Engineered long-acting platelet delivery system internally loaded with therapeutic drug as well as preparation method and application of engineered long-acting platelet delivery system

Abstract

The invention relates to the technical field of drugs, and relates to an engineered long-acting platelet delivery system internally loaded with a therapeutic drug as well as a preparation method and application of the engineered long-acting platelet delivery system. The system comprises platelets internally loaded with therapeutic drugs and nanoparticles containing platelet apoptosis inhibitors. The engineering long-acting platelet delivery system provided by the invention can realize in-vitro stable storage and in-vivo long-term circulation. Besides, according to the engineering long-acting platelet delivery system disclosed by the invention, accurate targeting of postoperative residual tumor cells and circulating tumor cells can be realized through efficient and natural wound tropism and tumor targeting of platelets, so that killer chemotherapeutic drugs are released, and postoperative tumor recurrence and metastasis are effectively avoided.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to an engineered long-acting platelet delivery system carrying therapeutic drugs, a preparation method and an application thereof. Background Art

[0002] Tumors seriously threaten human life and health, and surgical resection is one of the current mainstream treatments. After surgery, adjuvant therapy strategies are usually needed to inhibit residual tumor cells and circulating tumor cells, thereby improving the prognosis of cancer patients. However, systemic administration is very likely to cause adverse reactions, so specific targeted delivery systems are urgently needed to be developed.

[0003] Due to the unique wound tropism and tumor targeting of platelets, platelet-based drug delivery systems have been widely used and achieved good therapeutic effects. However, platelets are unstable and easily activated, which leads to apoptosis, seriously affecting the storage capacity of platelets in vitro and the circulation time in vivo, limiting their clinical application as transport carriers. Therefore, inhibiting platelet apoptosis and prolonging their storage time are necessary means to expand the prospects of clinical application of platelets.

[0004] The activity of serine / threonine protein kinase (PKA) in platelets decreases with the aging of platelets, and the activation of PKA can protect platelets from apoptosis and clearance caused by storage and external stimuli. In addition, compared with free drugs, the surface anchoring strategy can limit the spatial distance between the "backpack" and the carrier, ensuring that the carrier and the cargo have the same fate in vivo, that is, the cargo can effectively act on its carrier. Summary of the invention

[0005] The technical problem solved by the present invention is to overcome the existing technical defects, comprehensively consider the tumor targeting ability of platelets and the difficulties in production, storage and transportation, and provide an engineered long-acting platelet delivery system containing therapeutic drugs, and its preparation method and application.

[0006] To achieve the above purpose, the present invention adopts the following technical solution:

[0007] An engineered long-acting platelet delivery system loaded with therapeutic drugs, characterized in that the system is composed of platelets loaded with therapeutic drugs and nanoparticles containing platelet apoptosis inhibitors.

[0008] The platelets loaded with therapeutic drugs are co-incubated with platelet apoptosis inhibitors to obtain nanoparticles.

[0009] The therapeutic drug is one or more of anti-tumor drugs and cardiovascular disease therapeutic drugs.

[0010] The anti-tumor drugs are small molecule drugs, such as doxorubicin, paclitaxel, cisplatin, SN38, methotrexate and their derivatives.

[0011] The cardiovascular disease therapeutic drugs are lovastatin, metoprolol, enalapril, rapamycin and their derivatives.

[0012] The platelet apoptosis inhibitor is a substance that can inhibit the apoptosis and activation of platelets.

[0013] The platelet apoptosis inhibitor is one or more of quercetin, ticagrelor, and anti-CD38 monoclonal antibody.

[0014] A method for preparing an engineered long-acting platelet delivery system carrying a therapeutic drug, comprising: (1) preparing a nanosystem carrying a platelet apoptosis inhibitor;

[0015] (2) extracting and purifying platelets;

[0016] (3) loading the interior of the extracted platelets with therapeutic drugs;

[0017] (4) Anchoring the prepared nanosystem on the surface of platelets loaded with therapeutic drugs.

