Engineered cell membrane nanovesticle, preparation method therefor and use thereof

Abstract

An engineered cell membrane nanovesicle and a preparation method therefor and use thereof. A use of normal fibroblasts in the preparation of a drug or tumor diagnostic reagent or nano-drug delivery system or engineered vesicle for treating tumors. The vesicle prepared using the normal fibroblasts has high specificity and the ability to quickly target various types of tumor tissues, and can achieve tumor targeted diagnostic imaging or drug delivery; also, the accumulation in normal organs and non-targeted tissues is greatly reduced, the toxic side effects in vivo are minimized, and safety and effectiveness are greatly improved.

Description

Engineered cell membrane nanovesicles, preparation method and application thereof TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biological medicine, and relates to an engineered cell membrane nanovesicle, a preparation method and application thereof. BACKGROUND

[0002] Nanometer targeted drug delivery systems show obvious advantages in the field of treating tumor-related diseases: 1) Nanometer drugs can be passively targeted to tumor sites through the EPR effect (Enhanced permeability and retention effect, EPR). 2) The surface of nanometer drugs can be modified with targeting molecules or linked with drug molecules through environmentally sensitive bonds, achieving active targeting distribution of drugs and environmentally responsive release. Although nanoparticles can extend blood circulation time and improve targeting by surface modification, they still have many shortcomings as diagnostic media and drug delivery systems: first, most nanoparticles are artificial synthetic inorganic or organic substances, which have certain toxicity and are easily recognized by the immune system as foreign substances and removed, resulting in less accumulation at the tumor site; second, the surface modification technology required for nanoparticles to achieve targeting function is relatively complex and difficult to implement. In recent years, researchers have become increasingly interested in using natural biomimetic materials to prepare nanometer drug delivery systems. In complex biological systems, various cell lines exhibit "homing" targeting to specific tissues. For example, macrophages, as one of many immune cells, often actively target wound or inflammatory sites; bleeding wound sites can actively recruit platelets; in animal models of myocardial infarction, progenitor cells and transplanted stem cells have been shown to have myocardial homing effects; in addition, tumor cells tend to cluster together and form new tumor lesions near vascular epithelium, and this tendency of tumor cells to homologous tumor cells is called "homologous targeting".

[0003] Membrane nanovesicles (MNVs) are formed by coating the outer layer of synthetic nanoparticles (NPs) with cell membranes from natural sources. The advantage of MNVs is that they can completely replicate the surface antigens of the source cells, retaining the unique properties of natural cell membranes, including their ability to extend the circulation time, evade immunity, adhere, and target homologous targets, which greatly potentialize the new applications of cell membrane biomimetic technology in the field of nanomedicine. The types of cell membranes used in this technology include red blood cell membranes, white blood cell membranes, cancer cell membranes, stem cell membranes, and hybrid membranes, among others. This system has been shown to achieve effective drug delivery, tumor treatment, and immune regulation, among others. Among them, the high expression of specific proteins such as adhesion proteins, integrins, and adhesion kinases on the surface of cancer cell membranes (CCM) allows for strong adhesion between homologous tumor cells, enabling cell self-recognition. In-depth exploration of the key mechanisms of tumor cell membrane nanovesicles targeting homologous tumors will facilitate the design of efficient and precise engineered cell membrane delivery systems, improve their efficiency in targeting primary tumors and metastases, and provide new methods for clinical translation, which is of great significance for personalized diagnosis and treatment of future clinical cancer patients.

[0004] With the in-depth study of cancer cell (CC)-derived biomimetic membrane nanovesicles, their obvious targeting advantage and delivery capacity provide a solid foundation for their application in individualized precision medicine methods. However, there are still many challenges before their clinical translation. On the one hand, CC-MNVs coated with membranes from patients' own tumor cells still retain a large number of cancer-related proteins, only a few of which are closely related to targeting, while most of the other membrane proteins can cause adverse immune reactions. On the other hand, obtaining tumor tissue from patients through invasive procedures such as surgery to harvest primary tumor cells for extracting tumor cell membranes not only increases the risk of infection for patients but also may cause the dissemination and even metastasis of in situ tumor cells. More importantly, during the entire treatment cycle, the number of primary tumor cells from clinical cancer patients is insufficient to produce enough tumor cell membrane vesicles. Therefore, new methods are needed to alleviate these problems. SUMMARY

[0005] In some embodiments, the present disclosure provides the use of normal fibroblasts in the preparation of a medicament for treating a tumor or a tumor diagnostic reagent or a nano-drug delivery system or an engineered vesicle.

[0006] In some embodiments, the normal fibroblasts are selected from autologous normal fibroblasts.

[0007] In some embodiments, the normal fibroblasts are selected from fibroblast cell lines or strains.

[0008] In some embodiments, the normal fibroblast is selected from an established fibroblast cell line or strain.

[0009] In some embodiments, the normal fibroblast is selected from a fibroblast of skin or peritumoral tissue.

[0010] In some embodiments, the present disclosure provides a method for preparing an engineered vesicle, comprising: (1) overexpressing a gene in a normal fibroblast to obtain a gene-overexpressing fibroblast; (2) extracting a membrane of the fibroblast; (3) mixing the membrane of the fibroblast with a lipid nanoparticle to obtain the engineered vesicle.

[0011] In some embodiments, the gene-overexpressing fibroblast overexpresses an adhesion protein or a chemokine receptor.

[0012] In some embodiments, the gene-overexpressing fibroblast further overexpresses an immunosuppressive molecule or a cell-penetrating peptide.

[0013] In some embodiments, the gene comprises CD47 or CXCR4.

[0014] In some embodiments, the gene further comprises PD-1 or iRGD.

[0015] In some embodiments, the gene is PD-1, CD47 and iRGD.

[0016] In some embodiments, the gene is CXCR4, PD-1, CD47 and iRGD.

[0017] In some embodiments, the normal fibroblast is selected from an autologous normal fibroblast.

[0018] In some embodiments, the normal fibroblast is selected from a fibroblast cell line or strain.

[0019] In some embodiments, the normal fibroblast is selected from an established fibroblast cell line or strain.

[0020] In some embodiments, the normal fibroblast is selected from a fibroblast of skin or peritumoral tissue. In some embodiments, in step (2), the membrane solution of the fibroblast is obtained by sequentially using freeze-thaw method, gradient centrifugation and resuspension.

[0021] In some embodiments, in step (3), step (3.1) is included: the membrane of the fibroblast is formulated into a solution before mixing with the lipid nanoparticle to obtain a mixed solution of the membrane of the fibroblast and the lipid nanoparticle.

[0022] In some embodiments, in step (3), the step (3.2) is included: extruding and filtering the mixed solution of the fibroblast cell membrane and the lipid nanoparticle using a liposome extruder to obtain the engineered vesicle.

[0023] In some embodiments, in step (3.1), the concentration of the fibroblast cell membrane is 0.1-2 mg / mL.

[0024] In some embodiments, the fibroblast cell membrane solution is filtered through 300-500 nm and 100-350 nm polycarbonate filters in sequence before being mixed with the lipid nanoparticle.

[0025] In some embodiments, in step (3.2), the filtering of the mixed solution of the fibroblast cell membrane and the lipid nanoparticle is performed using 50-250 nm polycarbonate filters in sequence.

[0026] In some embodiments, in step (3.1), the mass ratio of the fibroblast cell membrane solution to the lipid nanoparticle is (0.2-3):1.

[0027] In some embodiments, in step (3.1), the mass ratio of the fibroblast cell membrane solution to the lipid nanoparticle is (0.5-3):1.

[0028] In some embodiments, in step (3.1), the mass ratio of the fibroblast cell membrane solution to the lipid nanoparticle is (0.5-2):1.

[0029] In some embodiments, the lipid nanoparticle is obtained by a solvent diffusion method.

[0030] In some embodiments, in step (3), the method for obtaining the lipid nanoparticle includes the steps of: (S1) mixing and reacting IR-780 iodide and NH2-PEG-DSPE to obtain a primary product IR780-PEG-DSPE; (S2) mixing IR780-PEG-DSPE, an anti-tumor drug, DPPC, and cholesterol into a solution and dispersing the solution into poloxamer 188 to prepare the lipid nanoparticle.

[0031] In some embodiments, the method for obtaining the lipid nanoparticle comprises the steps of: (A1) weighing IR-780 iodide and NH2-PEG-DSPE, dissolving in dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; (A2) adding triethylamine to the dichloromethane solution containing IR-780 iodide to react to obtain a reaction solution; (A3) adding the reaction solution to diethyl ether, centrifuging to remove by-products to obtain a precipitate, and drying the precipitate to obtain a primary product IR780-PEG-DSPE; (A4) dissolving an antitumor drug, IR780-PEG-DSPE, DPPC and cholesterol in ethanol, heating to obtain an ethanol phase solution; (A5) mixing the ethanol phase solution of step (A4) with a poloxamer 188 aqueous solution to react to prepare the lipid nanoparticle.

[0032] In some embodiments, in the ethanol phase solution of step (A4), the weight fractions of the antitumor drug, IR780-PEG-DSPE, DPPC and cholesterol are 5-30 parts of the antitumor drug, 10-30 parts of IR780-PEG-DSPE, 40-70 parts of DPPC and 1-15 parts of cholesterol.

[0033] In some embodiments, in the ethanol phase solution of step (A4), the weight fractions of the antitumor drug, IR780-PEG-DSPE, DPPC and cholesterol are 5-25 parts of the antitumor drug, 10-25 parts of IR780-PEG-DSPE, 45-70 parts of DPPC and 1-10 parts of cholesterol.

[0034] In some embodiments, in the ethanol phase solution of step (A4), the weight fractions of the antitumor drug, IR780-PEG-DSPE, DPPC and cholesterol are 5-20 parts of the antitumor drug, 10-20 parts of IR780-PEG-DSPE, 50-70 parts of DPPC and 3-6 parts of cholesterol.

[0035] In some embodiments, in the ethanol phase solution of step (A4), the mass percentages of the antitumor drug, IR780-PEG-DSPE, DPPC and cholesterol are 5-20% of the antitumor drug, 10-20% of IR780-PEG-DSPE, 50-70% of DPPC and 3-15% of cholesterol.

[0036] In some embodiments, in step (A5), the volume ratio of the poloxamer 188 aqueous solution to the ethanol phase solution is (5-20):1.

[0037] In some embodiments, in step (A5), the volume ratio of the poloxamer 188 aqueous solution to the ethanol phase solution is (8-15):1.

[0038] In some embodiments, in step (A5), the concentration of poloxamer 188 in the aqueous poloxamer 188 solution is 0.1-3 mg / mL.

[0039] In some embodiments, in step (A5), the concentration of poloxamer 188 in the aqueous poloxamer 188 solution is 0.5-1.5 mg / mL.

[0040] In some embodiments, the method for obtaining IR780-PEG-DSPE comprises the steps of: (B1) weighing IR-780 iodide and NH2-PEG-DSPE, dissolving them in dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; (B2) weighing triethylamine according to a molar ratio of triethylamine to IR-780 iodide of (0.5-5): 1, and adding it to the dichloromethane solution containing IR-780 iodide in step (B1), and stirring for 8-30 h; (B3) after the reaction is completed, the reaction solution is added to 8-12 times the volume of anhydrous ether, reacted, centrifuged, and the byproduct is removed, and then the precipitate is dried to obtain IR780-PEG-DSPE.

[0041] In some embodiments, in step (B2), the molar ratio of triethylamine to IR-780 iodide is (1-3): 1.

[0042] In some embodiments, the anti-tumor drug comprises RSL3 or doxorubicin.

[0043] In some embodiments, the present disclosure provides an engineered vesicle obtained by the method.

[0044] The present disclosure utilizes this diversified, safe and broad-spectrum cell membrane targeted delivery system platform MNVs, and has made a breakthrough in the establishment of the basic theory and scientific and technological system of applying biomimetic nanotechnology to diagnose and intervene metastatic tumors, thereby providing scientific basis and effective tools for the wide application of precise diagnosis and treatment of metastatic tumors.

[0045] In some embodiments, the present disclosure provides a biological membrane comprising a fibroblast membrane overexpressing a gene, wherein the gene comprises CD47 or CXCR4.

[0046] In some embodiments, the gene further comprises PD-1 or iRGD.

[0047] In some embodiments, the biological membrane is a fibroblast membrane overexpressing genes PD-1, CD47 and iRGD. In some embodiments, the biological membrane is a fibroblast membrane overexpressing genes CXCR4, PD-1, CD47 and iRGD.

[0048] In some embodiments, the fibroblast is selected from a normal fibroblast.

[0049] In some embodiments, the fibroblast is selected from a normal fibroblast of autologous origin.

