Nano-platform for co-delivery of small interfering RNA and small molecule drugs, and preparation method and application thereof

Abstract

The invention particularly relates to a glycosylated dendrimer for co-delivery of small interfering RNA (Ribonucleic Acid) and small molecular drugs as well as a preparation method and application of the glycosylated dendrimer. The nano platform provided by the invention is characterized in that an anti-inflammatory drug resveratrol (Res) and electrostatic adsorption small interfering RNA (si-Cp2e1) are loaded in a glycosylated dendrimer G5-Gal. The preparation method and the application comprise the following steps: preparation of G5-Gal, preparation of Res (at) G5-Gal, and preparation of Res (at) G5-Gal / si-Cp2e1. The preparation method is simple, the required raw materials are easy to obtain, the synthesis process is simple and convenient, separation and purification are easy, and the synthesized nano-drug has a liver targeting capability, can realize dual treatment of small molecule drug-small interfering RNA, and has a potential application prospect in the aspect of treatment of alcohol-related liver diseases.

Description

A nanoplatform for co-delivering small interfering RNA and small molecule drugs, its preparation method and application Technical Field

[0001] This invention belongs to the field of drug technology for treating alcoholic liver disease, specifically relating to a nanoplatform for co-delivering small interfering RNA and small molecule drugs, a method for preparing the nanoplatform, a pharmaceutical composition comprising the nanoplatform, and its application in the prevention and treatment of alcohol-related liver disease. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Alcohol abuse is a major global public health challenge. Long-term and / or excessive alcohol intake can damage multiple organs throughout the body, with the liver, as the core organ for ethanol metabolism, suffering particularly severe damage. This is considered a major cause of alcohol-associated liver disease (ALD). The pathological progression of ALD includes hepatic steatosis, alcoholic hepatitis, liver fibrosis, cirrhosis, and can further develop into hepatocellular carcinoma. This series of pathological changes seriously threatens the life and health of patients. Currently, the clinical treatment principles for ALD are mainly abstinence from alcohol and nutritional support. However, abstinence from alcohol may lead to alcohol withdrawal syndrome, and nutritional intervention cannot reverse the ongoing liver damage. It is worth noting that, to date, there are no drugs or therapies approved by the U.S. Food and Drug Administration (FDA) for the specific treatment of ALD.

[0004] In the pathogenesis of ALD, cytochrome P450 2E1 (CYP2E1)-mediated oxidative stress and inflammatory responses play a central role. As a key enzyme in ethanol metabolism, CYP2E1 expression is significantly upregulated under long-term alcohol abuse or high-concentration ethanol exposure. Its overactivation promotes the generation of large amounts of reactive oxygen species (ROS), exacerbating liver damage. Sustained ROS generation consumes large amounts of endogenous antioxidants such as glutathione (GSH), leading to an imbalance in the oxidation-antioxidant system, creating a vicious cycle that ultimately drives more severe inflammation and damage. Therefore, targeting and silencing the Cyp2e1 gene using small interfering RNA (siRNA) at its source is considered an effective strategy to reduce ROS generation and alleviate liver damage.

[0005] To address the limitations of single-modality treatments, the diversified development of nanocarrier systems offers new insights into overcoming the barriers to dual delivery of siRNA and small molecule drugs. Among these, amino-terminated fifth-generation (G5) poly(amidoamine) (PAMAM) dendritic macromolecules, as a commercially viable cationic nanocarrier, possess unique branched structures, abundant surface functional groups, and large internal cavities. These macromolecules can efficiently compress Cyp2e1 siRNA and encapsulate small molecule drugs through electrostatic adsorption. These properties give them significant advantages in targeted delivery of therapeutic agents to improve alcohol-related liver disease. In previous work, the inventors fully utilized the surface modification properties of amino-terminated PAMAM dendritic macromolecules to successfully construct a core-shell dendritic macromolecule nanoplatform for co-delivering the microRNA-21 inhibitor and the chemotherapeutic drug doxorubicin (Song C. et al. J. Mater. Chem. B, 2020, 8, 2768-2774). This work provides important evidence for synergistic delivery strategies of therapeutic components.

[0006] Building upon this foundation, this invention proposes that further combining anti-inflammatory and antioxidant drugs with siRNA could potentially construct a novel combined treatment system integrating "gene silencing at the source" and "synergistic effects of anti-inflammatory and antioxidant drugs," demonstrating significant potential for clinical translation. However, developing a carrier system capable of safely and efficiently co-delivering siRNA and drugs is the core bottleneck to overcoming the clinical translation challenges of this strategy and achieving targeted enrichment of drugs within hepatocytes. Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention provides the following technical solution: In a first aspect, a nanoplatform for co-delivering small interfering RNA and small molecule drugs is provided, wherein the nanoplatform uses a fifth-generation (G5) amino-terminated polyamide-amine dendritic macromolecule (G5-Gal) grafted with galactose as a carrier to load a small molecule anti-inflammatory drug and a small interfering RNA (si-Cyp2e1) targeting the Cyp2e1 gene.

[0008] This invention designs and provides a nanomedicine that enhances liver targeting and synergistically improves the therapeutic and gene-therapy activity of small molecule drugs. In the first aspect, G5 serves as a nanocarrier, possessing a precise nanostructure, a large drug-carrying cavity, and abundant surface-modifiable groups. Galactose (Gal) modified on the G5 surface can achieve active liver targeting of the nanomedicine by binding to desialylate glycoprotein receptors specifically expressed on the surface of hepatocytes. In vitro and in vivo experiments have confirmed that, compared to the unmodified carrier, G5-Gal exhibits stronger hepatocyte targeting ability. Simultaneously, G5-Gal has better anti-protein adsorption properties, lower hemolysis rate, and is safer to use. In some embodiments verified by this invention, the G5 is linked to Gal via a "COC," with 12 to 18 Gal molecules grafted onto the surface of each G5 molecule.

[0009] In addition, G5-Gal can also compress the stretched siRNA chain tightly through electrostatic interaction to form a structurally stable G5 / siRNA complex, thereby achieving efficient delivery of si-Cyp2e1 and avoiding the release or freeing of RNA chains during delivery. In some embodiments verified by the present invention, the ratio of Gal-G5 to si-Cyp2e1 is ≥16:1. At the above mass ratio, Gal-G5 has good compression ability.

[0010] G5-Gal can also encapsulate small-molecule anti-inflammatory drugs within its internal cavity. These small-molecule anti-inflammatory drugs can be selected from natural polyphenols, flavonoids, or their derivatives, such as paclitaxel, curcumin, silymarin, isoglycyrrhizin, paeonol, shikonin, scutellarin, dihydromyricetin, or resveratrol; they can also be synthetic small-molecule compounds, such as ZHB12 and ethyl lactate. In one embodiment verified by this invention, the small-molecule anti-inflammatory drug is resveratrol (Res). Res itself has poor water solubility and a short half-life. By loading it into the hydrophobic cavity inside G5 through hydrophobic interactions, the solubility, stability, and bioavailability of Res can be effectively improved. Verification showed that resveratrol delivered via the above-mentioned nanoplatform can effectively downregulate reactive oxygen species levels at the target site, and the combination of Res and si-Cyp2e1 has a synergistic effect, increasing the gene silencing ability of si-Cyp2e1. In this embodiment, the ratio of the number of Gal-G5 molecules to Res molecules is 12 to 16:1.

