Preparation method and application of cell membrane-derived nanovesicles for specific targeting of tumors and metastasis inhibition

Abstract

The application discloses a preparation method and application of cell membrane-derived nanovesicles with specific targeting tumor and metastasis inhibition. The CD82 overexpression lung epithelial cell HBE cell strain is constructed through gene engineering, cell membranes are extracted in vitro, AS1411 aptamer is modified, and a chemotherapeutic drug, doxorubicin (DOX), is loaded to prepare an engineered nanovesicle drug delivery system. The engineered nanovesicle drug delivery system prepared by the application is proved to have multiple effects such as targeting triple-negative breast cancer, inhibiting metastasis and inducing triple-negative breast cancer cell apoptosis through in-vitro and in-vivo experiments. The application fills the gap of the extracellular vesicles as a drug carrier, and provides a new thought and scientific basis for the clinical treatment of triple-negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an engineered nanovesicle that has multiple functions, including targeting malignant breast cancer, inhibiting metastasis, and inducing apoptosis in malignant breast cancer cells. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women, seriously threatening their health. Compared with other subtypes, triple-negative breast cancer (TNBC) has higher heterogeneity and malignancy, is more prone to metastasis and recurrence, and has a worse prognosis, with a mortality rate as high as 40% within the first 5 years after diagnosis. Due to the lack of important expressed receptors, novel interventional therapies, such as endocrine therapy, HER-2 targeted therapy, immunotherapy, and neoadjuvant chemotherapy, have limited effectiveness in treating it. Therefore, surgery-adjuvant chemotherapy has long been the main treatment. However, due to the lack of specific targets for chemotherapy, conventional postoperative adjuvant chemoradiotherapy is insufficient to eliminate all lesions, and the remaining small number of metastatic lesions will eventually lead to tumor recurrence. Therefore, there is an urgent need to find new therapeutic targets and treatment regimens.

[0003] Cell membrane-derived nanovesicles, similar to exosomes, are derived from cells and possess good biocompatibility. Furthermore, the cell membrane extraction process effectively removes potentially hazardous genetic material such as nucleic acids. In addition, compared to exosomes, extracting cell membranes and then processing them through ultrasonic disruption and extrusion allows for large-scale preparation of nanovesicles, enabling the standardization of drug carriers. Therefore, cell membrane-derived nanovesicles may replace exosomes as a promising next-generation drug carrier. However, the lack of specific targeting is a key bottleneck in the application of exosomes and cell membrane-derived nanovesicles as drug carriers. Recurrence and metastasis of malignant tumors are currently a major challenge in cancer treatment, urgently requiring new therapeutic targets and strategies. Summary of the Invention

[0004] The purpose of this invention is to provide a novel strategy for the treatment of malignant tumors by preparing cell membrane-derived nanovesicles that specifically target and inhibit metastasis to deliver chemotherapeutic drugs. This invention utilizes genetic engineering to construct a lung epithelial cell line overexpressing the tumor metastasis inhibitor molecule CD82. Cell membranes are extracted in vitro, the nucleic acid aptamer AS1411 is modified, and the chemotherapeutic drug DOX is loaded onto the nanovesicles to prepare engineered nanovesicles carrying chemotherapeutic drugs. In vivo and in vitro experiments have demonstrated that these nanovesicles possess multiple functions, including targeting malignant tumor cells, inhibiting metastasis, and inducing apoptosis in malignant tumor cells.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] Methods for preparing cell membrane-derived nanovesicles that specifically target tumors and inhibit metastasis include:

[0007] Step 1: Prepare HBE cell membranes of lung epithelial cells enriched with the tumor metastasis inhibitory molecule CD82;

[0008] The full-length cDNA fragment of the human CD82 molecule was cloned using genetic engineering and inserted into the lentiviral expression vector pLV-EF1α-MCS-IRES Bsd. Lentiviral cells were packaged and used to infect normal lung epithelial HBE cells. Stable HBE cell lines overexpressing the CD82 molecule were obtained through selection with the antibiotic blasticidin. Cell membranes extracted in vitro were prepared into a cell membrane suspension using sonication to obtain a lung epithelial HBE cell membrane suspension overexpressing the tumor metastasis inhibitory molecule CD82.

