Engineered cell membrane nano-vesicle as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to an engineered cell membrane nano-vesicle as well as a preparation method and application thereof. The invention provides application of normal fibroblasts in preparation of drugs for treating tumors or tumor diagnostic reagents or nano drug delivery systems or engineered vesicles. The vesicles prepared from the normal fibroblasts have high specificity and capability of rapidly targeting multiple types of tumor tissues, can realize tumor targeted diagnosis imaging or drug delivery, greatly reduce accumulation in normal organs and non-targeted tissues, maximally weaken toxic and side effects of the vesicles in vivo, and have good application prospects. And the safety and the effectiveness are greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to an engineered cell membrane nanovesicle, a preparation method thereof and an application thereof. Background Art

[0002] The nano-targeted drug delivery system has shown obvious advantages in the field of treating tumor-related diseases: 1) Nano-drugs can be passively targeted to tumor sites through the EPR effect (Enhanced permeability and retention effect, EPR). 2) Targeting molecules can be modified on the surface of nano-drugs or drug molecules can be linked by environment-sensitive bonds to achieve the active targeted distribution of drugs and the environment-responsive release. Although nanoparticles can be surface-modified to prolong the blood circulation time and improve the targeting property, there are still many disadvantages in using them as diagnostic media and drug delivery systems: First, most nanoparticles, as artificially synthesized inorganic or organic substances, have certain toxicity and are easily recognized as foreign substances by the immune system and cleared, resulting in a small accumulation amount at the tumor site; second, the surface modification technology required for nanoparticles to achieve the targeting function is relatively complex and difficult to implement. In recent years, researchers have become increasingly interested in using natural biomimetic materials to prepare nano-drug delivery systems. In a complex organism, various cell lines show a "homing" targeting phenomenon for specific tissues. For example, macrophages, as one of many immune cells, often actively target wounds or inflammatory sites; bleeding wound sites can actively recruit platelets; in an animal model of myocardial infarction, progenitor cells and transplanted stem cells have been proven to have a myocardial homing effect; in addition, tumor cells also tend to aggregate together to form new tumor lesions near the vascular epithelium, and this tendency of tumor cells towards homologous tumor cells is called "homologous targeting".

[0003] Coating the outer layer of synthetic nanoparticles (NPs) with cell membranes of natural origin can form membrane nanovesicles (MNVs), which have the advantage of being able to completely replicate the surface antigens of the source cells and retain the unique properties of natural cell membranes, including their ability to prolong circulation time, immune escape, adhesion, and homologous targeting, thus exerting great potential for 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 their hybrid membranes, etc. This system has been proven to achieve effective drug delivery, tumor treatment, immune regulation, etc. Among them, cancer cell membranes (CCM) highly express specific proteins such as adhesion proteins, integrins, and adhesion kinases on the surface, enabling strong adhesion between homologous tumor cells and realizing cell self-recognition. Deeply exploring the key mechanism of tumor cell membrane nanovesicles targeting homologous tumors will be conducive to designing an efficient and precise engineered cell membrane delivery system, improving its efficiency of specifically targeting primary tumors and metastases, providing a new method for its clinical transformation, and being of great significance for the 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 advantages and delivery capabilities provide a solid foundation for their application in personalized precision medicine methods, but still face many challenges before clinical translational applications. On the one hand, when CC-MNVs coated with the patient's own tumor cell membranes enter the human body, they still retain a large number of cancer-related proteins, and only a few membrane proteins are closely related to the targeting effect, while most other membrane proteins may cause adverse immune reactions. On the other hand, invasive means such as surgery are required to obtain the patient's tumor tissue and then harvest primary tumor cells to extract tumor cell membranes. This process not only increases the risk of patient infection 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 of clinical cancer patients is insufficient to prepare enough tumor cell membrane vesicles. Therefore, new methods are needed to alleviate these problems. Summary of the Invention

[0005] In some embodiments, the present invention provides the use of normal fibroblasts in the preparation of drugs for treating tumors, tumor diagnostic reagents, nano-drug delivery systems, or engineered vesicles.

[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 cell strains.

[0008] In some embodiments, the normal fibroblasts are selected from established fibroblast cell lines or cell strains.

[0009] In some embodiments, the normal fibroblasts are selected from fibroblasts of the skin or adjacent tissues of cancer.

[0010] In some embodiments, the present invention provides a method for preparing engineered vesicles, comprising: (1) overexpressing a gene in normal fibroblasts to obtain fibroblasts overexpressing the gene; (2) extracting the membranes of the fibroblasts; (3) mixing the membranes of the fibroblasts with lipid nanoparticles to prepare the obtained engineered vesicles.

[0011] In some embodiments, the fibroblasts overexpressing the gene overexpress adhesion proteins or chemokine receptors.

[0012] In some embodiments, the fibroblasts overexpressing the gene further overexpress immunosuppressive molecules or cell-penetrating peptides.

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

[0014] In some embodiments, the gene further includes 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 fibroblasts are selected from autologous normal fibroblasts.

[0018] In some embodiments, the normal fibroblasts are selected from fibroblast cell lines or cell strains.

[0019] In some embodiments, the normal fibroblasts are selected from established fibroblast cell lines or cell strains.

[0020] In some embodiments, the normal fibroblasts are selected from fibroblasts of the skin or adjacent tissues of cancer. In some embodiments, in step (2), it includes obtaining a membrane solution of fibroblasts by using the freeze-thaw method, gradient centrifugation and resuspension in sequence.

[0021] In some embodiments, in step (3), it includes step (3.1): formulating the membranes of fibroblasts into a solution and then mixing with lipid nanoparticles to obtain a mixed solution of the membranes of fibroblasts and lipid nanoparticles.

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

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

[0024] In some embodiments, the fibroblast membrane solution is filtered through polycarbonate membranes with pore sizes of 300 - 500 nm and 100 - 350 nm in sequence and then mixed with lipid nanoparticles.

[0025] In some embodiments, in step (3.2), the filtration after mixing the fibroblast membrane solution and lipid nanoparticles is carried out by sequentially using polycarbonate membranes with pore sizes of 50 - 250 nm.

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

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

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

[0029] In some embodiments, the lipid nanoparticles are obtained by the solvent diffusion method.

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

[0031] In some embodiments, 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 for reaction to obtain a reaction solution; (A3) adding the reaction solution to ether, centrifuging to remove by-products to obtain a precipitate, and then drying the precipitate to obtain the 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 an aqueous solution of poloxamer 188, and reacting to prepare lipid nanoparticles.

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

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

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

[0035] In some embodiments, 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 to 20% of the anti-tumor drug, 10 to 20% of IR780-PEG-DSPE, 50 to 70% of DPPC, and 3 to 15% of cholesterol.

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

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

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

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

[0040] In some embodiments, the method for obtaining IR780-PEG-DSPE includes 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 the molar ratio of triethylamine to IR-780 iodide of (0.5 to 5):1, and adding it to the dichloromethane solution containing IR-780 iodide described in step (B1), and stirring and reacting for 8 to 30 h; (B3) after the reaction is completed, adding the reaction solution to 8 to 12 times the volume of anhydrous ether for reaction, centrifuging to remove by-products, and then drying the precipitate 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 includes RSL3 or doxorubicin.

[0043] In some embodiments, the present invention provides the engineered vesicles prepared by the described method.

[0044] The present invention utilizes this diversified, safe and widely applicable cell membrane targeting delivery system platform MNVs, and has made breakthroughs in the establishment of the basic theory and scientific and technological system for diagnosing and intervening metastatic tumors by applying bionic nanotechnology, providing a scientific basis and effective tool for the wide application of precision diagnosis and treatment of metastatic tumors.

[0045] In some embodiments, the present invention provides a biological membrane, which includes a fibroblast cell membrane overexpressing a gene, and the gene includes CD47 or CXCR4.

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

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

[0048] In some embodiments, the fibroblasts are selected from normal fibroblasts.

[0049] In some embodiments, the fibroblasts are selected from normal fibroblasts of autologous origin.

[0050] In some embodiments, the normal fibroblasts are selected from fibroblast lines or cell strains.

[0051] In some embodiments, the normal fibroblasts are selected from established fibroblast lines or cell strains.

[0052] In some embodiments, the normal fibroblasts are selected from fibroblasts of the skin or para-cancerous tissues. In some embodiments, the present invention provides a method for preparing the biofilm, which comprises overexpressing a gene in fibroblasts and then extracting the fibroblasts; the gene includes CD47 or CXCR4.

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

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

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

[0056] In some embodiments, the fibroblasts are selected from normal fibroblasts.

[0057] In some embodiments, the fibroblasts are selected from normal autologous fibroblasts.

[0058] In some embodiments, the normal fibroblasts are selected from fibroblast lines or cell strains.

[0059] In some embodiments, the normal fibroblasts are selected from established fibroblast lines or cell strains.

[0060] In some embodiments, the normal fibroblasts are selected from fibroblasts of the skin or para-cancerous tissues.

[0061] In some embodiments, the present invention provides a vesicle, and the raw materials for preparing the vesicle include the biofilm described above.

[0062] The vesicle containing the biofilm has the ability of highly specific and rapid targeting of multiple types of tumor tissues, can achieve tumor-targeted diagnostic imaging or drug delivery, while greatly reducing the accumulation in normal organs and non-target tissues, minimizing its toxic and side effects in the body, and significantly improving the safety.

[0063] In some embodiments, the vesicles are genetically engineered cell membrane nanovesicles.

[0064] The present invention provides a method for alleviating these problems existing in the prior art. One way is to screen key target proteins closely related to targeting according to the tumor cell membrane homologous targeting recruitment adhesion mechanism, and modify the key target receptor proteins onto the surface of fibroblast cell membranes by genetic engineering methods to prepare novel and safe MNVs for tumor targeting.

[0065] In some embodiments, the raw materials for preparing the vesicles further include a thermosensitive liposome drug delivery system.

[0066] In some embodiments, the raw materials for preparing the thermosensitive liposome drug delivery system include IR-780 iodide, NH2-PEG-DSPE, triethylamine, an anti-tumor drug, and DPPC.

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

[0068] In some embodiments, the present invention provides the engineered vesicles prepared by the method described above.

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

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

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

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

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

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

[0075] In some embodiments, the present invention provides the use of the vesicles or engineered vesicles or the composition in the preparation of a drug for combined use with laser therapy.

[0076] In some embodiments, the present invention provides the applications of the engineered fibroblast membrane nanovesicles in tumor diagnostic imaging, in the preparation of photothermal-responsive release of ferroptosis inducers in tumor tissues, and in the inhibition of tumor growth by photothermal therapy in combination with other therapies.

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

[0078] In some embodiments, the present invention provides the applications of the biological membrane, the engineered vesicles or the composition in the preparation of drugs for treating tumors, tumor diagnostic reagents or nano-drug delivery systems.

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

[0080] In some embodiments, the present invention provides the application of the biological membrane or the engineered vesicles in the preparation of nano-drug delivery systems.