[0011] The second aspect provides a method for preparing the nanoplatform for co-delivering small interfering RNA and small molecule drug as described in the first aspect, wherein the small molecule drug is resveratrol (Res), and the preparation method includes the following steps: (1) Preparation of G5-Gal: G5 is added to activated Gal, and the reaction is carried out at room temperature for 2.5 to 3.5 days. The component with a molecular weight of 3500 Da or higher is retained and freeze-dried to obtain Gal-G5; (2) Preparation of Res@G5-Gal: Under light-protected conditions, the aqueous solution of Gal-G5 and the organic solution of Res are mixed and reacted overnight. The supernatant is retained by centrifugation to obtain Res@G5-Gal; (3) Preparation of Res@G5-Gal / si-Cyp2e1 complex: The aqueous solution of Res@G5-Gal is mixed with si-Cyp2e1 and allowed to stand at room temperature for 25 to 35 minutes to obtain the nanoplatform Res@G5-Gal / si-Cyp2e1.

[0012] The preparation method described in the second aspect above does not require high temperature and high pressure conditions, special reactor dishes, or complex purification or post-processing steps. The preparation process is simple, the raw materials are readily available and the cost is economical, and it has a high degree of operational safety for production technicians.

[0013] Furthermore, the present invention also provides the following preferred technical solution: In step (1) above, the core of Gal lies in the activation of the carboxyl group, and the activation method is such as adding an activating synergist, such as 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) + N-hydroxysuccinimide (NHS) or EDC. HCl + Sulfo-NHS or dicyclohexylcarbodiimide (DCC), etc. In one embodiment verified by this invention, the activation of Gal is specifically performed as follows: EDC·HCl is added to the Gal solution and stirred at room temperature for 25-35 min; then NHS is added and the reaction is stirred for 2-4 h for activation. In this embodiment, the molar ratio of Gal to EDC and NHS is 1:10-20:10-20, and the molar ratio of G5 to Gal is 1:30-40. A suitable solvent for this activation reaction is DMSO.

[0014] The filter membrane used for the interception is preferably a cellulose dialysis membrane, which is dialyzed in ultrapure water for 2 to 3 days, with the water changed 2 to 4 times a day, each time using 1.5 to 2.5 L of ultrapure water.

[0015] In step (2) above, the organic solution of Res can be a methanol solution or an ethanol solution, and the molar ratio of G5-Gal to Res is 1:15 ~ 25.

[0016] In step (3) above, the mass ratio of G5-Gal to si-Cyp2e1 is greater than or equal to 16:1.

[0017] During the research process of this invention, tests were also conducted on si-Cyp2e1 with different sequences. In the embodiment with the best effect, the sequence of si-Cyp2e1 is shown in SEQ ID NO:1.

[0018] Thirdly, a pharmaceutical composition is provided, wherein the composition comprises an active dose of the nanoplatform described in the first aspect.

[0019] In the third aspect, the "active dose" refers to the dosage at which the drug composition, when administered to a subject, can achieve the purpose of preventing, improving, treating the disease, or improving the prognosis. Since the present invention has demonstrated the in vitro and in vivo therapeutic activity of the above-mentioned co-delivered small interfering RNA and small molecule drug nanoplatform for alcohol-related liver disease, specifying the specific "active dose" value is technical content that can be obtained by those skilled in the art based on conventional research methods.

[0020] In some embodiments, the pharmaceutical composition described above may include, in addition to the glycosylated dendritic macromolecular nanoplatform that co-delivers small interfering RNA and small molecule drugs, other active ingredients, excipients, or pharmaceutically acceptable carriers for treating alcohol-related liver disease.

[0021] The active ingredients used to treat or alleviate alcohol-related liver disease include, but are not limited to, natural plant extracts (such as silymarin, puerarin, ginsenosides, and ursolic acid), small molecule compounds (prednisolone, N-acetylcysteine, and vitamin E), and targeted therapy drugs (obeticholic acid, sanistois, and anaspirin).

[0022] The auxiliary ingredients include antiemetics, granulocyte colony-stimulating factors, thrombopoietin, liver-protecting ingredients, immune enhancers, and intestinal flora regulators.

[0023] The pharmaceutically acceptable carriers include, but are not limited to, absorption enhancers, disintegrants, osmotic pressure regulators, solubilizers, emulsifiers, binders, diluents, wetting agents, pH adjusters, antioxidants, colorants, or flavoring agents.

[0024] Fourthly, the application of the co-delivery small interfering RNA and small molecule drug nanoplatform described in the first aspect and the pharmaceutical composition described in the third aspect is provided.

[0025] The applications described in the fourth aspect include, but are not limited to: (1) preparing drugs for alcohol-related liver disease; (2) preparing health products for improving alcoholic liver disease; and (3) preparing special medical foods suitable for patients with alcoholic liver disease.

[0026] The alcohol-related liver disease mentioned above refers to a series of liver-damaging diseases caused by long-term excessive drinking, specifically including alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, alcoholic cirrhosis, hepatitis, or liver cancer.

[0027] In (1) above, the purpose of administering the drug includes preventing, improving or treating the aforementioned diseases.

[0028] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention synthesizes a glycosylated dendritic macromolecular nanoplatform, Res@G5-Gal / si-Cyp2e1, which co-delivers small interfering RNA and small molecule drugs. Utilizing galactose, it achieves liver-targeting of therapeutic components, significantly increasing drug concentration at liver lesions and reducing systemic exposure and side effects. On one hand, the resveratrol loaded in this invention can directly neutralize existing ROS and exert anti-inflammatory effects, alleviating secondary inflammatory damage. On the other hand, the si-Cyp2e1 delivered in this invention can specifically silence the Cyp2e1 gene, inhibiting the overexpression of the key enzyme CYP2E1 in ethanol metabolism at its source, reducing ROS generation, and thus blocking the core initiation step of alcoholic liver injury. The two work synergistically in time and space, achieving multi-stage intervention in the pathological process of ALD.

[0029] Through the rational design and application of dendritic macromolecular nanomaterials, this invention overcomes the shortcomings of resveratrol, such as poor water solubility and instability, and protects siRNA from nuclease degradation. It also overcomes some limitations of dendritic macromolecules in targeting hepatocytes and in combination with other treatment modalities, and will have guiding significance for the clinical alleviation of alcohol-related liver disease, showing potential application prospects. Attached Figure Description

