GSH (glutathione) response type cell membrane bionic nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a GSH response type cell membrane bionic nano-drug, a preparation method and application, the cell membrane bionic nano-drug comprises cell membrane vesicles serving as a carrier, the cell membrane vesicles are prepared by performing cell membrane extraction and membrane extrusion on immune cells of which the membrane surfaces overexpress azide, and the cell membrane vesicles serve as a carrier. The azide on the cell membrane vesicles and the bio-coupling compound are bonded through a click chemical reaction to form-SH groups, and the-SH groups and the small-molecule inhibitor are combined together in a disulfide bond mode through the cross-linking agent. The drug is connected with a small-molecule inhibitor through a disulfide bond, so that the drug loading rate is relatively higher, the stability is better, and drug leakage in the blood circulation process is relatively less; the small-molecule inhibitor can be completely released only in a tumor site microenvironment, so that the tumor m6A methylation level is reduced, the EMT state is reversed, the tumor drug resistance is overcome, and a better tumor treatment effect is displayed in cooperation with chemotherapy drugs for tumor treatment.

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 methods for treating malignant tumors, but tumor cell resistance is the main factor leading to the failure of conventional chemotherapy for tumors. Its occurrence is related to multiple mechanisms, including reduced cell membrane uptake and increased efflux of drugs, changes in drug subcellular distribution that prevent drugs from approaching their targets, exocytosis of chemotherapeutic drugs by lung resistance-related proteins and breast cancer resistance proteins, enhanced cell detoxification and repair system functions that rapidly inactivate drugs and promptly repair drug-induced tumor cell DNA damage, qualitative and quantitative changes in drug targets, etc. Tumor cells that undergo epithelial to mesenchymal transition (EMT) can acquire apoptosis resistance, leading to tumor tolerance to radiotherapy and chemotherapy, and breast cancer cells that are resistant to chemotherapy also have EMT characteristics. EMT is closely related to tumor chemotherapy resistance, and the transformed mesenchymal phenotype tumor cells are more likely to be resistant than epithelial phenotype tumor cells. The important adhesion molecules that maintain the epithelial phenotype are replaced by mesenchymal phenotype molecules, resulting in reduced cell-to-cell 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 chemotherapeutic drugs by upregulating the expression of ABC transporters, thereby leading to the occurrence of drug resistance.

[0003] In recent years, studies have found that EMT plays an important role in inducing the formation of tumor cell resistance. The occurrence of tumor EMT is regulated by complex signaling pathways, which also play an important role in inducing tumor cell resistance. Related studies have found that tumors are in a highly methylated state under the EMT state. STM2457, as a small molecule inhibitor of METTL3, can effectively reduce its methylation level. Cell membrane bionic nanomedicines can effectively deliver drugs to tumor sites. Therefore, by reducing the level of tumor m6A methylation, the state of EMT is reversed, and overcoming tumor resistance has become a potential tumor treatment strategy. At the same time, the tumor microenvironment is a complex dynamic equilibrium system, which is mainly manifested as high expression of GSH and low pH value. This environment is suitable for the growth of tumor cells. Therefore, how to break this suitable environment and accurately regulate or utilize the unique substances in the tumor microenvironment (TME) is the key to anti-tumor research and an effective strategy to improve tumor treatment.

[0004] At present, drug delivery systems based on cell membrane nanomaterials mainly adopt two strategies: one method 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 use the hydrophobic structure of their bilayer membrane to encapsulate hydrophobic drugs. However, the limited bimolecular spatial structure often leads to a relatively low drug loading capacity; in addition, the inherent fluidity of lipids often leads to a large amount of leakage of drug molecules over time, which becomes one of the key factors limiting the further development and application of such drug delivery systems; another method is based on the formation of prodrugs by covalent bonds in drugs in nanocarriers. This method ensures the high stability of nanocarriers. 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 bionic nanomedicine not only has a certain stability, but also needs to be able to effectively release drugs under specific conditions to exert the efficacy of drugs. Summary of the invention

