CN116172972A

CN116172972A - Bionic nano-particle for targeting tumor as well as preparation method and application thereof

Info

Publication number: CN116172972A
Application number: CN202310110548.0A
Authority: CN
Inventors: 李博; 周倩; 杨童
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2023-02-10
Filing date: 2023-02-10
Publication date: 2023-05-30

Abstract

The invention discloses a bionic nano-carrier for targeting tumor, and aims to provide a bionic nano-carrier which can target specific tumor cells and is used for directional administration, so that the toxic and side effects of the medicine on normal cells and the harm on human bodies are reduced. The technical key point is that the cancer cells are engineered by utilizing genetic engineering, so that the cancer cells express immune check point molecules on immune cells, such as programmed death receptor 1 (PD 1) and the like. Bionic nano-particle P-NV is generated by utilizing an ultrasonic co-extrusion method, can be combined with mouse cancer cells in a homologous targeting way, and can be used for identifying immune checkpoint ligands on the cancer cells by utilizing immune checkpoint molecules, such as: programmed death ligand 1 (PD-L1) improves the biosafety, targeting and flexibility of the nanoparticle through dual targeting functions. Belongs to the field of medical biotechnology.

Description

Bionic nano-particle for targeting tumor as well as preparation method and application thereof

Technical Field

The invention relates to a bionic nano-carrier for targeting drug delivery at a specific tumor part, belonging to the technical field of medicines.

Background

The nano medicine has wide application prospect in early diagnosis and treatment of tumors, and the nano targeting drug delivery system has been vigorously developed since the rise, so that the nano targeting drug delivery system has great significance for treating refractory diseases (especially tumors). The nano targeting drug delivery platform can be divided into a chemically synthesized nano carrier and a naturally derived nano carrier according to the type of the nano carrier. Due to the unique physicochemical properties of nano-targeted drug delivery systems, strategies for treating incurable diseases (mainly cancers) using the same as a basic platform are being studied in great numbers.

The chemically synthesized nanocarriers can be classified into polymer nanocarriers (e.g., PLA, PLGA, PCL, PCA, PEG) and inorganic nanocarriers (metal nanocarriers (MNPs), carbon Nanotubes (CNTs), silicon-based nanocarriers, etc.). The anti-tumor nano drug delivery system generally depends on artificial synthetic materials with functional modification (such as ligand coupling, redox reaction and the like), has complex structure, short internal circulation period, is easy to be cleared by an immune system in vivo, particularly macrophages, and has a plurality of problems in the aspect of biological safety; meanwhile, only about 0.7% of the traditional targeted modified nano particles can reach the enrichment of tumor parts in vivo, which definitely increases the application difficulty of the nano material. Therefore, the development of conventional nano-drug delivery systems is transitioning.

Naturally derived nanocarriers comprise bio-organic components or cell-derived vesicles, such as exosomes, cell macrovesicles, oncolytic viruses, etc. Exosomes are nanoscale vesicles secreted by cells in an organism, useful for information transfer and substance delivery between cells and tissues. Since exosomes are derived from host cells, they are non-immunogenic and have unique advantages as targeted delivery systems for drugs. However, the natural exosome yield is extremely low, the acquisition and purification difficulty is high, and the enrichment is difficult to achieve the therapeutic dose; and the natural exosome has single component and cannot realize multiple functions. Research shows that after the surfaces of the nano materials with different properties are coated by cell membranes, the nano materials can be "disguised" to achieve the purpose of "deception" of an immune system, thereby avoiding phagocytosis by phagocytes and further prolonging the cycle of the nano materials. Compared with PEG modified nano particles, the in vivo-half life of the erythrocyte modified nano particles is prolonged by 2.5 times. The nanoparticle is 'worn' on cancer cell membrane, and can be 'positioned, navigated' and 'anchored' with each other by virtue of various adhesion molecules expressed on the cancer cell membrane and homologous cancer cells, so that the purpose of 'treating cancer with the cancer' is achieved by utilizing the 'homing' effect, and the adhesion efficiency is improved by nearly 20 times compared with that of the nanoparticle modified by erythrocyte membrane. In addition, some immune cell membranes are also used to disguise nanoparticles, which not only kill local tumors, but also activate the immune system to inhibit the growth of distant tumors. Therefore, the use of genetic engineering to engineer cell structures and thus to fabricate engineered exosome-like vesicle structures has become a new research hotspot. Such engineered exosome-like vesicles have non-immunogenic characteristics similar to exosomes, and can combine multiple components and achieve in vitro bulk amplification with the potential to achieve targeted delivery of therapy without triggering an adaptive immune response.

In addition, chemotherapy has great side effects, and cancer cell proliferation is inhibited by anticancer drugs (carboplatin, gemcitabine, molecular targeting drugs, etc.), so that tumor tissues are reduced and destroyed. However, anticancer drugs have an inhibitory effect on normal tissue cells of the human body while inhibiting cancer cells, and side effects associated with the therapy have a great influence on the daily life of the patient.

Disclosure of Invention

Aiming at the problems, the invention aims to provide a bionic nano particle for specifically targeting tumors, wherein the nano vesicle has the cell surface characteristics of lung cancer cells TC-1 and PD1 proteins, and can realize double targeting of lung cancer focus by utilizing the homing effect of cancer cells and the interaction between PD1 and the surface PD-L1 of the cancer cells; the excessive particles can saturate the PD-L1 molecules on the surface of the cancer cells, and block the combination of the excessive particles and the PD1 on the surface of the effector T cells, so that the immune response of the effector T cells on the tumor part is activated, and finally, the purposes of targeting the tumor cells, regulating the tumor immune microenvironment and eliminating the cancer cells are achieved.

For this purpose, the first technical solution provided by the present invention is as follows:

the bionic nano-particles for targeting tumor are mainly prepared from genetically engineered lung cancer cells.

Preferably, the bionic nanoparticle for targeting tumor is an immune checkpoint molecule PD1 protein which uses cancer cells and expresses immune cells on the surface of the cell membrane.

For this purpose, the second technical scheme provided by the invention is as follows:

1) Construction of lentiviral vector of pTRIP-mPD1-GFP

The lentiviral vector pTRIP-EGFP is digested by restriction enzymes NheI and BamHI, and simultaneously an mPD1 gene fragment with NheI and BamHI homology arms flanking the digested vector pTRIP-EGFP is synthesized, and mPD1 is cloned into the vector pTRIP-EGFP by homologous recombination enzymes to obtain the pTRIP-mPD1-GFP plasmid. Expression of the lentiviral vector of pTRIP-mPD1-GFP was verified by immunoblotting (WB).