[0081] In some embodiments, using the engineered fibroblast membrane nanovesicles as drug carriers can improve the bioavailability and tumor targeting of anti-tumor drugs, and can be used in combination with photothermal therapy. It can not only achieve the controlled release of anti-tumor drugs, but also accelerate the consumption of intracellular reduced glutathione and the accumulation of ROS. The inhibitory effect of anti-tumor drugs on GPX4 further exacerbates cellular oxidative stress and ferroptosis effects, bringing new hope for tumor treatment. Description of the Drawings

[0082] Figure 1 Optical photographs of autologous normal fibroblasts and genetically engineered fibroblasts;

[0083] Figure 2 Schematic diagram of the Western blot analysis results of protein expression of single-gene and multi-gene engineered fibroblasts and corresponding membrane vesicles.

[0084] Figure 3 In vivo distribution of genetically engineered fibroblast membrane nanovesicles X-Fb-MNVs with different mass ratios in MDA-MB-468 breast cancer-bearing mice; where: Figure 3 A shows the particle size changes of genetically engineered fibroblast membrane nanovesicles X-Fb-MNVs with different mass ratios; Figure 3 B shows the in vivo distribution maps of MDA-MB-468 breast cancer-bearing mice at 2 - 24 h after tail vein injection of X-Fb-MNVs with different mass ratios.

[0085] Figure 4Transmission electron microscopy image of genetically engineered fibroblast membrane nanovesicles X-Fb-MNVs.

[0086] Figure 5 Schematic diagrams 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.

[0087] Figure 6 Uptake of multi-gene engineered fibroblast membrane nanovesicles X-Fb-MNVs by macrophages;

[0088] Figure 7 In vivo distribution of X-Fb-MNVs or CXCR4-Fb-MNVs in a MDA-MB-468 breast cancer-bearing mouse model; where: Figure 7 A is the in vivo distribution map of MDA-MB-468 breast cancer-bearing mice at 2 - 48 h after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs; Figure 7 B is the schematic diagram of organ distribution of MDA-MB-468 breast cancer-bearing mice at 48 h after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-MNVs (in order of arrangement: heart, liver, spleen, lung, kidney, lymph nodes, tumor).

[0089] Figure 8 In vivo distribution of X-Fb-MNVs or CXCR4-Fb-MNVs in a 4T1 breast cancer-bearing mouse model; where: Figure 8 A is the in vivo distribution map of 4T1 breast cancer-bearing mice at 2 - 48 h after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs; Figure 8 B is the schematic diagram of organ distribution of 4T1 breast cancer-bearing mice at 48 h after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs (in order of arrangement: heart, liver, spleen, lung, kidney, lymph nodes, tumor).

[0090] Figure 9 In vivo distribution of X-Fb-MNVs or CXCR4-Fb-MNVs in a CT26 colon cancer-bearing mouse model; where: Figure 9 A is the in vivo distribution map of CT26 colon cancer-bearing mice at 2 - 48 h after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs; Figure 9Figure showing the organ distribution of CT26 colon cancer-bearing mice 48 h after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs (in the order of heart, liver, spleen, lung, kidney, tumor, lymph node).

[0091] Figure 10 Shows the in vivo distribution of X-Fb-MNVs or CXCR4-Fb-MNVs in the RIL175 hepatoma-bearing mouse model; among which: Figure 10 Figure A shows the in vivo distribution of RIL175 hepatoma-bearing mice 2 - 48 h after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs; Figure 10 Figure B shows the organ distribution of RIL175 hepatoma-bearing mice 48 h after tail vein injection of X-Fb-MNVs or CXCR4-Fb-MNVs or CTR-Fb-MNVs (in the order of heart, liver, spleen, lung, kidney, tumor, lymph node).

[0092] Figure 11 Shows the test results of the good photothermal conversion ability of gene-targeted fibroblast membrane nanovesicles; among which Figure 11 Figure A shows the temperature change results of engineered cell membrane nanovesicles MNVs irradiated with near-infrared laser; Figure 11 Figure B shows the temperature change curve of engineered cell membrane nanovesicles MNVs irradiated with near-infrared laser (808 nm, 1 W / cm 2 ) for 3 min.

[0093] Figure 12 Shows the cytotoxicity evaluation of engineered cell membrane vesicles on tumor cells MDA-MB-468 (12A) and KYSE-30 (12B).

[0094] Figure 13 Shows the level of lipid ROS induced by engineered cell membrane vesicles on tumor cells MDA-MB-468 (13A) and KYSE-30 (13B).

[0095] Figure 14 Shows the anti-tumor treatment effect of engineered cell membrane vesicles in the MDA-MB-468 breast cancer-bearing mouse model; among which: Figure 14 Figure A shows the tumor growth curve of MDA-MB-468 breast cancer-bearing mice within 21 days; Figure 14 Figure B shows the comparison chart of tumor tissue weights in different groups after the end of drug administration.

[0096] Figure 15 Shows the body weight change curve of MDA-MB-468 breast cancer-bearing mice within 21 days.

[0097] Figure 16 For the anti-tumor treatment effect of engineered cell membrane vesicles in the KYSE-30 esophageal cancer-bearing mouse model; among them: Figure 16 A is the tumor growth curve of KYSE-30 esophageal cancer-bearing mice within 21 days; Figure 16 B is the comparison chart of tumor tissue weights in different groups after the administration.

[0098] Figure 17 Is the weight change curve of KYSE-30 esophageal cancer-bearing mice within 21 days.

[0099] Figure 18 For the performance test results of engineered single-gene targeted NIH-3T3 cell membrane nanovesicles; among them, Figure 18 A-18B is the WB detection result of overexpressed membrane proteins in NIH-3T3 fibroblasts and the cell membrane nanovesicles obtained from them; Figure 18 C is the in vivo distribution map of MDA-MB-468 breast cancer-bearing mice at 2 - 48 h after tail vein injection of CXCR4-3T3-MNVs or CTR-3T3-MNVs; Figure 18 D is the schematic diagram of organ distribution of MDA-MB-468 breast cancer-bearing mice at 48 h after tail vein injection of CXCR4-3T3-MNVs or CTR-3T3-MNVs (in the order of heart, liver, spleen, lung, kidney, tumor, and lymph node).

[0100] Figure 19 For the anti-tumor treatment effect of engineered cell membrane vesicles in the 4T1 breast cancer-bearing mouse model, the comparison chart of tumor tissue weights in different groups after the administration.

[0101] Figure 20 For investigating the mRNA level of CD47 after gene transfection of different autologous cells by RT-qPCR.

[0102] Figure 21 For investigating the mRNA level of iRGD after gene transfection of different autologous cells by RT-qPCR.

[0103] Figure 22 For investigating the mRNA level of Pdcd1 after gene transfection of different autologous cells by RT-qPCR.

[0104] Figure 23 For investigating the mRNA level of CXCR4 after gene transfection of different autologous cells by RT-qPCR.

[0105] Figure 24 For the uptake of gene-engineered cell membrane nanovesicles from different cell sources by macrophages. Detailed implementation mode

[0106] The technical solutions of the present invention will be further described below through specific embodiments. The specific embodiments do not represent a limitation on the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0107] "Comprising" or "including" is intended to mean that a composition (such as a medium) and a method include the recited elements, but do not exclude other elements. When used to define a composition and a method, "consisting essentially of" means excluding other elements that are of any significant importance for the purpose of the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not substantially affect the basic and novel features of the claimed application. "Consisting of" means excluding trace elements of other components and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of this application. As used herein, the term "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and / or" means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a composition, combination, construct, etc. is described as including (or comprising) components A, B, C, and / or D, then the composition can include 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, B, C, and D used in combination.

[0108] In some embodiments, the "composition" is interpreted broadly. For example, a pharmaceutical composition, a drug set.

[0109] As used in the present invention, the term "pharmaceutical composition" refers to a product obtained by mixing or combining more than one active ingredient, including fixed combinations and non-fixed combinations of active ingredients. As used herein, terms such as "co-administering" or "combining administration" are intended to encompass administering selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the drugs are not necessarily administered by the same route or at the same time.

[0110] As used in the present invention, the term "fixed combination" means administering active ingredients (fibroblast cell membrane nanovesicles, and tumor therapeutic agents) to a patient simultaneously in the form of a single entity or dose.

[0111] As used herein, the term "non-fixed combination" means that the active ingredients are administered to a patient simultaneously, jointly or sequentially without a specific time limit as separate entities, wherein such administration can provide a therapeutically effective level of the two compounds in vivo, preferably at the same time. For example, a non-fixed combination can be two capsules each containing one active ingredient, which are intended to treat the patient in vivo with the two active ingredients together.

[0112] As used herein, "administered in combination" or "used in combination" means that two or more active substances or therapeutic means can be administered to a subject together, each as a single therapeutic means simultaneously or each as a single therapeutic means sequentially in any order (e.g., simultaneously, successively, intermittently). Among them, as mentioned in the present invention, "the use of the engineered vesicles in the preparation of a laser therapy in combination with a drug" refers to the use of the engineered vesicles in combination with a laser for anti-tumor treatment.

[0113] As used herein, the term "in combination with" when referring to administering a plurality of reagents to a subject means administering to the subject a first reagent and at least one other (i.e., second, third, fourth, fifth, etc.) reagent. As used herein, if, when administering the second reagent, the biological effect resulting from the administration of the first reagent persists in the subject such that the therapeutic effects of the first and second reagents are additive, then one reagent (engineered vesicles) is considered to be administered in combination with the second reagent (tumor therapeutic agent). The administration of the first reagent provides a therapeutic effect over a longer period of time, and the administration of the second reagent provides its therapeutic effect while the therapeutic effect of the first reagent is still ongoing. Therefore, the second reagent is considered to be administered in combination with the first reagent, even if the first reagent may have been administered at a time point far removed from the time of administration of the second reagent (such as days or weeks). In some embodiments, if the first and second reagents are administered simultaneously (within 30 minutes of each other), contemporaneously or sequentially, then the first reagent is considered to be administered in combination with the second reagent. In some embodiments, if the first reagent and the second reagent 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), then the first reagent is considered to be administered "contemporaneously" with the second reagent. The term "in combination with" should also be understood to apply to the situation where the first and second reagents are co-formulated in a single pharmaceutically acceptable formulation and the co-formulation is administered to the subject. In some embodiments, the engineered vesicles and the tumor therapeutic agent are administered or applied sequentially. For example, one or more other reagents are administered after administering one reagent. In some embodiments, they are administered simultaneously. For example, two or more reagents are administered at the same time or approximately the same time; the two or more reagents can be present in two or more separate formulations or combined into a single formulation (i.e., co-formulation). Regardless of whether these reagents are administered sequentially or simultaneously, as used herein, they are considered to be administered / used in combination.

[0114] In this article, "normal fibroblasts" refer to fibroblasts not derived from lesions.

[0115] All skin tissues or tissues adjacent to cancer are sourced in accordance with relevant standards, including the donor signing an informed consent form and undergoing donor screening and donor testing.

[0116] In this article, the CAS number of IR-780 iodide is 207399-07-3; the CAS number of RSL3 is 1219810-16-8; the CAS number of DPP is 63-89-8; the CAS number of poloxamer 188 is 9003-11-6; the CAS number of NH2-PEG-DSPE is 474922-26-4. Empty fibroblast membrane nanovesicles refer to the gene-targeted fibroblast membrane nanovesicles of the present invention without the addition of RSL3. Cell-penetrating peptides are also known as cell-penetrating peptides.

