Combination for the treatment of tumors

Abstract

The application discloses a combined drug for treating tumors; aims to provide a treatment scheme for targeting transport of a chemotherapeutic drug and a metabolic drug, activating a tumor microenvironment, and treating tumor patients by combining chemotherapy, metabolic therapy and immunotherapy; and the technical key point is that an immune-activated biomimetic nanovesicle is used to load a chemotherapeutic drug, doxorubicin hydrochloride (DOX), and a sugar metabolism inhibitor, 2-deoxy-D-glucose (2-DG); wherein: the activated biomimetic nanovesicle highly expresses PD1 molecules, can competitively interact with PD-L1, and can release the inhibition on immune cells; the DOX is a chemotherapeutic drug; the 2-DG is a glycolysis inhibitor, and can inhibit the glycolysis process in cells; and the application belongs to the field of medical biotechnology.

Description

TECHNICAL FIELD

[0001] The present application relates to a combination drug for treating tumors, belonging to the technical field of medicine. BACKGROUND

[0002] In China, cancer has been ranked in the top three of incurable diseases and is an important obstacle to improving people's life expectancy. Fast-paced, sub-healthy lifestyle and chronic infection, environmental exposure, etc. are the main causes of cancer. Among them, lung cancer is the most common cancer and the leading cause of cancer death in men, followed by prostate cancer, colorectal cancer and liver cancer. In women, breast cancer is the most common cancer and the leading cause of cancer death, followed by colorectal cancer and lung cancer. At present, the top few malignant tumors, such as lung cancer, esophageal cancer, gastric cancer, and liver cancer, have poor prognosis and high mortality. Mainly due to the extremely strong invasiveness of malignant tumors and the inefficiency of early screening methods, more than half of cancer patients are diagnosed at an advanced stage. At present, in addition to traditional surgery, radiotherapy and chemotherapy, targeted drug therapy and immunotherapy have developed in recent years. However, these treatment methods still face many challenges, such as the instability of targeted drug therapy; the uncertainty of immunotherapy suitable population; drug resistance and biological toxicity of chemotherapy drugs, etc.

[0003] Chemotherapy, as one of the most effective means of traditional cancer treatment, mainly refers to the elimination of cancer cells by chemotherapy drugs to treat cancer. However, since the chemotherapy drugs enter the human body, they cannot distinguish between cancer cells and normal cells, and will usually cause a series of abnormal reactions in the human body, the most common reactions are mainly nausea, vomiting, and reduced immunity. For example, the broad-spectrum antitumor drug doxorubicin hydrochloride (Doxorubicin, DOX) is a widely used cancer chemotherapy agent in clinical practice, mainly used for breast cancer, small cell lung cancer, leukemia, malignant lymphoma, etc. DOX, as an inhibitor of reverse transcriptase, RNA polymerase and DNA topoisomerase II, can induce DNA damage and apoptosis. However, due to the toxic side effects of DOX, such as bone marrow suppression, liver dysfunction, and severe cardiotoxicity, its clinical application is limited. In addition, the drug resistance caused by frequent use of DOX is another serious problem.

[0004] In the past decade, immunotherapy has made great progress in the treatment of cancer and has become an indispensable treatment for advanced cancer. Unlike experimental methods such as targeted therapy or chemotherapy, immunotherapy is to reshape the immune microenvironment of tumors and change the immunosuppressive tumor microenvironment into an activated immune microenvironment. For example: immunotherapy targeting the programmed death receptor 1 (PD1) / programmed death molecule 1 ligand (PD-L1) pathway. PD1 / PD-L1 pathway is abnormally activated in tumor tissues. PD-1 is mainly expressed on activated immune cells, including T cells, B cells, dendritic cells, monocyte-macrophage cells and other immune cells. PD-L1 is the ligand of PD1 and is highly expressed on tumor cells. The interaction of PD1 and PD-L1 can inhibit the function of effector T cells, help the tumor form an immunosuppressive tumor microenvironment, participate in tumor immune escape, and promote tumor formation and metastasis. Therefore, blocking the PD1 / PD-L1 pathway is an attractive target for treating cancer.

[0005] Like normal cells, cancer cells need energy to grow and reproduce. These energies mainly come from sugar and glucose (sugar present in the blood). But unlike normal cells, the sugar needed for the growth of cancer cells is more than 200 times that of normal cells. Cancer cells can obtain a large amount of ATP in the form of glycolysis, and create an acidic and hypoxic favorable survival environment for tumor cells. Metabolic glucose analog 2-deoxy-D-glucose (2-DG) is an inhibitor of glucose transport and glycolysis ATP production, which can reduce the energy and nutrients in tumor cells, and then change the metabolic environment of tumor cells, and then inhibit the Warburg effect of tumor cells. 2-DG also participates in inducing abnormal cell signal conduction, cell cycle arrest, DNA repair abnormalities, calcium ion influx and apoptosis, etc. In addition, due to the structural similarity of 2-DG to glucose, it can be used as a glucose analog to change N-linked glycosylation, and then cause protein misfolding and degradation. For example: 2-DG can induce the misfolding and degradation of PD-L1. Given that most malignant tumor cells prefer to use glycolysis, there is an opportunity to take advantage of this weakness to selectively eliminate cancer cells while not affecting untransformed normal cells. But clinical trials show that 2-DG has poor drug-like properties, requires high effective bioavailability and therapeutic concentration in vivo, which makes it challenging to use it for single-agent treatment to kill tumors.

[0006] It is worth noting that: immunotherapy and metabolic therapy in the clinical treatment of tumor application success, success has extended the survival of some cancer patients. Clinically, about 20 percent of patients with PD1 antibody immunotherapy is effective. But some cancer patients with driver gene mutations, less benefit from immunotherapy. For example: cancer patients with EGFR driver gene mutations have no response to PD1 antibody treatment. Therefore, there is an urgent need for some drugs to expand the scope of immunotherapy and increase the effect of immunotherapy.

[0007] At present, the development of nano-targeted drug delivery system also provides a new idea for tumor immunotherapy. The combination of nano-targeted drug delivery system and cancer immunotherapy can activate immune cells, enhance anti-tumor immunity and improve tumor microenvironment by nano-carrier wrapped immunomodulators, so as to achieve the elimination of malignant tumors. Programmed cell death protein 1 (PD1) / PD-L1 can silence the immune system through interaction between them, which is also a main immune checkpoint targeted by the nano-targeted drug delivery system. Therefore, it is of great significance to develop 2-DG loaded biomimetic nano-targeted drug delivery system as a therapeutic agent or adjuvant for tumor radiotherapy and chemotherapy, and combine with immunotherapy. SUMMARY

[0008] Based on the current chemotherapy drugs have great toxicity to patients, targeted therapy resistance and the outstanding problem that patients have little response to immunotherapy, the purpose of the present application is to provide a kind of drug for combination of chemotherapy drugs for targeting tumor cells, activating tumor microenvironment and delivering chemotherapy drugs, so as to achieve the purpose of treating tumor patients.

[0009] To this end, the technical scheme provided by the present application is as follows:

[0010] A kind of combined drug for treating cancer, mainly by biomimetic nanovesicle P-NV, 2-DG, DOX.

[0011] Among them, the biomimetic nanovesicle P-NV is prepared by lung cancer cell TC1 cell with high expression of PD1 after genetic engineering modification, which can recognize PD-L1 of tumor cells and the homing effect of tumor cells itself, target transport to tumor site, block the signal transduction of PD1 / PD-L1, and then achieve the effect of relieving the immune suppression of tumor microenvironment;2-deoxy-D-glucose (2-DG) is a glucose analogue, which can inhibit the glycolysis process and energy production of tumor cells, and can also degrade the expression of PD-L1 on tumor cells, which can achieve the effects of inhibiting tumor cell metabolism and relieving PD1 / PD-L1 inhibition at the same time. Doxorubicin (Doxorubicin, DOX) is a widely used cancer chemotherapy agent in clinic, which can induce DNA damage and apoptosis, and then inhibit the proliferation of cancer cells.

