Method for preparing platelet-lipidosome drug delivery system, platelet-lipidosome drug delivery system obtained by method and application of platelet-lipidosome drug delivery system

Abstract

The invention discloses a method for preparing a platelet-lipidosome drug loading system, the platelet-lipidosome drug loading system obtained by the method and application of the platelet-lipidosome drug loading system, a drug-loaded lipidosome is fused with a living platelet, and the drug on the lipidosome is transferred to the platelet in one step to obtain the living platelet-lipidosome drug loading system. The live platelet drug delivery system is prepared by fusing the lipidosome and the live platelets, and the method is simple, convenient, time-saving and efficient. According to the method, the platelet modification time is greatly shortened, only 5 minutes are needed, cytoplasm drug loading and surface modification can be achieved at the same time, and the highest drug loading efficiency is achieved. And moreover, various drugs can be loaded on the platelets at the same time, the time is not prolonged, and the flexibility is relatively high.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a platelet engineering method, and in particular to a method for preparing a platelet-liposome drug delivery system, a platelet-liposome drug delivery system and applications thereof. Background Art

[0002] Over the past few decades, cell-based systems have emerged as drug carriers, including red blood cells, platelets, stem cells, and dendritic cells, which have unique advantages such as long circulation time, low immunogenicity, targeted delivery properties, and the ability to penetrate natural physiological barriers. Among them, platelets produced by megakaryocytes have the natural property of targeting injured, inflamed tissues, and circulating tumor cells. Various platelet-based drug delivery systems have been developed for metastatic and postoperative cancer treatment.

[0003] Existing methods for loading drugs into platelets or binding drugs to platelet membranes include surface modification, cytoplasmic loading, and genetic engineering. Surface modification based on chemical coupling between thiol / amino groups of platelet membrane proteins and drugs such as functionalized antibodies / nanoparticles is the most common method for platelet engineering. However, this strategy is time-consuming (usually more than 2 hours) and can lead to undesirable platelet activation. In addition, modification of membrane proteins may also affect platelet migration, adhesion, activation and other functions. Cytoplasmic drug loading by electroporation may lead to impaired platelet function. In addition, methods based on genetic manipulation of megakaryocytes to produce platelets are limited by the unsatisfactory performance of the produced platelets and the difficulty in obtaining megakaryocytes. Platelet-coupled protein technology, according to the article by Wang et al. (DOI: 10.1038 / s41551-016-0011), platelet-coupled protein needs to be treated with Trauts' reagent for 30 minutes and mixed with PD-L1 antibody that was pre-thiolated for 2 hours for 2 hours before it can be loaded with protein. Regarding the loading of platelets with small molecule drugs, according to the research of Xu et al. (DOI: 10.1038 / srep42632), platelets need to be mixed with doxorubicin for 1 hour before they can effectively load drugs. If it is necessary to load multiple drugs in platelets at the same time, it is necessary to adopt a multi-step continuous loading method, which will bring many problems. First of all, platelets themselves are unstable, and the less operation they are subjected to, the better. Centrifugation and blowing will cause platelet activation, resulting in poor quality control (Semple et al., DOI: 10.1038 / nri2956). If two drugs are loaded at the same time, it will take at least 3.5 hours and multiple steps, and the drug leakage rate will also increase significantly during this process.

[0004] Therefore, there is an urgent need to engineer platelets in a rapid, effective, and safe manner. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of the existing platelet engineering process, such as time-consuming, cumbersome operation, and low efficiency. The present invention provides a method for preparing a platelet-liposome drug delivery system. The method adopts a liposome fusion method to prepare active platelets for multi-drug loading in one step. Multiple drugs can be coated inside the liposome or combined on the surface of the liposome, and cytoplasmic drug loading and surface modification can be achieved simultaneously in a short time, thereby improving drug loading efficiency and efficacy. At the same time, the application of platelet engineering in the drug delivery system is provided.