[0009] Step 2: Prepare cell membrane-derived nanovesicles that specifically target tumors and inhibit metastasis;

[0010] The nucleic acid aptamer AS1411 was synthesized chemically and covalently bound to cholesterol molecules. The synthesized AS1411 aptamer was then coupled to the CD82-enriched metastasis-inhibiting molecule-rich HBE cell membrane of lung epithelial cells prepared in step 1, yielding a cell membrane suspension specifically targeting tumors and inhibiting metastasis. The cell membrane suspension was then ultrasonically disrupted using a probe and stored at 4°C for 10-20 hours to facilitate cell membrane recovery. Using an Avanti liposome extruder, the cell membrane suspension was passed through a 200nm membrane 10-15 times to obtain 200nm diameter cell membrane-derived nanovesicles specifically targeting tumors and inhibiting metastasis.

[0011] This invention also provides the application of cell membrane-derived nanovesicles specifically targeting tumors and inhibiting metastasis as drug carriers for delivering chemotherapeutic drugs. The application involves using nanovesicles prepared by the above method as drug carriers to load chemotherapeutic drugs to prepare chemotherapeutic agents specifically targeting tumors and inhibiting metastasis in malignant breast cancer. Optionally, the chemotherapeutic drug used in this invention is doxorubicin (DOX). The drug loading method is as follows: At a nanovesicle to DOX mass ratio of 1:2, take 125 μL of 400 μg / mL DOX solution, add 200 μg of CD82-overexpressing lung epithelial cells (HBE cells) conjugated with the AS1411 aptamer, and add 1×PBS to a final concentration of 200 μg / mL (DOX). After mixing, incubate at room temperature for 4 hours, centrifuge at 14000g for 30 min at 4°C, resuspend the precipitate in 250 μL of 1×PBS, centrifuge at 14000g for 30 min at 4°C, and add 250 μL of precipitate to the resuspending solution. Resuspend in 1×PBS and store at 4°C; obtain CD82-overexpressing HBE cell membranes loaded with DOX-conjugated AS1411 aptamers; prepare cell membrane-derived drug-loaded nanovesicles loaded with DOX-conjugated AS1411 aptamers and CD82-overexpressing cells according to the method described in step 2, and obtain cell membrane-derived DOX-loaded nanovesicles that specifically target tumors and inhibit metastasis.

[0012] Advantages and beneficial effects of the present invention:

[0013] This invention discloses a method for preparing and applying cell membrane-derived nanovesicles that specifically target tumors and inhibit metastasis. The method involves constructing a CD82-overexpressing lung epithelial cell line (HBE) through genetic engineering, extracting the cell membrane in vitro, and modifying it with the AS1411 aptamer to obtain cell membrane-derived nanovesicles with specific tumor targeting and metastasis inhibition. These nanovesicles possess the function of specifically targeting tumors and effectively inhibiting tumor cell metastasis, effectively overcoming the current bottleneck in the treatment of malignant tumors—the lack of treatments targeting tumor metastasis. Furthermore, these nanovesicles can be used as drug carriers to load chemotherapy drugs; for example, they can effectively load the chemotherapy drug DOX. Specific targeting of tumors can effectively improve the therapeutic effect of chemotherapy drugs and reduce chemotherapy side effects. In vivo and in vitro experiments have demonstrated that these nanovesicles possess multiple functions, including specifically targeting malignant breast cancer, inhibiting metastasis, and inducing apoptosis in malignant breast cancer cells by loading the chemotherapy drug DOX. Attached Figure Description

[0014] Figure 1 The results of CD82 overexpression in Example 2: Western blot analysis was used to detect the expression of membrane proteins and exogenous CD82 in HBE-WT, HBE-CD82 cell lines overexpressing CD82, and cell membrane-derived nanovesicles.

