Drug delivery nano-vesicle based on self-activated T cell membrane as well as preparation method and application of drug delivery nano-vesicle

Abstract

The invention relates to the technical field of biology, in particular to a drug delivery nano-vesicle based on an autoactivated T cell membrane as well as a preparation method and an application of the drug delivery nano-vesicle. The drug delivery nano-vesicle comprises a cationic lipid type nano-particle, diaminobenzimidazole embedded in the cationic lipid type nano-particle, and a primary activated T cell membrane positioned on the outer surface of the cationic lipid type nano-particle. In the invention, the STING agonist diaminobenzimidazole (diABZI) is used for activating congenital and adaptive immune response, enhancing expression and secretion of IFN-beta by tumor cells and DC cells, inducing DC maturation and promoting T cells to infiltrate tumor; the activated T cell membrane simultaneously expressing multiple immune checkpoints competitively relieves the inhibition effect of multiple immune checkpoints, enhances the tumor killing ability of T cells, and realizes the synergistic anti-tumor curative effect.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to drug delivery nanovesicles based on autologous activated T cell membranes, and a preparation method and application thereof. Background Art

[0002] Biomacromolecule drugs refer to biomacromolecules produced by modern biotechnology and synthesized in vivo or in vitro for the diagnosis, prevention or treatment of diseases, mainly including proteins, peptides, antibodies, nucleic acids, etc. These biomacromolecule drugs are synthesized through biosynthesis, have unique multidimensional structures, and play an important role in biological life activities. Biomacromolecule drugs have strong targeting, high specificity, high efficacy, and have shown advantages such as definite efficacy and low toxicity and side effects in clinical treatment. They have gradually become an important research direction for drug development.

[0003] However, compared with small molecule drugs, biomacromolecule drugs are larger in size, larger in molecular weight, and have negative or positive charges on their surfaces, making it difficult for them to cross the cell membrane and enter the cell to exert their effects. In order to achieve efficient delivery of biomacromolecule drugs, people have developed many biomacromolecule drug delivery carriers, such as liposomes, cationic polymers, nanoparticles, etc. However, these carriers have problems such as low delivery efficiency and potential safety hazards. How to improve the delivery effect of biomacromolecule drugs while ensuring safety, make biomacromolecule drugs successfully compatible with various biological barriers in the body, and achieve efficient delivery into cells to exert their effects, is still a difficulty and development direction in the research of biomacromolecule drug delivery systems.

[0004] Biomacromolecule drugs have become one of the most promising areas in drug development in the 21st century. Significant progress has been made in the treatment of major diseases, but there are still many problems and obstacles that need to be solved in in vivo delivery. In recent years, through the development of various new dosage forms of biomacromolecule drugs, some progress has been made in solving the above problems, but the clinical effect is not good. In view of the importance of biomacromolecule drugs and their driving role in the overall development of the country's medicine, the construction of efficient in vivo delivery systems for biomacromolecule drugs is of great significance to solving the current bottleneck problems of biomacromolecule drugs and realizing industrialization. Constructing a multifunctional and synergistic efficient delivery system for biomacromolecule drugs will be an important direction for the future development of biomacromolecule drugs.

[0005] Lipid nanoparticles (LNPs) are the most mature drug carriers in clinical application and are expected to change the current dilemma of macromolecular drug therapy. Although many efforts have been made to improve the efficacy of cationic lipids used in lipid nanoparticles (LNPs), their safety issues cannot be ignored. Cationic lipids have long been successful in delivering nucleic acids, but due to safety risks, their applicability in treatment is greatly reduced and their clinical application is limited. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the present invention provides a drug delivery nanovesicle based on autologous activated T cell membrane and a preparation method and application thereof.

[0007] To achieve the above objectives, the scheme of this application is as follows:

[0008] In the first aspect, the present invention provides a drug delivery nanovesicle, which includes: cationic liposome nanoparticles, diaminobenzimidazole (diABZI, a STING agonist) embedded in the cationic liposome nanoparticles, and an autologous activated T cell membrane located on the outer surface of the cationic liposome nanoparticles.

[0009] Optionally, the average diameter of the cationic liposome nanoparticles is 100-200 nm.

[0010] Optionally, the cationic liposome nanoparticles have a zeta potential (ie, zeta potential) of -10 to -25 mV.

[0011] Optionally, the encapsulation efficiency of the cationic liposome nanoparticles is 55%-70%.

[0012] Optionally, the autologous activated T cell membrane is prepared by activating and extracting primary T cells isolated from the spleen or peripheral blood of Balb / c mice.

[0013] In a second aspect, the present invention also provides a method for preparing the drug delivery nanovesicles as described above, comprising the following steps:

[0014] S1. Activate primary T cells and extract membrane fragments to obtain activated T cell membranes;

[0015] S2. embedding the diaminobenzimidazole in the cationic liposome nanoparticles to obtain cationic liposome nanoparticles embedded with diaminobenzimidazole;

[0016] S3. Applying the activated T cell membrane to the outer surface of the cationic liposome nanoparticles embedded with diaminobenzimidazole to obtain the drug delivery nanovesicles.

[0017] Optionally, in step S1, activating primary T cells comprises:

[0018] Use monoclonal antibodies to activate primary T cells and screen activated T cells that express adhesion molecules and immune checkpoints;

[0019] Optionally, in step S1, extracting membrane fragments comprises:

[0020] The activated T cells are lysed with an isothiocyanate solution, and then ultrasonically disrupted to break the cell membranes of the activated T cells into membrane fragments, which are then centrifuged to obtain the activated T cell membranes.

[0021] Optionally, in step S2, the diaminobenzimidazole is embedded in the cationic liposome nanoparticles by a co-incubation method.

[0022] Optionally, in step S3, the activated T cell membrane is coated on the outer surface of the cationic liposome nanoparticles embedded with diaminobenzimidazole by extrusion method.

[0023] In a third aspect, the present invention also provides use of the drug delivery nanovesicles as described above and / or the drug delivery nanovesicles prepared according to the preparation method as described above in the preparation of anti-tumor drugs.

[0024] Beneficial effects of the present invention:

[0025] The present invention screens out activated T cell membranes expressing multiple adhesion molecules and immune checkpoints by studying the molecular expression of primary T cell membranes at different activation stages, thereby ensuring the effectiveness, stability and utilization of the vector.

