01), which is consistent with the in vitro hepatocyte uptake trend. This consistency across models suggests that PEG chain length may regulate ASGPR-mediated active targeting through a unified dual mechanism (GalNAc exposure and sugar cluster conformation).

[0087] To further elucidate the solvent exposure of the GalNAc groups in the four ligands, the present invention used Visual Molecular Dynamics (VMD) software to calculate the solvent accessible surface area (SASA) of the GalNAc groups at the lipid-water interface for each ligand. As shown in Table 5, during a 30-nanosecond simulation, the relative average SASA of the GalNAc groups in the four ligands ranked in ascending order: CHS-PEG2-6-GalNAc (49.92%) > CHS-PEG3-6-GalNAc (61.70%) > CHS-PEG4-6-GalNAc (73.38%) > CHS-PEG5-6-GalNAc (86.08%). Higher SASA values ​​indicate greater solvent exposure of the GalNAc group and less steric hindrance to binding to ASGPR. However, the liver-targeting efficiency of the four ligand-modified liposomes in vitro and in vivo showed different trends relative to SASA size, with the order being: LP-PEG3-6-GalNA > LP-PEG4-6-GalNA > LP-PEG5-6-GalNA > LP-PEG2-6-GalNA. Based on this, the present invention speculates that the solvent exposure level of the GalNAc group is one of the important factors affecting the affinity of the ligand for ASGPR, while the spatial geometric arrangement of the GalNAc group at the lipid-water interface may be another important factor affecting affinity.

[0088]

[0089] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0090] Since each ASGPR has three sugar binding sites that are triangularly distributed in space with a distance of 15-25 Å between the binding sites, multi-antennary GalNAc ligands, especially tri-antennary GalNAc (i.e., GalNAc3), significantly improve the ability to target and bind to ASGPR compared to mono-antennary GalNAc in small to medium molecular weight delivery systems. n -6-GalNAc is a single antenna ligand, but when it is distributed on the surface of liposomes, which are larger particles, when the length of the ligand linker, the hydrophilic-hydrophobic balance of the linker, and the distribution density in the bilayer are appropriate, a similar organizational structure to GalNAc3 may exist. To verify this hypothesis, the present invention combined three CHS-PEG n -6-GalNAc molecules are arranged in an equilateral triangle with a side length of 25 Å and placed in the bilayer membrane (e.g. Figure 3 The membrane dynamics simulation was performed for 60 ns, the spatial distance between the three sugar groups was measured every 0.1 ns, and the frequency of the three sugar groups spatially arranged into an optimal triangle (each side length = 19 ~32 Å) was counted.

[0091] As shown in Table 6, the shorter CHS-PEG2-6-GalNAc linker reduces the flexibility of the PEG chain, resulting in the highest frequency of the optimal triangular conformation in the spatial distribution of the sugar groups. However, the sugar group relative to SASA of CHS-PEG2-6-GalNAc is the lowest. At this point, the sugar group relative to SASA may dominate the affinity for ASGPR, resulting in the lowest affinity for CHS-PEG2-6-GalNAc. As the PEG chain lengthens, the sugar group relative to SASA gradually increases, but the PEG chain flexibility also gradually increases, resulting in a gradually decreasing frequency of the optimal triangular conformation. At this point, the geometric shape formed by the three sugar groups in space dominates the influence on ASGPR. Therefore, the affinity with ASGPR is in the following order: CHS-PEG3-6-GalNAc > CHS-PEG4-6-GalNAc > CHS-PEG5-6-GalNAc.

[0092]

[0093] * Frequency of occurrence of optimal sugar cluster (%) = number of occurrences of optimal triangle composed of 3 sugar groups / total number of statistics (600) * 100 In summary, this paper describes a method for anchoring galactose derivatives with cholesterol using PEG chains as steric groups and assessing the effects of varying PEG chain length and glycosyl type. The present study demonstrates that this liposomal formulation can target liver cancer cells with high specificity. These findings provide new insights into the development of more precise and effective liposomal drug delivery platforms that can achieve higher efficiency and selectivity in specific biological environments, thereby enhancing the efficacy of drug treatments.

