Ginsenoside liposome nucleic acid delivery system as well as preparation method and application thereof

Abstract

The invention provides a ginsenoside liposome nucleic acid delivery system as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the invention, a series of ginsenoside liposomes with anti-fibrosis activity are prepared by using ginsenoside loaded nucleic acid, and ginsenoside Rb1 with remarkable pharmacological activity is successfully screened out by optimizing a prescription. The ginsenoside liposome prepared by the invention is a nano drug delivery system with efficient delivery and synergistic treatment functions, and can significantly improve the transfection efficiency of nucleic acid and enhance the anti-hepatic fibrosis effect; the key problems of low in-vivo stability, non-specific distribution, low cell membrane penetration rate and the like of nucleic acid in hepatic fibrosis treatment are effectively solved, and the treatment effect of hepatic fibrosis is remarkably improved. The ginsenoside lipidosome nucleic acid delivery system prepared by the invention has a wide application prospect in preparation of medicines for treating hepatic fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a ginsenoside liposome nucleic acid delivery system and a preparation method and application thereof. Background Art

[0002] Fibrosis is a key pathological link in the development of multiple organ diseases, and its early intervention has become an important therapeutic direction for curbing the progression of end-stage organ diseases. Whether it is the liver, lungs, kidneys or heart, the fibrosis process involves excessive deposition of extracellular matrix and abnormal remodeling of tissue structure, leading to the gradual loss of organ function. In liver fibrosis, the activation of hepatic stellate cells is the core event; in pulmonary fibrosis, abnormal proliferation of fibroblasts and collagen deposition are the main pathological features. If these fibrotic diseases are not intervened, they may eventually lead to organ sclerosis, functional failure and even cancer. Therefore, early treatment of fibrosis can not only delay disease progression, but also significantly improve patient prognosis and reduce the medical burden.

[0003] With breakthroughs in molecular biology technology, gene therapy strategies have brought revolutionary ideas to this field. Studies have confirmed that by targeting and regulating the relevant signal pathways of fibrotic diseases, the fibrosis process can be effectively blocked. Therapeutic methods based on RNA interference (RNAi) have attracted much attention due to their precise gene regulation properties. However, the inherent properties of nucleic acid drugs make them easily degraded by nucleases in the body, and there are bottlenecks such as low transmembrane transport efficiency and poor tissue selectivity, which seriously restrict their clinical translation and application. Therefore, the development of nano-drug delivery systems with high transfection efficiency and targeted delivery capabilities has become a key breakthrough in improving the efficacy of RNAi.

[0004] Common nonviral vectors such as cationic liposomes and lipid nanoparticles are widely used due to their excellent nucleic acid loading capacity and transfection efficiency, demonstrating promising clinical efficacy. However, their clinical application is limited by cytotoxicity and inefficient intracellular release due to strong electrostatic binding between cationic charges and nucleic acids. To address these issues, liposome formulation optimization strategies can be used to improve performance. Steroid-like natural products can replace traditional cholesterol as membrane stabilizers. Their hydrophobic steroid cores embed into the phospholipid bilayer, while their hydrophilic sugar groups improve lipid membrane fluidity through hydrogen bonding, thereby promoting the intracellular dissociation of nucleic acid complexes. Furthermore, surface modification with PEGylated lipids coupled to targeting ligands can enhance the targeted accumulation of these vectors in fibrotic organs. This strategy not only significantly reduces the cytotoxicity of liposomes but also imparts synergistic therapeutic properties to the delivery system. Notably, these steroid-like natural products have dual effects—optimizing the physicochemical properties of the vectors while also directly intervening in fibrosis by inhibiting TGF-β pathway activation, downregulating the expression of key enzymes in collagen synthesis, and downregulating the expression of proinflammatory cytokines.

[0005] Natural active ingredients with a steroid backbone can replace cholesterol in the preparation of liposomes, which not only optimizes carrier performance but also imparts pharmacological activity to the delivery system. Steroid-backbone active ingredients, represented by ginsenosides, have an amphiphilic structure that can significantly improve the fluidity of the phospholipid bilayer: the hydrophobic steroid core is embedded in the lipid membrane to enhance stability, while the hydrophilic sugar groups are exposed on the carrier surface to enhance hydrophilicity. Ginsenoside liposomes have demonstrated significant therapeutic advantages in tumor models, including prolonged blood circulation time, cancer cell targeting, and significant inhibition of the growth of gastric cancer, glioma, and breast cancer. In recent years, pharmacological studies have systematically revealed the multi-target regulatory mechanisms of ginsenosides in anti-fibrotic therapy. As the main active ingredient in Panax plants, its representative monomers exert significant anti-fibrotic effects through the regulation of multiple cellular pathways.

