Functionalized platelet-based drug control system as well as preparation method and application thereof

Abstract

The invention discloses a functionalized platelet-based drug control system as well as a preparation method and application thereof, and relates to the technical field of pharmacy. The drug control system comprises a functional platelet base, a chemotherapeutic drug loaded in the functional platelet base, and protein sphere nanoparticles grafted on the surface of the functional platelet base, according to the invention, the reduction responsive functional platelet-based drug controlled release system of degrading enzyme HAase co-loaded with chemotherapeutic drug DOX, immunosuppressor Gal and ECM main component hyaluronic acid is prepared by using the advantages that platelets can be used as natural carriers without cell nucleuses, can escape from phagocytosis of macrophages, can target tumor cells, has good biological safety and the like, and can be used for preparing the reduction responsive functional platelet-based drug controlled release system of degrading enzyme HAase of the chemotherapeutic drug DOX, immunosuppressor Gal and ECM main component hyaluronic acid. The tumor chemotherapy and immune combined treatment is regulated and controlled.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a functionalized platelet-based drug control system and a preparation method and application thereof. Background Art

[0002] The proliferation of malignant tumors seriously endangers human health. As one of the most promising cancer treatments, tumor immunotherapy can restore the body's anti-tumor immune response and efficiently kill tumors. However, its clinical efficacy is severely limited by weak immunogenicity and suppressive immune microenvironment. Based on the fact that chemotherapeutic drugs and immunosuppressants can induce tumor immunogenic death (ICD), enhance immunogenicity, and suppress immune microenvironment, chemotherapy combined with immunotherapy strategy has significant advantages in the treatment of malignant tumors. For example, the chemotherapeutic drug doxorubicin (DOX) can effectively induce tumor ICD, transform "cold" tumors into "hot" tumors, enhance immunogenicity, and improve the body's anti-tumor immune response, but there is a restriction of chemotherapy resistance. The immunosuppressant Galunisertib is a TGF-β inhibitor. By inhibiting TGF-β signaling, it relieves the immunosuppression of TGF-β on immune cells in the tumor microenvironment, such as T cells, macrophages, neutrophils, etc., and blocks the growth and spread of tumor cells, but its clinical administration is limited by off-target effects and toxic side effects. Furthermore, DOX acts in the cell nucleus and should be accurately delivered to tumor cells to enhance the killing effect while avoiding damage to immune effector cells; while Gal should be accurately delivered to the extracellular matrix of tumor cells to effectively suppress immune tolerance. In addition, the abnormally dense extracellular matrix (ECM) barrier is another obstacle to the effectiveness of drug delivery. Therefore, how to overcome the constraints of graded drug delivery and dense ECM screen, and safely and effectively deliver the chemical drug DOX and the immunosuppressant Gal into tumor cells and the extracellular matrix, respectively, has important scientific significance and potential application prospects.

[0003] With the rapid development of nanotechnology, functionalized autologous cell drug delivery systems have received extensive attention in recent years. Among them, platelets can be specifically recruited and activated by tumor cells through the interaction of intercellular adhesion molecules, helping tumor cells to escape the attack of the body's immune system. At the same time, the integrin P-selectin highly expressed on the activated platelet membrane can specifically bind to the tumor cell surface antigen CD44, which is conducive to targeted uptake. However, in order to safely and efficiently overcome the dense ECM barrier and improve the bioavailability of drugs, and to responsively release loaded drugs in stages at the tumor lesions and activate tumor chemotherapy and immunotherapy, it is necessary to perform necessary functional modifications on the platelet base so that it has the characteristics of tumor penetration, graded release and in vivo degradability, thereby achieving efficient tumor killing. Summary of the invention

[0004] In view of the deficiencies in the above-mentioned background technology, the present invention provides a functionalized platelet-based drug control system and its preparation method and application. The system can load biological enzymes and chemotherapy and immunotherapy drugs with high fidelity, and overcome the dense ECM barrier in the body, deliver drugs to the corresponding sites of action in a graded manner, improve drug delivery efficiency and bioavailability, and help the drug control system to combine tumor chemotherapy and immunotherapy, effectively kill tumors and inhibit tumor growth and metastasis. The present invention constructs a functionalized platelet-based drug control system with a reduction response that enhances tumor penetration and graded drug release characteristics, co-loads DOX and Gal, combines chemotherapy and immunotherapy strategies, effectively kills tumors and inhibits tumor growth and metastasis, and provides a scientific basis and practical reference for the construction of a functionalized platelet-based drug control system and chemotherapy and immunotherapy combined therapy.

