CN115770294A - Biological liposome carrying antigen and cisplatin as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a bioliposome carrying antigen and cisplatin, a preparation method and application thereof, and belongs to the technical field of biological medicines. The biological liposome is formed by fusing a biological membrane and the liposome, the cisplatin is loaded in the vesicle, and the NK activatable target antigen is modified on the surface of the vesicle. The bioliposome of the invention takes the biomembrane fusion liposome as a carrier, targets tumor cells through the autonomous driving of the biomembrane, simultaneously realizes the implantation of the surface antigen of the tumor cells by utilizing the membrane fusion characteristic, enhances the expression of the tumor antigen, ensures that the tumor with insufficient antigen expression can be accurately identified and killed by NK cells, and provides a feasible method for solving the tumor heterogeneity.

Description

Biological liposome carrying antigen and cisplatin as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a bioliposome carrying antigen and cisplatin, and a preparation method and application thereof.

Background

Malignant tumor is one of the main death causes in the world at present, and has become a large class of diseases which seriously harm human life and health and restrict social and economic development. At present, the methods for treating tumors mainly comprise operations, chemotherapy, radiotherapy and the like, but the traditional treatment means has poor prognosis and large toxic and side effects and is easy to generate drug resistance. The immunotherapy of tumor has the advantages of high specificity, small toxic and side effects, long survival period and the like as an innovative treatment mode, and has become a hot spot in the research field of tumor therapy, including CAR-T, TCR-T, NK and the like. Natural Killer (NK) cells are important components of the innate immune system that have a direct killing effect on tumor cells, and unlike T cells, NK cells do not require any stimulation by antigen-presenting cells (APCs). In addition, NK cells produce cytokines and chemokines that recruit Dendritic Cells (DCs) and promote the maturation of DCs, enhancing the adaptive immune response.

Researches show that the NK cell infiltration of the in-situ tumor can effectively inhibit the growth of the tumor, and the NK cell infiltration of the high metastatic organ can effectively maintain the dormancy of the tumor cell and inhibit the tumor metastasis. These properties make NK cell therapy a new direction for current tumor immunotherapy research, including chimeric antigen receptor NK cell (CAR-NK) therapy and NK-targeted bispecific antibody therapy cells, among others.

The NK immune cell therapy has obvious effect on improving the immune function and the survival quality of malignant tumor patients, almost has no adverse reaction, is a safe and painless treatment scheme, has the advantages of non-cytotoxicity and targeting property, can complement the traditional treatment schemes of operations, radiotherapy, chemotherapy and the like, and provides a new treatment strategy for improving the prognosis and the survival quality of late-stage tumor patients. However, it was found in the studies that NK cell activity was affected by many factors, including imbalance of activating and inhibiting receptors on NK cells, abnormal binding of receptors to ligands, crosstalk of peripheral cell populations to NK cells in TME, etc. Tumor heterogeneity has been shown to limit the efficacy of NK therapy. I.e., tumor cells express different levels of target antigen, and most tumor cells contain target antigens well below the CAR-NK effective threshold, resulting in immune escape.

Disclosure of Invention

The invention aims to provide a bioliposome carrying antigen and cisplatin, which takes a bioliposome fusion liposome as a carrier, targets tumor cells through the autonomous driving of a biological membrane, simultaneously realizes the implantation of the antigen on the surface of the tumor cells by utilizing the membrane fusion characteristic, enhances the expression of the tumor antigen, ensures that the tumor with insufficient antigen expression can be accurately identified and killed by NK cells, and provides a feasible method for solving the tumor heterogeneity.

In order to realize the purpose of the invention, the following technical scheme is adopted:

a bioliposome is formed by fusing a biological membrane and the liposome, wherein cisplatin is loaded in the vesicle, and an NK activatable target antigen is modified on the surface of the vesicle;

the biological membrane is selected from the group consisting of neutrophil membrane, tumor cell membrane, stem cell membrane, macrophage membrane, platelet membrane, erythrocyte membrane, mitochondrial membrane and lysosome membrane, preferably platelet membrane.

In one embodiment of the invention, the NK activatable target antigen is an IgG antibody.

The preparation method of the bioliposome comprises the following steps:

(1) Cisplatin is added into water to form cisplatin-water suspension, hydrogen peroxide solution is added to react to obtain solution A, and the solution A is filtered and then recrystallized to obtain cisplatin prodrug Pt (IV).