[0006] Therefore, it is of great application value to develop a delivery system using ginsenoside liposomes that can synergistically promote transfection and anti-fibrosis. Summary of the Invention

[0007] The purpose of the present invention is to provide a ginsenoside liposome nucleic acid delivery system, its preparation method and use. By using ginsenosides instead of cholesterol to prepare liposomes, this structural optimization not only enhances its anti-fibrosis effect, but also improves the efficiency of nucleic acid delivery. The synergistic effect of ginsenosides with anti-fibrosis activity and nucleic acids has opened up a new dimension for the treatment of various fibrotic diseases. By rationally utilizing the structural and functional properties of natural active ingredients, a nano-drug delivery system with both efficient delivery and synergistic therapeutic functions has been constructed, providing an innovative solution to break through the bottleneck of existing gene therapy technology.

[0008] The present invention provides a ginsenoside liposome nucleic acid delivery system, which is composed of phospholipids, cationic liposomes, cophospholipids, ginsenosides and nucleic acids, wherein the molar ratio of the phospholipids, cationic liposomes, cophospholipids and ginsenosides is 1-5:20-40:20-40:20-50.

[0009] Furthermore, the molar ratio of the phospholipid, cationic liposome, cophospholipid and ginsenoside is 3:27.5-35:27.5-35:27-42, preferably 3:35:35:27.

[0010] Furthermore, the structural formula of the ginsenoside is shown in Formula I:

[0011]

[0012] wherein R1, R2, and R3 are each independently selected from hydrogen, hydroxy, -O-glc, -O-glc(2→1)glc, or -O-glc(6→1)glc;

[0013] Among them, glc is glucopyranose.

[0014] Furthermore, R1 is -O-glc(2→1)glc, R2 is hydrogen, and R3 is -O-glc(6→1)glc.

[0015] Furthermore, the phospholipid is phospholipid-polyethylene glycol;

[0016] The cationic liposome is 1,2-dioleoyl-3-trimethylammonium propane;

[0017] The phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;

[0018] The nucleic acid is siRNA.

[0019] The present invention also provides a method for preparing the above-mentioned ginsenoside liposome nucleic acid delivery system, the method comprising the following steps:

[0020] (1) preparing an organic phase solution: dissolving phospholipids, cationic liposomes, cophospholipids, and ginsenosides in a solvent to obtain an organic phase solution;

[0021] (2) preparing an aqueous phase solution: preparing a citrate buffer with a nuclease inhibitor and water, and dissolving the nucleic acid in the citrate buffer to obtain an aqueous phase solution;

[0022] (3) The organic phase solution and the aqueous phase solution are mixed and dialyzed to obtain ginsenoside liposomes.

[0023] Furthermore, in step (1), the solvent is an alcohol solvent;

[0024] In step (2), the nuclease inhibitor is diethyl pyrocarbonate; the pH of the citrate buffer is 2 to 6; the content of nucleic acid in the aqueous solution is 0.1 to 0.3 mg / mL;

[0025] In step (3), the volume ratio of the organic phase solution to the aqueous phase solution is 1 to 5:1; the mixing is performed by a microfluidic chip system, and the flow rate of the system is 1 to 3 mL / min.

[0026] Furthermore, in step (1), the solvent is ethanol;

[0027] In step (2), the pH of the citrate buffer is 4; the content of nucleic acid in the aqueous solution is 0.2 mg / mL;

[0028] In step (3), the volume ratio of the organic phase solution to the aqueous phase solution is 3:1; and the flow rate of the system is 1.6 mL / min.

[0029] The present invention also provides use of the ginsenoside liposome nucleic acid delivery system in preparing a drug for preventing and / or treating liver fibrosis.

[0030] Furthermore, the drug is a drug that inhibits the expression of YAP mRNA and YAP protein.

[0031] The present invention has achieved the following beneficial effects:

[0032] (1) The present invention utilizes ginsenoside liposomes loaded with nucleic acids to prepare ginsenoside liposomes with anti-fibrotic activity. The liposomes are a nano-drug delivery system with both efficient delivery and synergistic therapeutic functions.

[0033] (2) The present invention screened different types of ginsenosides (20(S)-protopanaxadiol (PPD), ginsenoside Rb1, and ginsenoside Rg1) in liposomes and successfully screened out ginsenoside Rb1 with significant pharmacological activity;

[0034] (3) The present invention constructs Rb1 cationic liposomes that specifically target activated hepatic stellate cells. By optimizing the formulation, the liposomes can significantly improve the transfection efficiency of siRNA and enhance the anti-liver fibrosis effect;

[0035] (4) The Rb1 cationic liposomes constructed by the present invention effectively solve the key problems faced by siRNA in the treatment of liver fibrosis, such as low in vivo stability, nonspecific distribution and low cell membrane penetration rate, and greatly improve the silencing efficiency of the target YAP, thereby inhibiting the activation of hepatic stellate cells, reversing the abnormal deposition of extracellular matrix, and significantly improving the therapeutic effect of liver fibrosis.