[0015] Figure 2 Example 3 shows the coupling results of the nucleic acid aptamer AS1411: (A) The coupling efficiency of nucleic acid aptamer AS1411 to cell membrane-derived nanovesicles. (B) The number of nucleic acid aptamers AS1411 coupled to a single cell membrane-derived nanovesicle. (C) Dark-field microscopy verification of the coupling of cell membrane-derived nanovesicles to nucleic acid aptamer AS1411; the cell membrane-derived nanovesicles are stained with Dil, and AS1411 contains FAM fluorescent molecules.

[0016] Figure 3 Example 4 shows the DOX loading results: (A) Loading efficiency of cell membrane-derived nanovesicles for the chemotherapy drug doxorubicin (DOX) and the encapsulation efficiency of the nanovesicles. (B) Drug release efficiency of DOX-loaded nanovesicles at pH 5.0 and pH 7.4.

[0017] Figure 4 Example 5: In vitro nanovesicle targeting results. In vitro fluorescence confocal microscopy was used to detect the targeting of CD82 cell membrane-enriched nanovesicles and CD82 cell membrane-enriched nanovesicles coupled with nucleic acid aptamer AS1411 to malignant breast cancer cells MDA-MB-231 and normal breast epithelial cells MCF-10A. Scale bar = 20 μm.

[0018] Figure 5 Example 6 shows the in vitro nanovesicle transfer inhibition results. An in vitro cell perforation and migration assay was used to detect the effect of CD82-enriched nanovesicles on the migration of MDA-MB-231 malignant breast cancer cells. Scale bar = 200 μm.

[0019] Figure 6 Example 7: Results of in vitro apoptosis induction in tumor cells using DOX-loaded nanovesicles. (A) In vitro immunofluorescence assay using Caspase-3 and TUNEL assays to detect apoptosis in MDA-MB-231 malignant breast cancer cells treated with DOX-loaded nanovesicles for 36 h. Scale bar = 20 μm. (B) In vitro Western blotting assay to detect apoptosis in MDA-MB-231 malignant breast cancer cells treated with DOX-loaded nanovesicles for 36 h.

[0020] Figure 7 Example 8 shows the results of in vivo nanovesicle targeting tumors in mice. (A) Schematic diagram of the in vivo mouse breast cancer orthotopic tumor model construction and the validation experiment of nanovesicles derived from the cell membrane of coupled nucleic acid aptamer AS1411 targeting tumor cells. (B) Tumor tissue and tumor weight of the in vivo mouse breast cancer orthotopic tumor model. (C) Imaging of mouse organ tissue samples showing the different conditions of nanovesicles in different tissues and the statistical results of relative fluorescence intensity of tumor tissue.

[0021] Figure 8 Example 9 shows the results of in vivo tumor metastasis inhibition by nanovesicles in mice. (A) Schematic diagram of the in vivo mouse breast cancer orthotopic tumor model construction and verification experiment of tumor metastasis inhibition by CD82-overexpressing cell membrane-derived nanovesicles. (B) Monitoring changes in mouse body weight and tumor volume in the in vivo mouse breast cancer orthotopic tumor model, and the weight of tumor tissue in the two groups. (C) Images of liver metastases and primary tumor tissue samples after treatment with wild-type and CD82-overexpressing cell membrane-derived nanovesicles, with white arrows indicating the location of liver metastases. (D) Results of HE staining of liver tissue and statistics of the number of metastases, with black arrows indicating the location of metastases, and statistics of metastases in the two groups.

[0022] Figure 9 Example 10: Results of tumor cell apoptosis induced by nanovesicles in mice. (A) Schematic diagram of the construction of an in vivo mouse breast cancer orthotopic tumor model and the use of DOX-derived cell membrane nanovesicles loaded with the coupled nucleic acid aptamer AS1411 to overexpress CD82. (B) Changes in mouse body weight and tumor volume during treatment. (C) Difference between orthotopic tumor tissue sample images and tumor weight after treatment.

[0023] Figure 10 Example 8 shows the results of nanovesicles targeting tumors in mice. Western blotting experiments were used to verify the expression of apoptosis-related proteins in tumor tissue.

