Preparation method and application of self-driven chemical-immunoassay combined drug system
By using a self-driven chemoimmunotherapy combined with a drug system that targets hypoxic areas of tumors with anaerobic bacteria, the problem of targeted delivery of chemotherapy and immunotherapy drugs in hypoxic tumor areas has been solved, achieving precision and enhanced safety in tumor treatment.
Patent Information
- Application Number
- CN202510950115.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-10
- Publication Date
- 2025-10-28
AI Technical Summary
Traditional chemotherapy drugs and immunotherapy drugs lack targeting selectivity in tumor treatment, making it difficult for them to simultaneously enter the hypoxic area of the tumor, resulting in limited treatment effects. Furthermore, the toxic side effects and biocompatibility issues after mixing have not been effectively resolved.
Employing a self-driven immunotherapy combination drug system, this system targets hypoxic areas of tumors with anaerobic bacteria, combining liposome nanoparticles and a polydopamine coating to load chemotherapy and immunotherapy drugs, achieving precise delivery and release.
It significantly improves the efficiency of tumor treatment, enhances drug accumulation in tumors, reduces toxicity to normal tissues, achieves the combined effect of chemotherapy and immunotherapy, and enhances the immune response through photothermal therapy.
Smart Images
Figure CN120837657A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for preparing and applying a self-driven immune-enhancing combined drug system. Background Technology
[0002] The hypoxic tumor microenvironment has a significant impact on multidrug resistance and cancer cell metastasis in malignant solid tumors. Simultaneously, the hypoxic tumor microenvironment also suppresses the immune response and recruits tumor-associated macrophages, providing protection for the survival of tumor stem cells. During traditional radiotherapy or chemotherapy, the tumor microenvironment can promote cancer cell survival, making it difficult for drugs to penetrate necrotic or hypoxic areas, thus limiting their efficacy. Traditional chemotherapy or immunotherapy drugs lack sufficient targeting selectivity at the tumor site; they also distribute to other organs in the body, and the insufficient accumulation of most therapeutic drugs at the tumor site limits the effectiveness of combined therapy. Furthermore, chemotherapy or immunotherapy drugs, being two different types of drugs, are generally difficult to mix into a single drug system. Mixing them presents significant challenges in terms of drug retention, toxicity, and biocompatibility. There are currently no reports of mixing them to achieve the combined effects of both drugs, let alone using targeted therapy to simultaneously exert the effects of chemotherapy or immunotherapy drugs. How to achieve the combined effects of two drugs within a single drug system with targeted therapy is a pressing problem that needs to be solved. Summary of the Invention
[0003] The purpose of this invention is to provide a method for preparing a self-driven chemotherapy-immunotherapy combined drug system that has tumor tissue targeting, can treat solid malignant tumors and inhibit tumor metastasis, and has good biocompatibility.
[0004] Another object of the present invention is to provide specific applications of the self-driven immune combination drug system prepared above.
[0005] This invention is achieved through the following technical solution: a method for preparing a self-driven immune-enhancing combined drug system, comprising the following steps: (1) Preparation of blank liposome nanoparticles; (2) Use blank liposome nanoparticles to load chemotherapy drugs to obtain liposome nanoparticles loaded with chemotherapy drugs; (3) Coating the surface of liposome nanoparticles loaded with chemotherapy drugs with a polydopamine coating; (4) Immunotherapy drugs are anchored on the surface of liposome nanoparticles loaded with chemotherapeutic drugs using a polydopamine coating to obtain nanoparticles loaded with both chemotherapeutic drugs and immunotherapy drugs. (5) Finally, anaerobic bacteria were combined with nanoparticles simultaneously loaded with chemotherapeutic drugs and immunotherapeutic drugs to obtain a self-driven chemotherapeutic-immunotherapy drug system that can deliver chemotherapeutic drugs and immunotherapeutic drugs to the hypoxic area of the tumor through anaerobic bacteria and release chemotherapeutic drugs and immunotherapeutic drugs to the hypoxic area of the tumor.