[0030] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0031] Figure 1 is a flowchart of the synthesis and treatment process of the nanoplatform for co-delivering small interfering RNA and small molecule drugs according to the present invention; Figure 1A is a flowchart of the synthesis process of the nanoplatform; Figure 1B is a schematic diagram of the principle by which the nanoplatform exerts gene-drug therapeutic effects; Figure 2 shows the characterization results of G5-Gal prepared according to the present invention; Figure 2A is... 1Figure 2 shows the 1H NMR spectrum, with B being the Fourier transform near-infrared spectrum. Figure 3 shows the biocompatibility-related properties of G5-Gal prepared in this invention under different concentration conditions; Figure 3A shows the anti-protein adsorption results, and Figure 3B shows the blood compatibility analysis. Figure 4 shows the phagocytosis of AML-12 cells treated with Cy5-G5 and Cy5-G5-Gal prepared in this invention for 24 hours under different concentration conditions; Figure 4A shows the flow cytometry results, and Figure 4B shows the quantitative analysis. Figure 5 shows the fluorescence imaging of ex vivo organs at different time points after tail vein injection of Cy5-G5 and Cy5-G5-Gal prepared in this invention. Figure 6 shows the G5-Gal prepared in this invention at different mass... The gene compression capacity of three small interfering RNAs (si-Cyp2e1-1, si-Cyp2e1-2, and si-Cyp2e1-3) was compared. Figure 6A shows the gel retardation experiment of G5-Gal / si-Cyp2e1-1, Figure 6B shows the gel retardation experiment of G5-Gal / si-Cyp2e1-2, and Figure 6C shows the gel retardation experiment of G5-Gal / si-Cyp2e1-3. Figure 7 shows the results of co-incubating AML-12 cells with G5-Gal / si-Cyp2e1-1, G5-Gal / si-Cyp2e1-2, and G5-Gal / si-Cyp2e1-3 prepared in this invention for 12 hours. The relative expression of Cyp2e1 mRNA; Figure 8 shows the gene compression ability of G5-Gal prepared in this invention to the selected small interfering RNA at different mass ratios. In Figure 8, A is the hydrodynamic diameter diagram of G5-Gal / si-Cyp2e1, and B is the surface potential diagram of G5-Gal / si-Cyp2e1; Figure 9 shows the cell viability diagrams of AML-12 cells treated with G5-Gal and G5-Gal / si-Cyp2e1 prepared in this invention at different concentrations for 24 hours. In Figure 9, A is the cell viability diagram of G5-Gal, and B is the cell viability diagram of G5-Gal / si-Cyp2e1; Figure 10 shows the gene compression ability of G5-Gal prepared in this invention to the selected small interfering RNA at different mass ratios. The phagocytosis of AML-12 cells after treatment with 5-Gal / si-Cyp2e1 at different mass ratios for 24 hours was shown in Figure 10. Figure 10A shows the results of flow cytometry, and Figure 10B shows the quantitative analysis. Figure 11 shows the UV-Vis spectra of Res@G5-Gal prepared in this invention and Res alone. Figure 12 shows the cytotoxicity analysis of AML-12 cells after treatment with Res@G5-Gal and Res@G5-Gal / si-Cyp2e1 prepared in this invention at different concentrations for 24 hours. Figure 12A shows the cell viability of Res@G5-Gal, and Figure 12B shows the cell viability of Res@G5-Gal / si-Cyp2e1.Figure 13 shows the ROS levels in ethanol-pretreated AML-12 cells after 24 hours of treatment with G5-Gal / si-Cyp2e1, Res@G5-Gal, and Res@G5-Gal / si-Cyp2e1 prepared according to this invention; Figure 14 shows the relative expression of Cyp2e1 mRNA in ethanol-pretreated AML-12 cells after 24 hours of treatment with G5-Gal / si-Cyp2e1, Res@G5-Gal, and Res@G5-Gal / si-Cyp2e1 prepared according to this invention; Figure 15 shows the ROS levels in ethanol-pretreated AML-12 cells after 24 hours of treatment with G5-Gal / si-Cyp2e1, Res@G5-Gal, and Res@G5-Gal / si-Cyp2e1 prepared according to this invention. Lipid deposition analysis after 24 hours of treatment with G5-Gal / si-Cyp2e1; Figure 15A shows Bodipy staining, Figure 15B shows triglyceride content, and Figure 15C shows total cholesterol content; Figure 16 shows the relative expression of ADRP / Perilipin2 mRNA in AML-12 cells pretreated with alcohol after 24 hours of treatment with G5-Gal / si-Cyp2e1, Res@G5-Gal, and Res@G5-Gal / si-Cyp2e1 prepared in this invention; Figure 17 shows the treatment results of in vivo treatment of alcohol-related liver disease with Res@G5-Gal / si-Cyp2e1 prepared in this invention; Figure 17A shows a mouse model of alcohol-related liver disease. The flowcharts for construction and treatment are shown in Figure 17. Figure 17B shows the mouse body weight change, Figure 17C shows the mouse liver weight change, Figure 17D shows the mouse liver weight / body weight ratio, Figure 17E shows the mouse alanine aminotransferase (ALT) level, and Figure 17F shows the mouse aspartate aminotransferase (AST) level. Figure 18 shows the lipid deposition and oxidative stress results after in vivo treatment of alcohol-related liver disease with Res@G5-Gal / si-Cyp2e1 prepared in this invention. Figure 18A shows triglyceride content, Figure 18B shows total cholesterol content, Figure 18C shows malondialdehyde (MDA) content, and Figure 18D shows hematoxylin-eosin (H&E) staining and Oil Red O staining. Figure 19 shows the Res@G5-Gal prepared in this invention. Figure 19 shows the expression of related genes after in vivo treatment of alcohol-related liver disease with / si-Cyp2e1; Figure 19A shows the relative expression of Cyp2e1 mRNA, Figure 19B shows the WB electrophoresis bands of different proteins, Figure 19C shows the relative expression of CYP2E1 protein, Figure 19D shows the relative expression of ADRP / Perilipin protein, and Figure 19E shows the relative expression of GPR78 protein; Figure 20 shows the H&E staining of heart, liver, spleen, lung, kidney, and brain tissue sections extracted from healthy mice after intravenous injection of G5-Gal / si-Cyp2e1, Res@G5-Gal, and Res@G5-Gal / si-Cyp2e1 prepared in this invention.Figure 21 shows the organ coefficient analysis of healthy mice after intravenous injection of G5-Gal / si-Cyp2e1, Res@G5-Gal, and Res@G5-Gal / si-Cyp2e1 prepared according to this invention; Figure 22 shows the results of blood routine related indicators in healthy mice after intravenous injection of G5-Gal / si-Cyp2e1, Res@G5-Gal, and Res@G5-Gal / si-Cyp2e1 prepared according to this invention; Figure 23 shows the results of blood biochemical related indicators in healthy mice after intravenous injection of G5-Gal / si-Cyp2e1, Res@G5-Gal, and Res@G5-Gal / si-Cyp2e1 prepared according to this invention. Detailed Implementation

[0032] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0034] As described in the background section, there is currently no specific treatment for ALD. This invention addresses the pathogenesis of ALD by designing a targeted delivery of anti-inflammatory drugs and using small interfering RNA to silence cytochrome P4502E1, thereby improving oxidative stress and lipid deposition caused by alcoholic liver disease.

[0035] The co-delivery system of the above-mentioned drug and siRNA uses amino-terminated fifth-generation (G5) polyamidoamine (PAMAM) dendritic macromolecules as nanocarriers. In order to verify the feasibility of the above-mentioned co-delivery system as a treatment for alcoholic liver disease, the present invention investigated the following scheme: (1) Preparation of G5-Gal: 50 mg G5, 11.04 mg Gal, 99.8 mg EDC·HCl and 59.9 mg NHS were weighed and dissolved in 3 mL, 2 mL, 5 mL and 2 mL DMSO, respectively. Under room temperature stirring conditions, EDC solution was added dropwise to the Gal solution in the reaction flask. After stirring at room temperature for 30 min, NHS solution was added dropwise to the above mixed solution. After stirring at room temperature for 3 h, the activated Gal solution was obtained. The G5 solution was then added dropwise to the activated Gal solution, and the reaction was continued at room temperature with stirring for 3 days. The resulting product was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in ultrapure water for 3 days (2 L × 3 times / day). After freeze-drying, G5-Gal powder was obtained and stored at -20 ℃ for later use.