2) Construction of a TC-1 cancer cell line stably and highly expressing PD1 on a cell membrane (abbreviation: TC-1P). The pTRIP-mPD1-GFP, viral packaging plasmid psPAX2 and PMD2.G are transfected together into 293T cells in a ratio of 2:2:1 by using a Vigofect cell transfection reagent, and cell supernatants are collected to obtain the pTRIP-mPD1-GFP lentivirus. The lentiviruses collected from pTRIP-mPD1-GFP were filtered through sterile 0.45 μm acetate filters, then mixed with fresh DMEM complete medium 1:1, and the cationic polymer polybrene added in a ratio of 1:2000 to infect TC-1 cells. After 24h of virus infection, the cells were changed and subjected to secondary infection, and the cells were cultured for 48h with fresh medium.

3) Flow cytometry was used to select cell TC-1P cell lines. TC1 cells infected with the recombinant lentivirus pTRIP-mPD1-GFP twice are respectively collected by pancreatin digestion, washed once by PBS, and GFP positive cell populations are detected by FACS to be further separated into mPD1 ⁺ The cell line of the monoclonal antibody is a monoclonal antibody,and (3) placing the monoclonal cells in a 96-well plate for culture, and finally obtaining the stable high-expression TC-1P cell line of the mPD 1. Expression and localization of PD1 on TC1-PD1 cells were verified by FACS and confocal laser microscopy.

4) The cell membrane of TC-1P cells was extracted. Culturing 1×10 ⁸ The cells were collected separately in ice PBS with cell scraping. After centrifugation at 700g for 10min at 4℃the supernatant was slowly decanted off and then centrifuged at 700g for 1min at 4 ℃. The supernatant in the upper centrifuge tube was carefully pipetted off with a pipette, followed by separation of the cell membrane using reagents in the cell membrane extraction kit (Biyun Tian-P0033). The method comprises the following specific steps: 1) The protease inhibitor PMSF is added into the membrane protein extraction reagent A according to the proportion, 1mL of the membrane protein extraction reagent A is added into every 2000 ten thousand cells, the cell sediment is fully resuspended, and then the ice bath is carried out for 10-15min. 2) At the end of the ice bath, the cell suspension was repeatedly freeze-thawed 5 times in liquid nitrogen and room temperature, followed by repeated 10 times of pushing in a 0.45uM syringe needle to thoroughly break the cells. 5) The cell suspension from the previous step was centrifuged at 700g for 10min at 4℃and the supernatant was carefully transferred to a new centrifuge tube. Finally, at 4 ℃,14000g is centrifuged for 60min, and cell membrane sediment is collected.

5) Preparing the cell membrane bionic nano vesicle P-NV. The cell membrane pellet of method A was fully resuspended in a proportion of 1mL of sterile water containing Protease Inhibitor (PI) per 2000 ten thousand cell membranes, and the cell membrane suspension was then passed through a contact sonicator, ice-bath sonicated for 2min (sonicator parameters 5 sOn, 5 sOff, power 150 wt. Times.30%), thereby turning the large cell membrane into a small volume phospholipid bilayer membrane. Finally, the membrane nanovesicles C-NV and P-NV having a particle size of about 160nm were produced by repeatedly subjecting the membrane to repeated extrusion 10 times or more through a 0.8 μm pore size filter, a 0.4 μm pore size filter and a 0.2 μm pore size filter in this order by using a liposome Extruder Avanti Mini-Extruder (KU: 610000-1 EA), and the total protein concentration of the cell membrane fragments and the membrane nanovesicles was measured by a bicinchoninic acid assay (BCA).

Assessment of cell membrane nanovesicle P-NV size and morphology. Particle size and morphology of cell membrane nanovesicles P-NV were detected using Transmission Electron Microscopy (TEM): the TEM-dedicated copper mesh was placed on a horizontally stretched filter paper, and the cell membrane nanovesicle P-NV (100. Mu.L) in Experimental example 2 was gently dropped onto the front surface of the TEM-dedicated copper mesh in a volume of 5 drops by a pipette, followed by standing at room temperature for 8-12 hours. 200 mu L of phosphomolybdic acid is dripped on the wiped glass table surface to form a water drop shape, and the front surface of the copper mesh dripped with the cell membrane nano vesicle P-NV is downwards obliquely inserted into the liquid drop for dyeing for 10min. The morphology and size of the cell membrane nanovesicles P-NV were observed by transmission electron microscopy. The observation by a transmission electron microscope shows that the cell membrane nano vesicle P-NV is in a hollow shell sphere shape, and the particle diameter of the lipid particle is less than about 100nm

Evaluation of the stability of the cell membrane nanovesicles P-NV. The aqueous P-NV solution was stored at 4℃and the freshly prepared or 4-and 6-day-old P-NV was subjected to a nanoparticle size change in a nanoparticle analyzer. The results show that: P-NV is soluble in water and has little effect on its structure when stored for 4 or 6 days at 4 ℃.

The cell membrane nano vesicle C-NV (control vesicle, nano particle prepared by TC-1C cell) and the P-NV have good homing targeting ability, and the P-NV has better targeting ability than the C-NV in vitro, and the cell membrane nano vesicle P-NV can be effectively enriched to the lung cancer cell transplantation tumor tissue of the mouse through blood circulation.

The technical scheme provided by the invention is that firstly, a vector capable of packaging lentiviruses and expressing PD1 is constructed by utilizing a molecular biology experimental means: pTRIP-PD1-GFP. pTRIP-PD1-GFP was prepared by co-transfecting 293T cells with the viral packaging plasmids psPAX2 and PMD2.G in a ratio of 2:2:1 by means of Vigofect cell transfection reagent, and then the TC-1 cell line was infected to obtain a cell line capable of overexpressing PD1-GFP (abbreviated as TC-1P-GFP). And (3) enriching to obtain the TC-1 cell line capable of stably and highly expressing the PD1-GFP through FACS sorting. TC-1P-GFP cells are lysed, ice bath ultrasound is carried out, and a liposome Extruder Avanti Mini-Extruder (KU: 610000-1 EA) is utilized to repeatedly push and squeeze a 0.8um aperture filter membrane, a 0.4 um aperture filter membrane and a 0.2 um aperture filter membrane for more than 10 times in sequence, so as to prepare the cell membrane nano vesicle (P-NV for short) with the particle size of 160nm and the PD1 high expression. The bionic nano-particle is formed by over-expressing immune check point molecules on the surface of a cancer cell engineered by gene engineering and generating the bionic nano-particle P-NV by utilizing an ultrasonic co-extrusion method. He has the cell surface characteristics of the lung cancer cell TC-1 and the PD1 protein, and can realize double targeting of lung cancer focus by utilizing the interaction of the cancer cell homing effect and the PD1 and the PD-L1 on the surface of the cancer cell; the bionic nano vesicle can be directionally administered, so that the damage of the medicine to cytotoxicity and human body is reduced, and the tumor immune microenvironment of the tumor part is activated, thereby providing a targeted transportation mode for later treatment. Wherein P-NV is the preparation of a PD1 molecule over-expressed by genetically engineered TC1 cells; C-NV is produced by TC-1C cells which are not genetically modified according to the preparation method of P-NV and is used as a control bionic nano vesicle.

