Melittin-loaded plga liposome nanoparticles and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a PLGA liposome nanoparticle loaded with bee venom and a preparation method and application thereof. The PLGA liposome nanoparticle takes a bee venom encapsulated by PLGA as a core, and the outer surface of the core is further coupled with RGD peptide and coated with a liposome. The application adopts a PLGA-liposome composite carrier to wrap the bee venom, effectively improves the drug encapsulation rate, realizes pH-responsive slow release, prolongs the in-vivo half-life of the drug, and overcomes the defects of easy degradation and poor stability of free bee venom. The application can effectively inhibit the growth of breast cancer in-vivo and in-vitro, simultaneously activates CD8+ T cell-mediated anti-tumor immunity, and has an inhibiting effect on lung fibrosis-related proteins. The carrier used is biodegradable, and the preparation process is mild and controllable. The application has the dual purposes of anti-tumor and anti-lung fibrosis, and has a good clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a PLGA liposome nanoparticle loaded with bee venom, its preparation method, and its application. Background Technology

[0002] Breast cancer is a common malignant tumor that seriously endangers women's health, and its incidence continues to rise globally. Currently, the main drugs used clinically to treat breast cancer include the following categories: First, traditional cytotoxic chemotherapy drugs such as anthracyclines (e.g., doxorubicin, epirubicin) and taxanes (e.g., paclitaxel, docetaxel), which kill rapidly proliferating tumor cells by interfering with DNA replication or microtubule function; second, endocrine therapy drugs, such as tamoxifen and aromatase inhibitors, suitable for hormone receptor-positive breast cancer; third, molecularly targeted drugs, such as trastuzumab (anti-HER2 monoclonal antibody) and pertuzumab, targeting patients with specific gene mutations or protein overexpression; and fourth, immune checkpoint inhibitors, such as PD-1 / PD-L1 antibodies, used for some triple-negative breast cancers.

[0003] However, the aforementioned drugs all have significant shortcomings in clinical application. Traditional chemotherapy drugs lack tumor selectivity, causing severe toxic side effects on normal tissues such as bone marrow, digestive tract, and heart while killing tumors. Endocrine therapy drugs are only effective for hormone-sensitive patients, and drug resistance is a prominent issue. Targeted drugs are limited by the target expression spectrum, have a narrow applicable population, and are accompanied by specific adverse reactions such as cardiotoxicity and interstitial pneumonia. Although immunotherapy can activate the body's anti-tumor immunity, the single-drug response rate is low, and it often causes autoimmune toxicity such as immune-related pneumonia and colitis. More challenging is that breast cancer patients often develop complications such as pulmonary fibrosis after chemotherapy or radiotherapy, and most existing drugs only target the tumor itself, lacking the ability to synergistically intervene in complications, leading to a decline in the long-term quality of life for patients. Therefore, there is an urgent need to provide a new strategy for the treatment of breast cancer. Summary of the Invention

[0004] The purpose of this invention is to provide PLGA liposome nanoparticles loaded with bee venom, which solves the problems existing in the prior art.

[0005] The technical solution adopted in this invention is: This invention provides PLGA liposome nanoparticles loaded with bee venom, wherein the PLGA liposome nanoparticles have bee venom encapsulated in PLGA as the core, and the outer surface is further coupled with RGD peptides and coated with liposomes. The mass ratio of liposomes to bee venom is 3-8:1.

[0006] Preferably, the mass ratio of liposomes to bee venom is 4:1.

[0007] Preferably, the encapsulation efficiency of the PLGA liposome nanoparticles is 60%~70%.

[0008] Preferably, the hydrodynamic diameter of the PLGA liposome nanoparticles is 90 nm to 120 nm.

[0009] A second aspect of this invention provides a method for preparing the PLGA liposome nanoparticles, comprising the following steps: The bee venom was mixed with PLGA at a mass ratio of 1:(0.8~1.5), and then encapsulated in PLGA. Liposomes were covalently coupled with RGD peptides at a mass ratio of (8~15):1 to obtain pH-sensitive liposomes; Using bee venom encapsulated within PLGA as the aqueous phase, pH-sensitive liposomes were driven to self-assemble in situ on the PLGA surface, thus obtaining the PLGA liposome nanoparticles.

[0010] Preferably, the mass ratio of bee venom to PLGA is 1:1.

[0011] Preferably, the mass ratio of liposomes to RGD peptides is 10:1.