[0050] In some embodiments, the normal fibroblast is selected from a fibroblast cell line or strain.

[0051] In some embodiments, the normal fibroblast is selected from an established fibroblast cell line or strain.

[0052] In some embodiments, the normal fibroblast is selected from a fibroblast of skin or para-cancerous tissue. In some embodiments, the present disclosure provides a method for preparing the biological membrane, overexpressing a gene in a fibroblast, and extracting the fibroblast; the gene includes CD47 or CXCR4.

[0053] In some embodiments, the gene further includes PD-1 or iRGD.

[0054] In some embodiments, the biological membrane is a fibroblast membrane overexpressing genes PD-1, CD47 and iRGD.

[0055] In some embodiments, the biological membrane is a fibroblast membrane overexpressing genes CXCR4, PD-1, CD47 and iRGD.

[0056] In some embodiments, the fibroblast is selected from a normal fibroblast.

[0057] In some embodiments, the fibroblast is selected from a normal fibroblast of autologous origin.

[0058] In some embodiments, the normal fibroblast is selected from a fibroblast cell line or strain.

[0059] In some embodiments, the normal fibroblast is selected from an established fibroblast cell line or strain.

[0060] In some embodiments, the normal fibroblast is selected from a fibroblast of skin or para-cancerous tissue.

[0061] In some embodiments, the present disclosure provides a vesicle, the raw material for preparing the vesicle including the biological membrane.

[0062] The vesicle containing the biological membrane has high specificity and the ability to rapidly target multiple types of tumor tissues, and can achieve tumor-targeted diagnostic imaging or drug delivery, while greatly reducing accumulation in normal organs and non-target tissues, minimizing its toxic side effects in the body, and greatly improving safety.

[0063] In some embodiments, the vesicle is a genetically engineered cell membrane nanovesicle.

[0064] The present disclosure provides a method for alleviating the problems existing in the prior art, which is screening a key target protein closely related to targeting according to tumor cell membrane homologous targeting recruitment adhesion mechanism, modifying the key target receptor protein to the surface of fibroblast cell membrane by genetic engineering method, preparing a new safe MNVs for tumor targeting.

[0065] In some embodiments, the preparation raw material of the vesicle further comprises a thermosensitive liposome drug delivery system.

[0066] In some embodiments, the preparation raw material of the thermosensitive liposome drug delivery system comprises IR-780 iodide, NH2-PEG-DSPE, triethylamine, an anti-tumor drug, and DPPC.

[0067] In some embodiments, the anti-tumor drug comprises RSL3 or doxorubicin.

[0068] In some embodiments, the present disclosure provides an engineered vesicle obtained by the method.

[0069] In some embodiments, the present disclosure provides a composition comprising the biological membrane or the engineered vesicle or the biological membrane or the vesicle, and a tumor therapeutic agent.

[0070] In some embodiments, the tumor therapeutic agent comprises an anti-tumor drug.

[0071] In some embodiments, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer, or nasopharyngeal cancer.

[0072] In some embodiments, the anti-tumor drug comprises RSL3 or doxorubicin.

[0073] In some embodiments, the present disclosure provides use of the engineered vesicle or the biological membrane or the vesicle or the composition in the preparation of an anti-tumor drug in combination with a tumor photothermal therapeutic agent.

[0074] In some embodiments, the present disclosure provides use of a tumor photothermal therapeutic agent in the preparation of an anti-tumor drug in combination with the engineered vesicle or the biological membrane or the vesicle or the composition.

[0075] In some embodiments, the present disclosure provides use of the vesicle or engineered vesicle or the composition in the preparation of a laser therapy combination drug.

[0076] In some embodiments, the present disclosure provides use of the engineered fibroblast cell membrane nanovesicle in tumor diagnosis imaging, in preparation of tumor tissue photothermal response release of ferroptosis inducer, and in photothermal therapy combined with other therapies to inhibit tumor growth.

[0077] In some embodiments, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer, or nasopharyngeal cancer.

[0078] In some embodiments, the present disclosure provides use of the biological membrane or the engineered vesicle or the composition in preparation of a drug for treating a tumor or a tumor diagnosis reagent or a nano-drug delivery system.

[0079] In some embodiments, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer, or nasopharyngeal cancer.

[0080] In some embodiments, the present disclosure provides a method for treating a tumor, comprising administering a fibroblast cell to a subject in need thereof.

[0081] In some embodiments, the present disclosure provides a method for treating a tumor, comprising administering an anti-tumor drug to a subject in need thereof, wherein the anti-tumor drug comprises a combination of a tumor photothermal therapy agent and the engineered vesicle or the biological membrane or the vesicle or the composition.

[0082] In some embodiments, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer, or nasopharyngeal cancer.

[0083] In some embodiments, the present disclosure provides a method for treating a tumor, comprising administering a laser therapy combination drug to a subject in need thereof, wherein the laser therapy combination drug comprises the engineered vesicle or the biological membrane or the vesicle or the composition.

[0084] In some embodiments, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer, or nasopharyngeal cancer.

[0085] In some embodiments, the present disclosure provides a method for treating a tumor, comprising administering a drug for treating a tumor to a subject in need thereof, wherein the drug for treating a tumor comprises the engineered vesicle or the biological membrane or the vesicle or the composition.

[0086] In some embodiments, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer, or nasopharyngeal cancer.

[0087] In some embodiments, the present disclosure provides use of the biological membrane or the engineered vesicle in preparation of a nano-drug delivery system.

[0088] In some embodiments, the engineered fibroblast membrane nanovesicles as drug carriers can improve the bioavailability and tumor targeting of antitumor drugs, can be used in combination with photothermal therapy, not only can achieve controlled release of antitumor drugs, but also can accelerate the consumption of intracellular reductive glutathione and the accumulation of ROS, and the inhibition of GPX4 by antitumor drugs further exacerbates cell oxidative stress and ferroptosis effect, bringing new hope for tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1 is an optical photograph of autologous normal fibroblasts and genetically engineered fibroblasts.

[0090] Figure 2 is a Western blot analysis result of protein expression of single-gene and multi-gene engineered fibroblasts and corresponding membrane vesicles.

[0091] Figure 3 is the in vivo distribution of different mass ratios of genetically engineered fibroblast membrane nanovesicles X-Fb-MNVs in MDA-MB-468 breast cancer tumor-bearing mice; wherein: Figure 3A is the particle size change of different mass ratios of genetically engineered fibroblast membrane nanovesicles X-Fb-MNVs; Figure 3B is the in vivo distribution of MDA-MB-468 breast cancer tumor-bearing mice after tail vein injection of different mass ratios of X-Fb-MNVs for 2-24h.

[0092] Figure 4 is a transmission electron micrograph of genetically engineered fibroblast membrane nanovesicles X-Fb-MNVs.

[0093] Figure 5 is a schematic diagram of the uptake of single-gene engineered fibroblast membrane nanovesicles CXCR4-Fb-MNVs and multi-gene engineered fibroblast membrane nanovesicles X-Fb-MNVs on 4T1 tumor cells and normal cells, respectively.

[0094] Figure 6 is the uptake of multi-gene engineered fibroblast membrane nanovesicles X-Fb-MNVs by macrophages;

[0095] Figure 7 is the in vivo distribution of X-Fb-MNVs or CXCR4-Fb-MNVs in MDA-MB-468 breast cancer tumor-bearing mouse models; wherein: Figure 7A is the in vivo distribution of MDA-MB-468 breast cancer tumor-bearing mice after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs for 2-48h; Figure 7B is a schematic diagram of organ distribution of MDA-MB-468 breast cancer tumor-bearing mice after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-MNVs for 48h (in order of arrangement, heart, liver, spleen, lung, kidney, lymph node, tumor).

[0096] Figure 8 is the in vivo distribution of X-Fb-MNVs or CXCR4-Fb-MNVs in 4T1 breast cancer tumor-bearing mouse models; wherein: Figure 8A is the in vivo distribution of 4T1 breast cancer tumor-bearing mice after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs for 2-48h; Figure 8B is the organ distribution diagram of 4T1 breast cancer tumor-bearing mice after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs for 48h (in order of arrangement, heart, liver, spleen, lung, kidney, lymph node, tumor).

[0097] Figure 9 is the in vivo distribution of X-Fb-MNVs or CXCR4-Fb-MNVs in CT26 colon cancer tumor-bearing mouse models; wherein: Figure 9A is the in vivo distribution of CT26 colon cancer tumor-bearing mice after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs for 2-48h; Figure 9B is the organ distribution diagram of CT26 colon cancer tumor-bearing mice after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs for 48h (in order of arrangement, heart, liver, spleen, lung, kidney, tumor, lymph node).

[0098] Figure 10 is the in vivo distribution of X-Fb-MNVs or CXCR4-Fb-MNVs in RIL175 liver cancer tumor-bearing mouse models; wherein: Figure 10A is the in vivo distribution of RIL175 liver cancer tumor-bearing mice after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs for 2-48h; Figure 10B is the organ distribution diagram of RIL175 liver cancer tumor-bearing mice after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs for 48h (in order of arrangement, heart, liver, spleen, lung, kidney, tumor, lymph node).

[0099] Figure 11 is the test results of the good photothermal conversion ability of the gene-targeted fibroblast membrane nanovesicles; wherein Figure 11A is the temperature change results of the engineered cell membrane nanovesicles MNVs after near-infrared laser irradiation; Figure 11B is the temperature change curve of the engineered cell membrane nanovesicles MNVs after near-infrared laser irradiation (808nm, 1W / cm 2 ) for 3min.

[0100] Figure 12 is the cytotoxicity evaluation of the engineered cell membrane vesicles on tumor cells MDA-MB-468 (12A) and KYSE-30 (12B).

[0101] Figure 13 is the level of lipid ROS induced by engineered cell membrane vesicles on tumor cells MDA-MB-468 (13A) and KYSE-30 (13B).

[0102] Figure 14 is the anti-tumor therapeutic effect of engineered cell membrane vesicles in MDA-MB-468 breast cancer tumor-bearing mouse models; wherein: Figure 14A is a tumor growth curve of MDA-MB-468 breast cancer tumor-bearing mice within 21 days; Figure 14B is a comparison chart of tumor tissue weights of different groups after administration.

[0103] Figure 15 is a body weight change curve of MDA-MB-468 breast cancer tumor-bearing mice within 21 days.

[0104] Figure 16 is the anti-tumor therapeutic effect of engineered cell membrane vesicles in KYSE-30 esophageal cancer tumor-bearing mouse models; wherein: Figure 16A is a tumor growth curve of KYSE-30 esophageal cancer tumor-bearing mice within 21 days; Figure 16B is a comparison chart of tumor tissue weights of different groups after administration.

[0105] Figure 17 is a body weight change curve of KYSE-30 esophageal cancer tumor-bearing mice within 21 days.

[0106] Figure 18 is the performance test results of engineered single-gene targeted NIH-3T3 cell membrane nanovesicles; wherein, Figures 18A-18B are the WB detection results of overexpressed membrane proteins of NIH-3T3 fibroblasts and cell membrane nanovesicles obtained therefrom; Figure 18C is the in vivo distribution of MDA-MB-468 breast cancer tumor-bearing mice after tail vein injection of CXCR4-3T3-MNVs or CTR-3T3-MNVs for 2-48 h; Figure 18D is a schematic diagram of organ distribution of MDA-MB-468 breast cancer tumor-bearing mice after tail vein injection of CXCR4-3T3-MNVs or CTR-3T3-MNVs for 48 h (in order of arrangement, heart, liver, spleen, lung, kidney, tumor and lymph node).

[0107] Figure 19 is the anti-tumor therapeutic effect of engineered cell membrane vesicles in 4T1 breast cancer tumor-bearing mouse models, and a comparison chart of tumor tissue weights of different groups after administration.

[0108] Figure 20 is the mRNA level of CD47 after gene transfection of different cells of autologous origin examined by RT-qPCR.

[0109] Figure 21 is the mRNA level of iRGD after gene transfection of different cells of autologous origin examined by RT-qPCR.

[0110] Figure 22 is the mRNA level of Pdcd1 after gene transfection of different cells of autologous origin examined by RT-qPCR.

[0111] Figure 23 is an RT-qPCR investigation of mRNA levels of CXCR4 after gene transfection of different autologous cell sources.

[0112] Figure 24 is the uptake of gene engineered cell membrane nanovesicles of different cell sources on macrophages. DETAILED DESCRIPTION

[0113] The technical solutions of the present disclosure are further illustrated below by specific examples, which do not represent a limitation on the protection scope of the present disclosure. Some non-essential modifications and adjustments made by others according to the concept of the present disclosure still fall within the protection scope of the present disclosure.