Preferably: cisplatin: water: the mass ratio of the hydrogen peroxide solution is 1:1-300:5-500; further preferably: cisplatin: water: the mass ratio of the hydrogen peroxide solution is 1:10-30:30-50 parts of;

preferably: the reaction time is 1-6h; further preferably: the reaction time is 3-5h.

In one embodiment of the invention, the concentration of the hydrogen peroxide solution is 3% to 30%.

(2) Dissolving the cisplatin prodrug Pt (IV) in a solvent, adding an acid anhydride acylating agent, and stirring for reaction under an inert atmosphere to obtain a solution B.

Preferably: cisplatin prodrug: the mass ratio of the acid anhydride acylating agent is 1-100:1-100; further preferably: cisplatin prodrug: the mass ratio of the acid anhydride acylating agent is 5-15:5-10;

preferably: stirring for 12-48h; further preferably: the stirring time is 20-24h.

In one embodiment of the invention, the solvent is N, N-Dimethylformamide (DMF); the acid anhydride acylating agent is succinic anhydride.

(3) Ether was added to solution B to precipitate the product, the precipitate was collected and resuspended in water and lyophilized to give the carboxylated cisplatin prodrug Pt (IV) -COOH.

(4) Cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC sodium salt) and antigen were dissolved in PBS solution, respectively, and the two solutions were mixed and adjusted to pH7.4 and shaken at room temperature to obtain solution C.

Preferably, the following components: the reaction time is 1-4h; further preferably: the reaction time is 2-3h;

preferably, the following components: the antigen is an NK activatable antigen; further preferably an IgG antibody.

(5) Dissolving phospholipid in a PBS solution, dripping the solution into the solution C, and performing oscillation reaction at room temperature to obtain phospholipid-antigen;

preferably, the following components: the reaction time is 2-8h; further preferably: the reaction time is 4-5h;

preferably, the following components: the phospholipid is natural phospholipid, semisynthetic phospholipid or synthetic phospholipid; further preferably: distearoyl phosphatidyl acetamide-polyethylene glycol-sulfhydryl (DSPE-PEG 5000-SH);

preferably, the following components: sulfo-SMCC sodium salt: antigen: the mass ratio of the phospholipid is 0.1-50:0.1-50:5-500; further preferably: sulfo-SMCC sodium salt: antigen: the mass ratio of the phospholipid is 1-10:1-10:10-50.

(6) Adding an anticoagulant into whole blood, centrifuging to obtain plasma containing platelets, continuously centrifuging to obtain platelets, adding a PBS (phosphate buffer solution) into the platelets, freezing at-80 ℃, unfreezing the platelets at room temperature, centrifuging for 5 minutes at 10000g to form precipitates, adding the PBS into the precipitates, freezing at-80 ℃, and repeating the freeze-thaw cycle for several times to obtain the platelet membrane.

(7) A blank liposome is prepared by adopting a film dispersion method, and the specific process comprises the steps of dissolving 1, 2-dioleoyl-sn-propanetriyl-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycerol-3-phosphocholine (HSPC) and Cholesterol Hemisuccinate (CHEMS) in an organic solvent, and then carrying out rotary evaporation and drying to form a film.

Preferably, the following components: the organic solvent is dichloromethane;

preferably: DOPE: HSPC: the mass ratio of CHEMS is 0.5-100:0.5-100:0.1 to 50; further preferably: DOPE: HSPC: the mass ratio of the CHEMS is 1-10:1-10:1-5.

(8) Thereafter, the carboxylated cisplatin previous medicinal PBS obtained in step (3) was dissolved in PBS, and the solution was added to the film obtained in step (7), hydrated at 37 ℃ for 30 minutes, and then sonicated with a homogenizer to obtain liposomes.

Preferably: the power of the refiner is preferably 10 to 70 percent, the time is 10 to 60 minutes, the ultrasonic time is 0.1 to 2s, and the time interval is 0.5 to 5s; further preferably: the power is 20 to 40 percent, the time is 25 to 35 minutes, the ultrasonic duration is 0.5 to 1s, and the time interval is 2 to 4s.

(9) To obtain bioliposomes carrying antigen and cisplatin, the phospholipid-antigen from step (5), the platelet membrane from step (6), and the liposomes from step (8) were mixed, sonicated for 30 minutes, and then the unencapsulated carboxylated cisplatin prodrug and phospholipid were removed by centrifugation to give Pt @ PL-IgG.

The application of the bioliposome in preparing tumor treatment medicines.