[0036] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0037] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Figure 2 shows the particle size distribution of R8-dGR modified with cholesterol, ginsenosides Rg1, Rb1 and PPD liposomes.

[0039] Figure 2 Figure 2 shows the particle size distribution of cholesterol, ginsenosides Rg1, Rb1 and PPD liposomes without R8-dGR modification.

[0040] Figure 3 The potential results of four liposomes before and after modification of R8-dGR (n=3, mean±SD): (A) after modification; (B) before modification.

[0041] Figure 4 These are the gel electrophoresis images of cholesterol (Chol)-LPs / siYAP, Rg1-LPs / siYAP, Rb1-LPs / siYAP and PPD-LPs / siYAP after incubation with serum.

[0042] Figure 5 The cytotoxicity of four liposomes containing different concentrations of DOTAP on (A) activated hepatic stellate cells (aHSC-T6) and (B) hepatocytes (AML12) after incubation for 24 h (n=3, mean±SD).

[0043] Figure 6 Real-time PCR analysis results of YAP mRNA in aHSC-T6 cells after incubation with different liposomes (n=3, mean±SD), ** represents p<0.01, ns represents no significant difference.

[0044] Figure 7 Effects of liposomes containing different proportions of ginsenosides on gene silencing efficiency: (A) Chol-LPs / siYAP containing different proportions of Chol; (B) Rg1-LPs / siYAP containing different proportions of Rg1; (C) Rb1-LPs / siYAP containing different proportions of Rb1 after incubation with aHSC-T6. The protein expression and semi-quantitative results of YAP (n=3, mean±SD), RE represents relative expression.

[0045] Figure 8 (A) YAP protein expression in aHSC-T6 cells after incubation with different liposomes. (B) Semi-quantitative results of YAP (n=3, mean±SD). ** represents p<0.01, *** represents p<0.001.

[0046] Figure 9 Semi-quantitative results of (A) α-SMA and Collagen I protein expression levels after incubation of aHSC-T6 cells with Rb1-R-LPs / siYAP (B) Collagen I (C) α-SMA (n=3, mean±SD), ** represents p<0.01, *** represents p<0.001, and (-) indicates no TGF-β1 treatment.

[0047] Figure 10Flow cytometry was used to detect the effect of liposomes on the reactive oxygen species level of aHSC-T6 (n=3, mean±SD), *** represents p<0.001, ns represents no significant difference, (-) represents no TGF-β1 treatment.

[0048] Figure 11 The figure shows the body weight change trend of mice in each experimental group (n=5, mean±SD).

[0049] Figure 12 Biochemical analysis of serum ALT and AST of mice in each group (n=3, mean±SD), * represents p<0.05, ** represents p<0.01, *** represents p<0.001, ns represents no significant difference.

[0050] Figure 13 (A) Expression of YAP and TNF-α proteins in the liver of mice in each group after treatment, (B) semi-quantitative results of YAP, (C) semi-quantitative results of TNF-α (n=3, mean±SD), * represents p<0.05, ** represents p<0.01, *** represents p<0.001, ns represents no significant difference compared with the normal group.

[0051] Figure 14 (A) Masson staining, Sirius red staining and immunohistochemical images of liver sections (B) Sirius red (C) Masson staining (D) semi-quantitative results of type I collagen, scale bar: 200 μm (n=3, mean±SD), * represents p<0.05, ** represents p<0.01, *** represents p<0.001, ns represents no significant difference. DETAILED DESCRIPTION

[0052] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0053] The structural formula of ginsenosides used in the embodiments of the present invention is shown in Formula I:

[0054]

[0055] Among them, the selection of R1, R2, and R3 substituents is shown in Table 1:

[0056] Table 1 Selection of R1, R2, and R3 substituents

[0057]

[0058] Among them, the structure of ginsenoside Rb1 is as follows:

[0059]