[0006] The first aspect of the present invention provides a method for preparing a platelet-liposome drug-carrying system, wherein drug-loaded liposomes are fused with live platelets, and the drugs on the liposomes are transferred to the platelets in one step to obtain live drug-carrying platelets.

[0007] In some embodiments, the fusion is mediated by a polyethylene glycol solution, and / or the mass ratio of the liposome to the drug is 1000:(1-50), and / or the mass ratio of the drug-loaded liposome to platelets is (1-2):(1-2).

[0008] In some embodiments, the molecular weight of the polyethylene glycol is 2000, 4000, 6000 or 8000; and / or, the polyethylene glycol solution is obtained by dissolving 50% polyethylene glycol in PBS; and / or, the pH of the solvent during fusion is 5.7-7.4.

[0009] In some embodiments, the method for preparing the drug-loaded liposomes comprises the following steps:

[0010] (1) preparing empty liposomes, the components of which include cholesterol, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, and distearoylphosphatidylethanolamine-polyethylene glycol maleimide, preferably in a molar ratio of (1.5-2):(0.8-1.5):(0.8-1.5):(0.1-0.3):(0.01-0.06), for example, 2:1:1:0.1:0.04;

[0011] (2) The drug is loaded inside the empty liposome or attached to the surface of the empty liposome to form drug-loaded liposomes.

[0012] In some embodiments, in step (1), empty liposomes are prepared by thin film hydration method, ultrasonic dispersion method, reverse evaporation method, solvent injection method, detergent removal method, freeze-thaw method or microfluidics method.

[0013] In some embodiments, in step (2), the drug is loaded into the liposome using an ammonium sulfate concentration gradient method, and / or the drug is linked to the liposome surface using a maleimide-thiol reaction.

[0014] In some embodiments, the drug is a protein drug, a small molecule drug, or a nanoparticle drug;

[0015] The protein drugs include antibodies, cytokines, peptides, albumin or enzymes, preferably IL-15;

[0016] The small molecule drugs include water-soluble drugs or fat-soluble drugs, preferably doxorubicin;

[0017] The nanoparticle drugs include inorganic nanoparticles or polymer nanoparticles, preferably gold nanoparticles.

[0018] In some embodiments, the platelets are separated and obtained by centrifugation, preferably differential centrifugation, and / or the platelets are derived from living cells of mammals, preferably living cells of rodents or primates, more preferably living cells of mice, rabbits, or humans.

[0019] The second aspect of the present invention provides a platelet-liposome drug delivery system prepared by the method described above.

[0020] The third aspect of the present invention provides the use of the platelet-liposome drug delivery system in the preparation of drugs.

[0021] The living platelets were first imaged by TEM ( Figure 2 ), confocal microscopy ( Figure 3 ) to observe its complete morphology, and most importantly, it can be activated by thrombin to release drugs ( Figure 4 ), which dead cells cannot do. Platelets can also adhere to tumor cells ( Figure 6 ), all of which indicate that platelets are living cells.

[0022] The positive and progressive effects of the present invention are:

[0023] 1. The present invention fuses liposomes and live platelets to prepare a live platelet drug-carrying system, which is simple, time-saving and efficient. The platelet transformation time is greatly shortened to only 5 minutes. Cytoplasmic drug loading and surface modification can be achieved simultaneously within 5 minutes, achieving the highest drug loading efficiency.

[0024] 2. The present invention mediates fusion through polyethylene glycol. By adjusting the molecular weight of polyethylene glycol and the fusion time, the drugs inside the liposomes can be quickly loaded onto the platelets.

[0025] 3. This method can load multiple drugs on platelets at the same time without prolonging the time, which is 5 minutes, and has high flexibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The drug loading efficiency results of the examples of the present invention after optimizing the reaction system, polyethylene glycol (PEG) molecular weight and reaction time;

[0027] Figure 2 This is a transmission electron microscopy image of platelets after being treated with polyethylene glycol in an example of the present invention. Gold nanoparticles are labeled on the membrane surface to prove that membrane fusion has occurred. The scale bar is 500 microns.