[0094] The Chinese-English translation and abbreviated descriptions of the present invention are shown in Table 7 below.

[0095]

[0096]

[0097] PEG3-divinyl ester: 1 H NMR (500 MHz, Pyr) δ 7.44 (dd, J = 14.0, 6.3 Hz, 2H, 2 × C H =CH2),4.93 (dd, J = 13.9, 1.7 Hz, 2H, 2 × CH=CH H ), 4.58 (dd, J = 6.3, 1.7 Hz, 2H,2 × CH=CH H ), 4.35 (s, 4H, 2 × CH2―C=O), 3.79 – 3.76 (m, 4H, 2 × O―C H2 ―C H2 ―O), 3.67 – 3.65 (m, 4H, 2 × O―C H2 ―C H2 ―O). 13 C NMR (126 MHz, Pyr) δ 168.56 (C=O), 141.56 ( C H=CH2), 98.69 (CH= C H2), 71.59 (CH2), 71.23 (CH2), 68.75 (CH2). PEG4-divinyl ester: 1 H NMR (500 MHz, Pyr) δ 7.44 (dd, J= 14.0, 6.3 Hz, 2H, 2 × C H =CH2),4.93 (dd, J = 13.9, 1.7 Hz, 2H, 2 × CH=CH H ), 4.58 (dd, J = 6.3, 1.7 Hz, 2H,2 × CH=CH H ), 4.35 (s, 4H, 2 × CH2―C=O), 3.78 (m, 4H, 2 × O―C H2 ―C H2 ―O),3.66 (m, 4H, 2 × O―C H2 ―C H2 ―O), 3.61 (s, 4H, 2 × O―C H2 ―C H2 ―O). 13 C NMR (126 MHz, Pyr) δ 168.55 (C=O), 141.52 ( C H=CH2), 98.67 (CH= C H2), 71.57 (CH2), 71.22 (CH2), 71.11(CH2), 68.83(CH2), 68.71 (CH2). PEG5-divinyl ester: 1 H NMR (500 MHz, Pyr) δ 7.44 (dd, J = 14.0, 6.3 Hz, 2H, 2 × C H =CH2),4.92 (dd, J = 13.9, 1.7 Hz, 2H, 2 × CH=CH H ), 4.58 (dd, J = 6.3, 1.7 Hz, 2H,2 × CH=CH H ), 4.35 (s, 4H, 2 × CH2―C=O), 3.78 (m, 4H, 2 × O―C H2 ―C H2 ―O),3.66 (m, 4H, 2 × O―C H2 ―C H2 ―O), 3.61 (s, 8H, 2 × O―C H2 ―CH2 ―O). 13 C NMR (126 MHz, Pyr) δ 168.55 (C=O), 141.53 ( C H=CH2), 98.68 (CH= C H2), 71.59 (CH2), 71.23 (CH2), 71.16(CH2), 71.15(CH2), 68.72 (CH2). CHS-PEG3-vinyl ester: 1 H NMR (500 MHz, Pyr) δ 7.46 (H-36, dd, J = 13.9, 6.3 Hz, 1H), 5.39(H-9, m, 1H), 4.95 (H-37a, dd, J = 13.9, 1.6 Hz, 1H), 4.87 (H-37b, m, 1H),4.60 (H-2, dd, J = 6.3, 1.7 Hz, 1H), 4.38 (H-29, s, 2H), 4.36 (H-34, s , 2H),3.87 (H-30, m, 2H), 3.82 (H-33, m, 2H), 3.73 (H-31,32, m, 4H), 2.47 (H-4,ddd, J = 17.2, 11.6, 6.6 Hz, 2H), 2.08 – 1.02 (m, 34H), 0.92 (H-24, 25, m,6H), 0.68 (H-26, s, 3H). 13C NMR (126 MHz, Pyr) δ 170.58 (C-28), 168.56(C-35), 141.58(C-36),140.22(C-6), 123.40(C-9), 98.69(C-37), 74.90(C-2), 71.66(C-30), 71.51(C-31),71.29(C-32), 71.27(C-33), 69.47(C-29), 68.80(C-34), 57.19(C-14), 56.78(C-15),50.63(C-7), 42.91(C-21), 40.35(C-12), 40.17(C-22), 38.85(C-4), 37.57(C-3),37.19(C-5), 36.92(C-18), 36.47(C-20), 32.55(C-10), 32.43(C-8), 28.93(C-16),28.68(C-23), 28.48(C-1), 24.92(C-17), 24.59(C-21), 23.38(C-24), 23.13(C-25),21.68(C-11), 