[0006] The principle of this technical solution is that anaerobic bacteria carry nanoparticles containing chemotherapy drugs and immunotherapy drugs. By leveraging the characteristic that anaerobic bacteria can actively target the anaerobic region of the tumor, the dual-drug nanoparticles are transported to the hypoxic area of the tumor, significantly enhancing the efficiency of drug accumulation in the tumor, increasing drug concentration, and achieving precise combined chemotherapy and immunotherapy, thereby greatly improving the efficacy of tumor treatment.
[0007] In this method, liposome nanoparticles loaded with chemotherapeutic drugs were prepared using an ammonium sulfate gradient method. After preparation, polydopamine was coated onto the surface of the nanoparticles through in-situ synthesis, thereby obtaining polydopamine nanoparticles encapsulating chemotherapeutic drugs. Anaerobic bacteria were tightly bound to the 5,6-dihydroxyindole and catechol molecular structures in the polydopamine molecule through intermolecular forces.
[0008] Anaerobic bacteria possess the ability to target hypoxic areas of tumors. They can also inhibit tumor cell growth through various mechanisms, such as submitting bacterial antigens to induce specific T-cell aggregation, thereby killing tumor cells at the bacterial aggregation site; inducing inflammatory responses and the release of inflammatory factors, resulting in non-specific tumor killing; competing with tumor cells for nutrients at the tumor site, preventing cancer cells from absorbing sufficient nutrients and thus limiting their growth; and having bacterial proteins phagocytosed by macrophages, leading to programmed cell death in tumor cells. Whether anaerobic bacteria function as drug carriers or possess inherent tumor-suppressive capabilities, they represent an important tool in targeted therapy for hypoxic tumor areas.
[0009] Liposomes, as novel drug carriers, offer significant advantages in anti-tumor therapy. Their phospholipid bilayer structure can simultaneously encapsulate both hydrophilic and hydrophobic drugs, passively targeting tumor tissues through enhanced permeability and retention (EPR) effects while reducing toxicity to normal cells. Surface modification with PEG prolongs cycle time, and linking antibodies or ligands enables active targeting, further enhancing efficacy. Therefore, liposomes, with their high biocompatibility and flexible drug loading capabilities, remain a core delivery system for targeted tumor therapy. With the integration of nanotechnology and precision medicine, functionalized liposomes hold promise for driving the development of personalized anti-cancer therapy.
[0010] Polydopamine is a novel, multifunctional biomimetic material with wide applications in biomedicine and other fields. Polydopamine offers several advantages in biomedical applications. Its preparation is simple and mild, requiring no organic solvents, greatly facilitating its research and application. Under alkaline conditions, dopamine can be polymerized into uniformly sized polydopamine nanoparticles. Due to its unique molecular structure, it exhibits excellent adhesion, allowing it to form a polydopamine layer on the surface of organic or inorganic nanoparticles through polymerization. Furthermore, polydopamine is also an excellent photothermal agent with significant photothermal conversion capabilities. It can generate heat energy under near-infrared laser irradiation, thereby killing tumor cells. Therefore, polydopamine is not only used as a drug carrier but also as a photothermal agent for tumor treatment.
[0011] To better prepare a self-driven immune-mediated drug system, further, in step (1), the specific process for preparing blank liposome nanoparticles is as follows: (1.1) Weigh out lecithin and cholesterol in a mass ratio of 3 to 10:1; (1.2) Dissolve the weighed lecithin and cholesterol in anhydrous ethanol, then transfer them to a flask and remove the organic solvent by rotary evaporation. (1.3) Add 250 mM ammonium sulfate to the flask and shake thoroughly to hydrate and detach the lipid membrane to form liposomes; (1.4) Blank liposome nanoparticles are obtained after sonication, filtration and dialysis.
[0012] To better prepare a self-driven chemoimmunotherapy combined drug system, further, in step (2), the specific preparation process of the liposome nanoparticles loaded with chemotherapeutic drugs is as follows: (2.1) Weigh out the small molecule chemotherapy drug according to the mass ratio of the blank liposome nanoparticles prepared, based on the mass ratio of the blank liposome nanoparticles to the small molecule chemotherapy drug, which is 5~10:1. (2.2) Add the weighed chemotherapy drug to the solution containing the blank liposome nanoparticles and mix well. Place the mixture in a constant temperature water bath at 45°C for 30 minutes. (2.3) Liposome nanoparticles loaded with chemotherapeutic drugs were obtained after dialysis.
