GSH-responsive cell membrane biomimetic nanomedicine, preparation method and application thereof

Abstract

The invention discloses a GSH-responsive cell membrane biomimetic nanomedicine, a preparation method and an application thereof, wherein the cell membrane biomimetic nanomedicine includes a cell membrane vesicle as a carrier, the cell membrane vesicle is prepared by cell membrane extraction and membrane extrusion of immune cells that overexpress azide compounds on membrane surface, the azide compounds on the cell membrane vesicles and bioconjugated compounds are bonded to form SH groups by click chemistry reactions, and the SH groups are combined with small molecule inhibitors in the form of disulfide bonds by a cross-linking agent. The medicine of the present invention is connected to small molecule inhibitors by disulfide bonds, and not only the drug loading rate is relatively higher, but also the stability is better, and drug leakage is relatively less during blood circulation; small molecule inhibitors will be completely released under the tumor site microenvironment, thereby reducing tumor m6A methylation levels, reversing EMT state, overcoming tumor drug resistance, and coordinating chemotherapy drugs to treat tumors to show better tumor treatment effects.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical preparations, and in particular to a GSH-responsive cell membrane bionic nanomedicine, a preparation method and an application thereof. Background Art

[0002] Chemotherapy is one of the most commonly used treatments for malignant tumors, but tumor cell resistance is a major factor contributing to the failure of conventional chemotherapy. This development is associated with multiple mechanisms, including reduced drug uptake and increased drug efflux through the cell membrane; altered drug subcellular distribution, preventing the drug from reaching its target; exocytosis of chemotherapeutic drugs by lung resistance-associated proteins and breast cancer resistance proteins; enhanced cellular detoxification and repair systems, enabling rapid drug inactivation and timely repair of drug-induced DNA damage in tumor cells; and qualitative and quantitative changes in drug targets. Tumor cells undergoing epithelial-mesenchymal transition (EMT) can acquire resistance to apoptosis, leading to resistance to radiotherapy and chemotherapy. Chemotherapy-resistant breast cancer cells also exhibit EMT characteristics. EMT is closely related to tumor chemotherapy resistance, and tumor cells with a mesenchymal phenotype after transformation are more likely to be resistant than tumor cells with an epithelial phenotype. Important adhesion molecules that maintain the epithelial phenotype are replaced by mesenchymal phenotype molecules, resulting in reduced intercellular adhesion, enhanced cell migration and invasion ability. The above two proteins are also closely related to tumor resistance, and EMT-inducing drugs can not only increase the invasiveness of tumor cells, but also increase the efflux of chemotherapy drugs by upregulating the expression of ABC transporters, thereby leading to the occurrence of drug resistance.

[0003] Recent studies have revealed that EMT plays a crucial role in inducing drug resistance in tumor cells. Tumor EMT is regulated by complex signaling pathways, which also play a crucial role in inducing drug resistance. Related studies have found that tumors are hypermethylated during EMT. STM2457, a small molecule inhibitor of METTL3, can effectively reduce its methylation level. Cell membrane-mimetic nanomedicines can effectively deliver drugs to tumors. Therefore, reducing tumor m6A methylation levels, thereby reversing EMT and overcoming drug resistance, has become a potential therapeutic strategy. The tumor microenvironment is also a complex dynamic equilibrium system, characterized by high GSH expression and a low pH value, which is conducive to tumor cell growth. Therefore, disrupting this favorable environment and precisely regulating or utilizing the unique substances in the tumor microenvironment (TME) are key to anti-tumor research and an effective strategy for improving tumor treatment.

[0004] Currently, drug delivery systems based on cell membrane nanomaterials mainly adopt two strategies: one is encapsulation (supramolecular) or non-covalent chemistry, that is, drug molecules are encapsulated in cell membrane nanocarriers through non-covalent bonds. The advantage of this method is that the drug is not chemically modified before and after release. The disadvantage is that cell membrane-derived lipid vesicles mainly utilize the hydrophobic structure of their bilayer membrane to encapsulate hydrophobic drugs. However, the limited bimolecular space often results in a relatively low drug loading capacity. In addition, the inherent fluidity of lipids often leads to a large amount of drug molecules leaking over time, which is one of the key factors limiting the further development and application of this type of drug delivery system. The other method is based on the covalent linkage of drugs within the nanocarrier to form prodrugs. This method ensures high stability of the nanocarrier. However, the disadvantage is that it may be accompanied by drug negative efficiency and incomplete cleavage of covalent bonds, resulting in reduced drug efficacy. Therefore, an ideal cell membrane biomimetic nanodrug, in addition to having a certain degree of stability, must also be able to effectively release the drug under specific conditions to exert the drug's efficacy. Summary of the Invention