[0028] Figure 3 This is a confocal microscope image of the platelets after fusion with liposomes with FITC-labeled albumin attached to the surface and doxorubicin encapsulated in the present invention. The upper right corner is the magnified image. The scale of the large image is 2 microns, and the scale of the magnified image is 1 micron.

[0029] Figure 4 FITC-IL-15 and doxorubicin release curves of platelets loaded with FITC-IL-15 and doxorubicin by polyethylene glycol fusion method in the presence and absence of thrombin, respectively;

[0030] Figure 5 Western blotting was used to measure the content of key proteins (CD41, CD47, CD61, CD62P) on the surface of platelets (DOX@PLT-IL-15) after loading IL-15 and DOX by polyethylene glycol fusion method in the present invention, and compared with unengineered platelets;

[0031] Figure 6 This is a confocal microscope image in an example of the present invention to prove that the engineered platelets manufactured by the polyethylene glycol fusion method can adhere to tumor cells (B16F10 cells) and thus play a killing role;

[0032] Figure 7 In the example of the present invention, the cytotoxicity of engineered platelets loaded with IL-15 and DOX by the polyethylene glycol fusion method was measured, wherein G1 was a PBS group, G2 was an untreated platelet group, G3 was a free IL-15 group, G4 was a free doxorubicin group, G5 was an engineered platelet group loaded with DOX by the polyethylene glycol fusion method, and G6 was an engineered platelet group loaded with IL-15 and DOX by the polyethylene glycol fusion method;

[0033] Figure 8 This example verifies that engineered platelets loaded with IL-15 by polyethylene glycol fusion can stimulate mouse CD8 + Flow cytometric images of T cell proliferation (CFSE labeling) and showing no significant difference in stimulation with free IL-15 and IL-2;

[0034] Fig. 9The in vivo pharmacokinetic curve of the engineered platelets produced by the polyethylene glycol fusion method after tail vein administration in the examples of the present invention;

[0035] Fig.10 The difference in the enrichment of engineered platelets loaded with IL-15 by the polyethylene glycol fusion method and free IL-15 in mouse lung tumor metastases after tail vein administration in the examples of the present invention;

[0036] Fig.11 Different drug-loaded liposomes can simultaneously load multiple drugs onto platelets. DETAILED DESCRIPTION

[0037] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0038] Example 1

[0039] The method for preparing drug-loaded platelets comprises the following steps:

[0040] (1) Separate platelets from mouse whole blood using differential centrifugation. Under anesthesia, collect orbital venous blood from mice and place it in a centrifuge tube with ACD anticoagulant (the volume ratio of whole blood to ACD anticoagulant is about 5:1. The formula of ACD anticoagulant is: 16mmol / L citric acid, 90mmol / L sodium citrate, 16mmol / L sodium dihydrogen phosphate, 142mmol / L glucose. After preparation, filter with a 0.22μm filter membrane and store in aliquots at 4°C). Centrifuge at room temperature at 150g for 10 minutes, remove the upper platelet-rich plasma, and then centrifuge at 1000g for 5 minutes to separate platelets. Approximately 5 to 7×10 8 Platelets.

[0041] (2) Blank liposomes were prepared by thin film hydration method. Cholesterol, dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), and distearoylphosphatidylethanolamine-polyethylene glycol maleimide (DSPE-PEG2000-Mal) were dissolved in chloroform at a molar ratio of 2:1:1:0.1:0.04. 10 mg of lipid was treated with 1 mL of chloroform, and all solvents were removed by slow rotary evaporation. After vacuuming with a water pump overnight, 250 mM (NH 4 ) 2 SO 4 The liposome mixture was hydrated at 37 °C for 1 h. After brief sonication of the liposomes in a water bath, the liposomes were extruded 10 times through a polycarbonate membrane with a pore size of 200 nm and dialyzed overnight.