[0012] Further, the combined medicine for treating tumor described above is characterized in that it is composed of the biomimetic nanovesicle P-NV, 2-DG and DOX.

[0013] Further, the combined medicine for treating tumor described above is characterized in that the biomimetic nanovesicle, the doxorubicin hydrochloride DOX and the sugar metabolism inhibitor 2-deoxy-D-glucose are composed according to the mass ratio of 10:6.9:5.8.

[0014] Further, the combined medicine for treating tumor described above is characterized in that the biomimetic nanovesicle, the doxorubicin hydrochloride DOX and the sugar metabolism inhibitor 2-deoxy-D-glucose are composed according to the mass ratio of 10:5:4.

[0015] Further, the combined medicine for treating tumor described above is characterized in that the biomimetic nanovesicle, the doxorubicin hydrochloride DOX and the sugar metabolism inhibitor 2-deoxy-D-glucose are composed according to the mass ratio of 10:4:3.

[0016] Further, the combined medicine for treating tumor described above is characterized in that the biomimetic nanovesicle, the doxorubicin hydrochloride DOX and the sugar metabolism inhibitor 2-deoxy-D-glucose are composed according to the mass ratio of 10:3:2.

[0017] Further, the combined medicine for treating tumor described above is characterized in that the biomimetic nanovesicle P-NV, 2-deoxy-D-glucose and doxorubicin hydrochloride DOX are administered according to the mass ratio described above, but are not limited to the mass ratio described above.

[0018] Further, the combined medicine for treating tumor described above is characterized in that the biomimetic nanovesicle P-NV, 2-deoxy-D-glucose and doxorubicin hydrochloride DOX are administered by tail vein injection of mice, and the administration is performed once every two days.

[0019] Further, the combined medicine for treating tumor described above is characterized in that the tumor is lung cancer, but is not limited to lung cancer.

[0020] Further, the combined medicine for treating tumor described above is characterized in that the administration is tail vein injection, but is not limited to this administration method.

[0021] Further, the combined medicine for treating tumor described above is characterized in that the tumor includes carcinoma in situ and metastatic cancer.

[0022] Compared with the prior art, the technical scheme provided by the present application has the following technical advantages:

[0023] The technical scheme provided by the application uses mouse lung cancer cells TC1 to construct an engineering cell with high expression of mPD1, and further ultrasonic co-extrusion to make it generate natural cell membrane nanovesicles (P-NV). The carrier is used to deliver broad-spectrum anticancer drugs doxorubicin hydrochloride (DOX) and metabolic anticancer drugs 2-deoxyglucose (2-DG), and the loading rate and release rate of the cell membrane nanovesicles on the two types of anticancer drugs are evaluated. The present application finds that the cell membrane nanovesicles loaded with DOX (PD-NV), the cell membrane nanovesicles loaded with 2-DG (PG-NV), and the cell membrane nanovesicles loaded with DOX and 2-DG (PDG-NV) can inhibit the growth of mouse lung cancer control cells TC1 (TC-1C) and TC1-mPD-L1 (TC-1L) with stable high expression of PD-L1 in vitro, and the inhibition level on TC1-mPD-L1 (TC-1L) cells is higher. The present application finds that the cell membrane nanovesicles loaded with 2-DG can inhibit the expression of mPD-L1 of mouse lung cancer cells TC-1C and TC-1L in vitro through the deglycosylation of PD-L1, thereby reversing the immunosuppression of tumor cells.

[0024] The present application finds that in small animal models, including mouse lung cancer xenograft models and primary tumor models, the treatment of the biomimetic nanovesicles P-NV, the biomimetic nanovesicles P-NV loaded with 2-DG (PG-NV), the biomimetic nanovesicles P-NV loaded with DOX (PD-NV), and the biomimetic nanovesicles P-NV loaded with 2-DG and DOX (PDG-NV) has no adverse effect on the body weight of the mice; after the treatment, the organs of the mice also have no obvious pathological abnormalities. The biomimetic nanovesicles P-NV have no obvious effect on the treatment of the mice; the biomimetic nanovesicles P-NV loaded with 2-DG (PG-NV) can slightly inhibit the growth of cancer in the mice; the biomimetic nanovesicles P-NV loaded with DOX (PD-NV) can inhibit the growth of cancer in the mice; when the nanovesicles loaded with 2-DG and DOX (PDG-NV) are used to treat the tumor models of the mice, the growth of the tumor in the mice can be significantly reduced, and there is an obvious synergistic effect, which basically achieves the effect of curing the tumor.

[0025] In summary, after the biomimetic nanovesicles P-NV are loaded with 2-DG and DOX, the PD-1 / PD-L1 interaction is inhibited, and the immunosuppression of the tumor is relieved, the infiltration of anti-tumor inflammatory immune cells is promoted, the immune microenvironment is improved, the aerobic glycolysis of tumor cells is inhibited, the DNA damage and apoptosis of tumor cells are induced, and a synergistic anticancer effect is produced. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is the effect of cell membrane nanovesicles P-NV on cell activity.

[0027] A: 50 μg / mL cell membrane nanovesicles P-NV had no obvious effect on the growth of 3T3 cells (mouse fibroblasts) and BEAS-2B cells (human lung bronchial epithelial cells). B: 100 μg / mL cell membrane nanovesicles P-NV had no obvious effect on the growth of 3T3 cells and BEAS-2B cells. C: 200 μg / mL cell membrane nanovesicles P-NV had no obvious effect on the growth of 3T3 cells and BEAS-2B cells. D: 200 μg / mL cell membrane nanovesicles P-NV had no effect on the growth of control TC1 cells (TC-1C) and engineered TC1-mPD-L1 cells (TC-1L);

[0028] Figure 2 is the loading and release efficiency of cell membrane nanovesicles P-NV on doxorubicin hydrochloride (DOX) and 2-deoxyglucose (2-DG).

[0029] A: Statistical results of the loading rate of cell membrane nanovesicles P-NV on doxorubicin hydrochloride (DOX) and 2-deoxyglucose (2-DG); B: Statistical results of the drug release rate of doxorubicin hydrochloride (DOX) and 2-deoxyglucose (2-DG) in cell membrane nanovesicles P-NV;

[0030] Figure 3 is the effect of P-NV loaded with different drugs on the viability of control TC1-1C cells and engineered TC-1L cells.

[0031] A: Lower concentration of cell membrane nanovesicles loaded with DOX (PD-NV) can dose-dependently inhibit the growth of control TC1-1C cells and engineered TC-1L cells in a short time (24 h), and the inhibition of TC-1L cells is more significant. B: Cell membrane nanovesicles loaded with 2-DG (PG-NV) gradually inhibit the cell viability of control TC1-1C cells and engineered TC-1L cells in a time-dependent manner, and the toxicity to cells is more obvious with the extension of time, and the inhibition of TC-1L cells is more significant. C: Lower concentration of cell membrane nanovesicles loaded with DOX and 2-DG (PDG-NV) can time- and dose-dependently inhibit the cell viability of control TC1-1C cells and engineered TC-1L cells, and almost all cells die after 48 h of co-incubation with target cells, and the inhibition of TC-1L cells is more significant.