[0117] Preparation method of fibroblast membrane (CXCR4-Fb-M) overexpressing a single gene in Example 1

[0118] Autologous normal skin tissue was excised, rinsed 3 times with sterile cold PBS buffer, and the rinsed normal tissue was minced into uniform fragments of 1-2 mm 2 and then rinsed 3 times with sterile cold PBS buffer. The washed tissue pieces were laid flat in a 10 cm cell culture dish, and 4 ml of primary cell culture medium (DMEM medium containing 10% fetal bovine serum and 100 ug / ml primocin) was added. Then, it was placed in an incubator at 37 °C and 5% CO2 for 7 days, waiting for fibroblasts to crawl out, adhere, and proliferate from the normal tissue. The state under the microscope is as Figure 1 shown (CTR-Fibroblast).

[0119] The fibroblasts were seeded into a 6-well plate at 6x10 4 cells / ml, placed in an incubator at 37 °C and 5% CO2 for 24 h to allow them to adhere, and a lentivirus solution overexpressing the target gene CXCR4 containing the resistance gene puromycin and a high-efficiency transfection solution were added. The cells were transfected in an incubator at 37 °C and 5% CO2 for 72 h. Among them, the fibroblasts overexpressing CXCR4 (CXCR4-Fibroblast) are as Figure 1 shown.

[0120] Then the culture medium was replaced with primary cell culture medium containing 2ug / ml puromycin. After the cells in the non-transfected group were completely killed by puromycin, the cells overexpressing the target gene CXCR4 were digested with trypsin and expanded to obtain CXCR4-overexpressing fibroblasts (CXCR4-Fb), and the overexpression of CXCR4 was verified by WB.

[0121] Fibroblasts overexpressing a single target gene CXCR4 (CXCR4-Fb) were collected, and the cell pellets were washed three times with pre-cooled PBS buffer. After centrifugation, the PBS buffer was aspirated, and a membrane protein extraction reagent containing 1mM PMSF protease inhibitor was added to the cell pellet. After thorough vortexing, it was placed on ice for 15 minutes, and then vortexed once every 5 minutes. The broken cell pellets were then placed in liquid nitrogen and 37°C water baths and repeatedly frozen and thawed four times until the solution was uniform. The uniform cell disruption solution was then centrifuged at 4°C, 700g for 10 minutes, and the supernatant after centrifugation was centrifuged at 4°C, 14000g for 30 minutes. The precipitate after centrifugation was the engineered single gene targeted fibroblast membrane protein (CXCR4-Fb-M).

[0122] Example 2 Preparation of fibroblast membrane (X-Fb-M) overexpressing multiple genes

[0123] Cut the normal skin tissue from the body, rinse it with sterile cold PBS buffer for 3 times, and cut the rinsed normal tissue into 1-2 mm pieces. 2 The washed tissue fragments were spread on a 10 cm cell culture dish, and 4 ml of primary cell culture medium (DMEM culture medium containing 10% fetal bovine serum and 100 ug / ml primocin) was added. Then, the dish was incubated in a 37°C, 5% CO2 incubator for 7 days to wait for fibroblasts to crawl out of normal tissues, adhere to the wall and proliferate.

[0124] Fibroblasts were cultured at 8 x 10 4 100 μg / ml were plated in a 6-well plate and placed in a 37°C, 5% CO2 incubator for 24 h to allow it to adhere to the wall. At the same time, a lentiviral solution containing the resistance gene puromycin and overexpressing the target gene CXCR4 and a lentiviral solution containing the resistance gene G418 and overexpressing three target genes PD-1, CD47 and iRGD fusion protein plasmids as well as a high-efficiency transfection solution (Ji Kai Gene) were added. The cells were placed in a 37°C, 5% CO2 incubator for transfection for 72 h. The fibroblasts (X-Fibroblast) that simultaneously overexpressed CXCR4, PD-1, CD47 and iRGD were as follows: Figure 1 shown.

[0125] Then, replace it with the primary cell culture medium containing 2 μg / ml puromycin and 1.2 mg / ml G418. After all the cells in the untransfected group are completely killed by puromycin and G418, trypsinize the fibroblasts overexpressing the target genes CXCR4, PD-1, CD47, and iRGD, and expand the culture to obtain fibroblasts overexpressing multiple target genes (CXCR4 / PD-1 / CD47 / iRGD-Fb, i.e., X-Fb), and verify the overexpression of CXCR4, PD-1, and CD47 / iRGD by WB.

[0126] Collect the fibroblasts overexpressing multiple target genes (X-Fb), wash the cell pellet 3 times with pre-cooled PBS buffer, aspirate the PBS buffer after centrifugation, add the membrane protein extraction reagent containing 1 mM PMSF protease inhibitor to the cell pellet, vortex thoroughly and place on ice for 15 min, then vortex once every 5 min. Then, freeze-thaw the disrupted cell pellet 4 times between liquid nitrogen and 37 °C water bath until the solution is homogeneous. Then, centrifuge the homogeneous cell disruption solution at 4 °C and 700 g for 10 min, and take the supernatant after centrifugation and centrifuge it at 4 °C and 14,000 g for 30 min. The precipitate after centrifugation is the engineered multi-gene targeted fibroblast membrane protein (X-Fb-M).

[0127] Preparation method of thermosensitive lipid nanoparticle drug delivery system (IR780 / DPPC / RSL3, NVs, or IR / RSL3 NPs) in Example 3

[0128] Weigh 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), accurately weigh them, dissolve them in dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; use triethylamine (TEA) as an acid-binding agent, weigh triethylamine (TEA) according to the molar ratio of triethylamine (TEA) to IR-780 iodide (IR780) of 2:1, and add it to the dichloromethane solution containing IR-780 iodide, stir and react at room temperature for 24 h; after the reaction is completed, add the reaction solution to 10 times the volume of anhydrous ether, let it stand overnight at 4 °C, then centrifuge at 3000 rpm for 5 min to remove by-products, and then dry the precipitate to obtain IR-780 iodide modified lipid conjugate (IR780-PEG-DSPE).

[0129] Using absolute ethanol as a solvent, prepare each component of the lipid nanoparticles and add them successively in descending order of mass percentage under the water bath condition of 60 °C, and stir 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 is 20%, the mass percentage of IR780-PEG-DSPE is 20%, the mass percentage of the thermosensitive lipid DPPC (CAS No.: 63-89-8) is 55%, and the mass percentage of cholesterol is 5%.

[0130] Using deionized water as a solvent, prepare a 1 mg / ml poloxamer 188 (CAS: 9003-11-6) aqueous solution, measure the poloxamer 188 aqueous solution according to the volume ratio of the poloxamer 188 aqueous solution to the ethanol-phase solution of 10:1, and under the condition of 60 °C, quickly disperse the ethanol-phase solution into the poloxamer 188 aqueous solution, and stir at a speed of 400 rpm for 8 min, and then cool to room temperature to prepare a thermosensitive lipid nanoparticle drug delivery system (IR780 / DPPC / RSL3, NVs).

[0131] Use high performance liquid chromatography (HPLC) to determine the content of RSL3 in 20% IR / RSL3 NPs (i.e., IR780 / DPPC / RSL3, NVs). The determination conditions are: C18 reverse-phase chromatographic column; the mobile phase is acetonitrile: water = 7:3 (v / v, 1% acetic acid); the flow rate is 1.0 mL / min; the injection volume is 20 μL; the column temperature is 25 °C. Here, 20% IR / RSL3 NPs refers to the mass percentage of RSL3 contained in the ethanol phase for synthesizing IR / RSL3 NPs being 20%.

[0132] Standard curve preparation: Prepare a 1.0 mg / mL RSL3 / DMSO solution and dilute it 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) with the mobile phase, and detect the ultraviolet absorption peak at 290 nm by HPLC. Use Agilent OpenLab software to analyze the data, with the drug concentration C (μg / mL) as the abscissa and the chromatographic peak area A as the ordinate to prepare a standard curve.

[0133] Determination of entrapment efficiency and drug loading: Precise transfer 50 μL of the IR / RSL3 NPs solution, dilute it 10 times with the mobile phase, ultrasonicate in a water bath for 30 min, detect the drug concentration by HPLC, and calculate the drug entrapment efficiency and drug loading:[[]]

[0134] Entrapment efficiency = mass of RSL3 in the drug-loaded lipid nanoparticle sample / mass of RSL3 dosed * 100%;

[0135] Drug loading = mass of RSL3 in the drug-loaded lipid nanoparticles sample / (mass of RSL3 in the drug-loaded lipid nanoparticles sample + mass of the lipid nanoparticles) * 100%;

[0136] After calculation, the encapsulation efficiency of the ferroptosis inducer RSL3 in NVs was 71.90%, and the drug loading was 16.92%.

[0137] Preparation method of thermosensitive lipid nanoparticle drug delivery system (IR780 / DPPC / RSL3, NVs) in Example 4

[0138] Weigh 5 mg of IR-780 iodide and 20 mg of NH2-PEG-DSPE, accurately weigh them, dissolve them in dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; use triethylamine (TEA) as an acid-binding agent, weigh triethylamine (TEA) according to the molar ratio of triethylamine (TEA) to IR-780 iodide (IR780) of 1:1, and add it to the dichloromethane solution containing IR-780 iodide, stir and react at room temperature for 8 h; after the reaction is completed, add the reaction solution to 8 times the volume of anhydrous ether, let it stand overnight at 3 °C, and then centrifuge at 2500 rpm for 4 min to remove by-products, and then dry the precipitate to obtain IR-780 iodide-modified lipid conjugate (IR780-PEG-DSPE).

[0139] Using absolute ethanol as a solvent, prepare each component of the lipid nanoparticles, and add them successively in descending order of mass percentage under the water bath condition of 50 °C, and stir at a speed of 600 rpm for 30 s; among them, 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%.

[0140] Using deionized water as a solvent, prepare a 1.5 mg / ml poloxamer 188 aqueous solution, measure the poloxamer 188 aqueous solution according to the volume ratio of the poloxamer 188 aqueous solution to the ethanol phase solution of 8:1, and quickly disperse the ethanol phase solution into the poloxamer 188 aqueous solution at 55 °C, and stir at a speed of 600 rpm for 10 min, and then cool to room temperature to prepare a thermosensitive lipid drug delivery system (IR780 / DPPC / RSL3, IR NVs).

[0141] After determination (by the same method as in Example 3), the encapsulation efficiency of the ferroptosis inducer RSL3 in NVs was 67.24%, and the drug loading was 7.81%.

[0142] Preparation Method of Thermosensitive Lipid Nanodrug Delivery System (IR780 / DPPC / RSL3, NVs) in Example 5

[0143] Weigh 15 mg of IR-780 iodide and 50 mg of NH2-PEG-DSPE, accurately weigh them, dissolve them in dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; use triethylamine (TEA) as an acid-binding agent, weigh triethylamine (TEA) according to the molar ratio of triethylamine (TEA) to IR-780 iodide (IR780) of 3:1, and add it to the dichloromethane solution containing IR-780 iodide, stir and react at room temperature for 30 h; after the reaction is completed, add the reaction solution to 12 times the volume of anhydrous ether, let it stand overnight at 5 °C, then centrifuge at a speed of 3500 rpm for 6 min to remove by-products, and then dry the precipitate to obtain IR-780 iodide-modified lipid conjugate (IR780-PEG-DSPE).