19.75(C-27), 19.38(C-19), 12.42(C-26). CHS-PEG4-vinyl ester: 1 H NMR (500 MHz, Pyr) δ 7.46 (H-38, dd, J = 13.9, 6.3 Hz, 1H), 5.38(H-9, m, 1H), 4.94 (H-39a, dd, J = 14.0, 1.6 Hz, 1H), 4.87 (H-39b, m, 1H),4.59 (H-2, dd, J = 6.3, 1.7 Hz, 1H), 4.36 (H-29, H-36, d, J = 7.8 Hz, 4H),3.86 (H-30, m, 2H), 3.80 (H-35, m, 2H), 3.72 (H-31,34, m, 4H), 3.68(H-32, m,2H), 3.65(H-33, m, 2H), 2.45 (H-4, m, 2H), 2.08 – 1.02 (m, 26H), 0.98(H-19,H-27, d, J= 7.9 Hz, 6H), 0.90 (H-24, 25, dd, J = 6.6, 1.3 Hz, 6H), 0.67 (H-26, s, 3H). 13 C NMR (126 MHz, Pyr) δ 170.58 (C-28), 168.56(C-37), 141.58(C-38),140.22(C-6), 123.40(C-9), 98.68(C-39), 74.88(C-2), 71.63(C-30), 71.52(C-31),71.28(C-32, C-33), 71.20(C-34), 71.19(C-35), 69.45(C-29), 68.77(C-36), 57.19(C-14), 56.78(C-15), 50.60(C-7), 42.89(C-21), 40.33(C-12), 40.15(C-22), 38.83(C-4), 37.55(C-3), 37.17(C-5), 36.90(C-18), 36.46(C-20), 32.55(C-10), 32.41(C-8), 28.91(C-16), 28.66(C-23), 28.46(C-1), 24.90(C-17), 24.57(C-21), 23.37(C-24), 23.11(C-25), 21.67(C-11), 19.74(C-27), 19.36(C-19), 12.40(C-26). CHS-PEG5-vinyl ester: 1 H NMR (500 MHz, Pyr) δ 7.46 (H-40, dd, J = 13.9, 6.3 Hz, 1H), 5.39(H-9, m, 1H), 4.94 (H-41a, dd, J = 14.0, 1.6 Hz, 1H), 4.87 (H-41b, m, 1H),4.60 (H-2, dd, J = 6.3, 1.7 Hz, 1H), 4.37 (H-29, H-38, d, J= 7.8 Hz, 4H),3.87 (H-30, m, 2H), 3.80 (H-37, m, 2H), 3.74 (H-31, m, 2H), 3.67(H-32, H-33,H-34, H-35, H-36, m, 10H) , 2.45 (H-4, m, 2H), 2.08 – 1.02 (m, 26H), 0.98 (H-19, H-27, d, J = 7.9 Hz, 6H), 0.90 (H-24, 25, dd, J = 6.6, 1.3 Hz, 6H), 0.67(H-26, s, 3H). 13 C NMR (126 MHz, Pyr) δ 170.58 (C-28), 168.57(C-39), 141.58(C-40),140.22(C-6), 123.40(C-9), 98.69(C-41), 74.89(C-2), 71.64(C-30), 71.54(C-37),71.29(C-31, C-36), 71.25(C-32, C-35), 71.22(C-33, C-34), 69.45(C-29), 68.77(C-38), 57.19(C-14), 56.78(C-15), 50.60(C-7), 42.89(C-21), 40.33(C-12), 40.15(C-22), 38.83(C-4), 37.55(C-3), 37.17(C-5), 36.90(C-18), 36.46(C-20), 32.55(C-10), 32.41(C-8), 28.91(C-16), 28.66(C-23), 28.46(C-1), 24.90(C-17), 24.57(C-21), 23.37(C-24), 23.11(C-25), 21.66(C-11), 19.74(C-27), 19.35(C-19),12.39(C-26). CHS-PEG3-6-GalNAc: ESI:[M+NH4]⁺: Observed m / z = 812.0 (Theoretical m / z = 794.04 + 18.04= 812.08). [M+Na]⁺: Observed m / z = 816.6 (Theoretical m / z = 794.04 + 22.99 =817.03). 