[0012] Preferably, the in-situ self-assembly condition is ultrasonic treatment for 10 minutes.

[0013] A third aspect of the present invention provides the application of the PLGA liposome nanoparticles, wherein the PLGA liposome nanoparticles are used in any of the following: 1) Preparation of anti-tumor drugs; 2) Prepare drugs to combat pulmonary fibrosis.

[0014] Preferably, the tumor is breast cancer.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention provides PLGA liposome nanoparticles loaded with bee venom. The PLGA liposome nanoparticles have a PLGA-encapsulated bee venom core, with an RGD peptide further coupled to the outer surface and coated with liposomes. The nanoparticles of this invention, with a PLGA-encapsulated bee venom core and an RGD peptide coupled to the surface and coated with a liposome shell, form a "core-shell" composite structure. The RGD peptide can actively recognize tumor angiogenesis and integrin receptors highly expressed by tumor cells. Combined with the inherent EPR passive targeting effect of PLGA-liposomes, this achieves high drug concentration at the tumor site, fundamentally improving the lack of selectivity of traditional chemotherapy drugs and significantly reducing non-specific damage to normal organs such as bone marrow, heart, and digestive tract. Meanwhile, the PLGA core significantly improves the encapsulation efficiency and stability of bee venom, while the liposome shell further prolongs the in vivo circulation half-life, effectively solving the inherent problems of easy degradation and short half-life of free bee venom. Moreover, the nanoparticles exhibit accelerated release behavior in the weakly acidic tumor microenvironment, achieving pH-responsive sustained release, which avoids the burst release of the drug in the blood circulation and can maintain the effective local concentration, helping to delay or reverse multidrug resistance, which is superior to the constant-rate release mode of traditional chemotherapy drugs.

[0016] At the level of anti-tumor effects, these nanoparticles not only directly kill breast cancer cells, but more importantly, they can activate the body's own anti-tumor immunity. This formulation can effectively stimulate cytotoxic T lymphocyte responses, compensating for the shortcomings of immune checkpoint inhibitors, such as low single-drug response rates and the tendency to induce autoimmune toxicity like immune-related pneumonia. More notably, while inhibiting tumors, these nanoparticles can significantly downregulate the expression of fibroblast markers αSMA and tissue matrix metalloproteinase inhibitor-1 (TIMP1), demonstrating a clear ameliorative effect on bleomycin-induced pulmonary fibrosis. This fills the gap in existing anti-breast cancer drugs' inability to synergistically intervene in the complication of pulmonary fibrosis secondary to radiotherapy and chemotherapy, thus improving patients' long-term quality of life. In vivo safety evaluation showed that after administration of these nanoparticles, inflammatory markers such as white blood cells, lymphocytes, and eosinophils all returned to normal levels, and no pathological increases were observed in serum biochemical indicators such as liver function, kidney function, and myocardial enzymes, confirming that it did not produce significant toxicity to the liver, kidneys, heart, or muscle tissue, exhibiting the advantages of high efficacy, low toxicity, and controllable risk.

[0017] The PLGA liposome nanoparticles loaded with bee venom provided by this invention overcome the shortcomings of existing breast cancer treatment drugs in terms of selectivity, stability, drug resistance, immune activation, and complication management through multiple mechanisms such as targeted delivery, pH-responsive release, immune regulation, and anti-fibrosis synergistic intervention. This provides a new strategy with great clinical translational potential for the efficient and low-toxicity treatment of breast cancer and the comprehensive management of its related complications. Attached Figure Description

[0018] Figure 1The particle size of the PLGA liposome nanoparticles loaded with bee venom prepared in Example 1 is shown.

[0019] Figure 2 Scanning electron microscopy morphology characterization of products from different steps; A: SEM image of PLGA nanospheres loaded with bee venom; B: SEM image of PLGA liposome nanoparticles loaded with bee venom.

[0020] Figure 3 In vitro cytotoxicity experiments of different cancer cells; A: In vitro cytotoxicity assay of CT26-Y cancer cells; B: In vitro cytotoxicity assay of CT26-Z cancer cells; C: In vitro cytotoxicity assay of Hepa1-6 cancer cells; D: In vitro cytotoxicity assay of 4T-1 cancer cells; E: In vitro cytotoxicity assay of EMT6 cancer cells; F: Repeat the in vitro cytotoxicity experiment of 4T-1 cancer cells.