[0114] “Comprise” or “comprising” is intended to mean that the compositions (e.g., media), and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, means excluding other elements of any importance to the combination claimed. Thus, a composition consisting essentially of the elements as defined herein, does not exclude other materials or steps which do not materially affect the basic and novel characteristic(s) of the claimed application. “Consisting of’ means excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this application. The term “and / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. The term “and / or” when used in a list of two or more items, means that any of the listed items can be employed by itself, or any combination of two or more of the listed items can be employed. For example, if a composition, combination, structure, etc. is described as comprising (or containing) components A, B, C, and / or D, then the composition can comprise A alone; B alone; C alone; D alone; a combination of A and B; a combination of A and C; a combination of A and D; a combination of B and C; a combination of B and D; a combination of C and D; a combination of A, B, and C; a combination of A, B, and D; a combination of A, C, and D; a combination of B, C, and D; or a combination of A, B, C, and D.

[0115] In some embodiments, the “composition” is interpreted broadly, for example, a pharmaceutical composition, a drug kit.

[0116] As used herein, the term “pharmaceutical composition” means the product that results from the mixing or combining of more than one active ingredient, and includes both fixed combinations and non-fixed combinations of active ingredients. The terms “co-administration” or “combined administration” and the like, as used herein, are meant to encompass administration of selected therapeutic agents to a single patient, and are intended to include therapy in which the drugs are not necessarily administered by the same route of administration or at the same time.

[0117] As used herein, the term "fixed combination" means that the active ingredients (fibroblast membrane nanovesicles, and a tumor therapeutic agent) are administered to a patient simultaneously in the form of a single entity or dosage.

[0118] As used herein, the term "non-fixed combination" means that the active ingredients are administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body, preferably at the same time. For example, a non-fixed combination can be two capsules each containing one active ingredient, with the intent that the two active ingredients together will provide a patient with therapy in the body.

[0119] As used herein, "in combination" or "in conjunction" means that two or more active agents or therapeutic means can be administered to a subject together, simultaneously as separate single therapeutic means or sequentially as separate single therapeutic means in any order (e.g., simultaneously, sequentially, spaced apart). Wherein, as mentioned in the disclosure, "use of the engineered vesicles in the preparation of a medicament for use in combination with laser therapy" refers to the use of the engineered vesicles in combination with laser for anti-tumor.

[0120] As used herein, the term "in combination with" when referring to administration of multiple agents to a subject means administration of a first agent to the subject with at least one other (i.e., second, third, fourth, fifth, etc.) agent. As used herein, an agent (engineered vesicle) is considered to be administered in combination with a second agent (tumor therapeutic) if the biological effect produced by administration of the first agent persists in the subject long enough for the therapeutic effects of the first agent and the second agent to overlap. Administration of the first agent provides therapeutic effect over an extended period of time, and administration of the second agent provides its therapeutic effect while the therapeutic effect of the first agent is still ongoing, and thus the second agent is considered to be administered in combination with the first agent even though the first agent can have been administered at a time point that is far removed from the time of administration of the second agent (e.g., days or weeks). In some embodiments, a first agent is considered to be administered in combination with a second agent if the first and second agents are administered simultaneously (within 30 minutes of each other), contemporaneously, or sequentially. In some embodiments, a first agent is considered to be administered "contemporaneously" with a second agent if the first and second agents are administered within about 24 hours of each other (e.g., within about 12 hours of each other, within about 6 hours of each other, within about 2 hours of each other, or within about 30 minutes of each other). The term "in combination with" is also understood to apply to situations in which the first agent and the second agent are co-formulated in a single pharmaceutically acceptable formulation and the co-formulation is administered to the subject. In some embodiments, the engineered vesicle and the tumor therapeutic are administered or applied sequentially, e.g., one agent is administered after one or more other agents. In some embodiments, administration is simultaneous, e.g., two or more agents are administered at or about the same time; the two or more agents can be present in two or more separate formulations, or combined into a single formulation (i.e., a co-formulation). Regardless of whether the agents are administered sequentially or simultaneously, as used herein, they are considered to be administered / used in combination.

[0121] As used herein, "normal fibroblast" refers to a fibroblast that is not derived from a lesion.

[0122] All sources of skin tissue or peritumoral tissue are in compliance with the relevant standards, which include that the donor has signed an informed consent and donor screening and donor testing are performed.

[0123] As used herein, the CAS number for IR-780 iodide is: 207399-07-3; the CAS number for RSL3 is: 1219810-16-8; the CAS number for DPPC is: 63-89-8; the CAS number for poloxamer 188 is: 9003-11-6; the CAS number for NH2-PEG-DSPE is: 474922-26-4. Empty fibroblast membrane nanovesicles refer to the base of the gene-targeted fibroblast membrane nanovesicles in the present disclosure without the addition of RSL3. Cell-penetrating peptides are also known as cell-penetrating peptides.

[0124] Preparation method of fibroblast membrane overexpressing single gene (CXCR4-Fb-M)

[0125] Autologous normal skin tissue was cut, washed with sterile cold PBS buffer for 3 times, and the washed normal tissue was cut into 1-2 mm uniform pieces, then washed with sterile cold PBS buffer for 3 times, and the washed tissue pieces were plated in a 10 cm cell culture dish, and 4 ml of primary cell culture solution (DMEM medium containing 10% fetal bovine serum and 100 ug / ml primocin) was added, and then placed in a 37°C, 5% CO2 incubator for 7 days, waiting for fibroblasts to grow out from the normal tissue, adhere and proliferate, and the microscopic state is shown in Figure 1 (CTR-Fibroblast). 2

[0126] The fibroblasts were plated in a 6-well plate at 6x10 4 The fibroblasts were plated in a 6-well plate at 6x10

[0127] Then replace with primary cell culture solution containing 2 ug / ml puromycin, and after the untransfected cells are completely killed by puromycin, the CXCR4 overexpressing cells are trypsinized and expanded, i.e. CXCR4 overexpressing fibroblasts (CXCR4-Fb) are obtained, and the overexpression of CXCR4 is verified by WB.

[0128] The CXCR4 overexpressing fibroblasts (CXCR4-Fb) were collected, the cell pellet was washed with pre-cooled PBS buffer for 3 times, and after centrifugation, the PBS buffer was aspirated, and membrane protein extraction reagent containing 1 mM PMSF protease inhibitor was added to the cell pellet, vortexed thoroughly, and placed on ice for 15 min, then vortexed every 5 min, then the broken cell pellet was placed in liquid nitrogen and 37°C water bath and repeatedly frozen and thawed 4 times until the solution was uniform, then the uniform cell breakage solution was centrifuged at 4°C, 700g for 10 min, and the supernatant after centrifugation was centrifuged at 4°C, 14000g for 30 min, and the precipitate after centrifugation was the engineered single gene targeted fibroblast membrane protein (CXCR4-Fb-M).

[0129] Preparation method of fibroblast membrane overexpressing multiple genes (X-Fb-M)

[0130] ​Normal skin tissue from the patient's own body was harvested and rinsed three times with sterile cold PBS buffer. The rinsed normal tissue was then minced into 1–2 mm pieces. 2 The tissue fragments were then rinsed three times with sterile cold PBS buffer. The rinsed tissue fragments were spread evenly in a 10cm cell culture dish, and 4ml of primary cell culture medium (DMEM medium containing 10% fetal bovine serum and 100ug / ml primocin) was added. The dish was then incubated at 37℃ and 5% CO2 for 7 days to allow fibroblasts to crawl out of the normal tissue, adhere to the dish, and proliferate.

[0131] Fibroblasts were arranged in 8x10 4 Cells were seeded at a density of 1 / ml into 6-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow them to adhere. Lentiviral solutions containing the resistance gene puromycin and overexpressing the target gene CXCR4, as well as lentiviral solutions containing the resistance gene G418 and simultaneously overexpressing three target genes PD-1, CD47, and iRGD fusion protein particles, along with high-efficiency transfection medium (Gikai Gene), were added. The cells were then transfected at 37°C with 5% CO2 for 72 hours. The X-Fibroblasts simultaneously overexpressing CXCR4, PD-1, CD47, and iRGD are shown in Figure 1.

[0132] Then, the culture medium was replaced with primary cell culture medium containing 2 μg / ml puromycin and 1.2 mg / ml G418. After the untransfected cells were completely killed by puromycin and G418, the fibroblasts that simultaneously overexpressed the target genes CXCR4, PD-1, CD47 and iRGD were digested with trypsin and cultured in a large scale to obtain fibroblasts that overexpressed multiple target genes (CXCR4 / PD-1 / CD47 / iRGD-Fb, i.e., X-Fb). The overexpression of CXCR4, PD-1 and CD47 / iRGD was verified by Western blotting.

[0133] Fibroblasts (X-Fb) overexpressing multiple target genes were collected. The cell pellet was washed three times with pre-cooled PBS buffer, centrifuged, and the PBS buffer was aspirated. Membrane protein extraction reagent containing 1 mM PMSF protease inhibitor was added to the cell pellet. After thorough vortexing, the pellet was placed on ice for 15 min, and then vortexed once every 5 min. The ruptured cell pellet was then subjected to repeated freeze-thaw cycles in liquid nitrogen and a 37°C water bath four times until the solution was homogeneous. The homogeneous cell rupture solution was then centrifuged at 700g for 10 min at 4°C. The supernatant was then centrifuged at 14000g for 30 min at 4°C. The pellet after centrifugation was the engineered multi-gene targeted fibroblast membrane protein (X-Fb-M).

[0134] Example 3 Preparation method of thermosensitive lipid nano-drug delivery system (IR780 / DPPC / RSL3, NVs, or IR / RSL3 NPs)

[0135] 10 mg of IR-780 iodide (CAS No.: 207399-07-3) and 40 mg of NH2-PEG-DSPE (i.e. DSPE-PEG-NH2, CAS No.: 474922-26-4) were weighed and accurately weighed, and dissolved in a dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; triethylamine (TEA) was used as an acid-binding agent, and triethylamine (TEA) was weighed according to a molar ratio of triethylamine (TEA) to IR-780 iodide (IR780) of 2:1, and added to the dichloromethane solution containing IR-780 iodide, and stirred at room temperature for 24 h; after the reaction was completed, the reaction solution was added to 10 times the volume of anhydrous ether, and left overnight at 4°C, and then centrifuged at a speed of 3000 rpm for 5 min to remove the by-product, and the precipitate was dried to obtain IR-780 iodide modified lipid grafting (IR780-PEG-DSPE).

[0136] The components of the lipid nanoparticles were prepared with anhydrous ethanol as the solvent, and were added in order of decreasing mass percentage at 60°C under water bath conditions, and stirred at a speed of 400 rpm for 1 min to obtain an ethanol phase solution; wherein the mass percentage of the ferroptosis inducer RSL3 (CAS No.: 1219810-16-8) in the ethanol phase solution was 20%, the mass percentage of IR780-PEG-DSPE was 20%, the mass percentage of the thermosensitive lipid DPPC (CAS No.: 63-89-8) was 55%, and the mass percentage of cholesterol was 5%.

[0137] A 1 mg / ml poloxamer 188 (CAS: 9003-11-6) aqueous solution was prepared with deionized water as the solvent, and the poloxamer 188 aqueous solution was measured according to a volume ratio of poloxamer 188 aqueous solution to ethanol phase solution of 10:1, and the ethanol phase solution was quickly dispersed into the poloxamer 188 aqueous solution under the condition of 60°C, and stirred at a speed of 400 rpm for 8 min, and then cooled to room temperature to prepare the thermosensitive lipid nano-drug delivery system (IR780 / DPPC / RSL3, NVs).

[0138] The content of RSL3 in 20% IR / RSL3 NPs (i.e. IR780 / DPPC / RSL3, NVs) was determined by high performance liquid chromatography (HPLC). The determination conditions were as follows: C18 reversed-phase chromatographic column; mobile phase: acetonitrile: water = 7:3 (v / v, 1% acetic acid); flow rate: 1.0 mL / min; injection volume: 20 μL; column temperature: 25 °C. Herein, 20% IR / RSL3 NPs refers to the mass percentage of RSL3 contained in the ethanol phase of the synthesized IR / RSL3 NPs.

[0139] Preparation of standard curve: a 1.0 mg / mL RSL3 / DMSO solution was prepared and diluted with the mobile phase to different concentration gradients (1 μg / mL, 3 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL, 300 μg / mL, 500 μg / mL), and the HPLC was used to detect the ultraviolet absorption peak at 290 nm. The data were analyzed using the Agilent OpenLab software, with the drug concentration C (μg / mL) as the abscissa and the chromatographic peak area A as the ordinate, to prepare a standard curve.