Compared with the prior art, the technical scheme provided by the invention has the following technical advantages:

1. the cell membrane nano drug delivery system has natural advantages in-vivo transportation and targeting of antitumor drugs, and unlike the traditional liposome drug delivery model, the bionic nano vesicle of the cell membrane vesicle can protect the loaded objects from the influence of enzymes and macrophages in blood, and prolong the half-life of the nano particles in vivo.

2. The nanometer vesicle coated by the lung cancer cell membrane can assist in targeting tumor cells of lung cancer, can assist in penetrating cell membrane barriers, and can promote uptake and endocytosis of the lung cancer cells to the vesicle. In order to further increase the targeting and flexibility of the bionic nano-vesicle, the technical scheme provided by the application utilizes a genetic engineering method to excessively express an immune checkpoint molecule PD1 on a cancer cell membrane, so that the bionic nano-vesicle is provided with the PD1 molecule at the same time, can be combined with a ligand PD-L1 of a epidemic checkpoint molecule on a tumor cell, can be combined with an exosome which is secreted into blood by the tumor cell and promotes tumor growth and metastasis, further enhances the targeting of the bionic nano-vesicle, and further realizes more systematic and efficient transportation of drugs, proteins or genes to target organs.

3. The invention develops a high drug-carrying bionic nano drug delivery system wrapped by a homologous cancer cell membrane for targeted treatment of lung cancer. According to the technical scheme provided by the application, the genetically modified mouse lung cancer cell TC-1P or TC-1C which overexpresses mPD1 is subjected to ultrasonic coextrusion to generate the bionic nano-particle P-NV or C-NV. In vitro, targeting of biomimetic nanoparticles was assessed using homologous mouse lung cancer cells TC1 and TC-1L overexpressing mPD-L1. The invention discovers that both C-NV and P-NV can be ingested by homologous mouse lung cancer cells TC-1C and TC-1L, and the longer the in vitro co-incubation time is, the higher the content of bionic nano particles ingested by target cells is along with the increase of the adding concentration; we also found that: compared with C-NV, P-NV targets TC-1L (TC 1 cells over-express PD-L1 to simulate cancer tissues of lung cancer patients with high expression of PD-L1, and is used as a target cell model) more strongly for a longer time. In addition, differences in the targeting of P-NV to TC-1L cells can be eliminated by mPD1 antibodies and mPD-L1 antibodies.

4. In order to further verify the targeting of our bionic nano vesicle P-NV, the technical scheme provided by the application utilizes a model of mouse transplantation tumor, and is shown by in vivo imaging of small animals, compared with a mouse injected with free Cy5 dye in a control group, after the P-NV is injected into the body of the mouse, the targeting can be finally and mainly targeted and enriched to a homologous TC1 lung cancer tumor site through blood circulation, and the targeting has obvious targeting and long retention time at the tumor site. Therefore, the bionic nano vesicle P-NV provided by the application can enhance the targeting sensitivity and stability of the bionic nano vesicle through double-function targeting, so that the medicine, protein or gene can be transported to a target organ more systematically and efficiently, and the therapeutic effect is finally achieved.

5. The nano vesicle has the surface characteristics of lung cancer cells and high expression of PD1 protein, can realize double targeting of lung cancer focus by utilizing the homing targeting effect of the cancer cells and the interaction of PD1 and PD-L1 on the surface of the cancer cells, and can target tumor cells with high expression of PD-L1 or low expression of PD-L1, thereby well overcoming tumor heterogeneity.

Drawings

FIG. 1 is a diagram of genetically engineering TC1 cells to overexpress the immune checkpoint molecule mPD1 (abbreviated as TC-1P) and fusion expressed Green Fluorescent Protein (GFP).

Wherein A: immunoblotting to detect the expression level of the total mPD1 protein in the constructed TC-1P cell line; b: flow cytometry was used to detect the expression level of the mPD1-GFP protein in the TC-1P cell line.

FIG. 2 is an evaluation of membrane localization of PD1 in a TC-1P cell line.

Wherein A: the laser confocal experiment observes the positioning of PD1 on the cell membrane; b: FACS detects PD1 expression on cell membranes.

FIG. 3 is an evaluation of the stability of biomimetic nanovesicles C-NV and P-NV.

Wherein A: observing the particle size and the morphology of the bionic nano vesicle P-NV by a Transmission Electron Microscope (TEM); b: stability of biomimetic nanovesicles C-NV and P-NV in solution; c: surface charges of bionic nano vesicles C-NV and P-NV; d: bionic nano vesicles C-NV and P-NV have protein characteristics of homologous TC1 cells; e: P-NV carries a large number of mPD1 proteins.

FIG. 4 is a diagram of a TC1 cell engineered to overexpress the immune checkpoint molecular ligand mPD-L1 (abbreviated as TC-1L) and PD-L1 is expressed on the cell membrane. The TC-1L cell line is used as a target cell to simulate the cancer tissue of a lung cancer patient with high-expression PD-L1.

Wherein A: immunoblotting to detect the expression level of the total mPD-L1 protein in the constructed TC-1L cell line; b: flow cytometry was used to detect the expression level of the mPD-L1 protein of the TC-1L cell line.

FIG. 5 is an evaluation of uptake of C-NV biomimetic nanovesicles by TC-1C cells at different time points.

Wherein A: incubating TC-1C cells and C-NV for 20min in vitro, fixing the cells, and observing the uptake of the C-NV by the TC-1C cells by laser confocal; b: TC-1C cells were incubated with C-NV in vitro for 40min and uptake of C-NV by TC-1C cells was observed by laser confocal.

FIG. 6 is an illustration of evaluation of uptake of P-NV biomimetic nanovesicles by TC-1L cells highly expressing PD-L1 protein at different time points

Wherein A: incubating TC-1L cells and P-NV for 20min in vitro, fixing the cells, and observing the uptake of the TC-1L cells to the P-NV by laser confocal; b: TC-1L cells were incubated with P-NV in vitro for 40min, and uptake of P-NV by TC-1C cells was observed by laser confocal.