[0042] (3) Doxorubicin-loaded liposomes were prepared by the ammonium sulfate concentration gradient method, and then IL-15 or FITC-BSA was coupled to the liposomes. The liposomes and platelets were mixed in 50% PEG2000 (the polyethylene glycol (PEG) involved in the present invention is prepared with pH=7.4 PBS containing 1 μM PGE1. All polyethylene glycol concentrations are expressed as mass concentrations, which refers to the ratio of the polyethylene glycol mass to the total mass. For example, the preparation method of 50% polyethylene glycol is to add 50 g polyethylene glycol to PBS until the final mass is 100 g. All PEGs of different molecular weights were purchased from Aladdin.) and diluted and then centrifuged. The specific process is to dialyze the liposomes after hydration in ammonium sulfate solution, then mix with doxorubicin (DOX) solution (DOX:lipids mass ratio is 1:10) at 45°C for 45min, purify the DOX-loaded liposomes with ultrafiltration device, and then mix with thiol-modified proteins (IL-15 or FITC-BSA). Both IL-15 and FITC-BSA must be thiol-modified before mixing with liposomes. The amount of IL-15 and BSA can vary within a certain range. It is possible to have less than 50μg of protein (IL-15 or BSA-FITC) per 1mg of lipid (1-50μg). Increasing the amount of protein may affect the drug loading efficiency. Incubate at 4°C for 24h, and couple the cytokine to the liposomes through a thiol-maleimide chemical bond. To prepare DOX@PLT-IL-15, the mass ratio of liposomes to platelets was 1:1, and incubated in 30μL 50% PEG2000 for 5 minutes. Then, the DOX@PLT-IL-15 was collected by diluting the solution with PBS containing 1 μM PGE1 and centrifuging at 1000 g for 5 min.

[0043] The performance test is as follows:

[0044] like Figure 1In order to explore the effects of different pH systems (the solvents were PBS at pH = 5.7 or 7.4, and the volume was maintained at 30 μL. Too large a volume would reduce the fusion efficiency. Preferably, the volume was 30 μL) and different PEG molecular weights on drug loading efficiency, under six conditions, namely pH = 5.7 PBS, pH = 7.4 PBS, PEG with a molecular weight of 2000, PEG with a molecular weight of 4000, PEG with a molecular weight of 6000, and PEG with a molecular weight of 8000 (all volumes were maintained at 30 μL) (the six conditions were: pH = 5.7 PBS (without polyethylene glycol, fusion for 2 hours), pH = 7.4 PBS (without polyethylene glycol, fusion for 2 hours), 50% PEG2000 (fusion for 5 minutes, the same applies later), 50% PEG4000, 50% PEG6000, and 50% PEG8000. After centrifugation, no solvent was added to the platelets in all PEG groups, and they were directly resuspended with 50% PEG2000, so the solvent system was 50% PEG2000. Combined with Figure 1 It can be explained that the drug loading efficiency of the PEG-free group was less than 20% after 2 hours, even though the pH-lowering method, which is traditionally believed to enhance the positive charge of liposomes, was used. However, the PEG groups had a higher drug loading efficiency within 5 minutes, and PEG2000 was preferred.

[0045] The fusion reaction of liposomes and platelets was performed, and then the concentration of proteins bound to platelets was measured. It was found that the drug loading efficiency of the PEG2000 group was the highest. Then, the effect of different treatment times on the drug loading efficiency was explored under the PEG2000 condition. The other conditions and reaction time were as described above. It was found that a 5-minute treatment was sufficient to achieve the highest efficiency (about 70%).