[0140] Determination of encapsulation efficiency and drug loading: 50 μL of the IR / RSL3 NPs solution was precisely pipetted, diluted 10 times with the mobile phase, and ultrasonicated in a water bath for 30 min. The drug concentration was determined by HPLC, and the drug encapsulation efficiency and drug loading were calculated:

[0141] Encapsulation efficiency = mass of RSL3 in the drug-loaded lipid nanoparticle sample / mass of RSL3 administered * 100%;

[0142] Drug loading = mass of RSL3 in the drug-loaded lipid nanoparticle sample / (mass of RSL3 in the drug-loaded lipid nanoparticle sample + mass of the lipid nanoparticle) * 100%;

[0143] It was calculated that the encapsulation efficiency of the ferroptosis inducer RSL3 in the NVs was 71.90%, and the drug loading was 16.92%.

[0144] Example 4 Preparation method of thermosensitive lipid nanoparticle drug delivery system (IR780 / DPPC / RSL3, NVs)

[0145] Take 5 mg of IR-780 iodide and 20 mg of NH2-PEG-DSPE, accurately weigh, dissolve in dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; take triethylamine (TEA) as an acid binding agent, and take triethylamine (TEA) in a molar ratio of 1:1 with IR-780 iodide (IR780), and add it to the dichloromethane solution containing IR-780 iodide, and stir at room temperature for 8 h; after the reaction is completed, the reaction solution is added to 8 times the volume of anhydrous ether, and is left overnight at 3°C, and then centrifuged at a speed of 2500 rpm for 4 min to remove the by-product, and then the precipitate is dried to obtain IR-780 iodide modified lipid grafting (IR780-PEG-DSPE).

[0146] Anhydrous ethanol is used as a solvent to prepare each component of the lipid nanoparticles, and is added in order of decreasing mass percentage at 50°C under water bath conditions, and is stirred at a speed of 600 rpm for 30 s; wherein the mass percentage of the ferroptosis inducer RSL3 in the ethanol phase solution is 10%, the mass percentage of IR780-PEG-DSPE is 20%, the mass percentage of the thermosensitive lipid DPPC is 64%, and the mass percentage of cholesterol is 6%.

[0147] Deionized water is used as a solvent to prepare a 1.5 mg / ml poloxamer 188 aqueous solution, and the poloxamer 188 aqueous solution is measured in a volume ratio of 8:1 with the ethanol phase solution, and the ethanol phase solution is quickly dispersed into the poloxamer 188 aqueous solution at 55°C, and is stirred at a speed of 600 rpm for 10 min, and is then cooled to room temperature to prepare the thermosensitive lipid drug delivery system (IR780 / DPPC / RSL3, IRNVs).

[0148] It is determined (in the same method as in Example 3) that the encapsulation efficiency of the ferroptosis inducer RSL3 in the NVs is 67.24%, and the drug loading amount is 7.81%.

[0149] Example 5 Preparation method of thermosensitive lipid nanodrug delivery system (IR780 / DPPC / RSL3, NVs)

[0150] Take 15 mg of IR-780 iodide and 50 mg of NH2-PEG-DSPE, accurately weigh and dissolve in dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; take triethylamine (TEA) as an acid binding agent, and take triethylamine (TEA) in a molar ratio of 3:1 with IR-780 iodide (IR780), and add it to the dichloromethane solution containing IR-780 iodide, and stir at room temperature for 30 h; after the reaction is completed, the reaction solution is added to 12 times the volume of anhydrous ether, and left overnight at 5°C, and then centrifuged at a speed of 3500 rpm for 6 min to remove the by-product, and then the precipitate is dried to obtain IR-780 iodide modified lipid grafting (IR780-PEG-DSPE).

[0151] Anhydrous ethanol is used as a solvent to prepare each component of the lipid nanoparticle, and is added in order of decreasing mass percentage at 70°C under water bath conditions, and stirred at a speed of 300 rpm for 2 min; wherein the mass percentage of ferroptosis inducer RSL3 in the ethanol phase solution is 15%, the mass percentage of IR780-PEG-DSPE is 20%, the mass percentage of thermosensitive lipid DPPC is 62%, and the mass percentage of cholesterol is 3%.

[0152] Deionized water is used as a solvent to prepare a 0.5 mg / ml poloxamer 188 aqueous solution, and the poloxamer 188 aqueous solution is measured in a volume ratio of 12:1 with the ethanol phase solution, and the ethanol phase solution is quickly dispersed into the poloxamer 188 aqueous solution at 65°C, and stirred at a speed of 300 rpm for 5 min, and then cooled to room temperature to prepare a thermosensitive lipid drug delivery system (IR780 / DPPC / RSL3, NVs).

[0153] It is determined (same as the method of Example 3) that the encapsulation efficiency of ferroptosis inducer RSL3 in IR NPs is 66.53%, and the drug loading is 11.74%.

[0154] Example 6 Preparation method of thermosensitive lipid nanodrug delivery system (IR780 / DPPC / RSL3, NVs)

[0155] Take 15 mg of IR-780 iodide and 20 mg of NH2-PEG-DSPE, accurately weigh and dissolve in dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; take triethylamine (TEA) as an acid binding agent, and take triethylamine (TEA) in a molar ratio of 3:1 with IR-780 iodide (IR780), and add it to the dichloromethane solution containing IR-780 iodide, and stir at room temperature for 8 h; after the reaction is completed, the reaction solution is added to 12 times the volume of anhydrous ether, and left overnight at 5°C, and then centrifuged at a speed of 3500 rpm for 4 min to remove the by-product, and then the precipitate is dried to obtain IR-780 iodide modified lipid grafting (IR780-PEG-DSPE).

[0156] Anhydrous ethanol is used as a solvent to prepare each component of the lipid nanoparticles, and is added successively at a mass percentage from high to low under the condition of a water bath at 60°C, and is stirred at a speed of 600 rpm for 1 min; wherein the mass percentage of the ferroptosis inducer RSL3 in the ethanol phase solution is 20%, the mass percentage of IR780-PEG-DSPE is 10%, the mass percentage of the thermosensitive lipid DPPC is 64%, and the mass percentage of cholesterol is 6%.

[0157] Deionized water is used as a solvent to prepare a 1.5 mg / ml poloxamer 188 aqueous solution, and the poloxamer 188 aqueous solution is measured in a volume ratio of 12:1 with the ethanol phase solution, and the ethanol phase solution is quickly dispersed into the poloxamer 188 aqueous solution under the condition of 60°C, and is stirred at a speed of 600 rpm for 8 min, and is then cooled to room temperature to prepare the thermosensitive lipid drug delivery system (IR780 / DPPC / RSL3, NVs).

[0158] It is determined (in the same method as in Example 3) that the encapsulation efficiency of the ferroptosis inducer RSL3 in the NVs is 66.6%, and the drug loading is 11.8%.

[0159] Example 7 Preparation method of engineered single-gene targeted fibroblast membrane nanovesicles (CXCR4-Fb-MNVs)

[0160] The engineered monogene-targeting fibroblast membrane protein (CXCR4-Fb-M) obtained in Example 1 was diluted with PBS to 0.6 mg / ml, and filtered through polycarbonate membranes of 400 nm and 200 nm in sequence using a liposome extruder, and the filtered engineered fibroblast membrane protein solution was mixed with the thermosensitive lipid nanodelivery system (IR780 / DPPC / RSL3, NVs) obtained in Example 3 according to a set mass ratio (CXCR4-Fb-M protein mass (referring to the mass of the 0.6 mg / ml cell membrane solution): NVs mass = 1:0.5), and co-extruded through a polycarbonate membrane of 100 nm using a liposome extruder to obtain engineered monogene-targeting fibroblast membrane nanovesicles (CXCR4-Fb-M NVs).

[0161] Example 8 Preparation method of engineered monogene-targeting fibroblast membrane nanovesicles (CXCR4-Fb-M NVs)

[0162] The engineered monogene-targeting fibroblast membrane protein (CXCR4-Fb-M) obtained in Example 1 was diluted with PBS to 0.6 mg / ml, and filtered through polycarbonate membranes of 400 nm and 200 nm in sequence using a liposome extruder, and the filtered engineered fibroblast membrane protein solution was mixed with the thermosensitive lipid nanodelivery system (IR780 / DPPC / RSL3, NVs) obtained in Example 3 according to a set mass ratio (CXCR4-Fb-M protein mass (referring to the mass of the 0.6 mg / ml cell membrane solution): NVs mass = 1:0.5), and co-extruded through a polycarbonate membrane of 100 nm using a liposome extruder to obtain engineered monogene-targeting fibroblast membrane nanovesicles (CXCR4-Fb-M NVs).

[0163] Example 9 Preparation method of engineered monogene-targeting fibroblast membrane nanovesicles (CXCR4-Fb-M NVs)

[0164] The engineered monogene-targeting fibroblast membrane protein (CXCR4-Fb-M) obtained in Example 1 was diluted with PBS to 0.6 mg / ml, and filtered through polycarbonate membranes of 400 nm and 200 nm in sequence using a liposome extruder, and the filtered engineered fibroblast membrane protein solution was mixed with the thermosensitive lipid nanodelivery system (IR780 / DPPC / RSL3, NVs) obtained in Example 3 according to a set mass ratio (CXCR4-Fb-M protein mass (referring to the mass of the 0.6 mg / ml cell membrane solution): NVs mass = 1:0.5), and co-extruded through a polycarbonate membrane of 100 nm using a liposome extruder to obtain engineered monogene-targeting fibroblast membrane nanovesicles (CXCR4-Fb-M NVs).

[0165] Example 10 Preparation method of engineered multi-gene targeted fibroblast membrane nanovesicles (X-Fb-MNVs)

[0166] The engineered multi-gene targeted fibroblast membrane protein solution (X-Fb-M) obtained in Example 2 was diluted with PBS to 0.6 mg / ml, and filtered through polycarbonate membranes of 400 nm and 200 nm in sequence using a liposome extruder. The filtered engineered fibroblast membrane protein solution was mixed with the thermosensitive lipid nanodrug delivery system (IR780 / DPPC / RSL3, NVs) obtained in Example 3 according to the set mass ratio (X-Fb-M protein mass (referring to the mass of the 0.6 mg / ml cell membrane solution): NVs mass = 1:0.5), and then co-extruded through a polycarbonate membrane of 100 nm using a liposome extruder to obtain engineered fibroblast membrane nanovesicles (X-Fb-MNVs).

[0167] Example 11 Preparation method of engineered multi-gene targeted fibroblast membrane nanovesicles (X-Fb-MNVs)

[0168] The engineered multi-gene targeted fibroblast membrane protein solution (X-Fb-M) obtained in Example 2 was diluted with PBS to 0.6 mg / ml, and filtered through polycarbonate membranes of 400 nm and 200 nm in sequence using a liposome extruder. The filtered engineered fibroblast membrane protein solution was mixed with the thermosensitive lipid nanodrug delivery system (IR780 / DPPC / RSL3, NVs) obtained in Example 3 according to the set mass ratio (X-Fb-M protein mass (referring to the mass of the 0.6 mg / ml cell membrane solution): NVs mass = 1:0.5), and then co-extruded through a polycarbonate membrane of 100 nm using a liposome extruder to obtain engineered fibroblast membrane nanovesicles (X-Fb-MNVs).

[0169] Example 12 Preparation method of engineered multi-gene targeted fibroblast membrane nanovesicles (X-Fb-MNVs)

[0170] The engineered multi-gene targeting fibroblast membrane protein solution (X-Fb-M) obtained in Example 2 was diluted with PBS to 0.6 mg / ml, and filtered through polycarbonate membranes of 400 nm and 200 nm in sequence using a liposome extruder, and the filtered engineered fibroblast membrane protein solution was mixed with the thermosensitive lipid nanodrug delivery system (IR780 / DPPC / RSL3, NVs) obtained in Example 3 according to the set mass ratio (X-Fb-M protein mass (referring to the mass of the 0.6 mg / ml cell membrane solution): NVs mass = 1:2), and then co-extruded through a polycarbonate membrane of 100 nm using a liposome extruder to obtain the engineered fibroblast membrane nanovesicle (X-Fb-M NVs).