The invention designs and successfully prepares the biological liposome which resists tumor heterogeneity and carries the NK activatable target antigen and the cisplatin prodrug, and can activate NK cells to realize the stable and controllable implantation of the antigen on the surface of the tumor cells, thereby accurately starting immunoreaction. The bioliposome targets tumor cells through the autonomous driving of platelet membranes, and simultaneously realizes the implantation of tumor cell surface antigen (IgG) by utilizing the membrane fusion characteristic. The antigen is stably and controllably implanted on the surface of the tumor cell, so that the immune recognition capability of the tumor cell is completely enhanced. In several mouse tumor models, the ITHAP strategy has successfully induced up to 50% of NK cell activation and resulted in tumor regression, significantly extending survival.

The invention has the beneficial effects that:

1. the present invention provides a bioliposome for enhancing tumor antigens to express and activate NK cells. The bioliposome carrying the NK activatable target antigen and the cisplatin prodrug can activate NK cells so as to realize the stable and controllable implantation of the antigen on the surface of a tumor cell, thereby accurately starting an immune reaction.

2. The bioliposomes of the invention initiate anti-tumor immune responses in three different directions: anti-tumor homogenized antigen characteristics (1) NK cell self-activation (3) release of a large amount of interferon, provide an active immune microenvironment for tumor cells, and further enhance NK therapy.

3. The biological liposome for resisting the tumor provided by the invention does not need too complex operation and equipment, has simple and convenient preparation method, and is a medicinal preparation with higher production cost performance, effectiveness and low toxicity.

4. The invention can simply, conveniently and efficiently prepare the safe and low-toxicity bioliposome with the anti-tumor heterogeneity of the combination therapy, and has potential medical prospect. Anchoring cancer cells with NK-activatable target antigens may provide a transformation pathway to address the challenges of tumor heterogeneity.

Drawings

FIG. 1 shows the structural formulas and preparation flow charts of cisplatin (Pt), cisplatin prodrug Pt (IV) and carboxylated cisplatin prodrug Pt (IV) -COOH.

FIG. 2 is a mass spectrum of cisplatin Pt (IV) and a mass spectrum of carboxylated cisplatin Pt (IV) -COOH.

FIG. 3 is a schematic diagram of the preparation and structure of cisplatin-entrapped IgG-platelet membrane bioliposome (Pt @ PL-IgG).

FIG. 4 is the particle size distribution and transmission electron microscope image of IgG-platelet membrane bioliposome entrapped with cisplatin.

FIG. 5 is a graph of the detection of platelet protein expression on Pt @ PL-IgG by Western blot analysis.

FIG. 6 is an SDS-PAGE protein analysis of platelets, platelet membranes, pt @ PL-IgG and IgG.

FIG. 7 shows IgG-FITC expression in 4T1 cells after different treatments.

FIG. 8 is an adhesion of NK cells to cisplatin-entrapped IgG-platelet membrane bioliposomes (Pt @ PL-IgG), cisplatin-entrapped IgG liposomes (Pt @ L-IgG), igG-platelet membrane bioliposomes (PL-IgG), cisplatin-entrapped platelet membrane bioliposomes (Pt @ PL), free IgG and PBS 4T1 cells treated by CLSM, with NK cells being encircled by dashed lines.

Detailed Description

The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples were carried out according to the conventional conditions in the art.

Example 1

(1) Taking 1.5g of cisplatin, adding 30mL of water into a 100mL double-neck flask to form cisplatin suspension, adding 50mL of 30% hydrogen peroxide solution, stirring for reacting for 4 hours, filtering by using a Buchner funnel, and recrystallizing a crude product to obtain a cisplatin prodrug (Pt (IV));

(2) Taking 1g of cisplatin prodrug and 1g of succinic anhydride, adding 10mL of N, N-dimethylformamide into a 50mL double-neck flask, and stirring the mixture under an inert atmosphere to react for 24 hours to obtain a carboxylated cisplatin prodrug primary solution;

(3) Adding diethyl ether into the primary solution of the carboxylated cisplatin prodrug obtained in the step (2), filtering and collecting precipitates by using a Buchner funnel, resuspending the product by using purified water, and freeze-drying by using a vacuum freeze dryer to obtain a carboxylated cisplatin prodrug Pt (IV) -COOH;

(4) 0.39mg of cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC sodium salt) was dissolved in 195. Mu.L of 1 XPBS (pH 7.4) solution in a 50mL flask, and then 200. Mu.g of IgG antibody solution dissolved in 900. Mu.L of 1 XPBS (pH 7.4) was added to adjust the pH of the mixture to 7.4, and the mixture was shaken at room temperature for 2 hours;