[0032] Figure 4 is that high concentration of free 2-DG and lower concentration of 2-DG loaded by cell membrane nanovesicles (PG-NV) can significantly inhibit the level of glycolysis in control TC1-1C cells and engineered TC-1L cells. The ability of PG-NV to inhibit ATP production is significantly better than that of free 2-DG.

[0033] A: High concentration of free 2-DG can dose-dependently inhibit the ATP level produced by glycolysis in both control TC1-1C cells and engineered TC-1L cells, and there is no difference in the inhibition of ATP level produced by the two target cells; B: Cell membrane nanovesicles loaded with lower concentration of 2-DG (PG-NV) can also dose-dependently inhibit the ATP level produced by glycolysis in both control TC1-1C cells and engineered TC-1L cells, and the inhibition effect on the ATP level of TC-1L cells is more significant.

[0034] Figure 5 High concentration of free 2-DG and cell membrane nanovesicles loaded with lower concentration of 2-DG (PG-NV) can promote the protein degradation of mPD-L1 in engineered TC-1L cells. The ability of PG-NV to degrade mPD-L1 protein is significantly better than that of free 2-DG.

[0035] A: High concentration of free 2-DG can dose-dependently promote the degradation of mPD-L1 protein in engineered TC-1L cells (top panel), and the statistics of mPD-L1 protein expression changes in TC-1L cells are shown in the bottom panel; B: Cell membrane nanovesicles P-NV have no effect on the degradation of mPD-L1 protein in TC-1L cells. However, cell membrane nanovesicles loaded with lower concentration of 2-DG (PG-NV) can dose-dependently promote the degradation of mPD-L1 protein in engineered TC-1L cells (top panel), and the statistics of mPD-L1 protein expression changes in TC-1L cells are shown in the bottom panel; C: Cell membrane nanovesicles loaded with lower concentration of 2-DG (PG-NV) can time-dependently promote the degradation of mPD-L1 protein in engineered TC-1L cells (top panel), and the statistics of mPD-L1 protein expression changes in TC-1L cells are shown in the bottom panel.

[0036] Figure 6 High concentration of free 2-DG and cell membrane nanovesicles loaded with lower concentration of 2-DG (PG-NV) can cause the reduction of mPD-L1 protein anchored on the cell membrane of both control TC1-1C cells and engineered TC-1L cells. The ability of PG-NV to unanchor mPD-L1 protein from the cell membrane is significantly better than that of free 2-DG.

[0037] A: High concentration of free 2-DG or low concentration of 2-DG loaded in cell membrane nanovesicles (PG-NV) can dose-dependently reduce the mPD-L1 protein on the cell membrane of engineered TC-1L cells (left), and the right is the statistical chart of the mean fluorescence intensity of the mPD-L1 expression change on the cell membrane of TC-1L cells; B: The mPD-L1 expression level of the cell membrane of the control group TC1-1C cells is low, but high concentration of free 2-DG or low concentration of 2-DG loaded in cell membrane nanovesicles (PG-NV) can still dose-dependently reduce the mPD-L1 protein on the cell membrane of the control group TC1-1C cells (left), and the right is the statistical chart of the mean fluorescence intensity of the mPD-L1 expression change on the cell membrane of the control group TC1-1C cells;

[0038] Figure 7 The cell membrane nanovesicles loaded with DOX (PD-NV) and 2-DG (PG-NV) can inhibit the growth of mouse lung cancer xenografts to different extents. The cell membrane nanovesicles loaded with DOX and 2-DG (PDG-NV) can significantly inhibit the growth of mouse lung cancer xenografts, with obvious synergistic effect, basically achieving the effect of curing lung cancer xenografts.

[0039] A: The body weight change of mice after receiving different treatments (PBS, P-NV, PG-NV, PD-NV or PDG-NV); B: Evaluation chart of P-NV loaded with different drug treatments on the growth inhibition of mouse subcutaneous xenografts; C: Tumor photos of mouse xenografts after two weeks of P-NV loaded with different drug treatments; D: Statistical chart of tumor weight of mouse xenografts; E: Ki67 staining chart of paraffin sections of mouse tumor tissues after two weeks of P-NV loaded with different drug treatments; F: Statistical chart of Ki67 staining positive cells of paraffin sections of mouse tumor tissues.

[0040] Figure 8 The HE staining (hematoxylin-eosin staining) sections of the heart, liver, spleen, lung and kidney of mice after receiving different treatments (PBS, P-NV, PG-NV, PD-NV or PDG-NV) were detected after two weeks, and there was no obvious abnormality.

[0041] Figure 9 It is the induction and later treatment of EGFR exon 19 deletion transgenic (abbreviated as: CC10-RTTA / EGFR-DEL) mouse lung cancer model.

[0042] A: The principle and process of inducing CC10-RTTA / EGFR-DEL transgenic mice to develop lung cancer in situ by tetracycline; B: The process diagram of the transgenic mice induced to develop lung cancer in situ, and then treated with different treatments (PBS, P-NV, PG-NV, PD-NV or PDG-NV) in the later stage.

[0043] Figure 10 The cell membrane nanovesicle P-NV can slightly inhibit the growth of lung cancer in situ in mice and improve the immunosuppressive microenvironment.

[0044] A: CT effect diagram of P-NV for treatment of lung cancer in CC10-RTTA / EGFR-DEL transgenic mouse model (left); statistical diagram of relative tumor infiltration area in lung tissue of mice before and after treatment (right); B: HE staining (hematoxylin-eosin staining) and Ki67 staining evaluation diagram of lung tissue after two weeks of P-NV treatment (left); statistical diagram of relative tumor area and number of Ki67 staining positive cells of lung tissue after two weeks of P-NV treatment (right); C: Immune fluorescence evaluation diagram of infiltrated CD4 positive (CD4+) and CD8 positive (CD8+) T cells in lung tissue after two weeks of P-NV treatment (left); statistical diagram of number of infiltrated CD4 positive (CD4+) and CD8 positive (CD8+) T cells (right).

[0045] Figure 11 The cell membrane nanovesicle loaded with 2-DG (PG-NV) can inhibit the growth of lung cancer in situ in transgenic mice to a certain extent, and can limitedly enhance the treatment effect of P-NV on lung cancer in situ in mice and improve the immunosuppressive microenvironment.

[0046] A: CT effect diagram of PG-NV for treatment of lung cancer in CC10-RTTA / EGFR-DEL transgenic mouse model (left); statistical diagram of relative tumor infiltration area in lung tissue of mice before and after treatment (right); B: HE staining (hematoxylin-eosin staining) and Ki67 staining evaluation diagram of lung tissue after two weeks of PG-NV treatment (left); statistical diagram of relative tumor area and number of Ki67 staining positive cells of lung tissue after two weeks of PG-NV treatment (right); C: Immune fluorescence evaluation diagram of infiltrated CD4 positive (CD4+) and CD8 positive (CD8+) T cells in lung tissue after two weeks of PG-NV treatment (left); statistical diagram of number of infiltrated CD4 positive (CD4+) and CD8 positive (CD8+) T cells (right).

[0047] Figure 12 Figure 11 is a CT image of a lung cancer primary tumor in a CC10-RTTA / EGFR-DEL transgenic mouse model treated with PD-NV (left) and a graph of the relative tumor area in the lung tissue of the mouse before and after treatment (right).

[0048] Figure 11 is a CT image of a lung cancer primary tumor in a CC10-RTTA / EGFR-DEL transgenic mouse model treated with PD-NV (left) and a graph of the relative tumor area in the lung tissue of the mouse before and after treatment (right).

[0049] Figure 13 Figure 12 is a CT image of a lung cancer primary tumor in a CC10-RTTA / EGFR-DEL transgenic mouse model treated with PDG-NV (left) and a graph of the relative tumor area in the lung tissue of the mouse before and after treatment (right).