[0026] The drug delivery nanovesicles of the present invention highly express immune checkpoint proteins PD-1, TIM-3, LAG-3 and TIGIT, implement effective combined administration, and deliver diaminobenzimidazole to tumor cells that highly express immune checkpoint ligands PD-L1, GAL-3, GAL-9 and CD155 to exert their effects, thereby achieving effective enrichment of the drug and thereby improving the bioavailability of the drug.

[0027] In the present invention, the innate and adaptive immune responses are activated by the STING agonist diaminobenzimidazole, the expression and secretion of IFN-β by tumor cells and DC cells are enhanced, DC maturation is induced, and T cell infiltration into tumors is promoted; at the same time, the activated T cell membrane expressing multiple immune checkpoints competitively relieves the inhibitory effects of multiple immune checkpoints, enhances the tumor-killing ability of T cells, and achieves a synergistic anti-tumor therapeutic effect.

[0028] The present invention uses autologous activated T cell membranes for drug delivery systems, which have high biosafety, express a variety of adhesion molecules and immunosuppressive molecules, can competitively bind to immune checkpoint ligands, and relieve the inhibition of immune checkpoints on T cell function. In addition, it has multi-target targeting, can more effectively gather at the tumor site, improve drug bioavailability, and has the characteristics of safety, efficacy enhancement, and toxicity reduction, in order to lay the foundation for basic research and technology development related to tumor immunotherapy targeting the tumor immune microenvironment, and provide theoretical references. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1is a flow chart of the method for preparing the drug delivery nanovesicles of the present invention, Cholesterol means cholesterol, Active T cell means activated T cell, Hypotonic lysis means hypotonic lysis, Membrance Isolation means extraction of membrane fragments, Active T cell Membrance vesicle means activated T cell vesicle, and Co-extrusion means co-extrusion;

[0030] Figure 2 2A is the result of the detection of immune checkpoints highly expressed by T cells on the third day of activation and the third day of secondary activation. 2B is the result of the detection of immune checkpoint LAG-3 expression level. 2C is the result of the detection of immune checkpoint PD-1 expression level. Day means day. Mean fluorescence intensity means mean fluorescence intensity. Value means numerical value.

[0031] Figure 3 Figure 3 is the preparation, characterization and biological function verification result of activated T cell membrane vesicles, 3A is the particle size detection result of activated T cell membrane nanovesicles (AM), 3B is the zeta potential detection result of activated T cell membrane nanovesicles (AM), 3C is the electron microscopy image of activated T cell membrane nanovesicles (AM), 3D is the expression level detection result of activated T cell membrane nanovesicles (AM) surface marker proteins CD3 and PD-1, 3E is the expression level detection result of immune checkpoint ligands PD-L1, GAL-3 and CD155, 3F is the cell viability detection result of CT26-Luci, 3G is the expression level detection result of IFN-γ, granzyme B (Granzyme B, abbreviated as GzmB) and perforin; Concentration means concentration, particals means particles, Daameter means particle size, Numers means numerical value, potential means potential, Lysis ofCT-26-Luci indicates the lysis ratio of CT-26-Luci, Cells indicates cells, Isotype indicates isotype control, and Control indicates control group, the same below;

[0032] Figure 4Figure 4 is the preparation and characterization result of drug-loaded activated T cell membrane nanovesicles, 4A is the particle size distribution diagram, 4B is the average particle size diagram, 4C is the Zeta potential detection result diagram, 4D is the polydispersity detection result diagram, 4E is the expression level detection result diagram of the marker proteins CD3, PD-1, TIM-3 and LAG-3, 4F is the electron microscope image of the blank liposome (AM-dLNP) coated with the activated T cell membrane, 4G is the encapsulation efficiency detection result diagram, 5H is the d particle size detection result diagram in the stability experiment, 5I is the Zeta potential detection result diagram of dLNP, M-dLNP, AM-LNP and AM-dLNP, T membrane represents T cell membrane, polydispersity represents polydispersity, Encapsulation Efficiency represents encapsulation efficiency, Granzyme B represents granzyme B, the same below;

[0033] Figure 5 Figure 5 is the result of the drug-loaded activated T cell membrane nanovesicles blocking immune checkpoint ligands in vitro and promoting T cell killing, 5A is the result of the expression level of PD-L1, 5B is the result of the expression level of GAL-3, 5C is the result of the expression level of CD155, 5D is the result of the viability of 4T1-Luci cells, 5E is the result of the staining of 4T1-Luci cells, 5F is the result of the expression level of the pro-inflammatory cytokine IFN-γ, 5G is the result of the expression level of the killing function molecule Perforin, and 5H is the result of the expression level of the killing function molecule Granzyme B;

[0034] Figure 6 Figure 6 is the result of in vitro cell uptake of drug-loaded activated T cell membrane nanovesicles, 6A is the result of flow cytometry of tumor cells CT-26 after 1 hour (the upper figure is the flow cytometry graph, and the lower figure is the result after convection cytometry analysis), 6A is the result of flow cytometry of tumor cells CT-26 after 4 hours (the upper figure is the flow cytometry graph, and the lower figure is the result after convection cytometry analysis), 6C is the result of flow cytometry of dendritic cells DC2.4 after 1 hour (the upper figure is the flow cytometry graph, and the lower figure is the result after convection cytometry analysis), 6D is the result of flow cytometry of dendritic cells DC2.4 after 4 hours (the upper figure is the flow cytometry graph, and the lower figure is the result after convection cytometry analysis), 6E is the result of IFN-β expression level in cell culture supernatant, 6F is the result of CD80 expression level in DCs, 6G is the result of CD86 expression level in DCs, and control represents the control group;

[0035] Figure 7The in vitro distribution diagram of drug-loaded activated T cell membrane nanovesicles is a fluorescence signal detection result diagram of different organs, where heart represents heart, liver represents liver, spleen represents spleen, lung represents lung, kidney represents kidney, and tumor represents tumor, the same below;

[0036] Figure 8 Figure 8 is a test result diagram of anti-tumor immunotherapy of drug-loaded activated T cell membrane nanovesicles, 8A is a flow chart of the dosing regimen, 8B is a test result diagram of body weight, 8C is a test result diagram of tumor volume, 8D is a test result diagram of CD8 perforin (i.e., perforin) cells, 8E is a test result diagram of CD8 granzyme B, 8F is a test result diagram of CD86 positive cell ratio, 8G is a test result diagram of NK cell ratio, 8H is a test result diagram of pro-inflammatory factor IFN-γ expression level, 8I is a test result diagram of pro-inflammatory factor TNF-α expression level; Drug administration means drug administration, Immune cells analysis means immune cell analysis, Time means time, days means days, orthortopic CT-26 tumor inoculation means CT-26 tumor vaccine, mouse means mouse, and means and, and Weight means body weight;

[0037] Fig. 9 This is a graph showing the results of the detection of drug-loaded activated T cell membrane nanovesicles blocking the expression levels of tumor immune checkpoint ligands PD-L1, GAL-3 and GAL-9;

[0038] Fig.10 This is the result of H&E staining of mouse organs. DETAILED DESCRIPTION

[0039] The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

[0040] One embodiment of the present invention provides a drug delivery nanovesicle, comprising: cationic liposome nanoparticles, diaminobenzimidazole embedded in the cationic liposome nanoparticles, and primary activated T cell membranes located on the outer surface of the cationic liposome nanoparticles. The cationic liposome nanoparticles have an average diameter of 100-200nm, an electrokinetic potential of -10 to -25mV, and an encapsulation rate of 55%-70%. The activated T cell membranes are prepared by activating and extracting primary T cells isolated from the spleen or peripheral blood of Balb / c mice.