[0005] The purpose of the present invention is to provide a GSH-responsive cell membrane bionic nanomedicine, a preparation method and an application thereof. The GSH-responsive cell membrane bionic nanomedicine of the present invention is to bond an azidation group on a cell membrane vesicle to a -SH group by utilizing the principle of click chemistry reaction, and to utilize a cross-linking agent to form a more structurally stable disulfide bond between a small molecule inhibitor and the -SH group, so that the drug loading rate of the small molecule inhibitor is relatively higher, and the drug leakage is relatively less with the passage of time, and the stability is strong; while having good biocompatibility, it also has a long blood circulation time; in addition, when the GSH-responsive cell membrane bionic nanomedicine is enriched in a tumor lesion area, the GSH rich in the tumor part can effectively open the disulfide bond of the cell membrane bionic nanomedicine, thereby releasing the small molecule inhibitor, and the released small molecule inhibitor can effectively reduce the methylation level of the tumor, thereby reversing the state of tumor EMT, overcoming the drug resistance of the tumor, and effectively reducing the use of chemotherapeutic 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 bionic nanomedicine comprises a cell membrane vesicle as a carrier, wherein the cell membrane vesicle is prepared by cell membrane extraction and membrane extrusion of immune cells overexpressing azide compounds on the membrane surface, the azide compounds on the cell membrane vesicles are bonded with bioconjugated compounds through click chemistry reactions to form -SH groups, and the -SH groups are connected to small molecule inhibitors through cross-linking agents.

[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 of the 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 bioconjugated 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 bionic 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 cell membranes of 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 to form a -SH group through a click chemistry reaction. The -SH group and the small molecule inhibitor are combined together by a cross-linking agent in the form of a disulfide bond to obtain a GSH-responsive cell membrane bionic nanomedicine.

[0018] As a further scheme 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, cultured in a 37°C incubator until the cell confluence reaches 75-85%, adding an azide compound to the DMEM culture medium, and incubating for 48 hours; finally, centrifuging at 1500-2500 rpm at 4°C for 5-15 minutes to obtain functionalized immune cells, collecting the functionalized immune cells, and washing them 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 a cell lysis buffer, and protease inhibitors were added. Subsequently, a 1 mL syringe was used to extract the cell suspension placed on ice, so that the cell membrane lysis buffer and the functionalized immune cells were fully mixed, and the extraction was performed every 5 minutes. The extraction steps were repeated 5-6 times, and the cells were placed in a -80°C refrigerator for 30 minutes, and then 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 later use;

[0021] The process of differential centrifugation 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 layer 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 are suspended in a buffer solution, and the suspended functionalized immune cell membrane fragments are ultrasonically treated using an ultrasonic generator in an ice bath, the ultrasonic generator power is 10 W, and the treatment time is 10 min. Then, a liposome extruder is used to extrude through a polycarbonate porous membrane to obtain functionalized cell membrane vesicles; wherein the pore size gradient of the polycarbonate porous membrane is 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 compounds and cross-linking agents; then a small molecule inhibitor is added thereto, and the reaction is carried out for 12-15 hours. After the reaction is completed, 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 inhibitors, and freeze-dried to obtain GSH-responsive cell membrane biomimetic nanomedicine powder, which is stored at -80°C for use;

[0026] The organic solvent is dimethyl sulfoxide, the mass ratio of the bioconjugate 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 bionic 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: first, the azide group is embedded into the immune cell membrane by using sugar metabolism technology, and then, the functionalized cell membrane vesicles are prepared, and the functionalized cell membrane vesicles modified with the azide group form -SH groups loaded on the surface of the cell membrane vesicles through a click chemistry reaction with the bioconjugated compound, and a structurally stable disulfide bond is formed between the small molecule inhibitor and the -SH group through a cross-linking agent, so that the drug loading rate of the small molecule inhibitor is relatively higher, the stability is better, and the drug leakage is relatively less over time;