[0144] Use anhydrous ethanol as a solvent to prepare each component of the lipid nanoparticles, and add them successively in descending order of mass percentage under the water bath condition of 70 °C, and stir at a speed of 300 rpm for 2 min; among them, the mass percentage of the ferroptosis inducer RSL3 in the ethanol phase solution is 15%, the mass percentage of IR780-PEG-DSPE is 20%, the mass percentage of the thermosensitive lipid DPPC is 62%, and the mass percentage of cholesterol is 3%;

[0145] Use deionized water as a solvent to prepare a 0.5 mg / ml poloxamer 188 aqueous solution, measure the poloxamer 188 aqueous solution according to the volume ratio of the poloxamer 188 aqueous solution to the ethanol phase solution of 12:1, and at 65 °C, quickly disperse the ethanol phase solution into the poloxamer 188 aqueous solution, and stir at a speed of 300 rpm for 5 min, then cool to room temperature to prepare a thermosensitive lipid drug delivery system (IR780 / DPPC / RSL3, NVs).

[0146] After measurement (using the same method as in Example 3), the encapsulation efficiency of the ferroptosis inducer RSL3 in IR NPs was 66.53%, and the drug loading was 11.74%.

[0147] Preparation Method of Thermosensitive Lipid Nanodrug Delivery System (IR780 / DPPC / RSL3, NVs) in Example 6

[0148] Weigh 15 mg of IR-780 iodide and 20 mg of NH2-PEG-DSPE accurately, dissolve them in dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; use triethylamine (TEA) as an acid-binding agent, weigh triethylamine (TEA) according to the molar ratio of triethylamine (TEA) to IR-780 iodide (IR780) of 3:1, and add it to the dichloromethane solution containing IR-780 iodide, and stir the reaction at room temperature for 8 h; after the reaction is completed, add the reaction solution to 12 times the volume of anhydrous ether, let it stand overnight at 5 °C, and then centrifuge for 4 min at a speed of 3500 rpm to remove by-products, and then dry the precipitate to obtain IR-780 iodide-modified lipid graft (IR780-PEG-DSPE).

[0149] Use absolute ethanol as a solvent to prepare each component of the lipid nanoparticles, and add them successively in descending order of mass percentage under the water bath condition of 60 °C, and stir at a speed of 600 rpm for 1 min; among them, 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%.

[0150] Use deionized water as a solvent to prepare a 1.5 mg / ml poloxamer 188 aqueous solution, measure the poloxamer 188 aqueous solution according to the volume ratio of the poloxamer 188 aqueous solution to the ethanol phase solution of 12:1, and at 60 °C, quickly disperse the ethanol phase solution into the poloxamer 188 aqueous solution, and stir at a speed of 600 rpm for 8 min, and then cool to room temperature to prepare a thermosensitive lipid drug delivery system (IR780 / DPPC / RSL3, NVs).

[0151] After measurement (the same method as in Example 3), the encapsulation efficiency of the ferroptosis inducer RSL3 in NVs was 66.6%, and the drug loading was 11.8%.

[0152] Preparation method of engineered single-gene targeted fibroblast membrane nanovesicles (CXCR4-Fb-MNVs) in Example 7

[0153] The engineered single-gene targeted fibroblast membrane protein (CXCR4-Fb-M) obtained in Example 1 was diluted with PBS to 0.6 mg / ml, and filtered successively through polycarbonate membranes with pore sizes of 400 nm and 200 nm 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 (mass of CXCR4-Fb-M protein (referring to the mass of the cell membrane solution at 0.6 mg / ml): mass of NVs = 1:0.5), and then co-extruded through a 100-nm polycarbonate membrane using a liposome extruder to obtain engineered single-gene targeted fibroblast cell membrane nanovesicles (CXCR4-Fb-MNVs).

[0154] Example 8 Preparation method of engineered single-gene targeted fibroblast cell membrane nanovesicles (CXCR4-Fb-MNVs)

[0155] The engineered single-gene targeted fibroblast membrane protein (CXCR4-Fb-M) obtained in Example 1 was diluted with PBS to 0.6 mg / ml, and filtered successively through polycarbonate membranes with pore sizes of 400 nm and 200 nm 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 (mass of CXCR4-Fb-M protein (referring to the mass of the cell membrane solution at 0.6 mg / ml): mass of NVs = 1∶1), and then co-extruded through a 100-nm polycarbonate membrane using a liposome extruder to obtain engineered single-gene targeted fibroblast cell membrane nanovesicles (CXCR4-Fb-MNVs).

[0156] Example 9 Preparation method of engineered single-gene targeted fibroblast cell membrane nanovesicles (CXCR4-Fb-MNVs)

[0157] The engineered single-gene targeted fibroblast membrane protein (CXCR4-Fb-M) obtained in Example 1 was diluted with PBS to 0.6 mg / ml, and filtered successively through polycarbonate membranes with pore sizes of 400 nm and 200 nm 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 (mass of CXCR4-Fb-M protein (referring to the mass of the cell membrane solution at 0.6 mg / ml): mass of NVs = 1:2), and then co-extruded through a 100-nm polycarbonate membrane using a liposome extruder to obtain engineered single-gene targeted fibroblast cell membrane nanovesicles (CXCR4-Fb-MNVs).

[0158] Preparation method of engineered multi-gene targeted fibroblast membrane nanovesicles (X-Fb-MNVs) in Example 10

[0159] Dilute the engineered multi-gene targeted fibroblast membrane protein solution (X-Fb-M) obtained in Example 2 with PBS to 0.6 mg / ml, and filter it successively through polycarbonate membranes with pore sizes of 400 nm and 200 nm using a liposome extruder. Mix the filtered engineered fibroblast membrane protein solution with the thermosensitive lipid nano-delivery system (IR780 / DPPC / RSL3, NVs) obtained in Example 3 according to the set mass ratio (mass of X-Fb-M protein (referring to the mass of the cell membrane solution at 0.6 mg / ml): mass of NVs = 1:0.5), and then co-extrude through a 100-nm polycarbonate membrane using a liposome extruder to obtain engineered fibroblast membrane nanovesicles (X-Fb-MNVs).

[0160] Preparation method of engineered multi-gene targeted fibroblast membrane nanovesicles (X-Fb-MNVs) in Example 11

[0161] Dilute the engineered multi-gene targeted fibroblast membrane protein solution (X-Fb-M) obtained in Example 2 with PBS to 0.6 mg / ml, and filter it successively through polycarbonate membranes with pore sizes of 400 nm and 200 nm using a liposome extruder. Mix the filtered engineered fibroblast membrane protein solution with the thermosensitive lipid nano-delivery system (IR780 / DPPC / RSL3, NVs) obtained in Example 3 according to the set mass ratio (mass of X-Fb-M protein (referring to the mass of the cell membrane solution at 0.6 mg / ml): mass of NVs = 1∶1), and then co-extrude through a 100-nm polycarbonate membrane using a liposome extruder to obtain engineered fibroblast membrane nanovesicles (X-Fb-MNVs).

[0162] Preparation method of engineered multi-gene targeted fibroblast membrane nanovesicles (X-Fb-MNVs) in Example 12

[0163] The engineered multi-gene targeted fibroblast membrane protein solution (X-Fb-M) obtained in Example 2 was diluted to 0.6 mg / ml with PBS and filtered successively through polycarbonate membranes with pore sizes of 400 nm and 200 nm using a liposome extruder. The filtered engineered fibroblast membrane protein solution was mixed with the thermosensitive lipid nano-delivery system (IR780 / DPPC / RSL3, NVs) obtained in Example 3 at a set mass ratio (mass of X-Fb-M protein (referring to the mass of the cell membrane solution at 0.6 mg / ml): mass of NVs = 1:2), and then co-extruded through a 100 nm polycarbonate membrane using a liposome extruder to obtain engineered fibroblast membrane nanovesicles (X-Fb-MNVs).

[0164] Example 13 WB Detection of Overexpressed Membrane Proteins in Fibroblasts and the Obtained Cell Membrane Nanovesicles

[0165] 1. Detection of Overexpressed Single Membrane Protein CXCR4 in Fibroblasts and the Obtained Cell Membrane Nanovesicles

[0166] To verify that the single-gene engineered fibroblasts obtained in the present invention successfully overexpress the membrane protein CXCR4, WB detection was performed using the fibroblasts (CXCR4-Fb or CXCR4-Fibroblast) obtained in Example 1 and the CXCR4-Fb-MNVs obtained in Example 7 as examples:

[0167] Take cell lysates of CXCR4-Fb cells, CTR-Fb (control fibroblasts) cells containing equal amounts of protein, and the corresponding membrane vesicle solutions CXCR4-Fb-MNVs and the control group CTR-Fb-MNVs solution. After adding 5x protein loading buffer, vortex and mix well, and heat in a 100 °C metal bath for 10 min. Load the prepared protein onto an SDS-PAGE gel and perform electrophoresis at a constant voltage of 120 V in an environment of 1x Tris-Glycine-SDS electrophoresis buffer. Transfer the separated proteins on the SDS-PAGE gel after electrophoresis to a PVDF membrane using the wet transfer method, in an environment of 1x TG transfer buffer, at a constant current of 280 mA for 2 h. Block the PVDF membrane after transfer with 5% skim milk for 1 h, then incubate overnight at 4 °C with anti-CXCR4 antibody, and incubate with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h the next day. Finally, detect the protein signal using a high-sensitivity chemiluminescent substrate and a ChemiDoc imaging system.

[0168] The results are as Figure 2As shown, the expression level of CXCR4 protein in the CXCR4-Fb group was significantly higher than that in the CTR-Fb group, indicating that the single-gene engineered fibroblasts successfully overexpressed the membrane protein CXCR4. In addition, the membrane protein CXCR4 was highly expressed on the fibroblast membrane nanovesicles CXCR4-Fb-MNVs, indicating that this method retained the engineered cell membrane proteins.

[0169] 2. Detection of overexpression of multiple membrane proteins in fibroblasts and the obtained cell membrane nanovesicles

[0170] To verify that the multi-gene engineered fibroblasts obtained in the present invention successfully overexpressed the membrane proteins CXCR4, PD-1, CD47, and iRGD, the WB detection was performed using the fibroblasts (X-Fb or X-Fibroblast) obtained in Example 2 and the X-Fb-MNVs obtained in Example 10 as examples:

[0171] Take the X-Fb cell lysate, CTR-Fb cell lysate containing equal amounts of cell membrane proteins, and the corresponding membrane vesicle solutions X-Fb-MNVs and the control group CTR-Fb-MNVs solution. After adding 5x protein loading buffer, vortex and mix well, and heat in a 100 °C metal bath for 10 min. Load the prepared proteins onto an SDS-PAGE gel and perform electrophoresis under the condition of a constant voltage of 120 V in an environment of 1x Tris-Glycine-SDS electrophoresis buffer. Transfer the separated proteins on the SDS-PAGE gel after electrophoresis to a PVDF membrane. Using the wet transfer method, perform membrane transfer under the condition of a constant current of 280 mA for 2 h in an environment of 1xTG transfer buffer. Block the PVDF membrane after membrane transfer with 5% skim milk for 1 h, then incubate overnight at 4 °C with anti-CXCR4, anti-PD-1, and anti-CD47 antibodies respectively. The next day, incubate with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h, and finally detect the protein signal using a high-sensitivity chemiluminescent substrate and a ChemiDoc imaging system.