[0050] Figure 12 is a CT image of a lung cancer primary tumor in a CC10-RTTA / EGFR-DEL transgenic mouse model treated with PDG-NV (left) and a graph of the relative tumor area in the lung tissue of the mouse before and after treatment (right). DETAILED DESCRIPTION

[0051] The application will be further described in conjunction with the experimental examples and the accompanying drawings, but the embodiments of the application are not limited thereto.

[0052] Example 1

[0053] The application provides a combined drug for treating tumors, which is composed of biomimetic nanovesicles P-NV, 2-DG and DOX in a mass ratio of 10:6.9:5.8.

[0054] Example 2

[0055] The application provides a combined drug for treating tumors, which is composed of biomimetic nanovesicles P-NV, 2-DG and DOX in a mass ratio of 10:6.9:5.8.

[0056] Example 3

[0057] The application provides a combined drug for treating tumors, which is composed of biomimetic nanovesicles P-NV, 2-DG and DOX in a mass ratio of 10:6.9:5.8.

[0058] Example 4

[0059] The application provides a combined drug for treating tumors, which is composed of biomimetic nanovesicles P-NV, 2-DG and DOX in a mass ratio of 10:6.9:5.8.

[0060] The following will be described according to the optimal mass ratio 10:6.9:5.8 of the biomimetic nanovesicles P-NV, 2-DG and DOX used in the application to the specific experimental examples of the application.

[0061] Experimental Example 1

[0062] Evaluation of the influence of cell membrane nanovesicles P-NV on normal cell and lung cancer cell activity

[0063] I. Experimental method

[0064] (A) Detection of the influence of 50 μg / mL P-NV treatment on the growth of 3T3 cells (mouse fibroblasts) and BEAS-2B cells (human lung bronchial epithelial cells). 3T3 or BEAS-2B were seeded at 2 × 10 4Individuals were seeded in 96-well plates, and after about 6 h of culture, the cells can grow adherently. Each well was added with complete DMEM medium (containing 10% FBS, 1% double-antibiotic) containing a final concentration of 50 μg / mL P-NV for incubation. At the time points of 12 h, 24 h and 48 h of incubation, 10 μL CCK8 (Cell Counting Kit) solution was added, and the reaction was allowed to proceed for 1 h. The activity of the cells was detected by a multifunctional enzyme label instrument (TECAN, Spark™ 10 M). (B) Detection of the effect of 100 μg / mL P-NV treatment on the growth of 3T3 cells and BEAS-2B cells. The method was the same as that in Experimental Example 1 Method A, except that the cells were treated with 100 μg / mL P-NV. (C) Detection of the effect of 200 μg / mL P-NV treatment on the growth of 3T3 cells and BEAS-2B cells. The method was the same as that in Experimental Example 1 Method A, except that the cells were treated with 200 μg / mL P-NV. (D) Detection of the effect of 200 μg / mL cell membrane nanovesicle P-NV on lung cancer cells TC1 cells (TC-1C) and engineered lung cancer cells TC-1L cells (TC-1L). The method was the same as that in Experimental Example 1 Method A, except that the TC-1C cells and TC-1L cells were treated with 200 μg / mL P-NV.

[0065] II. Experimental results and analysis

[0066] CCK8 experiments showed that 50, 100, 200 μg / mL cell membrane nanovesicle P-NV had no obvious effect on the growth of 3T3 cells and BEAS-2B cells Figure 1 A&B&C). Meanwhile, 200 μg / mL cell membrane nanovesicle P-NV had no effect on the growth of TC-1C and TC-1L Figure 1 D). The above results show that the cell membrane nanovesicles used for subsequent drug loading in this experiment have no toxicity to the cells themselves and do not affect the viability of the cells.

[0067] Experimental Example 2

[0068] Evaluation of the loading and release efficiency of cell membrane nanovesicle P-NV for doxorubicin hydrochloride (DOX) and 2-deoxyglucose (2-DG)

[0069] I. Experimental methods

[0070] (A) Loading 2-deoxyglucose (2-DG) onto cell membrane nanovesicles P-NV: 2 mL of P-NV aqueous solution with a particle size less than 0.4 μM and a protein concentration of 1 mg / mL prepared in advance was transferred to an Amicon® Ultra centrifugal filter (Millipore, UFC201024), centrifuged at 4°C / 6000 g for about 10 min, and ultrafiltrated to a volume of 400 μL. Then the ultrafiltration tube was fitted with a collection tube and inverted, and centrifuged at 4°C / 4000 g for 3 min to collect 400 μL of ultrafiltration supernatant. 200 μL of 2-DG aqueous solution with a concentration of 2.5 M was added to the 400 μL of P-NV collected by ultrafiltration, mixed gently, and incubated in a 4°C shaking incubator overnight. Then the incubation solution was transferred to an Amicon® Ultra centrifugal filter, centrifuged at 4°C / 6000 g for 15 min, and 530 μL of ultrafiltration supernatant was collected as free 2-DG that was not loaded onto P-NV. Then the ultrafiltration tube was fitted with a collection tube and inverted, and centrifuged at 4°C / 4000 g for 3 min to collect 60 μL of ultrafiltration supernatant as cell membrane nanovesicles P-NV loaded with 2-DG (referred to as PG-NV). Loading doxorubicin hydrochloride (DOX) onto cell membrane nanovesicles P-NV: The experimental method was the same as in Experimental Example 2, Method A, except that 200 μL of 20 mM DOX aqueous solution was directly diluted into 1.8 mL of P-NV aqueous solution with a particle size less than 0.4 μM and a protein concentration of 1 mg / mL prepared in advance, mixed gently, and incubated in a 4°C shaking incubator overnight. Then the incubation solution was centrifuged at 4°C / 4000 g for 60 min, and the supernatant was collected as free DOX that was not loaded onto P-NV. Then the cell membrane nanovesicles P-NV loaded with DOX (referred to as PD-NV) were resuspended in 200 μL of water.

[0071] (B) Measurement of 2-DG release from PG-NV in PBS: 50 μL of PG-NV (prepared by the method in Experimental Example 2, Method A) was resuspended in 8 mL of PBS (pH = 7.3) and mixed thoroughly, and then 7 mL was taken and divided into 7 portions, which were incubated at 37°C on a shaker (180 rpm / min) to detect the release rate of 2-DG from PG-NV in PBS at 0 h, 2 h, 4 h, 8 h, 16 h, and 24 h. At the corresponding time points, the PBS solution containing PG-NV was transferred to an Amicon® Ultra centrifugal filter, which was centrifuged at 4°C / 6000 g for 20 min, and the clear solution after centrifugation was collected for spectral analysis. Spectral analysis was performed using an ion chromatograph (Thermo science, ICS5000+), a DIONEX Carbo Pac PA10 chromatographic column (4 mm x 250 mm), a mobile phase of sodium hydroxide solution (c = 18 mmol / L, flow rate = 0.8 mL / min), and a pulsed amperometric detector to detect the content of 2-DG in the solution at different time points (0 h, 2 h, 4 h, 8 h, 16 h, and 24 h). The peak area was detected by the pulsed amperometric detector, and the peak area from 10 min to 13 min was calculated. Measurement of DOX release from PD-NV in water: the experimental method was the same as Experimental Example 2, Method A, except that 100 μL of PD-NV prepared in Method A was resuspended in 8 mL of deionized water, mixed thoroughly, and then 7 mL was taken and divided into 7 portions, which were incubated at 37°C on a shaker to detect the release rate of DOX from PD-NV in deionized water at 0 h, 2 h, 4 h, 8 h, 16 h, and 24 h. Then, at the specified time points, the incubation solution was centrifuged at 4°C / 14000 g for 60 min, and the supernatant was collected for later detection by a microplate reader (TECAN). DOX was quantitatively analyzed according to the absorbance of DOX at 595 nM.