[0060] Example 1: Preparation of ginsenoside liposomes

[0061] The microfluidic liposome construction method is as follows: First, the lipid material components, including phospholipid-polyethylene glycol (DSPE-PEG2000), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP, a cationic liposome), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, a phospholipid), and 20(S)-protopanaxadiol (PPD), were accurately weighed and dissolved in 200 μL of anhydrous ethanol at a molar ratio of 3:35:35:27 to prepare the organic phase solution. Simultaneously, a 25 mM sodium citrate buffer system (pH 4.0) was prepared in diethyl pyrocarbonate (DEPC)-treated water to dissolve the siRNA to a final concentration of 0.2 mg / mL as the aqueous phase solution. Liposome assembly was accomplished using a Y-shaped microfluidic chip system, with channel a connected to a 300 μL aqueous solution syringe, channel b to a 100 μL organic solution syringe, and the outlet connected to a 1.5 mL enzyme-free EP tubing as the product receiver. During the experiment, a total flow rate of 1.6 mL / min was set, and precise mixing was achieved by adjusting the volume ratio of the two phases (organic phase:aqueous phase) to 3:1. The liposomes were pre-diluted by adding PBS buffer (pH 7.4) prepared with DEPC water to the receiver tube, and then the syringe pump was activated to achieve controlled fusion of the two phases. The final product was purified by dialysis using a 3500 Da molecular weight cutoff (MWCO) dialysis bag. Dialysis was performed for 2 h in an ice bath against DEPC-PBS buffer (pH 7.4) to obtain liposomes with a uniform particle size distribution of the nucleic acid drug, designated PPD liposomes (PPD-LPs / siYAP).

[0062] Example 2: Preparation of ginsenoside liposomes

[0063] The preparation method of Reference Example 1 was used, except that 20(S)-protopanaxadiol (PPD) was replaced with ginsenoside Rb1. The obtained liposomes were designated as Rb1 liposomes (Rb1-LPs / siYAP).

[0064] Example 3: Preparation of ginsenoside liposomes

[0065] The preparation method of Reference Example 1 was used, except that 20(S)-protopanaxadiol (PPD) was replaced with ginsenoside Rg1. The obtained liposomes were designated as Rg1 liposomes (Rg1-LPs / siYAP).

[0066] Example 4: Preparation of surface-modified liposomes coupled with targeting ligands

[0067] The preparation method of reference example 1 is different only in that DSPE-PEG 2000 Replaced with DSPE-PEG2000 -R8-dGR, and obtain PPD liposomes with R8-dGR surface modified.

[0068] Example 5: Preparation of surface-modified liposomes coupled with targeting ligands

[0069] The preparation method of reference example 2 is different only in that DSPE-PEG 2000 Replaced with DSPE-PEG 2000 -R8-dGR, and obtained Rb1 liposomes surface-modified with R8-dGR (Rb1-R-LPs / siYAP).

[0070] Example 6: Preparation of surface-modified liposomes coupled with targeting ligands

[0071] The preparation method of Reference Example 3 was used, except that DSPE-PEG2000 was replaced with DSPE-PEG2000-R8-dGR to obtain Rg1 liposomes surface-modified with R8-dGR (Rg1-R-LPs / siYAP).

[0072] Example 7: Preparation of ginsenoside liposomes

[0073] With reference to the preparation method of Example 1, the molar ratio of 3:35:35:27 can be replaced with 3:27.5:27.5:42, 3:30:30:37, and 3:32.5:32.5:32 to prepare PPD liposomes with different ratios.

[0074] Example 8: Preparation of ginsenoside liposomes

[0075] Referring to the preparation method of Example 2, the molar ratio of 3:35:35:27 can be replaced with 3:27.5:27.5:42, 3:30:30:37, and 3:32.5:32.5:32 to prepare Rb1 liposomes with different ratios.

[0076] Example 9: Preparation of ginsenoside liposomes

[0077] Referring to the preparation method of Example 3, the molar ratio of 3:35:35:27 can also be replaced with 3:27.5:27.5:42, 3:30:30:37, and 3:32.5:32.5:32 to prepare Rg1 liposomes with different ratios.

[0078] The following is a control sample preparation.

[0079] Comparative Example 1: Preparation of cholesterol liposomes

[0080] The preparation method of Reference Example 1 was used, with the only difference being that ginsenoside was replaced with cholesterol to obtain cholesterol liposomes, which were designated as Chol-LPs / siYAP.

[0081] Comparative Example 2: Preparation of Surface-Modified Liposomes Coupled with Targeting Ligands

[0082] The preparation method of Reference Example 1 was referred to, with the only difference being that DSPE-PEG2000 was replaced with DSPE-PEG2000-R8-dGR to obtain cholesterol liposomes surface-modified with R8-dGR (Chol-R-LPs / siYAP).

[0083] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0084] Experimental Example 1: Characterization and performance testing of ginsenoside liposomes

[0085] 1. Characterization of ginsenoside liposomes

[0086] 1.1 Particle size distribution and potential investigation of ginsenoside liposomes

[0087] Malvern laser particle size analyzer was used to measure the particle size and potential of ginsenoside Rg1, Rb1, PPD liposomes and cholesterol liposomes. Figures 1 to 3 As shown, the particle size and potential of cholesterol liposomes are similar to those of ginsenoside Rg1, Rb1, and PPD liposomes. The particle size of the liposome modified with R8-dGR is 80-100nm, the potential is +8-12mV, and the particle size of the liposome without R8-dGR is 70-90nm, the potential is +5-7mV.

