Engineered platelets carrying platelet activation inhibitor nanoparticles and preparation method and application thereof

Abstract

The present invention discloses engineered platelets carrying platelet activation inhibitor nanoparticles, a preparation method and application thereof, which belongs to the field of pharmaceutical technology and relates to a preparation method of engineered platelets carrying ticagrelor nanoparticles and its therapeutic use in malignant solid tumors. The preparation of the engineered platelets described in the present invention includes the steps of extracting and purifying platelets, preparing gelatin nanoparticles loaded with platelet activation inhibitors, anchoring the nanoparticles to the outer surface of platelets, etc. The engineered platelets of the present invention are targeted to reach the tumor site by recruiting tumor-associated platelets in the body, and specifically respond to matrix metalloproteinases to release the carried platelet activation inhibitor, thereby inhibiting the activation of platelets in the tumor microenvironment, enhancing tumor vascular leakage, alleviating the immunosuppressive microenvironment, and effectively enhancing the effects of chemotherapy and immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to an engineered platelet carrying platelet activation inhibitor nanoparticles, a preparation method and an application thereof. Background Art

[0002] Cancer is currently one of the leading causes of death. Traditional treatments such as immunotherapy, chemotherapy, and radiotherapy remain unsatisfactory. Recent evidence indicates that platelets are closely linked to tumor development. Due to the rapid growth of tumors and the poor integrity of their vascular systems, nanomedicines can enter tumor tissues through leaky vasculature. However, damaged endothelium attracts resting platelets and initiates coagulation to protect the integrity of tumor vessels, impairing the enhanced permeability and retention (EPR) effect, reducing the accumulation of nanomedicines at the tumor site, and inhibiting their therapeutic efficacy.

[0003] Furthermore, it has been reported that resting platelets can interact with tumor cells and store tumor cell PD-L1 in α-granules. Upon platelet activation, stored PD-L1 is released from α-granules and transferred to the platelet surface. Platelets containing tumor-derived PD-L1 interact with T cells, significantly inhibiting the proliferation, activation, and cytotoxicity of CD8+ T cells, suppressing systemic antitumor immunity, and promoting immune escape. Therefore, increased platelet number and activity are associated with poor prognosis and decreased overall survival. Inhibiting platelet number and function has become a promising therapeutic strategy to alleviate tumor immunosuppression, disrupt tumor vascular integrity, and enhance targeted delivery of chemotherapeutic nanomedicines. However, systemic platelet consumption may lead to coagulopathy and increased systemic toxicity.

[0004] Platelets possess natural tissue-specific targeting properties and have been widely used in drug delivery systems in recent years. At the tumor site, platelets are activated due to the inflammatory microenvironment, which then recruits more platelets to the tumor through a cascade reaction. Consequently, platelet-based delivery systems have emerged to minimize off-target effects and side effects. Furthermore, activated platelets secrete matrix metalloproteinases, which specifically and rapidly degrade gelatin, releasing the entrapped drug. Summary of the Invention

[0005] The technical problem addressed by this invention is to overcome the shortcomings of existing technologies. Taking into account the tumor-targeting ability of platelets and the ability of gelatin particles to rapidly respond to matrix metalloproteinases (MMPs), a novel platelet activation inhibitor delivery system was developed. This system aims to minimize the systemic toxicity of antiplatelet drugs and enhance their clinical application. Specifically, we designed ticagrelor-loaded gelatin nanoparticles (TNPs) and conjugated them to the surface of resting platelets, forming engineered platelets (PTNPs) loaded with platelet activation inhibitor nanoparticles. These engineered platelets can target activated tumor-associated platelets and degrade the gelatin nanoparticles through secreted matrix metalloproteinases, subsequently releasing the entrapped platelet activation inhibitor, ticagrelor. This process maintains the quiescent state of newly recruited platelets, inhibiting the repair of the tumor vascular barrier and alleviating platelet-mediated tumor immunosuppression. These engineered platelets can be used synergistically with chemotherapeutic nanoparticles and immune checkpoint inhibitors to enhance the long-term efficacy of anti-tumor therapy and reduce side effects.

[0006] Specifically, the first purpose of the present invention is to address the pain points of poor efficacy of traditional anti-tumor nanoformulations and immune checkpoint inhibitors, overcome the shortcomings of traditional antiplatelet drugs such as multiple non-targeted distribution and large side effects, and provide a new dosage form targeting tumor-associated platelets, namely, engineered platelets (PTNPs) carrying platelet activation inhibitor nanoparticles.