1 H NMR (500 MHz, Pyr) δ 5.94 (H-1′, d, J = 3.2 Hz, 1H), 5.38 (H-9, d, J = 5.2 Hz, 1H), 5.31 – 5.24 (H-5′, m, 1H), 4.95 – 4.79 (m, 4H), 4.59 (H-3′,dd, J = 10.9, 3.2 Hz, 1H), 4.42 (H-4′, m , 1H), 4.36 (H-29, m, 2H), 4.26 (H-34, m, 2H), 3.85 (H-30, m, 2H), 3.77 (H-33, dd, J = 6.0, 3.3 Hz, 2H), 3.72(H-31, m, 2H), 3.67 (H-32, dd, J = 5.6, 3.8 Hz, 2H), 2.50 – 2.38 (H-4, m,2H), 2.08 (H-8′, m, 3H), 2.09 – 0.96 (m, 31H), 0.98 (H-19, H-27, d, J = 6.3Hz, 6H), 0.90 (H-24, H-25, dd, J = 6.6, 1.3 Hz, 6H), 0.67 (H-26, s, 3H) 13C NMR (126 MHz, Pyr) δ 171.35(C-7′), 171.14(C-35), 170.62(C-28),140.20(C-6), 123.39 (C-9), 93.26 (C-1′), 74.90 (C-2), 71.52(C-33), 71.25(C-30), 71.18(C-31,32), 70.76 (C-4′), 69.84(C-3′), 69.44(C-5′), 69.40 (C-34),69.14 (C-29), 66.01 (C-6′), 57.15(C-14), 56.73 (C-15), 52.51 (C-2′), 50.58(C-7), 42.87(C-21), 40.30(C-12), 40.13(C-22), 38.82 (C-4), 37.53(C-3), 37.16(C-5), 36.88(C-20), 36.43(C-18), 32.52 (C-10), 32.39 (C-8), 28.89 (C-16),28.64 (C-23), 28.45 (C-1), 24.88 (C-17), 24.54 (C-21), 23.65(C-8′), 23.34(C-25), 23.09(C-24), 21.65(C-11), 19.72(C-27), 19.34(C-19), 12.38(C-26). CHS-PEG4-6-GalNAc: ESI:[M+NH4]⁺: Observed m / z = 856.0 (Theoretical m / z = 838.09 + 18.04= 856.13). [M+Na]⁺: Observed m / z = 860.9 (Theoretical m / z = 838.09 + 22.99 =861.08). [M+K]⁺: Observed m / z = 876.9 (Theoretical m / z = 838.09 + 39.10 =877.19). 1 H NMR (500 MHz, Pyr) δ 5.95 (H-1′, s,1H), 5.39 (H-9, m, 1H), 5.29(H-5′, s, 1H), 4.89 (m, 4H), 4.59 (H-3′, d, J= 11Hz, 1H), 4.42 (H-4′, d, J =22.3 Hz, 1H), 4.36 (H-29, s, 2H), 4.27 (H-36, m, 2H), 3.85 (H-30, m, 2H),3.77 (H-35, m, 2H), 3.72 (H-31, m, 2H), 3.65 (H-32,33,34, m, 6H), 2.46 (H-4,m, 2H), 2.08 (H-8′, m, 3H), 2.09 – 0.96 (m, 26H), 0.98 (H-19, H-27, d, J =7.4 Hz, 6H), 0.90 (H-24, H-25, dd, J = 6.6, 1.3 Hz, 6H), 0.67 (H-26, s, 3H) 13 C NMR (126 MHz, Pyr) δ 171.35(C-7′), 171.14(C-37), 170.62(C-28),140.20(C-6), 123.39 (C-9), 93.26 (C-1′), 74.90 (C-2), 71.52(C-30, C-31),71.25(C-32), 71.18(C-33, C-34, C-35), 70.76 (C-4′), 69.84(C-3′), 69.44(C-29),69.40 (C-5′), 69.14 (C-36), 66.01 (C-6′), 57.15(C-14), 56.73 (C-15), 52.51(C-2′), 50.58 (C-7), 42.87(C-21), 40.30(C-12), 40.13(C-22), 38.82 (C-4),37.53(C-3), 37.16 (C-5), 36.88(C-20), 36.43(C-18), 32.52 (C-10), 32.39 (C-8),28.89 (C-16), 28.64 (C-23), 28.45 (C-1), 24.88 (C-17), 24.54 (C-21), 23.65(C-8′), 23.34(C-25), 23.09(C-24), 21.65(C-11), 19.72(C-27), 19.34(C-19), 12.38(C-26). CHS-PEG5-6-GalNAc: 1 H NMR (500 