[0021] Figure 4 To take pictures of the in vitro 4T-1 cytotoxicity experiment after drug administration.

[0022] Figure 5 After drug administration, Hoechest and PI staining were performed on 4T-1 cells in vitro, and the cells were photographed.

[0023] Figure 6 This is a flow cytometry diagram showing the cell volume after PI staining following drug administration.

[0024] Figure 7 The effect of PLGA liposome nanoparticles loaded with bee venom on bleomycin-induced pulmonary fibrosis; A: Photograph of mouse lung tissue; B: Statistical chart of mouse lung tissue weight; C: Detection of pulmonary fibrosis-related proteins; D: Representative image of lung tissue stained with HE.

[0025] Figure 8 These are the results of in vivo anti-tumor experiments; A: Statistical chart of tumor volume changes in vivo after treatment with different drugs; B: Statistical chart of tumor weight in vivo after treatment with different drugs; C~E: Analysis graph of tumor weight, spleen weight and tumor volume; F: Photograph of a mouse tumor; G: Photograph of mouse organs.

[0026] Figure 9This refers to the results of routine blood biochemical statistical analysis; A: Results of biochemical statistical analysis of white blood cells; B: Results of lymphocyte biochemical statistical analysis; C: Statistical analysis results of eosinophil biochemical analysis; D: Biochemical statistical analysis results of alanine aminotransferase; E: Biochemical statistical analysis results of aspartate aminotransferase; F: Biochemical statistical analysis results of creatine kinase; G: Biochemical statistical analysis results of creatinine; H: Biochemical statistical analysis results of urea nitrogen.

[0027] Figure 10 CD4 in mice + With CD8 + Flow cytometry analysis of changes in the proportion of T cells; A: CD4 levels after PBS treatment + With CD8 + Flow cytometry analysis of changes in the proportion of T cells; B: CD4 after Liposome group treatment + With CD8 + Flow cytometry analysis of changes in the proportion of T cells; C: CD4 after Melittin group treatment + With CD8 + Flow cytometry analysis of changes in the proportion of T cells; D: CD4 count after treatment with Melittin + Liposome + With CD8 + Flow cytometry statistical analysis of changes in the proportion of T cells. Detailed Implementation

[0028] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0029] The inventive concept of this invention is as follows: This invention addresses the problems of insufficient targeting, significant toxicity and side effects, and easy development of drug resistance in existing breast cancer treatments, as well as the lack of effective synergistic treatments for chemotherapy-related pulmonary fibrosis. Combining the shortcomings of free bee venom, such as poor stability, short half-life, significant hemolytic toxicity, and systemic inflammatory response, this invention designs a PLGA liposome nanoparticle loaded with bee venom. Using PLGA as the core improves drug encapsulation efficiency and stability; employing a liposome shell modified with DSPE-PEOz and RGD peptides prolongs in vivo circulation time and achieves active tumor targeting; utilizing the weakly acidic tumor microenvironment achieves pH-responsive sustained release, increasing local drug concentration and reducing systemic toxicity; while effectively inhibiting breast cancer, it activates CD8⁺ T cell immune responses and inhibits the expression of pulmonary fibrosis-related proteins, achieving dual anti-tumor and anti-pulmonary fibrosis effects; employing a combination of emulsification-solvent evaporation and thin-film dispersion methods, the process is mild and controllable with high biosafety, ultimately constructing a highly efficient, low-toxicity composite nanoparticle drug delivery system with both targeted delivery and immunomodulatory functions.

[0030] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments. In the description of the present invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0031] The raw materials used in this invention were purchased from the following sources: PLGA: R-PL1001-10K, ratio 75:25, purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0032] Melittin (Catalog No. SN-M-PEP28, purity 98%), purchased from Shanghai Shunna Biotechnology Co., Ltd.

[0033] DSPE-PEOz: SN-M-LP126, purchased from Shanghai Shunna Biotechnology Co., Ltd.

[0034] RGD peptide: Specification R-CW-RGD, purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0035] Example 1 The preparation method of PLGA liposome nanoparticles loaded with bee venom is as follows: S1. Preparation of PLGA nanospheres loaded with bee venom.