[0030] 2. The GSH-responsive cell membrane bionic nanomedicine of the present invention is enriched in the tumor lesion area. Under the action of the microenvironment of the tumor site, the disulfide bond 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, which is beneficial to cooperate with 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, and the preparation method is simple; and the prepared GSH-responsive cell membrane bionic nanomedicine has the advantages of good biocompatibility, high cancer cell uptake efficiency and low carrier toxicity, and is a drug preparation with high production cost performance, 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, but also better stability, and relatively less drug leakage during blood circulation; the small molecule inhibitor will be completely released in the microenvironment of the tumor site, thereby reducing the methylation level of 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 ultraviolet absorption full spectrum and loading rate variation diagram of the present invention;

[0036] Figure 4 The drug release results of the present invention in the environment of GSH and without GSH are shown;

[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 in overcoming tumor drug resistance and biosafety. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work 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 Bio-Tech Biotechnology Co., Ltd.

[0043] See also Figure 1 A GSH-responsive cell membrane bionic 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 bind to the m6A methylase METTL3 with high specificity, 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-linking agent is specifically succinimidyl 3-(2-pyridyldisulfide)-propionate (abbreviated as SPDP).

[0045] The present invention uses the principle of click chemistry to bond the -SH group through the azidation group on the cell membrane vesicle, and uses a cross-linking agent to form a more stable disulfide bond between the small molecule inhibitor and the -SH group, thereby improving the drug loading efficiency of the drug delivery system and avoiding the leakage of the small molecule inhibitor; the small molecule inhibitor is connected to the cell membrane carrier by means of 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 state of the tumor EMT, thereby overcoming the drug resistance of the tumor, and providing a new treatment strategy for treating tumor resistance.

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

[0047] (1) Construction of the Raw246.7 macrophage cell line modified with an azide group:

[0048] 1×10 6 The Raw246.7 macrophages were placed in a DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and seeded in a 10 cm culture dish and cultured in a 37°C incubator until the cell confluence reached 70-80%. Then, 40 μM concentration of N-azidoacetylgalactosamine-tetraacylated was added to the DMEM medium and incubated for 48 hours. The incubated cells 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 3 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 inhibitor (Shanghai Biyuntian) was added to the buffer before use. Subsequently, the cell suspension placed on ice was extracted with a 1 mL syringe to allow the cell membrane lysis buffer to be fully mixed with 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 for 30 minutes, taken out for thawing, and after 6 freeze-thaw cycles, the suspension was centrifuged at 700 g at 4°C for 10 minutes, the bottom precipitate was discarded, the supernatant solution was retained, and the lower cell membrane precipitate was obtained at 14,000 g for 30 minutes to obtain 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 Biotech), and then centrifuged at 12,000 rpm for 30 minutes to obtain membrane proteins. The membrane protein was quantitatively detected using the BCA protein concentration assay kit (Shanghai Biotech).

[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 power of the ultrasonic generator was 10 W, and the treatment time was 10 min. Then, the resuspended cell membrane suspension was extruded through a polycarbonate porous membrane 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, which 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-linking agent; then a small molecule inhibitor is added thereto, and the reaction is performed for 12-15 hours. After the reaction is completed, the product is transferred to a dialysis bag, and dialyzed in deionized water containing Tween80 for 30-50 hours to remove unreacted drugs, and freeze-dried to obtain GSH-responsive cell membrane bionic nano-drug 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-linking agent 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 microscope image and dynamic light scattering test results, the GSH-responsive cell membrane bionic 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 azidized cell membrane vesicles by forming a disulfide bond with the -SH group through a cross-linker, the cell membrane biomimetic nanomedicine (ACMS) was analyzed by UV-visible absorption spectroscopy, wherein 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 small molecule inhibitors and cell membrane fragments through a liposome extruder; ACM represents: azidized 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 is successfully bonded 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 bionic nanodrug (ACMS) can be effectively released in a GSH environment, the prepared GSH-responsive cell membrane bionic 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 released drug content. Figure 4 This shows that GSH-responsive cell membrane bionic nanomedicines can effectively release drugs in a GSH environment, but in a non-reducing environment, the drugs cannot be effectively released.

