Platelet drug-loaded vesicle loaded with pecetinib and application of platelet drug-loaded vesicle

Abstract

The invention provides a platelet drug-loaded vesicle loaded with pecetinib and application thereof, and relates to the technical field of biological medicines. On the premise of retaining the targeting property of the platelet vesicles, the properties of surface chemistry, charge, shape, size and the like of the platelet drug-loaded vesicles are controlled, so that the platelet drug-loaded vesicles can reach a tumor microenvironment through a vascular endothelial cell barrier to play a role. After intravenous injection of the platelet drug-loaded vesicle, migration and activation of bone marrow-derived suppressor cells in a tumor microenvironment can be significantly inhibited, which indicates that the platelet drug-loaded vesicle passes through a vascular endothelial cell barrier and successfully releases pecetinib in the tumor microenvironment; plasma protein in blood does not affect drug release of the platelet drug-loaded vesicle provided by the invention. Therefore, the platelet drug-loaded vesicle disclosed by the invention has good targeting property and drug release ability, is low in production cost and easy to prepare, and can save the research and development cost of a drug delivery system.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a platelet drug-loaded vesicle loaded with pesidatinib and an application thereof. Background Art

[0002] Cancer is a disease that seriously threatens human health. Current cancer treatments include surgery, radiotherapy, chemotherapy, immunotherapy and other methods. However, these methods still have a series of shortcomings when facing complex and changeable tumor diseases. First, traditional treatments such as surgery, radiotherapy and chemotherapy have limited efficacy for advanced tumors and are prone to recurrence and metastasis. Secondly, these treatments often have certain toxic side effects on normal tissues, affecting the quality of life of patients. Most importantly, tumor cells are often able to evade immune surveillance through a variety of mechanisms, resulting in the inability of the immune system to effectively eliminate cancer cells. Research on how to effectively kill tumors is an important public health goal worldwide, especially in developing countries.

[0003] The tumor immune microenvironment refers to the complex environment composed of cells, cytokines and signaling molecules around the tumor. The immune microenvironment plays a vital role in the occurrence, development and treatment response of the tumor. A good immune microenvironment helps to activate and enhance the immune system's killing ability against tumors and prevent the growth and spread of tumors. On the contrary, the infiltration of immunosuppressive cells, such as myeloid-derived suppressor cells and regulatory T cells, will hinder the normal function of the immune system. Among them, myeloid-derived suppressor cells can inhibit the activity of T cells and suppress natural immune responses through various mechanisms. The increase in myeloid-derived suppressor cells is closely related to the immune escape, drug resistance and recurrence of tumors. In particular, radiotherapy, chemotherapy and immunotherapy may all cause changes in the tumor immune microenvironment, and myeloid-derived suppressor cells will infiltrate the tumor area in large numbers after treatment. Therefore, regulating myeloid-derived suppressor cells has become an important strategy to improve the efficacy of tumor treatment.

[0004] Pecidinib mainly inhibits the JAK2 and FLT3 signaling pathways to reduce the generation, activation and immunosuppression of myeloid-derived suppressor cells, thereby making the tumor microenvironment more suitable for the anti-tumor effect of immune cells. Pecidinib, as a small molecule drug, mainly works through intravenous injection, but due to its lack of tumor targeting, it will be enriched in other organs in large quantities, and may cause lethal hepatotoxicity. At present, researchers have proposed a variety of different solutions to increase the tumor area concentration of Pecidinib and enhance the therapeutic effect of drugs. Lu et al. loaded Pecidinib into ZIF-8 nanoparticles and loaded the nanoparticles into macrophages to achieve targeted drug delivery. Li et al. encapsulated Pecidinib using liposomes and loaded it into hydrogels to achieve tumor immunotherapy. However, due to the poor water solubility of Pecidinib, hydrophilic polymer materials are difficult to load it efficiently. The existing artificially synthesized polymer nanoparticles have poor biocompatibility and are prone to other toxic side effects. Therefore, there is an urgent need for a Pecidinib carrier system with good tumor targeting and no toxic side effects. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a pesidatinib drug delivery system with good tumor targeting and no toxic side effects.