[0072] II. Experimental results and analysis

[0073] The 2-DG concentration was calculated from the ion chromatography 2-DG peak area detection result, and the DOX concentration was calculated from the microplate reader fluorescence intensity. Through analysis, we found that the loading rate of P-NV for 2-DG ≈ 58% (drug / protein weight ratio) and the loading rate of P-NV for DOX ≈ 69% (drug / protein weight ratio) Figure 2A). The same way to analyze the drug release rate of PG-NV and PD-NV at each time point, found that between 0 h to 8 h, with the growth of time PG-NV will release a large number of 2-DG, then gradually reach the release platform, the release rate of about 85%. While PD-NV in 2 h rapid release of about 21% DOX, then reach the release platform Figure 2 B).

[0074] Experimental Example 3

[0075] Evaluation of the cytotoxicity of different drug-loaded cell membrane nanovesicles on TC-1C and TC-1L cells

[0076] I. Experimental Methods

[0077] (A) Detection of the effect of different concentrations of PD-NV on the cell viability of TC-1C and TC-1L cells: The method is the same as that of Experimental Example 1 Method A, except that PD-NV (protein concentration 0, 1.5625 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5, 25 μg / mL) is used instead of P-NV (protein concentration 50 μg / mL), and the cell viability determination time is set to 24 h.

[0078] (B) Detection of the effect of PG-NV treatment at different time points on the cell viability of TC-1C and TC-1L cells: The method is the same as that of Experimental Example 1 Method A, except that PG-NV (protein concentration 100 μg / mL) is used instead of P-NV (protein concentration 50 μg / mL). The cell viability determination time is set to 1 day, 3 days, and 5 days.

[0079] (C) Detection of the effect of PDG-NV treatment on the cell viability of TC-1C and TC-1L cells: DOX and 2-DG are loaded into P-NV as PDG-NV according to Experimental Example 2 Methods A and B. Then the cell viability determination method is the same as that of Experimental Example 1 Method A, except that PDG-NV (protein concentration 6.25 μg / mL, 12.5 μg / mL) is used instead of P-NV (protein concentration 50 μg / mL). The cell viability determination time is set to 12 h, 24 h, and 48 h.

[0080] II. Experimental Results and Analysis

[0081] Analysis of the cell viability determination results showed that PD-NV could inhibit the cell viability of TC-1C and TC-1L cells in a dose-dependent manner at a lower concentration at 24 h, and the effect on TC-1L cells was more significant Figure 3A). 100 pg / mL of PG-NV could inhibit the cell viability of TC-1C cells and TC-1L cells in a time-dependent manner, and the effect on TC-1L cells was more significant Figure 2 B). Low doses (12.5 pg / mL, 6.25 pg / mL) of PG-NV could effectively inhibit the cell viability of TC-1C cells and TC-1L cells in a short time, and PG-NV had significant dose-dependent and time-dependent effects, and the effect on the cell viability of TC-1L cells was more significant Figure 3 C). In combination with Experimental Example 1, Figure 1 D. 200 pg / mL of P-NV had no effect on the cell viability of TC-1C cells and TC-1L cells. These results show that after the cell membrane nanovesicles are loaded with 2-DG and DOX, they can effectively inhibit the activity of tumor cells TC-1C and TC-1L cells at a lower concentration and in a shorter time, and have a very significant synergistic effect.

[0082] Experimental Example 4

[0083] Compared with high concentrations of free 2DG, low concentrations of PG-NV treatment can inhibit the ATP level generated by glycolysis in target cells, and the ATP level inhibition effect of PG-NV on TC-1L cells is stronger than that on TC-1C cells

[0084] I. Experimental Methods

[0085] (A) Detection of the effect of high concentrations of free 2-DG treatment on the ATP level of TC-1C cells and TC-1L cells: the method is the same as Method A of Experimental Example 1, except that higher concentrations of free 2-DG (0, 1.25 mM, 2.5 mM, 5 mM, 10 mM) are used instead of P-NV (protein concentration 50 pg / mL). After 24 h of treatment, the ATP level generated by the cells is evaluated by an ATP detection kit (promega, G7573).

[0086] (B) Detection of the effect of PG-NV treatment on the ATP level of TC-1C cells and TC-1L cells: the method is the same as Method A of Experimental Example 1, except that PG-NV (protein concentration 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 0) is used instead of P-NV (protein concentration 50 pg / mL). After conversion according to the loading efficiency of 2-DG Figure 2 A), different concentrations of protein correspond to 0.7 mM, 0.35 mM, 0.16 mM, 0.09 mM, 0.04 mM, and 0 of 2-DG, respectively.

[0087] II. Experimental Results and Analysis

[0088] By detecting the ATP levels produced by cells, we found that high concentrations of free 2-DG significantly inhibited the ATP levels produced by TC-1C and TC-1L cells with increasing drug concentration, but had no significant effect on the ATP levels produced by either target cell type. Figure 4 A). Low concentrations of PG-NV also significantly inhibited ATP production in TC-1C and TC-1L cells as vesicle concentration increased, with a more significant effect on ATP levels in TC-1L cells. Figure 4 B). The above results indicate that P-NV loaded with 2-DG (PG-NV) exhibits stronger cytotoxicity against lung cancer cells at lower concentrations and can more significantly inhibit intracellular glycolysis. PG-NV has a significant synergistic effect compared to P-NV and free 2-DG.

[0089] Experimental Example 5

[0090] Compared to high concentrations of free 2-DG, low concentrations of PG-NV can also promote the degradation of mPD-L1 protein in TC-1L cells.

[0091] I. Experimental Methods

[0092] (A) Western blot analysis of the effect of high concentrations of free 2-DG treatment on mPD-L1 degradation in TC-1L cells: TC-1L cells were prepared at 5 x 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 12-well plates and incubated overnight. 2-DG (2.5 mM, 5 mM, 10 mM) was added to the wells, and the cells were treated for 16 h. Cells were then collected by trypsin digestion, washed once with PBS (and centrifuged at 1300 rpm for 3 min, then the supernatant was discarded), and lysed on ice for 30 min with RIPA lysis buffer (Beyotime). 5xSDS containing 5% β-mercaptoethanol was added and mixed thoroughly, followed by boiling at 95°C for 5–10 min. The denatured protein solution was added to a 10% SDS-PAGE gel at a rate of 40 μg of protein per lane for denaturing gel electrophoresis. Immunoblot analysis was then performed using specified antibodies, including Anti-β-Actin and Anti-mPD-L1 (top figure). The bottom figure shows the quantitative analysis of mPD-L1 protein expression relative to β-Actin.

[0093] (B) Western blot analysis of the effect of low concentrations of PG-NV treatment on mPD-L1 degradation in TC-1L cells: The method was the same as in Experiment 5, Method A, except that free 2-DG was replaced with P-NV (protein concentration 100 μg / mL) or PG-NV (protein concentrations 50 μg / mL, 100 μg / mL, 200 μg / mL) (top figure). The bottom figure shows the quantitative analysis of mPD-L1 protein expression relative to β-Actin.

[0094] (C) Western blot analysis of the effect of low concentrations of PG-NV treatment at different time points on mPD-L1 degradation in TC-1L cells: The method was the same as in Experiment 5, Method A, except that TC-1L cells were treated with PG-NV (protein concentration 100 μg / mL) for 1, 2, 3, and 5 days (top figure). The bottom figure shows the quantitative analysis of mPD-L1 protein expression relative to β-Actin.

