Bionic nano preparation as well as preparation method and application thereof

Abstract

The invention discloses a bionic nano preparation as well as a preparation method and application thereof. Interleukin-2 and curcumin are encapsulated by polylactic acid-glycolic acid copolymer-polyethylene glycol, then the periphery of the formed nanoparticles is coated with a manganese oxide shell, the nano-composite is coated with a cell membrane, the nano-composite has a multi-layer composite structure, the cell membrane on the outermost layer provides tumor targeting, and the nano-composite has an anti-tumor effect. After reaching a tumor microenvironment, the manganese oxide shell is quickly degraded, the process is accompanied by a tumor killing effect so as to activate anti-tumor adaptive immunity, and manganese ions in a degradation product can activate anti-tumor innate immunity and enhance immune infiltration of T cells. When manganese oxide is degraded completely, interleukin-2 and curcumin begin to be slowly released so as to promote T cell proliferation in a tumor microenvironment and inhibit expression of an immune examination site PD-L1. Therefore, the multifunctional bionic nano preparation developed by the invention provides a new choice for tumor immunotherapy, and has good clinical application and development values.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical nanomaterials, and in particular to a bionic nano preparation and a preparation method and application thereof. Background Art

[0002] In recent years, the prevalence of cancer, a deadly disease, has continued to increase. Currently, surgery, radiotherapy, chemotherapy, immunotherapy or multi-strategy combined therapy are commonly used in clinical treatment of tumors. However, these treatments cannot reverse tumor progression and are accompanied by serious toxic side effects. Therefore, there is an urgent need to develop new anti-tumor drugs that can safely and effectively curb tumor progression. Increasing the targeting of anti-tumor drugs can improve their enrichment in the tumor microenvironment and reduce their accumulation in other organs, which helps to achieve the reduction of toxicity and enhancement of efficacy of anti-tumor drugs. The biomedical application of nanotechnology provides a new platform for tumor-targeted drug delivery. Common targeting strategies include coupling targeting ligands that can specifically recognize highly expressed tumor markers on the surface of nanoformulations, coating nanoformulations with biological materials (extracellular vesicles, cell membranes), and passive targeting based on the high permeability and retention (EPR) effect. Compared with other targeting strategies, nanocarriers coated with cell membrane carriers can inherit some functions of their parent cells due to membrane proteins. For example, CD47 expressed on the membrane can release a "don't eat me" signal to promote the long circulation of bionic nanocarriers. In tumor-targeted carriers, cell membranes from macrophages, tumor cells, etc. can also achieve chemotaxis to the tumor microenvironment and homologous targeted delivery through membrane proteins. Therefore, cell membrane bionic nanocarriers are widely used in the research of tumor targeted therapy due to their advantages such as long circulation and high targeting, and have extremely high clinical application value.

[0003] Immunotherapy has brought revolutionary changes to tumor treatment. Activating the body's anti-tumor immunity helps reduce tumor recurrence. This process first requires the release of tumor antigens to activate antigen-presenting cells, then activate tumor-specific T cells and complete immune infiltration of tumors, and finally achieve T cell proliferation in the tumor microenvironment and overcome T cell functional exhaustion caused by immune checkpoints. In addition, ideal immunotherapy also requires the initiation of an anti-tumor immune cycle, that is, tumor cells killed by T cells release tumor-associated antigens, thereby reactivating antigen-presenting cells in the tumor microenvironment. However, due to the lack of tumor antigens, low immune cell infiltration rate, rich in immunosuppressive cells, and tumor microenvironments that overexpress immune checkpoints, the effect of immunotherapy is usually poor, and the response rate of commonly used programmed cell death 1 / ligand 1 (PD-1 / PD-L1) inhibitors in clinical practice is low. Therefore, immunotherapy is usually combined with other anti-tumor strategies in basic research. However, most studies only focus on a few issues in tumor immunotherapy, and there is still a lack of strategies that can systematically overcome all of the above challenges. Therefore, it is necessary to develop a biomimetic targeted preparation that can overcome all challenges of tumor immunotherapy in an integrated manner. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing a bionic nanoparticle preparation.

[0005] Another object of the present invention is to provide a bionic nanoformulation. It is a bionic nanoformulation that can overcome all challenges of tumor immunotherapy in an integrated manner. After entering the systemic circulation, the outermost cell membrane of the prepared bionic nanoformulation can provide the drug with long circulation ability, and achieve precise targeted delivery to the tumor under chemotaxis, which plays a role in reducing toxicity and increasing efficacy. And after entering the tumor microenvironment, the bionic nanoformulation can programmatically release different drugs to meet the needs of different stages of tumor immunotherapy, thereby significantly improving the efficiency of tumor immunotherapy.

[0006] Another object of the present invention is to provide applications of the above-mentioned bionic nano preparation.