[0013] To better prepare a self-driven chemoimmunization combination drug system, the chemotherapeutic drug is further defined as a small molecule chemotherapeutic drug, specifically at least one of doxorubicin, doxorubicin hydrochloride, pirubicin, and paclitaxel.
[0014] To better prepare a self-driven chemoimmunotherapy combined drug system, further, in step (3), the specific process of coating the surface of the liposome nanoparticles loaded with chemotherapeutic drugs with a polydopamine coating is as follows: (3.1) The prepared liposome nanoparticles loaded with chemotherapeutic drugs were added to a Tris hydrochloric acid solution with a pH of 8.5; (3.2) After adding dopamine in an amount 1.5 to 2 times that of the liposome nanoparticles loaded with chemotherapy drugs, place the mixture on a magnetic stirrer and stir. (3.2) After a polydopamine coating is formed on the surface of the liposomes, liposome nanoparticles loaded with chemotherapeutic drugs are obtained with the polydopamine coating on the surface.
[0015] To better prepare a self-driven chemoimmunotherapy combined drug system, further, in step (4), the specific preparation process of the nanoparticles simultaneously loaded with chemotherapeutic drugs and immunotherapy drugs is as follows: (4.1) Add immunotherapy drugs to the solution containing liposome nanoparticles loaded with chemotherapy drugs coated with polydopamine and stir overnight; (4.2) Centrifugation and washing are performed to remove free immunotherapy drugs, thus obtaining nanoparticles loaded with both chemotherapeutic and immunotherapy drugs.
[0016] To better prepare a self-driven immunotherapy combination drug system, the immunotherapy drug is further defined as an immune checkpoint inhibitor, specifically at least one of a PD-1 antibody, a PD-L1 antibody, or a CTL4 antibody.
[0017] To better prepare the self-driven immune-mediated drug system, further, in step (5), the specific preparation process of the self-driven immune-mediated drug system is as follows: (5.1) Culture anaerobic bacteria at 37℃ in an anaerobic environment and collect anaerobic bacterial colonies in the logarithmic growth phase; (5.2) Add the collected anaerobic bacterial colonies to the solution containing the nanoparticles simultaneously loaded with chemotherapeutic drugs and immunotherapy drugs, and mix thoroughly; (5.3) Under anaerobic conditions, at 37°C and 100 rad / min, incubate with shaking for 2-6 hours; (5.4) Centrifuge at 2700 rad / min for 3 min to 10 min, wash with PBS solution, repeat centrifugation and washing three times, and collect the lower precipitate to obtain the self-driven chemoimmunization combined drug system.
[0018] To better prepare a self-driven immune-enhancing drug system, the anaerobic bacteria are further defined as anaerobic bacteria with anaerobic tropism and high biocompatibility, specifically at least one of Bifidobacterium, Lactobacillus, Escherichia coli, attenuated Salmonella, and Clostridium.
[0019] The application of the self-driven immunotherapy combined drug system prepared by the above method in the preparation of drugs for treating solid malignant tumors, including breast cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bile duct cancer, kidney cancer, cervical cancer, colorectal cancer, ovarian cancer, pancreatic cancer, and brain tumors.