[0041] like Figure 1 As shown, another embodiment of the present invention also provides a method for preparing the drug delivery nanovesicles as described above, comprising the following steps:

[0042] S1. Activate primary T cells with monoclonal antibodies, screen activated T cells expressing adhesion molecules and immune checkpoints, lyse activated T cells with lysis buffer, and then ultrasonically disrupt the cell membrane of activated T cells into membrane fragments, centrifuge, and obtain membrane fragments, i.e. activated T cell membranes;

[0043] S2. The diaminobenzimidazole is embedded in the cationic liposome nanoparticles by a co-incubation method to obtain cationic liposome nanoparticles embedded with diaminobenzimidazole;

[0044] S3. The activated T cell membrane is coated on the outer surface of cationic lipid nanoparticles embedded with diaminobenzimidazole by extrusion method to prepare drug delivery nanovesicles.

[0045] Another embodiment of the present invention also provides the use of the drug delivery nanovesicles as described above and / or the drug delivery nanovesicles prepared according to the preparation method as described above in the preparation of anti-tumor drugs.

[0046] In the following description, numerous details are discussed to provide a more thorough explanation of embodiments of the present invention; however, it is apparent to one skilled in the art that embodiments of the present invention may be practiced without these specific details.

[0047] (I) Isolation and activation of primary mouse T cells

[0048] S1.1 Remove the spleen of a Balb / c mouse under sterile conditions in a clean bench and place it in a 35 mm sterile culture dish. Add 5 mL of mouse lymphocyte separation solution (commercially available, return to room temperature and shake well before use) to the dish and grind to obtain a single cell suspension containing spleen cells.

[0049] S1.2 Blood was collected from the tail vein of the mouse. The mouse was placed in a fixator and the tail was repeatedly wiped with an alcohol cotton ball to make the tail vein of the mouse fully congested. The tip of the tail was cut with scissors, and blood flowed out from the tip of the tail. The blood was dripped into an anticoagulant test tube and diluted with 500ul PBS.

[0050] S1.3 The above suspension was used according to the instructions of mouse lymphocyte separation medium to extract lymphocytes.

[0051] S1.4 Lymphocytes were processed using the EasySepTM Mouse T Cell Isolation Kit to enrich mouse primary T cells (unactivated T cells);

[0052] S1.5 Coat a 24-well plate with 1 μg / mL anti-CD3 monoclonal antibody (clone 17A2) and place at 4°C for -24 h;

[0053] S1.6 After counting the primary mouse T cells, the primary mouse T cells were immediately resuspended in complete culture medium (i.e., RPMI1640 culture medium containing 10% FBS, 25 mM HEPES, 1×GlutaMax, 1% penicillin-streptomycin, 55 μM β-mercaptoethanol, 1×MEM NEAA, 1 mM sodium pyruvate, and 200 U / ml IL-2). Anti-CD28 monoclonal antibody was added to the complete culture medium to a final concentration of 1 μg / mL, and 2×10 6 The number of cells / well was seeded into antibody-coated well plates and activated for 48 h at 37°C and 5% CO2;

[0054] S1.7 Activated T cells were transferred to new wells every 2 days and maintained in complete culture medium only;

[0055] On day 6, the cells were rested for 24 h in complete medium without IL-2 (the complete medium was RPMI1640 medium, containing 10% FBS, 25 mM HEPES, 1×GlutaMax, 1% penicillin-streptomycin, 55 μM β-mercaptoethanol, 1×MEM NEAA and 1 mM sodium pyruvate);

[0056] Then, the cells were transferred to a new CD3 antibody-coated plate (24 h in advance, 200 uL of 1 μg / mL CD3 antibody was added to the 24-well plate and incubated overnight at 4°C to complete antibody coating), and activated for 48 h at 37°C and 5% carbon dioxide in complete medium (i.e., RPMI1640 medium containing 10% FBS, 25 mM HEPES, 1×GlutaMax, 1% penicillin-streptomycin, 55 μM β-mercaptoethanol, 1×MEM NEAA, 1 mM sodium pyruvate, and 200 U / ml IL-2) containing 1 μg / mL anti-CD28 antibody, and then transferred to a new plate and activated for 24 h in complete medium at 37°C and 5% carbon dioxide;

[0057] The expression levels of activated T cell immune checkpoints LAG-3, TIM-3, PD-1 and adhesion molecules were analyzed by flow cytometry (FCM) every day. The specific panels were CD3-FITC, CD4-Per5.5, CD8-Alex700, PD-1-BV421, LAG-3-APC, TIM-3-PE, TIGIT-PC7). The flow cytometry antibodies were prepared into a mixed solution according to the panel, and 5*10 6 For staining, the cells were resuspended by adding the prepared antibody mixture and incubated at room temperature in the dark for 20 min; washed once with PBS buffer (pH = 7.4), the cells were centrifuged at 300g for 5 min, filtered with a flow cytometer, detected and collected data using a FACS Celesta flow cytometer, and then processed with FlowJo software;

[0058] S1.8 Screening of activated T cells with high expression of immune checkpoints based on flow cytometry analysis. The results are as follows Figure 2 shown.