[0005] The first object of the present invention is to provide a functionalized platelet-based drug control system, the drug control system comprising a functionalized platelet-based, a chemotherapy drug loaded inside the functionalized platelet-based, and protein sphere nanoparticles grafted onto the surface of the functionalized platelet-based; The protein ball nanoparticles are cross-linked into spheres by NHS-SS-NHS and HAase, and immunosuppressants are loaded inside the spheres.

[0006] Preferably, the grafting rate of the protein sphere nanoparticles is 12-15%; and the loading rate of the chemotherapy drug is 4-8%.

[0007] Preferably, the protein sphere nanoparticles are prepared according to the following steps: HAase was dissolved in PBS solution to obtain solution A; NHS-SS-NHS was dissolved in DMSO to obtain solution B. After solution A and solution B were mixed, immunosuppressants were added, and after mixing evenly, the mixture was centrifuged and washed to obtain protein ball nanoparticles.

[0008] Preferably, the molar mass ratio of the HAase to NHS-SS-NHS is 1:10-20; and the mass ratio of the immunosuppressant to the functionalized platelet base is 0.3-0.7:1.

[0009] Preferably, the drug control system is spherical, and the particle size is 1.0-2.0 μm; the particle size of the protein spherical nanoparticles is 100-200 nm.

[0010] The second object of the present invention is to provide a method for preparing a functionalized platelet-based drug control system, comprising the following steps: Extract the functionalized platelet matrix; load the chemotherapy drug into the functionalized platelet matrix to obtain the functionalized platelet matrix loaded with the chemotherapy drug; prepare the protein sphere nanoparticles; dissolve the protein sphere nanoparticles and the cross-linking agent in a PBS solution, react at 1-5°C for 2-4 hours to obtain a cross-linked product; mix the cross-linked product with the functionalized platelet matrix loaded with the chemotherapy drug, react at room temperature for 1-2 hours, centrifuge and wash the reactant to obtain the functionalized platelet matrix drug control system.

[0011] Preferably, the functionalized platelet matrix loaded with chemotherapeutic drugs is prepared according to the following steps: The chemotherapy drug is dissolved in Tyrode buffer, a platelet PBS solution is added, and then a PGE1 solution and a triethylamine solution are added to react. The reactant is shaken at 37°C in the dark for 1 to 2 hours; the shaken product is then centrifuged and washed to obtain a functionalized platelet matrix loaded with chemotherapy drugs.

[0012] Preferably, the functionalized platelet matrix is ​​extracted according to the following steps: After the mice were anesthetized, whole blood was extracted from the mice and placed in a centrifuge tube containing the anticoagulant EDTA and mixed. The blood was centrifuged and the supernatant was taken. After the red blood cells were removed, the blood was centrifuged again and the supernatant was taken to obtain platelet-rich plasma. After centrifuging the platelet-rich plasma, discard the supernatant and the resulting precipitate is the functionalized platelet matrix.

[0013] Preferably, the cross-linking agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt.