[0095] II. Experimental Results and Analysis

[0096] Western blot results showed that high concentrations of free 2-DG treatment promoted the degradation of mPD-L1 protein in TC-1L cells, and the higher the concentration of 2-DG treatment, the more significant the change in mPD-L1 protein in TC-1L cells. Figure 5 A). Low-concentration PG-NV treatment also effectively promoted the degradation of mPD-L1 protein in TC-1L cells, and this promotion was significantly dose- and time-dependent. Figure 5 (B&C). The above results indicate that, compared to free 2-DG, PG-NV can degrade mPD-L1 protein in TC-1L cells at a lower concentration, exhibiting a significant synergistic effect.

[0097] Experimental Example 6

[0098] Compared to high concentrations of free 2-DG, low concentrations of PG-NV treatment also promote the degradation of mPD-L1 protein on the cell membranes of TC-1C and TC-1L cells.

[0099] I. Experimental Methods

[0100] (A) Flow cytometry detection of the effects of high concentrations of free 2-DG and low concentrations of PG-NV on mPD-L1 on the cell membrane of TC-1L cells: The method was the same as in Experiment 5, except that TC-1L cells were treated with 2-DG (1.25 mM, 2.5 mM, 5 mM, 10 mM) and PG-NV (protein concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL) for 16 h. The collected cells were washed once with PBS (and the supernatant was discarded after centrifugation at 1300 rpm for 3 min). Then, 100 μL of pre-diluted (1:100) PD-L1 flow cytometry antibody coupled with PE fluorescence (BioLegend) was added to each sample, and the mixture was gently mixed. The cells were incubated on ice in the dark for 20 min. After washing twice with pre-cooled PBS, the cells were resuspended in an appropriate amount of PBS. The proportion of PD-L1 positive cells in TC-1L cells was counted by FACS (left figure); right figure: PD-L1-PE average fluorescence intensity statistics.

[0101] (B) Flow cytometry analysis of the effects of high concentrations of free 2-DG and low concentrations of PG-NV on Mpd-L1 on the cell membrane of TC-1C cells: The method was the same as in Experiment 6, Method A, except that TC-1C cells were used instead of TC-1L cells (left figure); Right figure: Statistical analysis of the average fluorescence intensity of PD-L1-PE.

[0102] II. Experimental Results and Analysis

[0103] Flow cytometry analysis revealed high expression of mPD-L1 protein on the cell membrane of TC-1L cells. Both high concentrations of free 2-DG and low concentrations of PG-NV promoted the degradation of mPD-L1 protein on the cell membrane of TC-1L cells in a dose-dependent manner. Figure 6 A). The expression of mPD-L1 protein on the cell membrane of TC-1C cells is extremely low. Treatment with high concentrations of free 2-DG and low concentrations of PG-NV can also promote the degradation of mPD-L1 protein on the cell membrane of TC-1C cells. Figure 6 B). The above results indicate that, compared to free 2-DG, PG-NV can degrade mPD-L1 protein on the cell membranes of TC-1C and TC-1L at lower concentrations, exhibiting a significant synergistic effect.

[0104] Experimental Example 7

[0105] To evaluate the effects of cell membrane nanovesicles (P-NV) and P-NV loaded with different drugs (PG-NV, PD-NV, PDG-NV) on the growth of xenografts in mouse lung cancer and to conduct related pathological analysis.

[0106] I. Experimental Methods

[0107] (A) Mice transplanted tumor model: The prepared mouse lung cancer cells TC-1L were resuspended with a mixture of PBS and Matrigel matrix glue (CORNING) at a ratio of 1:1 (containing 8 x 10 5 cells per 100 μL of mixed solution), and placed on ice for standby. The mixture containing tumor cells was inoculated into the flank of 6-week-old C57BL / 6 mice, and 100 μL of tumor cell mixture was injected at each inoculation site. When the tumor volume reached about 80-90 mm3, the mice were randomly divided into PBS group (control), P-NV treatment group, PG-NV treatment group, PD-NV treatment group and PDG-NV treatment group, and the five groups of mice were given drugs synchronously (each time 1 mg / kg dose was injected into the tail vein, and the drug was given every two days) and the body weight of the mice was measured.

[0108] (B) After the tumor volume of the mice reached about 80-90 mm3, the size of the tumor was measured on day 0, 4, 8, 12, and 16, respectively. The specific method was as follows: the maximum diameter (denoted as D) and the minimum diameter (denoted as d) of the mouse tumor were measured using a vernier caliper, and the tumor volume (denoted as V) of the mouse was calculated using the formula: V (cm 3 ) = D x d 2 / 2. The calculated tumor volume was plotted using software GraphPad Prism, and the growth curve of the mouse tumor was drawn.

[0109] (C) After the treatment of the mice was completed, the mice were euthanized when the treatment endpoint was reached, the tumor was dissected and photographed, and the weight of the tumor was measured and statistically analyzed.

[0110] (D) Paraffin sections of mouse lung tissue were stained for Ki67: For the mice in Experimental Example 7A, at the experimental endpoint, the mice were euthanized and then dissected to remove the lung tissue from the mice. (1) The lung tissue was submerged in 10% formalin fixative and placed in a shaker room for 24 h at room temperature. After that, the lung lobes were removed and placed in an immunohistochemistry cassette and sequentially submerged in 70% ethanol, 80% ethanol, and 90% ethanol for 30 minutes each, then sequentially submerged in 1: 1 mixture of anhydrous ethanol and xylene, xylene I, xylene II for 15 minutes each, and then embedded with a paraffin embedding machine. The embedded paraffin block was sectioned using a tissue microtome (3-5 microns thick) and the tissue sections were stretched on a warm water surface, gently attached to a glass slide, and finally dried for use. (2) Refer to the Ki-67 antibody (immunohistochemical method) instruction manual. (a) The pre-configured antigen repair solution (1: 50) was placed in the full-automatic immunohistochemical pretreatment system (PT Link) and heated to 65°C, and the paraffin section was soaked in the antigen repair solution. (c) Adjust the instrument temperature to 95°C and maintain for 20 minutes, and then slowly reduce the temperature to 65°C. (d) Remove the section and soak it in elution buffer for 5 minutes, and then fix it to the tissue staining machine. (e) Treat with peroxidase blocker for 5 minutes, then elute for 1 minute, treat with primary antibody for 20 minutes, elute for 1 minute, treat with HRP-conjugated secondary antibody for 20 minutes, elute for 1 minute. (f) DAB color development for 10 minutes, ultrapure water rinse for 1 minute. (g) hematoxylin restain for 5 minutes, ultrapure water rinse for 1 minute, and then sequentially place in 70% ethanol for 2 minutes, 80% ethanol for 2 minutes, 90% ethanol for 2 minutes, anhydrous ethanol for 2 minutes, and then xylene for 5 minutes, xylene for 5 minutes. (h) Finally, the paraffin section was sealed with neutral resin, air-dried, and observed and photographed under a microscope.

[0111] II. Experimental results and analysis

[0112] The above results show that cell membrane nanovesicles loaded with DOX (PD-GV), 2-DG (PG-NV), or DOX and 2-DG (PDG-NV) have no effect on the body weight of tumor-bearing mice, and have low toxicity ( Figure 7 A) After the mice received different treatments (PBS, P-NV, PG-NV, PD-NV, or PDG-NV), cell membrane nanovesicles loaded with DOX (PD-GV) and 2-DG (PG-NV) can inhibit the growth of lung cancer transplanted tumors in mice to varying degrees. Cell membrane nanovesicles loaded with DOX and 2-DG (PDG-NV) can significantly inhibit the growth of lung cancer transplanted tumors in mice ( Figure 7B&C&D). Two weeks after mice received different treatments (PBS, P-NV, PG-NV, PD-NV or PDG-NV), the Ki67 staining of tumor sections of mice ( Figure 7 E&F) showed that the treatment of PDG-NV could significantly inhibit the proliferation of tumor cells in the transplanted tumor of mice, compared with P-NV, PG-NV and PD-NV. The above results collectively indicated that the cell membrane nanovesicles simultaneously loaded with DOX and 2-DG (PDG-NV) could significantly inhibit the growth of lung cancer transplanted tumor in mice, had obvious synergistic effect, and basically achieved the effect of curing lung cancer transplanted tumor.