[0172] As Figure 2 shown, the expression levels of CXCR4, PD-1, and CD47 proteins in the X-Fb group were up-regulated compared with the control group, indicating that the multi-gene engineered fibroblasts successfully overexpressed the membrane proteins CXCR4, PD-1, CD47, and iRGD proteins. In addition, the membrane proteins CXCR4, PD-1, CD47, and iRGD proteins were highly expressed on the membrane vesicles X-Fb-MNVs, indicating that this method retained the engineered cell membrane proteins.

[0173] Example 14 Performance Detection of Multi-Gene Engineered Cell Membrane Nanovesicles X-Fb-MNVs

[0174] 1. Detection of particle size and zeta potential of lipid delivery systems

[0175] Prepare thermosensitive lipid drug delivery systems modified with IR-780 iodide (IR / RSL3 NPs) with different RSL3 contents, and the mass ratios of RSL3 are 10%, 15% and 20% respectively (the mass ratio here refers to the content ratio of RSL3 in the ethanol-phase solution during the preparation process of IR / RSL3 NPs). Use a particle size and surface potential analyzer to measure the particle size and surface potential of the empty thermosensitive lipid drug delivery system (IRNPs) and IR / RSL3 NPs with three mass ratios respectively.

[0176] Among them, the preparation method of IR / RSL3 NPs is the same as that in Example 3.

[0177] The measurement 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 IRNPs 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 loading drugs, and increases with the increase of the concentration of RSL3. At the same time, the charge of IR / RSL3 NPs after loading drugs reverses from positive charge to negative charge and increases with the increase of the concentration of RSL3.

[0178] Table 1

[0179] Preparation Particle size (nm) Zeta potential (mV) IR NPs 67.54±0.93 6.17±1.60 10% IR / RSL3 NPs 72.67±5.33 -6.90±1.19 15% IR / RSL3 NPs 91.45±13.10 -9.90±1.43 20% IR / RSL3 NPs 98.72±6.65 -15.60±1.53

[0180] 2. Particle size detection of X-Fb-MNVs

[0181] Mix lipid nanoparticles NVs (20% IR / RSL3 NPs) with 0.6 mg / mL engineered X-Fb cell membrane solution at different mass ratios, and prepare engineered biomimetic membrane nanovesicles (X-Fb-MNVs, i.e., M / IR / RSL3 NPs) by probe sonication, and measure their particle size.

[0182] The results are as Figure 3 shown in A. When the mass ratio of the engineered cell membrane to the lipid nanoparticles (i.e., the thermosensitive lipid nanoparticle drug delivery system) NVs is 1:2, the particle size of X-Fb-MNVs is 130 ± 3.95 nm. The results prove that as the mass of the lipid nanoparticles NVs increases, the particle size of the cell membrane nanovesicles X-Fb-MNVs becomes smaller and more uniform and stable.

[0183] The remaining steps of the preparation method of X-Fb-MNVs are the same as those in Example 10.

[0184] 3. In vivo distribution of engineered cell membrane nanovesicles X-Fb-MNVs in 4T1 tumor-bearing mice

[0185] A small animal in vivo imager was used to observe 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.

[0186] As Figure 3 Shown in B, after intravenous injection of X-Fb-MNVs, when the mass ratio of lipid nanoparticles NVs to engineered cell membrane was 2:1, the distribution of X-Fb-MNVs at the tumor site was significantly stronger than that of other groups, indicating that when the mass ratio of lipid nanoparticles NVs to engineered cell membrane was 2.0, the engineered cell membrane nanovesicles X-Fb-MNVs had strong tumor targeting ability.

[0187] The remaining steps of the preparation method of X-Fb-MNVs are the same as those in Example 10.

[0188] 4. Transmission electron microscopy images of cell membrane nanovesicles X-Fb-MNVs

[0189] Solutions of lipid nanoparticles NVs and engineered cell membrane nanovesicles X-Fb-MNVs at 1 mg / mL were respectively diluted to 0.1 mg / mL with deionized water, dropped onto a copper mesh covered with a carbon film, stained with 2% (w / v) uranyl acetate, and the excess liquid was blotted with filter paper. The morphology and particle size of NVs and X-Fb-MNVs were observed by transmission electron microscopy respectively.

[0190] The morphology of the nanoparticles under transmission electron microscopy is as Figure 4 shown. 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 have an obvious "core-shell structure", which further proves that the cell membrane is successfully wrapped on the surface of the lipid nanoparticles NVs.

[0191] The preparation method of X-Fb-MNVs is the same as that in Example 10, and the preparation method of lipid nanoparticles NVs is the same as that in Example 3.

[0192] 5. Evaluation of the uptake ability of tumor cells and normal cells to engineered fibroblast cell membrane nanovesicles

[0193] 4T1 breast cancer cells and normal fibroblasts were respectively seeded at appropriate densities in 24-well plates with round cell culture slides and incubated in a 37 °C, 5% CO2 incubator for 24 h to allow them to adhere (the adhesion ratio was about 60% for both); after the cells adhered, engineered cell membrane vesicle solutions CXCR4-Fb-MNVs and X-Fb-MNVs labeled with FITC fluorescence with the same fluorescence amount and the control group CTR-Fb-MNVs were added to the culture medium, and incubation was continued for 12 h. Then, the culture medium was removed, and the slides were rinsed 3 times with PBS buffer. The cells on the slides were fixed with 4% paraformaldehyde fixative for 15 min. After removing the fixative, they were rinsed 3 more times with PBS buffer. The PBS residue on the slides was gently blotted dry, and the slides were covered face-down on glass slides with DAPI staining solution dropped on them, and then sealed with a mounting medium.

[0194] The fluorescence distribution of the prepared glass slides in the FITC band was observed through a laser confocal microscope, as Figure 5 shown. It could be clearly observed that compared with the fibroblast membrane nanovesicles CTR-Fb-MNVs of the control group, the multi-gene engineered cell membrane nanovesicles X-Fb-MNVs were significantly more taken up by tumor cells and less taken up by normal cells.

[0195] Among them, CXCR4-Fb-MNVs and X-Fb-MNVs were obtained by the methods of Example 9 and Example 12 respectively.

[0196] 6. Evaluate the ability of multi-gene engineered cell membrane nanovesicles to evade macrophage phagocytosis

[0197] The macrophage cell line RAW264.7 was seeded at a density of 1×10 5 cells / ml in 24-well plates with round cell culture slides and incubated in a 37 °C, 5% CO2 incubator for 24 h to allow them to adhere; after the cells adhered, FITC-fluorescently labeled multi-gene engineered cell membrane nanovesicles X-Fb-MNVs and a CD47 blocker (MCE, HY-143224B) were added to the culture medium, and incubation was continued for 4 h. Then, the culture medium was removed, and the slides were rinsed 3 times with PBS buffer. The cells on the slides were fixed with 4% paraformaldehyde fixative for 15 min. After removing the fixative, they were rinsed 3 more times with PBS buffer. The PBS residue on the slides was gently blotted dry, and the slides were covered face-down on glass slides with DAPI staining solution dropped on them, and then sealed with a mounting medium.

[0198] The fluorescence distribution of the prepared glass slides in the FITC band was observed through a laser confocal microscope, as Figure 6As shown, it can be clearly observed that compared with the fibroblast cell membrane nanovesicles CTR-Fb-MNVs of the control group, the multi-gene engineered cell membrane nanovesicles X-Fb-MNVs are significantly less likely to be taken up by macrophages. 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 being phagocytosed by macrophages due to overexpression of the membrane protein CD47.

[0199] X-Fb-MNVs were prepared by the method of Example 12.

[0200] 7. Evaluate the targeting ability of engineered fibroblast cell membrane nanovesicles on multiple models of breast cancer, colon cancer, and liver cancer:

[0201] 7.1 Targeting ability on breast cancer

[0202] 8×10 6 MDA-MB-468 breast cancer cells were inoculated at the left lower breast of 4-week-old female nude mice. After 6 weeks, an MDA-MB-468 breast cancer orthotopic tumor-bearing mouse model was successfully established. The tumor-bearing mice were randomly divided into three groups, and equal amounts of engineered cell membrane vesicle solutions CXCR4-Fb-MNVs and X-Fb-MNVs labeled with the near-infrared fluorescent probe IR780 and the control group CTR-Fb-MNVs were injected via the tail vein. A small animal in vivo imager was used to observe the fluorescence distribution in MDA-MB-468 tumor-bearing mice within 2 - 48 h after injection (the fluorescence imaging band captured in this experiment was 720 - 845 nm, which coincides with the emission band of IR780).

[0203] The results are as Figure 7 shown in A. The fluorescence intensity at the tumor sites of the multi-gene engineered fibroblast cell membrane nanovesicles X-Fb-MNVs group and the single-gene engineered fibroblast cell membrane nanovesicles CXCR4-Fb-MNVs group of mice was significantly higher than that of the control group CTR-Fb-MNVs, and the aggregation level of X-Fb-MNVs in the tumor region was higher than that of CXCR4-Fb-MNVs.

[0204] After 48 h, 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 nodes, and tumor. The fluorescence distribution in the organs was observed through a small animal imager. Figure 7 B shows that the fluorescence at the tumor site of the X-Fb-MNVs group was significantly stronger than that of other groups, and no obvious accumulation was seen in the liver, kidney, and other organs. 5×10 54T1 breast cancer cells were used to successfully establish an orthotopic tumor-bearing mouse model of 4T1 breast cancer after 4 weeks. The tumor-bearing mice were randomly divided into three groups and were respectively injected via the tail vein with engineered cell membrane vesicle solutions CXCR4-Fb-MNVs and X-Fb-MNVs labeled with an equal amount of near-infrared fluorescent probe IR780 and the control group CTR-Fb-MNVs. A small animal in vivo imager was used to observe the fluorescence distribution in 4T1 tumor-bearing mice within 2 - 48 h after injection. As Figure 8 shown in A, the fluorescence intensity at the tumor sites of 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 of the control group CTR-Fb-MNVs, and the aggregation level of X-Fb-MNVs in the tumor region was higher than that of CXCR4-Fb-MNVs.

[0205] After 48 h, the mice were sacrificed by cervical dislocation, and their internal organs were taken and placed in the order of heart, liver, spleen, lung, kidney, axillary lymph nodes, and tumor. The fluorescence distribution in the internal organs was observed through a small animal imager. Figure 8 B shows that the fluorescence at the tumor site in the X-Fb-MNVs group was significantly stronger than that in other groups, and X-Fb-MNVs mainly aggregated in the tumor tissue and less accumulated in the metabolic organs liver and kidney.

[0206] Among them, CXCR4-Fb-MNVs and X-Fb-MNVs were respectively prepared by the methods of Example 9 and Example 12.