[0005] The present invention aims to provide a GSH-responsive cell membrane biomimetic nanodrug, a preparation method, and an application thereof. The GSH-responsive cell membrane biomimetic nanodrug of the present invention is achieved by bonding azide groups on cell membrane vesicles to -SH groups using the principle of click chemistry reaction, and using a cross-linking agent to form a more structurally stable disulfide bond between the small molecule inhibitor and the -SH group. The drug loading rate of the small molecule inhibitor is relatively higher, and drug leakage is relatively less over time, and the stability is strong. In addition to having good biocompatibility, it also has a long blood circulation time. In addition, when the GSH-responsive cell membrane biomimetic nanodrug is enriched in the tumor lesion area, the GSH rich in the tumor part can effectively open the disulfide bond of the cell membrane biomimetic nanodrug, thereby releasing the small molecule inhibitor. The released small molecule inhibitor can effectively reduce the methylation level of the tumor, thereby reversing the EMT state of the tumor, overcoming the drug resistance of the tumor, and effectively reducing the use of chemotherapy drugs, thereby solving the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A GSH-responsive cell membrane biomimetic nanomedicine includes cell membrane vesicles as a carrier. The cell membrane vesicles are prepared by extracting and extruding the cell membranes of immune cells that overexpress azide compounds on their membrane surfaces. The azide compounds on the cell membrane vesicles are bonded to bioconjugated compounds through a click chemistry reaction to form -SH groups, which are then linked to small molecule inhibitors through a cross-linking agent.

[0008] As a further embodiment of the present invention, the immune cells are one or more of macrophages, neutrophils or lymphocytes.

[0009] As a further embodiment of the present invention, the small molecule inhibitor is specifically one or more METTL3 methylation inhibitors.

[0010] As a further embodiment of the present invention, the azide compound is specifically N-azidoacetylgalactosamine-tetraacylated.

[0011] As a further embodiment of the present invention, the bioconjugate compound is specifically diphenylcyclooctyne-thiol;

[0012] The cross-linking agent is specifically succinimide 3-(2-pyridyldisulfide)-propionate.

[0013] A method for preparing a GSH-responsive cell membrane biomimetic nanomedicine comprises the following steps:

[0014] S1. Select at least one immune cell and incubate it with an azide compound to obtain an immune cell modified with an azide group, namely a functionalized immune cell;

[0015] S2. extracting the cell membrane of the functionalized immune cells to obtain corresponding functionalized immune cell membrane fragments;

[0016] S3, extruding the functionalized immune cell membrane fragments through a liposome extruder to obtain corresponding functionalized cell membrane vesicles;

[0017] S4, the azide group and the bioconjugated compound are bonded through a click chemistry reaction to form a -SH group, and the -SH group is combined with the small molecule inhibitor in the form of a disulfide bond through a cross-linker to obtain a GSH-responsive cell membrane biomimetic nanomedicine.

[0018] As a further embodiment of the present invention, the method for preparing functionalized immune cells in step S1 is specifically as follows: at least one selected immune cell is inoculated into DMEM culture medium and cultured in a 37°C incubator until the cell confluence reaches 75-85%, an azide compound is added to the DMEM culture medium, and the culture is incubated for 48 hours; finally, the cells are centrifuged at 1500-2500 rpm at 4°C for 5-15 minutes to obtain functionalized immune cells, the functionalized immune cells are collected, and the cells are washed multiple times with PBS buffer for later use.

[0019] As a further embodiment of the present invention, the method for extracting the cell membrane in step S2 is specifically as follows:

[0020] The collected functionalized immune cells were suspended in cell lysis buffer and protease inhibitors were added. Subsequently, the cell suspension placed on ice was extracted with a 1 mL syringe to fully mix the cell membrane lysis buffer and the functionalized immune cells. The extraction was repeated every 5 minutes for 5-6 times, and the cells were placed in a -80°C refrigerator for 30 minutes, taken out and thawed. After 4 freeze-thaw cycles, differential centrifugation was performed using a centrifuge to obtain the corresponding functionalized immune cell membrane fragments, which were stored at -80°C for use.