[0059] (II) Extraction and characterization of activated T cell membranes

[0060] S2.1 Collect the activated T cells screened in (a) and wash them three times with PBS buffer (pH = 7.4);

[0061] S2.2 The cells were suspended in 20 mM Tris-HCl (pH 7.5), 10 mM KCl, 2 mM MgCl 2 and Pierce protease and phosphatase inhibitors (EDTA-free, 1 tablet per 10 mL solution) in hypotonic lysis buffer (commercially available), incubate on ice for 3 h, and then sonicate on ice at 80 W for 5 min, specifically sonicating for 3 s, stopping for 5 s, and repeating this cycle several times until 5 min;

[0062] The homogenate obtained after ultrasound was then centrifuged at 7000 g and 4°C for 10 min to remove intact cells and mitochondria, and the supernatant obtained by centrifugation was centrifuged at 148000 g and 4°C for 45 min, and the membrane precipitate obtained by centrifugation was washed twice with PBS buffer (pH = 7.4);

[0063] S2.3 was sequentially extruded through a 0.4 μm liposome extruder and a 0.2 μm liposome extruder 11 times each to obtain uniform cell membrane vesicles, followed by ultracentrifugation at 148,000 g and 4°C for 45 min, and the centrifuged precipitate was washed twice with PBS buffer (pH = 7.4), and the precipitate was collected and resuspended with 0.2 mM EDTA to obtain activated T cell membrane nanovesicles (AM);

[0064] S2.4 extracting unactivated T cell membranes according to the methods of S2.1 to S2.3, that is, treating the mouse primary T cells (unactivated T cells) obtained in S1.6 according to the methods of S2.1 to S2.3 to obtain unactivated T cell membrane nanovesicles (M), which are stored at -80°C for future use;

[0065] S2.5 Determination of cell membrane vesicle concentration: The cell membrane vesicle concentration of activated T cell membrane nanovesicles (AM) and non-activated T cell membrane nanovesicles (M) was determined using a BCA protein concentration determination kit (commercially available) according to the instructions.

[0066] After testing, for activated T cell membrane nanovesicles (AM), 1×10 8 The protein content in the purified membrane of each cell was 0.3 mg.

[0067] S2.6 Particle size distribution and Zeta potential: Determine the particle size distribution and Zeta potential of activated T cell membrane nanovesicles (AM), the specific steps are as follows:

[0068] Take about 0.2 mL of sample and place it in a 5 mL centrifuge tube. Add 1 mL of water to the centrifuge tube and ultrasonically disperse for 5 minutes. Take 1 mL of the upper solution and slowly add it to the sample pool to avoid bubbles in the sample pool.

[0069] Nanoparticle size and Zeta potential analyzer (Brookhaven Instruments, NanoBrook Omni) was used for testing. The instrument was preheated for 30 minutes. The test software was started and the Zeta (or particle size) test program was selected. Water was used as the solvent and the test was performed after preheating at 25°C for 30 seconds. Each sample was tested three times in parallel and the average value was taken as the final result. The results are shown in the figure. Figure 3 A and Figure 3 As shown in B.

[0070] Depend on Figure 3 A and Figure 3 B shows that the particle size of activated T cell membrane nanovesicles (AM) is 100-200 nm, and the Zeta potential is -25 to -35 mV.

[0071] S2.7 Observe the morphology of activated T cell membrane nanovesicles (AM) and unactivated T cell membrane nanovesicles (M) by transmission electron microscopy. The specific steps are as follows:

[0072] Sample preparation: Spread the sealing film flat on the glass slide, use tweezers to place the copper mesh on the sealing film, use a pipette to take 10 μL of sample and drop it on the copper mesh, time it for 10 minutes, and use a small piece of filter paper to absorb the excess liquid;

[0073] Use a pipette to take 10 μL of 2% uranyl acetate and drop it on the copper grid. After timing for 3 minutes, use a small piece of filter paper to absorb the excess liquid; dry it, time it for 5 minutes, load the sample, and observe it under a transmission electron microscope (the electron microscope model is Thermo Fisher Scientific CD Ltd, Talos F200S). The results are as follows Figure 3 As shown in C.

[0074] like Figure 3 As shown in C, activated T cell membrane nanovesicles (AM) have a typical membrane morphology.

[0075] S2.8 The expression levels of CD3 and PD-1, the marker proteins on the surface of activated T cell membrane nanovesicles (AM), were detected by Western Blot. The specific steps were as follows: extracting the protein solution and performing protein quantitative analysis using the BCA method; boiling and denaturing, followed by electrophoresis separation, membrane transfer and blocking, adding primary antibodies (anti-CD3 and anti-PD-1) and incubating overnight at 4°C, eluting the next day, adding the corresponding secondary antibodies, incubating at room temperature for 1 hour, and exposing and developing the samples by chemiluminescence after sufficient elution. The results are shown in the figure. Figure 3 As shown in D.

[0076] like Figure 3 As shown in D, the surface marker proteins CD3 and PD-1 on the surface of activated T cell membrane vesicles were well retained.

[0077] S2.9 activated T cell membrane vesicles relieve T cell immunosuppression and restore T cell killing:

[0078] To explore the mechanism of activated T cell membrane vesicles, the specific steps are:

[0079] CT26-Luci cells were pretreated with 20 ng / ml IFN-γ for 24 h, and the expression levels of immune checkpoint ligands PD-L1, GAL-3 and CD155 were detected by flow cytometry. The specific steps were as follows: 5*10 6Cells were used for staining, and the corresponding antibody mixture was added to resuspend the cells, and incubated at room temperature in the dark for 20 minutes; washed once with PBS buffer (pH = 7.4), the cells were centrifuged at 300g for 5 minutes), filtered with a flow filter, and then detected and collected data with a FACS Celesta flow cytometer, and the data were processed with FlowJo software. A control group was set up, which was CT26-Luci cells not treated with IFN-γ. The results are shown in Figure 3 As shown in E.

[0080] like Figure 3 As shown in E, CT26-Luci pretreated with IFN-γ highly expressed immune checkpoint ligands PD-L1, GAL-3, and CD155.

[0081] IFN-γ pretreated CT26-Luci were co-incubated with T cells, activated T cell membranes (AM), unactivated T cell membranes (M), and PBS buffer (pH = 7.4) at 37°C and 5% carbon dioxide for 24 h (CT26-Luci cells / activated T cells = 1 / 5, number ratio, n = 3), i.e., IFN-γ pretreated CT26-Luci and T cell co-incubation group (referred to as T treatment group), IFN-γ pretreated CT26-Luci and activated T cell membrane (AM) co-incubation group (referred to as AM+T treatment group), IFN-γ pretreated CT26-Luci and unactivated T cell membrane (M) co-incubation group (referred to as M+T treatment group), and IFN-γ pretreated CT26-Luci and PBS buffer (pH = 7.4) co-incubation group (referred to as M group);

[0082] Subsequently, the cell viability of CT26-Luci was detected by firefly luciferase method. The specific steps were as follows: TM The assay was performed according to the instructions of the Cytotoxicity Assay Kit (Progmega), i.e., after the incubation of CT26-Luci and T cells, Cytotoxicity Assay Reagent was added, and the cells were incubated at 37°C and 5% carbon dioxide for 15 min. The results were then detected using a multi-function microplate reader (Varioskan Lux, Thermo). The results were as follows: Figure 3 As shown in F;

[0083] Flow cytometry was used to detect the expression levels of T cell killing functional molecule Granzyme B, T cell killing functional molecule Perforin and pro-inflammatory cytokine IFN-γ. The method was the same as above. The results are as follows Figure 3 As shown in G.