[0046] like Figure 2 In order to prove that liposomes and platelets are fused by a fusion mechanism, liposomes (biotin-coupled lipids were added during preparation) were surface-labeled with 15 nm streptavidin-tagged gold nanoparticles, fused with platelets in 2.5% glutaraldehyde at 4°C overnight under the conditions described above, and then stained with 1% osmic acid and 2% lead acetate, respectively, and then dehydrated in 50%, 70%, 90%, and 100% ethanol, respectively. The sections embedded in epoxy resin on the copper grid were observed under a transmission electron microscope. The localization of the gold nanoparticles showed that the liposome membrane was evenly dispersed on the surface of the platelets.

[0047] like Figure 3The morphology of fused platelets was photographed under a confocal microscope. FITC-labeled bovine serum albumin was used as the connecting protein. Doxorubicin with red fluorescence was encapsulated inside the liposomes and fused with platelets under the above conditions. Then, the platelets were washed twice with PBS containing PGE1, dropped on a glass-bottomed culture dish, and observed under a confocal microscope. The magnified image shows that FITC-labeled bovine serum albumin is distributed on the surface of platelets.

[0048] like Figure 4 In order to simulate the wound bleeding environment in vivo, the release curves of fused platelets loaded with drugs (DOX and IL-15) were drawn in the presence / absence of thrombin. Specifically, 0.5U / mL thrombin or no thrombin was added to the fused platelets prepared in the above manner, and the supernatant was taken to measure the absorbance or fluorescence intensity at 1000g for 5 minutes every 0.5, 1, 1.5, 2, and 4 hours. The experiment was repeated three times in parallel and the drug release rate at each time point was calculated. This experiment verified that the fused platelets can still respond to thrombin and basically release the drug completely within 2 hours, while the drug leakage rate is low in the absence of thrombin.

[0049] like Figure 5 In order to verify whether the surface proteins of fused platelets were damaged, a Western blot test was performed. After making fused platelets, untreated platelets were used as controls and lysed at 4°C for 20 minutes with RIPA lysis buffer containing 1mM PMSF (a serine protease activity inhibitor). After centrifugation at 10000rpm for 10 minutes, the supernatant was collected and quantified with a BCA kit to make the total protein content consistent. The obtained proteins were denatured by heating and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 160V. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with skim milk powder for 2h, and incubated with CD41, CD47, CD61, and CD62P primary antibodies at 4°C for 12h. After washing 3 times to remove the antibodies, they were incubated with HRP-labeled secondary antibodies for 2h. After removing all unbound antibodies, the labeled membrane was treated with an enhanced chemiluminescence kit to display protein bands. The results showed that key functional proteins on the platelet surface, such as CD41, CD47, CD61 and CD62P, were intact, especially CD62P, which is a key protein for platelet adhesion to tumor cells.

[0050] like Figure 6 In order to explore whether fused platelets can produce adhesion with tumor cells, 5×10 4A number of B16F10 cells were inoculated in a 24-well cell culture plate and cultured for 12 hours, followed by incubation with Cy5-NHS-labeled fused platelets for 24 hours. After centrifugation and washing twice to remove unbound fused platelets, B16F10 cell nuclei were labeled with 10μg / mL Hoechst 33342 for 10 minutes, and B16F10 cell membranes were labeled with 5μg / mL DiO for 15 minutes. After washing away the unbound dye, confocal microscopy revealed that platelets can adhere to tumor cells.

[0051] like Figure 7 To measure the cytotoxicity of engineered platelets loaded with IL-15 and DOX by the polyethylene glycol fusion method, B16F10 cells were seeded in 96-well plates at a density of 5000 cells per well. After 48 h of culture, the original culture medium was replaced with fresh culture medium containing different formulas (G1: PBS, G2: free platelets, G3: free IL-15, G4: DOX, G5: fused platelets loaded with DOX, G6: fused platelets loaded with DOX and IL-15, and the control IL-15 concentration of each group was 50 ng / mL and the DOX concentration was 0.1 μg / mL). After incubation for 48 h, 10 μL of CCK-8 solution was added to each well and cultured in a CO2 cell culture incubator for 1 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader. The results showed that there was no significant difference in the cytotoxicity of the preparations in groups G4, G5, and G6 to B16F10 cells, which indicates that the fused platelets loaded with doxorubicin have a killing effect equivalent to that of free doxorubicin at 48 hours, and IL-15 does not affect this killing effect.