[0024] Figure 11 Example 8 shows the results of nanovesicle targeting tumors in mice. In vivo immunofluorescence assays (Caspase 3 and TUNEL assays) were used to detect apoptosis in malignant breast cancer tumor tissue treated with DOX-derived nanovesicles overexpressing CD82 via AS1411. Scale bar = 20 μm. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to embodiments and accompanying drawings:

[0026] Example 1: Preparation of cell membrane-derived nanovesicles for metastasis inhibition

[0027] Using genetic engineering methods, the full-length cDNA of the human tumor metastasis molecule CD82 was cloned and overexpressed in normal lung epithelial HBE cells to construct a CD82-overexpressing cell line. Cell membranes of wild-type and CD82-overexpressing lung epithelial HBE cells were extracted in vitro to obtain wild-type HBE cell membranes and a suspension of CD82-enriched lung epithelial HBE cell membranes. The cell membrane suspensions were then sonicated using a probe and stored at 4°C for 10-20 hours to facilitate cell membrane recovery. Using an Avanti liposome extruder, the cell membrane suspensions were passed through a 200nm membrane 10-15 times to obtain 200nm diameter nanovesicles, including wild-type and metastasis-inhibiting cell membrane-derived nanovesicles, providing control and experimental materials for the following examples.

[0028] Example 2: Detection of CD82 molecule expression in the transfer-inhibiting cell membrane-derived nanovesicles prepared in Example 1.

[0029] Western blotting was used to detect the expression of CD82 molecules in wild-type and CD82-enriched transfer-inhibiting cell membrane-derived nanovesicles prepared in Example 1, with wild-type HBE cell membranes and their derived nanovesicles as controls. The results showed that CD82 overexpression was highly enriched in the cell membrane and its derived transfer-inhibiting nanovesicles. Figure 1 .

[0030] Example 3: Preparation of cell membrane-derived nanovesicles specifically targeting tumors and inhibiting metastasis

[0031] This invention provides a method for conjugating the nucleic acid aptamer AS1411 to a cell membrane suspension. Optionally, the method of conjugating the AS1411 aptamer to the cell membrane suspension via cholesterol is as follows: Order the nucleic acid aptamer AS1411 synthesized by Sangon Biotech (Shanghai) Co., Ltd., covalently bind cholesterol molecules, and conjugate the nucleic acid aptamer AS1411 to the cell membranes of wild-type and CD82-overexpressing HBE cells to prepare cell membrane-derived nanovesicles specifically targeting tumors (wild-type) and inhibiting metastasis. Dilute the ordered chemically synthesized AS1411 aptamer to a 10 μM working solution with ddH2O. Add 2.5 μL, 5 μL, 10 μL, and 20 μL of the working solution to 250 μg of cell membrane suspension, respectively, to a final volume of 200 μL. After mixing, incubate at 37°C for 15 min; centrifuge at 14000g for 30 min at 4°C; add 200 μL of the precipitate. Resuspended in 1×PBS and stored at 4°C to obtain wild-type and CD82-enriched cell membrane suspensions conjugated with the AS1411 aptamer. The cell membrane suspensions were then placed in a beaker filled with ice water and subjected to probe-guided sonication to disrupt the membranes. The disrupted cell membrane suspensions were stored at 4°C for 10-20 hours to facilitate cell membrane recovery. Using an Avanti liposome extruder, the cell membrane suspensions were passed through a 200nm membrane 10-15 times to obtain approximately 200nm AS1411-conjugated wild-type and CD82-enriched cell membrane-derived nanovesicles. The preparation of wild-type and metastasis-inhibiting cell membrane-derived nanovesicles specifically targeting tumors was conducted. The connection efficiency between the AS1411 aptamer and the nanovesicles, and the number of AS1411 aptamers connected to a single nanovesicle, were verified by the following experiments: Figure 2 As shown, 0.02 nM aptamer AS1411 can connect 100 μg of nanovesicles with a connection efficiency of over 95%, and the number of AS1411 aptamers connected to a single nanovesicle exceeds 1000, which is sufficient to achieve the ability to specifically target tumor cells.