[0084] like Figure 3As shown in F, the tumor cell killing rate in the AM+T treatment group was the highest compared with other groups. This result indicates that activated T cell membrane (AM) can enhance the tumor cell killing ability of T cells.

[0085] like Figure 3 As shown in G, the expression levels of T cell killing functional molecule Granzyme B, T cell killing functional molecule Perforin and proinflammatory cytokine IFN-γ in the AM+T treatment group were the highest. This result shows that activated T cell membrane (AM) can enhance the killing ability of T cells. Obviously, the present invention successfully prepared drug delivery nanovesicles (AM) based on activated T cell membranes that stably express immune checkpoints.

[0086] (III) Preparation of diABZI-loaded cationic liposomes coated with activated T cell membrane

[0087] S3.1 prepare a precursor solution for preparing stable micronized liposomes, namely, an ammonium sulfate solution as an aqueous phase component and a lipid molecule alcohol solution as a lipid phase component, wherein the aqueous phase solution is an ammonium sulfate solution with a concentration of 25 mmol / L, and the lipid phase solution is an ethanol solution of lipid components, wherein the lipid components are cationic liposome SM102, cholesterol, cationic liposome DMG-PEG2000, DODA and PDODAP in a molar ratio of 6:6:2:1, and the concentration of cholesterol in the ethanol solution is 43 mg / 25 mL;

[0088] The ammonium sulfate solution and the ethanol solution were respectively introduced into the water phase injection port and the lipid phase injection port of the microfluidic chip (commercially available), and a collection component was placed at the outlet;

[0089] The ammonium sulfate solution and the ethanol solution were mixed through the mixing channel of the microfluidic chip, and the power of the mixed raw material solution was provided by a micro-injection pump system (Chemyx micro-injection pump, USA). The flow rates of the ammonium sulfate solution and the ethanol solution were 0.1 mL / min and 2 mL / min, respectively. The products were collected at the outlet of the main microchannel of the microfluidic chip, and the final liquid product was collected by an ultrafiltration centrifuge tube. The final liquid product was centrifuged at a speed of 4000 r / min for 20 min, and the obtained precipitate was the prepared blank cationic liposome (LNP);

[0090] S3.2 Preparation of cationic liposome dLNP loaded with diABZI: 0.09 mg diABZI was dissolved in 9 μL DMSO, and then 300 μL LNP was added to DMSO, heated in a 65°C water bath for 60 min, and dialyzed to remove free diABZI not encapsulated by liposomes to obtain dLNP;

[0091] S3.3 Activated T cell membrane coated with diABZI-loaded cationic liposomes (AM-dLNP): The activated T cell membrane (AM) was coated on the dLNP by extrusion method. The specific steps are as follows:

[0092] 1×10 8 AM purified from each cell was mixed with 200ul dLNP (diABZI 0.9mg / mL), and then extruded through a polycarbonate membrane with a pore size of 400nm and a polycarbonate membrane with a pore size of 200nm in sequence to prepare drug-loaded cationic liposomes coated with activated T cell membranes (i.e., AM-dLNP);

[0093] The same method is used to prepare drug-loaded cationic liposomes coated with non-activated T cell membranes (M-dLNP), blank liposomes coated with activated T cell membranes (AM-dLNP) and cationic liposomes coated with activated T cell membranes (AM-dLNP), wherein the blank liposomes coated with activated T cell membranes (AM-LNP) refer to blank cationic liposomes (LNP) coated with activated T cell membranes in the same manner as AM-dLNPs except that the drug is not loaded, and the cationic liposomes coated with activated T cell membranes (AM-dLNP) refer to dLNPs coated with activated T cell membranes in the same manner as AM-dLNPs except that the drug is not loaded;

[0094] S3.4 Particle size distribution and Zeta potential:

[0095] The particle size distribution and Zeta potential of dLNP, M-dLNP, AM-LNP and AM-dLNP were determined by the same method as S2.6. The results are shown in Figure 4 A to Figure 4 As shown in D;

[0096] like Figure 4 A to Figure 4 As shown in D, the particle size of the nanovesicles in each group was 100-200 nm, the Zeta potential of dLNP was 15.67 mV, the Zeta potential of M-dLNP was -20.73 mV, the Zeta potential of AM-LNP was -17.20 mV, and the Zeta potential of AM-dLNP was -13.62 mV.

[0097] S3.5 The expression levels of marker proteins CD3, PD-1, TIM-3 and LAG-3 of blank liposomes (AM-dLNP) coated with activated T cell membrane were detected by Western Blot. The method was the same as above. The results were as follows Figure 4 As shown in E;

[0098] like Figure 4As shown in E, the signature protein molecules CD3, PD-1, TIM-3, and LAG-3 on the surface of activated T cell membrane nanovesicles are well preserved.

[0099] S3.6 Observation of the morphology of nanomedicine by transmission electron microscopy: The morphology of dLNP, M-dLNP, AM-LNP and AM-dLNP was observed by the same method as S2.7. The results are shown in Figure 4 As shown in F;

[0100] like Figure 4 As shown in F, blank liposomes (AM-dLNP) coated with activated T cell membrane have a typical core-shell structure.