[0018] For example, the engineered long-acting platelets with therapeutic drugs loaded inside and platelet apoptosis inhibitors anchored outside, the platelets are loaded with doxorubicin inside, and liposomes loaded with quercetin are anchored outside by reacting N-hydroxysuccinimide with amino groups. The liposomes loaded with quercetin are prepared by thin film hydration method. The platelets loaded with doxorubicin are prepared by incubation method.

[0019] Furthermore, doxorubicin is loaded into the platelets as a drug, and the quercetin-loaded liposomes are anchored on the surface of the platelets. The preparation method comprises the following steps:

[0020] (1) preparing quercetin-loaded liposomes;

[0021] (2) Extracting platelets;

[0022] (3) loading doxorubicin into the interior of platelets;

[0023] (4) Anchoring liposomes on the platelet surface.

[0024] Wherein, the liposomes described in step (1) are prepared by thin film hydration method. 2000 -NHS, cholesterol and phospholipids were mixed in a mass ratio of 1:6:36, and then quercetin (which is similar to DSPE-PEG 2000-NHS (mass ratio: 1:1) was completely dissolved in a mixed solution of chloroform and methanol, and the organic phase was removed by evaporation under reduced pressure. An appropriate amount of PBS solution was added, and the liposome film was hydrated in a water bath at 60°C for 1 h. After ultrasonication in an ice bath to a suitable particle size, the liposome loaded with quercetin was obtained by centrifugation for later use.

[0025] Among them, phospholipids can be selected from DMPC, DSPC, DHPC, etc.

[0026] DSPE-PEG 2000 -NHS can be replaced by other linkages, including but not limited to DSPE-PEG 2000 -DBCO,DSPE-PEG 2000 -N 3 ,DSPE-PEG 2000 -NH 2 ,DSPE-PEG 2000 -SH, etc. The nanosystem can be anchored on the surface of platelets loaded with therapeutic drugs through click reactions such as sulfone chloride and amine, azide and alkyne, N-hydroxysuccinimide and amino, maleimide and thiol.

[0027] Meanwhile, quercetin can be replaced by other platelet activation or apoptosis inhibitors, including but not limited to ticagrelor, aspirin, etc.

[0028] In step (2), fresh blood after anticoagulation is taken, and platelets are obtained by gradient centrifugation, which are then washed and purified for later use.

[0029] In step (3), doxorubicin (the mass ratio of doxorubicin to inhibitor is 1:1) is dissolved in PBS containing 1% DMSO, incubated with platelets for 1 hour, and centrifuged to remove free doxorubicin to obtain doxorubicin-loaded platelets for later use.

[0030] In step (4), the quercetin-loaded liposomes in step (1) are incubated with the doxorubicin-loaded platelets in step (3) for 1 hour, and then centrifuged to remove the uncoupled liposomes.

[0031] An application of the engineered long-acting platelet delivery system carrying therapeutic drugs, and an application of the engineered long-acting platelet delivery system carrying therapeutic drugs in a drug delivery system.

[0032] The engineered long-acting platelet delivery system loaded with therapeutic drugs is used in preventing tumor growth, metastasis, and postoperative recurrence.

[0033] The application of the engineered long-acting platelet delivery system loaded with therapeutic drugs in inhibiting cardiovascular diseases.

[0034] The platelets are of mouse origin and extracted from fresh mouse plasma.

[0035] The present invention prepares engineered long-acting platelets (PDQLs) carrying therapeutic drugs and externally anchoring platelet apoptosis inhibitors, and investigates their properties. At the same time, engineered platelets (PQLs) externally anchoring quercetin liposomes, platelets (PLs) externally anchoring blank liposomes, engineered platelets (PDs) carrying doxorubicin, and physical mixtures of engineered platelets carrying doxorubicin and quercetin liposomes (PD&QLs) are prepared, and then the above substances are evaluated:

[0036] 1) The physical and chemical properties of the engineered long-acting platelets loaded with therapeutic drugs and anchored with platelet apoptosis inhibitors were characterized, such as the morphological structure of the engineered platelets, protein expression, retention time of liposomes on the platelet surface, and drug release.

[0037] 2) The adhesion, uptake and cytotoxicity of engineered long-acting platelets loaded with therapeutic drugs and anchored with platelet apoptosis inhibitors were investigated at the cellular level.