[0021] The differential centrifugation process is as follows: first, centrifuge at a speed of 700g for 10 minutes to obtain the supernatant, and then centrifuge at a speed of 14000g for 30 minutes to obtain the lower cell membrane precipitate.

[0022] As a further embodiment of the present invention, the specific steps of extracting functionalized cell membrane vesicles in step S3 are as follows:

[0023] The purified functionalized immune cell membrane fragments were suspended in a buffer solution and ultrasonically treated in an ice bath using an ultrasonic generator with a power of 10 W and a treatment time of 10 min. Then, the fragments were extruded through a polycarbonate porous membrane using a liposome extruder to obtain functionalized cell membrane vesicles. The pore size gradient of the polycarbonate porous membrane was 1 μm, 800 nm, 400 nm, and 200 nm.

[0024] As a further solution of the present invention, the specific steps of step S4 are:

[0025] First, a cross-linking agent and a bioconjugation compound are dissolved in an organic solvent at room temperature, and the bioconjugation compound and the cross-linking agent are added to the functionalized cell membrane vesicle dispersion, stirred and mixed, and reacted for 12 hours. After the reaction, the product is transferred to a dialysis bag and dialyzed in deionized water containing Tween 80 for 30-50 hours to remove unreacted bioconjugation compound and cross-linking agent. Then, a small molecule inhibitor is added thereto and reacted for 12-15 hours. After the reaction, the product is transferred to a dialysis bag and dialyzed in deionized water containing Tween 80 for 30-50 hours to remove unreacted small molecule inhibitor. The product is freeze-dried to obtain GSH-responsive cell membrane biomimetic nanoparticle drug powder, which is stored at -80°C for use.

[0026] The organic solvent is dimethyl sulfoxide, the mass ratio of the bioconjugation compound, the cross-linking agent and the small molecule inhibitor is 1:2:2, and the mass fraction of Tween 80 is 1 / 1000.

[0027] Application of GSH-responsive cell membrane biomimetic nanomedicine combined with the chemotherapy drug paclitaxel.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The process of the GSH-responsive cell membrane biomimetic nanomedicine of the present invention is as follows: first, azide groups are embedded into immune cell membranes using sugar metabolism technology. Then, functionalized cell membrane vesicles are prepared. The functionalized cell membrane vesicles modified with azide groups undergo a click chemistry reaction with a bioconjugated compound to form -SH groups loaded on the surface of the cell membrane vesicles. A cross-linking agent is used to form a structurally stable disulfide bond between the small molecule inhibitor and the -SH group. This results in a relatively higher drug loading rate and better stability of the small molecule inhibitor, and relatively less drug leakage over time.

[0030] 2. The GSH-responsive cell membrane biomimetic nanomedicine of the present invention is enriched in the tumor lesion area. Under the influence of the tumor microenvironment, the disulfide bonds will be opened, thereby efficiently releasing small molecule inhibitors, ensuring good drug efficacy. The released small molecule inhibitors can effectively reduce the methylation level of tumor cells and reverse the state of tumor EMT, thereby overcoming the drug resistance of the tumor and facilitating the synergistic effect of chemotherapy drugs to inhibit tumor growth.

[0031] 3. The present invention adopts cell metabolic engineering to prepare functionalized immune cell membranes and adopts functionalized bioorthogonal reactions to bond chemical groups on the surface of cell membrane vesicles, which makes the preparation method simple. The prepared GSH-responsive cell membrane biomimetic nanomedicine has the advantages of good biocompatibility, high cancer cell uptake efficiency and low carrier toxicity. It is a pharmaceutical preparation with high production cost-effectiveness, high efficiency and low toxicity.

[0032] In summary, the drug of the present invention connects small molecule inhibitors through disulfide bonds, which not only has a relatively higher drug loading rate and better stability, but also has relatively less drug leakage during blood circulation. The small molecule inhibitors will be completely released only in the microenvironment of the tumor site, thereby reducing the methylation level of the tumor EMT, overcoming tumor resistance, and synergizing with chemotherapy drugs to treat tumors to show more effective biological effects, providing broad application prospects for the treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the preparation process of the present invention;

[0034] Figure 2 The transmission electron microscope image and hydrated particle size test results of the present invention are shown;

[0035] Figure 3 It is the full spectrum of ultraviolet absorption and the graph of loading rate change of the present invention;