[0006] In order to solve the above technical problems, the present invention provides a platelet drug-loaded vesicle loaded with pesidatinib and its application. The platelet drug-loaded vesicle of the present invention has good biosafety and can avoid the occurrence of side effects; at the same time, the platelet drug-loaded vesicle can be delivered to the tumor area after radiotherapy, chemotherapy and immunotherapy by intravenous injection, so as to achieve efficient delivery of pesidatinib. After the drug-loaded vesicle is injected into the blood vessel through intravenous injection, it flows through the body through the blood circulation. At this time, the drug-loaded vesicle needs to pass through the vascular endothelial cell barrier to reach the target location. The size and charge properties of the drug-loaded vesicle may affect the efficiency of the drug-loaded vesicle passing through the vascular endothelial cell barrier. When the target location is the tumor microenvironment, the irregular vascular structure near the tumor microenvironment and the abnormal hemodynamics will also affect the delivery of the drug-loaded vesicle in the tumor microenvironment. In addition, once the drug-loaded vesicle enters the blood, it will be exposed to high concentrations of plasma proteins and will inevitably interact with plasma proteins. These plasma proteins adhere to the surface of the drug-loaded vesicle through van der Waals forces, electrostatic contacts, hydrogen bonds, and hydrophilic / hydrophobic interactions to form protein coronas. The formation of protein coronas will affect the stability and targeting of the drug-loaded vesicles, and thus affect the drug-loading capacity and the effect of drug release. The formation of protein coronas depends largely on the surface chemistry, charge, shape, and size of the drug-loaded vesicles. Therefore, the present invention, while retaining the tumor targeting of platelet vesicles, controls the surface chemistry, charge, shape, size, and other properties of platelet drug-loaded vesicles, so that the platelet drug-loaded vesicles can reach the tumor area through the vascular endothelial cell barrier to play a role. Intravenous injection of the platelet drug-loaded vesicles of the present invention can significantly inhibit the migration and activation of bone marrow-derived suppressor cells in the tumor, indicating that the platelet drug-loaded vesicles of the present invention can pass through the vascular endothelial cell barrier and successfully release Pesidatinib in the tumor microenvironment, and the plasma proteins in the blood do not affect the platelet drug-loaded vesicles of the present invention. Therefore, the platelet drug-loaded vesicles of the present invention have good targeting and drug release capabilities, and have low production costs, are easy to prepare, can be mass-produced, stored and transported, and can save the research and development costs of the drug delivery system.

[0007] The first object of the present invention is to provide a platelet drug-loaded vesicle loaded with pecidinib, co-incubate pecidinib with platelets to obtain drug-loaded platelets, activate the drug-loaded platelets, remove unactivated drug-loaded platelets, and separate the platelet drug-loaded vesicles.

[0008] Furthermore, the co-incubation temperature is 0-8°C, and the co-incubation time is 10-14 hours.

[0009] Furthermore, the drug-loaded platelet activation is to co-incubate the drug-loaded platelets with a platelet activator.

[0010] Furthermore, the co-incubation temperature is 30-40° C., and the co-incubation time is 20-40 minutes.

[0011] Furthermore, the platelet activator is selected from one or more of thrombin, epinephrine, thromboxane, 5-hydroxytryptamine, angiotensin, tissue plasminogen activator, urokinase plasminogen activator, and platelet activating factor.

[0012] Furthermore, the particle size of the platelet drug-loaded vesicles is 100-200 nm, and the surface charge is -4 to -8 mV.

[0013] Furthermore, the present invention first loads Pecidinib on platelets, then activates the platelets and secretes platelet vesicles, and Pecidinib is wrapped inside the platelet vesicles to form platelet drug-loaded vesicles. This preparation method can improve the loading rate of Pecidinib. The platelet drug-loaded vesicles transport Pecidinib to the tumor microenvironment through the tumor targeting of the platelet vesicles and release Pecidinib to exert its effect. Because the platelet drug-loaded vesicles are small in size, they have better biodistribution and permeability, and can reach the tumor microenvironment more effectively.