[0113] Experimental Example 8

[0114] The effects of cell membrane nanovesicles P-NV and P-NV loaded with different drugs (PG-NV, PD-NV, PDG-NV) on the physiological state of mice were evaluated.

[0115] I. Experimental Methods

[0116] For the mice in Experimental Example 7A, at the experimental endpoint, the mice were euthanized, and the mice were dissected to remove the heart, liver, spleen, lung, and kidney tissues of the mice. According to the method E of Experimental Example 7, except that H&E staining was used instead of Ki67 staining. The specific method of H&E is as follows: The paraffin sections of each tissue were pre-incubated at 60°C for half an hour, and then the hematoxylin-eosin (H&E) staining kit (Mesgen, ME9200) was used according to the instructions. (a) Paraffin section deparaffinization and hydration: deparaffinization in xylene for 5 minutes, deparaffinization in xylene for 5 minutes, anhydrous ethanol immersion for 5 minutes, 95% ethanol immersion for 2 minutes, 80% ethanol immersion for 2 minutes, 70% ethanol immersion for 2 minutes, and distilled water immersion for 2 minutes. (b) Staining of paraffin sections: hematoxylin staining for 8 minutes, ultrapure water washing for 1 minute, ultrapure water immersion for 15 minutes, followed by eosin staining for 2 minutes, and ultrapure water washing for 1 minute. (d) Dehydration, transparency and mounting of paraffin sections: the tissue sections were sequentially placed in 95% ethanol for 1 minute, 95% ethanol for 1 minute, anhydrous ethanol for 1 minute, xylene-carbonic acid mixture (3:1) for 1 minute, xylene for 1 minute, and xylene for 1 minute. Finally, the paraffin sections were mounted with neutral balsam, air-dried, and observed and photographed under a microscope.

[0117] II. Experimental Results and Analysis

[0118] After the mice received different treatments (PBS, P-NV, PG-NV, PD-NV or PDG-NV), the H&E staining of the heart, liver, spleen, lung and kidney sections of the mice was detected two weeks later ( Figure 8). The above results show that the cell membrane nanovesicle P-NV and P-NV loaded with different drugs (PG-NV, PD-NV, PDG-NV) do not have obvious damage to the tissues of mice, and basically do not affect the life and physiological state of mice.

[0119] Experimental Example 9

[0120] The induction and later treatment flow chart of EGFR exon 19 deletion transgene (abbreviation: CC10-RTTA / EGFR-DEL) mouse lung cancer model.

[0121] I. Experimental method

[0122] (A) This method uses a lung cancer orthotumor model of CC10-RTTA-EGFR cancer driver gene mutation transgenic mice to evaluate the therapeutic effect of P-NV loaded with different drugs on mouse orthotumor lung cancer. CC10 protein is a protein specifically expressed in the lung, and the Tet-on system is a relatively mature eukaryotic foreign gene induction expression system. RTTA protein can bind to a specific TRE sequence with the help of doxycycline (Dox), thereby realizing the transcriptional expression regulation of the target gene EGFR connected by the TRE element. In short: CC10-RTTA / EGFR-DEL mice can form orthotumor lung cancer tumors in the lungs after being fed with Dox for about 12 weeks, thereby providing a mouse model for later treatment.

[0123] (B) Use the mouse model induced in method A of Experimental Example 9 to treat the mice. Before treatment, perform computer tomography (pingseng HEALTHCARE) to record the tumor burden. When the mouse has formed a tumor in the lung, treatment can be performed. Randomly divide the lung cancer mice into a PBS group (Control) and a P-NV treatment group, a PG-NV treatment group, a PD-NV treatment group, and a PDG-NV treatment group, and simultaneously perform a two-week drug treatment (nanovesicles are injected into the tail vein at a dose of 1 mg / kg each time, and the drug is administered every two days). During the treatment period, record the tumor burden at 7 days and 14 days by computer tomography (Computer Tomography, CT).

[0124] Experimental Example 10

[0125] Evaluate the therapeutic effect of P-NV on the lung cancer orthotumor of CC10-RTTA / EGFR-DEL transgenic mice.

[0126] I. Experimental method

[0127] (A) P-NV treatment effect evaluation on CC10-RTTA / EGFR-DEL transgenic mouse lung cancer orthotumor. Using the mouse model in Experimental Example 9 Method A, the CC10-RTTA / EGFR-DEL transgenic lung cancer mice were treated according to the treatment method in Experimental Example 9 Method B, and CT scans were performed on the 7th day and 14th day of treatment to evaluate the tumor-bearing condition of the mice (left). The right is the statistics of the lung cancer shadow part of the mouse lung CT picture.

[0128] (B) P-NV treatment of CC10-RTTA / EGFR-DEL transgenic mice, lung H&E staining analysis and Ki67 positive cell analysis. According to the method of Experimental Example 7 Method E and Experimental Example 8, the lung tissue of the mouse was stained (left). The difference is that the primary lung cancer model of CC10-RTTA / EGFR-DE transgenic mice replaces the lung cancer xenograft model of TC-1L. The right is the statistics and analysis of the microphotograph results of the tissue sections.

[0129] (C) P-NV treatment of CC10-RTTA / EGFR-DEL transgenic mice, lung tissue infiltration of CD4 positive (CD4+) and CD8 positive (CD8+) T cells immunofluorescence evaluation. Using the mouse model in Experimental Example 9 Method A, according to the treatment method in Experimental Example 9 Method B, after 14 days of treatment, the mouse was immunofluorescence stained according to Experimental Example 7 Method E (left). The difference is that the donkey anti-goat-FITC secondary antibody (recognizes CD8 positive T cells), the donkey anti-goat-CY3 secondary antibody (recognizes CD4 positive T cells) is used instead of HRP secondary antibody staining, DAPI is used instead of Ki67 staining, and fluorescence microscope is used instead of ordinary bright field photography. The right is the number statistics of lung tissue infiltration of CD4 positive (CD4+) and CD8 positive (CD8+) T cells.

[0130] II. Experimental results and analysis

[0131] After two weeks of treatment, CT scans of the lungs of mice in different treatment groups found that compared with the Control group mice (tumor area increased by about 25% compared with before treatment), the P-NV treatment group mice had no obvious growth of lung tumors, and the area was slightly reduced, and the lung cancer mouse tumor area was reduced by less than 10% compared with before treatment Figure 10 A). HE staining of pathological sections, Ki67 staining showed slight weakening relative to before treatment Figure 10 B); lung infiltration of CD4 T and CD8 T cells was slightly elevated Figure 10C). So the treatment effect of orthotopic tumor model showed that PG-NV did not significantly regulate the tumor microenvironment of mice, although it could inhibit the growth of lung cancer in mice, but the tumor regression was not obvious.

[0132] Experimental Example 11

[0133] The treatment effect of PG-NV on lung cancer orthotopic tumor of CC10-RTTA / EGFR-DEL transgenic mice was evaluated.