[0036] Figure 4 The graphs are drug release results of the present invention in GSH and GSH-free environments;

[0037] Figure 5 It is the SDS-PAGE characterization diagram of the present invention;

[0038] Figure 6 This is a graph showing the MTT test results of the present invention combined with the chemotherapy drug paclitaxel;

[0039] Figure 7 Confocal microscopy images of induced 4T1 cells co-incubated with GSH-responsive cell membrane biomimetic nanomedicine (ACMS);

[0040] Figure 8 This is a test result diagram of the present invention's overcoming of tumor drug resistance and biosafety. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] The cell lysis buffer, protease inhibitors, membrane protein extraction reagents and BCA protein concentration determination kit in the examples of the present invention were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0043] See also Figure 1 A GSH-responsive cell membrane biomimetic nanomedicine includes cell membrane vesicles as carriers. The cell membrane vesicles are prepared by cell membrane extraction and membrane extrusion of immune cells that overexpress azide compounds on their membrane surfaces. The azide compounds on the cell membrane vesicles are bonded to bioconjugated compounds through click chemistry reactions to form -SH groups, and the -SH groups are connected to small molecule inhibitors through cross-linking agents.

[0044] Among them, the immune cells are specifically Raw246.7 macrophages; the small molecule inhibitor is specifically the STM2457 type METTL3 methylation inhibitor, STM2457 is a small molecule drug that can highly specifically bind to the m6A methylase METTL3, and can reduce its mRNA methylation level; the azide compound is specifically N-azidoacetylgalactosamine-tetraacylated (abbreviated as Ac4GalNAz); the bioconjugated compound is specifically diphenylcyclooctyne-thiol (abbreviated as DBCO-SH); the cross-linker is specifically succinimide 3-(2-pyridyldisulfide)-propionate (abbreviated as SPDP).

[0045] The present invention utilizes the principle of click chemistry to bond the azide groups on the cell membrane vesicles to the -SH groups, and uses a cross-linking agent to form a more structurally stable disulfide bond between the small molecule inhibitor and the -SH group, thereby improving the drug loading efficiency of the drug delivery system and preventing leakage of the small molecule inhibitor. The small molecule inhibitor is connected to the cell membrane carrier via a disulfide bond and can be effectively released in a reducing environment. The small molecule inhibitor STM2457 is used to reduce the methylation level of the tumor, thereby reversing the EMT state of the tumor and overcoming the drug resistance of the tumor, providing a new treatment strategy for treating tumor resistance.

[0046] The method for preparing the GSH-responsive cell membrane biomimetic nanomedicine comprises the following steps:

[0047] (1) Construction of the Azide-Modified Raw246.7 Macrophage Cell Line:

[0048] 1×10 6 The Raw246.7 macrophages were placed in DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and seeded into 10 cm culture dishes and cultured in a 37°C incubator until the cell confluence reached 70-80%. N-azidoacetylgalactosamine-tetraacylated was added to the DMEM culture medium at a concentration of 40 μM and incubated for 48 hours. The cells after co-incubation were collected into PBS buffer using a cell scraper. Finally, the cells were centrifuged at 2000 rpm at 4°C for 5 minutes to obtain functionalized immune cells. The functionalized immune cells were collected and washed three times with PBS buffer for later use.

[0049] (2) Preparation of functionalized immune cell membrane fragments (ACM):

[0050] The collected functionalized immune cells were suspended in 1 mL of cell lysis buffer (Shanghai Biyuntian), and 10 μL of protease inhibitors (Shanghai Biyuntian) were added to the buffer before use. Subsequently, the cell suspension placed on ice was extracted with a 1 mL syringe to fully mix the cell membrane lysis buffer and the cells. The extraction was performed every 5 minutes, and the steps were repeated 5-6 times. The extracted cell suspension was then placed in a -80°C refrigerator and frozen for 30 minutes. It was taken out and thawed. After 6 freeze-thaw cycles, it was centrifuged at 700 g at 4°C for 10 minutes. The bottom sediment was discarded, and the supernatant solution was retained. The solution was centrifuged at 14,000 g for 30 minutes to obtain the lower cell membrane precipitate, i.e., the functionalized immune cell membrane fragments (ACM) modified with azide groups. The functionalized immune cell membrane fragments (ACM) were stored at -80°C for use.