[0036] (2) Preparation of G5-Gal / si-Cyp2e1 complex: Weigh 2 mg of G5-Gal powder obtained in step (1) and dissolve it in 1 mL of ultrapure water. Mix it with 1 μg of si-Cyp2e1 according to different mass ratios (m(G5-Gal): m(si-Cyp2e1) are 2, 4, 8, 16, 32, 48, 64:1). Let it stand at room temperature for 25 to 35 minutes to obtain G5-Gal / si-Cyp2e1 complexes with different proportions.

[0037] (3) Preparation of Res@G5-Gal: Weigh 10 mg of G5-Gal obtained in step (1) above and dissolve it in 1 mL of ultrapure water, then transfer it to a brown reaction flask; under light-protected conditions, weigh 1.45 mg of Res and dissolve it in 500 μL of methanol solution. Add the Res methanol solution dropwise to the G5-Gal solution in the brown reaction flask. After completion, wrap the mouth of the flask with tin foil, tie it tightly with a rubber band, and then poke a hole with a needle. Let the reaction proceed overnight at half the rated capacity. The next day, aspirate the solution from the reaction flask into a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 15 min, and aspirate the supernatant to obtain the Res@G5-Gal solution, which can be stored at 4 ℃ for later use.

[0038] (4) Preparation of Res@G5-Gal / si-Cyp2e1 complex: Take the Res@G5-Gal solution obtained in step (3), add si-Cyp2e1 at a mass ratio of G5-Gal to si-Cyp2e1 of 16:1, mix gently, and let stand at room temperature for 25 to 35 minutes to obtain the Res@G5-Gal / si-Cyp2e1 complex.

[0039] To verify whether the nanomaterials obtained in steps (1)-(4) above can achieve liver targeting and exert antioxidant therapeutic activity, this application also verified the performance of the above four nanomaterials respectively: (I) G5-Gal1, Morphological characterization The G5-Gal prepared in step (1) of Example 1 above was subjected to nuclear magnetic resonance hydrogen spectroscopy (NMR 1H N ... 1 Characterization using H NMR and Fourier transform infrared spectroscopy (FI-IR) techniques. 1 The 1H NMR characterization results are shown in Figure 2A: 2.2 ~ 3.4 ppm are the characteristic proton peaks of G5.NH2, and 3.5 ~ 4.5 ppm are the characteristic proton peaks of Gal. Based on their integral area ratio, it was calculated that each G5 is linked to 15 Gal molecules. The FI-IR characterization results are shown in Figure 2B: G5-Gal at 1092 cm⁻¹ -1 The COC absorption peak was observed, while the G5 dendritic macromolecule alone did not. These results demonstrate the successful synthesis of G5-Gal.

[0040] 2. Biocompatibility Characterization (1) Characterization of Anti-protein Adsorption Performance The anti-protein adsorption performance of G5-Gal prepared in step (1) of Example 1 was characterized. Bovine serum albumin aqueous solution (2 mg / mL) was mixed with G5 and G5-Gal aqueous solutions of different concentrations (0.25, 0.5 and 1 mg / mL) at a volume ratio of 1:1. After being placed at 37 ℃ for 4 h, the mixture was centrifuged (8000 rpm / min, 5 min) and the supernatant was collected. The protein content in the supernatant was detected using a BCA quantitative kit according to standard specifications. The results are shown in Figure 3A. Compared with G5 alone, G5-Gal had the highest supernatant protein concentration at all tested concentrations, indicating that Gal-G5 has excellent anti-protein adsorption performance.

[0041] (2) Blood compatibility characterization: The blood compatibility of the G5-Gal prepared in step (1) of Example 1 was characterized. In the material preparation section, G5-Gal solutions with concentration gradients of 50, 100, 200, 500 and 1000 nM were prepared using PBS as solvent. PBS and 1% Triton X-100 were used as negative and positive controls, respectively. In the blood preparation section, blood was collected from the eyeballs of healthy mice, and 1.5 mL of blood was collected in an anticoagulant tube. After washing three times with PBS, the blood was resuspended in 5 mL of PBS to prepare a red blood cell suspension. 100 μL of the red blood cell suspension was thoroughly mixed with 900 μL of each of the above concentrations of solution, incubated at 37 ℃ for 2 h, and then centrifuged at 2000 rpm for 5 min to obtain the hemolyzed sample. Finally, the supernatant was taken, and its absorbance at a wavelength of 540 nm was measured using a UV-Vis spectrophotometer. As shown in Figure 3B, the hemolysis rate of G5-Gal was less than 5% within the experimental concentration range. This indicates that G5-Gal does not fuse with blood within the test concentration range and meets the safety requirements for in vivo experiments.

[0042] 2. Targeting Test (1) In vitro targeting performance characterization Using mouse liver parenchyma (AML-12) cells as model cells, the targeting performance of G5-Gal was characterized in vitro. The targeting effect was evaluated by detecting the amount of G5-Gal phagocytosed by cells. First, AML-12 cells were seeded in 12-well plates at a density of 100,000 cells / well. DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin (100 U / mL penicillin, 100 U / mL streptomycin) was used and cultured overnight at 37 ℃ and 5% CO2. The medium was then replaced with medium containing fluorescent dye Cy5-labeled G5 and Gal-G5 (denoted as Gy5-G5 and Gy5-G5-Gal, respectively) (material concentrations of 1 μM and 2 μM, respectively), and PBS was used as a negative control. The medium was co-cultured with AML-12 cells at 37 ℃ for 4 h. Cells were then washed twice with PBS buffer, digested with trypsin, centrifuged (1200 rpm, 3 min), collected, and resuspended in PBS. Finally, the fluorescence intensity of the cells was detected by flow cytometry. The results are shown in Figure 4. Compared with the PBS-treated group, the fluorescence intensity of Gy5-G5 and Gy5-G5-Gal treatments was significantly increased, and the increase was directly proportional to the concentration. Compared with the Gy5-G5-treated group, AML-12 cells treated with both concentrations of Gy5-G5-Gal showed higher fluorescence intensity, indicating that G5-Gal has the ability to target AML-12 cells.

[0043] (2) In vivo targeting performance characterization: 6-8 week old male C57BL / 6 healthy mice were selected (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China, Beijing). All animal experiments were strictly conducted in accordance with the standards of the Animal Protection Association, the same below). First, the healthy mice were randomly divided into two groups and injected with 100 μL of the same concentration of Gy5-G5 and Gy5-G5-Gal via the tail vein, respectively. The major organs (including heart, liver, spleen, lung, kidney and brain) were dissected at 0, 15, 30, 45, 60, 90, 120, 240 and 600 min after injection, and the fluorescence distribution was observed using a mouse in vivo fluorescence imaging system (IVIS SPECTRUM CT, PerkinElmer, USA). As shown in Figure 5, fluorescent signals were observed in the lungs of both groups of mice 15 minutes after injection. The fluorescent signals gradually weakened over time, but the fluorescence intensity of the liver region of Gy5-G5-Gal was always higher than that of the Cy5-G5 group within a certain observation time (0 ~ 600 min). This proves that Gal can carry G5 to the liver region and achieve liver-targeting effects in vivo.