[0014] Furthermore, the platelets are from autologous or allogeneic sources.

[0015] The second object of the present invention is to provide an application of the above-mentioned platelet drug-loaded vesicles in the preparation of anti-tumor drugs.

[0016] Furthermore, the anti-tumor drug is administered by intravenous injection.

[0017] Furthermore, the anti-tumor drug exerts its effect by acting on immunosuppressive cells in the tumor immune microenvironment.

[0018] Furthermore, the immunosuppressive cells include myeloid-derived suppressor cells.

[0019] The third object of the present invention is to provide a tumor-targeted drug, which comprises the above-mentioned platelet drug-loaded vesicles.

[0020] Beneficial effects of the present invention:

[0021] (1) The present invention, while retaining the tumor targeting property of platelet vesicles, controls the surface chemistry, charge, shape, size and other properties of platelet drug-loaded vesicles, so that the platelet drug-loaded vesicles can pass through the vascular endothelial cell barrier to reach the tumor area to exert their effects. Intravenous injection of the platelet drug-loaded vesicles of the present invention can significantly inhibit the migration and activation of bone marrow-derived suppressor cells in the tumor, indicating that the platelet drug-loaded vesicles of the present invention can pass through the vascular endothelial cell barrier and successfully release pecidinib in the tumor microenvironment, and the plasma proteins in the blood have no effect on the platelet drug-loaded vesicles of the present invention;

[0022] (2) The platelet drug-loaded vesicles loaded with Pexidinib of the present invention can target the tumor area, reduce the proportion of myeloid-derived suppressor cells in the tumor area, restore the cell killing function of T cells in the tumor area, activate the anti-tumor immune response, and thus inhibit tumor growth. Compared with extracellular vesicles from other cell sources, the platelet drug-loaded vesicles of the present invention retain proteins on the surface of platelets, such as CD41, P-selectin, CXCR2, etc., which can more efficiently target the tumor immune microenvironment after treatment. In addition, the platelet drug-loaded vesicles of the present invention have low production costs, are easy to prepare, can be mass-produced, stored and transported, and save the research and development costs of the drug delivery system. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0024] Figure 1 It is a representative graph and statistical graph of tumor myeloid-derived suppressor cells obtained by flow cytometry experiment in Example 1 (I);

[0025] Figure 2 This is a statistical diagram of the tumor CD8+T cells and the ratio of CD8+T cells to myeloid-derived suppressor cells obtained by the flow cytometry experiment in Example 1 (I);

[0026] Figure 3 The representative graph and statistical graph of tumor myeloid-derived suppressor cells obtained by flow cytometry experiment in Example 1 (ii);

[0027] Figure 4 This is a statistical diagram of tumor region drive factors obtained by the enzyme-linked immunosorbent assay in Example 1 (III);

[0028] Figure 5 The flow cytometry analysis graph and statistical graph of chemokine receptors in the tumor area obtained by the flow cytometry experiment in Example 3 (I);

[0029] Figure 6The in vivo fluorescence imaging images obtained for the biodistribution of different materials in Example 3 (I);

[0030] Figure 7 The in vitro fluorescence imaging diagrams and statistical diagrams of various organs obtained for the biodistribution of different materials in Example 3 (I);

[0031] Figure 8 The confocal imaging and statistical diagram of tumor tissue after being treated with different materials in Example 3 (II);

[0032] Fig. 9 The imaging graph and statistical graph obtained by the Western blotting experiment in Example 5;

[0033] Fig.10 This is an electron microscope image of the platelet-derived drug-loaded vesicles in Example 5;

[0034] Fig.11 is the ultraviolet absorption spectrum of the platelet-derived drug-loaded vesicles in Example 5;