[0038] 3) Engineered long-acting platelets that load therapeutic drugs internally and anchor platelet apoptosis inhibitors externally resist apoptosis caused by external stimuli and long-term storage.

[0039] 4) Pharmacokinetic behavior and in vivo distribution of engineered long-acting platelets that internally load therapeutic drugs and externally anchor platelet apoptosis inhibitors.

[0040] 5) The ability of engineered long-acting platelets that load therapeutic drugs internally and anchor platelet apoptosis inhibitors externally to inhibit tumor recurrence and metastasis after surgery.

[0041] The advantages of the present invention are:

[0042] The drug delivery system of the present invention is highly efficient and low-toxic, and overcomes the pain points that severely limit the clinical application of platelets, such as poor in vitro transport stability, harsh storage conditions, and difficulty in long-term storage. Specifically:

[0043] 1. The preparation of the engineered long-acting platelet delivery system of the present invention includes extracting and purifying platelets, loading chemotherapeutic drugs into the platelets, preparing and optimizing liposomes carrying apoptosis inhibitors, and anchoring the liposomes on the surface of platelets. The engineered long-acting platelet delivery system of the present invention can achieve stable storage in vitro and long-term circulation in vivo. In addition, the engineered long-acting platelet delivery system of the present invention can achieve precise targeting of postoperative residual tumor cells and circulating tumor cells through the efficient and natural wound tropism and tumor targeting of platelets, thereby releasing lethal chemotherapeutic drugs and effectively avoiding the recurrence and metastasis of postoperative tumors.

[0044] 2. The drug delivery system based on platelets of the present invention is designed to enhance the targeting ability of drugs while enhancing the storage and transportation stability of drugs, and to maximize the clinical application potential of platelet-related preparations. The present invention is a bioengineered long-acting platelet system with adriamycin internally anchored with quercetin liposomes externally. Due to the specific tumor targeting and capture ability, the system can specifically deliver chemotherapeutic drugs to the tumor site to inhibit postoperative recurrence and metastasis of tumors. In addition, the externally anchored quercetin liposomes with sustained release effect continuously release quercetin, synchronously prolonging the in vitro storage and in vivo circulation time, and not affecting the platelet carrier activity.

[0045] In summary, the engineered long-acting platelets prepared by the present invention, which internally carry therapeutic drugs and externally anchor platelet apoptosis inhibitors, can achieve the effects of stable storage in vitro and long-term circulation in vivo while targeting residual tumor cells and circulating tumor cells after surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a diagram of the platelet extraction process;

[0047] Figure 2 Schematic diagram of the preparation of PDQLs;

[0048] Figure 3 It is the morphological representation of PDQLs;

[0049] A is a transmission electron microscopy image of quercetin-loaded liposomes (QLs); B is a transmission electron microscopy image of PDQLs and blank platelets; C is a scanning electron microscopy image of PDQLs and blank platelets; D is a confocal microscopy image of PDQLs, in which red is DiR-labeled liposomes, green is doxorubicin, and blue is Alexa Fluor. 350 labeled platelets;

[0050] Figure 4 The pharmaceutical characterization for PDQLs;

[0051] A is the SDS-PAGE protein analysis of PDQLs, PDs, PQLs, and platelets, and the arrows indicate the changes in platelet surface proteins after QLs modification; B is the protein imprint analysis of PDQLs, PDs, PQLs, and platelet-specific proteins CD41, CD61, and P-selectin; C is a representative image of QLs fluorescence on the platelet surface; D is the relative quantification of QLs fluorescence on the platelet surface; E is the cumulative release curve of doxorubicin, and the release medium is PBS containing 4% DMSO; F is the cumulative release curve of quercetin, and the release medium is PBS containing 1% Tween 80.

[0052] Figure 5 for the therapeutic effects of PDQLs at the cellular level;

[0053] A is a confocal image of DiR-labeled platelets, PLs, and PQLs adhering to the surface of tumor cells, B is a semi-quantitative analysis of Figure A, C is a confocal image of tumor cells taking up free doxorubicin, PDs, and PDQLs, D is a representative flow cytometry image of tumor cells taking up free doxorubicin, PDs, and PDQLs, E is a quantitative analysis of Figure D, and F is an in vitro cytotoxic study of different concentrations of free doxorubicin, PDs, PQLs, and PDQLs on tumor cells.