[0036] The nanosphere solution was prepared using an emulsification-solvent evaporation method: 10 mg PLGA was dissolved in 2 mL of dichloromethane; 10 mg bee venom was dissolved in 5 mL of distilled water; the two solutions were thoroughly mixed. Then, the mixture was added dropwise to 5 mL of distilled water while stirring at high speed for 2 hours to ensure uniform dispersion and complete evaporation of the dichloromethane. The final result was 10 mL of a 1 mg / mL nanosphere solution, which was stored in a sealed container at 4 °C protected from light.

[0037] S2. Preparation of PLGA liposome nanoparticles loaded with bee venom.

[0038] PLGA liposome nanoparticles were prepared using a thin-film dispersion method: liposomes were prepared according to a 4:1 mass ratio of DSPE-PEOz to bee venom. First, 50 mg of DSPE-PEOz was dissolved in 25 mL of chloroform, and 5 mg of RGD peptide was dissolved in 2.5 mL of anhydrous ethanol. Then, 19.2 mL of the DSPE-PEOz solution in chloroform and 1.92 mL of the RGD peptide solution in anhydrous ethanol were mixed in a pear-shaped flask and thoroughly mixed. The solvent was removed by rotary evaporation at 40ºC. After film formation, 9.6 mL of the bee venom-loaded PLGA nanosphere solution prepared in the first step was repeatedly washed for hydration, followed by sonication for 10 min. This yielded pH-sensitive bee venom liposome nanoparticles with a concentration of 1 mg / mL. The sample was then dialyzed in distilled water for 1 h. The dialysis bag used had a molecular weight cutoff of 5 kDa, resulting in 9.4 mL of bee venom-loaded PLGA liposome nanoparticle sample (molar concentration of 340 mol / mL). Finally, seal the liquid sample and store it at 4°C for later use.

[0039] The average hydrodynamic diameter of the PLGA liposome nanoparticles loaded with bee venom prepared in Example 1 was 105 nm, as determined by dynamic light scattering (DLS). Figure 1 In contrast, scanning electron microscopy (SEM) at 100,000x magnification showed that the average diameter of the melitoxin-loaded liposomes was approximately 30 nm. Figure 2 As shown, this difference in particle size is likely attributed to dehydration during SEM sample preparation, which caused shrinkage of the PLGA-liposome matrix. Regarding encapsulation efficiency (EE), the results showed that the preparation of 75:25 PLGA and DSPE-PEOz, with a mass ratio of 4:1 to melitoxin, exhibited a relatively high encapsulation efficiency of 63.59%.

[0040] The release of the PLGA liposome nanoparticles loaded with bee venom prepared in Example 1 was studied, as follows: (1) Take 1 mL of two portions of PLGA liposome nanoparticles loaded with bee venom prepared in Example 1 and place them in a pre-soaked dialysis bag (soaked in deionized water for 2 hours in advance). Clamp the two ends of the dialysis bag with clips (bind one bottle with an elastic band to keep it vertical). Place them in 250 mL of PBS solution at pH 6.0 and pH 7.4 respectively. Under light-protected conditions, set the rotation speed to 700 r / min and take samples at different time periods.

[0041] (2) Samples were taken at intervals of 1h, 2h, 3h, 5h, 10h, 22h, 24h, 47h and 48h, and their absorbance was measured at a wavelength of 214nm.

[0042] (3) Data analysis: After deducting points with large errors, we get Table 1 below.

[0043] Table 1 Release rate of PLGA liposome nanoparticles loaded with bee venom Table 1 shows that the release rate of bee venom peptide-loaded liposome nanoparticles is slightly accelerated under acidic conditions.

[0044] Example 2 The application of PLGA liposome nanoparticles loaded with bee venom was verified by evaluating the effect of the PLGA liposome nanoparticles loaded with bee venom prepared in Example 1, as detailed below: 1. In vitro cytotoxicity experiment.

[0045] Cells in the logarithmic growth phase were digested, collected, and counted, then diluted to a specific concentration. 100 μL of the diluted cell suspension (5000 cells / well) was added to each 96-well plate, with three replicates per group. Cells were incubated overnight at 37°C and 5% CO2 to allow cell adhesion. The next day, the original culture medium was aspirated, and 100 μL of a drug culture system with different concentration gradients (0–2.5 μg / mL) prepared in medium was added. The cells were then incubated at 37°C and 5% CO2 for 48 hours. The drug culture systems used in this experiment were divided into the following three groups:

[0046] Liposome nanoparticles: PLGA liposome nanoparticles without bee venom loading prepared according to the method described in Example 1.