[0207] 7.2 Targeting ability to colon cancer

[0208] 1 * 10 6 CT-26 colon cancer cells were subcutaneously inoculated on the left side of 6-week-old female BALB / c mice. After 3 weeks, a subcutaneous tumor-bearing mouse model of CT-26 colon cancer was successfully established. The tumor-bearing mice were randomly divided into 3 groups and were respectively injected via the tail vein with engineered cell membrane vesicle solutions CXCR4-Fb-MNVs and X-Fb-MNVs labeled with an equal amount of near-infrared fluorescent probe IR780 and the control group CTR-Fb-MNVs.

[0209] A small animal in vivo imager was used to observe the fluorescence distribution in CT-26 tumor-bearing mice within 2 - 48 h after injection. As Figure 9 shown in A, the fluorescence intensity at the tumor sites of 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 of the control group CTR-Fb-MNVs, and the aggregation level of X-Fb-MNVs in the tumor region was higher than that of CXCR4-Fb-MNVs.

[0210] After 48 h, the mice were sacrificed by cervical dislocation. Their organs were taken out and arranged 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 imager. Figure 9 B shows that the fluorescence in the tumor site of the X-Fb-MNVs group was significantly stronger than that in other groups, and no obvious accumulation was seen in the liver, kidney, and other organs.

[0211] Among them, CXCR4-Fb-MNVs and X-Fb-MNVs were prepared by the methods of Example 9 and Example 12, respectively.

[0212] 7.3 Targeting ability to liver cancer

[0213] 1×10 6 RIL-175 liver cancer cells were subcutaneously inoculated on the left side of 4-week-old male C57(BL / 6N) mice. After 3 weeks, a subcutaneous tumor-bearing mouse model of RIL-175 liver cancer was successfully established. The tumor-bearing mice were randomly divided into 3 groups and were respectively injected via the tail vein with engineered cell membrane vesicle solutions CXCR4-Fb-MNVs and X-Fb-MNVs labeled with an equal amount of near-infrared fluorescent probe IR780 and the control group CTR-Fb-MNVs.

[0214] A small animal in vivo imager was used to observe the fluorescence distribution in RIL-175 tumor-bearing mice within 2 - 48 h after injection. As Figure 10 shown in A, the fluorescence intensity at the tumor site in the mice of 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.

[0215] After 48 h, the mice were sacrificed by cervical dislocation. Their organs were taken out and arranged 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 imager. Figure 10 B shows that the fluorescence in the tumor site of the X-Fb-MNVs group was significantly stronger than that in other groups, and no obvious accumulation was seen in the liver, kidney, and other organs.

[0216] Among them, CXCR4-Fb-MNVs and X-Fb-MNVs were prepared by the methods of Example 9 and Example 12, respectively.

[0217] According to the in vitro and in vivo experimental results shown above, both the single-gene engineered fibroblast membrane nanovesicles (CXCR4-Fb-MNVs) and multi-gene engineered fibroblast membrane nanovesicles (X-Fb-MNVs) prepared by using the method of the present invention have the ability of high specificity and rapid targeting of various types of tumor tissues, while avoiding being phagocytosed by macrophages during blood circulation, greatly reducing the accumulation in normal organs and non-target tissues, and minimizing their toxic and side effects in vivo.

[0218] Example 15 Killing Ability of Engineered Fibroblast Membrane Nanovesicles against Tumor Cells

[0219] In order to evaluate the killing ability of the engineered fibroblast membrane nanovesicles obtained in the present invention against tumor cells, now taking the engineered fibroblast membrane nanovesicles CXCR4-Fb-MNVs (i.e., M / IR / RSL3 NPs or MNVs) obtained in Example 9 as an example, the following tests are carried out:

[0220] 1. Evaluation of the photothermal effect of engineered fibroblast membrane nanovesicles

[0221] Take equal volumes of PBS and the aqueous solution of single-gene targeted fibroblast membrane nanovesicles with an IR-780 content of 5.0 μg / mL, and place them in an incubator to equilibrate the temperature. Use a near-infrared laser with a wavelength of 808 nm to irradiate each sample at a power of 1 W / cm 2 for 3 min, and use an infrared thermal imager to record the temperature change trend of the samples.

[0222] As Figure 11 shown in Figure 11 A and

[0223] B, after being irradiated with near-infrared laser, the temperature of the aqueous solution of single-gene targeted fibroblast membrane nanovesicles rises to a maximum of 50.6 °C, and still remains at 45.2 °C at the 3rd min, while the temperature of the control group (PBS) hardly changes and remains at about 31 °C. This result indicates that the single-gene targeted fibroblast membrane nanovesicles have good photothermal conversion ability.

[0224] 2. Evaluation of the killing effect of engineered fibroblast membrane nanovesicles on in vitro tumor cells:

[0225] Seed breast cancer cells MDA-MB-468 and esophageal cancer cells KYSE-30 at 1*10 4Inoculate at a density of [number] / 100 μl into 96-well plates. After the cells adhered to the wall, each cell line was divided into 8 groups (control (DMSO), control + laser (DMSO + Laser), small molecule drug RSL3 (RSL3), RSL3 + laser (RSL3 + Laser), fibroblast cell membrane nanovesicles without drug loading (M / IR NPs), fibroblast cell membrane nanovesicles without drug loading + laser (M / IR NPs + Laser), fibroblast cell membrane nanovesicles loaded with drug (M / IR / RSL3 NPs), and fibroblast cell membrane nanovesicles loaded with drug + laser (M / IR / RSL3 NPs + Laser)). Add drugs according to the groups (the concentration of RSL3 in the groups containing RSL3 in MDA-MB-468 cells was 3 μM, and the concentration of RSL3 in the groups containing RSL3 in KYSE-30 cells was 1 μM), and place them in an incubator at 37 °C and 5% CO2 for 4 h. Irradiate each laser group with a near-infrared laser with a wavelength of 808 nm at a power of 1 W / cm 2 for 3 min, then continue to incubate the cells in each group for 20 h, and use a CCK8 kit (Meilun) and a microplate reader to detect the cell viability of each group.

[0226] The detection results are as Figure 12 shown in Figures A and 12B. The small molecule drug RSL3 can inhibit the proliferation of MDA-MB-468 and KYSE-30 cells to a certain extent, but laser irradiation does not promote its anti-tumor effect; the fibroblast cell membrane nanovesicles without drug loading itself do not have obvious toxicity to tumor cells, but under laser irradiation, due to the generation of a photothermal effect, the death of tumor cells is accelerated; while the group of fibroblast cell membrane nanovesicles loaded with drug shows the most significant cell death phenomenon under laser irradiation.

[0227] 2.2 Detection of lipid peroxide levels in cells of each group

[0228] Inoculate MDA-MB-468 and KYSE-30 cells at a density of 3×10 5 cells / ml into 6-well plates. After the cells adhered to the wall, each cell line was divided into 8 groups according to the above cell toxicity test, add drugs according to the groups (the concentration of RSL3 in the groups containing RSL3 in MDA-MB-468 cells was 4 μM, and the concentration of RSL3 in the groups containing RSL3 in KYSE-30 cells was 2 μM), and place them in an incubator at 37 °C and 5% CO2 for 4 h. Irradiate each laser group with a near-infrared laser with a wavelength of 808 nm at a power of 1 W / cm 2 for 3 min, then continue to incubate the cells in each group for 2 h, and use BODIPY 581 / 591C11 probe (Invitrogen) and flow cytometry to detect the lipid peroxide levels in cells of each group.

[0229] The detection results are as follows Figure 13 shown. Both the small molecule drug RSL3 and the drug-free fibroblast cell membrane nanovesicles after laser irradiation can induce ferroptosis in breast cancer and esophageal cancer cells to a certain extent. However, when the two are combined, that is, the drug-loaded fibroblast cell membrane nanovesicles combined with near-infrared laser can induce the ferroptosis effect of tumor cells to the greatest extent.

[0230] 3. Evaluate the in vivo efficacy and safety of engineered fibroblast cell membrane nanovesicles in tumor-bearing mice:

[0231] Inoculate 8×10 6 MDA-MB-468 breast cancer cells at the left lower breast of 4-week-old female nude mice. When the tumors of the mice grow to 80-100 mm 3 , randomly divide the tumor-bearing mice into 8 groups (PBS, PBS+laser, RSL3, RSL3+laser, drug-free fibroblast cell membrane nanovesicles, drug-free fibroblast cell membrane nanovesicles+laser, drug-loaded fibroblast cell membrane nanovesicles, and drug-loaded fibroblast cell membrane nanovesicles+laser). Inject the corresponding drugs (the dose of RSL3 in the groups containing RSL3 is 1 mg / kg) through the tail vein 5 times continuously, with an interval of 48 h each time. During each administration, 24 h later, irradiate the tumors of the mice in the laser group with near-infrared laser at a wavelength of 808 nm with a power of 0.8 W / cm 2 for 3 min. Continuously record the body weight of the mice and the volume of the tumors during the drug administration period until the 21-day observation period, and sacrifice the mice on the 21st day to weigh their tumors.

[0232] Inoculate 5×10 6 KYSE-30 esophageal cancer cells subcutaneously on the left side of 4-week-old male nude mice. When the tumors of the mice grow to 80-100 mm 3 , randomly divide the tumor-bearing mice into 8 groups according to the above in vivo experiment of MDA-MB-468 mice. Inject the corresponding drugs (the dose of RSL3 in the groups containing RSL3 is 1 mg / kg) through the tail vein 5 times continuously, with an interval of 48 h each time. During each administration, 24 h later, irradiate the tumors of the mice in the laser group with near-infrared laser at a wavelength of 808 nm with a power of 0.8 W / cm 2 for 3 min. Continuously record the body weight of the mice and the volume of the tumors during the drug administration period until the 21-day observation period, and sacrifice the mice on the 21st day to weigh their tumors.

[0233] The results of the anti-tumor treatment experiment are as follows Figure 14 (MDA-MB-468 breast cancer mouse model) and Figure 16(KYSE-30 esophageal cancer mouse model) As shown, in all groups, the drug-loaded fibroblast cell membrane nanovesicles combined with near-infrared laser treatment achieved the best in vivo anti-tumor effect; the tumors of mice in the negative control (PBS group) continued to grow, while the tumor growth of mice in the PBS combined with laser group and the non-drug-loaded fibroblast cell membrane nanovesicle treatment group was not effectively inhibited, demonstrating that single near-infrared laser irradiation and single empty fibroblast cell membrane nanovesicles have no therapeutic effect on tumors; and the anti-tumor effect of the drug-loaded fibroblast cell membrane nanovesicle treatment group was better than that of the RSL3 treatment group and the RSL3 combined with laser group, proving that fibroblast cell membrane nanovesicles improved the solubility of RSL3 in blood circulation and its targeting to tumors; we also found that although the non-drug-loaded fibroblast cell membrane nanovesicles combined with laser treatment could also inhibit tumor growth to a certain extent, indicating that photothermal therapy can effectively kill tumor cells, but due to its lack of loading of the ferroptosis inducer RSL3, it could not induce ferroptosis of tumor cells and could not achieve the best therapeutic effect. After the treatment, the change trend of the tumor weight of mice was consistent with that of the tumor volume.