[0044] (II) G5-Gal / si-Cyp2e1 1. Gene compression capacity The G5-Gal / si-Cyp2e1 complex prepared in step (2) above was subjected to a gel retardation experiment to evaluate the gene compression capacity of G5-Gal in step (1). An 8-well agarose gel (1.0% w / v) containing ethidium bromide (1 mg / mL) was prepared and allowed to solidify at room temperature. G5-Gal solution was mixed with 1 μg si-Cyp2e1 at different mass ratios (2, 4, 8, 16, 32, 64:1) and incubated for 15-20 min to prepare the G5-Gal / si-Cyp2e1 complex, with DNA marker and naked si-RNA as controls. Then, the corresponding G5-Gal / si-Cyp2e1 complex dyes were mixed and added to the wells of agarose gels. The migration of si-Cyp2e1 in the gel was analyzed using a gel imaging system (80 V, 40 min). Three different gene sequences of si-Cyp2e1 (named si-Cyp2e1-1, si-Cyp2e1-2, and si-Cyp2e1-3) were selected, and gel retardation experiments were performed on all of them. Referring to Figure 7, for the three sequences si-Cyp2e1-1, si-Cyp2e1-2, and si-Cyp2e1-3, when the mass ratio of G5-Gal to si-Cyp2e1 is greater than or equal to 16:1, G5-Gal can prevent the migration of si-Cyp2e1 in the agarose gel, that is, G5-Gal completely compresses si-Cyp2e1, forming a G5-Gal / si-Cyp2e1 complex.

[0045] 2. Gene Optimization Screening: Using AML-12 cells as the model cell and the mRNA expression level of the CYP2E1 gene as the evaluation criterion, three genes, si-Cyp2e1-1, si-Cyp2e1-2, and si-Cyp2e1-3, were screened. First, AML-12 cells were seeded in 12-well plates at a density of 100,000 cells / well. After overnight culture, the culture medium was replaced with a G5-Gal / si-Cyp2e1 complex at a mass ratio of 16:1, with si-NC as a negative control (si-NC is a disordered RNA gene sequence with the same number of base pairs as si-Cyp2e1 and cannot silence the expression of the CYP2E1 gene). The cells were co-cultured with AML-12 cells at 37°C for 12 h. Cells were lysed with cell lysis buffer, and total RNA was extracted. The mRNA expression level of the Cyp2e1 gene in the cells was detected by reverse transcription and quantitative real-time PCR. The results are shown in Figure 7. G5-Gal / si-NC showed high Cyp2e1 expression, while the expression level of the G5-Gal / si-Cyp2e1 complex was reduced, especially in si-Cyp2e1-2 and si-Cyp2e1-3, which showed significant differences compared to the control group. This indicates that G5-Gal is an excellent gene vector that can effectively load si-Cyp2e1 to inhibit Cyp2e1 gene expression in AML-12 cells. Since the si-Cyp2e1-3 treatment group had the lowest mRNA expression level, si-Cyp2e1-3 was selected for subsequent experiments.

[0046] 3. Hydrodynamic Diameter and Surface Potential: The hydrodynamic diameter and surface potential of the G5-Gal / si-Cyp2e1 complex prepared in step (2) above were characterized. Specifically, the G5-Gal / si-Cyp2e1 complex (mass ratios of 16, 32, 64, 96, and 128:1, with si-Cyp2e1 dosage of 5 μg) was diluted with ultrapure water to a final volume of 1 mL and characterized using a Malvern laser particle size analyzer (Malvern, MK, 633 nm laser). Referring to Figure 8, under different mass ratios, the hydrodynamic particle size of the complex was approximately between 230 and 272 nm, and the surface potential was between 36 and 49 mV. The particle size and potential generally showed a stable state and were within a suitable range for gene delivery, making them suitable for cell adsorption and endocytosis, which is beneficial for intracellular gene delivery.

[0047] 4. In vitro evaluation: Using AML-12 cells as a model cell, G5-Gal and G5-Gal / si-Cyp2e1 were evaluated in vitro.

[0048] (1) Cell viability assay: The cell viability of G5-Gal prepared in step (1) and G5-Gal / si-Cyp2e1 prepared in step (2) was tested using the CCK-8 assay. AML-12 cells were loaded at 1×10⁻⁶ cells per cell line. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured overnight at 37 °C with 5% CO2 in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin (100 U / mL penicillin and 100 U / mL streptomycin). The medium was then replaced with different concentrations of G5-Gal and G5-Gal / si-Cyp2e1, with G5-Gal concentrations of 0, 10, 25, 50, 75, 100, 150, and 200 μM, and cultured for another 24 h. The medium was then discarded, and cell viability was assessed using the CCK-8 assay kit according to the manufacturer's instructions. As shown in Figure 9, G5-Gal and G5-Gal / si-Cyp2e1 showed no significant toxicity to AML-12 cells at all tested concentrations, with cell viability greater than 85%, indicating good cell compatibility. Notably, the cell viability of G5-Gal / si-Cyp2e1 was slightly higher than that of G5-Gal, which may be because the loading of si-Cyp2e1 reduced the positive charge of G5-Gal, making it more conducive to cell growth.

[0049] (2) Cellular uptake of G5-Gal / si-Cyp2e1: si-Cyp2e1 labeled with FAM (green fluorescent) was used. The phagocytic efficiency of the G5-Gal / si-Cyp2e1 complex at different mass ratios was detected by flow cytometry. AML-12 cells were seeded in 12-well plates at a density of 100,000 cells / well and cultured overnight at 37 ℃ and 5% CO2 using DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin (100 U / mL penicillin and 100 U / mL streptomycin). The culture medium (containing 2% fetal bovine serum) was changed to different mass ratios (0, 8, 16, 32, 64:1) of the G5-Gal / si-Cyp2e1 complex (1 μg si-Cyp2e1 per well), and AML-12 cells were co-cultured at 37°C for 4–6 h with PBS and naked si-Cyp2e1 as negative controls. Afterward, the cells were washed twice with PBS buffer, digested with trypsin, centrifuged (1200 rpm, 3 min), collected, and resuspended in PBS. Finally, the fluorescence intensity of the cells was detected by flow cytometry. The results are shown in Figures 10A and 10B. Compared with the PBS and naked si-Cyp2e1 treatment groups, the fluorescence intensity of the G5-Gal / si-Cyp2e1 treatment group was significantly increased, reaching a maximum at a 16:1 ratio. This indicates that AML-12 cells took up the most G5-Gal / si-Cyp2e1 at this mass ratio.

[0050] (III) Res@G5-Gal and Res@G5-Gal / si-Cyp2e11, UV-Vis Spectroscopy Characterization: The Res@G5-Gal prepared in step (1) above and Res alone were subjected to UV-vis testing. Both were prepared into a homogeneous solution of 0.1 mg / mL and scanned in the full wavelength range of 200-500 nm using a UV spectrophotometer (Agilent Cary 5000). The results are shown in Figure 11. Both Res@G5-Gal and Res alone showed significant characteristic absorption peaks in the wavelength range of 270-360 nm, with the maximum absorption peak located at around 306 nm. This is the characteristic absorption peak of Res, which proves that Res was successfully loaded into G5-Gal. In addition, the precipitate after centrifugation was dissolved in methanol and its ultraviolet absorption at 306 nm was tested. According to the standard curve and calculation formula, one G5-Gal was loaded with 20 Res molecules, the drug loading efficiency was 55%, and the drug loading capacity was 14.4%.