[0047] Bee venom liposome nanoparticles: PLGA liposome nanoparticles loaded with bee venom prepared in Example 1.

[0048] Bee venom: Product No. SN-M-PEP28, purity 98%, purchased from Shanghai Shunna Biotechnology Co., Ltd.

[0049] After drug administration, 50 μL / well of pre-chilled trichloroacetic acid solution (10%, w / v) was added to the original culture medium, and the mixture was fixed at 4°C for 2 h. The cells were then rinsed 5 times with tap water and air-dried. Next, 100 μL / well of 0.4% (w / v) SRB staining solution (prepared with 1% glacial acetic acid) was added and stored in the dark. The cells were stained with low-speed shaking on a horizontal shaker for 20 min, then the staining solution was discarded. The cells were rinsed 5 times with 1% (v / v) glacial acetic acid to remove unbound dye and air-dried. Finally, 100 μL / well of unbuffered Tris-base solution (10 mM, pH=10.5) was added, and the cells were shaken on a horizontal shaker for 20 min to fully dissolve the dye. The absorbance at 540 nm was measured using a microplate reader. IC50 curves were plotted using Origin, and the resistance index was calculated. The formula for calculating the resistance index is: Resistance Index = IC50 of resistant cells / IC50 of parental cells. Figure 3 As shown, in vitro cytotoxicity experiments revealed that 4T-1 breast cancer cells showed the best effect, with an IC50 of 32 μg / mL. Therefore, this tumor cell line was selected for subsequent experiments.

[0050] 2. In vitro 4T-1 cell experiment.

[0051] Logarithmic-phase breast cancer cells (4T-1) were digested, collected, and counted, then diluted to a specific concentration. 2 mL of the diluted cell suspension (40,000 cells / well) was added to each well of a 6-well plate, with two replicates per group. The plates were incubated overnight at 37°C with 5% CO2 to allow cell adhesion. The next day, the original culture medium was aspirated using a pipette, and 2 mL of PLGA liposome nanoparticles loaded with bee venom prepared in Example 1 were added. Drug concentrations of 0 μg / mL, 10 μg / mL, and 12.5 μg / mL were administered. The culture was then incubated at 37°C with 5% CO2 for 48 hours. Finally, photographs were taken. Figure 4 As shown, when the concentration of bee venom liposome nanoparticles administered was 12.5 μg / mL, significant cell death was clearly observed.

[0052] Logarithmic-phase 4T-1 cells were digested, collected, and counted, then diluted to a specific concentration. 2 mL of the diluted cell suspension (100,000 cells / well) was added to each 6-well plate, with two replicates per group. Cells were incubated overnight at 37°C with 5% CO2 to allow cell adhesion. The next day, the original culture medium was aspirated using a pipette, and 2 mL of 20 μg / mL of prepared PLGA liposome nanoparticles loaded with bee venom was added. The plates were then incubated at 37°C with 5% CO2 for 24 hours. Finally, the cells were stained with hoechest and PI, and photographed under different fluorescence conditions. Figure 5 As shown, cells in the drug-treated group (the bee venom liposome nanoparticle group prepared in Example 1) were clearly stained with PI, indicating that the cells were in an apoptotic state. However, cells in the control group (blank group and liposome nanoparticle group) showed less apoptosis.

[0053] Logarithmic-phase cells were digested, collected, and counted, then diluted to a specific concentration. 2 mL of the diluted cell suspension (100,000 cells / well) was added to each 6-well plate, with two replicates per group. Cells were incubated overnight at 37°C with 5% CO2 to allow cell adhesion. The next day, the original culture medium was aspirated using a pipette, and 2 mL of a prepared 20 μg / mL drug culture system was added. Cells were incubated at 37°C with 5% CO2 for 48 hours. Cells were then digested, filtered through a 40 μm filter to obtain single cells, centrifuged, and resuspended in 100 μL of PBS (1% FBS, PI 2 μg / mL) dye working solution. Staining was performed for half an hour. Finally, the cells were analyzed by flow cytometry. Results are shown below. Figure 6 As shown, the number of cells stained with PI in the bee venom liposome nanoparticle group was three times that in the control group, the blank liposome nanoparticle group, and the bee venom group, indicating that the bee venom liposome nanoparticle group accelerated cell apoptosis after administration.

[0054] 3. Effects of bee venom liposome nanoparticles on bleomycin-induced pulmonary fibrosis.

[0055] 3.1 Experimental methods.