[0007] The second object of the present invention is to provide a method for preparing the engineered platelets carrying the platelet activation inhibitor nanoparticles.

[0008] The third object of the present invention is to provide applications of the engineered platelets carrying the platelet activation inhibitor nanoparticles.

[0009] To achieve the first objective, the present invention employs a technical solution: providing engineered platelets loaded with platelet activation inhibitor nanoparticles. The engineered platelets are externally coupled to the platelet activation inhibitor-containing nanoparticles via sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sodium salt (Sulfo-SMCC). The platelet activation inhibitor-containing nanoparticles can be prepared by methods such as emulsification solvent evaporation, secondary desolvation, nanoprecipitation, and reverse microemulsion.

[0010] Specifically, the engineered platelets carrying platelet activation inhibitor nanoparticles comprise ticagrelor, gelatin, oleic acid, synthetic phospholipids, Sulfo-SMCC, and platelets, wherein ticagrelor, gelatin, oleic acid, and synthetic phospholipids together constitute the nanoparticles, with one platelet loaded with 1-14 nanoparticles. Preferably, one platelet is loaded with 10 nanoparticles, containing 15 picograms of ticagrelor.

[0011] The ticagrelor of the present invention may also be other platelet activation inhibitors, such as one or more drugs that inhibit platelet activation, such as aspirin, clopidogrel, tirofiban, cilostazol, or their derivatives.

[0012] The gelatin can also be other organic carriers sensitive to matrix metalloproteinases, such as collagen, matrix metalloproteinase-sensitive peptides or one or more of their derivatives, which can be specifically degraded by matrix metalloproteinases.

[0013] The oleic acid and synthetic phospholipids may also be other emulsifiers and stabilizers. The synthetic phospholipid is preferably 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0014] The Sulfo-SMCC can also be modified with other cross-linking agents, such as NHS-SS-NHS and other protein linkers, and coupled to the platelet surface.

[0015] The platelet activation inhibitor-loaded nanoparticles are spherical with a particle size of 70-110 nm. They are primarily composed of gelatin, with oleic acid and synthetic phospholipids acting as emulsifiers and stabilizers. The platelet activation inhibitor is the encapsulated drug. The weight percentages of the components are: 3%-5% platelet activation inhibitor, 1%-3% synthetic phospholipid, 30%-40% oleic acid, and the remainder gelatin.

[0016] The platelets are of mouse origin and are extracted from fresh mouse plasma.

[0017] In order to achieve the second objective, the present invention adopts a technical solution of loading nanoparticles loaded with platelet activation inhibitors onto the outside of platelets. The preparation method comprises the following steps:

[0018] (1) preparing nanoparticles encapsulating platelet activation inhibitors;

[0019] (2) Extraction of platelets;

[0020] (3) Coupling nanoparticles to the platelet surface.

[0021] The nanoparticles described in step (1) are prepared by an emulsified solvent evaporation method. Specifically, ticagrelor, oleic acid, and synthetic phospholipids are dissolved in dichloromethane to prepare an organic phase, which is then transferred to a gelatin aqueous solution at a volume ratio of 1:2 to 1:4. Nanoparticles are prepared by ultrasonication in an ice bath. The prepared nanoparticles are spherical and have a particle size of 70-110 nm. The organic solvent is then evaporated by a suspension evaporation method and set aside.

[0022] In step (2), fresh blood anticoagulated with sodium citrate is centrifuged to obtain platelet-rich plasma, and the platelet-rich plasma is mixed with equal volumes of blood preservation solution ACD, centrifuged at 800 g for 10 minutes to obtain pure platelets, which are then placed in Ren's solution containing prostacyclin for later use.

[0023] In step (3), the nanoparticles prepared in step (1) are first mixed with an excess of Sulfo-SMCC in PBS and stirred at room temperature -37°C for 0.5 hours. The nanoparticles are purified using an ultrafiltration tube and washed three times in PBS to remove excess Sulfo-SMCC. The Sulfo-SMCC-coupled nanoparticles are then incubated with the platelets extracted in step (2) in Ren's solution for 0.5-1 hour. The platelets are centrifuged at 800g for 10 minutes and washed three times in Ren's solution to remove uncoupled nanogels.