[0007] The purpose of the present invention is achieved through the following technical solutions: A method for preparing a bionic nanoparticle preparation comprises the following steps: (1) dissolving carboxyl-terminated poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA) and curcumin in an organic solvent 1, then adding dropwise to an organic solvent 2 containing interleukin-2, preparing colostrum by ultrasonication, then adding dropwise the obtained colostrum to an organic solvent 3, finally stirring the obtained emulsion to volatilize the organic solvent therein, thereby obtaining PLGA nanoparticles loaded with interleukin-2 and curcumin; (2) removing large particles and materials that have not formed nanoparticles from the solution obtained in (1) by centrifugation, then collecting PLGA nanoparticles by centrifugation again, and resuspending them in deionized water, adding potassium permanganate to the obtained PLGA nanoparticle solution, stirring in the dark, collecting nanoparticles by centrifugation, and washing and resuspending them in deionized water to obtain a manganese oxide-PLGA nanocomposite; (3) Collecting cells cultured in vitro, lysing the cells by hypotonic method, and collecting cell membranes by gradient centrifugation; (4) The nanocomposite obtained in (2) is mixed with the cell membrane obtained in (3), and the cell membrane-wrapped manganese oxide-PLGA nanocomposite is prepared by ultrasonication to obtain the bionic nanoformulation.

[0008] Preferably, the molecular weight of the carboxyl-terminated poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) in step (1) is 5,000 to 30,000.

[0009] More preferably, the molecular weight of the carboxyl-terminated poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) in step (1) is 20,000 to 30,000.

[0010] Preferably, the organic solvent 1 in step (1) is a mixture of ethyl acetate and chloroform, and the organic solvent 2 and the organic solvent 3 are aqueous solutions of polyvinyl alcohol.

[0011] More preferably, the organic solvent 1 in step (1) is a 1:1 mixture of ethyl acetate and chloroform, the organic solvent 2 is a 1% (v / v) polyvinyl alcohol aqueous solution, and the organic solvent 3 is a 0.5% (v / v) polyvinyl alcohol aqueous solution.

[0012] Preferably, the ratio of the carboxyl-terminated poly(lactic acid-co-glycolic acid)-polyethylene glycol, curcumin, interleukin-2 described in step (1) to the potassium permanganate described in step (2) is 15-25 mg: 1.5-2.5 mg: 1.5-2.5 μg: 10-25 mg by mass.

[0013] More preferably, the ratio of the carboxyl-terminated poly(lactic acid-co-glycolic acid)-polyethylene glycol, curcumin, interleukin-2, and the potassium permanganate described in step (2) is 20 mg: 2 mg: 2 μg: 20 mg by mass.

[0014] In actual preparation: Preferably, the amount of polylactic acid-co-glycolic acid-polyethylene glycol added in step (1) is calculated based on the ratio of polylactic acid-co-glycolic acid-polyethylene glycol to organic solvent 1 of 15-25 mg:0.5-1 mL, and the amount of curcumin added is calculated based on the ratio of curcumin to organic solvent 1 of 1.5-2.5 mg:0.5-1 mL.

[0015] More preferably, the amount of polylactic acid-co-glycolic acid-polyethylene glycol added in step (1) is calculated based on a ratio of 20 mg:0.6 mL to the organic solvent 1, and the amount of curcumin added is calculated based on a ratio of 2 mg:0.6 mL to the organic solvent 1.

[0016] Preferably, the ratio of interleukin-2, organic solvent 2, and organic solvent 3 in step (1) is 1.5-2.5 μg: 3-5 mL: 9-11 mL.

[0017] More preferably, the ratio of interleukin-2, organic solvent 2, and organic solvent 3 in step (1) is 2 μg: 5 mL: 11 mL.

[0018] Preferably, the ultrasound method described in step (1) is probe ultrasound with an ultrasound intensity of 100 W and a duty cycle of 3 seconds:3 seconds.

[0019] Preferably, the stirring speed in step (1) is 1000-1400 rpm, and the stirring time is overnight (8-14 h).

[0020] Preferably, in step (2), when removing the large particles and the material that has not formed nanoparticles in (1), the centrifugation conditions are 1000-1500 × g for 8-12 minutes, and when collecting the PLGA nanoparticles, the centrifugation conditions are 12000-15000 × g for 15-20 minutes.

[0021] Preferably, in step (2), collecting PLGA nanoparticles refers to collecting PLGA nanoparticles of appropriate size (less than 400 nanometers).

[0022] More preferably, in step (2), collecting PLGA nanoparticles refers to collecting PLGA nanoparticles below 400 nanometers.

[0023] Preferably, the light-shielded stirring condition in step (2) is 1000-1400 rpm for 4-8 h.

[0024] Preferably, the cells in step (3) include any one or more of red blood cells, platelets, macrophages, and tumor cells.

[0025] More preferably, the cells in step (3) include M1 macrophages.

[0026] Most preferably, the cells in step (3) include M1 macrophage cell line RAW264.7 cells.

[0027] Preferably, the hypotonic method in step (3) is to use TM buffer containing 1% PMSF for lysis in an ice-water bath at 4°C for 8 to 14 h.