[0054] Figure 6 To investigate the effect of QLs on inhibiting platelet apoptosis;

[0055] A is a representative flow cytometric image of platelets, PDs, PQLs, and PDQLs apoptosis at room temperature for 0, 2, and 4 days. B is a representative flow cytometric image of platelets, PDs, PQLs, and PDQLs apoptosis at room temperature for 0, 2, and 4 days. -1 A is a photograph of thrombin-treated platelets, PDs, PQLs, and PDQLs, and C is a Western blot analysis of apoptotic proteins Bcl-2 and Bax in platelets, PDs, PQLs, and PDQLs after 0 and 2 days at room temperature.

[0056] Figure 7 The molar concentration-time curves of free doxorubicin, PDs, and PDQLs, i.e., the pharmacokinetic study of PDQLs.

[0057] Figure 8 The organizational distribution of PDQLs;

[0058] A is the fluorescence distribution image of free DiR, DiR-labeled PDs, and DiR-labeled PDQLs immediately injected into postoperative mice; B is the in vitro fluorescence imaging of major organs and surviving tumors 24 hours after administration; and C is the quantitative analysis of Figure B.

[0059] Fig. 9 This is an investigation of the in vivo inhibitory effect of PDQLs on tumor recurrence and metastasis; A is the timeline of PDQLs therapy administration, B is the in vivo bioluminescence imaging of fluorescently labeled tumor cells remaining on days 10, 11, 16, and 21 after tumor inoculation, C is the tumor growth curve of mice in different treatment groups, D is the average tumor growth curve, E is the survival time of mice in different treatment groups, F is the weight change of mice, G is the image of the tumor on the 20th day after tumor inoculation, H is the weight of the mouse tumor, and I is a representative image of the tumor lung in the lung metastasis model on the 20th day and the H&E staining results. DETAILED DESCRIPTION

[0060] The present invention is further described below by way of examples, but the invention is not limited to the scope of the examples.

[0061] The engineered platelet system of the present invention has natural wound tropism and tumor targeting, and can kill postoperative residual tumor cells and circulating tumor cells by targeted delivery of the loaded drugs. The externally anchored platelet apoptosis inhibitor will be continuously released to delay the apoptosis time of platelets, so as to achieve the in vitro stable storage of engineered platelets and long-term circulation in vivo, thereby significantly inhibiting postoperative tumor recurrence and metastasis.

[0062] Example 1: Preparation and characterization of an engineered long-acting platelet system that internally loads therapeutic drugs and externally anchors platelet apoptosis inhibitors.

[0063] Platelet extraction, such as Figure 1 As shown, 10 mL of fresh anticoagulated mouse blood was taken and centrifuged at 200 g for 4 min to obtain platelet-rich plasma, which was mixed with an equal amount of blood preservation solution (commercially available product) and centrifuged at 800 g for 10 min. The platelets were respun in PBS containing 1 μM prostacyclin and set aside.

[0064] Preparation of engineered long-acting platelet systems with therapeutic drugs loaded internally and platelet apoptosis inhibitors anchored externally, such as Figure 2 As shown,

[0065] Quercetin-loaded liposomes were prepared by thin film hydration method. Specifically, 36 mg DSPC, 6 mg cholesterol, 1 mg DSPE-PEG 2000 -NHS and 1 mg of quercetin were dissolved in a methanol-chloroform mixed solvent (methanol: chloroform = 1:9, volume ratio). The residual organic solvent was completely removed by a rotary evaporator to form a thin layer of light yellow lipid film. Sterile PBS was used as a hydration solution, hydrated at 60°C for 1 hour, and then ultrasonicated in an ice bath, and gradient centrifuged (4000r, 5min, discard the precipitate, 13000r, 15min, collect the precipitate) to obtain uniform liposomes (QLs). QLs were resuspended in PBS for later use.