[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The system prepared by the present invention can simultaneously load chemotherapeutic drugs and immunotherapy drugs. By taking advantage of the characteristic that anaerobic bacteria can actively target the anaerobic region of the tumor, the dual-drug nanoparticles of chemotherapy and immunotherapy are transported to the hypoxic region of the tumor, which significantly enhances the accumulation efficiency of the therapeutic drugs in the tumor, increases the drug concentration, and achieves precise combined chemotherapy and immunotherapy, thereby improving the efficacy of tumor treatment to a greater extent. (2) In this invention, dopamine is used as a raw material for surface modification of drug-loaded nanoparticles. After self-polymerization, polydopamine is formed, which contains catechol structure, thus having strong adhesion. This allows the prepared drug-loaded nanoparticles to adhere more stably to the surface of bacteria, have good biocompatibility, and disintegrate in the acidic and reducing (high glutathione concentration) microenvironment of tumors, thus playing a role in slow drug release. (3) The polydopamine used in this invention is an excellent photothermal agent with significant photothermal conversion ability. It can generate heat energy under near-infrared laser irradiation and can be used for photothermal therapy of tumors. In addition, when combined with immunostimulatory cytokine therapy, it can further enhance the immune effect stimulated by photothermal therapy, form long-term immune memory, and produce a long-term killing effect on tumor cells. It is superior to traditional photothermal therapy and immunotherapy. It can not only achieve more precise delivery of photothermal therapy agents, but also produce a brand-new immune enhancement therapy effect. (4) The preparation process of this invention is simple and has high biosafety. It can effectively kill tumor cells, improve the hypoxic microenvironment of tumor tissue, significantly improve the effect of conventional chemotherapy and immunotherapy, provide more ideas and means for clinical tumor treatment, provide a new direction for solid tumor treatment, and promote the further development of comprehensive treatment combining chemotherapy and immunotherapy in clinical applications. Attached Figure Description
[0021] Other features, objects, and advantages of the invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a transmission electron microscope image of the self-driven immunotherapy combined drug system prepared in Example 1 of this invention. Figure 2 This is a transmission electron microscope (TEM) image of the blank liposome nanoparticles prepared in Example 1 of this invention. Figure 3This is a transmission electron microscope image of the liposome nanoparticles loaded with doxorubicin prepared in Example 1 of this invention. Figure 4 These are photographs of the hemolysis experiments in Example 2 of the present invention; Figure 5 These are microscope images of each group after co-incubation with mouse red blood cell suspension in Example 2 of the present invention; Figure 6 The bacterial distribution in major organs and tumor tissues of tumor-bearing mice after intravenous injection of the self-driven immunization combined drug system prepared in Example 3 of the present invention at 1, 4 and 7 days. Figure 7 This is a line graph showing the relative tumor volume changes in each group of mice in Example 4 of the present invention; Figure 8 This is a line graph showing the weight changes of mice in each group in Example 4 of the present invention; Figure 9 This is a drug distribution diagram in the bodies of mice in groups three, four, and five after treatment in Example 4 of the present invention; Figure 10 Images of the major organs of mice in each group after treatment in Example 4 of this invention; Figure 11 These are Masson staining images of the heart tissue of mice in each group after treatment in Example 4 of the present invention; Figure 12 This is a graph showing the analysis of various blood routine indicators of mice in each group after treatment in Example 5 of the present invention; Figure 13 This is a graph showing the analysis of liver and kidney function indicators in each group of mice after treatment in Example 5 of the present invention. Detailed Implementation
[0022] The present invention is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for more detailed and specific illustration and should not be construed as limiting the present invention in any way. This section provides a general description of the materials and test methods used in the experiments of the present invention. Although many materials and operating methods used to achieve the objectives of the present invention are well known in the art, the present invention is still described in as much detail as possible herein. Those skilled in the art will understand that the present invention is further illustrated below with specific embodiments; however, it should be understood that these embodiments are merely for more detailed and specific illustration and should not be construed as limiting the present invention in any way.
[0023] Example 1: This embodiment provides a method for preparing a self-driven immune-enhancing combined drug system, as detailed below: Raw materials for preparation: lecithin, cholesterol, doxorubicin, anhydrous ethanol, ammonium sulfate, Tris hydrochloric acid solution at pH 8.5, dopamine, aPD-1 antibody, Bifidobacterium infantis, bacterial culture medium, and PBS solution.