[0028] Preferably, the ratio of the nanocomplex to the cell membrane in step (3) is 0.5-2 mg corresponding to 1×10 7 cells.

[0029] More preferably, the ratio of the nanocomplex to the cell membrane in step (3) is 1 mg corresponding to 1×10 7 cells.

[0030] Preferably, the ultrasound method described in step (3) is probe ultrasound with an ultrasound intensity of 100 W and a duty cycle of 3 seconds:3 seconds.

[0031] A bionic nano preparation is obtained by the above preparation method.

[0032] Application of the above bionic nano preparation in the preparation of anti-tumor drugs.

[0033] Preferably, the tumor comprises breast cancer.

[0034] The present invention uses cell membrane bionic nanocarriers to deliver manganese oxide, curcumin and interleukin-2 in a targeted manner, and through reasonable design, it first releases manganese ions quickly and then slowly releases interleukin-2 and curcumin, which can provide an integrated solution to a series of problems faced by tumor immunotherapy, such as tumor antigen loss, difficulty in T cell infiltration into the tumor microenvironment, limited T cell proliferation, and impaired T cell function. Specifically: After reaching the tumor microenvironment, the bionic nanoformulation will quickly degrade its manganese oxide shell, which is accompanied by a tumor-killing effect to produce tumor-associated antigens through immunogenic cell death pathways, thereby activating anti-tumor adaptive immunity, and the manganese ions in the degradation products can upregulate the cGAS-STING pathway to activate anti-tumor innate immunity and enhance the immune infiltration of T cells. When the degradation of the manganese oxide shell is complete, the PLGA nanoparticles at the core of the nanoformulation begin to slowly degrade, thereby slowly releasing the encapsulated interleukin-2 and curcumin to promote T cell proliferation in the tumor microenvironment and inhibit the expression of the immune checkpoint PD-L1. Therefore, the present invention has developed a multifunctional bionic nanoformulation that can activate T cells, enhance T cell tumor infiltration, promote T cell proliferation, and prevent T cell function exhaustion, providing a new option for tumor immunotherapy, with good clinical application and development value.

[0035] Compared with the prior art, the present invention has the following advantages and effects: 1. The biomimetic nanoformulation prepared by the present invention has a multilayer structure, wherein the outermost cell membrane coat provides long circulation and tumor targeting capabilities, the inner portion is a manganese oxide shell of a nanocomposite, and the innermost portion is interleukin-2 and curcumin encapsulated by PLGA nanoparticles. This unique multilayer structure enables the prepared biomimetic nanoformulation to have the ability to programmatically release different drugs, i.e., rapid release of manganese ions, sustained release of interleukin-2 and curcumin.

[0036] 2. The bionic nanoformulation provided by the present invention can quickly degrade the manganese oxide shell in the tumor microenvironment, and the released manganese ions help to enhance the tumor infiltration of T cells. When T cells arrive at the tumor microenvironment, the sustained-release interleukin-2 and curcumin can help T cells overcome the problems of blocked proliferation and functional exhaustion. Therefore, the bionic nanoformulation prepared by the present invention is more in line with the needs of different stages of tumor immunotherapy, and provides an integrated solution to a series of challenges faced by tumor immunotherapy.

[0037] 3. The bionic nanoparticle preparation prepared by the present invention is composed of a cell membrane of biological origin, interleukin-2, trace element manganese and biodegradable hydrocarbons, has good biocompatibility, and is suitable for in vivo drug administration.

[0038] 4. The preparation method of the bionic nanoformulation provided by the present invention has a relatively simple production process. The solvent required for the synthesis is mostly water, and only contains a small amount of volatile organic solvent. It is environmentally friendly and can achieve low-cost, large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a transmission electron microscopy image of the failed preparation of manganese oxide-drug-loaded PLGA nanoparticles of the present invention.

[0040] Figure 2 Transmission electron microscopy images and particle size distribution diagrams of PLGA nanoparticles (PLGA NPs), PLGA nanoparticles loaded with curcumin and interleukin-2 (PCI), and manganese oxide-drug-loaded PLGA nanoparticles (MPCI) prepared in the present invention.

[0041] Figure 3 Transmission electron microscopy images of the bionic nanoparticle preparation (MMPCI) prepared in the present invention under normal physiological environment and weakly acidic environment containing hydrogen peroxide.

[0042] Figure 4 This is a graph showing the anti-tumor activity test results of the bionic nanoformulation prepared by the present invention; wherein A is the effect of different concentrations of bionic nanoformulations on the activity of mouse breast cancer 4T1 cells; B is the apoptosis rate of mouse breast cancer 4T1 cells induced by different concentrations of bionic nanoformulations; and C is the immunogenic cell death of mouse breast cancer 4T1 cells induced by different concentrations of bionic nanoformulations.

[0043] Figure 5 This is a graph showing the results of a study on the ability of the bionic nanoformulation prepared in the present invention to activate the cGAS-STING pathway in vitro.