[0134] I. Experimental Methods

[0135] (A) Evaluation of the treatment effect of PG-NV on lung cancer orthotopic tumor of CC10-RTTA / EGFR-DEL transgenic mice. The experimental method referred to the method A of Experimental Example 10, except that PG-NV was used instead of P-NV for treatment.

[0136] (B) Lung H&E staining analysis and Ki67 positive cell analysis of CC10-RTTA / EGFR-DEL transgenic mice after treatment with PG-NV. The experimental method referred to the method B of Experimental Example 10, except that PG-NV was used instead of P-NV for treatment.

[0137] (C) Evaluation of lung tissue infiltration of CD4 positive (CD4+) and CD8 positive (CD8+) T cells of CC10-RTTA / EGFR-DEL transgenic mice after treatment with PG-NV. The experimental method referred to the method C of Experimental Example 10, except that PG-NV was used instead of P-NV for treatment.

[0138] II. Experimental Results and Analysis

[0139] After two weeks of treatment, the lung tumor of mice in different treatment groups was found by CT scan. Compared with the Control group of mice (the tumor area increased by about 25% compared with before treatment), the lung tumor of mice in the PG-NV treatment group was inhibited to a certain extent, and the area was slightly reduced (the lung cancer mouse tumor area decreased by about 30% compared with before treatment) Figure 11 A). And the results were consistent with the CT scan from the HE staining photos and Ki67 photos of pathological sections Figure 11 B). Lung infiltration of CD4 T and CD8 T cells was slightly improved Figure 11 C). So the treatment effect of orthotopic tumor model showed that PG-NV did not significantly regulate the tumor microenvironment of mice, although it could inhibit the growth of lung cancer in mice, but the tumor regression was not obvious.

[0140] Experimental Example 12

[0141] To evaluate the therapeutic effect of PD-NV on lung cancer orthotopic tumor in CC10-RTTA / EGFR-DEL transgenic mice.

[0142] I. Experimental Methods

[0143] (A) Evaluation of the therapeutic effect of PD-NV on lung cancer orthotopic tumor in CC10-RTTA / EGFR-DEL transgenic mice. The experimental method refers to the method A of Experimental Example 10, except that PD-NV is used instead of P-NV for treatment.

[0144] (B) Analysis of lung H&E staining and Ki67 positive cells after treatment of PD-NV in CC10-RTTA / EGFR-DEL transgenic mice. The experimental method refers to the method B of Experimental Example 10, except that PD-NV is used instead of P-NV for treatment.

[0145] (C) Evaluation of lung tissue infiltration of CD4 positive (CD4+) and CD8 positive (CD8+) T cells by immunofluorescence after treatment of PD-NV in CC10-RTTA / EGFR-DEL transgenic mice. The experimental method refers to the method C of Experimental Example 10, except that PD-NV is used instead of P-NV for treatment.

[0146] II. Experimental Results and Analysis

[0147] After two weeks of treatment, CT scans of the lungs of mice in different treatment groups showed that compared with the Control group mice (tumor area increased by about 25% compared with before treatment), the lung tumor of the PD-NV treatment group mice was inhibited to a certain extent, and the area was slightly reduced (lung cancer mouse tumor area decreased by about 45% compared with before treatment) Figure 12 A). And from the HE staining photos and Ki67 photos of the pathological sections, the results consistent with the CT scan were obtained Figure 12 B). Lung infiltration of CD4 T and CD8 T cells was slightly increased Figure 12 C). Lung infiltration of CD4 T and CD8 T cells was significantly increased Figure 11 C). Therefore, the treatment effect of the orthotopic tumor model shows that PD-NV improves the tumor microenvironment of mice to a certain extent and has a certain therapeutic effect on lung cancer in CC10-RTTA / EGFR-DEL transgenic mice.

[0148] Experimental Example 13

[0149] To evaluate the therapeutic effect of PDG-NV on lung cancer orthotopic tumor in CC10-RTTA / EGFR-DEL transgenic mice.

[0150] I. Experimental Methods

[0151] (A) Evaluation of the therapeutic effect of PDG-NV on lung cancer orthotopic tumor in CC10-RTTA / EGFR-DEL transgenic mice. The experimental method refers to the method A of experimental example 10, except that PDG-NV is used instead of P-NV for treatment.

[0152] (B) Analysis of lung H&E staining and Ki67 positive cells after treatment of PDG-NV on CC10-RTTA / EGFR-DEL transgenic mice. The experimental method refers to the method B of experimental example 10, except that PDG-NV is used instead of P-NV for treatment.

[0153] (C) Evaluation of lung tissue infiltration of CD4 positive (CD4+) and CD8 positive (CD8+) T cells by immunofluorescence after treatment of PDG-NV on CC10-RTTA / EGFR-DEL transgenic mice. The experimental method refers to the method C of experimental example 10, except that PDG-NV is used instead of P-NV for treatment.

[0154] II. Experimental results and analysis

[0155] After two weeks of treatment, the lung tumor of mice in different treatment groups was found by CT scan. Compared with the control group of mice (the tumor area increased by about 25% compared with before treatment), the lung tumor area of PDG-NVG treated mice was slightly reduced (the lung cancer mouse tumor area was reduced by about 70% compared with before treatment) Figure 13 A). HE staining of pathological sections showed that the tumor area of mice was reduced by about 90% compared with the control group; and Ki67 staining showed that the tumor growth activity was also reduced by about 85% Figure 13 B). The proportion of lung infiltrating CD4 T and CD8 T cells was also significantly improved Figure 13 C). Therefore, the treatment effect of the orthotopic tumor model showed that P-NV loaded with DOX and 2-DG (PDG-NV) could greatly improve the tumor immune microenvironment of lung tissue in mice, and had more significant therapeutic effect on EGFR-DEL transgenic mouse lung cancer, basically achieving the purpose of cure. The more significant anti-tumor effect achieved by PDG-NV indicates the synergistic therapeutic effect of P-NV, 2-DG and DOX on tumor tissue.

Claims

1. A combination for use in the treatment of a tumor, characterized in that, The biomimetic nanovesicle is loaded with chemotherapeutic drug doxorubicin hydrochloride DOX and sugar metabolism inhibitor 2-deoxy-D-glucose. The biomimetic nanovesicle is constructed by using mouse lung cancer cells TC1 to construct high expression mPD1 engineering cells, and further ultrasonic co-extrusion to generate natural cell membrane nanovesicle; and the biomimetic nanovesicle is an activated biomimetic nanovesicle.

2. The combination for use in the treatment of a tumor according to claim 1, wherein, The biomimetic nanovesicle, doxorubicin hydrochloride DOX and sugar metabolism inhibitor 2-deoxy-D-glucose are composed according to a mass ratio of 10:6.9:5.

8.

3. The combination of claim 1 for treating a tumor, wherein The biomimetic nanovesicle, doxorubicin hydrochloride DOX and sugar metabolism inhibitor 2-deoxy-D-glucose are composed according to a mass ratio of 10:5:

4.

4. The combination of claim 1 for treating a tumor, wherein The biomimetic nanovesicle, doxorubicin hydrochloride DOX and sugar metabolism inhibitor 2-deoxy-D-glucose are composed according to a mass ratio of 10:4:

3.

5. The combination of claim 1 for treating a tumor, wherein The biomimetic nanovesicle, doxorubicin hydrochloride DOX and sugar metabolism inhibitor 2-deoxy-D-glucose are composed according to a mass ratio of 10:3:

2.

6. The combination of claim 1 for treating a tumor, wherein The administration modes of the biomimetic nanovesicle P-NV, 2-deoxy-D-glucose and doxorubicin hydrochloride DOX are all tail vein injection.

7. The combination of claim 1 for treating a tumor, wherein The lung cancer includes carcinoma in situ and metastatic cancer.

Images