[0024] The specific preparation process is as follows: (1) Preparation of liposome nanoparticles loaded with chemotherapeutic drugs by ammonium sulfate gradient method: A certain amount of lecithin and cholesterol were weighed at a ratio of 10~3:1 (w / w), dissolved in anhydrous ethanol, and transferred to a flask. The organic solvent was removed by rotary evaporation. 250 mM ammonium sulfate was added to the flask and shaken thoroughly to hydrate and detach the lipid membrane, forming liposomes. After sonication, filtration, and dialysis, liposome nanoparticles were obtained. A certain amount of doxorubicin (liposomes:doxorubicin = 5~10:1) was weighed and added to the prepared blank liposome solution and mixed well. The mixture was incubated in a 45℃ constant temperature water bath for 30 minutes, and after dialysis, doxorubicin-loaded liposome nanoparticles were obtained. (2) Preparation of polydopamine-coated liposome nanoparticles via the self-polymerization reaction of dopamine under alkaline conditions: Doxorubicin-loaded liposome nanoparticles were added to a Tris hydrochloric acid solution at pH 8.5, then dopamine was added, and the mixture was placed on a magnetic stirrer and stirred to form a polydopamine coating on the surface of the liposomes. This is the polydopamine-coated doxorubicin-loaded liposome nanoparticles. (3) Anchoring immunotherapy drugs to the surface of liposome nanoparticles via polydopamine: The prepared polydopamine-coated doxorubicin-loaded liposome nanoparticles were added to the aPD-1 antibody and stirred overnight. The mixture was then centrifuged and washed to remove the free drug, thus obtaining liposome nanoparticles co-loaded with doxorubicin and aPD-1 antibody. (4) Preparation of anaerobic bacterial colony solution: Bifidobacterium infantis was cultured at 37°C in an anaerobic environment, and colonies in the logarithmic growth phase were collected to obtain an anaerobic bacterial solution. (5) The collected anaerobic bacterial solution was thoroughly mixed with the prepared liposome nanoparticle solution co-loaded with doxorubicin and aPD-1 antibody, and shaken for 2-3 hours under anaerobic conditions at 37°C and 100 rad / min. Then, the mixture was centrifuged at 2700 rpm for 4 minutes to remove unloaded nanoparticles, washed with PBS solution and centrifuged twice to obtain a self-driven chemoimmunotherapy drug system that can target hypoxic areas of tumors and release doxorubicin and aPD-1 antibody.
[0025] The transmission electron microscopy morphology of the prepared self-driven immunotherapy combined drug system, such as... Figure 1 As shown.
[0026] The transmission electron microscopy morphology of the blank liposome nanoparticles prepared in step (1) is as follows: Figure 2 As shown, the transmission electron microscopy morphology of the prepared doxorubicin-loaded liposome nanoparticles is as follows: Figure 3 As shown.
[0027] Example 2: This embodiment uses the self-driven immune-enhancing combined drug system prepared in the above embodiment to conduct an in vitro blood safety experiment, as detailed below: 1. Selecting experimental subjects: Mouse blood was collected and prepared into a 2% (v / v0) red blood cell suspension.
[0028] 2. Preparation of the medicine solution: The same amount of blank liposome nanoparticles, liposome nanoparticles loaded with doxorubicin, liposome nanoparticles loaded with both doxorubicin and aPD-1 antibody, and the prepared self-driven chemoimmunization combined drug system were each prepared into 1 mL solutions with physiological saline.
[0029] Prepare 1 mL of bacterial suspension of anaerobic bacteria in the self-driven immunization combined drug system using physiological saline.
[0030] 3. Experimental Grouping: Group A: 1 mL of double-distilled water, used as a positive control group to induce complete hemolysis; Group B: 1 mL of normal saline, serving as a negative control group to simulate conditions without hemolysis; Group C: 1 mL of physiological saline containing blank liposome nanoparticles; Group d: 1 mL of physiological saline containing liposome nanoparticles loaded with doxorubicin; Group e: 1 mL of physiological saline containing a bacterial suspension of anaerobic bacteria from the self-driven immune combined drug system; Group f: 1 mL of physiological saline containing liposome nanoparticles simultaneously loaded with doxorubicin and aPD-1 antibody; Group g: 1 mL of physiological saline containing a self-driven immune-enhancing drug system.
[0031] 4. Experimental Procedure Each group was thoroughly mixed with 1 mL of 2% (v / v0) mouse erythrocyte suspension and incubated at 37°C for 4 hours. After 4 hours, the hemolysis of erythrocytes in each group was observed, and the rupture of erythrocytes in each group was observed under a microscope.