[0044] Figure 6 This is a diagram showing the tumor targeting evaluation results of the bionic nanoformulation prepared by the present invention; wherein A is the distribution of fluorescently labeled MPCI (Cy5-MPCI) and the bionic nanoformulation (Cy5-MMPCI) in various organs; and B is the quantitative analysis structure of A.

[0045] Figure 7 This is a diagram showing the evaluation results of the anti-tumor activity of the bionic nanoformulation prepared by the present invention; wherein A is the tumor volume growth curve of different groups; and B is the in vitro imaging of tumors of different groups.

[0046] Figure 8 This is a diagram showing the evaluation results of the bionic nanopreparation prepared by the present invention in reshaping the tumor immune microenvironment; wherein A is the content of MDSC cells in the tumor microenvironment; B is the content of mature DC cells in the tumor microenvironment; C is the content of M1 macrophages in the tumor microenvironment; and D is the content of tumor-specific toxic T cells in the tumor microenvironment.

[0047] Fig. 9 The figure is a study result of the effect of CD8 monoclonal antibody inhibitor on the anti-tumor activity of the bionic nanoformulation prepared by the present invention; wherein A is the volume of mouse tumor; and B is the weight of mouse tumor and its imaging.

[0048] Fig.10 This is a safety evaluation diagram of the bionic nanoformulation prepared in the present invention. DETAILED DESCRIPTION

[0049] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments, but the embodiments of the present invention are not limited thereto.

[0050] Example 1 An embodiment of the nanoformulation of the present invention, the preparation method of the nanoformulation of this embodiment comprises the following steps: 1. Dissolve 20 mg of carboxyl-terminated poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) (PLGA, molecular weight 30,000) and 2 mg of curcumin in 0.6 mL of a 1:1 solution of ethyl acetate and chloroform, then add dropwise to 5 mL of a 1% polyvinyl alcohol solution containing 2 μg of interleukin-2, prepare colostrum by probe ultrasound (ultrasonic power 100 W, duty cycle 3 s:3 s), then add dropwise the obtained colostrum to 11 mL of a 0.5% polyvinyl alcohol solution, finally stir the obtained emulsion at 1200 rpm overnight to evaporate the organic solvent, and obtain PLGA nanoparticles loaded with interleukin-2 and curcumin; 2. Centrifuge at 1000 × g for 10 minutes to remove large particles and materials that have not formed nanoparticles in the solution obtained in (1), then centrifuge at 12000 × g for 15 minutes again to collect PLGA nanoparticles of appropriate size, and resuspend them in deionized water to 5 mL. Add 20 mg of potassium permanganate to the PLGA nanoparticle solution, protect from light and stir at 1200 rpm. After 6 hours, collect the nanoparticles by centrifugation, wash them three times with deionized water and resuspend them to obtain manganese oxide-PLGA nanocomplexes; 3. Collect M1 macrophage cell line RAW264.7 cells induced by lipopolysaccharide and interferon-γ, then lyse them with TM buffer containing protease inhibitors in an ice-water bath at 4 degrees overnight, and then centrifuge at 10,000 × g for 30 minutes to collect the resulting cell membranes; 4. The nanocomplex obtained in (2) and the cell membrane obtained in (3) were mixed at a ratio of 1 mg PLGA to 1 × 10 7 The membranes obtained from the cells were mixed in a certain proportion, and the cell membrane-wrapped manganese oxide-PLGA nanocomplex was prepared by probe ultrasound method (ice water bath, ultrasound power 100 W, duty cycle 3 s:3 s) to obtain the biomimetic nanoparticle preparation (MMPCI).

[0051] Comparative Example 1 1. Dissolve 20 mg of carboxyl-terminated poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA) and 2 mg of curcumin in 0.6 mL of a 1:1 solution of ethyl acetate and chloroform, then add dropwise to 5 mL of a 1% polyvinyl alcohol solution containing 2 μg of interleukin-2, prepare colostrum by probe ultrasound (ultrasonic power 100 W, duty cycle 3 s:3 s), then add dropwise the obtained colostrum to 11 mL of a 0.5% polyvinyl alcohol solution, finally stir the obtained emulsion at 1200 rpm overnight to evaporate the organic solvent, and obtain PLGA nanoparticles loaded with interleukin-2 and curcumin; 2. Centrifuge at 1000 × g for 10 minutes to remove large particles and materials that have not formed nanoparticles in the solution obtained in (1), then centrifuge at 12000 × g for 15 minutes to collect PLGA nanoparticles of appropriate size and resuspend them in deionized water to 5 mL. Add 20 mg of potassium permanganate to the PLGA nanoparticle solution, protect from light and stir at 1200 rpm. After 2 hours, collect the nanoparticles by centrifugation, wash them three times with deionized water and resuspend them to obtain manganese oxide-PLGA nanocomplexes.