[0051] At the same time, in order to characterize the proteins retained on the functionalized immune cell membrane fragments (ACM), the functionalized immune cell membrane fragments (ACM) were lysed on ice for 20 minutes using a membrane protein extraction reagent (Shanghai Biyuntian), and then centrifuged at 12000 rpm for 30 minutes to obtain membrane proteins. The membrane protein was quantitatively detected using a BCA protein concentration assay kit (Shanghai Biyuntian).

[0052] (3) Preparation of functionalized cell membrane vesicles:

[0053] Functionalized immune cell membrane fragments (ACM) were resuspended in PBS buffer (pH = 7.4), and the cell membrane fragments were ultrasonically treated using an ultrasonic generator in an ice bath. The ultrasonic generator power was 10 W and the treatment time was 10 min. Then, the resuspended cell membrane suspension was extruded through polycarbonate porous membranes with different pore size ranges (1 μm, 800 nm, 400 nm and 200 nm, respectively) using a liposome extruder to obtain functionalized cell membrane vesicles. The vesicles were freeze-dried at low temperature for 24 h to obtain ACM lyophilized powder, which was then dispersed in PBS buffer (pH = 7.4) when used.

[0054] (4) Preparation of GSH-responsive cell membrane biomimetic nanomedicines:

[0055] First, the cross-linking agent and the bioconjugation compound are dissolved in an organic solvent at room temperature, and the bioconjugation compound and the cross-linking agent are added to the functionalized cell membrane vesicle dispersion, stirred and mixed, and reacted for 12 hours. After the reaction, the product is transferred to a dialysis bag and dialyzed in deionized water containing Tween 80 for 30-50 hours to remove unreacted bioconjugation compound and cross-linker. Then, a small molecule inhibitor is added and reacted for 12-15 hours. After the reaction, the product is transferred to a dialysis bag and dialyzed in deionized water containing Tween 80 for 30-50 hours to remove unreacted drug. The product is freeze-dried to obtain GSH-responsive cell membrane biomimetic nanodrug powder, which is stored at -80°C for use.

[0056] The organic solvent is dimethyl sulfoxide, the mass ratio of the bioconjugate compound, the cross-linker and the small molecule inhibitor is 1:2:2, and the mass fraction of Tween 80 is 1 / 1000.

[0057] The morphology of GSH-responsive cell membrane biomimetic nanomedicine (ACMS) was observed by transmission electron microscopy using phosphotungstic acid negative staining. Figure 2 A shows a transmission electron microscope image of GSH-responsive cell membrane biomimetic nanomedicine (ACMS); a dynamic light scattering instrument is used to measure the hydrodynamic size of the GSH-responsive cell membrane biomimetic nanomedicine. Figure 2 B shows the hydrated particle size of GSH-responsive cell membrane biomimetic nanomedicine.

[0058] According to the transmission electron microscopy images and dynamic light scattering test results, the GSH-responsive cell membrane biomimetic nanomedicine (ACMS) prepared in the present invention is in the form of vesicles with a diameter of about 100-200 nm.

[0059] In order to verify that the small molecule inhibitor (STM2457) was successfully bonded to the azidated cell membrane vesicles through the cross-linker and -SH group to form a disulfide bond, the cell membrane biomimetic nanomedicine (ACMS) was analyzed by UV-visible absorption spectroscopy, where STM2457 represents: free small molecule inhibitor STM2457; ACMS represents: GSH-responsive cell membrane biomimetic nanomedicine; NPS represents: cell membrane biomimetic nanomedicine NPS obtained by extruding the small molecule inhibitor and cell membrane fragments through a liposome extruder; ACM represents: azidated cell membrane vesicles. Figure 3 A shows the UV-visible absorption spectra of GSH-responsive cell membrane biomimetic nanomedicine (ACMS) and free small molecule inhibitor (STM2457), indicating that the small molecule inhibitor was successfully bound to the azidated cell membrane vesicles; Figure 3 B shows the drug loading rate of STM2457 in NP@STM2457 and ACMS components at different time periods. Obviously, ACMS has a relatively higher drug loading rate and relatively less drug leakage over time.

[0060] In order to verify that the prepared GSH-responsive cell membrane biomimetic nanodrug (ACMS) can be effectively released in a GSH environment, the prepared GSH-responsive cell membrane biomimetic nanodrug was loaded into a dialysis bag, and the dialysis bag was placed in an environment containing 10 mM GSH and without GSH, respectively. The concentration of the drug in the external environment was measured at different times to calculate the drug release content. Figure 4 This shows that GSH-responsive cell membrane biomimetic nanomedicines can effectively release drugs in a GSH environment, but in a non-reducing environment, the drugs cannot be effectively released.