MHz, Pyr) δ 5.95 (H-1′, t, J = 3.1 Hz, 1H), 5.39 (H-9,dd, J = 2.9, 2.3 Hz, 1H), 5.29 (H-5′, m, 1H), 4.88 (m, 4H), 4.60 (H-3′, dd, J = 10.9, 3.0 Hz, 1H), 4.43 (H-4′, m, 1H), 4.36 (H-29, s, 2H), 4.26 (H-38, m,2H), 3.87 (H-30, m, 2H), 3.74 (H-37, H-31, m, 4H), 3.65 (H-32, 33, 34, 35,36, m, 10H), 2.45 (H-4, m, 2H), 2.08 (H-8′, m, 3H), 2.09 – 0.96 (m, 26H),0.98 (H-19, H-27, d, J = 8.1 Hz, 6H), 0.90 (H-24, H-25, m, 6H), 0.67 (H-26,s, 3H) CHS-PEG3-6-Lac: 13C NMR (126 MHz, Pyr) δ 171.34(C-7′), 171.14(C-39), 170.61(C-28),140.19(C-6), 123.39 (C-9), 93.26 (C-1′), 74.90 (C-2), 71.53(C-30), 71.51 (C-37), 71.22(C-31), 71.21(C-36), 71.19(C-32, C-33, C-34, C-35), 70.76 (C-4′),69.84(C-3′), 69.44(C-29), 69.40 (C-5′), 69.14 (C-38), 66.01 (C-6′), 57.15(C-14), 56.73 (C-15), 52.51 (C-2′), 50.58 (C-7), 42.87(C-21), 40.30(C-12), 40.13(C-22), 38.82 (C-4), 37.53(C-3), 37.16 (C-5), 36.88(C-20), 36.43(C-18), 32.52(C-10), 32.39 (C-8), 28.89 (C-16), 28.64 (C-23), 28.45 (C-1), 24.88 (C-17),24.54 (C-21), 23.65(C-8′), 23.34(C-25), 23.09(C-24), 21.65(C-11), 19.72(C-27), 19.34(C-19), 12.38(C-26). 1 H NMR (400 MHz, Pyr) δ 5.38 (H-9, d, J = 4.5 Hz, 1H), 5.20 (H-1'',d, J = 7.9 Hz, 1H), 4.98 – 4.21 (m, 18H), 4.17 – 4.04 (m, 2H), 3.89 – 3.83(m, 2H), 3.81 – 3.75 (m, 2H), 3.74 – 3.63 (m, 4H), 2.52 – 2.35 (H-4, m, 2H),2.02 – 0.87 (m, 40H), 0.66 (H-26, s, 3H). 13C NMR (101 MHz, Pyr) δ 171.26 (C-35), 170.60 (C-28), 140.13 (C-6), 123.33 (C-9), 106.95 (C-1''), 85.27 (C-4'), 77.45 (C-5''), 75.62 (C-3''),74.85 (C-2), 73.39 (C-2'), 73.24 (C-2''), 72.07 (C-5'), 71.43(C-30), 71.39(C-33), 71.13(C-31), 71.05(C-32), 70.59 (C-3'), 70.25(C-4''), 69.37(C-34), 69.04(C-29), 67.51 (C-6'), 64.27(C-1'), 62.20 (C-6''), 57.08 (C-14), 56.65 (C-15),50.51(C-7), 42.80 (C-4), 40.24(C-13), 40.07(C-12), 38.75(C-22), 37.46 (C-3), 37.10 (C-5), 36.82 (C-20), 36.38 (C-18), 32.47 (C-10), 32.32(C-8), 28.85 (C-1), 28.60 (C-16), 28.38(C-23), 24.83(C-17), 24.49(C-21), 23.31(C-25), 23.05(C-24), 21.59(C-11), 19.67(C-27), 19.29(C-19), 12.32(C-26). It should be noted that the workflow described above is merely illustrative and does not limit the scope of protection of the present invention. In practical applications, technicians in this field can select part or all of it according to actual needs to achieve the purpose of the embodiment scheme, and no limitation is made here.