[0024] Engineered platelets loaded with platelet activation inhibitor nanoparticles were successfully prepared.

[0025] To achieve the third purpose mentioned above, the technical solution adopted by the present invention is: to prepare an engineered platelet carrying platelet activation inhibitor nanoparticles, and to investigate its application in assisting nanomedicine or immune checkpoint inhibitors in anti-tumor treatment.

[0026] The application of the engineered platelets carrying platelet activation inhibitor nanoparticles in the preparation of a drug delivery system.

[0027] The use of the engineered platelets carrying platelet activation inhibitor nanoparticles in the preparation of drugs used in combination with anti-PD-L1, anti-PD-1, and anti-CTLA-4 immune checkpoint inhibitors.

[0028] The engineered platelets carrying platelet activation inhibitor nanoparticles are used in the preparation of drugs used in combination with adriamycin liposomes and paclitaxel albumin nanoparticles and nano-chemotherapeutic drugs.

[0029] Specifically, the present invention prepared engineered platelets loaded with platelet activation inhibitor nanoparticles (PTNPs) and investigated their properties. Engineered platelets loaded with blank nanoparticles (PNPs), platelet activation inhibitor-loaded nanoparticles (TNPs), and a platelet activation inhibitor solution (Ticagrelor) were also prepared. The detailed investigations are as follows:

[0030] 1) The physicochemical properties of engineered platelets loaded with platelet activation inhibitor nanoparticles were characterized, such as the particle size of the nanoparticles, the morphology of the functionalized platelets, the adhesion efficiency, protein expression, and drug release.

[0031] 2) The targeting ability of engineered platelets loaded with platelet activation inhibitor nanoparticles in melanoma-bearing mice was investigated.

[0032] 3) The ability of engineered platelets loaded with platelet activation inhibitor nanoparticles to enhance the efficacy of anti-tumor immunotherapy was investigated.

[0033] 4) The ability of engineered platelets loaded with platelet activation inhibitor nanoparticles to enhance the anti-tumor efficacy of nanomedicines was investigated.

[0034] The results showed that the engineered platelets loaded with platelet activation inhibitor nanoparticles prepared by the present invention can target tumor-associated platelets and inhibit the activation of remaining platelets, destroy the tumor vascular endothelial barrier, alleviate the immunosuppressive microenvironment, and promote the anti-tumor effects of nanomedicines and immune checkpoint inhibitors.

[0035] Beneficial effects of the present invention:

[0036] The engineered platelets loaded with platelet activation inhibitor nanoparticles provided by the present invention can target platelets associated with tumor sites. Matrix metalloproteinases released by activated platelets specifically degrade the loaded nanoparticles, releasing the platelet activation inhibitor and inhibiting platelet activation. This reduces PD-L1 expression on the platelet surface, thereby alleviating the tumor's immunosuppressive microenvironment, maintaining tumor vascular incompleteness, enhancing nanodrug penetration efficiency, and promoting the anti-tumor effects of immune checkpoint inhibitors and nano-chemotherapeutic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the preparation of PTNPs;

[0038] Nanoparticles (TNPs) containing platelet activation inhibitors were prepared by the emulsification solvent evaporation method and then cross-linked to the platelet surface via Sulfo-SMCC.

[0039] Figure 2 For the characterization of PTNPs;

[0040] A is the transmission electron microscopy characterization of nanoparticles (TNPs) containing platelet activation inhibitors, the scale bar is 100 nm;

[0041] B is a western-blot analysis of key proteins in engineered platelets;

[0042] C is the transmission electron microscopy characterization of engineered platelets, scale bar: 1 μm, scale bar of the magnified part: 100 nm;

[0043] D is the scanning electron microscopy characterization of engineered platelets, scale bar: 300 nm;

[0044] E, confocal microscopy analysis of engineered platelets;

[0045] F is a picture of the appearance of engineered platelets;

[0046] G is the retention of nanoparticles on the platelet surface;

[0047] H is the quantitative analysis of Figure G;

[0048] I is the in vitro release of ticagrelor under different conditions;

[0049] J shows the adhesion of platelets to B16-F10 cells after treatment with different preparations.