[0032] Example 4: Preparation of cell membrane-derived drug-loaded nanovesicles specifically targeting tumors and inhibiting metastasis

[0033] This invention provides a method for preparing drug-loaded nanovesicles that specifically target tumors and inhibit metastasis. Optionally, the nanovesicles serve as excellent carriers for chemotherapeutic drugs, possessing a triple effect of specifically targeting malignant tumors, inhibiting metastasis, and promoting cell apoptosis by loading chemotherapeutic drugs. For loading the chemotherapeutic drug, optionally, the chemotherapeutic drug used in this invention is doxorubicin (DOX). The drug loading method is as follows: Take a DOX solution at a mass ratio of 1:2, add 200 μg of CD82-overexpressing lung epithelial cells (HBE cells) conjugated with the AS1411 aptamer to the DOX solution, add 1×PBS to a final volume of 250 μL (i.e., a final DOX concentration of 200 μg / mL), mix well, incubate at room temperature for 4 hours, centrifuge at 14000g for 30 min at 4°C, resuspend the precipitate in 250 μL of 1×PBS, centrifuge at 14000g for 30 min at 4°C, and add 250 μL of PBS to the precipitate again. Resuspended in 1×PBS and stored at 4°C; CD82-overexpressing HBE cell membranes loaded with DOX and conjugated with the AS1411 aptamer were obtained; using an Avanti liposome extruder, the cell membrane suspension was propelled through a 200 nm membrane to prepare cell membrane-derived nanovesicles loaded with DOX and conjugated with the AS1411 aptamer and enriched with CD82 molecules. Multifunctional nanovesicles were obtained to deliver chemotherapy drugs for adjuvant tumor therapy. For the determination of DOX loading efficiency, nanovesicle encapsulation efficiency, and release efficiency of DOX loaded by nanovesicles under different pH conditions, see [link to documentation]. Figure 3 .

[0034] Example 5: In vitro experimental verification of chemotherapy targeting nanovesicles that specifically target tumors and inhibit metastasis.

[0035] The targeting ability of the AS1411 aptamer to different cell types was detected by nanovesicle phagocytosis assay. The AS1411 aptamer was modified with a green FAM fluorescent group at its 3' end. Two types of nanovesicles from two cell lines (HBE-CD82-Exo and AS1411-HBE-CD82-Exo) were stained with DiI and then incubated with MDA-MB-231 malignant breast cancer cells and MCF-10A normal breast cells at 37°C for 60 min. The results showed that nanovesicles connected to the AS1411 aptamer (AS1411-HBE-CD82-Exo) significantly improved the targeting ability to MDA-MB-231 malignant breast cancer cells, but had no change in the targeting ability to MCF-10A normal breast epithelial cells. Nanovesicles without AS1411 modification (HBE-CD82) showed no significant difference in targeting ability to MDA-MB-231 and MCF-10A cells. Figure 4 .

[0036] Example 6: In vitro experiments verify the inhibitory effect of nanovesicles specifically targeting tumors and metastasis on cell migration.

[0037] Serum-free cultured MDA-MB-231 malignant breast cancer cells were treated with wild-type nanovesicles and nanovesicles overexpressing the metastasis inhibitor CD82 for 24 hours. The effect of nanovesicles on inhibiting cell migration was then assessed using a Transwell assay. The results showed that the number of migrating cells in the wild-type nanovesicle HBE-WT-Exo group was consistent with the control group, indicating that nanovesicles derived from normal epithelial cell membranes had no significant effect on cell migration. However, the number of migrating cells in the HBE-CD82-Exo group, which overexpressed the metastasis inhibitor CD82, was significantly reduced. This demonstrates that CD82-enriched nanovesicles can effectively inhibit the migration of MDA-MB-231 malignant breast cancer cells in vitro. Figure 5 .

[0038] Example 7: In vitro experimental verification of the specific targeting of tumors and inhibition of metastasis by DOX-loaded nanovesicles inducing tumor cell apoptosis.