[0234] By observing the weight changes of tumor-bearing mice in different groups during the treatment period, the in vivo safety of the engineered fibroblast cell membrane nanovesicles was evaluated. The results are as Figure 15 (MDA-MB-468 breast cancer mouse model) and Figure 17 (KYSE-30 esophageal cancer mouse model) As shown, the body weight of tumor-bearing mice in the engineered fibroblast cell membrane nanovesicle laser irradiation group increased slightly, similar to the weight change in the normal saline group, indicating that the engineered fibroblast cell membrane nanovesicles have low systemic toxicity, and local laser irradiation of tumors has little impact on the body and high safety.

[0235] Example 16 Preparation Method and Application of Engineered Single-Gene Targeted NIH-3T3 Cell Membrane Nanovesicles

[0236] 16.1 Preparation Method of NIH-3T3 Cell Membrane Overexpressing a Single Gene

[0237] The NIH-3T3 fibroblast cell line was seeded into a 6-well plate at a density of 6x10 4 cells / ml, placed in an incubator at 37 °C and 5% CO2 for 24 h to allow it to adhere, then a lentivirus solution overexpressing the target gene CXCR4 containing the resistance gene puromycin and a high-efficiency transfection solution were added, and the cells were transfected in an incubator at 37 °C and 5% CO2 for 72 h to obtain fibroblasts overexpressing CXCR4 (CXCR4-3T3 or NIH-3T3).

[0238] Then, the engineered single-gene targeted cell membrane protein was prepared using CXCR4-3T3 according to the method of Example 1.

[0239] 16.2 Preparation method of engineered single-gene targeted NIH-3T3 cell membrane nanovesicles

[0240] The cell membrane protein used was the cell membrane protein obtained in Example 16.1. The remaining methods were the same as those in Example 9, and finally CXCR4-3T3-MNVs were obtained.

[0241] 16.3 WB detection of overexpressed membrane proteins in NIH-3T3 fibroblasts and the cell membrane nanovesicles obtained therefrom

[0242] The detection method was the same as that in Example 13. Results Figure 18 A and Figure 18 B are shown.

[0243] 16.4 Targeting ability of engineered cell membrane nanovesicles to breast cancer

[0244] 8×10 6 MDA-MB-468 breast cancer cells were inoculated at the right lower mammary gland of 4-week-old female nude mice. After 6 weeks, an MDA-MB-468 breast cancer orthotopic tumor-bearing mouse model was successfully established. The tumor-bearing mice were randomly divided into two groups, and equal amounts of engineered cell membrane vesicle solution CXCR4-3T3-MNVs labeled with the near-infrared fluorescent probe IR780 and the control group CTR-3T3-MNVs were injected via the tail vein. A small animal in vivo imager was used to observe the fluorescence distribution in MDA-MB-468 tumor-bearing mice within 2 - 48 h after injection.

[0245] The results are as Figure 18 shown in C. The fluorescence intensity at the tumor site in the group of single-gene engineered fibroblast cell membrane nanovesicles CXCR4-3T3-MNVs was significantly higher than that in the control group CTR-3T3-MNVs.

[0246] After 48 h, the mice were sacrificed by cervical dislocation, and their organs were placed in the order of heart, liver, spleen, lung, kidney, tumor, and axillary lymph nodes. The fluorescence distribution in the organs was observed through a small animal imager. Figure 18 D shows that the fluorescence at the tumor site in the CXCR4-3T3-MNVs group was significantly stronger than that in other groups, and no obvious accumulation was seen in the liver, kidney, and other organs.

[0247] Example 17 Killing ability of engineered cell membrane nanovesicles loaded with DOX (doxorubicin) on tumor cells

[0248] 5×10 5 4T1 breast cancer cells were inoculated at the right lower mammary gland of 6-week-old female BALB / c mice. When the tumors in the mice grew to 80 - 100 mm 3When the tumor-bearing mice were randomly divided into 6 groups (PBS, DOX, multi-gene fibroblast membrane nanovesicles without drug loading, multi-gene fibroblast membrane nanovesicles without drug loading + laser, fibroblast membrane nanovesicles loaded with DOX, and fibroblast membrane nanovesicles loaded with DOX + laser), and this was done continuously for 5 times, with an interval of 48 h each time. The corresponding drugs were injected via the tail vein (the dose of DOX in the groups containing DOX was 2 mg / kg). During this period, 24 h after each drug administration, the tumors of the mice in the laser group were irradiated with near-infrared laser with a wavelength of 808 nm at a power of 0.8 W / cm 2 for 3 min. During the drug administration period, the body weight of the mice and the volume of the tumors were continuously recorded until the 21-day observation period, and the mice were sacrificed on the 21st day, and their tumors were weighed.

[0249] The results of the anti-tumor treatment experiment are as Figure 19 (4T1 breast cancer mouse model) shown. Among all groups, the combination of fibroblast membrane nanovesicles loaded with DOX and 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 fibroblast membrane nanovesicles loaded with the drug DOX treatment group was better than that of the free DOX group, demonstrating that the fibroblast membrane nanovesicles improved the targeting and inhibitory effect of DOX on tumors; at the same time, the combination of fibroblast membrane nanovesicles without drug loading and laser treatment could also inhibit tumor growth to a certain extent, indicating that photothermal therapy could effectively kill tumor cells, but due to its lack of loading of the anti-tumor drug DOX, the best treatment effect was not achieved.

[0250] Among them, Figure 19 in which, the X-Fb-MNVs is the group of multi-gene fibroblast membrane nanovesicles without drug loading; X-Fb-MNVs(+) is the group of multi-gene fibroblast membrane nanovesicles without drug loading + laser; X-Fb-MNVs / DOX is the group of fibroblast membrane nanovesicles loaded with DOX; X-Fb-MNVs / DOX(+) is the group of fibroblast membrane nanovesicles loaded with DOX + laser. Among them, the preparation method of X-Fb-MNVs / DOX is the same as that in Example 10, only replacing RLS3 with DOX therein; the preparation method of X-Fb-MNVs is the same as that of X-Fb-MNVs / DOX in this example, only without drug loading.

[0251] Example 18 Detection of the in vitro transfection efficiency of autologous normal cells and the ability of the corresponding cell membrane vesicles to avoid phagocytosis by systemic macrophages

[0252] 1. Detection of the in vitro transfection efficiency of autologous normal cells

[0253] Autologous immune cells (monocytes, T cells), stem cells (umbilical cord blood mesenchymal stem cells, MSC), and fibroblasts (Fb) were selected. Each type of cell was seeded at a density of 6×10 4 cells / ml in a 6-well plate and incubated in a 37°C, 5% CO2 incubator for 24 h to allow cell attachment. Then, a lentiviral solution containing an overexpressed target gene PD-1 and a CD47 / iRGD fusion protein plasmid with the resistance gene G418, along with a high-efficiency transfection reagent, was added. The cells were transfected in a 37°C, 5% CO2 incubator for 72 h, and then the primary cell culture medium containing 1.2 mg / ml G418 was replaced. After all the cells in the untransfected group were killed by G418, the cells overexpressing the target gene PD-1 / CD47 / iRGD were digested with trypsin and expanded in culture, thus obtaining cells overexpressing multiple target genes (PD-1 / CD47 / iRGD-cell, namely PCI-monocyte, PCI-T cell, PCI-MSC, PCI-Fb). The mRNA level of CD47 in these cells was verified by RT-qPCR.

[0254] Cells overexpressing multiple target genes (PD-1 / CD47 / iRGD-cell, namely PCI-monocyte, PCI-T cell, PCI-MSC, PCI-Fb) were selected. Total RNA was isolated using Trizol reagent, and then reverse transcribed into cDNA using the PrimeScript TM 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 gene was quantified using ACTIN as an internal reference.

[0255] As determined by RT-PCR, as Figures 20 - 23 shown, under the same transfection conditions, the relative expression levels of CD47 mRNA, iRGD mRNA, and PD-1 mRNA in autologous fibroblasts after gene transfection were relatively high. Compared with the control group, the fold change was the largest, and in some cases, it even reached the ten-thousand-fold level, much higher than that in autologous monocytes, T cells, and mesenchymal stem cells after gene transfection. This indicates that autologous fibroblasts are more suitable as cells for in vitro expansion and gene transfection, and thus can be used for engineering cell membrane carriers.

[0256] PCI refers to the simultaneous overexpression of PD-1, CD47, and iRGD.

[0257] Figures 20 - 23In the text: 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, and CTR-T cell refers to control T cells; PCI-MSC refers to MSC cells overexpressing PD-1, CD47, and iRGD simultaneously, and CTR-MSC refers to control MSC cells; PCI-Fb or PCI-Fibroblast refers to fibroblasts overexpressing PD-1, CD47, and iRGD simultaneously, and CTR-Fibroblast refers to control fibroblasts, and X-Fibroblast refers to fibroblasts overexpressing CXCR4, PD-1, CD47, and iRGD simultaneously, that is, the fibroblasts (X-Fb) overexpressing multiple target genes obtained in Example 2. Among them, Figure 23 In the text, CXCR4-Fibroblast is the fibroblast (CXCR4-Fb) overexpressing the target gene CXCR4 obtained in Example 1.

[0258] 2. Detection of the ability of cell membrane vesicles obtained from autologous normal cells to avoid phagocytosis by systemic macrophages

[0259] Collect the above cells overexpressing multiple target genes (PCI-monocyte, PCI-T cell, PCI-MSC, PCI-Fb), wash the cell pellet 3 times with pre-cooled PBS buffer, aspirate the PBS buffer after centrifugation, add a membrane protein extraction reagent containing 1 mM PMSF protease inhibitor to the cell pellet, vortex thoroughly and place on ice for 15 min, then vortex once every 5 min, and then place the disrupted cell pellet in liquid nitrogen and 37 °C water bath and freeze-thaw repeatedly 4 times until the solution is homogeneous. Then, centrifuge the homogeneous cell disruption solution at 4 °C and 700 g for 10 min, take the supernatant after centrifugation and centrifuge at 4 °C and 14,000 g for 30 min. The precipitate after centrifugation is the engineered multi-gene cell membrane protein (PCI-monocyte-M, PCI-T cell-M, PCI-MSC-M, PCI-Fb-M). Prepare the engineered multi-gene cell membrane protein into cell membrane nanovesicles (MNVs) according to the method of Example 10 to obtain PCI-Fb-MNVs, PCI-Monocyte-MNVs, PCI-T-MNVs, and PCI-MSC-MNVs.

[0260] Seed the macrophage cell line RAW264.7 at 1*10 5Inoculate at a density of

[0261] cells / ml into a 24-well plate with round cell culture slides and incubate in a 37°C, 5% CO2 incubator for 24 h to allow them 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 and continue to incubate for 2 h. Remove the culture medium, rinse the slides 3 times with PBS buffer, fix the cells on the slides with 4% paraformaldehyde fixative for 15 min, remove the fixative and continue to rinse 3 times with PBS buffer, gently dry the PBS residue on the slides, cover the slides face down on a glass slide with DAPI stain dropped on it, and then mount the slides with a mounting medium. Figure 24 As shown, it can be clearly observed that compared with the cell membrane nanovesicles CTR-Fb-MNVs of the control group, the green fluorescence of the multi-gene engineered fibroblast cell membrane nanovesicles X-Fb-MNVs and PCI-Fb-MNVs on macrophages is significantly weakened and is not easily taken up by macrophages, while the green fluorescence of the gene engineered monocyte cell membrane nanovesicles PCI-Monocyte-MNVs, T cell membrane nanovesicles PCI-T-MNVs, and mesenchymal stem cell membrane nanovesicles PCI-MSC-MNVs in macrophages is stronger and is easily taken up and phagocytosed by macrophages, indicating that the multi-gene engineered fibroblast cell membrane nanovesicles X-Fb-MNVs and PCI-Fb-MNVs have good ability to avoid being phagocytosed by macrophages.