[0050] Figure 3 To investigate the ability of PTNPs to target tumor sites in vivo;

[0051] A is the in vivo fluorescence imaging of fluorescently labeled engineered platelets injected into the tail vein at different times;

[0052] B is the in vitro fluorescence imaging of major tissues and tumors;

[0053] C is the semi-quantitative fluorescence intensity analysis of major tissues and tumors.

[0054] Figure 4 To analyze how PTNPs reduce the expression of tumor-derived PD-L1 on the platelet surface and enhance the efficacy of anti-tumor immunotherapy;

[0055] A is immunofluorescence staining of PD-L1 on the platelet surface;

[0056] B is the flow cytometric analysis of the relative fluorescence intensity of PD-L1 on the platelet surface;

[0057] C is the ELISA analysis of PD-L1 on the platelet surface of tumor-bearing mice;

[0058] D is a schematic diagram of drug administration and treatment of B16-F10 tumor-bearing mice;

[0059] E is the mouse weight curve;

[0060] F is the mouse tumor volume curve;

[0061] G is the tumor weight of mice on the tenth day.

[0062] Figure 5 This is the anti-tumor immune response in mice after combined administration of PTNPs and aPD-L1;

[0063] A is the flow cytometry image analysis of the changes in the number of CD8 positive T cells and its quantitative analysis of different drug administration types;

[0064] B is the flow cytometry image analysis of the changes in the number of CD4-positive Foxp3-positive T cells and its quantitative analysis of different drug administration types;

[0065] C is the flow cytometry image analysis of the changes in the number of M1-like macrophages and its quantitative analysis of different drug administration types;

[0066] D is the flow cytometry image analysis of the changes in the number of M2-like macrophages caused by different drug administration types and its quantitative analysis.

[0067] Figure 6 To analyze how PTNPs enhance the tumor accumulation of doxil liposomes and enhance their chemotherapy efficacy;

[0068] A shows the in vivo fluorescence imaging at different times after injection of different preparations into the tail vein followed by injection of fluorescently labeled Doxil;

[0069] B is the in vitro fluorescence imaging of major tissues and tumors;

[0070] C is the semi-quantitative fluorescence intensity analysis of major tissues and tumors;

[0071] D is the accumulation of Doxil in the tumor;

[0072] E is a schematic diagram of drug administration and treatment of 4T1 tumor-bearing mice;

[0073] F is the mouse tumor volume curve;

[0074] G is the tumor weight of mice on day 11;

[0075] H is the concentration of doxorubicin in the tumor;

[0076] I is the mouse body weight curve.

[0077] Figure 7 The following are the hematological parameters of mice after treatment with different drugs: alanine aminotransferase (ALT); aspartate aminotransferase (AST); blood urea nitrogen (BUN); and creatinine (CREA). DETAILED DESCRIPTION

[0078] The present invention is further described below by way of examples, but the invention is not limited to the scope of the examples.

[0079] Example 1: Preparation and characterization of engineered platelets (PTNPs) carrying platelet activation inhibitor nanoparticles.

[0080] Synthesis diagram see Figure 1 .

[0081] The nanoparticles serving as backpacks were prepared using an emulsification solvent evaporation method. Specifically, 10 mg of ticagrelor, 5 mg of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 100 μL of oleic acid were dissolved in 1 mL of dichloromethane to prepare an organic phase. This phase was slowly added dropwise to a 5% gelatin solution and sonicated at 450 W for 3 minutes in an ice bath. The resulting emulsion was rotary evaporated to remove the organic solvent and incubated with an excess of Sulfo-SMCC (molar ratio of gelatin:Sulfo-SMCC = 1:15). After 30 minutes, the excess linker was removed by ultrafiltration.

[0082] Fresh mouse blood was anticoagulated with sodium citrate and centrifuged at 200 g for 4 minutes to remove red blood cells. The supernatant was mixed with an equal amount of ACD solution and centrifuged at 800 g for 10 minutes. Platelets were resuspended in Ringer's solution containing prostacyclin and set aside.

[0083] The Sulfo-SMCC-coupled nanoparticles were incubated with platelets for 1 hour, centrifuged at 800 g for 10 minutes, and washed three times with Ringer's solution to remove unloaded nanoparticles, thereby obtaining engineered platelets loaded with platelet activation inhibitor nanoparticles (PTNPs).