[0051] 2. In vitro evaluation: Using AML-12 cells as a model cell, G5-Gal / si-Cyp2e1, Res@G5-Gal, and Res@G5-Gal / si-Cyp2e1 were evaluated in vitro.

[0052] (1) Cell viability assay: The Res@G5-Gal and Res@G5-Gal / si-Cyp2e1 prepared in steps (2) and (3) were subjected to cell viability assay using the CCK-8 assay. AML-12 cells were sputtered at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured overnight. The medium was then replaced with different concentrations of Res@G5-Gal and Res@G5-Gal / si-Cyp2e1, with Res concentrations of 0, 15, 25, 50, 75, 100, 150, and 200 μM, and cultured for another 24 h. The medium was then discarded, and cell viability was assessed using the CCK-8 assay kit according to the manufacturer's instructions. The results are shown in Figure 12. Cell viability decreased with increasing Res@G5-Gal and Res@G5-Gal / si-Cyp2e1 concentrations. However, even at the maximum tested concentration, the cell viability after Res@G5-Gal treatment was still greater than 70%, and the cell viability after Res@G5-Gal / si-Cyp2e1 treatment was greater than 80%.

[0053] (2) ROS level test: The intracellular ROS level was detected using the DCFH-DA reactive oxygen species probe. AML-12 cells were seeded in 12-well plates at a density of 100,000 cells / well and cultured overnight at 37 ℃ and 5% CO2 in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Fresh medium containing 100 mM ethanol (containing 2% fetal bovine serum) was added, and after 24 hours, the medium was replaced with a complex containing G5-Gal / si-Cyp2e1, Res@G5-Gal, or Res@G5-Gal / si-Cyp2e1 (G5-Gal to si-Cyp2e1 mass ratio of 16:1, 1 μg si-Cyp2e1 per well). The experimental group treated with PBS instead of ethanol served as the negative control. After culturing for another 24 h, the cells were incubated with culture medium containing the DCFH-DA probe in a dark incubator for 20 minutes. Finally, the culture medium was discarded, and the cells were washed with PBS buffer. 300 μL of PBS was added to each well to maintain cell morphology, and ROS expression was observed using an inverted fluorescence microscope. The results are shown in Figure 13. Compared to the PBS group, the reactive oxygen species (ROS) level in the alcohol-treated group was significantly increased. This is due to oxidative stress generated by alcohol metabolism itself, mitochondrial damage caused by alcohol, and the production of ROS by the strong oxidase CYP2E1 induced by alcohol. Treatment with G5-Gal / si-Cyp2e1 and Res@G5-Gal reduced the intracellular ROS level to some extent, indicating that downregulation of the Cyp2e1 gene and Res can partially downregulate ROS levels. The ROS level decreased most significantly after Res@G5-Gal / si-Cyp2e1 treatment, indicating that si-Cyp2e1 and Res can work simultaneously to scavenge ROS.

[0054] (3) Cyp2e1 mRNA expression was detected using RT-qPCR. AML-12 cells were seeded in 12-well plates at a density of 100,000 cells / well and cultured overnight at 37 ℃ and 5% CO2 in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Fresh medium containing 100 mM ethanol (containing 2% fetal bovine serum) was added, and after 24 hours, the medium was replaced with a complex containing G5-Gal / si-Cyp2e1, Res@G5-Gal, or Res@G5-Gal / si-Cyp2e1 (G5-Gal to si-Cyp2e1 mass ratio of 16:1, 1 μg si-Cyp2e1 per well). The cells were cultured for another 24 h. The experimental group treated with PBS instead of ethanol served as the negative control. Cells were lysed using cell lysis buffer, and total RNA was extracted. The mRNA expression level of the CYP2E1 gene was detected by reverse transcription and quantitative real-time PCR. The results are shown in Figure 14. Compared with the PBS group, the expression level of the CYP2E1 gene was significantly increased after alcohol pretreatment. This is because excessive alcohol induces activation of the CYP2E1 pathway, thereby upregulating CYP2E1 gene expression. Res@G5-Gal also showed a significant increase, with no significant difference compared to the alcohol group. The treatment group contained si-Cyp2e1, which can silence CYP2E1 expression, thus effectively reducing the mRNA level of the CYP2E1 gene. Notably, the expression level of the CYP2E1 gene was significantly lower after Res@G5-Gal / si-Cyp2e1 treatment than in the G5-Gal / si-Cyp2e1 treatment group, indicating a synergistic effect between Res and si-Cyp2e1, increasing the gene silencing ability of si-Cyp2e1.

[0055] (4) Lipid deposition analysis: Lipid deposition levels of lipid droplets, triglycerides, and total cholesterol in AML-12 cells were detected using Bodipy dye and an ELISA kit. AML-12 cells were seeded in 12-well plates at a density of 100,000 cells / well and cultured overnight at 37 °C and 5% CO2 in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Fresh medium containing 100 mM ethanol (containing 2% fetal bovine serum) was added, and after 24 hours, the medium was replaced with a complex containing G5-Gal / si-Cyp2e1, Res@G5-Gal, or Res@G5-Gal / si-Cyp2e1 (G5-Gal to si-Cyp2e1 mass ratio of 16:1, 1 μg si-Cyp2e1 per well). The cells were cultured for another 24 h. The experimental group treated with PBS instead of ethanol served as the negative control. Cells were incubated with culture medium containing the Bodipy probe at room temperature for 15 minutes under light-protected conditions. The culture medium was then discarded, and the cells were washed with PBS buffer. 300 μL of PBS was added to each well to maintain cell morphology. Lipid droplet expression was observed using an inverted fluorescence microscope. Cells were then digested, collected, resuspended in 200 μL of PBS, and sonicated. After incubation with working solution at 37 °C for 10 min, absorbance was measured at 500 nm. The results are shown in Figure 15. Compared to the PBS group, the levels of lipid droplets, triglycerides, and total cholesterol were significantly increased after alcohol pretreatment, which is because alcohol disrupts lipid metabolism homeostasis. The levels of these three substances decreased after treatment with G5-Gal / si-Cyp2e1 and Res@G5-Gal. Notably, the levels of these three substances were lowest after treatment with Res@G5-Gal / si-Cyp2e1.

[0056] (5) The mRNA expression of ADRP / Perilipin2 mRNA was further evaluated by RT-qPCR to assess the inhibitory effect of Res@G5-Gal / si-Cyp2e1 on lipid droplet formation. AML-12 cells were seeded in 12-well plates at a density of 100,000 cells / well and cultured overnight at 37 ℃ and 5% CO2 in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin (100 U / mL penicillin and 100 U / mL streptomycin). Fresh culture medium containing 100 mM ethanol (containing 2% fetal bovine serum, the same below) was added. After 24 hours, the medium was replaced with a complex containing G5-Gal / si-Cyp2e1, Res@G5-Gal, or Res@G5-Gal / si-Cyp2e1 (G5-Gal to si-Cyp2e1 mass ratio of 16:1, 1 μg si-Cyp2e1 per well), and cultured for another 24 h. Cells were lysed with cell lysis buffer, and total RNA was extracted. The mRNA expression level of the Cyp2e1 gene in the cells was detected by reverse transcription and quantitative real-time PCR. The results are shown in Figure 16 of the instruction manual. Compared with the PBS group, the expression level of ADRP / Perilipin2 gene was significantly increased after alcohol pretreatment, indicating that alcohol leads to a significant increase in lipid droplet levels. The ADRP / Perilipin2 gene level in the G5-Gal / si-Cyp2e1 treatment group decreased but was not significantly different. However, the ADRP / Perilipin2 gene level was significantly decreased in cells treated with Res@G5-Gal and Res@G5-Gal / si-Cyp2e1, and the difference was significant compared with the alcohol group. This indicates that Res has a direct effect on inhibiting lipid droplet formation.