[0088] 1.2 Investigation of ginsenoside liposome encapsulation efficiency

[0089] In order to evaluate its drug loading performance, the present invention established an ultrafiltration centrifugation detection system based on fluorescence labeling: using FAM-siYAP as a tracer molecule, four groups of drug-loaded liposomes (containing cholesterol liposomes and ginsenosides Rg1, Rb1, and PPD liposomes) were prepared by microfluidic technology. An equal volume of sample was added to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa, and centrifuged at 5000 rpm for 10 minutes at a low temperature of 4°C to remove unencapsulated free FAM-siYAP. The retentate phase was collected and diluted to 1 mL with DEPC water in equal proportions. The fluorescence spectrophotometer (λ ex =494nm,λ em =518 nm) was used to measure the fluorescence intensity (F1). A separate, unfiltered, crude liposome sample was processed in the same manner and the total fluorescence intensity (F2) was measured. Encapsulation efficiency was calculated using the formula EE (%) = (F1 / F2) × 100%. The results showed that the siRNA encapsulation efficiency of all four systems exceeded 90% (Table 2), confirming the excellent nucleic acid loading capacity of the preparation process.

[0090] Table 2 Encapsulation efficiency of cholesterol liposomes and three ginsenoside liposomes (n=3)

[0091]

[0092] 1.3 Investigation of antiserum degradation

[0093] To verify whether ginsenoside liposomes loaded with siRNA can protect siRNA from ribonuclease degradation in serum, ginsenoside liposomes loaded with siRNA (Rg1-LPs / siYAP, Rb1-LPs / siYAP, and PPD-LPs / siYAP), cholesterol liposomes (Chol-LPs / siYAP), and free siYAP were mixed with serum and incubated on a shaker at 37°C and 70 rpm for 2 hours. After incubation, the emulsions were broken using Triton X-100, and then agarose gel electrophoresis was performed, and the results were imaged and photographed using a gel imager. Experimental Results Figure 4 The results showed that in the presence of fetal bovine serum (FBS), Triton X-100 completely disrupted siRNA entrapment in liposomes without interfering with gel imaging. Free siYAP was completely degraded in serum, while the fluorescence intensity of free siYAP in the absence of serum was close to that of the four liposomes. This indicates that ginsenoside Rg1, Rb1, and PPD liposomes, as well as cholesterol liposomes, effectively protect siYAP from degradation by nucleases in serum.

[0094] 1.4 Cytotoxicity study

[0095] The CCK-8 assay was used to investigate the cytotoxicity of blank ginsenoside liposomes without siYAP encapsulation on activated hepatic stellate cells (aHSC-T6) and hepatocytes (AML12). Figure 5 It was shown that, at the set concentration, except for PPD-R-LPs, the cell viabilities of the other three liposomes were all above 70% after incubation with aHSC-T6 and AML12 cells, indicating that the other three liposomes can be used for subsequent experiments.

[0096] Experimental Example 2: Investigation of the in vitro gene silencing effect of liposomes containing different ginsenosides

[0097] 1. Experimental methods

[0098] (1) Transfection of ginsenoside liposomes

[0099] HSC-T6 cells were seeded in 12-well or 24-well plates. Ginsenoside liposomes loaded with siYAP were prepared using microfluidics. When cell confluency reached 60%-80%, the cells were treated with ginsenoside liposomes diluted in serum-free medium. After a 6-hour incubation, the medium was replaced with 10 ng / mL TGF-β1. After 36-48 hours, cellular RNA was extracted, and mRNA levels were measured by RT-qPCR. After another 48-60 hours of culture, total cell protein was extracted, and protein expression was analyzed by Western blotting. Furthermore, cholesterol was substituted for ginsenoside, and cholesterol liposomes were prepared using the same method. A commercially available transfection Mate loaded with siYAP was used as a positive control.

[0100] (2) RT-qPCR detection of YAP mRNA expression

[0101] The relative expression of YAP mRNA was calculated based on the cycle threshold value detected by the instrument, using GAPDH as the internal reference and the commercially available transfection reagent Mate as the positive control.

[0102] (3) Western Blot detection of YAP protein expression

[0103] Western blot experiments were performed to investigate the gene silencing effects of liposomes containing different ginsenosides, with cholesterol liposomes serving as a control group and the commercially available transfection reagent Mate serving as a positive control.

[0104] 2. Experimental results

[0105] The results are as follows Figure 6 As shown, ginsenoside Rg1 liposome and ginsenoside Rb1 liposome groups can significantly downregulate YAPmRNA. There are significant differences between ginsenoside Rb1 liposome and cholesterol liposome. Ginsenoside Rb1 liposome has the best silencing effect, with the expression level of YAP mRNA being 40.07%.