[0084] Transmission electron microscopy ( Figure 2 A) shows that the prepared nanoparticles loaded with platelet activation inhibitors are uniformly spherical with a diameter of about 70-110 nanometers. When each platelet is loaded with 10 nanoparticles, i.e., 14.98 picograms of ticagrelor, membrane proteins that regulate platelet adhesion and aggregation, such as CD41, CD61, and P-selectin, are still expressed on the platelet surface ( Figure 2 B). Transmission electron microscopy ( Figure 2 C) and scanning electron microscopy ( Figure 2 D) The image further confirms the successful preparation of engineered platelets. In addition, the morphology of platelets did not change significantly after the attachment of nanoparticles, indicating that the modification of nanoparticles does not induce platelet activation. This property is conducive to the targeted aggregation of engineered platelets and the specific release of ticagrelor. Confocal microscopy ( Figure 2E) further demonstrated the successful binding of nanoparticles to platelets, and the engineered platelets prepared showed a uniform milky white texture ( Figure 2 F). In addition, when engineered platelets were incubated with mouse plasma, more than 85% of the nanoparticles remained anchored on the platelet surface within 24 hours ( Figure 2 G, H), demonstrating that platelet-borne nanoparticles can stably accompany platelets to the tumor site without off-target effects. Activated platelets can secrete matrix metalloproteinases, which can specifically degrade gelatin. When the nanoparticles were co-incubated with the supernatant of activated platelets, the nanoparticles rapidly stratified, with 53.2% of ticagrelor released within 2 hours. However, when the nanoparticles were co-incubated with the supernatant of resting platelets, very little ticagrelor was released ( Figure 2 I) It was demonstrated that activated platelets can specifically trigger the release of nanoparticles.

[0085] Tumor-secreted substances can activate platelets, and activated platelets can adhere to the surface of tumor cells, while resting platelets rarely adhere. To explore the interaction between tumor cells and engineered platelets, we used engineered platelets to pretreat tumor cells for 2 hours and then added DiR-labeled resting platelets. We found that the newly introduced platelets hardly adhered to the surface of tumor cells ( Figure 2 J), this is because tumor-secreted substances activate engineered platelets, and the matrix metalloproteinases secreted by them cause the rapid release of ticagrelor, thereby inhibiting the activation of newly added platelets.

[0086] Example 2: Targeting ability of engineered platelets loaded with platelet activation inhibitor nanoparticles (PTNPs) to tumor-associated platelets.

[0087] To investigate the ability of PTNPs to target tumor-associated platelets (tumor sites) in vivo, we injected melanoma-bearing mice with fluorescent dye DiR-labeled PTNPs, DiR-labeled TNPs, and a mixed solution of DiR and ticagrelor. In vivo imaging was performed at different time points, such as Figure 3 As shown in A, DiR-labeled PTNPs showed stronger fluorescence signals at the tumor site. In contrast, DiR-labeled TNPs and free solution were mainly distributed in the liver and lungs, and accumulated less in the tumor ( Figure 3 B, C), demonstrating that PTNPs can specifically deliver ticagrelor to tumor sites.

[0088] Example 3: PTNPs reduce the expression of tumor-derived PD-L1 on the platelet surface and enhance the anti-tumor immune response triggered by aPD-L1.

[0089] PD-L1 protein can interact with PD-1 protein on the surface of T cells, triggering immune escape. It has been reported that platelets can interact with tumor cells and express PD-L1 on their surface, a process that can be weakened by inhibiting platelet activation. To evaluate the effect of PTNPs on the expression of PD-L1 on the platelet surface, we incubated PTNPs, TNPs, ticagrelor solution, and PBS with B16-F10 tumor cells for 2 hours, then added new platelets and incubated for 1 hour, observing the expression of PD-L1 on the surface of the newly added platelets, and used untreated platelets as a control. Figure 4 As shown in A, platelets treated with PBS and TNPs showed higher green fluorescence intensity. Flow cytometry analysis showed ( Figure 4 B), the fluorescence intensity of PTNPs-treated platelets was similar to that of normal platelets, indicating that PTNPs can reduce the expression of PD-L1 on the platelet surface by inhibiting platelet activation. We further verified this finding in mice. Mice bearing B16-F10 melanoma were treated with saline, TNPs, ticagrelor, and PTNPs. After 8 hours of administration, platelets were extracted from the blood and the expression of PD-L1 on their surface was analyzed. Compared with the saline control group, the expression of PD-L1 on the platelet surface of mice treated with PTNPs was reduced by 3.2 times ( Figure 4 C), which further confirmed that PTNPs can reduce the expression of PD-L1 on the platelet surface, suggesting that PTNPs may improve the therapeutic effect of aPD-L1 monoclonal antibody.