[0035] Fig.12 It is a statistical diagram of the particle size, zeta potential and stability of the platelet-derived drug-loaded vesicles in Example 5;

[0036] Fig.13 The loading diagram and drug release statistics diagram of the platelet-derived drug-loaded vesicles obtained by high-speed liquid chromatography analysis in Example 5;

[0037] Fig.14 The figure is a statistical graph of the tumor growth curve, mouse survival rate and weight of tumor-bearing mice in Example 6 (I);

[0038] Fig.15 The tumor immune cell map and statistical graph obtained from the single-cell sequencing analysis experiment in Example 6 (ii);

[0039] Fig.16 The representative graph and statistical graph of myeloid-derived suppressor cells and T cells in the tumor area obtained in the flow cytometry experiment in Example 6 (ii);

[0040] Fig.17 This is an image of myeloid-derived suppressor cells and T cells in the tumor area obtained in the immunofluorescence experiment in Example 6 (ii);

[0041] Fig.18 The volcano plot, violin plot and bubble plot of the representative genes of myeloid-derived suppressor cells obtained in the tumor single-cell sequencing analysis experiment in Example 6 (ii);

[0042] Fig.19The T cell distribution map, proportion statistical map and representative gene bubble map obtained in the tumor single cell sequencing analysis experiment in Example 6 (ii);

[0043] Fig. 20 The representative graph and statistical graph of effector T cells obtained in the flow cytometry experiment in Example 6 (ii);

[0044] Fig.21 These are H&E slice images of the main tissues obtained in the safety evaluation experiment in Example 7. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0046] Example 1: Assessment of Tumor Infiltration of Myeloid-Derived Suppressor Cells

[0047] (I) Evaluation of infiltration of myeloid-derived suppressor cells after immune checkpoint inhibitor therapy

[0048] The immune checkpoint inhibitor (programmed death ligand antibody, aPD-L1) was injected intratumorally into the mouse tumors once every two days for ten days. The proportion of myeloid-derived suppressor cells in the tumor area was evaluated by flow cytometry. Figure 1 As shown in Figure 2, the proportion of myeloid-derived suppressor cells (MDSCs) increased significantly after treatment. Flow cytometry experiments measured the proportion of CD8+T cells in the tumor area at different time points, and calculated the ratio of CD8+T cells to myeloid-derived suppressor cells. The results are shown in Figure 2. Figure 2 As shown, compared with the untreated group, although the proportion of CD8+T cells in the tumor area was increased after treatment, the ratio of CD8+T cells to myeloid-derived suppressor cells was significantly decreased on the 6th day and was lower than that of the untreated group.

[0049] 2. Evaluation of infiltration of myeloid-derived suppressor cells after radiotherapy and chemotherapy

[0050] The chemotherapy drug doxorubicin (DOX) was injected intratumorally into mice, or radiofrequency electrodes were inserted into the tumor area of ​​mice to perform radiofrequency ablation (RFA) on the mouse tumors. Flow cytometry was used to assess the proportion of myeloid-derived suppressor cells in the tumor area. Figure 3 As shown, the proportion of myeloid-derived suppressor cells in the tumor area increased significantly after treatment.

[0051] (III) Mechanistic studies on infiltration of bone marrow-derived suppressor cells

[0052] Enzyme-linked immunosorbent assay was used to measure the levels of chemokines associated with myeloid-derived suppressor cell infiltration in the tumor region. Figure 4 As shown in the figure, the ratio of CXCL1, CXCL2 and CXCL3 in the treated tumor area increased significantly on day 6. At the same time, the ratio of chemokine receptor (CXCR2) associated with myeloid-derived suppressor cells in the tumor area was also evaluated by flow cytometry. Figure 5 As shown, compared with the untreated group, the expression of the chemokine receptor was significantly upregulated on the 3rd, 6th and 15th days, and the expression level increased most significantly on the 6th day. This shows that after treatment with immune checkpoint inhibitors, myeloid-derived suppressor cells are recruited by related chemokines and infiltrate more into the tumor area, limiting the therapeutic effect of immune checkpoint inhibitors. After treatment with immune checkpoint inhibitors, the substantial increase in CXCL1, CXCL2 and CXCL3 in the tumor area can recruit other cells and vesicles expressing related receptors while recruiting myeloid-derived suppressor cells. The above-mentioned chemokine receptor, CXCR2, is expressed on the surface of platelets, and CXCR2 is also expressed on the surface of platelet vesicles. Therefore, platelet vesicles can actively target the tumor area after treatment.