[0056] C57 mice were used to establish a pulmonary fibrosis model by intratracheal infusion of bleomycin (5 mg / kg body weight, dissolved in physiological saline). Two weeks later, the mice were treated with PLGA liposome nanoparticles loaded with bee venom prepared in Example 1 (the drug concentration was diluted with culture medium and injected intraperitoneally at a concentration of 1.0 mg / mL, with an interval of 3 days per injection). Two weeks later, the mice were treated by exsanguination and euthanasia, and lung tissue was harvested, weighed, photographed, and a portion of lung tissue from the same location was fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE).

[0057] 3.2 Extraction of lung tissue protein and Western blot analysis.

[0058] (1) Sample preparation: 500 μl of RIPA strong lysis buffer (with protease inhibitor added) was used to lyse lung tissue. The lysed liquid was transferred to a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 20 min at 4°C. The supernatant was used to determine the protein concentration using the BCA method. Adding... β The samples were strongly diluted with 5× mercaptoethanol loading buffer and RIPA to achieve a final concentration of 2 μg / μL for each sample. After vortexing, the samples were denatured at 95°C for 10 min.

[0059] (2) Preparation of SDS-polyacrylamide gel: Add the prepared 9% polyacrylamide separating gel between glass plates with a thickness of 1 mm. Stop adding the separating gel when the liquid level reaches 1 cm below the bottom edge of the sample comb. Press the gel with isopropanol and let it stand at room temperature for about 1 h. After the separating gel has polymerized, pour out the isopropanol, rinse with deionized water, and blot dry the residual liquid with filter paper. Add the stacking gel mixture, insert the sample comb, and let it stand at room temperature for 45 min to allow the stacking gel to polymerize.

[0060] (3) Electrophoresis: Install the electrophoresis apparatus and add Tris-glycine electrophoresis buffer to the electrophoresis tank. Add the sample and marker protein solution in sequence. Adjust the voltage to 120V, and adjust the voltage according to the number of gels at 15mA / block. Stop electrophoresis when bromophenol blue migrates to the bottom of the separating gel.

[0061] (4) Transfer: Gently pry open the gel plate, cut off the stacking gel, and soak the remaining gel in transfer buffer; cut one PVDF membrane (first soak the PVDF membrane in methanol for 15 seconds to activate it, then soak it in transfer buffer for later use) and 6 sheets of filter paper the same size as the separating gel, and soak the filter paper in transfer buffer for later use. Place the gel in the following order: anode, sponge, three layers of filter paper, PVDF membrane, gel, three layers of filter paper, sponge. Remove air bubbles between each layer with a glass rod, clamp the transfer clips, and place the gel and ice pack together in the transfer tank. Add transfer buffer and maintain a constant current of 120mA for 2 hours to transfer the protein onto the PVDF membrane.

[0062] (5) Blocking and antibody incubation: Rinse the PVDF membrane in TBST solution and incubate it in blocking solution (5% bovine serum albumin BSA prepared in TBST) at room temperature for 1 hour. After blocking, transfer the PVDF membrane to a container containing primary antibody working solution (TBST diluted with 5% BSA) and incubate it overnight at 4°C with shaking. Wash the membrane with TBST solution for 10 minutes each time, for a total of 4 washes. Place the membrane in working solution containing horseradish peroxidase-labeled secondary antibody (1:5000, TBST diluted with 5% BSA) and incubate it at room temperature for 1 hour. Wash the membrane with TBST solution 4 times, every 10 minutes.

[0063] (6) Development: Mix equal volumes of ECL luminescent solution A and solution B, distribute them evenly on the PVDF film, incubate at room temperature for several seconds, and then expose. The exposure time depends on the signal strength; select a suitable exposure image for analysis.

[0064] 3.3 HE staining.

[0065] (1) Sampling and fixation: Take fresh tissue blocks from animals (since lung tissue is easy to float, use cotton to press it below the liquid surface to avoid uneven fixation), place them in 4% paraformaldehyde solution for fixation, the ratio of fixative to tissue block should be greater than 10:1, the fixation time is 4h to 24h, the fixation time should not be too long or too short, the fixative needs to fully penetrate the tissue, so that the tissue protein denatures and coagulates, terminates endogenous or exogenous enzyme reaction, prevents tissue autolysis or heterolysis, so as to maintain the original structure and morphology.