[0090] We then further evaluated the anti-tumor activity of aPD-L1 in B16-F10 mice. Figure 4 As shown in D, seven days after tumor inoculation, the tumor volume reached about 50 mm 3 The mice were divided into six groups and injected with normal saline, PTNPs, aPD-L1, TNPs&aPD-L1 (TNPs and aPD-L1 were injected separately, 8 hours apart), ticagrelor&aPD-L1 (ticagrelor and aPD-L1 were injected separately, 8 hours apart), and PTNPs&aPD-L1 (PTNPs and aPD-L1 were injected separately, 8 hours apart) into the tail vein every other day. Within ten days, the mice receiving ticagrelor&aPD-L1 lost weight slightly, while the weight of mice in the other groups increased steadily ( Figure 4 E) Compared with other groups, the PTNPs&aPD-L1 group showed lower tumor growth rate and tumor volume, indicating the effective anti-tumor effect of the combination of PTNPs and aPD-L1 ( Figure 4 F, G).

[0091] To confirm the immunotherapeutic effect of PTNPs&aPD-L1, we analyzed tumor-infiltrating lymphocytes and tumor-associated macrophages extracted from tumors by flow cytometry. + Effector T cells showed a moderate increase in both PTNPs (G2) and aPD-L1 (G3) treatments, indicating that strategies to reduce platelet surface PD-L1 expression and aPD-L1 can moderately activate T cell-mediated immune responses ( Figure 5 A), it is noteworthy that PTNPs&aPD-L1 triggered a more effective immune response, CD8 + Effector T cells increased by 2.31 times, and suppressor T cells decreased by 0.39 times ( Figure 5 A, B). In addition, compared with the control group, the number of pro-inflammatory M1 macrophages increased significantly and the number of M2 macrophages decreased significantly after PTNPs&aPD-L1 treatment ( Figure 5 Figures C and D demonstrate the potent immune response induced by the combination of PTNPs and aPD-L1. Overall, PTNPs can inhibit the activation of tumor-associated platelets, reduce the expression of PD-L1 on their surface, and alleviate immunosuppression. Combined with an aPD-L1 monoclonal antibody, they exhibit potent anti-tumor effects.

[0092] Example 5: Investigating the effect of PTNPs on regulating the tumor retention of Doxil (commercially available doxorubicin liposomes) and enhancing its chemotherapy effect.

[0093] Defects in the tumor vasculature allow nanoparticles to penetrate and remain in the tumor interstitium, however, activated platelets make a significant contribution to maintaining the integrity of tumor blood vessels, thereby limiting the tumor perfusion of nanomedicines. In order to evaluate the ability of PTNPs to enhance the penetration and retention of nanomedicines, eight hours after the infusion of saline, TNPs, ticagrelor, and PTNPs, DiR-labeled Doxil was infused into the tail vein, and the fluorescence distribution was observed at specific time points. Figure 6 As shown in AC, the fluorescence signal at the tumor site of mice treated with PTNPs gradually increased within 24 hours, while in the TNPs or ticagrelor groups, the fluorescence intensity at the tumor site reached a peak after 12 hours, indicating that Doxil remained at the tumor site for a longer time. This may be due to the strong PTNPs recruitment ability of platelets. PTNPs continuously target and release ticagrelor to inhibit platelet activation at the tumor site and maintain tumor vascular-specific leakage for a long time, which contributes to the accumulation of Doxil at the tumor site. We then analyzed the distribution of Doxil in the tumor by immunofluorescence, as shown in Figure 5. Figure 6 As shown in D, the red fluorescence of the saline & Doxil group was negligible, while PTNPs & Doxil showed the most efficient Doxil penetration, confirming that PTNPs can indeed promote the accumulation and retention of Doxil in the tumor site.