FIG. 7 is an evaluation of uptake of biomimetic nanovesicle P-NV by P-NV treated TC-1C cells and TC-1L cells under different conditions. Wherein A: the higher the concentration of P-NV incubated with the target cells in vitro, the more P-NV is taken up by the target cells, and under the same conditions, the higher the uptake of P-NV by TC-1L cells is than by TC-1C cells (left panel), the right panel is a statistic of the fluorescence level of P-NV taken up by the target cells; b: the longer the time that P-NV was incubated with the target cells in vitro, the more P-NV was taken up by the target cells, and under the same conditions TC-1L cells had a higher uptake of P-NV than TC-1C cells (left panel), the right panel is a statistic of the level of fluorescence taken up P-NV by the target cells.

FIG. 8 is an evaluation of the effect of mPD1 antibodies and mPD-L1 antibodies on the ability of cells to capture biomimetic nanoparticles.

Wherein A: the mPD1 antibody and the mPD-L1 antibody can weaken the uptake of TC-1C cells to the bionic nano vesicle P-NV; b: the mPD1 antibody and the mPD-L1 antibody can weaken the uptake of TC-1L cells to the bionic nano vesicle P-NV; c: statistics of the fluorescent levels of uptake of P-NV (A and B) by target cells.

FIG. 9 is a graph showing that P-NV binding to target cells is dependent to some extent on immune checkpoint PD1/PD-L1 interactions.

Wherein A: FACS detection of TC-1L ^Low 、TC-1L ^Middle 、TC-1L ^High The amount of PDL1 expressed on the cell membrane; b: TC1 cells with different mPD-L1 expression levels have different P-NV uptake capacities. C: different concentrations of mPD-L1 antibody to TC-1L ^High Effects of the ability of cells to uptake P-NV.

FIG. 10 is an evaluation of P-NV targeting in mice.

Wherein A: detecting changes in fluorescence signals (fluorescence signals within red circles) of free Cy5 or Cy 5-labeled P-NV at 0h, 1h, 4h, and 12h at mouse tumor sites by a small animal biopsy instrument; b: detecting changes in free Cy5 or Cy 5-labeled P-NV in mouse tissue and in transplanted tumors by a small animal in vivo imager; c: statistics of fluorescence intensity in fig. 10A; d: statistics of fluorescence intensity in FIG. 10B

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

Example 1

TC-1 cells were genetically engineered to overexpress the immune checkpoint molecule mPD1-GFP (TC-1P).

1. Experimental method

(A) (1) construction of pTRIP-mPD1-GFP lentiviral vector: the lentiviral vector pTRIP-EGFP is digested by restriction enzymes NheI and BamHI, and simultaneously an mPD1 gene fragment with NheI and BamHI homology arms flanking the digested vector pTRIP-EGFP is synthesized, and mPD1 is cloned into the vector pTRIP-EGFP by homologous recombination enzymes to obtain the pTRIP-mPD1-GFP plasmid. (2) production of recombinant lentiviruses and infection of TC1 cells: and (3) co-transfecting 293T cells with the recombinant lentiviral vector pTRIP-EGFP constructed in the method A, virus packaging plasmid psPAX2 and PMD2.G according to the ratio of 2:2:1 by using a Vigofect cell transfection reagent, and collecting cell supernatant to obtain the pTRIP-mPD1-GFP lentivirus. The collected lentiviruses of pTRIP-mPD1-GFP were filtered through sterile 0.45um acetate filters, then mixed with fresh DMEM complete medium 1:1, and the cationic polymer polybrene was added in a ratio of 2000:1 to infect TC-1 cells. After 24h of virus infection, the cells were changed and subjected to secondary infection, and the cells were cultured for 48h with fresh medium. (3) Flow cytometry was used to select cell lines TC-1P and TC-1L: TC-1 cells infected with the recombinant lentivirus pTRIP-mPD1-GFP twice in method B were collected by pancreatin digestion, washed once with PBS, and FITC was detected by FACS ⁺ The cell population is further separated into monoclonal cells expressing mPD1-GFP, and the monoclonal cells are placed in a 96-well plate for expansion culture, so that the TC-1P monoclonal cell strain with the mPD1 stable and high expression is finally obtained. (4) Expression of mPD1 in stable overexpressing cell lines constructed by western immunoblotting detection: collecting TC-1P monoclonal cell strain and TC-1C cells obtained in method C, lysing with RIPA lysate on ice for 30min, adding 5 XSDS containing 5% beta-mercaptoethanol, mixing, and decocting at 95deg.C for 5-10min. The denatured protein solution was applied to 10% SDS-PAGE gel in an amount of 40ug protein per lane, and subjected to denaturing gel electrophoresis. Subsequent use of specified anti-cancer agentsThe body was subjected to immunoblot analysis, including Anti- β -action, anti-mPD1.

(B) Flow cytometry detects mPD1-GFP expression in the constructed stable overexpressing cell line: similarly, TC-1P monoclonal cell lines and TC-1C cells in method C were collected by pancreatin digestion, washed once with PBS, TC-1C cells and TC-1P cells were added to pre-chilled PBS in the same cell number, washed twice (supernatant was discarded after centrifugation at 1300rpm for 3 min), and finally, after appropriate amount of PBS was added to resuspend cells, the proportion of GFP positive cells in TC-1P cells was counted by FACS.

2. Experimental results and analysis

From the western blot results, it was confirmed that constructed TC-1P cells were able to stably express mPD1 protein at high levels (fig. 1A). GFP was detected by flow cytometry and found from the TC-1P monoclonal cell line as compared to TC-1C cells ⁺ The cell populations were all significantly offset overall, indicating that constructed TC-1P cells were able to significantly express GFP-tagged mPD1 (FIG. 1B).

Example 2

The mPD1 protein expressed by TC-1P cell line can be localized on cell membrane in large quantity.

1. Experimental method

(A) Observing the localization of mPD1 protein on cell membrane in genetically engineered TC-1P monoclonal cell strain by laser confocal microscope: a sterile confocal culture-dedicated microscope cover slip was spread on a 24-well plate, and then the TC-1P monoclonal cell line obtained in method A was plated at 1X 10 ⁵ Is inoculated into 24-well plates and cultured overnight in an incubator. After the cells were firmly attached, PBS was washed once, 300. Mu.L of the biofilm fluorescent dye Deep Red (10. Mu.M) pre-diluted in PBS was added to the wells, and the wells were stained in a cell incubator for 5min, followed by washing 3 times with PBS, 300. Mu.L of 4% paraformaldehyde was added to each well, and the wells were allowed to stand at room temperature for 20-30min to fix the cells. DAPI was dissolved in PBS at a final concentration of 2 μm, followed by aspiration of 24-well plate paraformaldehyde and washing with PBS 5 times, addition of 500 μl of DAPI solution to each well, standing at room temperature for 1min, aspiration of the staining solution, washing with PBS 3 times, and sealing. 15 mu L of anti-quenching agent is dripped in the middle of a clean glass slide, and a cover glass in a 24-hole plate is gently clamped by using a tip forceps The cover glass is gently covered at the center of the liquid drop in the direction of the front face downwards, then nail polish is coated around the cover glass, and the cover glass is placed on a horizontal table top at room temperature in a dark place. Finally observing the localization of the cell mPD1 protein on the cell by a laser confocal microscope.