[0171] Example 13 WB detection of overexpression of membrane proteins by fibroblasts and cell membrane nanovesicles obtained therefrom

[0172] 1. Detection of overexpression of single membrane protein CXCR4 by fibroblasts and cell membrane nanovesicles obtained therefrom

[0173] In order to verify that the single-gene engineered fibroblasts obtained in the present disclosure successfully overexpress the membrane protein CXCR4, WB detection was performed on the fibroblasts obtained in Example 1 (CXCR4-Fb or CXCR4-Fibroblast) and the CXCR4-Fb-MNVs obtained in Example 7 as examples:

[0174] Equal amounts of protein were taken from the CXCR4-Fb cell lysate, the CTR-Fb (control fibroblast) cell lysate, and the corresponding membrane vesicle solutions CXCR4-Fb-MNVs and the control group CTR-Fb-MNVs solution, 5x protein loading buffer was added and vortexed to mix, and a 100°C metal bath was used for heating for 10 min. The prepared protein was loaded onto an SDS-PAGE gel, and electrophoresis was performed at a constant voltage of 120 V in an environment of 1x Tris-Glycine-SDS electrophoresis buffer. The proteins separated by the SDS-PAGE gel after electrophoresis were transferred to a PVDF membrane, and wet transfer was performed in an environment of 1x TG transfer solution at a constant current of 280 mA for 2 h. The PVDF membrane after transfer was blocked with 5% skim milk for 1 h, then anti-CXCR4 antibody was used for incubation at 4°C overnight, the next day horseradish peroxidase-conjugated secondary antibody was used for incubation at room temperature for 1 h, and finally high-sensitivity chemiluminescent substrate and a ChemiDoc imaging system were used to detect the protein signal.

[0175] The results are shown in Figure 2. The expression of CXCR4 protein in the CXCR4-Fb group was significantly higher than that in the CTR-Fb group, indicating that the single gene engineered fibroblast successfully overexpressed the membrane protein CXCR4. In addition, the membrane nanovesicles CXCR4-Fb-MNVs highly expressed the membrane protein CXCR4, indicating that the method retained the engineered cell membrane protein.

[0176] 2. Detection of overexpression of multiple membrane proteins by fibroblasts and cell membrane nanovesicles obtained therefrom

[0177] To verify that the multiple gene engineered fibroblasts obtained by the present disclosure successfully overexpress the membrane proteins CXCR4, PD-1, CD47 and iRGD, the fibroblasts obtained in Example 2 (X-Fb or X-Fibroblast) and the X-Fb-MNVs obtained in Example 10 were subjected to WB detection:

[0178] The X-Fb cell lysate, CTR-Fb cell lysate and corresponding membrane vesicle solution X-Fb-MNVs and control group CTR-Fb-MNVs solution containing equal amounts of cell membrane proteins were added with 5x protein loading buffer and vortexed, and heated in a 100°C metal bath for 10 min. The prepared protein was loaded onto SDS-PAGE gel, and electrophoresis was performed at a constant voltage of 120V in the presence of 1x Tris-Glycine-SDS electrophoresis buffer. The separated proteins on the SDS-PAGE gel after electrophoresis were transferred to a PVDF membrane using a wet transfer method in the presence of 1x TG transfer solution at a constant current of 280mA for 2h. The PVDF membrane after transfer was blocked with 5% skim milk for 1h, and then anti-CXCR4, anti-PD-1 and anti-CD47 antibodies were used for incubation at 4°C overnight, and the next day horseradish peroxidase-conjugated secondary antibody was used for incubation at room temperature for 1h, and finally high-sensitivity chemiluminescent substrate and ChemiDoc imaging system were used for detection of protein signal.

[0179] As shown in Figure 2, the expression of CXCR4, PD-1 and CD47 proteins in the X-Fb group was up-regulated relative to the control group, indicating that the multiple gene engineered fibroblasts successfully overexpressed the membrane proteins CXCR4, PD-1, CD47 and iRGD. In addition, the membrane vesicles X-Fb-MNVs highly expressed the membrane proteins CXCR4, PD-1, CD47 and iRGD, indicating that the method retained the engineered cell membrane proteins.

[0180] Example 14 Performance detection of multiple gene engineered cell membrane nanovesicles X-Fb-MNVs

[0181] 1. Lipid drug delivery system particle size and zeta potential detection

[0182] IR-780 iodide modified thermosensitive lipid drug delivery system (IR / RSL3 NPs) with different RSL3 contents were prepared, and the mass ratio of RSL3 was 10%, 15% and 20% respectively (the mass ratio here refers to the content ratio of RSL3 in the ethanol phase solution during the preparation of IR / RSL3 NPs). The particle size and surface potential of unloaded thermosensitive lipid drug delivery system (IR NPs) and three mass ratios of IR / RSL3 NPs were determined by particle size and surface potential instrument.

[0183] wherein, the preparation method of IR / RSL3 NPs is the same as that of Example 3.

[0184] The determination results are shown in Table 1. It can be seen that the particle size of IR NPs is 67.54±0.93 nm, and the particle sizes of 10%, 15% and 20% IR / RSL3 NPs are 72.67±5.33 nm, 91.45±13.10 nm and 98.72±6.65 nm respectively; the potential of IR NPs is 6.17±1.60 mV, and the potentials of 10%, 15% and 20% IR / RSL3 NPs are -6.90±1.19 mV, -9.90±1.43 mV and -15.60±1.53 mV respectively. The results show that the particle size of IR / RSL3 NPs increases after drug loading, and increases with the increase of the concentration of RSL3. At the same time, the charge of IR / RSL3 NPs after drug loading is reversed from positive charge to negative charge, and increases with the increase of the concentration of RSL3.

[0185] Table 1

[0186] 2, Particle size detection of X-Fb-MNVs

[0187] The lipid nanoparticles NVs (20% IR / RSL3 NPs) were mixed with 0.6 mg / mL of engineered X-Fb cell membrane solution according to different mass ratios to prepare engineered biomimetic membrane nanovesicles (X-Fb-MNVs, i.e. M / IR / RSL3 NPs) by probe sonication, and the particle size was determined.

[0188] The results are shown in Figure 3A. When the engineered cell membrane and the lipid nanoparticles (i.e. thermosensitive lipid nanoparticle drug delivery system) NVs are mixed at a mass ratio of 1:2, the particle size of X-Fb-MNVs is 130±3.95 nm. The results prove that with the increase of the mass of lipid nanoparticles NVs, the particle size of cell membrane nanovesicles X-Fb-MNVs becomes smaller and more uniform and stable.

[0189] The preparation method of X-Fb-MNVs is the same as that of Example 10.

[0190] 3. In vivo distribution of engineered cell membrane nanovesicles X-Fb-MNVs in 4T1 tumor-bearing mice

[0191] The in vivo distribution of engineered cell membrane nanovesicles X-Fb-MNVs with different mass ratios in 4T1 (breast cancer cell line) tumor-bearing mice was observed by a small animal live imaging instrument.

[0192] As shown in FIG. 3B, after tail vein injection of X-Fb-MNVs, when the mass ratio of lipid nanoparticles NVs to engineered cell membrane was 2:1, X-Fb-MNVs showed a significantly stronger distribution in the tumor site than other groups, indicating that when the mass ratio of lipid nanoparticles NVs to engineered cell membrane was 2.0, engineered cell membrane nanovesicles X-Fb-MNVs had a stronger tumor targeting ability.

[0193] The preparation method of X-Fb-MNVs was the same as that of Example 10.

[0194] 4. Transmission electron microscopy (TEM) image of cell membrane nanovesicles X-Fb-MNVs

[0195] 1 mg / mL of lipid nanoparticles NVs and engineered cell membrane nanovesicles X-Fb-MNVs solutions were diluted with deionized water to 0.1 mg / mL, dropped onto carbon-coated copper grids, stained with 2% (w / v) uranyl acetate, and the excess liquid was absorbed with filter paper. The morphology and particle size of NVs and X-Fb-MNVs were observed by transmission electron microscopy, respectively.

[0196] The morphology of nanoparticles under transmission electron microscopy is shown in FIG. 4. It can be seen that the lipid nanoparticles NVs are spherical structures with a diameter of about 50 nm, and the X-Fb-MNVs have a diameter of about 150 nm and a clear "core-shell structure". This result further proves that the cell membrane is successfully wrapped on the surface of the lipid nanoparticles NVs.

[0197] The preparation method of X-Fb-MNVs was the same as that of Example 10, and the preparation method of lipid nanoparticles NVs was the same as that of Example 3.

[0198] 5. Evaluation of the uptake ability of tumor cells and normal cells to engineered fibroblast cell membrane nanovesicles

[0199] 4T1 breast cancer cells and normal fibroblasts were seeded in 24-well plates with round cell-crawling slides at appropriate densities and incubated at 37°C in a 5% CO2 incubator for 24 h to adhere (adhesion ratio was about 60%); after cell adhesion, the fluorescently labeled engineered cell membrane vesicle solution FITC CXCR4-Fb-MNVs and X-Fb-MNVs and the control group CTR-Fb-MNVs were added to the culture medium at a consistent fluorescence, and incubated for 12 h, the culture medium was removed, the PBS buffer was used to rinse the crawling slides 3 times, the cells on the crawling slides were fixed with 4% paraformaldehyde fixing solution for 15 min, after removing the fixing solution, PBS buffer was continued to be used to rinse 3 times, the PBS residue on the crawling slides was gently wiped off, the crawling slides were covered on the glass slides with DAPI dye drops on the front, and then the slides were sealed with a sealing agent.

[0200] The prepared glass slides were observed by laser confocal microscope for fluorescence distribution in the FITC band, as shown in FIG. 5, it can be obviously observed that compared with the control group of fibroblast membrane nanovesicles CTR-Fb-MNVs, the multi-gene engineered cell membrane nanovesicles X-Fb-MNVs were obviously taken up by tumor cells more, and were taken up by normal cells less.

[0201] wherein CXCR4-Fb-MNVs and X-Fb-MNVs were prepared by the methods of Example 9 and Example 12, respectively.

[0202] 6. Evaluation of the ability of multi-gene engineered cell membrane nanovesicles to escape phagocytosis by macrophages

[0203] The macrophage cell line RAW264.7 was seeded in 24-well plates with round cell-crawling slides at a density of 1*10 5 The macrophage cell line RAW264.7 was seeded in 24-well plates with round cell-crawling slides at a density of 1*10

[0204] The prepared glass slides were observed by laser confocal microscope for fluorescence distribution in the FITC band, as shown in Figure 6. It can be obviously observed that the multi-gene engineered cell membrane nanovesicles X-Fb-MNVs are not easily taken up by macrophages compared with the control group of fibroblast membrane nanovesicles CTR-Fb-MNVs. When the CD47 protein is competitively blocked, the amount of X-Fb-MNVs taken up by macrophages increases, indicating that the multi-gene engineered cell membrane nanovesicles X-Fb-MNVs have good ability to avoid phagocytosis by macrophages due to overexpression of the membrane protein CD47.

[0205] X-Fb-MNVs were prepared by the method of Example 12.

[0206] 7. Evaluation of the targeting ability of engineered fibroblast membrane nanovesicles to various models of breast cancer, colon cancer, and liver cancer:

[0207] 7.1 Targeting ability to breast cancer

[0208] 8-week-old female nude mice were inoculated with 8*10 6 MDA-MB-468 breast cancer cells in the left lower breast, and after 6 weeks, a MDA-MB-468 breast cancer orthotopic tumor-bearing mouse model was successfully established. The tumor-bearing mice were randomly divided into three groups, and were injected with equal amounts of near-infrared fluorescent probe IR780-labeled engineered cell membrane vesicle solution CXCR4-Fb-MNVs and X-Fb-MNVs and control group CTR-Fb-MNVs via the tail vein. A small animal live imaging instrument was used to observe the fluorescence distribution in the MDA-MB-468 tumor-bearing mice within 2-48 h after injection (the fluorescence imaging graph captured in this experiment was in the wave band of 720-845 nm, which matched the emission wave band of IR780).

[0209] The results are shown in Figure 7A. The fluorescence intensity of the tumor site of the mice in the multi-gene engineered fibroblast membrane nanovesicle X-Fb-MNVs group and the single-gene engineered fibroblast membrane nanovesicle CXCR4-Fb-MNVs group was significantly higher than that of the control group CTR-Fb-MNVs, and the aggregation level of X-Fb-MNVs in the tumor area was higher than that of CXCR4-Fb-MNVs.