(5) Dissolving 4.5mg of distearoyl phosphatidyl acetamide-polyethylene glycol-sulfhydryl (DSPE-PEG 5000-SH) in 900 μ L of 1 XPBS (pH7.4), dripping into the flask containing the mixed solution in the step (4), and oscillating and reacting at room temperature for 4h to obtain DSPE-IgG2b;

(6) A4% sodium citrate solution was added to whole mouse blood, and the mixture was centrifuged at 300g for 15 minutes to obtain platelet-containing plasma, and then at 800g for 10 minutes to obtain platelets. Adding 1 XPBS (pH7.4) buffer solution into the platelets, freezing at-80 ℃, unfreezing the platelets at room temperature, centrifuging for 5 minutes at 10000g to form precipitates, adding the 1 XPBS (pH7.4) buffer solution into the precipitates, and repeating the freeze-thaw cycle for a plurality of times to obtain platelet membranes;

(7) Preparing blank liposome by adopting a film dispersion method, wherein the specific process comprises the steps of dissolving 25mg1, 2-dioleoyl-sn-propanetriyl-3-phosphoethanolamine (DOPE), 12.5mg1, 2-dioleoyl-sn-glycerol-3-phosphocholine (HSPC) and 3.12mg Cholesterol Hemisuccinate (CHEMS) in dichloromethane, and then spin-drying by using a rotary evaporator to remove a solvent dichloromethane to form a film;

(8) Dissolving the carboxylated cisplatin prodrug obtained in step (3) with 5mL of 1XPBS (pH 7.4), adding the solution to the film obtained in step (7), hydrating at 37 ℃ for 30 minutes, and then sonicating with a homogenizer for 30 minutes to obtain liposomes;

(9) Mixing the DSPE-IgG2b obtained in the step (5), the platelet membrane obtained in the step (6) and the liposome obtained in the step (8), and carrying out ultrasonic treatment for 30 minutes to obtain the cisplatin-encapsulated IgG-platelet membrane bioliposome Pt @ PL-IgG.

FIG. 1 shows the structural formulas and preparation flow charts of cisplatin (Pt), cisplatin prodrug Pt (IV) and carboxylated cisplatin prodrug Pt (IV) -COOH.

FIG. 2 is a mass spectrum of cisplatin Pt (IV) and a mass spectrum of carboxylated cisplatin Pt (IV) -COOH. The successful synthesis thereof was demonstrated.

FIG. 3 is a schematic diagram of the preparation and structure of cisplatin-entrapped IgG-platelet membrane bioliposome (Pt @ PL-IgG).

FIG. 4 is a particle size distribution and TEM image of cisplatin-loaded IgG-platelet membrane bioliposome (Pt @ PL-IgG). The particle size distribution of Pt @ PL-IgG measured by dynamic light scattering is shown in the figure, the particle size of the Pt @ PL-IgG is about 120 nm, and the Pt @ PL-IgG presents a typical liposome and vesicle structure, and is beneficial to in vivo drug delivery.

FIG. 5 is detection of platelet protein expression on Pt @ PL-IgG by Western blot analysis. Platelets participate in the critical step of malignant tumor progression by adhering to tumor cells. At the same time, integrin proteins (CD 41 and CD 62P) in platelets fulfill an adhesion function, being fully retained on Pt @ PL-IgG.

FIG. 6 is an SDS-PAGE protein analysis. Wherein G1 is a marker, G2 is a platelet, G3 is a platelet membrane, G4 is Pt @ PL-IgG and G5 is IgG. Most of the membrane proteins on platelet membranes were extensively retained on Pt @ -PL-IgG as observed by SDS-PAGE. Meanwhile, a band corresponding to IgG (37 kDa) was found in Pt @ PL-IgG, indicating successful entrapment of IgG in the membrane of the bioliposome.

FIG. 7 shows IgG-FITC expression in 4T1 cells after different treatments. 4T1 cells were plated on 37 ℃ confocal culture dishes for 24 hours, then co-cultured with Pt @ PL-IgG, pt @ L-IgG, PL-IgG, pt @ PL, free IgG and PBS for 4 hours (2. Mu.g/mL for IgG2b and 10. Mu.g/mL for Pt (IV) -COOH in each group). IgG expression on cells was detected by flow cytometry. As shown in FIG. 7, the expression level of IgG in tumor cells increased from 3.59% (PBS) to 58.7% after Pt @PL-IgG treatment. At the same time, pt @ PL-IgG treatment showed 2.6 times higher IgG expression in tumors than the Pt @ L-IgG group.