[0039] The TUNEL assay was used to detect overall cell apoptosis. The results showed that almost all MDA-MB-231 malignant breast cancer cells treated with Free-DOX and AS1411-HBE-CD82-DOX-Exo nanovesicles loaded with DOX exhibited green fluorescent apoptotic signals, with the apoptotic cell rate approaching 100%. Figure 6 Immunofluorescence assays were performed to detect the expression and localization of the apoptosis molecule Cleaved caspase-3 within cells. Compared with the control group, the expression of Cleaved caspase-3 in cells of the Free-DOX and AS1411-HBE-CD82-DOX-Exo groups was significantly upregulated, demonstrating that DOX-loaded nanovesicles specifically targeting tumors and inhibiting metastasis can exert similar pharmacological effects to free DOX in vitro. Figure 6 Western blot analysis was used to detect changes in specific protein levels during apoptosis. Compared with the control group, the expression levels of pro-apoptotic proteins PARP, cleaved PARP, and caspase-3, and cleaved caspase-3 were significantly increased in MDA-MB-231 cells treated with Free-DOX and AS1411-HBE-CD82-DOX-Exo groups, while the expression of anti-apoptotic protein Bcl-2 was significantly downregulated, significantly inducing apoptosis. Figure 6 .

[0040] This invention provides the application of nanovesicles that specifically target tumors and inhibit metastasis as good chemotherapeutic drug carriers. Optionally, in in vitro experiments, the nanovesicles can specifically target malignant breast cancer cells while having almost no targeting on normal breast epithelial cells, significantly inhibit the migration of malignant breast cancer cells, and effectively induce apoptosis of malignant breast cancer cells.

[0041] Example 8: In vivo mouse experiments to verify the tumor-specific targeting and metastasis-inhibiting effects of nanovesicles.

[0042] The experimental mice were BALB / c nude mice (nu / nu) (4-5 weeks old, female), purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and stored in the pathogen-free facility of Nankai University. All animal experiments were approved by the Animal Protection and Use Committee of Nankai University and handled in accordance with the "Nankai University Animal Protection Guidelines". A mouse tumor xenograft model was constructed using 1.5 × 10⁻⁶ mice. 6 MDA-MB-231 malignant breast cancer cells were subcutaneously injected into the third pair of mammary fat pads of 4-5 week old female nude mice (n=3 per group). Three weeks after cell injection, DiR-labeled nanovesicles HBE-CD82-Exo and AS1411-HBE-CD82-Exo were injected via the tail vein, with each mouse receiving 30 μg of cell membrane-derived nanovesicles (approximately 7.5 × 10⁻⁶). 8 Mice were sacrificed after 24 hours (using a 100 μL system), and liver, lung, spleen, heart, kidney, small intestine, stomach, and tumor tissues were isolated. Fluorescence imaging analysis of the small animal organ samples was performed to detect the distribution of nanovesicles in different organs. Results showed that the AS1411 aptamer-modified nanovesicles (AS1411-HBE-CD82-Exo) efficiently targeted tumor tissues in a mouse tumor-bearing model, while the control group without AS1411 aptamer modification showed less targeting of tumor tissues. Nanovesicles were enriched in liver, lung, and spleen tissues, with no significant differences among different nanovesicle groups. Therefore, AS1411 aptamer-modified nanovesicles can significantly improve specific targeting of tumor tissues. Figure 7 .

[0043] Example 9: In vivo mouse experiments to verify the tumor metastasis inhibitory ability of nanovesicles specifically targeting tumors and inhibiting metastasis.