[0066] 1 mg of doxorubicin was dissolved in PBS containing 1% DMSO and mixed with 1×10 8 The platelets obtained by the above extraction were incubated at 37°C for 1 hour, centrifuged at 800g for 10 minutes to remove free doxorubicin, and then the doxorubicin-loaded platelets (PDs) were obtained. Then, the platelets were incubated with the prepared QLs for 1 hour, and centrifuged to obtain engineered long-acting platelets (PDQLs) with quercetin liposomes anchored by chemotherapy drugs inside.

[0067] At the same time, blank platelets were co-incubated with QLs as described above to prepare an engineered platelet system with externally anchored quercetin liposomes (PQLs).

[0068] The above-obtained materials were measured, and the encapsulation efficiency of the prepared quercetin-loaded liposomes was 85.3%. The liposomes were spherical in structure and about 150 nm in size. It was determined that each platelet contained 3.6 pg of drug and 1.6 pg of inhibitor ( Figure 3 A). Transmission electron microscopy ( Figure 3 B) and scanning electron microscopy ( Figure 3 C) all show that liposomes can be covalently anchored on the platelet surface, and the morphological structure of platelets does not change significantly. In addition, laser confocal scanning microscopy images ( Figure 3 D) The fluorescence co-localization results further demonstrated the successful preparation of engineered long-lasting platelets, in which platelets were treated with Alexa Fluor 350 labeled (blue), liposomes were labeled with DiR (red), and doxorubicin itself has green fluorescence. Due to physical factors such as blood flow and collision, liposomes anchored on the platelet surface may fall off, so flow cytometry was used to verify the retention of liposomes on the platelet surface. The liposomes were labeled with coumarin 6, and the results showed that 78% of the liposomes were still anchored on the platelet surface after 24 hours ( Figure 4 A, B).

[0069] The results of SDS-PAGE gel electrophoresis showed that most proteins on the platelet surface were expressed on the surface of PDQLs ( Figure 4 C), however, some differences in protein expression were observed around 42 kDa and 190 kDa, which may be due to the modification of QLs. Western blot analysis experiments demonstrated that neither doxorubicin encapsulation nor QLs modification would affect the expression of key proteins related to platelet surface adhesion and aggregation ( Figure 4 D). That is, the PDQLs prepared by the present invention have the same physiological function as normal platelets. In addition, quercetin and doxorubicin can be gradually released ( Figure 4 E, F), in which the released doxorubicin can be taken up by tumor cells to play a cell-killing role, while quercetin directly acts on the carrier platelets to help them resist the negative effects of external stimuli.

[0070] Example 2: Effects of PDQLs on tumor cells at the in vitro cellular level.

[0071] First, platelets and blank liposome-loaded platelets (PLs) were used as controls, and the adhesion of engineered platelet systems (PQLs) with quercetin liposomes (PQLs) to wild-type B16-F10 tumor cells was investigated by confocal fluorescence imaging. 4 ) were cultured on a confocal dish. After 24 h, fresh serum-free medium was replaced and the cells were placed in a 4°C refrigerator for 20 min. DiR-labeled platelets, PLs, and PQLs (4 × 108 To avoid the interference of doxorubicin's own green fluorescence, PQLs were used instead of PDQLs. The adhesion results showed that compared with unmodified platelets, only some PQLs could adhere to the surface of tumor cells ( Figure 5 A), this is because the liposomes on the platelet surface continuously release quercetin, and quercetin can inhibit platelet activation and reduce the expression of P-selectin on the platelet surface, thereby affecting platelet adhesion. The present invention prepared blank liposome-anchored platelets (PLs) as a control group for adhesion experiments, and found that the number of PLs on the tumor cell surface was no significantly different from that of the platelet group ( Figure 5 B), further confirming that the reduction in the adhesion level of PQLs was caused by quercetin, while the surface anchoring strategy itself did not affect its adhesion.