[0032] 5. Experimental Results The hemolysis status of each group of red blood cells, such as Figure 4 As shown. The hemolysis of red blood cells under a microscope, as shown... Figure 5 As shown.
[0033] according to Figure 4 , Figure 5 As shown, compared to completely hemolyzed double-distilled water, after mixing with the red blood cell suspension in each group, the morphology of the red blood cells was similar to that of the negative control. The cells were spherical, structurally intact, and did not show an obvious dark red color, indicating that no hemolysis occurred. This shows that blank liposome nanoparticles, liposome nanoparticles loaded with doxorubicin, liposome nanoparticles loaded with both doxorubicin and aPD-1 antibody, and even anaerobic bacteria that play a targeting role, do not cause hemolysis. This also fully demonstrates that the self-driven chemoimmunotherapy combined drug system prepared in this invention has good blood compatibility.
[0034] Example 3: In this embodiment, the self-driven immunotherapy combined with drug system prepared in the above embodiment was used to conduct an in vivo targeting experiment on tumor tissue. The experimental procedure is as follows: A tumor-bearing mouse model was established in accordance with ethical requirements for animal experiments. Female BALB / c mice, approximately 4-6 weeks old, were subcutaneously injected with 4T1 cancer cells. Once the tumors formed and grew to a volume of 50-100 mm³, 0.2 ml of a saline suspension of the self-driven immunotherapy combined drug system prepared according to this invention was intravenously injected. Mice were sacrificed on days 1, 4, and 7 post-injection, and the tumors were dissected, along with vital organs (heart, liver, spleen, lungs, and kidneys) for later use. The vital organ tissues and tumor tissues were minced, homogenized, and evenly spread onto solid LB agar plates and cultured at 37°C under anaerobic conditions. After 48 hours, images of the agar plates were taken and bacteria counted to determine the distribution of bacteria in the organs and tumors, verifying the targeting ability of this invention to tumor tissue.
[0035] The result is as follows Figure 6 As shown in the figure. The results showed that the bacteria were mainly distributed in the liver, kidneys, and tumor tissues. As time went on, the number of bacteria in the liver and kidneys decreased, but the number of bacteria in the tumor tissues remained high. This proves that the self-driven chemoimmunotherapy combined drug system obtained in this invention has good targeting ability to tumor tissues, while it is less distributed or rapidly metabolized in normal organs, and has little impact on normal organs.
[0036] Example 4: This embodiment uses the self-driven immunotherapy combined drug system prepared in the above embodiment to verify its antitumor effect. The specific experimental process is as follows: Female BALB / c mice aged 4-6 weeks with similar body size and weight and living in the same environment were selected and subcutaneously injected with 10 7 4T1 cells were used to establish multiple breast cancer-bearing mouse models. Cells with an average growth volume of 90 mm² were selected. 3Mice with tumors (ensuring sufficient hypoxic areas are formed within the tumor tissue) and roughly the same body weight were used as experimental subjects.
[0037] The selected experimental mice were divided into groups, and each group received the following intravenous injections every 3 days: Group 1: Blank control group, injected with normal saline; Group 2: Injection of bacterial suspension of anaerobic bacteria in the self-driven immunization combined drug system; Group 3: Injection of free doxorubicin and aPD-1 antibody drugs; Group 4: Injection of liposome nanoparticle solutions co-loaded with doxorubicin and aPD-1 antibody; Group 5: Self-driven immunomodulatory combined drug system solution prepared by injection.
[0038] The treatment plan was completed after three administrations, and the tumor volume and body weight of the mice were recorded regularly.
[0039] Experimental results are as follows Figure 8 , Figure 9 As shown, the tumor volume growth rate in the fifth group was the lowest, and there was no significant change in body weight, indicating that its toxicity was within a safe range. This also demonstrates that the present invention has the advantage of good safety while ensuring efficacy.
[0040] After each group of mice was treated, the tumors were removed and the vital organs (heart, liver, spleen, lungs, and kidneys) were taken out for later use.