[0052] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is: In Example 3, 20 mg of carboxyl-terminated poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA) was dissolved in 0.6 mL of a 1:1 solution of ethyl acetate and chloroform, and then added dropwise to 5 mL of a 1% polyvinyl alcohol solution, and colostrum was prepared by probe ultrasound (ultrasonic power 100 W, duty cycle 3 s:3 s), and the obtained colostrum was added dropwise to 11 mL of a 0.5% polyvinyl alcohol solution, and finally the obtained emulsion was stirred at 1200 rpm overnight to evaporate the organic solvent therein, thereby obtaining PLGA nanoparticles (PLGANPs).

[0053] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that in Comparative Example 3, PLGA nanoparticles (PCI) loaded with curcumin and interleukin-2 were prepared in the same manner as in Example 1, but no subsequent operations were performed.

[0054] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that in Comparative Example 4, manganese oxide-PLGA nanocomposite loaded with interleukin-2 and curcumin (MPCI) was prepared in the same manner as in Example 1, but no subsequent operation was performed.

[0055] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that in Comparative Example 5, curcumin is not added to ethyl acetate and chloroform when preparing drug-loaded PLGA NPs, and the other operations are the same, and finally a manganese oxide-interleukin-2-loaded PLGA nanocomposite (MPI) without curcumin is obtained.

[0056] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that in Comparative Example 6, interleukin-2 is not added to the 1% polyvinyl alcohol solution when preparing drug-loaded PLGA NPs, and the other operations are the same, and finally a manganese oxide-curcumin-loaded PLGA nanocomposite (MPC) without interleukin-2 is obtained.

[0057] Effect Example 1 The transmission electron microscopy characterization of the product prepared in Comparative Example 1 of the present invention shows that Figure 1 As shown, the sample obtained after the PLGA NPs loaded with curcumin and interleukin-2 reacted with potassium permanganate for 2 hours was spherical, and the surface of the PLGA NPs was not completely wrapped by the manganese oxide shell, and the requirements for programmed release of different drugs (i.e., rapid release of manganese ions, sustained release of interleukin-2 and curcumin) could not be met, and the further cell membrane wrapping efficiency was affected. Therefore, the nanoformulation provided in Comparative Example 1 was a failed product.

[0058] Effect Example 2 Characterization and performance testing of the products prepared in the examples and comparative examples of the present invention: 1. The products prepared in the examples and comparative examples of the present invention were subjected to transmission electron microscopy imaging and particle size distribution analysis. The results are as follows: Figure 2 As shown, PLGA NPs and PCI are similar in morphology, both are spherical with smooth surface and particle size below 200 nanometers. The particle size of MPCI is also below 200 nanometers, but compared with PLGA NPs and PCI, it has a layer of manganese oxide shell on its periphery, and compared with the nanoformulation provided in Comparative Example 1, it has a more complete manganese oxide shell, which can meet the requirements of programmed release of different drugs (i.e., rapid release of manganese ions, sustained release of interleukin-2 and curcumin), so the MPCI provided in Example 1 is an optimization of Comparative Example 1.

[0059] 2. The product prepared in Example 1 of the present invention was imaged by transmission electron microscopy. The specific implementation scheme was as follows: the MMPCI of the same batch was resuspended in two buffer systems, one of which was a buffer system with a pH of 7.4 and no hydrogen peroxide, and the other was a buffer system with a pH of 6.3 and containing 10 mM hydrogen peroxide. After incubation for 1 hour, the MMPCI in the two buffer systems were collected and imaged by transmission electron microscopy respectively. The results are shown in FIG. Figure 3As shown in the figure, in a buffer system with a pH of 7.4 and no hydrogen peroxide, MMPCI presents a distinct multilayer structure, with the cell membrane at the outermost periphery, followed by a manganese oxide shell, and spherical nanoparticles inside. However, in a buffer system with a pH of 6.3 and 1 mM hydrogen peroxide, the core-shell structure of MMPCI is damaged, the peripheral cell membrane and manganese oxide shell are obviously detached and degraded, and only a more significant spherical structure can be observed. This result shows that MMPCI has the potential to rapidly degrade the peripheral manganese oxide shell in a weakly acidic and hydrogen peroxide-rich tumor microenvironment, and then slowly release curcumin and interleukin-2 in the innermost part.

[0060] Effect Example 3 The in vitro anti-tumor activity of the product prepared in Example 1 of the present invention was tested, and the anti-tumor activity test was performed using the mouse breast cancer 4T1 cell line as a model. The specific operation is as follows: 1. MMPCI was diluted to 40, 20, 10, 5, 2.5, and 1.25 μg / mL in a gradient manner using the basal culture medium DMEM. The cells were then added to a 96-well plate in which 4T1 cells had grown to 80% density. The 4T1 cells were incubated with MMPCI at different concentrations for 24 hours. The residual MMPCI in the wells was then washed and 10 μL of CCK-8 reagent was added. The cells were incubated again in the incubator for 3 hours and the absorbance of each group at 450 nm was detected using an ELISA reader. In this experiment, DMEM without MMPCI was used as the control group. The results are shown in the figure. Figure 4 As shown in Figure A, different concentrations of MMPCI had a significant effect on the activity of 4T1 cells, indicating that MMPCI has strong anti-tumor activity.