[0072] Using free doxorubicin as control, the uptake of doxorubicin in PDs and PDQLs by wild-type B16-F10 tumor cells was observed by laser confocal microscopy. 4 Cells were inoculated in confocal culture dishes, and free doxorubicin, PDs, and PDQLs (equivalent dose of 5 μg mL -1 ) were incubated for 0.5h and 2h, respectively. The results showed that with the increase of incubation time, the fluorescence intensity in the cells gradually increased, confirming that doxorubicin can be internalized by tumor cells in a time-dependent manner ( Figure 5 C). In addition, the tumor fluorescence signal in the PDs-treated group was significantly higher than that in the free doxorubicin-treated group, and the tumor uptake in the PDQLs group was lower ( Figure 5 D, E).

[0073] B16-F10 melanoma cells (2×10 3 The 48h cytotoxicity of free doxorubicin, PDs, PQLs, and PDQLs was investigated using a 48h cell model. Similar to the results of cell uptake, PDs showed the strongest cytotoxicity, while PDQLs showed weaker cytotoxicity. In the absence of the cytotoxic drug doxorubicin, PQLs had almost no cell killing effect ( Figure 5 F).

[0074] Example 3: Effect of quercetin-loaded liposomes in delaying platelet apoptosis.

[0075] In order to verify whether QLs can protect platelet apoptosis caused by long-term storage, platelets, PDs, PQLs, PDQLs (1×10 8Platelets) were stored at room temperature for 4 days, and the changes in mitochondrial membrane potential were measured by flow cytometry. The results showed that with the extension of storage time, the mitochondrial membrane potential of platelets gradually decreased, and the presence of quercetin liposomes could effectively alleviate the apoptosis rate of platelets ( Figure 6 A). Western blot analysis showed that the level of Bcl-2 in platelets was significantly increased in the presence of QLs ( Figure 6 C), further confirmed that QLs can inhibit platelet apoptosis. In addition, quercetin also has an anti-platelet activation effect. Therefore, in the platelet, PDs, PQLs, PDQLs (1×10 8 0.5 U mL -1 After 1 hour, a large number of pure platelets and PDs aggregated into spheres, while QLs-modified platelets (PQLs, PDQLs) did not aggregate until 4 hours later ( Figure 6 B), and the aggregated platelets formed a loose film, which further confirmed that QLs can delay the activation and apoptosis of platelets caused by negative factors.

[0076] To further verify the effect of QLs on platelets in vivo, free doxorubicin, PDs, and PDs & QLs (a physical mixture of PDs and QLs, in which DSPE-PEG was used in the preparation of QLs) were investigated. 2000 Replace DSPE-PEG 2000 -NHS), the pharmacokinetic characteristics of PDQLs. Specifically, healthy C57BL / 6 mice were injected with the corresponding preparations (doxorubicin equivalent dose of 2 mg kg -1 , quercetin equivalent dose 0.89 mg kg -1 ), and 20 μL of blood was collected from the tail vein at specific time points, and the fluorescence of doxorubicin was measured by an enzyme-labeled instrument. The results showed that free doxorubicin was rapidly cleared from the body, while the area under the drug-time curve of doxorubicin in the PDs, PD&QLs, and PDQLs groups was significantly increased ( Figure 7 ), which were 2.98 times, 4.21 times and 11.63 times higher than those in the solution group. This is because platelets are autologous cells that express CD47 receptors on their surface, sending a "don't eat me" signal to macrophages, thereby prolonging their half-life. In addition, QLs anchored on the surface of platelets can continuously release quercetin to act on carrier platelets, inhibiting their apoptosis and further prolonging blood circulation time.

[0077] Tissue distribution experiments were then performed to verify the ability of PDQLs to target residual tumor cells after surgery. The tumor was partially resected (approximately 30% residual) and DiR solution, DiR-labeled PDs, DiR-labeled PDQLs, and DiR equivalent doses of 1 mg kg were injected into the tail vein immediately after surgery.-1 , and the fluorescence distribution of the preparations was observed by small animal in vivo imaging at specific time points. Compared with the free DiR group, both PDs and PDQLs showed higher fluorescence intensity at the tumor wound site ( Figure 8 ). In addition, the peak fluorescence signal of PDs appeared at 6h, while the fluorescence signal of PDQLs continued to increase and reached a peak at 12h. This may be because PDQLs have a longer blood circulation time, allowing more PDQLs to be distributed in the tumor site.