[0041] The vital organ tissues and tumor tissues of some treated mice from groups three, four, and five were minced, ground, and homogenized. The concentrations of doxorubicin in the vital organ tissues and tumor tissues were then measured. The experimental results are as follows: Figure 9 As shown, the highest concentration of chemotherapeutic drugs was observed in the tumor tissue of mice treated with this invention, indicating that this invention can significantly increase the accumulation of chemotherapeutic drugs in tumor tissue. Furthermore, the distribution in other normal organs was significantly lower than that of other doxorubicin drug systems, indicating that this invention can reduce distribution in normal organs and thus alleviate toxic side effects.
[0042] After H&E staining of vital organ tissues from each group of treated mice, the results are as follows: Figure 10 As shown, no obvious abnormalities were observed in the structure of important organs such as the heart, liver, spleen, lungs, and kidneys in any of the groups of mice, especially in the fifth group.
[0043] Masson-stained sections of heart tissue from each group of treated mice yielded the following results: Figure 11As shown, the third group of treated mice had clear blue collagen deposits in their hearts, indicating fibrotic lesions in the hearts of these mice, one of the most common toxic side effects of the chemotherapy drug doxorubicin. In contrast, no collagen fiber deposits were found in the hearts of mice treated with this invention, demonstrating that this invention significantly reduces the cardiotoxicity of the chemotherapy drug doxorubicin, ensuring its safety in vivo.
[0044] Example 5: This embodiment evaluates the safety of the self-driven immune combination drug system prepared in the above embodiment in animal experiments.
[0045] Healthy female BALB / C mice aged 4-6 weeks with the same body size, weight, and living in the same environment were selected as experimental subjects.
[0046] The selected experimental mice were divided into groups, and each group received the following intravenous injections every 3 days: Control group: injected with 0.2 mL of normal saline; Experimental group: 0.2 mL of physiological saline suspension of the self-driven immunization combined drug system was injected.
[0047] After three administrations, the treatment plan was completed. Complete blood counts and liver and kidney function indicators were measured in each group of mice. The average values of the measured indicators for each group were taken.
[0048] Results of routine blood tests in mice as follows Figure 12 As shown, the self-driven immune-enhancing combined drug system prepared by the invention did not significantly affect the various blood routine indicators of mice when used for treatment.
[0049] Results of liver and kidney function tests in mice as follows Figure 13 As shown, the self-driven immune-enhancing combined drug system prepared by the invention did not have a significant impact on the liver and kidney function of mice when used for treatment.
[0050] This demonstrates that the self-driven immune-mediated drug combination system prepared in this invention has good biosafety.
[0051] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims
1. A method for preparing a self-driven immune-enhancing combined drug system, characterized in that, The following steps are involved: (1) Preparation of blank liposome nanoparticles; (2) Use blank liposome nanoparticles to load chemotherapy drugs to obtain liposome nanoparticles loaded with chemotherapy drugs; (3) Coating the surface of liposome nanoparticles loaded with chemotherapy drugs with a polydopamine coating; (4) Immunotherapy drugs are anchored on the surface of liposome nanoparticles loaded with chemotherapeutic drugs using a polydopamine coating to obtain nanoparticles loaded with both chemotherapeutic drugs and immunotherapy drugs. (5) Finally, anaerobic bacteria were combined with nanoparticles simultaneously loaded with chemotherapeutic drugs and immunotherapeutic drugs to obtain a self-driven chemotherapeutic-immunotherapy drug system that can deliver chemotherapeutic drugs and immunotherapeutic drugs to the hypoxic area of the tumor through anaerobic bacteria and release chemotherapeutic drugs and immunotherapeutic drugs to the hypoxic area of the tumor.
2. The method for preparing a self-driven immune-enhancing combined drug system according to claim 1, characterized in that, In step (1), the specific process for preparing blank liposome nanoparticles is as follows: (1.1) Weigh out lecithin and cholesterol in a mass ratio of 3 to 10:1; (1.2) Dissolve the weighed lecithin and cholesterol in anhydrous ethanol, then transfer them to a flask and remove the organic solvent by rotary evaporation. (1.3) Add 250 mM ammonium sulfate to the flask and shake thoroughly to hydrate and detach the lipid membrane to form liposomes; (1.4) Blank liposome nanoparticles are obtained after sonication, filtration and dialysis.