(B) Flow cytometry detects the expression of mPD1 on cell membranes in the constructed stable overexpressing cell lines: similarly, TC-1P monoclonal cell lines and TC-1C cells in method A were collected by pancreatin digestion, washed once with PBS, TC-1C cells and TC-1P cells were added to equal amounts of pre-diluted PD1 flow antibody (1:100) coupled with BV421 fluorescence, incubated on ice for 20min in the absence of light, washed twice with pre-chilled PBS (supernatant after centrifugation at 1300rpm for 3 min), and finally resuspended with an appropriate amount of PBS, and the proportion of PD1-BV421 positive cells in TC-1P cells was counted by FACS.

2. Experimental results and analysis

Significant co-localization of the mPD1-EGFP fusion protein of TC-1P cells with Deep Red-labeled cell membranes was observed by laser confocal microscopy (FIG. 2A), while FACS thus further confirmed the expression of large amounts of mPD1 protein on TC-1P cell membranes (FIG. 2A).

Example 3

Preparation of bionic nanovesicles C-NV and P-NV, and evaluation of the characterization properties of the bionic nanovesicles C-NV and P-NV

1. Experimental method

(A) Cell membranes of TC-1 and TC-1P cells were collected: cells were cultured in 15cm dishes, prepared approximately 1X 10 ⁸ TC-1 and about 1x 10 of (C) ⁸ The cells were collected separately in ice PBS with cell scraping. After centrifugation at 700g for 10min at 4℃the supernatant was slowly decanted off and then centrifuged at 700g for 1min at 4 ℃. The supernatant in the upper centrifuge tube was carefully pipetted off with a pipette, followed by separation of the cell membrane using reagents in the cell membrane extraction kit (Biyun Tian-P0033). The method comprises the following specific steps: 1) The protease inhibitor PMSF is added into the membrane protein extraction reagent A according to the proportion, 1mL of the membrane protein extraction reagent A is added into every 2000 ten thousand cells, the cell sediment is fully resuspended, and then the ice bath is carried out for 10-15min. 2) At the end of the ice bath, the cell suspension is repeatedly cooled in liquid nitrogen and at room temperatureFreeze thawing 5 times followed by repeated 10 times of pushing in a 0.45um syringe needle to thoroughly break the cells. 3) The cell suspension from the previous step was centrifuged at 700g for 10min at 4℃and the supernatant was carefully transferred to a new centrifuge tube. Finally, at 4 ℃,14000g is centrifuged for 60min, and cell membrane sediment is collected. And (B) preparation of bionic nano vesicles C-NV and P-NV: the cell membrane pellet of method A was fully resuspended in a proportion of 1mL of sterile water containing Protease Inhibitor (PI) per 2000 ten thousand cell membranes, and the cell membrane suspension was then passed through a contact sonicator, ice-bath sonicated for 2min (sonicator parameters 5 sOn, 5 sOff, power 150 wt. Times.30%), thereby turning the large cell membrane into a small volume phospholipid bilayer membrane. Finally, the biomimetic nanovesicles C-NV and P-NV with particle size of about 160nm were produced by repeatedly pushing through a 0.8 μm pore size filter, a 0.4 μm pore size filter and a 0.2 μm pore size filter in this order by using a liposome Extruder Avanti Mini-Extruder (KU: 610000-1 EA) 10 times or more, and the total concentration of cell membrane fragments and the protein of the biomimetic nanovesicles was measured by a bicinchoninic acid assay (BCA). (C) And detecting the particle size and the morphology of the bionic nano vesicle P-NV by a Transmission Electron Microscope (TEM): and (a) tabletting. The TEM-dedicated copper mesh was placed on a horizontally stretched filter paper, and the biomimetic nanovesicle P-NV (100. Mu.L) in Experimental example 2 was gently dropped onto the front surface of the TEM-dedicated copper mesh in a volume of 5. Mu.L per drop by a pipette, followed by standing at room temperature for 8-12 hours. (b) negative dyeing. 200 mu L of phosphomolybdic acid is dripped on the wiped glass table surface to form a water drop shape, the front surface of the copper mesh dripped with the bionic nano vesicle P-NV is downwards inclined and inserted into the liquid drop, and the glass table surface is dyed for 10min. (c) imaging. The morphology and size of the bionic nano vesicle P-NV were observed by transmission electron microscopy. (D) The nanometer particle size analyzer detects the hydration particle size of bionic nanometer vesicles C-NV and P-NV in solution: the above aqueous P-NV solution was stored at 4℃and the hydrated particle size of the nanoparticles was measured in a nanoparticle analyzer with freshly prepared P-NV and P-NV stored for 4 days and 6 days, and the hydrated particle size of freshly prepared C-NV was measured in the same manner. (E) Bionic nanovesicles C-NV and P-NV surface potential measurement: freshly prepared C-NV and P-NV were each added to a Markov particle size analyzer potential sample cell and their surface potentials were measured using a Markov particle size analyzer. (F) Coomassie brilliant blue staining detection imitation Protein expression of the nanovesicles C-NV and P-NV compared to total protein of intact cells: TC-1P and TC-1L stable cell lines and TC-1C cells were prepared as denatured protein samples according to the procedure of Experimental example 1, method F. Similarly, 2X SDS containing 5% beta-mercaptoethanol was added to the TC-1C and TC-1P cell membrane suspensions obtained in Experimental example 3, method A and the biomimetic nanovesicles C-NV and P-NV obtained by subsequent ultrasonic co-extrusion, and the mixture was denatured by boiling at 95℃for 5-10 min. The above samples were sequentially subjected to denaturing gel electrophoresis in 10% SDS-PAGE gels of 15mm in 10 wells at 70. Mu.g protein per lane. After electrophoresis, placing the protein gel in coomassie brilliant blue staining solution, performing shaking table staining for 6 hours at room temperature, then pouring off the coomassie staining solution, adding a decolorizing solution, decolorizing on the shaking table at room temperature until the outer part of the protein gel lane is decolorized cleanly. And finally scanning the protein gel by a high-resolution scanner.