[0061] To verify that the prepared GSH-responsive cell membrane biomimetic nanomedicine (ACMS) retained the parent cell membrane proteins, gel electrophoresis (SDS-PAGE) was used to analyze the protein bands of CM, ACM, and ACMS, where CM represents: cell membrane without azidation treatment; GSH-responsive cell membrane biomimetic nanomedicine (ACMS) was mixed with SDS buffer, heated at 95°C for 10 minutes, and then added to 10% SDS-PAGE. The sample was run at a voltage of 100V for 2 hours. The resulting gel was stained with Coomassie blue and washed with PBS buffer. The protein bands were then imaged and observed using a gel imaging camera system. Figure 5The SDS-PAGE characterization diagram of the GSH-responsive cell membrane biomimetic nanomedicine is shown. Most of the protein bands of CM were observed on the GSH-responsive cell membrane biomimetic nanomedicine ACMS and ACM, indicating that the membrane proteins of CM were effectively retained on the surface of ACMS and ACM.

[0062] In order to verify the ability of the prepared GSH-responsive cell membrane biomimetic nanodrug ACMS and the chemotherapy drug paclitaxel (PTX) to kill tumor cells, the MTT method was used to test the cell activity of the GSH-responsive cell membrane biomimetic nanodrug ACMS at inhibitor STM2457 concentrations of 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 and 64 μg / mL; the cell activity at paclitaxel (PTX) concentrations of 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 and 64 μg / mL; and the cell activity at ACMS concentration of 10 μg / mL and paclitaxel (PTX) concentrations of 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 and 64 μg / mL.

[0063] The test process is as follows: 4T1 cells (mouse breast cancer cells) induced by TGF-β were suspended in 100 μL culture medium containing 10% fetal bovine serum, and 1×10 4 The cells were seeded in a 96-well plate at a density of 100 cells / mL and incubated at 37°C for 24 hours. Subsequently, the above-mentioned drugs at different concentrations were added to the 96-well plate and incubated for another 24 hours. 20 μL of MTT (5 mg / mL) was added to each well and incubated at 37°C for another 4 hours. Subsequently, 150 μL of DMSO was added. Finally, the OD value of the solution at 570 nm was measured using a multifunctional microplate reader (Bio-Rad 550). Cell viability was calculated as OD 处理 / OD 对照 × 100%, where OD 处理 was obtained from cells treated with different methods. 对照 It is obtained from cells that have not been treated in any way.

[0064] Figure 6 It shows that GSH-responsive cell membrane biomimetic nanomedicine (ACMS) has low toxicity. Under the action of GSH-responsive cell membrane biomimetic nanomedicine (ACMS), the sensitivity of tumor cells to paclitaxel is improved.

[0065] In order to verify that the prepared GSH-responsive cell membrane biomimetic nanomedicine ACMS can effectively reverse tumor EMT, immunofluorescence technology was used to verify the content of EMT characteristic proteins E-cadherin and Vimentin before and after tumor cell treatment: First, the cells were adhered to the bottom of a 12-well plate, and then the induced 4T1 cells (mouse breast cancer cells) were suspended in a 12-well plate containing 10% fetal bovine serum and 1 mL RPM I-1640 medium (1×10 cells per well). 5 cells), incubated at 37°C for 24 h; after the cells attached, ACMS (10 μg / mL) was added to a 12-well plate, incubated at 37°C for 24 h, then washed three times with PBS buffer, 0.5 mL of 4% paraformaldehyde was added to the well plate and fixed at room temperature for 15-30 min, and then washed three times with PBS buffer. Next, the cells on the slides were blocked with 10% goat serum for 1-2 hours. After blocking, the prepared E-cadherin and Vimentin antibodies (diluted with 10% goat serum at a dilution ratio of 1:100 according to the antibody instructions) were evenly applied to the slides. The slides were placed in a dark box and incubated at low temperature for 12 hours. Then, the slides were washed three times with PBS buffer. The prepared fluorescent-labeled secondary antibody (diluted with 10% goat serum at a dilution ratio of 1:100) was evenly applied to the slides and incubated at room temperature for 1 hour. Then, a drop of DAPI staining solution containing a fluorescence quencher was added to the slides. The slides were evenly sealed on a coverslip with nail polish. The fluorescence of the two proteins in the cells was observed by CLSM. Cells not treated with ACMS were defined as the PBS group and served as a control.