[0101] S3.7 Encapsulation efficiency test:

[0102] The encapsulation efficiency of dLNP, M-dLNP and AM-dLNP was detected by the following specific steps:

[0103] DiABZI was prepared into a mother solution with a concentration of 1 mg / mL using dimethyl sulfoxide (DMSO), and then diluted with acetonitrile to a working solution with a gradient concentration of 10 ug / mL, 5 ug / mL, 1 ug / mL, 0.5 ug / mL and 0.25 ug / mL;

[0104] Four volumes of acetonitrile were added to dLNP, M-dLNP, and AM-dLNP, respectively, vortexed, centrifuged at 12,000 g for 30 min, and then diluted 10 times with acetonitrile;

[0105] The working solution and diluted samples were detected by high performance liquid chromatography HPLC (SHIMADZU Corporation, LC-20A, chromatographic column: C18 column (250 mm × 4.6 mm, 5 μm)), with an injection volume of 20 uL, mobile phase: aqueous phase acetonitrile: water = 90:10 (v / v), flow rate of 1 mL / min, column temperature of 30 °C, and detection wavelength of 214 nm;

[0106] Draw a concentration-peak area standard curve based on the test results, and calculate the encapsulation efficiency according to the following formula. The results are as follows: Figure 4 As shown in G;

[0107] Encapsulation efficiency = drug content in liposomes / dose × 100%;

[0108] In the formula, the drug content in the liposomes represents the product of the concentration obtained according to the standard curve and the corresponding solution volume and the corresponding dilution multiple;

[0109] like Figure 4As shown in G, the encapsulation efficiency of dLNP is 75.86%, the encapsulation efficiency of M-dLNP is 62.62%, and the encapsulation efficiency of AM-dLNP is 58.05%.

[0110] S3.8 Stability test:

[0111] dLNP, M-dLNP, AM-LNP and AM-dLNP were resuspended in 10% FBS and placed at 37°C for 5 days. Every day, an appropriate amount of sample solution was taken from each group to measure the particle size and Zeta potential to evaluate the stability of each group of samples over time. The test method was the same as above. The results are shown in Figure 4 H and 4I.

[0112] like Figure 4 As shown in H and 4I, the stability of each group of nanovesicles in 10% FBS was good and the morphology and structure could be maintained for a long time.

[0113] (IV) Study on the biological functions of drug-loaded activated T cell membrane nanovesicles in vitro

[0114] The in vitro biological functions of drug-loaded activated T cell membrane nanovesicles were studied. The specific steps are as follows:

[0115] S4.1 Drug-loaded activated T cell membrane vesicles block tumor cell immune checkpoint ligands: 4T1-Luci cells were pretreated with IFN-γ at a concentration of 20 ng / ml for 24 h;

[0116] Subsequently, IFN-γ-pretreated 4T1-Luci cells were incubated with AM-dLNP, AM-LNP, M-dLNP, dLNP and PBS buffer (pH = 7.4) at 37°C and 5% carbon dioxide for 4 h, and the cells were collected, washed three times with PBS buffer and stained with the corresponding antibodies, and then analyzed by flow cytometry. The specific staining steps were the same as before. The results are shown in Figure 5 As shown in A, 5B and 5C;

[0117] like Figure 5 As shown in A, 5B and 5C, the expression levels of PD-L1, GAL-3 and CD155 on the surface of 4T1-Luci cells in the AM-dLNP and AM-LNP treatment groups were significantly reduced compared with the other groups. This result shows that the drug-loaded activated T cell membrane nanovesicles blocked the immune checkpoint ligands PD-L1, GAL-3 and CD155 highly expressed in 4T1-Luci;

[0118] S4.2 Drug-loaded activated T cell membrane vesicles relieve T cell membrane immunosuppression and restore T cell killing:

[0119] 4T1-Luci cells were pretreated with IFN-γ at a concentration of 20 ng / ml for 24 h;

[0120] Mouse primary T cells (unactivated T cells) were activated for 72 h according to the method in (a);

[0121] IFN-γ pretreated 4T1-Luci and activated 72h T cells were incubated with diABZI, dLNP, M-dLNP, AM-LNP, AM-dLNP and PBS buffer (pH=7.4) at 37°C and 5% carbon dioxide for 24h (4T1-Luci cells / activated T cells=1 / 5, diABZI=5ug / mL, n=3), for a total of 6 groups; then the activity of 4T1-Luci cells was detected by firefly luciferase method, the specific steps were the same as before, and the results were shown in Figure 3. Figure 5 D and 5E;

[0122] FCM was used to detect the expression levels of the killing function molecule Granzyme B, the killing function molecule Perforin, and the pro-inflammatory cytokine IFN-γ to evaluate whether AM-dLNP can enhance the killing function of T cells. The method was the same as above. The results are shown in Figure 5 As shown in F, 5G, and 5H;

[0123] like Figure 5 As shown in D and 5E, the AM-dLNP+T cell group had the highest tumor cell killing rate compared with the other groups.

[0124] like Figure 5 As shown in F, 5G, and 5H, the expression levels of T cell killer molecule Granzyme B, killer molecule Perforin, and proinflammatory cytokine IFN-γ were the highest. This result shows that activated T cell membrane (AM) can block the immune checkpoint ligands on the surface of 4T1-Luci, relieve T cell membrane immunosuppression, and thus enhance the killing ability of T cells.

[0125] S4.3 In vitro uptake of drug-loaded activated T cell membrane vesicles:

[0126] The uptake efficiency of AM-dLNP, AM-LNP, M-dLNP and dLNP by tumor cells CT-26 and dendritic cells DC2.4 was compared. The specific steps are as follows:

[0127] The IFN-γ-pretreated tumor cells CT-26 (pretreated with IFN-γ at a concentration of 20 ng / ml for 24 h) were divided into two portions and inoculated into a confocal culture dish and a 24-well plate, respectively, with 1.0 × 10 5 The cells seeded in the confocal culture dish were used for subsequent laser confocal photography, and the cells seeded in the 24-well plate were used for subsequent FCM analysis.

[0128] Dendritic cells DC2.4 were inoculated into two portions, and the two portions were inoculated into a confocal culture dish and a 24-well plate, with 1.0×10 cells per well. 5 The cells seeded into the confocal culture dish were used for subsequent laser confocal photography, and the cells seeded into the 24-well plate were used for subsequent FCM analysis.

[0129] Tumor cells CT-26 pretreated with IFN-γ were mixed with AM-dLNP, AM-LNP, M-dLNP and dLNP respectively, and co-cultured at 37°C and 5% carbon dioxide for 1 h (tumor cells CT-26 / activated T cells = 1 / 5, quantity ratio, diABZI = 5ug / mL, n = 3), and a control group was set up, which was CT-26 cells pretreated with IFN-γ without adding any reagents;

[0130] Dendritic cells DC2.4 were mixed with AM-dLNP, AM-LNP, M-dLNP and dLNP respectively, and co-cultured for 4 h at 37°C and 5% carbon dioxide (dendritic cells DC2.4 / activated T cells = 1 / 5, diABZI = 5ug / mL, n = 3), and a control group was set up, which consisted of DC2.4 cells without any reagent treatment;

[0131] The cell uptake of nanomedicine was observed by FCM and confocal laser scanning (CLSM). The FCM method was the same as above. The results are shown in Figure 6 As shown in A to 6D;

[0132] like Figure 6 As shown in A to 6D, AM-dLNP and AM-LNP were more easily taken up by tumor cells CT-26 and dendritic cells DC2.4 compared with the other groups.