[0014] The third object of the present invention is to provide a functionalized platelet-based drug control system for use in the preparation of drugs for tumor chemotherapy or combined immunotherapy.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a functionalized platelet-based drug control system and a preparation method and application thereof. With the help of EPR passive targeting and platelet active targeting of tumors, the platelet-based drug control system can efficiently and specifically accumulate in tumor lesions, thereby improving the safety and effectiveness of delivery. The surface of the functionalized platelet-based drug control system prepared by the present invention is grafted with reduction-responsive protein nanospheres synthesized by cross-linking the reduction-sensitive bond NSH-SS-NHS with the ECM regulator-HAase, which can respond to the highly reducing microenvironment outside the tumor cells, rapidly degrade and release the loaded drug in situ, and has good biosafety. The drug control system also has the characteristics of graded drug delivery, can overcome tumor penetration and ECM barriers, and deliver DOX and Gal to corresponding sites of action respectively, thereby improving delivery efficiency. The Pts@DOX / HANGs@Gal platelet-based drug control system prepared by the present invention can enhance the immune response in vivo through combined chemotherapy and immunotherapy. On the one hand, DOX-mediated tumor cell ICD can enhance cellular immunogenicity and immune response; on the other hand, Gal-mediated TGF-β inhibition relieves immune tolerance, and the two jointly cascade amplify the body's anti-tumor immune response, thereby efficiently killing tumors; the Pts@DOX / HANGs@Gal platelet-based drug control system prepared by the present invention achieves personalized tumor chemotherapy and immunotherapy effects under the joint action of the above mechanisms. It not only overcomes off-target effects and improves the safety and effectiveness of delivery, but also overcomes tumor heterogeneity and broadly enhances the anti-tumor immune response level in different patients.

[0016] The present invention utilizes the advantages of platelets that they have no cell nucleus and can be used as natural carriers, can escape macrophage phagocytosis, can target tumor cells, and have good biosafety to prepare a reduction-responsive functionalized platelet-based drug controlled release system that co-loads the chemotherapy drug DOX, the immunosuppressant Gal, and the ECM main component hyaluronic acid degrading enzyme HAase to regulate tumor chemotherapy and immunotherapy. The drug control system can respond to the reductive tumor microenvironment, induce the external protein sphere nanoparticles HANGs@Gal to degrade in the extracellular matrix and release Gal and HAase, and at the same time use the PMP-DOX released by activated platelets to deliver DOX into the tumor cells, achieving precise graded drug delivery and improving drug utilization. More importantly, the chemotherapy and immunotherapy mediated by the drug control system can effectively enhance cellular immunogenicity to suppress immune tolerance, and enhance the body's anti-tumor immune response, thereby effectively inhibiting tumor growth, metastasis and recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The transmission electron microscopy, scanning electron microscopy and particle size distribution diagrams of the products of the present invention are as follows: A is the TEM image of Pts@DOX and its local magnified image; B is the TEM image of HANGs@Gal; C is the TEM image of Pts@DOX / HANGs@Gal and its local magnified image; D is the SEM image of Pts@DOX and Pts@DOX / HANGs@Gal; E is the DLS image of Pts@DOX, HANGs@Gal and Pts@DOX / HANGs@Gal, A scale: 1 μm and 200 nm, B scale: 500 nm, C scale: 500 nm and 200 nm, D scale: 1 μm.

[0018] Figure 2 2 is a transmission electron micrograph of the product of the present invention after activation: wherein A is a TEM image of the activated Pts@DOX and a magnified TEM image of the released DOX-PMPs; B is a schematic diagram of the activation of Pts@DOX, A scale: 1 μm and 200 nm.

[0019] Figure 3 The Western-blot result graph and its quantitative statistical graph and SDS-PAGE electrophoresis Coomassie Brilliant Blue staining result graph of the product of the present invention: wherein A is the WB result graph of Pts, Pts@DOX, Pts@DOX / HANGs, and Pts@DOX / HANGs@Gal; B is the quantitative statistical result graph of A; C is the Coomassie Brilliant Blue staining result graph of Pts, Pts@DOX, Pts@DOX / HANGs, and Pts@DOX / HANGs@Gal.

[0020] Figure 4 These are the DOX UV absorption graph, the relative cell viability graph, the circular dichroism graph and the Elisa graph of the product of the present invention: A is the UV absorption graph of Pts@DOX / HANGs@Gal; B is the relative cell viability graph of Pts@DOX / HANGs@Gal, C is the circular dichroism graph of HAase and HAase released from Pts@DOX / HANGs@Gal; D is the Elisa result graph of HAase and HAase released from Pts@DOX / HANGs@Gal.