FIG. 8 shows the adhesion of NK cells to 4T1 cells treated with Pt @ PL-IgG, pt @ L-IgG, PL-IgG, pt @ PL, free IgG and 1 XPBS (pH 7.4) by CLSM, with NK cells being circled by dotted lines. 4T1 cells seeded on confocal culture dishes were co-cultured with Pt @ -PL-IgG, pt @ -L-IgG, PL-IgG, pt @ -PL, free-IgG and PBS at 37 ℃ for 4 hours. Each group had an IgG2b concentration of 2. Mu.g/mL and a Pt (IV) -COOH concentration of 10. Mu.g/mL. Then, NK cells were added to 4T1 cells at a ratio of 1. The results showed that no NK cell adhesion was observed in the PBS group, whereas a large amount of NK cell adhesion was observed in the Pt @PL-IgG group. The number of adherent NK cells of the Pt @ PL-IgG group is similar to that of adherent NK cells of the PL-IgG group, and the adherent NK cells are obviously superior to that of the Pt @ L-IgG group, so that the function of the bioliposome is amplified by the platelet membrane.

Example 2

(1) Adding 2g of cisplatin into a 100mL double-neck flask, adding 35mL of water to form cisplatin suspension, adding 55mL of 30% hydrogen peroxide solution, stirring for reacting for 4 hours, filtering by using a Buchner funnel, and recrystallizing a crude product to obtain a cisplatin prodrug (Pt (IV));

(2) 1.2g of cisplatin prodrug and 1.3g of succinic anhydride were put in a 50mL two-necked flask, and 12mLN, N-dimethylformamide was added thereto, and the mixture was stirred under an inert atmosphere to react for 24 hours to obtain a carboxylated cisplatin prodrug preliminary solution;

(3) Adding diethyl ether into the primary solution of the carboxylated cisplatin prodrug obtained in the step (2), filtering and collecting precipitates by using a Buchner funnel, re-suspending the product by using purified water, and freeze-drying by using a vacuum freeze dryer to obtain a carboxylated cisplatin prodrug Pt (IV) -COOH;

(4) 0.4mg of cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC sodium salt) was dissolved in 200. Mu.L of 1 XPBS (pH 7.4) solution in a 50mL flask, and then 200. Mu.g of IgG antibody solution dissolved in 900. Mu.L of 1 XPBS (pH 7.4) was added to adjust the pH of the mixture to 7.4, and the mixture was shaken at room temperature for 2 hours;

(5) Dissolving 4.5mg distearoyl phosphatidyl acetamide-polyethylene glycol-sulfhydryl (DSPE-PEG 5000-SH) in 900 μ L1X PBS (pH7.4), dropping into the flask containing the mixed solution in the step (4), and reacting at room temperature for 4h with shaking to obtain DSPE-IgG2b;

(6) A4% sodium citrate solution was added to whole mouse blood, and centrifugation was carried out at 300g for 15 minutes to obtain platelet-containing plasma, and centrifugation at 800g for 10 minutes was continued to obtain platelets. Adding 1 XPBS (pH7.4) buffer solution into platelets, freezing at-80 ℃, thawing the platelets at room temperature and centrifuging for 5 minutes at 10000g to form precipitates, adding 1 XPBS (pH7.4) buffer solution into the precipitates, and repeating the freeze-thaw cycle for a plurality of times to obtain platelet membranes;

(7) Preparing blank liposome by adopting a film dispersion method, and specifically, dissolving 30mg1, 2-dioleoyl-sn-propanetriyl-3-phosphoethanolamine (DOPE), 15mg1, 2-dioleoyl-sn-glycerol-3-phosphocholine (HSPC) and 4mg Cholesterol Hemisuccinate (CHEMS) in dichloromethane, and then spin-drying by using a rotary evaporator to remove a solvent dichloromethane to form a film;

(8) Dissolving the carboxylated cisplatin prodrug obtained in step (3) with 5mL1X PBS (ph 7.4), adding the solution to the film obtained in step (7), hydrating at 37 ℃ for 30 minutes, and then sonicating with a homogenizer for 30 minutes to obtain liposomes;

(9) Mixing the DSPE-IgG2b obtained in the step (5), the platelet membrane obtained in the step (6) and the liposome obtained in the step (8), and carrying out ultrasonic treatment for 30 minutes to obtain the cisplatin-loaded IgG-platelet membrane bioliposome Pt @ PL-IgG.