[0210] After 48 h, the mice were sacrificed by cervical dislocation, and their organs were removed and placed in the order of heart, liver, spleen, lung, kidney, axillary lymph node, and tumor. The fluorescence distribution in the organs was observed by a small animal imaging instrument. Figure 7B shows that the fluorescence of the tumor site in the X-Fb-MNVs group was significantly stronger than that in the other groups, and no obvious accumulation was observed in the liver, kidney, and other organs. 5*10 5A 4T1 breast cancer cell, 4 weeks later, successfully established a 4T1 breast cancer orthotopic tumor-bearing mouse model. The tumor-bearing mice were randomly divided into three groups, and tail vein injection of equal amounts of near-infrared fluorescent probe IR780-labeled engineered cell membrane vesicle solution CXCR4-Fb-MNVs and X-Fb-MNVs and control group CTR-Fb-MNVs. The fluorescence distribution in the 4T1 tumor-bearing mice was observed within 2-48 h after injection using a small animal live imaging instrument. As shown in Figure 8A, the fluorescence intensity of the tumor site of the mice in the multi-gene engineered fibroblast cell membrane nanovesicle X-Fb-MNVs group and the single-gene engineered fibroblast cell membrane nanovesicle CXCR4-Fb-MNVs group was significantly higher than that in the control group CTR-Fb-MNVs, and the aggregation level of X-Fb-MNVs in the tumor area was higher than that of CXCR4-Fb-MNVs.

[0211] 48 h later, the mice were sacrificed by cervical dislocation, and their organs were taken and placed in the order of heart, liver, spleen, lung, kidney, axillary lymph node, and tumor. The fluorescence distribution in the organs was observed by a small animal imaging instrument. Figure 8B shows that the fluorescence of the tumor site in the X-Fb-MNVs group was significantly stronger than that in the other groups, and X-Fb-MNVs mainly accumulated in the tumor tissue, while less accumulated in the metabolic organs liver and kidney.

[0212] wherein CXCR4-Fb-MNVs and X-Fb-MNVs are prepared by the methods of Example 9 and Example 12, respectively.

[0213] 7.2 Targeting ability to colon cancer

[0214] A 6-week-old female BALB / c mouse was inoculated subcutaneously with 1*10 6 CT-26 colon cancer cells, 3 weeks later, successfully established a CT-26 colon cancer subcutaneous tumor-bearing mouse model. The tumor-bearing mice were randomly divided into three groups, and tail vein injection of equal amounts of near-infrared fluorescent probe IR780-labeled engineered cell membrane vesicle solution CXCR4-Fb-MNVs and X-Fb-MNVs and control group CTR-Fb-MNVs.

[0215] The fluorescence distribution in the CT-26 tumor-bearing mice was observed within 2-48 h after injection using a small animal live imaging instrument. As shown in Figure 9A, the fluorescence intensity of the tumor site of the mice in the multi-gene engineered fibroblast cell membrane nanovesicle X-Fb-MNVs group and the single-gene engineered fibroblast cell membrane nanovesicle CXCR4-Fb-MNVs group was significantly higher than that in the control group CTR-Fb-MNVs, and the aggregation level of X-Fb-MNVs in the tumor area was higher than that of CXCR4-Fb-MNVs.

[0216] 48h after the mice were sacrificed by cervical dislocation, their organs were taken and placed in the order of heart, liver, spleen, lung, kidney, tumor, and axillary lymph nodes. The fluorescence distribution in the organs was observed by a small animal imaging instrument. As shown in FIG. 9B, the fluorescence of the tumor site in the X-Fb-MNVs group was significantly stronger than that in the other groups, and no obvious accumulation was observed in the liver, kidney and other organs.

[0217] CXCR4-Fb-MNVs and X-Fb-MNVs were prepared by the methods of Example 9 and Example 12, respectively.

[0218] 7.3 Targeting ability for liver cancer

[0219] At the age of 4 weeks, male C57 (BL / 6N) mice were inoculated subcutaneously with 1*10 6 After 3 weeks, a RIL-175 liver cancer subcutaneous tumor-bearing mouse model was successfully established. The tumor-bearing mice were randomly divided into 3 groups, and were injected with equal amounts of near-infrared fluorescent probe IR780-labeled engineered cell membrane vesicle solution CXCR4-Fb-MNVs and X-Fb-MNVs and control group CTR-Fb-MNVs via the tail vein.

[0220] A small animal live imaging instrument was used to observe the fluorescence distribution in the RIL-175 tumor-bearing mice within 2-48h after injection. As shown in FIG. 10A, the fluorescence intensity of the tumor site in the mice of the X-Fb-MNVs group and the CXCR4-Fb-MNVs group was significantly higher than that in the control group CTR-Fb-MNVs, and the aggregation level of X-Fb-MNVs in the tumor area was higher than that of CXCR4-Fb-MNVs.

[0221] 48h after the mice were sacrificed by cervical dislocation, their organs were taken and placed in the order of heart, liver, spleen, lung, kidney, tumor, and axillary lymph nodes. The fluorescence distribution in the organs was observed by a small animal imaging instrument. As shown in FIG. 10B, the fluorescence of the tumor site in the X-Fb-MNVs group was significantly stronger than that in the other groups, and no obvious accumulation was observed in the liver, kidney and other organs.

[0222] CXCR4-Fb-MNVs and X-Fb-MNVs were prepared by the methods of Example 9 and Example 12, respectively.

[0223] According to the results of the above in vitro and in vivo experiments, the single-gene engineered fibroblast membrane nanovesicles (CXCR4-Fb-MNVs) and the multi-gene engineered fibroblast membrane nanovesicles (X-Fb-MNVs) prepared by the method of the present disclosure both have high specificity and the ability to quickly target multiple types of tumor tissues, while avoiding phagocytosis by macrophages during blood circulation, greatly reducing the accumulation in normal organs and non-target tissues, and minimizing the toxic side effects in the body.

[0224] Example 15 Killing ability of engineered fibroblast membrane nanovesicles on tumor cells

[0225] To evaluate the killing ability of the engineered fibroblast membrane nanovesicles obtained by the present disclosure on tumor cells, the following tests were performed using the engineered fibroblast membrane nanovesicles CXCR4-Fb-MNVs (i.e., M / IR / RSL3 NPs or MNVs) obtained in Example 9 as an example:

[0226] 1. Evaluation of the photothermal effect of engineered fibroblast membrane nanovesicles

[0227] An equal volume of PBS and a single-gene targeted fibroblast membrane nanovesicle aqueous solution with an IR-780 content of 5.0 μg / mL were placed in an incubator to equilibrate the temperature. Each sample was irradiated with a near-infrared laser with a wavelength of 808 nm at a power of 1 W / cm 2 for 3 min, and the temperature change trend of the sample was recorded using an infrared thermal imager.

[0228] As shown in FIGS. 11A and 11B, after near-infrared laser irradiation, the temperature of the single-gene targeted fibroblast membrane nanovesicle aqueous solution rose to a maximum of 50.6°C, and at the 3rd min, it still maintained at 45.2°C, while the temperature of the control group (PBS) hardly changed, maintaining at about 31°C. This result indicates that the single-gene targeted fibroblast membrane nanovesicles have good photothermal conversion ability.

[0229] 2. Evaluation of the killing effect of engineered fibroblast membrane nanovesicles on tumor cells in vitro:

[0230] 2.1 Cell activity detection of each group

[0231] Breast cancer cells MDA-MB-468 and esophageal cancer cells KYSE-30 were seeded in 96-well plates at a density of 1*10 4Each cell line was seeded at 1,000 cells / 100 ul in 96-well plates. After the cells adhered, each cell line was divided into 8 groups (control (DMSO), control + laser (DMSO + Laser), small molecule drug RSL3 (RSL3), RSL3 + laser (RSL3 + Laser), unloaded fibroblast membrane nanovesicles (M / IR NPs), unloaded fibroblast membrane nanovesicles + laser (M / IR NPs + Laser), drug-loaded fibroblast membrane nanovesicles (M / IR / RSL3 NPs), and drug-loaded fibroblast membrane nanovesicles + laser (M / IR / RSL3 NPs). Drugs were added according to the groups (the concentration of RSL3 in the groups containing RSL3 was 3 mM for MDA-MB-468 cells and 1 mM for KYSE-30 cells) and incubated in a 37°C, 5% CO2 incubator for 4 h. Each laser group was irradiated with a near-infrared laser with a wavelength of 808 nm at a power of 1 W / cm 2 for 3 min, and then each group of cells was incubated for another 20 h. The activity of each group of cells was detected using a CCK8 kit (Meilun) and a microplate reader.

[0232] The results are shown in FIGS. 12A and 12B. The small molecule drug RSL3 can inhibit the proliferation of MDA-MB-468 and KYSE-30 cells to some extent, but laser irradiation does not promote its anti-tumor effect. Unloaded fibroblast membrane nanovesicles do not have obvious toxicity to tumor cells, but under laser irradiation, they accelerate tumor cell death due to the photothermal effect. The most significant cell death phenomenon occurs in the drug-loaded fibroblast membrane nanovesicle group under laser irradiation.

[0233] 2.2 Lipid peroxide level detection of each group of cells

[0234] MDA-MB-468 and KYSE-30 cells were seeded at 3*10 5 cells / ml in 6-well plates. After the cells adhered, each cell line was divided into 8 groups according to the above cytotoxicity test, and drugs were added according to the groups (the concentration of RSL3 in the groups containing RSL3 was 4 mM for MDA-MB-468 cells and 2 mM for KYSE-30 cells) and incubated in a 37°C, 5% CO2 incubator for 4 h. Each laser group was irradiated with a near-infrared laser with a wavelength of 808 nm at a power of 1 W / cm 2 for 3 min, and then each group of cells was incubated for another 2 h. The lipid peroxide level of each group of cells was detected using a BODIPY 581 / 591 C11 probe (Invitrogen) and a flow cytometer.

[0235] The results are shown in Figure 13. The small molecule drug RSL3 and the fibroblast membrane nanovesicles without drug after laser irradiation can induce ferroptosis in breast cancer and esophageal cancer cells to some extent, but the combination of the two, i.e., drug-loaded fibroblast membrane nanovesicles combined with near-infrared laser, can maximize the induction of ferroptosis in tumor cells.

[0236] 3. Evaluate the in vivo efficacy and safety of engineered fibroblast membrane nanovesicles in tumor-bearing mice:

[0237] At 4 weeks of age, 8*10 6 MDA-MB-468 breast cancer cells were inoculated into the left lower breast of female nude mice, and when the tumor grew to 80-100mm 3 in size, the tumor-bearing mice were randomly divided into 8 groups (PBS, PBS+laser, RSL3, RSL3+laser, fibroblast membrane nanovesicles without drug, fibroblast membrane nanovesicles without drug+laser, drug-loaded fibroblast membrane nanovesicles, and drug-loaded fibroblast membrane nanovesicles+laser). Continuous 5 times, 48h interval by tail vein injection of the corresponding drugs (RSL3 dose in groups containing RSL3 is 1mg / kg), during which the tumor of the laser group mice was irradiated with near-infrared laser of wavelength 808nm at a power of 0.8W / cm 2 for 3min 24h after each administration, the body weight and tumor volume of the mice were recorded continuously during the administration period until the 21-day observation period, and the mice were sacrificed on day 21, and the tumor was weighed.

[0238] At 4 weeks of age, 5*10 6 KYSE-30 esophageal cancer cells were inoculated subcutaneously into the left side of male nude mice, and when the tumor grew to 80-100mm 3 in size, the tumor-bearing mice were randomly divided into 8 groups according to the above MDA-MB-468 mouse in vivo experiment, continuous 5 times, 48h interval by tail vein injection of the corresponding drugs (RSL3 dose in groups containing RSL3 is 1mg / kg), during which the tumor of the laser group mice was irradiated with near-infrared laser of wavelength 808nm at a power of 0.8W / cm 2 for 3min 24h after each administration, the body weight and tumor volume of the mice were recorded continuously during the administration period until the 21-day observation period, and the mice were sacrificed on day 21, and the tumor was weighed.

[0239] The results of anti-tumor therapy experiments are shown in FIG. 14 (MDA-MB-468 breast cancer mouse model) and FIG. 16 (KYSE-30 esophageal cancer mouse model). In all groups, the drug-loaded fibroblast membrane nanovesicles combined with near-infrared laser treatment achieved the best in vivo anti-tumor effect; the tumors of the mice in the negative control (PBS group) continued to grow, and the tumor growth of the mice in the PBS combined with laser group and the non-drug-loaded fibroblast membrane nanovesicle treatment group was also not effectively inhibited, proving that near-infrared laser irradiation alone and empty fibroblast membrane nanovesicles alone have no therapeutic effect on tumors; the anti-tumor effect of the drug-loaded fibroblast membrane nanovesicle treatment group is better than that of the RSL3 treatment group and the RSL3 combined with laser group, proving that the fibroblast membrane nanovesicle improves the solubility of RSL3 in blood circulation and the targeting of tumors; we also found that although the non-drug-loaded fibroblast membrane nanovesicle combined with laser treatment can inhibit tumor growth to some extent, it shows that photothermal therapy can effectively kill tumor cells, but because it does not load the ferroptosis inducer RSL3, it cannot induce tumor cell ferroptosis and achieve the best therapeutic effect. After the treatment ended, the change trend of the tumor weight and tumor volume of the mice was consistent.