[0052] like Figure 8 To verify whether engineered platelets loaded with IL-15 by polyethylene glycol fusion can stimulate CD8 + T cell proliferation to play an immunotherapy role, and the mouse CD8 + T cells and labeled with CFSE dye. The cells were treated with each group of preparations (PBS, IL-2 (free IL-2), IL-15 (free IL-15), PLT-IL-15 (fused platelets loaded with IL-15 prepared under the above conditions)) for 2 days (the IL-15 concentration was controlled at 50 ng / mL), and the cell proliferation was analyzed by flow cytometry. Specifically, the spleen of C57BL / 6 mice was taken, ground with a 70μm cell filter, and the red blood cells were lysed with red blood cell lysis buffer (2mL / spleen) at 4°C for 10 minutes. CD8 + T cells from EasySep mice CD8 + Positive T cell enrichment kit is isolated by magnetic bead negative selection. Purified CD8 + T cells were 1×10 6The cells were resuspended in RPMI-1640 medium containing mouse CD3 / CD28 activation magnetic beads and different formulations per milliliter. After 2 days of culture, T cells were isolated and the proliferation of T cells was analyzed by flow cytometry after live-dead staining. The results showed that the mean fluorescence intensity of CFSE dye had no significant difference in the IL-2, IL-15 and PLT-IL-15 groups, but they were all significantly lower than that in the PBS group, indicating that the activation effect of PLT-IL-15 on CD8+T cells was consistent with that of free IL-2 and IL-15, and both had significant activation effects.

[0053] like Fig. 9 In order to study the pharmacokinetic behavior of liposome-fused platelets in vivo, free Cy5-NHS-labeled IL-15 (referred to as free IL-15) and PLT-Cy5-IL-15 (fused platelets obtained by fusion of liposomes with surface-bound Cy5-IL-15 and platelets, referred to as PLT-IL-15) were injected into the tail vein of C57BL / 6 mice. 100 μL of solution was injected into the tail vein of each mouse, wherein the free IL-15 group was a PBS solution containing 2 μg of Cy5-IL-15, and the PLT-IL-15 group was a PBS solution of fused platelets (on which 2 μg of Cy5-IL-15 was loaded). Blood was collected from the eye sockets at 0.05, 1, 2, 3, 6, 24, and 48 hours, and the fluorescence intensity in the blood was measured. The images showed that Cy5-IL-15 was rapidly cleared within 8 hours, while PLT-Cy5-IL-15 could circulate for up to 48 hours, showing a longer blood retention time. This indicates that fusion platelets have excellent sustained-release properties for delivering IL-15.

[0054] like Fig.10 In order to study the targeting effect of liposome-fused platelets on B16F10 metastatic tumor model, 5×10 5 B16F10 tumor cells were suspended in 100 μL PBS and injected intravenously into C57BL / 6 mice as described above. Each mouse was injected into the tail vein with 100 μL of 5×10 5 B16F10 tumor cells in PBS solution. The mice were randomly divided into two groups and intravenously administered free Cy5-IL-15 and PLT-Cy5-IL-15 on the first day. The dosage was as described above. Each mouse was injected with 100 μL of solution through the tail vein. The free IL-15 group was a PBS solution containing 2 μg of Cy5-IL-15, and the PLT-IL-15 group was a PBS solution of fused platelets (on which 2 μg of Cy5-IL-15 was loaded). (Preparation method as described above Fig. 9). The lungs of mice were harvested 2 hours after injection, and drug accumulation was detected by fluorescence imaging. The results showed that the Cy5 fluorescence intensity in the lungs of the group injected with Cy5-PLT-IL-15 by tail vein was significantly stronger than that of the group injected with Cy5-IL-15 by tail vein, indicating that IL-15 delivered by fused platelets can effectively target the lung metastasis sites of B16F10 cells.