[0133] S4.4 Drug-loaded activated T cell membrane vesicles enhance the expression of IFN-β in tumor cells:

[0134] Detect the expression level of IFN-β in the cell culture supernatant. The specific steps are as follows:

[0135] IFN-γ pretreated tumor cells CT-26 (2×10 5 / well, n=3) were cultured with AM-dLNP, AM-LNP, M-dLNP, dLNP, diABZI (5 μg / ml) and PBS buffer (pH=7.4) at 37°C and 5% carbon dioxide for 24 h, and the cell culture supernatant was collected. The expression level of IFN-β in the cell culture supernatant was detected by ELISA kit (commercially available). The results are shown in Figure 6 As shown in E;

[0136] like Figure 6As shown in E, the expression level of IFN-β in tumor cells CT-26 treated with AM-dLNP was significantly increased compared with other groups.

[0137] S4.5 Drug-loaded activated T cell membrane vesicles promote DCs maturation:

[0138] The tibiae of Balb / C mice were washed with PBS buffer (pH = 7.4), and then the cell suspension was passed through a 70 μm cell strainer to obtain bone marrow cells;

[0139] The cells were then cultured in 6-well plates at 37°C and 5% carbon dioxide in RPMI1640 medium containing 2 mM L-glutamine, 10% FBS, 0.4 mM sodium pyruvate, 50 μM β-mercaptoethanol, 20 ng / ml IL-4, and 20 ng / mL GM-CSF to induce differentiation into BMDCs;

[0140] Fresh culture medium was added on the 2nd, 4th, and 7th days, and the cells were transferred to a 12-well plate on the 8th day, with 105 cells per well, and AM-dLNP, AM-LNP, M-dLNP, dLNP, diABZI (diABZI 5 μg / ml), and PBS buffer (pH = 7.4) were added respectively;

[0141] After 24 h, the cells were scraped and washed three times with PBS buffer (pH = 7.4);

[0142] DC maturation marker antibodies (CD80, CD86) were used for staining, and FCM was used to detect the expression levels of maturation marker molecules CD80 and CD86 in DCs. The method was the same as above. The results were as follows: Figure 6 F and 6G;

[0143] like Figure 6 As shown in Figures 6F and 6G, the expression levels of maturation marker molecules CD80 and CD86 in DCs treated with AM-dLNP were significantly increased compared with those in other groups.

[0144] (V) Study on the anti-tumor effect of drug-loaded activated T cell membrane nanovesicles in vivo

[0145] The steps for studying the anti-tumor effect of drug-loaded autologous activated T cell membrane nanovesicles in vivo are as follows:

[0146] S5.1 Accumulation of drug-loaded activated T cell membrane nanovesicles in tumor tissues: After review and approval by the Ethics Committee of Chongqing Medical University (approval number: IACUC-CQMU-2024-0567), experimental animals were used in relevant experiments, and a CT-26 colon cancer tumor-bearing model was constructed on Balb / c mice (half male and half female, 16±2g, 6-8 weeks old). The specific steps for constructing the CT-26 colon cancer tumor-bearing model were as follows: 5×10 5 CT-26 cells were injected subcutaneously into Balb / c mice (half male and half female, 16±2 g, 6-8 weeks old), and the tumor volume was measured every 2-3 days. The tumor volume was calculated as follows: V = (length × width 2 ) / 2, the tumor grows to 100mm 3 , tumor-bearing mice were obtained;

[0147] Tumor-bearing mice were randomly divided into 4 groups and intravenously injected with 100 μL of AM-dLNP, AM-LNP, M-dLNP, and dLNP labeled with Cy5.5 (1.5 mg diABZI / kg, n = 4), respectively;

[0148] At 2h, 4h, 6h and 24h after injection, the IVIS Spectrum imaging system was used to detect the fluorescence signals in the mice and in the tumors. Specifically, after in vivo imaging, one mouse in each group was taken to collect the tumor and other major organs (heart, liver, spleen, lung, and kidney) for ex vivo imaging, and the fluorescence signals of different organs were analyzed using IVIS software. The results are shown in Figure 7 As shown;

[0149] like Figure 7 As shown, AM-dLNP accumulated most in the tumor site compared with other groups. This result shows that the drug delivery nanovesicles based on activated T cell membrane in the present invention have excellent targeting.

[0150] S5.2 Anti-tumor therapy with drug-loaded autologous activated T cell membrane nanovesicles:

[0151] To study the anti-tumor effect of AM-dLNP, in the CT-26 colon cancer model constructed above, when the tumor volume was about 50-100mm 3 The tumor-bearing mice were randomly divided into 6 groups and intravenously injected with 100 μL AM-dLNP, AM-LNP, M-dLNP, dLNP and free diABZI (1.5 mg diABZI / kg) on ​​the 1st, 4th and 8th day, respectively, and PBS injection (pH = 7.4) was used as control (n = 5) (the dosing schedule flow chart is shown in Figure 8 The tumor volume and body weight of the mice were recorded every two days. Figure 8As shown in B, the tumor volume was calculated according to the formula volume = long diameter * short diameter * short diameter * 1 / 2. The result is as follows Figure 8 As shown in C;

[0152] If the mouse dies, loses more than 15% of its body weight, or has a tumor volume greater than 2000 mm 3 The mice were judged dead at 3 pm. Three days after the last injection, the tumors and spleens were collected and weighed. A portion of the tumors was fixed in 4% paraformaldehyde for preservation. A portion of the tumors and spleens were cut into small pieces and gently crushed with a nylon mesh to prepare a single-cell suspension.

[0153] A portion of the cell suspension was taken for flow cytometry staining with the corresponding antibodies, using the same method as above, to analyze CD45+ immune cells, CD3+T lymphocytes, CD4+T cells, CD8+T cells, NK cells and DCs in the tumor and spleen. The specific detection panel is shown in the following table:

[0154] Table 1 Detection panel

[0155]

[0156] Some tumor cell suspensions were quickly frozen in liquid nitrogen and stored at -80°C for cytokine detection. Cytokine levels were detected using the Bio-Plex ProTM assay (commercially available kit). The results are as follows: Figure 8 D to 8G;

[0157] Another part of the tumor cell suspension was quickly frozen in liquid nitrogen and stored at -80°C for cytokine detection. The expression levels of pro-inflammatory factors IFN-γ and TNF-α were determined by Bio-Plex ProTM analysis (using commercially available kits). The results are as follows: Figure 8 H and 8I;

[0158] like Figure 8 As shown in B, no obvious weight loss was found in the mice in each group.