[0066] (2) Dehydration and clearing: Use ethanol of varying concentrations, from low to high, as a dehydrating agent to gradually remove water from the tissue block. Then place the tissue block in xylene as a clearing agent to clear it, replacing the alcohol in the tissue block with xylene.

[0067] (3) Embedding and sectioning: Place the tissue block in paraffin at a suitable temperature and keep it warm in a paraffin melting box. After the paraffin has completely immersed the tissue block, embed it and let it cool and solidify into a block. The embedded paraffin block is generally 8 μm thick. The cut slices are placed in water at a suitable temperature to flatten them, then attached to a glass slide and dried in a 45°C constant temperature oven.

[0068] (4) Dewaxing: Remove the paraffin from the sections with xylene, then soak them in ethanol of varying concentrations, and finally wash with distilled water to hydrate the sections. The specific procedure is as follows: xylene (I) 8 min → xylene (II) 10 min → xylene (III) 10 min → 100% ethanol 2 min → 100% ethanol 2 min → 95% ethanol 2 min → 80% ethanol 2 min → wash with distilled water for 1 min.

[0069] (5) Staining: Stain with hematoxylin aqueous solution for 10 min. Select a suitable acidic solution and immerse for 30 s for color separation. Rinse with running water for 30 min and then soak in distilled water for 15 min. Dehydrate in 70% and 90% alcohol for 10 min each. Stain with eosin staining solution for 3 min.

[0070] (6) Dehydration, clearing and mounting: The stained sections were dehydrated with 95% alcohol and then cleared with xylene. Then, they were mounted with neutral resin.

[0071] (7) Use a panoramic scanner for pathological slides to acquire images.

[0072] 3.4 Discussion of Results.

[0073] This invention sets up three groups, namely: BLM: Bleomycin infusion; Saline: Infuse an equal volume of saline solution into normal mice; Melittin: After successful bleomycin modeling, PLGA liposome nanoparticles loaded with bee venom prepared in Example 1 were dripped in.

[0074] See results Figure 7 After administration of bee venom liposome nanoparticles, lung weight increased. HE staining results showed increased inflammatory cell infiltration, significant alveolar cavity shrinkage, and pulmonary interstitial fibrosis in the bleomycin model group. Compared with the control group, the bee venom intervention group showed increased inflammatory cell infiltration and alveolar cavity shrinkage. HE results alone cannot determine whether bee venom has a protective effect against bleomycin-induced pulmonary fibrosis. However, protein detection results showed significant inhibition of fibroblast marker αSMA and tissue matrix metalloproteinase inhibitor-1 (TIMP1), both of which are indicators of pulmonary fibrosis. This suggests that bee venom liposome nanoparticles may have an ameliorative effect on bleomycin-induced pulmonary fibrosis.

[0075] 4. In vivo anti-tumor experiment.

[0076] 4.1 Experimental subjects.

[0077] Laboratory animals: Wild-type male C57 mice (7-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in an SPF environment with a 12-hour light / dark cycle, fed with sterile standard feed, and provided with free access to food and water. All experimental protocols complied with the regulations of the Animal Ethics Committee of Peking University School of Medicine.

[0078] 4.2 Experimental methods.

[0079] Male mice weighing 20-26g were selected and divided into 4 groups. 5 × 10⁶ breast cancer 4T1 cells were subcutaneously seeded into the left side of each mouse. 5 / each, when the tumor volume reaches 100-200 mm after inoculation. 3 Administering was initiated using PBS to prepare the medication. All groups received the first two injections at a concentration of 1.0 mg / mL, followed by 1.5 mg / mL, administered every two days. The treatments for the four groups are as follows:

[0080] PBS: Inject an equal volume of PBS; Liposome: Injection of PLGA liposome nanoparticles without bee venom loading, prepared according to the method described in Example 1.

[0081] Melittin: Injection of bee venom.

[0082] Melittin+Liposome: Injection of PLGA liposome nanoparticles loaded with bee venom prepared in Example 1.

[0083] 4.3 Discussion of Results.

[0084] like Figure 8As shown, the Liposome group showed no tumor inhibition, while the Melittin+Liposome group showed significant tumor inhibition. The body weight of the mice was not affected. In addition, there was a significant difference in tumor inhibition effect between the Melittin+Liposome group and other groups.