[0240] The body weight changes of the tumor-bearing mice in different groups during the treatment period were observed to evaluate the in vivo safety of the engineered fibroblast membrane nanovesicles. The results are shown in FIG. 15 (MDA-MB-468 breast cancer mouse model) and FIG. 17 (KYSE-30 esophageal cancer mouse model). The body weight of the tumor-bearing mice in the engineered fibroblast membrane nanovesicle laser irradiation group increased slightly, similar to the body weight change of the physiological saline group, indicating that the engineered fibroblast membrane nanovesicle has low systemic toxicity, and the local laser irradiation of the tumor has little effect on the body and is highly safe.

[0241] Example 16 Preparation method of engineered single-gene targeted NIH-3T3 cell membrane nanovesicles and application thereof

[0242] 16.1 Preparation method of NIH-3T3 cell membrane overexpressing a single gene

[0243] The NIH-3T3 fibroblast cell line was plated at 6x10 4 cells / ml in a 6-well plate and incubated in a 37°C, 5% CO2 incubator for 24 h to adhere. The lentiviral solution overexpressing the target gene CXCR4 containing the resistance gene puromycin and the high-efficiency transfection solution were added, and the cells were transfected in a 37°C, 5% CO2 incubator for 72 h to obtain fibroblasts overexpressing CXCR4 (CXCR4-3T3 or NIH-3T3).

[0244] The CXCR4-3T3 was used to prepare the engineered single-gene targeted cell membrane protein according to the method of Example 1.

[0245] 16.2 Preparation method of engineered monogenic targeting NIH-3T3 cell membrane nanovesicles

[0246] The cell membrane protein used is the cell membrane protein obtained in Example 16.1, and the remaining methods are the same as in Example 9, and finally CXCR4-3T3-MNVs are obtained.

[0247] 16.3 WB detection of overexpressed membrane proteins of NIH-3T3 fibroblasts and cell membrane nanovesicles obtained therefrom

[0248] The detection method is the same as in Example 13. The results are shown in FIG. 18A and FIG. 18B.

[0249] 16.4 Targeting ability of engineered cell membrane nanovesicles to breast cancer

[0250] 8*10 6 MDA-MB-468 breast cancer cells were inoculated into the right lower breast of 4-week-old female nude mice, and after 6 weeks, a MDA-MB-468 breast cancer orthotopic tumor-bearing mouse model was successfully established. The tumor-bearing mice were randomly divided into two groups, and were injected with equal amounts of near-infrared fluorescent probe IR780-labeled engineered cell membrane vesicle solution CXCR4-3T3-MNVs and control group CTR-3T3-MNVs, respectively. A small animal live imaging instrument was used to observe the fluorescence distribution in the MDA-MB-468 tumor-bearing mice within 2-48 h after injection.

[0251] The results are shown in FIG. 18C. The fluorescence intensity of the tumor site of the monogenic engineered fibroblast membrane nanovesicle CXCR4-3T3-MNV group mice was significantly higher than that of the control group CTR-3T3-MNVs.

[0252] The mice were sacrificed by cervical dislocation after 48 h, and their organs were taken and placed in the order of heart, liver, spleen, lung, kidney, tumor and axillary lymph node. The fluorescence distribution in the organs was observed by a small animal imaging instrument. FIG. 18D shows that the fluorescence of the tumor site of the CXCR4-3T3-MNV group was significantly stronger than that of the other groups, and no obvious accumulation was observed in the liver, kidney and other organs.

[0253] Example 17 Killing ability of engineered cell membrane nanovesicles loaded with DOX (doxorubicin) to tumor cells

[0254] 5*10 5 4T1 breast cancer cells were inoculated into the right lower breast of 6-week-old female BALB / c mice, and when the tumors of the mice grew to 80-100 mm 3The tumor-bearing mice were randomly divided into 6 groups (PBS, DOX, non-drug-loaded multi-gene fibroblast membrane nanovesicles, non-drug-loaded multi-gene fibroblast membrane nanovesicles + laser, DOX-loaded fibroblast membrane nanovesicles, and DOX-loaded fibroblast membrane nanovesicles + laser), and the corresponding drugs were injected into the tail vein of the mice 5 times at an interval of 48 h, with a dosage of 2 mg / kg of DOX in the DOX-containing groups. During the administration, the tumor of the laser group mice was irradiated with near-infrared laser at a wavelength of 808 nm at a power of 0.8 W / cm 2 The body weight and tumor volume of the mice were recorded during the administration, and the mice were sacrificed on the 21st day, and the tumors were weighed.

[0255] The results of the anti-tumor treatment experiment are shown in FIG. 19 (4T1 breast cancer mouse model). In all groups, the DOX-loaded multi-gene fibroblast membrane nanovesicles combined with near-infrared laser treatment achieved the best in vivo anti-tumor effect; the tumor weight of the mice in the negative control (PBS group) increased significantly; and the anti-tumor effect of the drug-loaded DOX fibroblast membrane nanovesicles treatment group was better than that of the free DOX group, which proved that the fibroblast membrane nanovesicles improved the targeting and inhibitory effect of DOX on tumors; at the same time, the non-drug-loaded fibroblast membrane nanovesicles combined with laser treatment could also inhibit the growth of tumors to a certain extent, which showed that photothermal therapy could effectively kill tumor cells, but it did not achieve the best treatment effect because it did not load the anti-tumor drug DOX.

[0256] In FIG. 19, the X-Fb-MNVs are the non-drug-loaded multi-gene fibroblast membrane nanovesicles group; X-Fb-MNVs(+) is the non-drug-loaded multi-gene fibroblast membrane nanovesicles + laser group; X-Fb-MNVs / DOX is the DOX-loaded fibroblast membrane nanovesicles group; and X-Fb-MNVs / DOX(+) is the DOX-loaded fibroblast membrane nanovesicles + laser group. The preparation method of X-Fb-MNVs / DOX is the same as that of Example 10, except that RLS3 is replaced by DOX; and the preparation method of X-Fb-MNVs is the same as that of X-Fb-MNVs / DOX in this embodiment, except that it is not loaded with drugs.

[0257] Example 18 Detection of in vitro transfection efficiency of autologous normal cells and the ability of corresponding cell membrane vesicles to avoid phagocytosis by macrophages in the body circulation

[0258] 1. Detection of in vitro transfection efficiency of autologous normal cells

[0259] Autologous immune cells (monocytes, T cells), stem cells (umbilical cord blood mesenchymal stem cells, MSCs), and fibroblasts (Fb) were selected and each cell type was divided into 6*10 groups. 4 Cells were seeded at a density of 1 / ml into 6-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow them to adhere. Lentiviral solution containing overexpressed target genes PD-1 and CD47 / iRGD fusion protein particles with the resistance gene G418, along with high-efficiency transfection medium, was added. Cells were transfected at 37°C with 5% CO2 for 72 hours. Then, the medium was replaced with primary cell culture medium containing 1.2 mg / ml G418. After the untransfected cells were completely killed by G418, the cells overexpressing the target genes PD-1 / CD47 / iRGD were digested with trypsin and cultured to obtain cells overexpressing multiple target genes (PD-1 / CD47 / iRGD-cell, i.e., PCI-monocyte, PCI-T cell, PCI-MSC, PCI-Fb). The mRNA level of CD47 was verified by RT-qPCR.

[0260] Cells overexpressing multiple target genes (PD-1 / CD47 / iRGD-cells, i.e., PCI-monocytes, PCI-T cells, PCI-MSCs, and PCI-Fb) were selected. Total RNA was isolated using Trizol reagent and then reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit (TakaRa). Quantitative PCR was performed using the SYBR Green PCR Kit (Qiagen, 208054) according to the manufacturer's instructions. The mRNA expression level of the target genes was quantified using ACTIN as an internal control.

[0261] As shown in Figures 20-23, RT-PCR analysis revealed that, under the same transfection conditions, the relative expression levels of CD47 mRNA, iRGD mRNA, and PD-1 mRNA in gene-transfected autologous fibroblasts were significantly higher than those in the control group, with some showing fold increases of tens of thousands of times. These levels were far higher than those in gene-transfected autologous monocytes, T cells, and mesenchymal stem cells. This indicates that autologous fibroblasts are more suitable as cells for in vitro expansion and gene transfection, thus enabling their use as engineered cell membrane carriers.

[0262] PCI refers to the simultaneous overexpression of PD-1, CD47, and iRGD.

[0263] In FIGS. 20-23: PCI-monocyte refers to monocytes overexpressing PD-1, CD47 and iRGD simultaneously; CTR-monocyte refers to control monocytes; PCI-T cell refers to T cells overexpressing PD-1, CD47 and iRGD simultaneously, CTR-T cell refers to control T cells; PCI-MSC refers to MSC cells overexpressing PD-1, CD47 and iRGD simultaneously, CTR-MSC refers to control MSC cells; PCI-Fb or PCI-Fibroblast refers to fibroblasts overexpressing PD-1, CD47 and iRGD simultaneously, CTR-Fibroblast refers to control fibroblasts, X-Fibroblast refers to fibroblasts overexpressing CXCR4, PD-1, CD47 and iRGD simultaneously, i.e. fibroblasts overexpressing multiple target genes obtained in Example 2 (X-Fb). In FIG. 23, CXCR4-Fibroblast refers to fibroblasts overexpressing target gene CXCR4 obtained in Example 1 (CXCR4-Fb).

[0264] 2. Detection of the ability of cell membrane vesicles obtained from autologous normal cells to avoid phagocytosis by macrophages in the systemic circulation

[0265] The above-mentioned cells overexpressing multiple target genes (PCI-monocyte, PCI-T cell, PCI-MSC, PCI-Fb) were collected, washed with pre-cooled PBS buffer for 3 times, and after centrifugation, the PBS buffer was absorbed, and the membrane protein extraction reagent containing 1 mM PMSF protease inhibitor was added to the cell precipitate, vortexed thoroughly, and then placed on ice for 15 min, and then vortexed every 5 min, and then the broken cell precipitate was repeatedly frozen and thawed in liquid nitrogen and 37°C water bath for 4 times until the solution was uniform, and then the uniform cell broken solution was centrifuged at 4°C, 700g for 10 min, and then the supernatant after centrifugation was centrifuged at 4°C, 14000g for 30 min, and then the precipitate after centrifugation was the engineered multi-gene cell membrane protein (PCI-monocyte-M, PCI-T cell-M, PCI-MSC-M, PCI-Fb-M). The engineered multi-gene cell membrane protein was prepared into cell membrane nanovesicles (MNVs) according to the method of Example 10, and PCI-Fb-MNVs, PCI-Monocyte-MNVs, PCI-T-MNVs, PCI-MSC-MNVs were obtained.

[0266] The macrophage cell line RAW264.7 was seeded at 1*10 5The density of 1x104 / ml is inoculated in a 24-well plate coated with round cell slides, and incubated at 37°C, 5% CO2 incubator for 24h to adhere; after the cells adhere, add FITC fluorescently labeled engineered multi-gene cell membrane nanovesicles PCI-Fb-MNVs, PCI-Monocyte-MNVs, PCI-T-MNVs, PCI-MSC-MNVs to the culture medium, continue to incubate for 2h, remove the culture medium, use PBS buffer to rinse the slides 3 times, the cells on the slides are fixed with 4% paraformaldehyde fixing solution for 15min, after removing the fixing solution, continue to rinse 3 times with PBS buffer, gently wipe off the PBS residue on the slides, cover the slides face down on the glass slide with DAPI staining solution, then use mounting medium to mount the slides.

[0267] The prepared glass slides are observed by laser confocal microscope for fluorescence distribution in the FITC band, as shown in FIG. 24, it can be obviously observed that compared with the cell membrane nanovesicles CTR-Fb-MNVs of the control group, the green fluorescence of the multi-gene engineered fibroblast membrane nanovesicles X-Fb-MNVs and PCI-Fb-MNVs on the macrophages is obviously weakened, and it is not easy to be taken up by macrophages, while the green fluorescence of the genetically engineered monocyte membrane nanovesicles PCI-Monocyte-MNVs, T cell membrane nanovesicles PCI-T-MNVs, and mesenchymal stem cell membrane nanovesicles PCI-MSC-MNVs in the macrophages is strong, and it is easy to be taken up and phagocytosed by macrophages, indicating that the multi-gene engineered fibroblast membrane nanovesicles X-Fb-MNVs and PCI-Fb-MNVs have good ability to avoid phagocytosis by macrophages.