[0061] 2. MMPCI was diluted to 10, 5, and 2.5 μg / mL in the basic culture medium DMEM, and then added to a 6-well plate where 4T1 cells had grown to 80% density. Different concentrations of MMPCI were incubated with 4T1 cells for 24 hours. Subsequently, the cells were collected and the apoptosis rate was detected using a cell apoptosis kit. In this experiment, DMEM without MMPCI was used as the control group. The results are shown in Figure 2. Figure 4 As shown in B, as the MMPCI administration concentration increased, the apoptosis rate of 4T1 cells increased significantly, indicating that MMPCI can induce apoptosis of 4T1 cells.

[0062] 3. Dilute MMPCI in a gradient with the basal culture medium DMEM to a concentration of 10, 5, and 2.5 μg / mL, and then add it to a 12-well plate slide where 4T1 cells have grown to 80% density, so that different concentrations of MMPCI are incubated with 4T1 cells. After 24 hours, discard the culture medium and fix the cells with 4% paraformaldehyde. Then treat the cells with a permeabilization solution, and then add primary antibodies for calreticulin and high-mobility group protein B1, respectively, and incubate overnight at 4 degrees. The next day, continue to incubate the cells with a fluorescent secondary antibody, and stain the nucleus with DAPI. Finally, seal the slide with a sealing agent to prevent fluorescence quenching and perform imaging. In this experiment, DMEM without MMPCI was used as the control group. The results are as follows Figure 4 As shown in C, MMPCI can induce upregulation of calreticulin expression and membrane translocation in 4T1 cells, while reducing the expression of intracellular high-mobility group protein B1, indicating that MMPCI induces immunogenic cell death in 4T1 cells.

[0063] 4. MMPCI was diluted to a concentration of 5 μg / mL in the basal culture medium DMEM, and then added to a 6-well plate where 4T1 cells had grown to 80% density, and different concentrations of MMPCI were incubated with 4T1 cells for 24 hours. In this experiment, DMEM without MMPCI was used as the control group. Subsequently, the cells were collected and the protein was extracted with RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors, and the protein was denatured at 100 degrees for ten minutes after adding loading buffer, and the resulting protein was stored at -20 degrees. Subsequently, the expression rates of key proteins p-STING, p-TKB1 and p-IRF3 of the cGAS-STING pathway in the control group and the MMPCI-treated group were detected by western blot technology. β-actin was used as the internal reference protein in this experiment. The results are as follows Figure 5 As shown, the cGAS-STING pathway of 4T1 cells was significantly upregulated after MMPCI treatment.

[0064] Effect Example 4 The in vivo distribution of the products prepared in Example 1 and Comparative Example 4 of the present invention was evaluated. The fluorescent labeling of the nanoformulation was designed. The specific method was to add 10 mg of fluorescent dye Cy5 to a 1% polyvinyl alcohol solution when preparing PLGA NPs loaded with curcumin and interleukin-2. The rest of the preparation scheme was the same. The specific operation was: Six-week-old female BABL / c mice were injected with 100 μl of 1×10 6 The cells were randomly divided into two groups and the dorsal transplanted tumors were grown to 500 mm 3Fluorescently labeled MPCI (Cy5-MPCI) and fluorescently labeled MMPCI (Cy5-MMPCI) were injected into the tail vein of mice at the same time. The fluorescence intensity of the nanoformulations injected into the two groups of mice was equal. After 24 hours, the mice were killed, and the hearts, livers, spleens, lungs, kidneys and tumors of the mice were removed. The fluorescence intensity in different organs was detected by a small animal in vivo imaging device, and the fluorescence was quantitatively analyzed. The results are shown in Figure 6 As shown, the fluorescence intensity of the Cy5-MMPCI group at the tumor site was significantly higher than that of the Cy5-MPCI group, indicating that the M1-type RAW264.7 cell membrane involved in Example 1 helps to enhance the targeted delivery of the nanoformulation to the tumor.

[0065] Effect Example 5 The in vivo antitumor activity of the nanoformulations prepared in Example 1 of the present invention and Comparative Examples 3-6 was evaluated. The specific operation is as follows: 1. Inject 100 μL of 1×10 6 The cells were randomly divided into six groups and the dorsal transplanted tumors were grown to 100 mm 3 The nanoformulations obtained in Example 1 and Comparative Examples 3-6 were injected into the tail vein of mice at the same time, and the control group was injected with an equal volume of PBS. The first administration date was regarded as the first day, and the volume of the back transplanted tumors of each group of mice was measured every two days, and the drug was administered once every three days. In this embodiment, the method for calculating the volume of mouse tumors is: tumor volume = (long diameter of tumor × short diameter of tumor × short diameter of tumor) / 2. The growth curves of the tumors of each group of mice are shown in Figure 7 As shown in A, the tumor in the control group grew the fastest, and the other drug-treated groups all showed varying degrees of tumor inhibition. The final tumor volumes of each group were: 1342.6 ± 325 in the control group, 702.2 ± 161.9 in the MPI group, 929.9 ± 211 in the PCI group, 591.8 ± 172.7 in the MPC group, 577.1 ± 267.7 in the MPCI group, and 489.1 ± 159.7 in the MMPCI group. This result shows that the tumor inhibition effect of the MMPCI group was the most significant. The mice were killed on the thirteenth day, and the tumors of the mice in each group were removed for in vitro imaging, as shown in Figure 2. Figure 7 As shown in B, the MMPCI group also showed the most significant anti-tumor effect.