[0106] Liposomes containing different proportions of ginsenosides were prepared for cell transfection to investigate the gene silencing efficiency. Figure 7 As shown, ginsenoside Rg1 liposomes and ginsenoside Rb1 liposomes at different concentrations (42%, 37%, 32%, and 27%) significantly downregulated YAP protein, with significant differences compared to the cholesterol liposome group. The ginsenoside Rb1 liposome group had the best silencing effect at 27%, with YAP protein expression at 24.99%.

[0107] The results are as follows Figure 8As shown in the results, ginsenoside Rg1 liposomes and ginsenoside Rb1 liposomes significantly downregulated YAP protein, with significant differences compared to the cholesterol liposome group. The ginsenoside Rb1 liposome group had the best silencing effect, with YAP protein expression at 27.91%.

[0108] Experimental Example 3: In vitro anti-hepatic fibrosis effects of liposomes containing different types of ginsenosides

[0109] 1. Western Blot Detection of α-SMA and Collagen I Protein Expression

[0110] Western blot analysis was used to evaluate the ability of ginsenoside liposomes to downregulate the expression of activation markers α-SMA and fibrosis marker Collagen I in activated hepatic stellate cells (aHSC-T6). A control group of cells not stimulated with TGF-β1 and not treated with the drug was set up. Figure 9 The results showed that after stimulation with TGF-β1, HSC-T6 cells were fully activated, and the expression of α-SMA and Collagen I proteins increased significantly. All three liposomes significantly reduced the expression levels of α-SMA and Collagen I, restoring them to the state before TGF-β1 stimulation. Compared with cholesterol liposomes, ginsenoside Rg1 and Rb1 liposomes showed more significant effects in reducing the expression of α-SMA and Collagen I. Among them, the ginsenoside Rb1 liposome group, which can effectively silence YAP, showed the strongest in vitro anti-fibrosis ability, indicating that silencing YAP can effectively inhibit the activation of aHSC-T6 cells, thereby achieving an anti-fibrosis effect.

[0111] 2. Investigation of the ability of ginsenoside liposomes to reduce reactive oxygen species levels in aHSC-T6 cells

[0112] Liver fibrosis is accompanied by an increase in cellular reactive oxygen species (ROS) levels. Nucleic acid drugs entering cells will activate the STING pathway and also increase the level of reactive oxygen species (ROS). Ginsenosides have the ability to reduce cellular ROS levels, mainly due to their diverse antioxidant mechanisms. In order to evaluate the regulatory effect of ginsenoside liposomes on the ROS level of aHSC-T6, the present invention used flow cytometry and a cell reactive oxygen species kit for detection. The results of flow cytometry showed that ( Figure 10), compared with cholesterol liposomes (Chol-R-LPs / siYAP) and the commercially available transfection reagent Mate / siYAP group, although they could reduce ROS levels in aHSC-T6 cells to some extent, the levels were still significantly higher than the baseline levels without TGF-β1 stimulation. In contrast, Rg1-R-LPs / siYAP, Rb1-R-LPs / siYAP, and Rb1-LPs / siYAP all significantly reduced ROS levels in aHSC-T6 cells, with the Rb1-R-LPs / siYAP group having the lowest ROS levels. This indicates that the liposomes not only have good anti-inflammatory function in vitro, but can also synergistically achieve anti-liver fibrosis effects by reducing ROS levels.

[0113] Experimental Example 4: Evaluation of the Anti-liver Fibrosis Ability of Liposomes Containing Different Types of Ginsenosides in Vivo

[0114] 1. Establishment of Hepatic Fibrosis Mice and Dosage Scheme

[0115] C57BL / 6 male mice were housed under standard conditions and injected intraperitoneally with 10% (w / v) carbon tetrachloride in olive oil at a dose of 1 mL / kg, starting on day 1 and every other day for 4 weeks, to induce liver fibrosis. Starting on day 6 of liver fibrosis modeling, the mice were randomly divided into six groups, with five mice in each group: a normal liver control group (normal), a liver fibrosis model injected with PBS, a liver fibrosis model injected with R8-dGR-modified Chol-R-LPs / siYAP, a liver fibrosis model injected with nonsense scrambled siRNA (siNC), a liver fibrosis model injected with Rb1-R-LPs / siNC, a liver fibrosis model injected with unmodified R8-dGR, and a liver fibrosis model injected with Rb1-R-LPs / siYAP. Starting on day 6, siYAP was administered via the tail vein at a concentration of 1 mg / kg every two days for a total of seven doses. On day 27, the mice were killed and their major organs were collected for subsequent experiments.