[0053] Example 2: Preparation of platelet extracellular vesicles

[0054] Blood was collected from the ophthalmic sinus vein of the mouse, and after standing, it was centrifuged at 0.1G for 20 minutes at 4°C to remove the red blood cell pellet, followed by centrifugation at 0.8G for 20 minutes to obtain the platelet pellet and remove the plasma supernatant. EDTA buffer solution was added to resuspend the platelet extracellular vesicles, and 5U / mL of thrombin was added, and the platelets were activated and secreted in a constant temperature oscillator at 37°C for 30 minutes to activate the platelets and secrete platelet cell vesicles. After activation, 0.8G centrifugation was performed for 20 minutes to remove the unactivated platelets, collect the supernatant, and centrifuged at 14800rpm for 20 minutes to collect platelet extracellular vesicles (PEVs).

[0055] Example 3: Evaluation of tumor targeting of platelet extracellular vesicles

[0056] (I) Distribution of platelet extracellular vesicles in treated mice

[0057] Platelet extracellular vesicles were labeled with the fluorescent dye Cy5.5-NHS, and the fluorescently labeled platelet extracellular vesicles (Cy5.5-PEVs) and pure dye were delivered to the tumor area of ​​mice after radiotherapy, chemotherapy and aPD-L1 treatment through the tail vein, and observed by a small animal in vivo imager at 0.5, 4, 8, 12, and 24 hours after the tail vein. At the same time, after 24 hours, the mice were euthanized, and their hearts, livers, spleens, lungs, kidneys and tumors were collected for in vitro imaging. The tumor tissue was embedded in OCT embedding agent and made into 10μm slices using a freezing microtome (model: CM1860). The samples were fixed and stained with 4% paraformaldehyde and DAPI, respectively, and photographed with a confocal microscope. Figure 5 , Figure 6 , Figure 7 As shown, compared with pure dyes, platelet extracellular vesicles are retained more in the tumor area for a longer time. Compared with untreated tumors, platelet vesicles are retained more in the tumor area after radiotherapy, chemotherapy and aPD-L1 treatment. This is because after aPD-L1, radiotherapy and chemotherapy, a microenvironment enriched with driving factors is formed in the tumor area, and the expression of chemokines including CXCL1, CXCL2, and CXCL3 is upregulated, and the chemokine receptor CXCR2 expressed on platelet vesicles can bind to the above-mentioned driving factors, thereby driving the tumor area after active targeted therapy of platelet vesicles. Therefore, the platelet drug-loaded vesicles of the present invention can deliver targeted drugs to tumors. Moreover, compared with pure dyes, the enrichment of platelet vesicles in the heart, liver, spleen, lungs, and kidneys has been greatly reduced, because platelet vesicles are more enriched in the tumor area under the same dose. Therefore, the platelet drug-loaded vesicles of the present invention can reduce the side effects of pesidatinib.

[0058] (II) Mechanistic studies on tumor targeting of platelet extracellular vesicles

[0059] Platelets and platelet extracellular vesicles were disrupted and analyzed for surface proteins using gel electrophoresis and Western blotting. Figure 8 As shown, CXCR2 is expressed on both platelets and platelet extracellular vesicles, indicating that it can be recruited by a variety of driving factors in the tumor area, thus laying the foundation for targeted delivery of drugs into the tumor area after treatment.