(G) Protein immunoblotting detection of protein expression of biomimetic nanovesicles C-NV and P-NV: the protein sample of method D of example 3 was subjected to denaturing gel electrophoresis by sequentially applying the above samples to 15-well 15mm 10% SDS-PAGE gel in an amount of 40. Mu.g protein per lane. Subsequent immunoblot analysis using the indicated antibodies, including Anti-beta-action, anti-mPD1, anti-mPD-L1, anti-Na ⁺ /K ⁺ ATPase。

2. Experimental results and analysis

The biomimetic nano vesicles P-NV prepared by ultrasonic co-extrusion are all hollow spheres and lipid particles with particle diameters of less than about 100nm (figure 3A) as observed by a transmission electron microscope. P-NV was soluble in water and stored for 6 days at 4℃had no effect on its structural stability (FIG. 3B). Both C-NV and P-NV were negatively charged as demonstrated by surface potential analysis (FIG. 3C). By coomassie blue staining and western blotting, it was demonstrated that C-NV and P-NV contained a protein component on the membrane of homologous lung cancer cell TC1 (fig. 3D), and that P-NV still expressed a large amount of mPD1 protein on the membrane (fig. 3E)).

Example 4

TC-1L cell line (TC 1 cell over-expresses PD-L1, simulates cancer tissue of lung cancer patient with high expression of PD-L1, and is used as a target cell model for constructing and verifying TC-1L for short).

1. Experimental method

Experimental methods reference Experimental example 1, except that TC-1L monoclonal cell lines were used in place of TC-1P cell lines.

2. Experimental results and analysis

From the Western immunoblotting results, it was confirmed that the constructed TC-1L cells stably and highly expressed mPD-L1 protein (FIG. 4A). By flow cytometry, it was found that the mPD-L1 of the TC-1L monoclonal cell line was compared to the TC-1C cell line ⁺ The apparent global shift of the cell population of (a) suggests that constructed TC-1L cells can significantly express mPD-L1 and that the mPD-L1 protein is mainly expressed on the cell membrane (FIG. 4B).

Example 5

TC-1C cells can perform time-dependent uptake on C-NV bionic nano vesicles

1. Experimental method

(1) Dyeing the bionic nano vesicle C-NV by using a biomembrane fluorescent dye Cy5.5: 1mL of C-NV having a particle diameter of about 0.2mm and a protein concentration of 2mg/mL was prepared in advance by the method of Experimental example 2, and the C-NV was prepared as a vesicle solution and a cell membrane dye 700: 1.43mL of Cy5.5 fluorochrome at a concentration of 7mM was added in proportion, gently mixed, incubated in a shaker at 37℃for 1h, followed by 12h rotation on a shaker at 4 ℃. Transferring the incubation liquid to

Ultra centrifugal filter, 4 ℃/6000g centrifugation 15min. The ultrafiltration tube was then capped and inverted, 4 ℃/4000g, and the ultrafiltration supernatant, membrane dye-stained C-NV, was collected by centrifugation for 3 min. (2) Laser confocal microscopy observed uptake of C-NV by TC-1C cells at various time points: experimental methods referring to Experimental example 2, the difference is that the cell membrane of the target cell is stained by the biofilm fluorescent dye Dil, and then C-NV is added to incubate with TC-1C cells for 20min or 40 min. Specifically, after the medium in the 24-well plate on which the target cells were spread was aspirated, 300mL of serum-free DMEM medium, which was warmed at 37℃in advance, was added to each well, and the cells were starved for 2 to 3 hours. Biofilm fluorescent dye D il was diluted into PBS, 24-well plate medium on which target cells were spread was aspirated, washed three times with PBS, 300. Mu.L of diluted Dil staining solution was added, and incubated in a cell incubator for 5min, followed by washing 3 times with PBS, and Cy 5-labeled C-NV collected by ultrafiltration in method A of Experimental example 5 was diluted into 1% serum-containing DMEM medium warmed at 37℃in advance to give a final vesicle concentration of 100mg/mL. Then adding the culture medium containing the vesicle into each hole of a 24-hole plate sequentially according to a time gradient of 20min and 40min in a liquid changing mode, and covering cells according to 500 mu L of each hole. Finally, referring to the experimental method of experimental example 2, the sample is fixed, the cell nucleus is stained, and the chip is sealed. Finally, the co-localization of the vesicles with the target cells was observed by laser confocal microscopy.

2. Experimental results and analysis

From laser confocal microscopy observations, it can be demonstrated that biomimetic nanovesicle C-NV can be significantly taken up by homologous TC-1C cells, and that this level of uptake exhibits a time-dependent increase, suggesting that C-NV has good "homing" targeting ability (fig. 5).

Example 6

TC-1L cells with high expression of PD-L1 protein can perform time-dependent uptake on P-NV bionic nano vesicles, and the uptake capacity of the TC-1L cells on P-NV is far higher than that of TC-1C cells.

1. Experimental method

Experimental method referring to example 5, except that the biomimetic nanovesicles replaced C-NV with P-NV and the target cells replaced TC-1C cells with TC-1L cells highly expressing PD-L1 protein.

2. Experimental results and analysis

From the observation of a laser confocal microscope, it can be demonstrated that the bionic nanovesicle P-NV can be obviously ingested by homologous TC-1L cells which highly express the PD-L1 protein, and the ingestion level shows a time-dependent increase, which indicates that the P-NV has good homing targeting ability (figure 6). Furthermore, it can be observed that under the same conditions, TC-1L cells that highly express PD-L1 protein uptake P-NV that highly expresses PD1 is significantly more than TC-1C cells uptake C-NV.

Example 7

Compared with TC-1C cells, the bionic nano vesicle P-NV with high expression of PD1 protein has stronger targeting to TC-1L cells with high expression of PD-L1

1. Experimental method

(A) Flow cytometry detection as the concentration of biomimetic nanovesicles P-NV increases, so does the level of binding of P-NV on TC-1C cells or TC-1L cells. Will be 5x 10 ⁵ TC-1C cells and 5X 10 ⁵ The TC-1L cells were individually inoculated into six-well plates and placed in a cell incubator for overnight culture. The medium was then aspirated from the 6-well plate and 500. Mu.L of serum-free DMEM medium, warmed at 37℃beforehand, was added to each well to starve the cells for 2-3h. When the aim was to flow test the trend of the binding of P-NV to target cells with increasing co-incubation time, the fluorescence labeling method of the biomimetic nanovesicle P-NV was referred to Experimental example 4 method A, except that Cy5.5 was replaced with the cell membrane dye Cy5, cy 5-labeled biomimetic nanovesicle P-NV collected by ultrafiltration was diluted to 200. Mu.g/mL, 100. Mu.g/mL, 50. Mu.g/mL with pre-37℃warm DMEM medium containing 1% serum, and the incubation time of P-NV to target cells was 1h. Cells were collected by pancreatin digestion, washed twice with ice PBS, centrifuged at 1300rpm for 3min, the supernatant was discarded, the cells were resuspended with an appropriate amount of PBS, and then FACS was performed to count the binding of P-NV on the cell membranes of each of TC-1C and TC-1L cells (APC ⁺ Cell populations).