[0044] The experimental mice were BALB / c nude mice (nu / nu) (4-5 weeks old, female). A mouse tumor xenograft model was established, and 1.5 × 10⁻⁶ tumor grafts were used. 6 One malignant breast cancer cell, MDA-MB-231, was subcutaneously injected into the fat pad of the third pair of mammary glands in 4-5 week old female nude mice. The tumors reached a size of approximately 400 mm. 3 Approximately 3 weeks after tumor cell injection, patients were randomly divided into two groups: the AS1411-HBE-WT-Exo group and the AS1411-HBE-CD82-Exo group (n=3 in each group). Every three days, 50 μg of different cell membrane-derived nanovesicles were injected via the tail vein. Body weight and tumor volume were measured every three days, and the volume was calculated using the standard equation: V = 1 / 2 × L × W2 Where V is the tumor volume, L is the tumor length, and W is the tumor width. Mice were sacrificed after 6 weeks, and major tissues were collected for tumor cell metastasis analysis. Results showed a significant difference in liver metastasis between the two groups. The liver tissue of the AS1411-HBE-WT-Exo nanovesicle treatment group showed multiple clearly observable metastatic lesions, while the liver tissue of the AS1411-HBE-CD82-Exo nanovesicle group had a smooth surface and no obvious metastatic lesions. Furthermore, no significant tumor metastasis was observed in the lung tissue of either group. To further determine the tumor metastasis status, liver tissue was stained with hematoxylin and eosin (HE) to detect and count the number of metastatic lesions. Results showed that all three mice in the AS1411-HBE-WT-Exo nanovesicle group showed significant metastatic lesions in their liver tissue, while only one mouse in the AS1411-HBE-CD82-Exo nanovesicle group showed mild metastatic lesions. The specific number of metastatic lesions showed a significant difference; relevant experimental data can be found in [link to relevant data]. Figure 8 .

[0045] Example 10: In vivo mouse experiments to verify the specific targeting of tumors and the inhibition of metastasis by nanovesicles inducing tumor cell apoptosis.

[0046] The experimental mice were BALB / c nude mice (nu / nu) (4-5 weeks old, female). A mouse tumor xenograft model was established, and 1.5 × 10⁻⁶ tumor grafts were used. 6 One malignant breast cancer cell, MDA-MB-231, was subcutaneously injected into the fat pad of the third pair of mammary glands in 4-5 week old female nude mice. The tumors were allowed to grow to approximately 100 mm in size. 3 Approximately 10 days later, mice were randomly divided into three groups (n=4 per group). Every three days, mice were injected via tail vein with PBS, 3 mg / kg Free-DOX, or an equivalent amount of DOX-loaded nanovesicles (AS1411-HBE-CD82-DOX-Exo) at a dose equivalent to 3 mg / kg DOX, for a total of five injections. Mouse weight and tumor volume were recorded during these injections. Results showed that compared to the control group, the tumor volume was significantly reduced in the Free-DOX group and the AS1411-HBE-CD82-DOX-Exo group. The reduction was more pronounced in the AS1411-HBE-CD82-DOX-Exo group than in the Free-DOX group, indicating that the AS1411-HBE-CD82-DOX-Exo group significantly induced tumor cell apoptosis. The DOX-loaded nanovesicles showed better therapeutic effects than free DOX. Related experimental results are shown in [link to relevant experimental results]. Figure 9 .

[0047] Furthermore, Western blotting and immunofluorescence experiments were used to verify the specific changes in apoptosis-related proteins in tumor tissues at the protein level. The results showed that the expression levels of pro-apoptosis-related proteins Cleaved caspase-3, Cleaved PARP, and Bax were significantly increased in the AS1411-HBE-CD82-DOX-Exo group. The expression of the anti-apoptosis-related protein Bcl-2 also showed a downward trend in the AS1411-HBE-CD82-DOX-Exo group. The expression of p53 was significantly increased in the AS1411-HBE-CD82-DOX-Exo group, indicating a significant promotion of cell apoptosis. Related experimental results are shown in […]. Figure 10 .

[0048] Finally, TUNEL assays and immunofluorescence assays were used to verify tumor cell apoptosis. The results showed that the proportion of apoptotic cells in the AS1411-HBE-CD82-DOX-Exo group was significantly higher than that in the Free-DOX group and the control group. Immunofluorescence staining of cleaved caspase-3 in tumor tissues showed that the expression level of cleaved caspase-3 was significantly increased in the AS1411-HBE-CD82-DOX-Exo group. This demonstrates that the AS1411-HBE-CD82-DOX-Exo group significantly induced tumor cell apoptosis. Figure 11 .