[0060] Example 4: Preparation of platelet-derived drug-loaded vesicles

[0061] Blood was collected from the ophthalmic sinus vein of the mouse, and after standing, it was centrifuged at 0.1G for 20 minutes at 4°C to remove the red blood cell precipitate, followed by centrifugation at 0.8G for 20 minutes to obtain the platelet precipitate and remove the plasma supernatant. The platelets were then incubated with PLX at 4°C for 12 hours, with the concentration of PLX being 300μg / mL. After the co-incubation, an anticoagulant buffer solution was added, and the 0.8G centrifugation was repeated three times for 20 minutes to remove free PLX. Subsequently, EDTA buffer solution was added to resuspend the drug-loaded platelets, and 5U / mL of thrombin was added, and the platelets were activated and secreted in a constant temperature oscillator at 37°C for 30 minutes to activate the platelets and secrete platelet-derived drug-loaded vesicles. After activation, the platelets were centrifuged at 0.8G for 20 minutes to remove the unactivated platelets, collect the supernatant, and collect the platelet-derived drug-loaded vesicles (PLX-PEVs) by centrifugation at 14800rpm for 20 minutes.

[0062] Example 5: Characterization of platelet-derived drug-loaded vesicles

[0063] The internal structure of platelet-derived drug-loaded vesicles (abbreviated as PLX-PEVs) was characterized by field emission transmission electron microscopy (model: FEI TF20). Fig. 9 As shown in Figure 2, the platelet-derived drug-loaded vesicles have a particle size of about 150 nm, an ellipsoidal appearance, and are evenly distributed. A Zeta potential meter (model: Nano ZS90, manufacturer MALVERN) was used to evaluate the hydrodynamic size, surface charge, and stability of PLX-PEVs. Fig.10 As shown in the figure, the hydrodynamic size of PLX-PEVs is about 130nm; the surface charge is about -6mV, and it can be stably stored at 4℃ for at least 7 days, indicating that it has good stability. A UV spectrophotometer (model: Genesys 150, manufacturer: Thermo Fisher) was used to evaluate the absorbance of PLX-PEVs. The results are shown in Fig.11 As shown in the figure, the absorption peak of platelet-derived drug-loaded vesicles is significantly offset compared with that of platelet extracellular vesicles, indicating that the drug is successfully loaded on the platelet-derived drug-loaded vesicles. High-speed liquid chromatography (model: ultimate 3000, manufacturer: Thermo Fisher) was used to further evaluate the loading and release of the drug. The results are shown in the figure. Fig.13 As shown, under the condition of a dosing concentration of 300 μg / mL, the drug loading amount was about 33 μg, the loading rate was about 11%, and within 50 hours, the drug release rate of the platelet-derived drug-loaded vesicles was about 80%.

[0064] Example 6: Study on the anti-tumor effect of platelet drug-loaded vesicles

[0065] (I) Effects of platelet-derived drug-loaded vesicles on tumor growth

[0066] 2*10 6 4T1 tumor cells were subcutaneously injected into the right back of the mouse to construct 4T1 tumor-bearing mice. Subsequently, when the tumor volume reached about 100 mm 3 PBS buffer solution or 100 μg of aPD-L1 were injected into the tumor once every two days for six days. On the sixth day of intratumoral injection, platelet-derived drug-loaded vesicles or PBS buffer solution were injected into the tumor-bearing mice through the tail vein. During this process, tumor size and mouse weight were measured every two days. Fig.14 As shown, compared with the untreated group and the single administration of pesidatinib, platelet-derived drug-loaded vesicles can significantly inhibit tumor growth, prolong the survival time of tumor-bearing mice, and have no significant effect on the body weight of mice.