(B) Flow cytometry detects uptake levels of biomimetic nanovesicles P-NV by TC-1C cells or TC-1L cells over time: experimental methods referring to Experimental example 7 method A, cy 5-labeled biomimetic nanovesicles P-NV collected by ultrafiltration were diluted to a final vesicle concentration of 100. Mu.g/mL in a pre-warmed DMEM medium containing 1% serum at 37 ℃. Then adding the culture medium containing the vesicle into each hole of a six-hole plate sequentially according to time gradient of 0.5h, 1h and 2h in a liquid changing mode, and covering cells according to 500 mu L of each hole. Cells were collected by pancreatin digestion, washed twice with ice PBS, centrifuged at 1300rpm for 3min, the supernatant was discarded, the cells were resuspended with an appropriate amount of PBS, and then FACS was performed to count the binding of P-NV on the cell membranes of each of TC-1C and TC-1L cells (APC ⁺ Cell populations).

2. Experimental results and analysis

From the flow statistics it was found that the fluorescence intensity of Cy 5-labeled P-NV detected on TC-1C cells and TC-1L cells increased in a significant gradient with increasing P-NV concentration in the added incubation. And fluorescence intensity of Cy 5-labeled P-NV detected on TC-1L cells highly expressing PD-L1 protein was significantly higher than TC-1C cells (FIG. 7A). While the fluorescence intensity of Cy 5-labeled P-NV detected on TC-1C cells and TC-1L cells increased significantly with increasing P-NV co-incubation time with target cells when treated with the same concentration of P-NV, the fluorescence intensity of Cy 5-labeled P-NV detected on TC-1L cells was significantly higher compared to TC-1C cells with high expression of PD-L1 protein (FIG. 7B). These two sets of results demonstrate that P-NV has good "homing" targeting ability, and also that P-NV with high expression of mPD1 can rely on protein interactions of PD1/PD-L1 to bind more strongly to the target cell TC-1L with high expression of mPD-L1.

Example 8

The mPD1 antibody and the mPD-L1 antibody can inhibit the binding of the bionic nano-particle P-NV and the target cell to a certain extent

1. Experimental method

Experimental method referring to experimental example 7, except that the mPD-L1 antibody was incubated with the target cells, or the mPD1 antibody was incubated with the biomimetic nanovesicle P-NV. Specifically, in the mPD-L1 antibody-treated group, the mPD-L1 antibody was diluted to 5. Mu.g/mL in advance, then the antibody diluted solution was incubated with designated TC-1C cells and TC-1L cells highly expressing the PD-L1 protein at 4℃for 1 hour, washed three times with PBS, and after that, cy 5-labeled P-NVs (100. Mu.g/mL) was added thereto and incubated at 4℃for 1 hour; whereas in the mPD1 antibody-treated group, cy 5-labeled P-NV was diluted to 100. Mu.g/mL, incubated with mPD1 antibody (5. Mu.g/mL) at 4℃for 1 hour, followed by incubation of the above vesicles with designated TC-1C cells and TC-1L cells highly expressing PD-L1 protein at 4℃for 1 hour. After the antibody was not blocked or blocked, FACS detected changes in the amount of P-NV ingested by TC-1C (FIG. 9A) or TC-1L (FIG. 9B) cells.

2. Experimental results and analysis

From the flow-through and related statistics, it was found that TC-1L cells highly expressing PD-L1 protein uptake of P-NV was higher than TC-1C cells, while addition of mPD-L1 antibody incubated with TC-1C cells or TC-1L cells, or addition of mPD1 antibody incubated with P-NV significantly reduced uptake of P-NV by TC-1C cells or TC-1L cells, and significantly eliminated the difference in binding capacity of P-NV to TC-1C cells or TC-1L cells (FIG. 9). This suggests that P-NV binding to target cells is affected by other effects, including immune checkpoint PD1/PD-L1 interactions, in addition to relying on the "homing effect", whereas PD1 antibodies or PD-L1 antibodies can undo this effect.

Example 9

Binding of P-NV to target cells depends to some extent on immune checkpoint PD1/PD-L1 interactions.

1. Experimental method

(A) Experimental methods reference Experimental example 1, except that mPD-L1 (abbreviated as TC-1L) was expressed under TC-1L ^Low ) Express mPD-L1 (abbreviation: TC-1L ^Middle ) High expression mPD-L1 (abbreviation: TC-1L ^High ) The cell line replaces the TC-1P cell line. FACS analysis at TC-1L ^Low 、TC-1L ^Middle 、TC-1L ^High Amount of PDL1 expressed by the membrane on the cell.

(B) Experimental methods reference Experimental example 7, except that TC-1L was used ^Low 、TC-1L ^Middle 、TC-1L ^High The cells replace TC-1C or TC-1L cells. The difference in P-NV uptake capacity of TC1 cells with different mPD-L1 expression levels was detected by FACS.

(C) Experimental methods reference Experimental example 7, except that TC-1L was used ^High Cell replacement TC-1C or TC-1L cells, and increasing incubation of different concentrations of mPD-L1 antibody (0, 10. Mu.g/mL, 25. Mu.g/mL) with target cells. Detection of varying concentrations of mPD-L1 antibody versus TC-1L by FACS ^High Effects of the ability of cells to uptake P-NV.

2. Experimental results and analysis

We successfully constructed TC-1L cell lines expressing different amounts of mPD-L1 protein using genetic engineering means (FIG. 9A). The higher the expression level of the mPD-L1 protein, the more P-NV is taken up (fig. 9B), and this uptake can be attenuated dose-dependently by blocking the PD1/PD-L1 interaction (fig. 9C). These results illustrate: binding of P-NV to target cells depends to some extent on immune checkpoint PD1/PD-L1 interactions.

Example 10

Bionic nano vesicle P-NV can be effectively enriched into lung cancer cell transplantation tumor tissue of mice through blood circulation

1. Experimental method

(A) Establishment of a mouse transplantation tumor model: prepared mouse lung cancer cells TC-1 and TC-1L were resuspended in a 1:1 mixture of PBS and Matrigel (CORNING, 356237) (8X 10 per 100. Mu.L of the mixture) ⁵ Cells), and placed on ice for use. The mixture containing tumor cells was inoculated onto the back of 6-week-old C57BL/6 mice, and 100. Mu.L of the tumor cell mixture was injected at the inoculation point. Experimental methods referring to Experimental example 7 method A, biomimetic nanovesicles P-NV were stained with the biofilm fluorescent dye Cy 5. When the tumor volume reaches about 80-90mm ³ On the left and right, the free Cy5 dye was observed and recorded by a small animal biopsy imager, and a fluorescence image before P-NV injection of Cy5 label was recorded. The mice were then dosed by tail intravenous injection with Cy 5-labeled P-NV (1 mg/kg, protein weight), and the same concentration of free Cy5 dye. The fluorescence intensity of free Cy5 dye and Cy 5-labeled P-NV enriched in TC-1L transplanted tumor tissue after 1h, 4h, 12h of administration was then recorded by observation with a small animal biopsy.