[0057] 7. In vivo activity evaluation (1) Evaluation of the therapeutic effect of Res@G5-Gal / si-Cyp2e1 on alcohol-related liver disease: Male C57BL / 6 mice aged 6-8 weeks were selected. Then, according to the established protocol (Figure 17A), an alcohol-related liver disease mouse model (NIAAA model, also known as Gao-Binge model) was constructed and Res@G5-Gal / si-Cyp2e1 was injected to evaluate the therapeutic effect of Res@G5-Gal / si-Cyp2e1 on alcohol-related liver disease. The model mice were randomly divided into four groups of 6 mice each: alcohol group, G5-Gal / si-Cyp2e1 group, Res@G5-Gal group and Res@G5-Gal / si-Cyp2e1 group. Healthy mice were used as the negative control group. PBS or different therapeutic drugs were injected via tail vein (100 μL, [Res] = 5 mg / kg). The weight of the mice was recorded every 2 days. The results are shown in Figure 17B. Except for the alcohol group, the body weight of the other groups steadily increased, indicating that continuous alcohol intake is harmful to the health of mice and causes weight loss. All treatment groups can alleviate the harm of alcohol intake, and G5-Gal / si-Cyp2e1, Res@G5-Gal and Res@G5-Gal / si-Cyp2e1 do not produce significant side effects in mice.

[0058] Eleven days after treatment, blood was collected from the eyes of the mice, and liver tissue was dissected and weighed. The results are shown in Figures 17C and D. Although the liver weight of the mice did not change significantly, the liver weight / body weight ratio was increased in the alcohol group, but decreased after treatment in all different groups. Serum was extracted from the collected blood, and ALT and AST were detected using an ELISA kit. The results are shown in Figures 17E and F. Compared with the control group, both ALT and AST levels in the alcohol group were significantly increased, indicating that excessive alcohol intake can impair liver function. For ALT, treatment with G5-Gal / si-Cyp2e1, Res@G5-Gal, and Res@G5-Gal / si-Cyp2e1 all showed significant decreases and statistically significant differences. For AST, the treatment groups with Res@G5-Gal and Res@G5-Gal / si-Cyp2e1 showed significant decreases and statistically significant differences. Therefore, Res@G5-Gal / si-Cyp2e1 has the best effect in alleviating alcohol-related liver disease.

[0059] (3) Analysis of lipid deposition and oxidative stress results: The serum samples from (2) above were tested for the levels of triglycerides, total cholesterol and malondialdehyde. The results are shown in Figure 18, A, B and C. Compared with the control group, the levels of triglycerides, total cholesterol and malondialdehyde in the alcohol group were significantly increased, indicating that excessive alcohol intake caused oxidative stress in the liver, which led to lipid deposition. After treatment with Res@G5-Gal / si-Cyp2e1, the levels of triglycerides, total cholesterol and malondialdehyde were significantly reduced, indicating that Res@G5-Gal / si-Cyp2e1 has the effect of relieving oxidative stress and reducing lipid deposition.

[0060] Frozen sections of the liver tissue described in (2) above were used for H&E and Oil Red O staining analysis. The results are shown in Figures 18, D and E. Compared with the normal group, white circular gaps appeared in both the H&E and Oil Red O images of the alcohol group, and the Oil Red O color was significantly redder, indicating the formation of lipid droplets. After treatment, the number of white gaps was significantly reduced and the color of Oil Red O was significantly lighter, especially in the Res@G5-Gal / si-Cyp2e1 treatment group. This indicates that Res@G5-Gal / si-Cyp2e1 can reduce the formation of lipid droplets and improve lipid deposition.

[0061] (4) Analysis of related gene expression In order to further explore the related molecular mechanisms of alleviating alcohol-related liver disease, molecular detection was performed on the liver tissue in (2) above, and the relative expression of Cyp2e1 mRNA was analyzed by RT-qPCR technology. The results are shown in Figure 19A. Compared with the PBS group, the expression level of CYP2E1 gene was significantly increased after alcohol pretreatment. This is because excessive alcohol will induce activation of the CYP2E1 pathway, thereby upregulating the expression of CYP2E1 gene. Res@G5-Gal was also significantly increased, and there was no significant difference compared with the alcohol group. After treatment, the mRMA level of CYP2E1 gene was significantly reduced. It is worth noting that the expression levels of G5-Gal / si-Cyp2e1 and Res@G5-Gal / si-Cyp2e1 treatment groups were lower and there was no significant difference. This indicates that the main reason for the reduction of the mRMA level of Cyp2e1 gene is the role played by the small interfering gene si-Cyp2e1.

[0062] Western blotting was used to detect the relative expression levels of CYP2E1, ADRP / Perilipin2, and GPR78 proteins. The results are shown in Figures 19 (B, C, and D). Compared to the PBS group, the protein expression levels of CYP2E1, ADRP / Perilipin2, and GPR78 were significantly increased after alcohol pretreatment. This is because excessive alcohol induces activation of the CYP2E1 pathway, leading to lipid deposition and endoplasmic reticulum stress. For CYP2E1 protein, similar to the RT-qPCR results, all three preparations downregulated CYP2E1 protein expression levels. Treatment with G5-Gal / si-Cyp2e1 and Res@G5-Gal / si-Cyp2e1 showed the highest expression levels. The expression levels in the three groups were lower and there was no significant difference. For ADRP / Perilipin2 protein, the Res@G5-Gal and Res@G5-Gal / si-Cyp2e1 treatment groups showed significant decreases and there was no significant difference. For GRP78 protein, all three formulations significantly downregulated GRP78 protein expression levels, and there was no significant difference among the three formulations. Therefore, Res@G5-Gal / si-Cyp2e1 has the best effect in downregulating CYP2E1 protein and reducing lipid deposition and endoplasmic reticulum stress.

[0063] (5) Biosafety Evaluation: Six- to eight-week-old male C57BL / 6 healthy mice were randomly divided into four groups (PBS group, G5-Gal / si-Cyp2e1 group, Res@G5-Gal group, and Res@G5-Gal / si-Cyp2e1 group, respectively). The mice were injected via tail vein on days 3, 6, and 9 (100 μL, [Res] = 5 mg / kg). On day 11, blood was collected from the mice's eyes for complete blood count and blood biochemistry tests. Major organs (heart, liver, spleen, lung, kidney, and brain) were extracted through dissection for H&E staining and organ coefficient analysis. The results are shown in Figures 20-23. No significant cardiotoxicity, liver or kidney damage, lung toxicity, spleen infiltration, or brain damage were observed in any of the formulations. Complete blood count indicators in all formulations were within the normal range. Blood biochemistry indicators in all formulations showed no significant difference compared to the PBS group. This demonstrates that the G5-Gal / si-Cyp2e1, Res@G5-Gal, and Res@G5-Gal / si-Cyp2e1 prepared in this invention have good biocompatibility.