[0061] In order to verify that the prepared GSH-responsive cell membrane biomimetic nanomedicine (ACMS) retains the mother cell membrane protein, 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℃ for 10min, and then added to 10% SDS-PAGE respectively. The sample was run at a voltage of 100V for 2h, and the obtained gel was stained with Coomassie blue and washed with PBS buffer, and the protein bands were imaged and observed using a gel imaging camera system. Figure 5The SDS-PAGE characterization diagram of the GSH-responsive cell membrane bionic nanomedicine is shown. Most of the protein bands of CM were observed on the GSH-responsive cell membrane bionic 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 bionic nanodrug ACMS and the combined chemotherapy drug paclitaxel (PTX) to kill tumor cells, the MTT method was used to test the cell activity of the GSH-responsive cell membrane bionic 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 h. Subsequently, the above drugs at different concentrations were added to the 96-well plate. After further incubation for 24 h, 20 μL of MTT (5 mg / mL) was added to each well. The cells were further incubated at 37°C for 4 h. 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 is obtained from cells treated with different methods, OD 对照 It is obtained from cells that have not been treated in any way.

[0064] Figure 6 It is shown that the GSH-responsive cell membrane bionic nanomedicine (ACMS) has low toxicity, and under the action of the GSH-responsive cell membrane bionic nanomedicine (ACMS), the sensitivity of tumor cells to paclitaxel is increased.

[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 attached 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 per well). 5 cells), incubated at 37°C for 24h; after the cells adhered, ACMS (10μg / mL) was added to a 12-well plate, incubated at 37°C for 24h, then washed three times with PBS buffer, 0.5mL of 4% paraformaldehyde was added to the well plate and fixed at room temperature for 15-30min, and then washed three times with PBS buffer. Next, the cells on the slide were blocked with 10% goat serum for 1-2 hours. After blocking, the prepared E-cadherin and Vimentin antibodies (according to the antibody manual, the antibodies were diluted with 10% goat serum at a dilution ratio of 1:100) were evenly covered on the slide, and the slide was placed in a dark box for low-temperature incubation for 12 hours, and then washed three times with PBS buffer. The prepared fluorescent-labeled secondary antibody (the antibody was diluted with 10% goat serum at a dilution ratio of 1:100) was evenly covered on the slide, incubated at room temperature for 1 hour, and then a drop of DAPI staining solution containing a fluorescence quencher was dropped on the slide, and the slide was evenly sealed on the cover glass with nail polish, and the fluorescence of the two proteins in the cells was observed by CLSM. Cells not treated with ACMS were defined as the PBS group as a control.

[0066] Figure 7 The confocal microscopy images of 4T1 cells co-incubated with ACMS after induction are shown. The CLSM results show that the expression of E-cadherin protein in cells treated with ACMS increased, while the expression of Vi mentin protein decreased. ACMS effectively reversed the EMT of 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 until the tumor volume reached 100-200mm 3At 14:00, mice were injected with ACMS (5 mg / kg) through the tail vein and paclitaxel PTX (10 mg / kg) through the abdominal cavity, and the drugs were given every 2 days for a total of 7 times. The mice were divided into PBS group, PTX group, ACMS group and P+ACMS group (both ACMS and PTX were given), and the weight and tumor volume of the mice were recorded. After the treatment cycle, the main organs of the mice were stained with HE.