(B) Mice reaching the experimental endpoint were euthanized, dissected out of heart, liver, spleen, lung, kidney tissue and transplanted tumors, and then observed for distribution of free Cy5 dye and Cy 5-labeled P-NV in each tissue by a small animal biopsy imager.

(C) Example fluorescent statistical analysis of fig. 10A.

(D) Example fluorescent statistical analysis of fig. 10B.

2. Experimental results and analysis

As a result of in vivo imaging of mice, it was found that fluorescence enrichment was significantly observed at tumor sites of transplanted tumor mice with engineered TC-1L cells, whereas in contrast to free Cy5, cy 5-labeled P-NV, the fluorescence intensity peaked and then gradually decreased at about 4 hours after administration (FIG. 9). This suggests that P-NV can be targeted to accumulate in tumor tissue of tumor-bearing mice by blood circulation, and that accumulated P-NV in tumor tissue reaches saturation when circulating in blood for about 4 hours. Meanwhile, after 12 hours, the Cy5 marked P-NV is still stronger at the tumor tissue part, and the long-acting property of the P-NV targeting tumor part is reflected.

Claims

1. The bionic nano-particles for targeting tumor are characterized by being prepared from genetically engineered lung cancer cells.

2. The biomimetic nanoparticle for targeting a tumor according to claim 1, wherein the biomimetic nanoparticle for targeting a tumor is an immune checkpoint molecule PD1 that expresses immune cells using lung cancer cells.

3. The method for preparing the tumor-targeted bionic nanoparticle according to claim 1, which comprises the following steps in sequence:

1) Construction of lentiviral vector of pTRIP-mPD1-GFP

Cloning mPD1 into a vector pTRIP-EGFP by homologous recombinase to obtain a pTRIP-mPD1-GFP plasmid;

2) Construction of TC-1 cancer cell line stably and highly expressing PD1 on cell membrane

The pTRIP-mPD1-GFP, virus packaging plasmid psPAX2 and PMD2.G are transfected together into 293T cells through a Vigofect cell transfection reagent, and cell supernatant is collected to obtain pTRIP-mPD1-GFP lentivirus; the lentiviruses collected in pTRIP-mPD1-GFP were filtered and then mixed 1:1 with fresh DMEM complete medium, 1:2000, cationic polymer polybrene is added to infect TC1 cells; after 24 hours of virus infection, the cells are subjected to liquid exchange and secondary infection, and fresh culture medium is replaced to culture the cells for 48 hours;

3) Flow cytometry to select cell TC-1P cell lines

TC1 cells infected with the recombinant lentivirus pTRIP-mPD1-GFP twice are collected by pancreatin digestion, After washing with PBS, GFP positive cell populations were detected by FACS and then sorted to mPD1 ⁺ The monoclonal cells are placed on a plate for culture, and finally a TC-1P cell line with stable and high expression of mPD1 is obtained; observing by using FACS and a laser confocal microscope, and verifying the expression and positioning conditions of PD1 on TC1-PD1 cells;

4) Extraction of cell membranes of TC-1P cells

Culture 1X10 ⁸ Respectively collecting the cells into PBS (phosphate buffer solution) of ice by using a cell scraper, centrifuging, slowly pouring out the supernatant after centrifuging, centrifuging again, carefully sucking the supernatant in the centrifuge tube in the last step by using a pipettor, and finally centrifuging again to collect cell membrane sediment;

5) Preparation of cell membrane bionic nanovesicle P-NV

The cell membrane sediment extracted in the step 4) is fully resuspended according to the proportion of adding 1mL of sterile water containing protease inhibitor into every 2000 ten thousand cell membranes, then the cell membrane suspension is passed through a contact ultrasonic cell disruption instrument, ice bath ultrasonic treatment is carried out for 2min to change the large-volume cell membrane into a small-volume phospholipid bilayer membrane, and the cell membrane is repeatedly pushed for more than 10 times by using a liposome Extruder Avanti Mini-Extruder to sequentially pass through a 0.8 mu m pore size filter, a 0.4 mu m pore size filter and a 0.2 mu m pore size filter, so that the cell membrane nanovesicles C-NV and P-NV with the particle size of about 160nm are manufactured, and the total concentration of protein in cell membrane fragments and the cell membrane nanovesicles is measured by a bicinchoninic acid assay (BCA).

4. The method for preparing the tumor-targeted bionic nanoparticle according to claim 1, wherein the mass ratio of pTRIP-mPD1-GFP to viral packaging plasmids psPAX2 and PMD2.G is 2:2:1.

5. The method of claim 1, wherein the filtration is performed using a sterile 0.45 μm acetate filter.

6. The method for preparing the tumor-targeted bionic nanoparticle according to claim 1, wherein the step of separating cell membranes by the reagent in the cell membrane extraction kit is as follows:

1) Adding a protease inhibitor PMSF into the membrane protein extraction reagent A according to a proportion, fully resuspending the cell sediment according to a proportion of adding 1mL of the membrane protein extraction reagent A into every 2000 ten thousand cells, and then carrying out ice bath for 10-15min;

2) At the end of the ice bath, repeatedly freezing and thawing the cell suspension in liquid nitrogen and at room temperature for 5 times, and then repeatedly pushing the cell suspension in a syringe needle of 0.45 mu m for 10 times to thoroughly break the cells;

3) The cell suspension from the previous step was centrifuged at 700g for 10min at 4℃and the supernatant was carefully transferred to a new centrifuge tube.

7. The method for preparing the tumor-targeted biomimetic nanoparticle according to claim 1, wherein step 4) is to centrifuge 700g for 10min at 4 ℃, slowly pour out the supernatant after centrifugation, and then centrifuge again 700g for 1min at 4 ℃; carefully sucking up the supernatant in the centrifuge tube in the last step by using a pipette, then separating cell membranes by using reagents in a cell membrane extraction kit, centrifuging 700g of the cell suspension at 4 ℃ for 10min, and carefully transferring the supernatant into a new centrifuge tube; finally, at 4 ℃,14000g is centrifuged for 60min, and cell membrane sediment is collected.

8. The method for preparing the tumor-targeted biomimetic nanoparticles according to claim 1, wherein the ultrasonic instrument parameters are 5s on,5s Off, and the power is 150wt×30%.