[0094] To determine whether PTNPs can enhance the efficacy of chemotherapy, we evaluated the antitumor activity of PTNPs and Doxil in mice bearing 4T1 tumors (breast cancer). 3 At the same time, normal saline, PTNPs, Doxil, TNPs & Doxil (TNPs and Doxil were injected separately, 8 hours apart), ticagrelor & Doxil (ticagrelor and Doxil were injected separately, 8 hours apart), PTNPs & Doxil (PTNPs and Doxil were injected separately, 8 hours apart) were injected into the tail vein every other day to observe the changes in tumor volume ( Figure 6 E). Mice treated with PTNPs & Doxil showed significant tumor growth inhibition, while PTNPs alone only slightly delayed tumor growth ( Figure 6 F, G), which indicates that the main function of PTNPs is to enhance the accumulation of Doxil in the tumor site, and Doxil mainly kills the tumor. We measured the concentration of Doxil in the tumor by fluorescence spectroscopy. The Doxil concentration in the tumor of mice in the PTNPs & Doxil group was 2.06, 2.40, and 5.49 times higher than that in the ticagrelor & Doxil group, the TNPs & Doxil group, and the Doxil group, respectively ( Figure 6 H), confirming the powerful ability of PTNPs to promote Doxil tumor penetration by inhibiting platelet activation. In addition, the body weight of mice in the Ticagrelor & Doxil group was slightly reduced ( Figure 6 I), which may be due to systemic hematological toxicity. We also conducted a hepatotoxicity and renal toxicity assessment of the formulation ( Figure 7 ), the results showed that the levels of alanine transaminase (ALT), aspartate transaminase (AST) and blood urea nitrogen (BUN) in mice in the ticagrelor & Doxil group were slightly increased, indicating the potential toxicity of free drugs, while PTNPs & Doxil had a strong safety profile.

Claims

1. An engineered platelet carrying platelet activation inhibitor nanoparticles, characterized in that: The nanoparticles comprise a platelet carrier and a platelet activation inhibitor-loaded nanoparticle. The nanoparticles are based on gelatin that can be specifically degraded by matrix metalloproteinases, and are stabilized by oleic acid and synthetic phospholipids. The nanoparticles are modified with Sulfo-SMCC and NHS-SS-NHS linkers and are anchored to the outside of the platelet membrane. The particle size of the nanoparticles is 70-110 nm. The platelets are of mouse origin and extracted from fresh mouse plasma. The platelet activation inhibitor is ticagrelor. The weight percentage composition of the components is: 3%-5% platelet activation inhibitor, 1%-3% synthetic phospholipids, 30%-40% oleic acid, and the remainder is gelatin. One platelet is loaded with 10 nanoparticles.

2. The engineered platelet carrying platelet activation inhibitor nanoparticles according to claim 1, characterized in that: The nanoparticles loaded with platelet activation inhibitors are prepared by an emulsification solvent evaporation method, a secondary desolvation method, a nanoprecipitation method or a reverse microemulsion method.

3. The method for preparing engineered platelets carrying platelet activation inhibitor nanoparticles according to any one of claims 1 to 2, characterized in that: The steps include: (1) Preparation of nanoparticles loaded with platelet activation inhibitors; The platelet activation inhibitor ticagrelor, oleic acid, and synthetic phospholipids were dissolved in dichloromethane to prepare an organic phase, which was then transferred to a gelatin aqueous solution at a ratio of 1:2 to 1:

4. Ultrasonication was performed in an ice bath to prepare nanoparticles. The prepared nanoparticles were spherical and had a particle size of 70-110 nm. (2) Extraction of platelets; Fresh blood anticoagulated with sodium citrate is centrifuged to obtain platelet-rich plasma, which is then mixed with equal volumes of blood preservation solution ACD and centrifuged to obtain pure platelets. (3) Coupling the nanoparticles to the platelet surface; mixing the nanoparticles prepared in step (1) with the linker in PBS, incubating at 37°C, and purifying the nanoparticles using an ultrafiltration tube to remove excess linkers; then incubating the nanoparticles with the platelets purified in step (2) in Ren's solution containing prostaglandins, centrifuging, washing, and removing uncoupled nanogels.

4. Use of the engineered platelets carrying platelet activation inhibitor nanoparticles according to any one of claims 1 to 2 in the preparation of a drug delivery system.

5. Use of the engineered platelets carrying platelet activation inhibitor nanoparticles according to any one of claims 1-2 in the preparation of a drug for use in combination with anti-PD-L1.

6. Use of the engineered platelets carrying platelet activation inhibitor nanoparticles according to any one of claims 1 to 2 in the preparation of a drug for use in combination with doxorubicin liposomes.

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