[0085] Figure 9 To evaluate the in vivo biosafety of PLGA liposome nanoparticles loaded with melitoxin, this invention tested the complete blood count and serum biochemical parameters of mice in different treatment groups. The complete blood count results showed that, compared with the PBS group, the white blood cell (WBC), lymphocyte (Lym), and eosinophil (Eos) counts in mice in the free melitoxin group showed an increasing trend, with Eos levels significantly upregulated (p<0.001), suggesting that free melitoxin can induce inflammation and allergic reactions. In contrast, the above parameters in the PLGA liposome nanoparticle-loaded group all returned to normal levels, and the WBC level was significantly lower than that in the free melitoxin group (p<0.05), indicating that liposome encapsulation can effectively alleviate the systemic inflammatory response induced by melitoxin. Regarding serum biochemical indicators, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which reflect liver function, creatinine (CREA-S) and blood urea nitrogen (UREA), which reflect kidney function, and creatine kinase (CK), which reflects myocardial / skeletal muscle damage, showed no pathological elevation in any group. Among them, the AST and CK levels in the PLGA liposome nanoparticle group loaded with bee venom were significantly lower than those in the PBS group (p<0.05, p<0.01), and the CREA-S level was significantly lower than that in the Liposome group (p<0.05). This suggests that the PLGA liposome nanoparticles loaded with bee venom did not cause significant toxic damage to the liver, kidneys, and muscle tissues and have good in vivo safety.

[0086] To investigate the effects of different treatments on T cell subsets in tumor draining lymph nodes (DLNs), this invention used flow cytometry to detect changes in the ratio of CD4⁺ to CD8⁺ T cells. The results are as follows: Figure 10 The results showed that, compared with the PBS group, treatment with free bee venom had a limited effect on increasing the proportion of CD8⁺ cytotoxic T cells; however, the proportion of CD8⁺ T cells in the Melittin+Liposome group increased significantly, from 9.66% in the PBS group to 14.77%, suggesting that this preparation can effectively activate effector T cell responses in draining lymph nodes. Meanwhile, with the increase in the proportion of CD8⁺ T cells, the proportion of CD4⁺ helper T cells in all groups showed a decreasing trend, with the proportion of CD4⁺ T cells in the Melittin+Liposome group decreasing to 29.98%, significantly lower than the 34.53% in the PBS group. These results indicate that liposome-encapsulated bee venom nanoparticles can effectively enhance the anti-tumor T cell immune response in draining lymph nodes of tumor-bearing mice, providing immunological evidence to support its anti-tumor activity.

[0087] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0088] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A PLGA liposome nanoparticle loaded with bee venom, characterized in that, The PLGA liposome nanoparticles have PLGA-encapsulated bee venom as their core, and their outer surface is further coupled with RGD peptides and coated with liposomes. The mass ratio of liposomes to bee venom is 3-8:

1.

2. The PLGA liposome nanoparticles as described in claim 1, characterized in that, The mass ratio of liposomes to bee venom is 4:

1.

3. The PLGA liposome nanoparticles as described in claim 1, characterized in that, The encapsulation efficiency of the PLGA liposome nanoparticles is 60%~70%.

4. The PLGA liposome nanoparticles as described in claim 1, characterized in that, The hydrodynamic diameter of the PLGA liposome nanoparticles is 90 nm to 120 nm.

5. The method for preparing PLGA liposome nanoparticles as described in claim 1, characterized in that, The steps are as follows: The bee venom was mixed with PLGA at a mass ratio of 1:(0.8~1.5), and then encapsulated in PLGA. Liposomes were covalently coupled with RGD peptides at a mass ratio of (8~15):1 to obtain pH-sensitive liposomes; Using bee venom encapsulated within PLGA as the aqueous phase, pH-sensitive liposomes were driven to self-assemble in situ on the PLGA surface, thus obtaining the PLGA liposome nanoparticles.

6. The preparation method according to claim 5, characterized in that, The mass ratio of bee venom to PLGA is 1:

1.

7. The preparation method according to claim 5, characterized in that, The mass ratio of liposomes to RGD peptides was 10:

1.

8. The preparation method according to claim 5, characterized in that, The condition for in-situ self-assembly was ultrasonic treatment for 10 minutes.

9. The application of the PLGA liposome nanoparticles as described in claim 1, characterized in that, The PLGA liposome nanoparticles are used in any of the following: 1) Preparation of anti-tumor drugs; 2) Prepare drugs to combat pulmonary fibrosis.

10. The application as described in claim 9, characterized in that, The tumor is breast cancer.

Images