[0262] CTR-Fb-MNVs: Refers to fibroblast cell membrane nanovesicles that do not overexpress genes. Except for not overexpressing genes, the remaining obtaining steps are the same as in Example 10.

[0263] PCI-Fb-MNVs: Fibroblast cell membrane nanovesicles that simultaneously overexpress PD-1, CD47, and iRGD.

[0264] PCI-Monocyte-MNVs: Monocyte cell membrane nanovesicles that simultaneously overexpress PD-1, CD47, and iRGD.

[0265] PCI-T-MNVs: T cell membrane nanovesicles that simultaneously overexpress PD-1, CD47, and iRGD.

[0266] PCI-MSC-MNVs: MSC cell membrane nanovesicles that simultaneously overexpress PD-1, CD47, and iRGD.

[0267] X-Fb-MNVs: fibroblast cell membrane nanovesicles obtained for Preparation Example 10.

Claims

1. Application of normal fibroblasts in the preparation of drugs or tumor diagnostic reagents or nano drug delivery systems or engineered vesicles for treating tumors.

2. The use according to claim 1, characterized in that The normal fibroblasts are selected from normal fibroblasts of autologous origin; Preferably, the normal fibroblasts are selected from fibroblast cell lines or cell strains; Preferably, the normal fibroblasts are selected from established fibroblast cell lines or cell strains; Preferably, the normal fibroblasts are selected from fibroblasts of skin or paracancerous tissue.

3. A method for preparing engineered vesicles, characterized in that: include: (1) overexpressing a gene in normal fibroblasts to obtain gene-overexpressing fibroblasts; (2) extracting the membrane of the fibroblasts; (3) mixing the membrane of the fibroblasts with lipid nanoparticles to prepare the engineered vesicles; Preferably, the gene-overexpressing fibroblasts overexpress adhesion proteins or chemokine receptors; Preferably, the gene-overexpressing fibroblasts also overexpress immunosuppressive molecules or cell-penetrating peptides; Preferably, the gene includes CD47 or CXCR4; Preferably, the gene further includes 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 fibroblasts are selected from normal fibroblasts of autologous origin; Preferably, the normal fibroblasts are selected from fibroblast cell lines or cell strains; Preferably, the normal fibroblasts are selected from established fibroblast cell lines or cell strains; Preferably, the normal fibroblasts are selected from fibroblasts of skin or paracancerous tissue; Preferably, step (2) comprises sequentially adopting freeze-thaw method, gradient centrifugation and resuspension to obtain a membrane solution of fibroblasts; Preferably, step (3) comprises step (3.1): preparing the fibroblast membrane into a solution and then mixing it with lipid nanoparticles to obtain a mixed solution of the fibroblast membrane and lipid nanoparticles; Preferably, step (3) comprises step (3.2): using a liposome extruder to extrude and filter the mixed solution of the fibroblast membrane and lipid nanoparticles to obtain the engineered vesicles; Preferably, in step (3.1), the membrane concentration of the fibroblasts is 0.1 to 2 mg / mL; Preferably, the fibroblast membrane solution is filtered through 300-500 nm and 100-350 nm polycarbonate filter membranes in sequence before being mixed with the lipid nanoparticles; Preferably, in step (3.2), the filtration of the fibroblast membrane solution and the lipid nanoparticles after mixing is performed by sequentially filtering using a 50-250 nm polycarbonate filter membrane; Preferably, in step (3.1), the mass ratio of the fibroblast membrane solution to the liposome nanoparticles is (0.2-3):1; Preferably, in step (3.1), the mass ratio of the fibroblast membrane solution to the liposome nanoparticles is (0.5-3):1; Preferably, in step (3.1), the mass ratio of the fibroblast membrane solution to the liposome nanoparticles is (0.5-2):1; Preferably, the lipid nanoparticles are obtained by a solvent diffusion method; Preferably, in step (3), the method for obtaining lipid nanoparticles comprises the steps of: (S1) mixing IR-780 iodide and NH2-PEG-DSPE to react to obtain a primary product IR780-PEG-DSPE; (S2) mixing IR780-PEG-DSPE, anti-tumor drugs, DPPC and cholesterol into a solution and dispersing the solution into poloxamer 188 to prepare lipid nanoparticles; Preferably, the method for obtaining the lipid nanoparticles comprises the steps of: (A1) weighing IR-780 iodide and NH2-PEG-DSPE, and dissolving them in a dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; (A2) adding triethylamine to the dichloromethane solution containing IR-780 iodide to react and obtain a reaction solution; (A3) adding the reaction solution to diethyl ether, removing byproducts by centrifugation to obtain a precipitate, and then drying the precipitate to obtain the primary product IR780-PEG-DSPE; (A4) dissolving the 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 the aqueous solution of poloxamer 188 to react and prepare lipid nanoparticles; Preferably, in the ethanol phase solution of step (A4), the weight proportions of the anti-tumor drug, IR780-PEG-DSPE, DPPC and cholesterol are 5 to 30 parts of the anti-tumor drug, 10 to 30 parts of IR780-PEG-DSPE, 40 to 70 parts of DPPC and 1 to 15 parts of cholesterol, respectively; Preferably, in the ethanol phase solution of step (A4), the weight proportions of the anti-tumor drug, IR780-PEG-DSPE, DPPC and cholesterol are 5 to 25 parts of the anti-tumor drug, 10 to 25 parts of IR780-PEG-DSPE, 45 to 70 parts of DPPC and 1 to 10 parts of cholesterol, respectively; Preferably, in the ethanol phase solution of step (A4), the weight proportions of the anti-tumor drug, IR780-PEG-DSPE, DPPC and cholesterol are 5 to 20 parts of the anti-tumor drug, 10 to 20 parts of IR780-PEG-DSPE, 50 to 70 parts of DPPC and 3 to 6 parts of cholesterol, respectively; Preferably, in the ethanol phase solution of step (A4), the mass percentages of the anti-tumor drug, IR780-PEG-DSPE, DPPC and cholesterol are 5-20% of the anti-tumor drug, 10-20% of IR780-PEG-DSPE, 50-70% of DPPC and 3-15% of cholesterol, respectively; 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 to 3 mg / mL; Preferably, in step (A5), the concentration of poloxamer 188 in the poloxamer 188 aqueous solution is 0.5 to 1.5 mg / mL; Preferably, the method for obtaining IR780-PEG-DSPE comprises the steps of: (B1) weighing IR-780 iodide and NH2-PEG-DSPE, and dissolving them in a dichloromethane solution to obtain a dichloromethane solution containing IR-780 iodide; (B2) weighing triethylamine at a molar ratio of triethylamine to IR-780 iodide of (0.5-5):1, adding the triethylamine to the dichloromethane solution containing IR-780 iodide in step (B1), and stirring the reaction for 8-30 hours; (B3) After the reaction is completed, the reaction solution is added to 8 to 12 times the volume of anhydrous ether for reaction, centrifuged to remove byproducts, and then the precipitate is dried to obtain IR780-PEG-DSPE; Preferably, in the step (B2), the molar ratio of triethylamine to IR-780 iodide is (1-3): 1; Preferably, the anti-tumor drug includes RSL3 or doxorubicin.

4. The engineered vesicle prepared by the method according to claim 3.

5. A biofilm, characterized in that The biofilm includes a fibroblast membrane that overexpresses genes, wherein the genes include CD47 or CXCR4; Preferably, the gene further includes PD-1 or iRGD; Preferably, the biomembrane is a fibroblast membrane overexpressing genes PD-1, CD47 and iRGD; Preferably, the biomembrane is a fibroblast membrane overexpressing genes CXCR4, PD-1, CD47 and iRGD; Preferably, the fibroblasts are selected from normal fibroblasts; Preferably, the fibroblasts are selected from normal fibroblasts of autologous origin; Preferably, the normal fibroblasts are selected from fibroblast cell lines or cell strains; Preferably, the normal fibroblasts are selected from established fibroblast cell lines or cell strains; Preferably, the normal fibroblasts are selected from fibroblasts of skin or paracancerous tissue.

6. A method for preparing the biofilm according to claim 5, characterized in that: Overexpressing genes in fibroblasts, and then extracting the fibroblasts; the genes include CD47 or CXCR4; Preferably, the gene further includes PD-1 or iRGD; Preferably, the biomembrane is a fibroblast membrane overexpressing genes PD-1, CD47 and iRGD; Preferably, the biomembrane is a fibroblast membrane overexpressing genes CXCR4, PD-1, CD47 and iRGD; Preferably, the fibroblasts are selected from normal fibroblasts; Preferably, the fibroblasts are selected from normal fibroblasts of autologous origin. Preferably, the normal fibroblasts are selected from fibroblast cell lines or cell strains; Preferably, the normal fibroblasts are selected from established fibroblast cell lines or cell strains; Preferably, the normal fibroblasts are selected from fibroblasts of skin or paracancerous tissue.

7. A vesicle, characterized in that The raw material for preparing the vesicles includes the biofilm according to claim 5; Preferably, the vesicles are genetically engineered cell membrane nanovesicles; Preferably, the raw materials for preparing the vesicles further include lipid nanoparticles; Preferably, the lipid nanoparticles include a thermosensitive liposome drug delivery system; Preferably, the raw materials for preparing the thermosensitive liposome drug delivery system include IR-780 iodide, NH2-PEG-DSPE, triethylamine, anti-tumor drugs and DPPC; Preferably, the anti-tumor drug includes RSL3 or doxorubicin.

8. A composition, characterized in that Comprising the engineered vesicle according to claim 4 or the biofilm according to claim 5 or the vesicle according to claim 7, and a tumor therapeutic agent; Preferably, the tumor therapeutic agent includes an anti-tumor drug; Preferably, the tumor includes breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer; Preferably, the anti-tumor drug includes RSL3 or doxorubicin.

9. Use of the engineered vesicle according to claim 4, the biofilm according to claim 5, the vesicle according to claim 7, or the composition according to claim 8 in the preparation of an anti-tumor drug used 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 used in combination with the engineered vesicle according to claim 4, the biofilm according to claim 5, the vesicle according to claim 7, or the composition according to 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 according to claim 4, the biofilm according to claim 5, the vesicle according to claim 7, or the composition according to claim 8 in the preparation of a drug for combined use in laser therapy; Preferably, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer.

12. Use of the engineered vesicle according to claim 4, the biofilm according to claim 5, the vesicle according to claim 7, or the composition according to claim 8 in the preparation of a drug for treating tumors, a tumor diagnostic agent, or a nano drug delivery system; Preferably, the tumor comprises breast cancer, colon cancer, esophageal cancer, liver cancer, lung cancer or nasopharyngeal cancer.

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