3. The method for preparing a self-driven immune-enhancing combined drug system according to claim 1 or 2, characterized in that, In step (2), the specific preparation process of the liposome nanoparticles loaded with chemotherapy drugs is as follows: (2.1) Weigh out the small molecule chemotherapy drug according to the mass ratio of the blank liposome nanoparticles prepared, based on the mass ratio of the blank liposome nanoparticles to the small molecule chemotherapy drug, which is 5~10:
1. (2.2) Add the weighed chemotherapy drug to the solution containing the blank liposome nanoparticles and mix well. Place the mixture in a constant temperature water bath at 45°C for 30 minutes. (2.3) Liposome nanoparticles loaded with chemotherapeutic drugs were obtained after dialysis.
4. The method for preparing a self-driven immune-enhancing combined drug system according to claim 3, characterized in that, The chemotherapy drug is a small molecule chemotherapy drug, specifically at least one of doxorubicin, doxorubicin hydrochloride, pirubicin, and paclitaxel.
5. A method for preparing a self-driven immune-enhancing combined drug system according to claim 1 or 2, characterized in that, In step (3), the specific process of coating the surface of the liposome nanoparticles loaded with chemotherapy drugs with a polydopamine coating is as follows: (3.1) The prepared liposome nanoparticles loaded with chemotherapeutic drugs were added to a Tris hydrochloric acid solution with a pH of 8.5; (3.2) After adding dopamine in an amount 1.5 to 2 times that of the liposome nanoparticles loaded with chemotherapy drugs, place the mixture on a magnetic stirrer and stir. (3.2) After a polydopamine coating is formed on the surface of the liposomes, liposome nanoparticles loaded with chemotherapeutic drugs are obtained with the polydopamine coating on the surface.
6. The method for preparing a self-driven immune-enhancing combined drug system according to claim 1 or 2, characterized in that, In step (4), the specific preparation process of the nanoparticles simultaneously loaded with chemotherapy drugs and immunotherapy drugs is as follows: (4.1) Add immunotherapy drugs to the solution of liposome nanoparticles loaded with chemotherapy drugs coated with polydopamine and stir overnight; (4.2) Centrifugation and washing are performed to remove free immunotherapy drugs, thus obtaining nanoparticles loaded with both chemotherapeutic and immunotherapy drugs.
7. The method for preparing a self-driven immune-enhancing combined drug system according to claim 6, characterized in that, The immunotherapy drug is an immune checkpoint inhibitor, specifically at least one of PD-1 antibody, PD-L1 antibody, or CTL4 antibody.
8. A method for preparing a self-driven immune-enhancing combined drug system according to claim 1 or 2, characterized in that, In step (5), the specific preparation process of the self-driven immune combined drug system is as follows: (5.1) Culture anaerobic bacteria at 37℃ in an anaerobic environment and collect anaerobic bacterial colonies in the logarithmic growth phase; (5.2) Add the collected anaerobic bacterial colonies to the solution containing the nanoparticles simultaneously loaded with chemotherapeutic drugs and immunotherapy drugs, and mix thoroughly; (5.3) Under anaerobic conditions, at 37°C and 100 rad / min, incubate with shaking for 2-6 hours; (5.4) Centrifuge at 2700 rad / min for 3 min to 10 min, wash with PBS solution, repeat centrifugation and washing three times, and collect the lower precipitate to obtain the self-driven chemoimmunization combined drug system.
9. The method for preparing a self-driven immune-enhancing combined drug system according to claim 8, characterized in that, The anaerobic bacteria are anaerobic bacteria with anaerobic tendency and high biosafety, specifically at least one of Bifidobacterium, Lactobacillus, Escherichia coli, attenuated Salmonella, and Clostridium.
10. The use of the self-driven immunotherapy combined drug system prepared by the method according to any one of claims 1 to 9 in the preparation of drugs for treating solid malignant tumors, wherein the solid malignant tumors include breast cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bile duct cancer, kidney cancer, cervical cancer, colorectal cancer, ovarian cancer, pancreatic cancer, and brain tumors.