[0116] 2. Mouse weight detection

[0117] During the treatment period, the mice were weighed before each tail vein administration, and the weight data were plotted as a trend graph ( Figure 11 Compared with the mice in the liver fibrosis model, the healthy mice in the Normal group that were not injected with carbon tetrachloride were slightly heavier. However, there was no significant change in the weight of the mice in each group during the entire treatment period, indicating that the injection of liposomes in each group had no obvious toxicity to the mice.

[0118] 3. Biochemical analysis of serum ALT and AST

[0119] The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were measured by biochemical analysis to evaluate the liver health of each group of mice. On the third day after the last administration (day 26), blood was collected from the eye socket for testing. The results showed (see Figure 12 ), compared with the normal liver control group (Normal), the ALT and AST levels of the liver fibrosis model group (PBS group) were significantly increased, indicating the presence of liver damage. The groups treated with different liposomes, including the Chol-R-LPs / siYAP group, the Rb1-R-LPs / siNC group, the Rb1-LPs / siYAP group, and the Rb1-R-LPs / siYAP group, showed varying degrees of decrease in ALT and AST levels, indicating that these liposomes can alleviate liver fibrosis to a certain extent. In particular, the ginsenoside Rb1 liposomes modified with R8-dGR (Rb1-R-LPs / siYAP group) and the ginsenoside Rb1 liposomes without R8-dGR modification (Rb1-LPs / siYAP group) performed better in reducing ALT and AST levels, with no significant difference from the normal group, indicating that ginsenoside Rb1 liposomes have stronger anti-liver fibrosis ability. Among these liposomes, the Rb1-R-LPs / siYAP group had the most significant anti-hepatic fibrosis effect. Furthermore, the Rb1-R-LPs / siNC group was able to reduce serum ALT and AST levels in mice with hepatic fibrosis to a certain extent, further confirming that ginsenoside Rb1 achieves its anti-hepatic fibrosis effect by reducing plasma transaminase activity.

[0120] 4. Investigation of liver YAP and TNF-α protein expression levels

[0121] The expression levels of YAP and TNF-α proteins in the liver were detected by Western Blot. Figure 13 ), the YAP protein expression levels in each liposome-treated group were reduced, indicating that these liposomes can achieve a certain degree of gene silencing effect. Among them, the YAP expression levels in the ginsenoside liposome and cholesterol liposome-treated groups were comparable to those in the normal liver group (Normal), showing no significant differences. In particular, the Rb1-R-LPs / siYAP group had the lowest YAP expression level, showing the best gene silencing effect. In addition, the YAP expression level in the Rb1-R-LPs / siNC group also decreased, indicating that ginsenoside Rb1 itself has anti-hepatic fibrosis effects and can synergize with nucleic acid drugs to exert therapeutic effects.

[0122] In terms of TNF-α protein expression levels, the TNF-α expression levels in the Rb1-R-LPs / siNC group, the Rb1-LPs / siYAP group, and the Rb1-R-LPs / siYAP group were significantly reduced, approaching the levels of normal healthy mice (Normal). This result indicates that liposomes prepared with ginsenosides instead of cholesterol can significantly exert anti-inflammatory effects, thereby synergistically achieving anti-liver fibrosis effects. Compared with Rb1-LPs / siYAP without R8-dGR modification, Rb1-R-LPs / siYAP modified with R8-dGR showed a more significant effect in reducing YAP and TNF-α expression levels.

[0123] 5. Masson staining, Sirius red staining and immunohistochemistry of liver sections

[0124] To further evaluate the in vivo anti-fibrotic effect of ginsenoside Rb1 liposomes, we collected liver samples, fixed them with 4% paraformaldehyde, embedded them in paraffin, and sectioned them. The sections were then stained with Masson's stain, Sirius red stain, and immunohistochemistry. The results were observed under a microscope and photographed. Finally, semi-quantitative analysis was performed using Image J software.

[0125] The staining results showed (see Figure 14 ), cholesterol liposomes and ginsenoside Rb1 liposomes can significantly reduce the collagen level in mouse fibrotic liver. Ginsenoside Rb1 liposomes showed a significant reduction in the content of red collagen in Sirius red staining, blue collagen in Masson staining, and brown-yellow type I collagen in immunohistochemical staining, and there was no significant difference in the collagen level of normal healthy mouse liver. Compared with Rb1-LPs / siYAP without R8-dGR modification, Rb1-R-LPs / siYAP modified with R8-dGR showed a more significant effect in reducing collagen deposition in mouse fibrotic liver. The Rb1-R-LPs / siNC group can also reduce collagen levels, which indicates that ginsenoside Rb1 itself has anti-liver fibrosis effects and can synergize with nucleic acid drugs to exert therapeutic effects.