[0066] Figure 7 Confocal microscopy images of induced 4T1 cells co-incubated with ACMS are shown. CLSM results show that ACMS-treated cells showed increased E-cadherin protein expression and decreased Vimentin protein expression. ACMS effectively reversed EMT in tumors.

[0067] In order to verify that the prepared GSH-responsive cell membrane biomimetic nanomedicine ACMS can effectively overcome tumor drug resistance and has good biosafety: mouse 4T1 cells (5×10 6 ) were inoculated into the lower flank of 5-6 week old female Balb / c mice and the tumor volume was 100-200 mm 3At 14:00 p.m., mice were injected with ACMS (5 mg / kg) via the tail vein and paclitaxel (PTX) (10 mg / kg) intraperitoneally, every two days for a total of seven doses. Mice were divided into PBS, PTX, ACMS, and P+ACMS groups (administering both ACMS and PTX). Body weights and tumor volumes were recorded. Following the completion of the treatment cycle, HE staining of the main organs of the mice was performed.

[0068] Figure 8 A shows the curve of changes in tumor volume in small animals; Figure 8 B shows the curve of changes in animal weight during the treatment period; Figure 8 C shows HE staining of major organs in mice in the PBS, PTX, ACMS, and P+ACMS groups after treatment. These data demonstrate that ACMS can effectively overcome tumor resistance while maintaining good biosafety.

[0069] From the above preparation process and experimental data, it can be seen that:

[0070] 1. The GSH-responsive cell membrane nanomedicine of the present invention does not require complex design and preparation processes and will not cause functional biological toxicity;

[0071] 2. The GSH-responsive cell membrane nanodrug of the present invention has a relatively higher drug loading rate and better stability. Over time, drug leakage is relatively small. While having good biocompatibility, it also has a long blood circulation time.

[0072] 3. The GSH-responsive cell membrane nanomedicine of the present invention can effectively release the drug in the tumor microenvironment;

[0073] 4. The GSH-responsive cell membrane nanomedicine of the present invention can effectively reverse the EMT state of tumors and overcome the drug resistance of tumors.

[0074] The GSH-responsive cell membrane nanomedicine of the present invention can effectively enter tumor tissue and, under the reducing conditions of the tumor microenvironment, effectively open disulfide bonds to release the drug, overcoming tumor drug resistance. Combined with paclitaxel, it can effectively inhibit tumor growth. Furthermore, the GSH-responsive cell membrane nanomedicine of the present invention has excellent biosafety and no toxic side effects on major organs. These encouraging results confirm the transformative potential of these GSH-responsive cell membrane biomimetic nanomedicines in cancer treatment.

[0075] Although the embodiments of the present invention only disclose the use of macrophage cell membranes to prepare functionalized cell membrane vesicles, and the use of cell membrane vesicles to bind small molecule inhibitors to form cell membrane biomimetic nanomedicines, and demonstrate a model for the treatment of breast cancer cells with cell membrane biomimetic nanomedicines, according to the teachings of the present invention, those skilled in the art can anticipate the use of cell membranes such as neutrophil membranes, lymphocyte membranes, endothelial cell membranes, and red blood cell membranes and expand to tumor models such as lung cancer and liver cancer.

[0076] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A GSH-responsive cell membrane biomimetic nanomedicine, characterized in that: The invention comprises cell membrane vesicles as carriers. The cell membrane vesicles are prepared by extracting and extruding the cell membranes of immune cells that overexpress azide compounds on their membrane surfaces. The azide compounds on the cell membrane vesicles are bonded to the bioconjugated compounds through click chemistry reactions to form -SH groups. The -SH groups are then bonded to the small molecule inhibitors via disulfide bonds through a cross-linking agent. The small molecule inhibitor is STM2457.

2. A GSH-responsive cell membrane biomimetic nanomedicine according to claim 1, characterized in that: The immune cells are one or more of macrophages, neutrophils or lymphocytes.

3. A GSH-responsive cell membrane biomimetic nanomedicine according to claim 1, characterized in that: The azide compound is specifically N-azidoacetylgalactosamine-tetraacylated; The bioconjugated compound is specifically diphenylcyclooctyne-thiol; The cross-linking agent is specifically succinimide 3-(2-pyridyldisulfide)-propionate.