[0078] Example 4: The effect of PDQLs on anti-tumor recurrence and metastasis after surgery.

[0079] In order to verify the anti-tumor recurrence and metastasis effect of PDQLs, we established an incomplete tumor resection model and a metastasis model ( Fig. 9 A). Luciferase-labeled B16-F10 (B16-F10-luc) tumors were inoculated into the abdomen of mice. When the tumors grew to 500 mm 3 After surgery, the tumor was incompletely removed (approximately 1% remained). Immediately after surgery, the mice were injected with saline, free doxorubicin, PDs, PQLs, PDs&QLs, and PDQLs via the tail vein. The doxorubicin equivalent dose was 2 mg kg -1 , quercetin equivalent dose 0.89 mg kg -1 , monitoring tumor recurrence by bioluminescent signals ( Fig. 9 B). Among them, PDQLs showed better ability to inhibit tumor recurrence after surgery ( Fig. 9 C, D). However, there was no significant difference in the therapeutic effect between the PD&QLs group and the PDs group. This is because PDs and QLs have different fates in the body, and the quercetin released by QLs cannot accurately act on PDs, thereby failing to inhibit platelet apoptosis. The survival period of mice treated with PDQLs can reach 39 days, while all mice in the saline group died on the 22nd day ( Fig. 9 E). The body weight of all mice remained stable during the treatment period, confirming that the engineered long-acting platelets prepared by the present invention have good safety. The same phenomenon was also observed in the wild-type melanoma mouse postoperative model, and the recurrent tumors in the PDQLs group were the smallest ( Fig. 9 G, H). Based on the postoperative model, 5×10 5 platelets to construct a lung metastasis model, and the treatment results showed that the number of tumor metastases in the PDQLs group was the lowest ( Fig. 9 I). The above results confirmed the powerful anti-tumor efficacy of PDQLs in many aspects and further demonstrated the importance of inhibiting platelet apoptosis through surface anchoring strategy.

Claims

1. An engineered long-acting platelet delivery system loaded with therapeutic drugs, characterized in that: The system is platelets loaded with therapeutic drugs and nanoparticles containing platelet apoptosis inhibitors.

2. The engineered long-acting platelet delivery system containing therapeutic drugs according to claim 1, characterized in that: The platelets loaded with therapeutic drugs are co-incubated with platelet apoptosis inhibitors to obtain nanoparticles.

3. The engineered long-acting platelet delivery system containing therapeutic drugs according to claim 1 or 2, characterized in that: The therapeutic drug is one or more of anti-tumor drugs and cardiovascular disease therapeutic drugs.

4. The engineered long-acting platelet delivery system containing therapeutic drugs according to claim 1 or 2, characterized in that: The platelet apoptosis inhibitor is a substance that inhibits the apoptosis and activation of platelets.

5. The engineered long-acting platelet delivery system containing therapeutic drugs according to claim 4, characterized in that: The platelet apoptosis inhibitor is one or more of quercetin, cyclic adenosine monophosphate, aminophylline, cilostazol, enoximone, ticagrelor, and anti-CD38 monoclonal antibody.

6. A method for preparing the engineered long-acting platelet delivery system containing therapeutic drugs as claimed in claim 1, characterized in that: (1) Preparation of nanosystems loaded with platelet apoptosis inhibitors; (2) extracting and purifying platelets; (3) loading the interior of the extracted platelets with therapeutic drugs; (4) Anchoring the prepared nanosystem on the surface of platelets loaded with therapeutic drugs.

7. Use of the engineered long-acting platelet delivery system containing therapeutic drugs as claimed in claim 1, characterized in that: The application of the engineered long-acting platelet delivery system loaded with therapeutic drugs in a drug delivery system.

8. Use of the engineered long-acting platelet delivery system containing therapeutic drugs according to claim 1, characterized in that: The engineered long-acting platelet delivery system loaded with therapeutic drugs is used in preventing tumor growth, metastasis, and postoperative recurrence.

9. Use of the engineered long-acting platelet delivery system containing therapeutic drugs according to claim 1, characterized in that: The application of the engineered long-acting platelet delivery system loaded with therapeutic drugs in inhibiting cardiovascular diseases.

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