[0066] 2. Quickly cut the tumor tissue obtained above into pieces of 1-2 mm 3, and transferred to a C tube containing tissue digestion solution, then treated the tissue with a tissue dissociator, then took out the C tube and placed it in a 37-degree constant temperature shaker for half an hour, and then repeated the tissue dissociator and shaker incubation operation. Then, the obtained tissue suspension was passed through a 70-micron and 40-micron pore size filter, and the tumor tissue remaining on the filter was appropriately ground during the process to prepare a single cell suspension. The obtained single cell suspension was centrifuged at 300 × g for 5 minutes, the supernatant was removed, and resuspended with flow buffer. Subsequently, CD45 magnetic beads were added to the resuspended single cell suspension to sort out the immune cells in the tumor microenvironment, and then the flow antibody mixture corresponding to CD45, CD11b, CD11c, Gr-1, F4 / 80, CD86, CD3, and CD8 was added to each sample, and samples with larger cell amounts were selected to prepare flow single positive tubes. Each sample was incubated at room temperature for half an hour to allow the flow cytometry antibodies to fully react with the cells, then centrifuged again and the cells were resuspended in flow cytometry buffer to remove the unbound flow cytometry antibodies. Finally, the filter was filtered again to remove larger cell clumps. The content of MDSC, mature DC cells, M1 macrophages, and cytotoxic T cells in the tumor microenvironment was analyzed by flow cytometry. The results are shown in Figure 2. Figure 8 As shown in the figure, MMPCI can reduce the proportion of MDSC in the tumor microenvironment and increase the content of mature DC cells, M1 macrophages and cytotoxic T cells, indicating that MMPCI can reshape the tumor immunosuppressive microenvironment and initiate anti-tumor immunity.

[0067] Effect Example 6 The effect of CD8 monoclonal antibody inhibitor on the anti-tumor activity of the biomimetic nanoformulation prepared in Example 1 of the present invention is as follows: Six-week-old female BABL / c mice were injected with 100 μL of 1×10 6 The cells were then randomly divided into six groups and the dorsal transplanted tumors were grown to 50 mm 3 The mice were randomly divided into two groups. One group of mice was intraperitoneally injected with 200 μg of CD8 monoclonal antibody inhibitor, and the administration of CD8 monoclonal antibody inhibitor was maintained once every three days. The first administration date was recorded as the first day. From the second day, the bionic nanoformulation obtained in Example 2 was injected into the tail vein of each mouse, and the bionic nanoformulation was injected once every three days. Starting from the first day, the tumor volume of the mice was recorded every two days. Finally, on the eleventh day, the mice were killed, the tumor tissues were removed, and the tumor tissues were imaged and weighed in vitro. The results are shown in the figure. Fig. 9 As shown, CD8 monoclonal antibody inhibitors significantly weakened the antitumor efficacy of MMPCI, indicating that cytotoxic T cell-mediated tumor suppression is crucial for the antitumor activity of MMPCI.

[0068] Effect Example 7 The safety of the bionic nanoparticle preparation prepared in Example 1 of the present invention was evaluated. The specific operation is as follows: The biomimetic nanoformulation prepared in Example 1 was injected into the caudal vein of a six-week-old female BABL / c mouse. The mouse was killed on the third day after administration. The heart, liver, spleen, lung, and kidney of the mouse were removed and fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin, and finally made into 4-micron thick tissue sections. The obtained tissue sections were stained with hematoxylin-eosin, and images were collected with a tissue pathology scanner. The experiment used untreated six-week-old female BABL / c mice as controls. The results are shown in Figure 2. Fig.10 As shown, no abnormalities were found in the heart, liver, spleen, lung, and kidney three days after MMPCI treatment, indicating that MMPCI has good safety and potential for further clinical application.