4. The method for preparing a GSH-responsive cell membrane biomimetic nanomedicine according to any one of claims 1 to 3, characterized in that: The steps include: S1. Select at least one immune cell and incubate it with an azide compound to obtain an immune cell modified with an azide group, namely a functionalized immune cell; S2. extracting the cell membrane of the functionalized immune cells to obtain corresponding functionalized immune cell membrane fragments; S3, extruding the functionalized immune cell membrane fragments through a liposome extruder to obtain corresponding functionalized cell membrane vesicles; S4, the azide group and the bioconjugated compound are bonded through a click chemistry reaction to form a -SH group, and the -SH group is combined with the small molecule inhibitor in the form of a disulfide bond through a cross-linker to obtain a GSH-responsive cell membrane biomimetic nanomedicine.

5. The method for preparing a GSH-responsive cell membrane biomimetic nanomedicine according to claim 4, characterized in that: The method for preparing functionalized immune cells in step S1 is specifically as follows: at least one selected immune cell is inoculated into DMEM culture medium and cultured in a 37°C incubator until the cell confluence reaches 75-85%, then an azide compound is added to the DMEM culture medium and incubated for 48 hours; finally, the cells are centrifuged at 1500-2500 rpm at 4°C for 5-15 minutes to obtain functionalized immune cells, and the functionalized immune cells are collected and washed multiple times with PBS buffer for later use.

6. The method for preparing a GSH-responsive cell membrane biomimetic nanomedicine according to claim 5, characterized in that: The method for extracting the cell membrane in step S2 is specifically as follows: The collected functionalized immune cells were suspended in cell lysis buffer and protease inhibitors were added. Subsequently, the cell suspension placed on ice was extracted with a 1 mL syringe to fully mix the cell membrane lysis buffer and the functionalized immune cells. The extraction was repeated every 5 minutes for 5-6 times, and the cells were placed in a -80°C refrigerator for 30 minutes, taken out and thawed. After 4 freeze-thaw cycles, differential centrifugation was performed using a centrifuge to obtain the corresponding functionalized immune cell membrane fragments, which were stored at -80°C for use. The differential centrifugation process was as follows: first, centrifuge at 700 g for 10 min to obtain the supernatant, and then centrifuge at 14,000 g for 30 min to obtain the lower cell membrane precipitate.

7. The method for preparing a GSH-responsive cell membrane biomimetic nanomedicine according to claim 6, characterized in that: The specific steps of extracting functionalized cell membrane vesicles in step S3 are: The purified functionalized immune cell membrane fragments were suspended in a buffer solution and ultrasonically treated in an ice bath using an ultrasonic generator with a power of 10 W for 10 min. Then, they were extruded through a polycarbonate porous membrane using a liposome extruder to obtain functionalized cell membrane vesicles. The pore size gradient of the polycarbonate porous membrane was 1 μm, 800 nm, 400 nm, and 200 nm.

8. The method for preparing a GSH-responsive cell membrane biomimetic nanomedicine according to claim 7, characterized in that: The specific steps of step S4 are: First, a cross-linking agent and a bioconjugation compound are dissolved in an organic solvent at room temperature, and the bioconjugation compound and the cross-linking agent are added to the functionalized cell membrane vesicle dispersion, stirred and mixed, and reacted for 12 hours. After the reaction, the product is transferred to a dialysis bag and dialyzed in deionized water containing Tween 80 for 30-50 hours to remove unreacted bioconjugation compound and cross-linking agent. Then, a small molecule inhibitor is added to the dispersion and reacted for 12-15 hours. After the reaction, the product is transferred to a dialysis bag and dialyzed in deionized water containing Tween 80 for 30-50 hours to remove unreacted small molecule inhibitor. The product is freeze-dried to obtain GSH-responsive cell membrane biomimetic nanoparticle drug powder, which is stored at -80°C for use. The organic solvent is dimethyl sulfoxide, the mass ratio of the bioconjugation compound, the cross-linking agent and the small molecule inhibitor is 1:2:2, and the mass fraction of Tween 80 is 1 / 1000. 9 . Use of the GSH-responsive cell membrane biomimetic nanomedicine according to any one of claims 1 to 3 in combination with the chemotherapy drug paclitaxel in the preparation of a drug for treating breast cancer.

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