[0159] like Figure 8 As shown in Figure C, compared with other groups, the tumor volume of mice treated with AM-dLNP was significantly reduced, that is, the development was significantly delayed. This result shows that the drug delivery nanovesicles based on activated T cell membranes of the present invention can inhibit tumor growth.

[0160] like Figure 8As shown in D to 8G, the expression of CD8+T cell killing functional molecules Granzyme B and Perforin in the tumor treated with AM-dLNP was the highest (Granzyme B and Perforin are key molecules produced by CD8+T cells to kill tumor cells), and the number of mature DCs and NK cells was the largest. This result shows that the drug delivery nanovesicle based on activated T cell membrane of the present invention has excellent anti-tumor activity.

[0161] like Figure 8 As shown in H and 8I, the expression levels of pro-inflammatory factors IFN-γ and TNF-α in the tumors of the AM-dLNP treatment group were significantly increased. This result shows that the drug delivery nanovesicles based on activated T cell membranes of the present invention have excellent anti-tumor activity by increasing the expression levels of the promoting factor IFN-γ and the pro-inflammatory factor TNF-α;

[0162] S5.3 Immunohistochemistry and immunofluorescence analysis of immune checkpoint ligand expression in tumors:

[0163] In the above-constructed CT-26 colon cancer tumor-bearing model, the collected tumor was placed in 4% paraformaldehyde, and after fixation, the tumor was dehydrated, embedded and sliced, then deparaffinized and soaked in 3% methanol hydrogen peroxide at 25°C for 10 minutes. It was washed three times with PBS buffer (pH=7.4), and the slices were immersed in citric acid buffer (0.01M, pH=6.0) and heated to boiling in a microwave for antigen retrieval;

[0164] Rinse three times with PBS buffer (pH = 7.4), add goat serum, and incubate at 25°C for 30 min. Rinse three times with PBS buffer (pH = 7.4), add primary antibodies diluted with 5% goat serum: PD-L1 antibody (1:50), Gal-3 antibody (1:100), and Gal-9 antibody (1:100), and incubate at 4°C overnight;

[0165] On the second day, add poly-HRP secondary antibody (goat anti-rabbit), incubate at 25°C for 30 minutes, add TSA fluorescent dye reaction solution, incubate at room temperature in the dark for 8 minutes; rinse with PBS buffer (pH = 7.4) 3 times, 5 minutes each time; add DAPI staining solution, incubate at room temperature for 5 minutes; rinse with PBS buffer (pH = 7.4) 3 times, 5 minutes each time; after drying the slices, seal them with anti-fluorescence attenuation sealing agent, and observe the expression of PD-L1 under a fluorescence microscope. Immunohistochemistry, that is, after the completion of the secondary antibody incubation, perform DAB staining, hematoxylin staining, dehydrate and seal the slices, and observe the expression levels of GAL-3 and GAL-9 under a microscope. The results are as follows Fig. 9 As shown;

[0166] like Fig. 9 As shown, compared with other groups, the expression levels of immune checkpoint ligands PD-L1, GAL-3 and GAL-9 in mouse tumors treated with AM-dLNP and AM-LNP were significantly reduced. This result shows that the drug delivery nanovesicles based on activated T cell membranes of the present invention can block immune checkpoints, relieve T cell immunosuppression, and thus exert anti-tumor effects.

[0167] S5.4 Study on the biosafety of drug-loaded autologous activated T cell membrane nanovesicles:

[0168] The organs in 5.2 were subjected to histological examination. The heart, liver, spleen, lung and kidney of each group of mice were stained with H&E to observe whether there was tissue damage. The results are as follows: Fig.10 shown.

[0169] like Fig.10 As shown, no obvious histological damage was found in each group. This result shows that the drug delivery nanovesicle based on activated T cell membrane of the present invention has good biosafety.

[0170] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A drug delivery nanovesicle, It is characterized in that The drug delivery nanovesicle comprises: cationic liposome nanoparticles, diaminobenzimidazole embedded in the cationic liposome nanoparticles, and autologous activated T cell membranes located on the outer surface of the cationic liposome nanoparticles.

2. The drug delivery nanovesicle according to claim 1, It is characterized in that The average diameter of the cationic liposome nanoparticles is 100-200 nm.

3. The drug delivery nanovesicle according to claim 1, It is characterized in that The cationic liposome nanoparticles have an electrokinetic potential of -10 to -25 mV.

4. The drug delivery nanovesicle according to claim 1, It is characterized in that The encapsulation rate of the cationic liposome nanoparticles is 55%-70%.

5. The drug delivery nanovesicle according to claim 1, It is characterized in that The activated T cell membrane is prepared by extracting and activating primary T cells separated from the spleen or peripheral blood of Balb / c mice.

6. A method for preparing the drug delivery nanovesicles according to any one of claims 1 to 5, It is characterized in that The following steps are involved: S1. Activate primary T cells and extract membrane fragments to obtain activated T cell membranes; S2. embedding the diaminobenzimidazole in the cationic liposome nanoparticles to obtain cationic liposome nanoparticles embedded with diaminobenzimidazole; S3. Applying the activated T cell membrane to the outer surface of the cationic liposome nanoparticles embedded with diaminobenzimidazole to obtain the drug delivery nanovesicles.

7. The method for preparing the drug delivery nanovesicles according to claim 6, It is characterized in that In step S1, extracting membrane fragments comprises: The activated T cells are lysed with an isothiocyanate solution, and then ultrasonically disrupted to break the cell membranes of the activated T cells into membrane fragments, which are then centrifuged to obtain the activated T cell membranes.

8. The method for preparing the drug delivery nanovesicles according to claim 6, It is characterized in that In step S2, the diaminobenzimidazole is embedded in the cationic liposome nanoparticles by a co-incubation method.

9. The method for preparing the drug delivery nanovesicles according to claim 6, It is characterized in that In step S3, the activated T cell membrane is coated on the outer surface of the cationic liposome nanoparticles embedded with diaminobenzimidazole by extrusion method.

10. Use of the drug delivery nanovesicle according to any one of claims 1 to 5 and / or the drug delivery nanovesicle prepared according to the preparation method according to any one of claims 6 to 9 in the preparation of anti-tumor drugs.

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