[0064] In summary, Res@G5-Gal / si-Cyp2e1 demonstrates good biocompatibility, liver-targeting ability, and antioxidant activity. It can target the liver in vivo, exerting antioxidant effects to mitigate alcohol-induced lipid metabolism abnormalities and repair liver damage caused by alcohol intake. Furthermore, Res@G5-Gal / si-Cyp2e1 exhibits good loading capacity for both si-Cyp2e1 and Res drugs, does not affect normal cell activity, and shows a more significant effect on improving lipid deposition.

[0065] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, all chemical reagents are commercially available and can be used directly without further purification. Among them, the fifth-generation amino-terminated polyamide-amine dendritic macromolecule G5.NH2 was purchased from Weihai Chenyuan Molecular New Materials Co., Ltd. (Weihai, China). Unless otherwise specified, the other raw materials or processing techniques are all conventional commercially available raw materials or conventional processing techniques in the art.

[0066] Example 1 In this example, a nanoplatform for co-delivering small interfering RNA and small molecule drugs is provided, and the preparation method of the nanoplatform includes the following steps: (1) Preparation of G5-Gal: 50 mg G5, 11.04 mg Gal, 99.8 mg EDC·HCl and 59.9 mg NHS are weighed and dissolved in 3 mL, 2 mL, 5 mL and 2 mL DMSO respectively. Under room temperature stirring conditions, EDC solution is added dropwise to the Gal solution in the reaction flask. After stirring at room temperature for 30 min, NHS solution is added dropwise to the above mixed solution. After stirring at room temperature for 3 h, an activated Gal solution is obtained. Then, G5 solution is added dropwise to the activated Gal solution. After stirring at room temperature for 3 days, the product is transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in ultrapure water for three days (2 L × 3 times / day). After freeze drying, G5-Gal powder is obtained and stored at -20 ℃ for later use.

[0067] (2) Preparation of Res@G5-Gal: Weigh 10 mg of G5-Gal obtained in step (1) above and dissolve it in 1 mL of ultrapure water, then transfer it to a brown reaction flask; under light-protected conditions, weigh 1.45 mg of Res and dissolve it in 500 μL of methanol solution. Add the Res methanol solution dropwise to the G5-Gal solution in the brown reaction flask. After completion, wrap the mouth of the flask with tin foil, tie it tightly with a rubber band, and then poke a hole with a needle. Let the reaction proceed overnight at half the rated capacity. The next day, aspirate the solution from the reaction flask into a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 15 min, and aspirate the supernatant to obtain the Res@G5-Gal solution, which can be stored at 4 ℃ for later use.

[0068] (3) Preparation of Res@G5-Gal / si-Cyp2e1 complex: Take the Res@G5-Gal solution obtained in step (2), add si-Cyp2e1 at a mass ratio of G5-Gal to si-Cyp2e1 of 16:1, mix gently, and let stand at room temperature for 25 to 35 minutes to obtain the Res@G5-Gal / si-Cyp2e1 complex.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanoplatform for co-delivering small interfering RNA and small molecule drugs, characterized in that, The nanoplatform uses G5-Gal as a carrier to load small molecule anti-inflammatory drugs and si-Cyp2e1.

2. The nanoplatform for co-delivering small interfering RNA and small molecule drugs as described in claim 1, characterized in that, The G5 molecules are connected to Gal via COC bonds, and 12 to 18 Gal molecules are grafted onto the surface of each G5 molecule.

3. The nanoplatform for co-delivering small interfering RNA and small molecule drugs as described in claim 1, characterized in that, The mass ratio of Gal-G5 to si-Cyp2e1 is ≥16:

1.

4. The nanoplatform for co-delivering small interfering RNA and small molecule drugs as described in claim 1, characterized in that, The small molecule anti-inflammatory drug is Res, and the molecular ratio of Gal-G5 to Res is 12 to 16:

1.

5. The method for preparing the co-delivery small interfering RNA and small molecule drug nanoplatform according to any one of claims 1-4, characterized in that, The small molecule drug is resveratrol, and the preparation method includes the following steps: (1) Preparation of G5-Gal: G5 is added to activated Gal, and the reaction is carried out at room temperature for 2.5 to 3.5 days. The component with a molecular weight of 3500 Da or above is retained and freeze-dried, which is Gal-G5; (2) Preparation of Res@G5-Gal: Under light-protected conditions, the aqueous solution of Gal-G5 and the organic solution of Res are mixed and reacted overnight. The supernatant is retained by centrifugation to obtain Res@G5-Gal; (3) Preparation of Res@G5-Gal / si-Cyp2e1 complex: The aqueous solution of Res@G5-Gal is mixed with si-Cyp2e1 and then allowed to stand at room temperature for 25 to 35 minutes to obtain the nano-platform Res@G5-Gal / si-Cyp2e1.

6. The preparation method according to claim 5, characterized in that, In step (1), the activation of Gal is specifically performed as follows: EDC·HCl is added to the Gal solution and stirred at room temperature for 25-35 min, and NHS is added and stirred for 2-4 h for activation; in this embodiment, the molar ratio of Gal to EDC and NHS is 1:10-20:10-20, and the molar ratio of G5 to Gal is 1:30-40; the filter membrane used for retention is preferably a cellulose dialysis membrane, which is dialyzed in ultrapure water for 2-3 days, with water changed 2-4 times a day, each time using 1.5-2.5 L of ultrapure water; or, in step (2), the organic solution of Res is a methanol solution, and the molar ratio of G5-Gal to Res is 1:15-25; or, in step (3), the mass ratio of G5-Gal to si-Cyp2e1 is greater than or equal to 16:1; or, the sequence of si-Cyp2e1 is as shown in SEQ ID. NO:1 is shown.

7. A pharmaceutical composition comprising an active dose of the nanoplatform of any one of claims 1-4.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition, in addition to the glycosylated dendritic macromolecular nanoplatform that co-delivers small interfering RNA and small molecule drugs, also includes other active ingredients, auxiliary ingredients, or pharmaceutically acceptable carriers for treating alcohol-related liver disease; the active ingredients for treating or alleviating alcohol-related liver disease include, but are not limited to, natural plant extracts, small molecule compounds, and targeted therapeutic drugs; the auxiliary ingredients include, but are not limited to, antiemetic drugs, granulocyte colony-stimulating factors, thrombopoietin, hepatoprotective ingredients, immune enhancers, or intestinal flora regulators; the pharmaceutically acceptable carriers include, but are not limited to, absorption enhancers, disintegrants, osmotic pressure regulators, solubilizers, emulsifiers, binders, diluents, wetting agents, pH adjusters, antioxidants, colorants, or flavoring agents.

9. Use of the co-delivery small interfering RNA and small molecule drug nanoplatform according to any one of claims 1-4, or the pharmaceutical composition according to claim 7 or 8.

10. The application as described in claim 9, characterized in that, Including but not limited to: (1) drugs for preparing alcohol-related liver disease; (2) health products for improving alcoholic liver disease; (3) special medical foods suitable for patients with alcoholic liver disease; the alcohol-related liver disease includes, but is not limited to, alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, alcoholic cirrhosis, hepatitis or liver cancer.

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