[0069] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a bionic nanoparticle preparation, characterized in that: The following steps are involved: (1) dissolving carboxyl-terminated poly(lactic acid-co-glycolic acid)-polyethylene glycol and curcumin in an organic solvent 1, then adding dropwise to an organic solvent 2 containing interleukin-2, preparing colostrum by ultrasonication, then adding dropwise the obtained colostrum to an organic solvent 3, finally stirring the obtained emulsion to volatilize the organic solvent therein, thereby obtaining PLGA nanoparticles loaded with interleukin-2 and curcumin; (2) removing large particles and materials that have not formed nanoparticles from the solution obtained in (1) by centrifugation, then collecting PLGA nanoparticles by centrifugation again, and resuspending them in deionized water, adding potassium permanganate to the obtained PLGA nanoparticle solution, stirring in the dark, collecting nanoparticles by centrifugation, and washing and resuspending them in deionized water to obtain a manganese oxide-PLGA nanocomposite; (3) Collecting cells cultured in vitro, lysing the cells by hypotonic method, and collecting cell membranes by gradient centrifugation; (4) The nanocomposite obtained in (2) is mixed with the cell membrane obtained in (3), and the cell membrane-wrapped manganese oxide-PLGA nanocomposite is prepared by ultrasonication to obtain the bionic nanoformulation.

2. The method for preparing the bionic nanoparticle preparation according to claim 1, characterized in that: The molecular weight of the carboxyl-terminated poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) in step (1) is 5,000 to 30,000; The organic solvent 1 in step (1) is a mixture of ethyl acetate and chloroform, and the organic solvent 2 and the organic solvent 3 are aqueous solutions of polyvinyl alcohol; The ratio of the carboxyl-terminated polylactic acid-co-glycolic acid-polyethylene glycol, curcumin, interleukin-2, and the potassium permanganate described in step (2) is 15-25 mg: 1.5-2.5 mg: 1.5-2.5 μg: 10-25 mg by mass.

3. The method for preparing the bionic nanoparticle preparation according to claim 2, characterized in that: The molecular weight of the carboxyl-terminated poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) in step (1) is 20,000 to 30,000; The organic solvent 1 in step (1) is a 1:1 mixture of ethyl acetate and chloroform, the organic solvent 2 is a 1% polyvinyl alcohol aqueous solution, and the organic solvent 3 is a 0.5% polyvinyl alcohol aqueous solution; The ratio of the carboxyl-terminated poly(lactic acid-co-glycolic acid)-polyethylene glycol, curcumin, interleukin-2, and the potassium permanganate described in step (2) is 20 mg: 2 mg: 2 μg: 20 mg by mass.

4. The method for preparing the bionic nanoparticle preparation according to any one of claims 1 to 3, characterized in that: The amount of the polylactic acid-co-glycolic acid-polyethylene glycol added in step (1) is calculated based on the ratio of 15-25 mg: 0.5-1 mL to the organic solvent 1, and the amount of the curcumin added is calculated based on the ratio of 1.5-2.5 mg: 0.5-1 mL to the organic solvent 1; The ratio of interleukin-2, organic solvent 2 and organic solvent 3 in step (1) is 1.5-2.5 μg: 3-5 mL: 9-11 mL.

5. The method for preparing the bionic nanoparticle preparation according to claim 4, characterized in that: The amount of the polylactic acid-co-glycolic acid-polyethylene glycol added in step (1) is calculated based on a ratio of 20 mg:0.6 mL to the organic solvent 1, and the amount of the curcumin added is calculated based on a ratio of 2 mg:0.6 mL to the organic solvent 1; The ratio of interleukin-2, organic solvent 2, and organic solvent 3 described in step (1) is 2 μg: 5 mL: 11 mL; The ultrasound method described in step (1) is probe ultrasound with an ultrasound intensity of 100 W and a duty cycle of 3 seconds:3 seconds; The stirring speed in step (1) is 1000-1400 rpm and the time is 8-14 h.

6. The method for preparing the bionic nano preparation according to any one of claims 1 to 3, characterized in that: In step (2), when removing the large particles and the material that has not formed nanoparticles in (1), the centrifugation conditions are 1000-1500 × g for 8-12 minutes, and when collecting PLGA nanoparticles, the centrifugation conditions are 12000-15000 × g for 15-20 minutes; In step (2), collecting PLGA nanoparticles refers to collecting PLGA nanoparticles smaller than 400 nanometers; The light-shielded stirring condition in step (2) is 1000-1400 rpm for 4-8 h.

7. The method for preparing the bionic nano preparation according to any one of claims 1 to 3, characterized in that: The cells described in step (3) include any one or more of red blood cells, platelets, macrophages, and tumor cells; The hypotonic method described in step (3) is to lyse the cells in TM buffer containing 1% PMSF in an ice-water bath at 4°C for 8 to 14 h; The ratio of the nanocomplex to the cell membrane in step (3) is 0.5-2 mg corresponding to 1×10 7 Cells The ultrasound method described in step (3) is probe ultrasound with an ultrasound intensity of 100 W and a duty cycle of 3 seconds:3 seconds.

8. A bionic nano preparation, characterized in that: The method is obtained by the preparation method described in any one of claims 1 to 7.

9. Use of the bionic nano preparation described in claim 8 in the preparation of anti-tumor drugs.

10. The use of the bionic nano preparation according to claim 9 in the preparation of anti-tumor drugs, characterized in that: The tumor includes breast cancer.

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