Claims (9)

1. A bioliposome, characterized in that: a biomembrane and liposome are fused to form a vesicle, cisplatin is loaded in the vesicle, and an NK activatable target antigen is modified on the surface of the vesicle;

the biological membrane is selected from the group consisting of neutrophil membrane, tumor cell membrane, stem cell membrane, macrophage membrane, platelet membrane, erythrocyte membrane, mitochondrial membrane and lysosome membrane.

2. The bioliposome of claim 1, wherein: the NK activatable target antigen is an IgG antibody.

3. The bioliposome of claim 1, wherein: the biological membrane is a platelet membrane.

4. The method for preparing bioliposomes according to claim 1, wherein: the method comprises the following steps:

(1) Adding cisplatin into water to form cisplatin-water suspension, adding hydrogen peroxide solution, reacting to obtain solution A, filtering the solution A, and recrystallizing to obtain cisplatin prodrug Pt (IV);

wherein: cisplatin: water: the mass ratio of the hydrogen peroxide solution is 1:1-300:5-500, the reaction time is 1-6h;

(2) Dissolving a cisplatin prodrug Pt (IV) in a solvent, adding an acid anhydride acylating agent, and then stirring to react under an inert atmosphere to obtain a solution B;

wherein: cisplatin prodrug: the mass ratio of the acid anhydride acylating agent is 1-100:1-100; the stirring reaction time is 12-48h;

(3) Adding ether to solution B to precipitate the product, collecting the precipitate and resuspending in water, and freeze-drying to give carboxylated cisplatin prodrug Pt (IV) -COOH;

(4) Respectively dissolving cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt and an antigen in a PBS solution, mixing the two solutions, adjusting the pH value to 7.4, and carrying out oscillation reaction at room temperature to obtain a solution C;

wherein: the reaction time is 1-4h;

(5) Dissolving phospholipid in a PBS solution, dripping the solution into the solution C, and performing oscillation reaction at room temperature to obtain phospholipid-antigen;

wherein: the reaction time is 2-8h; cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt: antigen: the mass ratio of the phospholipid is 0.1-50:0.1-50:5-500;

(6) Adding an anticoagulant into whole blood, centrifuging to obtain plasma containing platelets, continuously centrifuging to obtain platelets, adding a PBS (phosphate buffer solution) into the platelets, freezing at-80 ℃, unfreezing the platelets at room temperature, centrifuging for 5 minutes at 10000g to form precipitates, adding the PBS into the precipitates, freezing at-80 ℃, and repeating the freeze-thaw cycle to obtain platelet membranes;

(7) Preparing blank liposome by adopting a film dispersion method, dissolving 1, 2-dioleoyl-sn-propanetriyl-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol hemisuccinate in an organic solvent, and then performing rotary evaporation and drying to form a film;

wherein: 1, 2-dioleoyl-sn-propanetriyl-3-phosphoethanolamine: 1, 2-dioleoyl-sn-glycero-3-phosphocholine: the mass ratio of the cholesterol hemisuccinate is 0.5-100:0.5-100:0.1 to 50;

(8) Thereafter, the carboxylated cisplatin prodrug Pt (IV) -COOH obtained in step (3) was dissolved with PBS, and the solution was added to the film obtained in step (7), hydrated at 37 ℃ for 30 minutes, and then sonicated with a homogenizer to obtain liposomes;

(9) Mixing the phospholipid-antigen obtained in step (5), the platelet membrane obtained in step (6) and the liposome obtained in step (8), sonicating for 30 minutes, and then removing unencapsulated carboxylated cisplatin prodrug and phospholipid by centrifugation to obtain the bioliposome.

5. The method of claim 4, wherein: the concentration of the hydrogen oxide solution is 3% -30%.

6. The method of claim 4, wherein: the acid anhydride acylating agent is succinic anhydride.

7. The method of claim 4, wherein: the phospholipid is natural phospholipid, semisynthetic phospholipid or synthetic phospholipid.

8. The method of claim 7, wherein: the phospholipid is distearoyl phosphatidyl acetamide-polyethylene glycol-sulfhydryl DSPE-PEG5000-SH.

9. Use of the bioliposome of claim 1 for the preparation of a medicament for the treatment of tumors.

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