[0098] In addition, for technical details not fully described in this embodiment, please refer to the parameter operation method provided in any embodiment of the present invention, and will not be repeated here.

[0099] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or system comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or system. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or system comprising the element.

[0100] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0101] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing liver-targeted drug-loaded liposomes, characterized in that: The method comprises: Synthetic liver-targeting carbohydrate ligands: S1: Palladium acetate catalyzes the vinyl exchange between dicarboxyl PEG and vinyl acetate to form divinyl ester PEG; S2: Enzymatically catalyze the reaction of cholesterol (CHS) with PEG n The vinyl end group of the -divinyl ester undergoes selective esterification to generate single-end vinyl-modified CHS-PEG n -vinyl ester; S3: Enzymatically catalyze the reaction of the C-6 hydroxyl group of GalNAc with PEG n The vinyl end group of the -vinyl ester undergoes esterification to generate GalNAc-modified CHS-PEG n -6-GalNAc;PEG n where n=3, 4, 5; and / or The C-6 hydroxyl group of Lac and CHS-PEG n The vinyl end group of the -vinyl ester undergoes esterification to generate Lac-modified CHS-PEG n -6-Lac, PEG n where n=2, 3, 4, 5; Preparation of liver-targeted drug-loaded liposomes; S4: Using thin film dispersion method, CHS-PEG n -6-GalNAc and / or CHS-PEG n -6-Lac, phospholipids and cholesterol were mixed in proportion to prepare GalNAc or Lac-modified liposomes.

2. The method for preparing liver-targeted drug-loaded liposomes according to claim 1, wherein: The S1 uses palladium acetate and 1,10-phenanthroline to catalyze the formation of bifunctional PEG n -divinyl ester. PEG n where n=2, 3, 4, 5.

3. The method for preparing liver-targeted drug-loaded liposomes according to claim 1, wherein: The enzyme catalyst used in S2 is Novozyme 435, a lipase obtained from Candida antarctica.

4. The method for preparing liver-targeted drug-loaded liposomes according to claim 1, wherein: The enzyme catalyst used in S3 is lipase obtained from Thermomyces lanuginosus.

5. The method for preparing liver-targeted drug-loaded liposomes according to claim 1, wherein: In the S4, the ratio of phospholipid to cholesterol is 1-10:1 mol.

6. A liver-targeted drug-loaded liposome, characterized in that: It is composed of hydrogenated soybean phosphatidylcholine, cholesterol and liver-targeting sugar ligand; the liver-targeting sugar ligand is CHS-PEG n -X; PEG n where n=2, 3, 4, 5; The CHS is a cholesterol skeleton; The PEG n is a polyethylene glycol chain, n = 2, 3, 4, 5; The X is 6-GalNAc or 6-Lac.

7. The liver-targeted drug-loaded liposome according to claim 6, characterized in that: The particle size of the liposome is 80-150 nm.

8. The liver-targeted drug-loaded liposome according to claim 6, characterized in that: The liposome is prepared by the preparation method according to any one of claims 1 to 5.

9. Use of the liver-targeted drug-loaded liposomes according to claims 6-8 in preparing medicines.

10. The use according to claim 9, characterized in that The drug is a hydrophobic drug or a hydrophilic drug; and the drug is encapsulated in the liposome by freeze-drying reconstitution or thin film dispersion method using an ammonium sulfate gradient method to achieve active drug loading.

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