[0068] Figure 8 A shows the curve of the change of tumor volume in small animals; Figure 8 B shows the curve of changes in the weight of small animals during the treatment period; Figure 8 C shows HE staining of the main organs of mice in the PBS group, PTX group, ACMS group and P+ACMS group after treatment. The data results show that ACMS can effectively overcome tumor resistance and has 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 complicated 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. As time goes by, the drug leakage is relatively small. While having good biocompatibility, it also has a longer 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 the tumor and overcome the drug resistance of the tumor.

[0074] The GSH-responsive cell membrane nanomedicine of the present invention can effectively enter the tumor tissue, effectively open the disulfide bond under the reducing conditions of the tumor microenvironment, thereby releasing the drug, overcoming the drug resistance of the tumor, and can effectively inhibit the growth of the tumor in combination with paclitaxel. At the same time, the GSH-responsive cell membrane nanomedicine of the present invention has good biosafety and has no toxic side effects on major organs. These encouraging results guarantee the transformation potential of these GSH-responsive cell membrane bionic nanomedicines in cancer treatment.

[0075] Although the embodiments of the present invention only disclose the use of macrophage cell membranes to make functionalized cell membrane vesicles, and the use of cell membrane vesicles to bond small molecule inhibitors to form cell membrane bionic nanomedicines, and demonstrate a model in which cell membrane bionic nanomedicines are used to treat breast cancer cells, according to the teachings of the present invention, those skilled in the art can expect to use 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 invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered 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, wherein the cell membrane vesicles are prepared by cell membrane extraction and membrane extrusion of immune cells overexpressing azide compounds on the membrane surface, the azide compounds on the cell membrane vesicles are bonded with the bioconjugated compounds through click chemistry reactions to form -SH groups, and the -SH groups are bonded with the small molecule inhibitors through disulfide bonds through a cross-linking agent.

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 bionic nanomedicine according to claim 1, characterized in that: The small molecule inhibitor is specifically one or more of the METTL3 methylation inhibitors.

4. 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.

5. A method for preparing a GSH-responsive cell membrane bionic nanomedicine, 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 cell membranes of 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 to form a -SH group through a click chemistry reaction. The -SH group and the small molecule inhibitor are combined together by a cross-linking agent in the form of a disulfide bond to obtain a GSH-responsive cell membrane bionic nanomedicine.

6. The method for preparing a GSH-responsive cell membrane bionic nanomedicine according to claim 5, characterized in that: The method for preparing functionalized immune cells in step S1 is specifically as follows: inoculating at least one selected immune cell into a DMEM culture medium, culturing in a 37°C incubator until the cell confluence reaches 75-85%, adding an azide compound to the DMEM culture medium, and incubating for 48 hours; finally, centrifuging at 1500-2500 rpm at 4°C for 5-15 minutes to obtain functionalized immune cells, collecting the functionalized immune cells, and washing them multiple times with PBS buffer for later use.

7. The method for preparing a GSH-responsive cell membrane bionic nanomedicine according to claim 6, 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 a cell lysis buffer, and protease inhibitors were added. Subsequently, a 1 mL syringe was used to extract the cell suspension placed on ice, so that the cell membrane lysis buffer and the functionalized immune cells were fully mixed, and the extraction was performed every 5 minutes. The extraction steps were repeated 5-6 times, and the cells were placed in a -80°C refrigerator for 30 minutes, and then 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 later use; The differential centrifugation process is 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.

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

9. The method for preparing a GSH-responsive cell membrane bionic nanomedicine according to claim 8, 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 compounds and cross-linking agents; then a small molecule inhibitor is added thereto, and the reaction is carried out for 12-15 hours. After the reaction is completed, 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 inhibitors, and freeze-dried to obtain GSH-responsive cell membrane biomimetic nanomedicine powder, which is stored at -80°C for use; The organic solvent is dimethyl sulfoxide, the mass ratio of the bioconjugate compound, the cross-linking agent and the small molecule inhibitor is 1:2:2, and the mass fraction of Tween 80 is 1 / 1000.

10. Application of GSH-responsive cell membrane bionic nanomedicine combined with the chemotherapy drug paclitaxel.

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