[0126] These results demonstrate that the present invention successfully constructed ginsenoside Rg1, Rb1, PPD liposomes, and cholesterol liposomes, all with siRNA encapsulation efficiencies exceeding 90% and excellent nucleic acid loading capacity. These liposomes effectively protected siYAP from degradation by serum nucleases. Ginsenosides Rg1, Rb1, and cholesterol liposomes exhibited minimal toxicity to aHSC-T6 and AML12 cells. In in vitro gene silencing experiments, ginsenoside Rb1 liposomes demonstrated the best silencing effect on YAP mRNA and YAP protein. In in vitro and in vivo anti-fibrosis experiments, the ginsenoside Rb1 liposome group exhibited the strongest in vitro anti-fibrosis activity.

[0127] In summary, the present invention provides a ginsenoside liposome nucleic acid delivery system, and its preparation method and use. The present invention utilizes ginsenoside loaded nucleic acid to prepare a series of ginsenoside liposomes with anti-fibrosis activity, and by optimizing the prescription, successfully screened out ginsenoside Rb1 with significant pharmacological activity. The ginsenoside liposome prepared by the present invention is a nano drug delivery system with both efficient delivery and synergistic therapeutic functions, which can significantly improve the transfection efficiency of nucleic acids and enhance the anti-liver fibrosis effect, effectively solving the key problems of low in vivo stability, non-specific distribution and low cell membrane penetration rate faced by nucleic acids in the treatment of liver fibrosis, and significantly improving the therapeutic effect of liver fibrosis. The ginsenoside liposome nucleic acid delivery system prepared by the present invention has broad application prospects in the preparation of drugs for the treatment of liver fibrosis.

Claims

1. A ginsenoside liposome nucleic acid delivery system, characterized in that: The invention is composed of phospholipid, cationic liposome, phospholipid, ginsenoside and nucleic acid, wherein the molar ratio of the phospholipid, cationic liposome, phospholipid and ginsenoside is 1-5:20-40:20-40:20-50.

2. The ginsenoside liposome nucleic acid delivery system according to claim 1, characterized in that: The molar ratio of the phospholipid, cationic liposome, cophospholipid and ginsenoside is 3:27.5-35:27.5-35:27-42, preferably 3:35:35:

27.

3. The ginsenoside liposome nucleic acid delivery system according to claim 1, characterized in that: The structural formula of the ginsenoside is shown in Formula I: wherein R1, R2, and R3 are each independently selected from hydrogen, hydroxy, -O-glc, -O-glc(2→1)glc, or -O-glc(6→1)glc; Among them, glc is glucopyranose.

4. The ginsenoside liposome nucleic acid delivery system according to claim 1, characterized in that: R1 is -O-glc(2→1)glc, R2 is hydrogen, and R3 is -O-glc(6→1)glc.

5. The ginsenoside liposome nucleic acid delivery system according to claim 1, characterized in that: The phospholipid is phospholipid-polyethylene glycol; The cationic liposome is 1,2-dioleoyl-3-trimethylammonium propane; The phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; The nucleic acid is siRNA.

6. A method for preparing the ginsenoside liposome nucleic acid delivery system according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) preparing an organic phase solution: dissolving phospholipids, cationic liposomes, cophospholipids, and ginsenosides in a solvent to obtain an organic phase solution; (2) preparing an aqueous phase solution: preparing a citrate buffer with a nuclease inhibitor and water, and dissolving the nucleic acid in the citrate buffer to obtain an aqueous phase solution; (3) The organic phase solution and the aqueous phase solution are mixed and dialyzed to obtain ginsenoside liposomes.

7. The method according to claim 6, characterized in that In step (1), the solvent is an alcohol solvent; In step (2), the nuclease inhibitor is diethyl pyrocarbonate; the pH of the citrate buffer is 2 to 6; the content of nucleic acid in the aqueous solution is 0.1 to 0.3 mg / mL; In step (3), the volume ratio of the organic phase solution to the aqueous phase solution is 1 to 5:1; the mixing is performed by a microfluidic chip system, and the flow rate of the system is 1 to 3 mL / min.

8. The method according to claim 7, characterized in that In step (1), the solvent is ethanol; In step (2), the pH of the citrate buffer is 4; the content of nucleic acid in the aqueous solution is 0.2 mg / mL; In step (3), the volume ratio of the organic phase solution to the aqueous phase solution is 3:1; and the flow rate of the system is 1.6 mL / min.

9. Use of the ginsenoside liposome nucleic acid delivery system according to any one of claims 1 to 5 in the preparation of a medicament for preventing and / or treating liver fibrosis.

10. The use according to claim 9, characterized in that The drug is a drug that inhibits the expression of YAP mRNA and YAP protein.

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