[0268] CTR-Fb-MNVs: refers to fibroblast membrane nanovesicles without overexpressing genes, except that the genes are not overexpressed, the rest of the steps are the same as in Example 10.

[0269] PCI-Fb-MNVs: fibroblast membrane nanovesicles overexpressing PD-1, CD47 and iRGD at the same time.

[0270] PCI-Monocyte-MNVs: monocyte membrane nanovesicles overexpressing PD-1, CD47 and iRGD at the same time.

[0271] PCI-T-MNVs: T cell membrane nanovesicles overexpressing PD-1, CD47 and iRGD at the same time.

[0272] PCI-MSC-MNVs: MSC cell membrane nanovesicles overexpressing PD-1, CD47 and iRGD at the same time.

[0273] X-Fb-MNVs: Fibroblast membrane nanovesicles prepared as obtained for Example 10.

Claims

1. Use of normal fibroblast cells in the preparation of a medicament for treating a tumor or a tumor diagnostic reagent or a nano-drug carrier system or an engineered vesicle.

2. The use of claim 1, wherein, The normal fibroblast cells are selected from autologous normal fibroblast cells; Preferably, the normal fibroblast cells are selected from a fibroblast cell line or strain; Preferably, the normal fibroblast cells are selected from an established fibroblast cell line or strain; Preferably, the normal fibroblast cells are selected from skin or para-cancer tissue fibroblasts.

3. A method of making an engineered vesicle, wherein, Comprise: (1) overexpressing genes in normal fibroblast cells to obtain fibroblasts overexpressing genes; (2) extracting the membranes of the fibroblasts; (3) mixing the membranes of the fibroblasts with lipid nanoparticles to prepare the engineered vesicles; Preferably, the fibroblasts overexpressing genes overexpress adhesion proteins or chemokine receptors; Preferably, the fibroblasts overexpressing genes also overexpress immunosuppressive molecules or cell-penetrating peptides; Preferably, the genes comprise CD47 or CXCR4; Preferably, the genes also comprise PD-1 or iRGD; Preferably, the genes are PD-1, CD47 and iRGD; Preferably, the genes are CXCR4, PD-1, CD47 and iRGD; Preferably, the normal fibroblast cells are selected from autologous normal fibroblast cells; Preferably, the normal fibroblast cells are selected from a fibroblast cell line or strain; Preferably, the normal fibroblast cells are selected from an established fibroblast cell line or strain; Preferably, the normal fibroblast cells are selected from skin or para-cancer tissue fibroblasts. Preferably, in step (2), the membranes of the fibroblasts are obtained by sequentially using freeze-thawing, gradient centrifugation and resuspension; Preferably, in step (3), it comprises step (3.1): the membranes of the fibroblasts are formulated into a solution before being mixed with the lipid nanoparticles to obtain a mixed solution of the membranes of the fibroblasts and the lipid nanoparticles; Preferably, in step (3), it comprises step (3.2): using a liposome extruder to extrude and filter the mixed solution of the membranes of the fibroblasts and the lipid nanoparticles to obtain the engineered vesicles; Preferably, in step (3.1), the concentration of the membranes of the fibroblasts is 0.1-2 mg / mL; Preferably, the membrane solution of the fibroblasts is filtered through 300-500 nm and 100-350 nm polycarbonate filters before being mixed with the lipid nanoparticles; Preferably, in step (3.2), the filtration of the mixed solution of the membranes of the fibroblasts and the lipid nanoparticles is performed by sequentially using 50-250 nm polycarbonate filters; Preferably, in step (3.1), the mass ratio of the membrane solution of the fibroblasts to the lipid nanoparticles is (0.2-3):1; Preferably, in step (3.1), the mass ratio of the membrane solution of the fibroblasts to the lipid nanoparticles is (0.5-3):1; Preferably, in step (3.1), the mass ratio of the membrane solution of the fibroblasts to the lipid nanoparticles is (0.5-2):1; Preferably, the lipid nanoparticles are obtained by solvent diffusion method; Preferably, in step (3), the method for obtaining the lipid nanoparticles comprises the steps of: (S1) mixing and reacting IR-780 iodide and NH2-PEG-DSPE to obtain a primary product IR780-PEG-DSPE; (S2) dispersing a solution of IR780-PEG-DSPE, an anti-tumor drug, DPPC and cholesterol into poloxamer 188 to prepare the lipid nanoparticles; Preferably, the method for obtaining the lipid nanoparticles comprises the steps of: (A1) weighing IR-780 iodide and NH2-PEG-DSPE, dissolving them in dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; (A2) adding triethylamine to the dichloromethane solution containing IR-780 iodide to react, to obtain a reaction solution; (A3) adding the reaction solution into diethyl ether, centrifuging to remove by-products, obtaining a precipitate, and drying the precipitate to obtain a primary product IR780-PEG-DSPE; (A4) dissolving an anti-tumor drug, IR780-PEG-DSPE, DPPC and cholesterol in ethanol, and heating to obtain an ethanol phase solution; (A5) mixing the ethanol phase solution of step (A4) with a poloxamer 188 aqueous solution to react to prepare the lipid nanoparticles; Preferably, in the ethanol phase solution of step (A4), the weight fractions of the anti-tumor drug, IR780-PEG-DSPE, DPPC and cholesterol are respectively 5-30 parts of the anti-tumor drug, 10-30 parts of IR780-PEG-DSPE, 40-70 parts of DPPC and 1-15 parts of cholesterol; Preferably, in the ethanol phase solution of step (A4), the weight fractions of the anti-tumor drug, IR780-PEG-DSPE, DPPC and cholesterol are respectively 5-25 parts of the anti-tumor drug, 10-25 parts of IR780-PEG-DSPE, 45-70 parts of DPPC and 1-10 parts of cholesterol; Preferably, in the ethanol phase solution of step (A4), the weight fractions of the anti-tumor drug, IR780-PEG-DSPE, DPPC and cholesterol are respectively 5-20 parts of the anti-tumor drug, 10-20 parts of IR780-PEG-DSPE, 50-70 parts of DPPC and 3-6 parts of cholesterol; Preferably, in the ethanol phase solution of step (A4), the mass percentages of the anti-tumor drug, IR780-PEG-DSPE, DPPC and cholesterol are respectively 5-20% of the anti-tumor drug, 10-20% of IR780-PEG-DSPE, 50-70% of DPPC and 3-15% of cholesterol; Preferably, in step (A5), the volume ratio of the poloxamer 188 aqueous solution to the ethanol phase solution is (5-20) : 1; Preferably, in step (A5), the volume ratio of the poloxamer 188 aqueous solution to the ethanol phase solution is (8-15) : 1; Preferably, in step (A5), the concentration of poloxamer 188 in the poloxamer 188 aqueous solution is 0.1-3 mg / mL; Preferably, in step (A5), the concentration of poloxamer 188 in the aqueous poloxamer 188 solution is 0.5-1.5 mg / mL; Preferably, the method for obtaining the IR780-PEG-DSPE comprises the steps of: (B1) weighing IR-780 iodide and NH2-PEG-DSPE, and dissolving them in dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; (B2) weighing triethylamine according to a molar ratio of (0.5-5) : 1 of triethylamine to IR-780 iodide, and adding it to the dichloromethane solution containing IR-780 iodide in step (B1), and stirring for 8-30 h; (B3) after the reaction is completed, the reaction solution is added to 8-12 times the volume of anhydrous ether for reaction, centrifuged to remove by-products, and the precipitate is dried to obtain IR780-PEG-DSPE; Preferably, in step (B2), the molar ratio of triethylamine to IR-780 iodide is (1-3) : 1; Preferably, the anti-tumor drug comprises RSL3 or doxorubicin.

4. The engineered vesicle obtained by the method of claim 3.

5. A biofilm, wherein, The biological membrane comprises a fibroblast membrane overexpressing a gene, the gene comprising CD47 or CXCR4; Preferably, the gene further comprises PD-1 or iRGD; Preferably, the biological membrane is a fibroblast membrane overexpressing genes PD-1, CD47 and iRGD; Preferably, the biological membrane is a fibroblast membrane overexpressing genes CXCR4, PD-1, CD47 and iRGD; Preferably, the fibroblast is selected from a normal fibroblast; Preferably, the fibroblast is selected from a normal fibroblast of autologous origin; Preferably, the normal fibroblast is selected from a fibroblast cell line or strain; Preferably, the normal fibroblast is selected from an established fibroblast cell line or strain; Preferably, the normal fibroblast is selected from a fibroblast of skin or paracancerous tissue.

6. A method of preparing the biofilm of claim 5, wherein, The fibroblast is overexpressed with a gene, and the fibroblast is extracted; The gene comprises CD47 or CXCR4; Preferably, the gene further comprises PD-1 or iRGD; Preferably, the biological membrane is a fibroblast membrane overexpressing genes PD-1, CD47 and iRGD; Preferably, the biological membrane is a fibroblast membrane overexpressing genes CXCR4, PD-1, CD47 and iRGD; Preferably, the fibroblast is selected from a normal fibroblast; Preferably, the fibroblast is selected from a normal fibroblast of autologous origin Preferably, the normal fibroblast is selected from a fibroblast cell line or strain; Preferably, the normal fibroblast is selected from an established fibroblast cell line or strain; Preferably, the normal fibroblast is selected from a fibroblast of skin or paracancerous tissue.

7. A vesicle, wherein, The preparation raw material of the vesicle comprises the biological membrane of claim 5; Preferably, the vesicle is a genetically engineered cell membrane nanovesicle; Preferably, the preparation raw material of the vesicle further comprises a lipid nanoparticle; Preferably, the lipid nanoparticle comprises a thermosensitive liposome drug delivery system. Preferably, the thermosensitive liposome drug delivery system preparation raw materials comprise IR-780 iodide, NH2-PEG-DSPE, triethylamine, an anti-tumor drug and DPPC. Preferably, the anti-tumor drug comprises RSL3 or doxorubicin.

8. A composition wherein, The engineered vesicle of claim 4 or the biological membrane of claim 5 or the vesicle of claim 7, and a tumor therapeutic agent; Preferably, the tumor therapeutic agent comprises an anti-tumor drug. Preferably, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer. Preferably, the anti-tumor drug comprises RSL3 or doxorubicin.

9. Use of the engineered vesicle of claim 4 or the biological membrane of claim 5 or the vesicle of claim 7 or the composition of claim 8 in the preparation of an anti-tumor drug for use in combination with a tumor photothermal therapeutic agent; Preferably, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer.

10. Use of a tumor photothermal therapeutic agent in the preparation of an anti-tumor drug for use in combination with the engineered vesicle of claim 4 or the biological membrane of claim 5 or the vesicle of claim 7 or the composition of claim 8; Preferably, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer.

11. Use of the engineered vesicle of claim 4 or the biological membrane of claim 5 or the vesicle of claim 7 or the composition of claim 8 in the preparation of a laser therapy combination drug; Preferably, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer.

12. Use of the engineered vesicle of claim 4 or the biological membrane of claim 5 or the vesicle of claim 7 or the composition of claim 8 in the preparation of a tumor treatment drug or a tumor diagnostic reagent or a nano-drug delivery system; Preferably, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer.

13. A method of treating a tumor, wherein, Preferably, the fibroblast is administered to a subject in need thereof.

14. A method of treating a tumor, wherein, Preferably, the anti-tumor drug comprises RSL3 or doxorubicin.

9. Use of the engineered vesicle of claim 4 or the biological membrane of claim 5 or the vesicle of claim 7 or the composition of claim 8 in the preparation of an anti-tumor drug for use in combination with a tumor photothermal therapeutic agent; 15. A method of treating a tumor, wherein, Preferably, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer.

10. Use of a tumor photothermal therapeutic agent in the preparation of an anti-tumor drug for use in combination with the engineered vesicle of claim 4 or the biological membrane of claim 5 or the vesicle of claim 7 or the composition of claim 8; 16. A method of treating a tumor, wherein, Preferably, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer.

11. Use of the engineered vesicle of claim 4 or the biological membrane of claim 5 or the vesicle of claim 7 or the composition of claim 8 in the preparation of a laser therapy combination drug; Preferably, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer.

12. Use of the engineered vesicle of claim 4 or the biological membrane of claim 5 or the vesicle of claim 7 or the composition of claim 8 in the preparation of a tumor treatment drug or a tumor diagnostic reagent or a nano-drug delivery system; Preferably, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer. Preferably, the tumor comprises a breast cancer, a colon cancer, an esophageal cancer, a liver cancer, a lung cancer or a nasopharyngeal cancer. Preferably, the tumor comprises a breast cancer, a colon cancer, an esophageal cancer, a liver cancer, a lung cancer or a nasopharyngeal cancer.

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