[0067] (II) Effects of platelet-derived drug-loaded vesicles on the tumor immune microenvironment

[0068] The above tumor tissues were collected and the changes in the tumor immune microenvironment were analyzed by single-cell sequencing. Fig.15 As shown in the figure, the results of single-cell sequencing analysis showed that after tail vein injection of platelet-derived drug-loaded vesicles, the proportion of bone marrow-derived suppressor cells in the tumor area of ​​mice was significantly reduced, and the proportion of T cells was significantly increased. Fig.16 , 17 As shown in the figure, flow cytometry and immunofluorescence results also confirmed that platelet-derived drug-loaded vesicles can significantly reduce the proportion of myeloid-derived suppressor cells and increase the proportion of T cells in the tumor area. Further analysis of the gene expression and gene enrichment pathways of myeloid-derived suppressor cells revealed that Fig.18 As shown in the figure, the expression of effector-related genes S100A8, S100A9 and migration-related gene CXCR2 was significantly reduced, and gene enrichment pathway analysis showed high enrichment of pathways such as apoptosis and cell senescence. Single-cell sequencing technology and flow cytometry were used to further analyze tumor T cells, such as Fig.19 , 20 As shown in the figure, the results of T cell regrouping analysis showed that the proportion of effector T cells increased significantly after tail vein injection of platelet-derived drug-loaded vesicles, and the expression of representative genes related to effector T cells, such as interferon γ, increased significantly. The above results indicate that tail vein injection of platelet-derived drug-loaded vesicles can inhibit the migration and activation of bone marrow-derived suppressor cells in tumors, thereby activating the anti-tumor effect of T cells.

[0069] Example 7: Safety evaluation of platelet-derived drug-loaded vesicles on major organs

[0070] In order to investigate whether platelet-derived drug-loaded vesicles could damage major organs including the heart, liver, spleen, lungs, and kidneys, we collected the hearts, livers, spleens, lungs, and kidneys of the above-mentioned treated mice and performed H&E sections on them. Fig.21 As shown in the figure, the results of H&E sections showed that the platelet-derived drug-loaded vesicles did not affect the normal physiological structure of the above organs. The above results indicate that the platelet-derived drug-loaded vesicles have good safety.

[0071] In summary, the present invention relates to the application of platelet-derived drug-loaded vesicles for targeting myeloid-derived suppressor cells and enhancing the therapeutic response of immune checkpoint inhibitors in tumor-bearing mice. The platelet-derived drug-loaded vesicles administered by tail vein regulate the tumor immune microenvironment of mice, block the function and migration of myeloid-derived suppressor cells in the tumor area, and activate the function of T cells, thereby achieving anti-tumor effects.

[0072] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A platelet drug-loaded vesicle loaded with pecidinib, characterized in that: Pesidatinib is co-incubated with platelets to obtain drug-loaded platelets, the drug-loaded platelets are activated, and the platelet drug-loaded vesicles are separated.

2. The platelet drug-loaded vesicle according to claim 1, characterized in that: The co-incubation temperature is 0-8°C, and the co-incubation time is 10-14 hours.

3. The platelet drug-loaded vesicle according to claim 1, characterized in that: The drug-loaded platelet activation is to co-incubate the drug-loaded platelets with a platelet activator.

4. The platelet drug-loaded vesicle according to claim 3, characterized in that: The co-incubation temperature is 30-40° C., and the co-incubation time is 20-40 minutes.

5. The platelet drug-loaded vesicle according to claim 3, characterized in that: The platelet activator is selected from one or more of thrombin, epinephrine, thromboxane, 5-hydroxytryptamine, angiotensin, tissue plasminogen activator, urokinase plasminogen activator, and platelet activating factor.

6. The platelet drug-loaded vesicle according to claim 1, characterized in that: The particle size of the platelet drug-loaded vesicle is 100-200 nm, and the surface charge is -4 to -8 mV.

7. Use of the platelet drug-loaded vesicles according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The anti-tumor drug is administered by intravenous injection.

9. The use according to claim 7, characterized in that: The anti-tumor drug exerts its effects by acting on immunosuppressive cells in the tumor immune microenvironment.

10. A tumor targeting drug, characterized in that: It comprises the platelet drug-loaded vesicles as described in any one of claims 1-6.

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