[0055] In order to demonstrate the flexibility of the liposome-fused platelet preparation method, liposomes loaded with BSA-FITC and liposomes loaded with BSA-Cy5 were mixed in a 1:1 ratio and fused with platelets (the specific operation is as described above, and the overall ratio, time and other parameters are the same), centrifuged at 1000g for 5 minutes, washed once with PBS containing 1μM PGE1, and dropped on a confocal culture dish for observation. The results showed that BSA-FITC and BSA-Cy5 signals appeared simultaneously on the platelets in the field of view, indicating that these two proteins were loaded on the platelets at the same time. This shows that this technology can load multiple drugs on platelets at the same time by pre-mixing different drug-loaded liposomes. It has good scalability and does not increase the processing time of platelets, which is still only 5 minutes. See the results. Fig.11 .

Claims

1. A method for preparing a platelet-liposome drug delivery system, It is characterized in that The drug-loaded liposomes are fused with live platelets, and the drugs on the liposomes are transferred to the platelets in one step to obtain a live platelet-liposome drug-loading system.

2. The method according to claim 1, It is characterized in that The fusion is mediated by polyethylene glycol solution, virus method, cationic liposome fusion method or electrical stimulation, wherein the virus method is preferably Sendai virus method or herpes virus method, and / or the mass ratio of the liposome to the drug is 1000:(1-50), and / or the mass ratio of the drug-loaded liposome to platelets is (1-2):(1-2).

3. The method according to claim 2, It is characterized in that The molecular weight of the polyethylene glycol solution is 2000, 4000, 6000 or 8000; and / or, the polyethylene glycol solution is obtained by dissolving 50% polyethylene glycol in PBS; And / or, the pH of the solvent during fusion is 5.7-7.

4.

4. The method according to claim 1, It is characterized in that The method for preparing the drug-loaded liposome comprises the following steps: (1) preparing empty liposomes, the components of which include cholesterol, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, and distearoylphosphatidylethanolamine-polyethylene glycol maleimide, preferably in a molar ratio of (1.5-2):(0.8-1.5):(0.8-1.5):(0.1-0.3):(0.01-0.06), for example, 2:1:1:0.1:0.04; (2) The drug is loaded inside the empty liposome or attached to the surface of the empty liposome to form drug-loaded liposomes.

5. The method according to claim 4, It is characterized in that In step (1), empty liposomes are prepared by thin film hydration method, ultrasonic dispersion method, reverse evaporation method, solvent injection method, detergent removal method, freeze-thaw method or microfluidics method.

6. The method according to claim 4, It is characterized in that In step (2), the drug is loaded into the liposome using an ammonium sulfate concentration gradient method, and / or the drug is linked to the surface of the liposome using a maleimide-thiol reaction.

7. The method according to claim 1, It is characterized in that The drug is a protein drug, a small molecule drug or a nanoparticle drug; The protein drugs include antibodies, cytokines, polypeptides, albumin or enzymes, preferably IL -15； The small molecule drugs include water-soluble drugs or fat-soluble drugs, preferably doxorubicin; The nanoparticle drugs include inorganic nanoparticles or polymer nanoparticles, preferably gold nanoparticles.

8. The method according to claim 1, It is characterized in that The platelets are separated and obtained by centrifugation, preferably differential centrifugation, and / or the platelets are derived from living cells of mammals, preferably living cells of rodents or primates, more preferably living cells of mice, rabbits, or humans.

9. The platelet-liposome drug delivery system prepared by the method according to any one of claims 1 to 8.

10. Use of the platelet-liposome drug delivery system according to claim 9 in the preparation of drugs.

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