[0049] In summary, the results of in vitro experiments and in vivo mouse tumor models demonstrate that the specific targeting aptamer AS1411 modified with tumor-specific and metastasis-inhibiting nanovesicles can effectively promote the specific targeting of tumor cells by nanovesicles; enrichment of the metastasis-inhibiting molecule CD82 can effectively inhibit the in vitro migration and in vivo metastasis of tumor cells; and DOX-loaded tumor-specific and metastasis-inhibiting nanovesicles can effectively improve the therapeutic effect of tumor treatment while inhibiting tumor metastasis. Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0050] This invention provides the application of nanovesicles that specifically target tumors and inhibit metastasis as good carriers for chemotherapeutic drugs. Optionally, by constructing a mouse tumor-bearing model, the anti-tumor effect of nanovesicles in vivo experiments is verified. The results show that nanovesicles can efficiently target malignant breast cancer tumor lesions, inhibit the formation of malignant breast cancer metastases, efficiently deliver anti-tumor drugs to malignant breast cancer tumor lesions to inhibit tumor growth and induce tumor cell apoptosis.

Claims

1. A method for preparing cell membrane-derived nanovesicles that specifically target tumors and inhibit metastasis, characterized in that, The nucleic acid aptamer AS1411 was synthesized chemically and covalently bound to cholesterol molecules. The synthesized AS1411 aptamer was then coupled to the cell membrane of lung epithelial cells (HBE) enriched with the tumor metastasis inhibitory molecule CD82, yielding a suspension of AS1411-coupled and CD82-enriched cell membranes. The cell membrane suspension was then ultrasonically disrupted using a probe and stored at 4°C for 10-20 hours to facilitate cell membrane recovery. Using an Avanti liposome extruder, the cell membrane suspension was passed through a 200 nm membrane 10-15 times to obtain 150-250 nm cell membrane-derived nanovesicles specifically targeting tumors and inhibiting metastasis. The cell membrane preparation process for the enriched CD82 tumor metastasis inhibitory molecule in lung epithelial cells (HBE) is as follows: The full-length cDNA fragment of the CD82 molecule was cloned and inserted into the lentiviral expression vector pLV-EF1α-MCS-IRESBsd. The lentivirus was packaged and used to infect lung epithelial cells (HBE cells). CD82-overexpressing HBE cells were obtained by screening with Blasticidin antibiotic. The cell membranes of CD82-overexpressing HBE cells were extracted in vitro to obtain a suspension of HBE cell membranes enriched with the tumor metastasis inhibitor molecule CD82.

2. The application of the cell membrane-derived nanovesicles specifically targeting tumors and inhibiting metastasis prepared by the preparation method of claim 1 as drug carriers for delivering chemotherapeutic drugs, characterized in that, The application involves using the nanovesicles as drug carriers to load the chemotherapy drug doxorubicin (DOX) to prepare chemotherapy agents that specifically target tumors and inhibit metastasis in malignant breast cancer.

3. The application according to claim 2, characterized in that, The method for loading the chemotherapeutic drug doxorubicin using the nanovesicles is as follows: Take 125 μL of 400 μg / mL DOX solution at a nanovesicle to DOX mass ratio of 1:2, add 200 μg of CD82-overexpressing lung epithelial cells (HBE cells) conjugated with the AS1411 aptamer, and add 1×PBS to a final volume of 250 μL, i.e., a final DOX concentration of 200 μg / mL. After mixing, incubate at room temperature for 4 hours, centrifuge at 14000 g for 30 min at 4°C, resuspend the precipitate in 250 μL of 1×PBS, centrifuge at 14000 g for 30 min at 4°C, and add 250 μL of precipitate to the resuspending solution. Resuspend in 1×PBS and store at 4°C; obtain CD82-overexpressing HBE cell membranes loaded with DOX-conjugated AS1411 aptamers; prepare cell membrane-derived drug-loaded nanovesicles loaded with DOX-conjugated AS1411 aptamers and CD82-overexpressing cells using the method described in claim 2; obtain cell membrane-derived DOX-loaded nanovesicles that specifically target tumors and inhibit metastasis.

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