[0021] Figure 5 2 are drug release diagrams of the products of the present invention under different conditions: wherein A is the cumulative release diagram of DOX of Pts@DOX and Pts@DOX / HANGs@Gal under pH 7.4; B is the cumulative release diagram of Gal of HANGs@Gal and Pts@DOX / HANGs@Gal nanoparticles under PBS and 1 mM GSH conditions, respectively.

[0022] Figure 6 : It is a cell activity diagram of the Pts@DOX / HANGs@Gal drug control system of the present invention: wherein A is a cell activity diagram of tumor cells treated with PBS, DOX, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal for 24 h and 48 h respectively; B is a cell viability diagram of cells treated with Pts@DOX / HANGs@Gal for different time periods.

[0023] Figure 7 : It is the in vitro tumor penetration diagram of the Pts@DOX / HANGs@Gal drug control system of the present invention: A is the CLSM diagram of tumor multicellular spheroids incubated with the system for 4 h and 12 h respectively with or without Thrombin, and B is the fluorescence quantification statistics; A scale bar: 100 µm.

[0024] Figure 8 The functional platelet drug control system of the present invention is a CLSM image of tumor cell uptake after different treatment times: A is the CLSM image of B16F10 cells incubated with DOX, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal for 4 h and 12 h, respectively, and the cell nucleus and cytoskeleton are stained with DAPI (blue) and ActinGreen, respectively. TM 488 (green) staining; B is the quantitative statistical analysis of FCM under the above treatment conditions; Scale bar in A: 50 µm.

[0025] Fig. 9 CLSM images of targeted adhesion of the product of the present invention to tumor cells: A is the CLSM image of targeted adhesion of Pts and Pts / HANGs@Gal to tumor cells under the conditions of HA pre-treatment and without HA pre-treatment; B is fluorescence quantification statistics; A scale bar: 50 µm.

[0026] Fig.10 CLSM images of the products of the present invention promoting ICD-related proteins in tumor cells: A is a CLSM image of CRT protein induced by treatment with different nanoparticles; B is a CLSM image of HMGB1 protein induced by treatment with different nanoparticles; the cell nucleus is stained with DAPI (blue), the scale bar of A: 40 µm, the scale bar of B: 40 µm.

[0027] Fig.11The in vivo treatment diagram of the functionalized platelet drug-controlled system of the present invention: A is the tumor size of tumor-bearing mice after treatment with normal saline, DOX, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal for different time periods; B is the relative tumor volume change during the drug treatment; C and D are the mouse survival rate curve and body weight change diagram.

[0028] Fig.12 The immunofluorescence analysis diagram of the functionalized platelet drug control system of the present invention: A, B and C are immunofluorescence staining of TUNEL, Ki-67 and TGF-β in tumor tissue, respectively; scale bar: 100 μm.

[0029] Fig.13 The functional platelet drug control system of the present invention regulates the immune response of T cells, DC and NK cells in vivo. A is CD8 + Cytotoxic T cells and CD4 + A is the flow cytometry detection of helper T cells; B is the flow cytometry detection of tumor-infiltrating DC cells; C is the flow cytometry detection of tumor-infiltrating NK cells.

[0030] Fig.14 Figures 2 and 3 are the in vivo treatment pathological analysis and biosafety test diagrams of the functionalized platelet drug-controlled system of the present invention: A is the blood routine and liver and kidney function results of mice after drug treatment; B is the H&E staining results of the main organs of tumor-bearing mice after treatment with normal saline, DOX, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal; Scale bar: 150 µm.

[0031] Fig.15 It is an in vivo anti-tumor metastasis diagram of the platelet-based drug control system of the present invention: wherein A and B are lung tumor metastasis nodules and quantitative statistical diagrams of B16-F10 malignant tumor lung metastasis model mice in each drug treatment group.

[0032] Fig.16 This is the in vivo drug distribution of the functionalized platelet drug control system of the present invention: wherein A is the distribution of DOX in various organs and tumors after treatment in different treatment groups; and B is the related fluorescence quantitative statistics of A.

[0033] Fig.17 It is a schematic diagram of the Pts@DOX / HANGs@Gal drug control system of the present invention combined with chemotherapy and immunotherapy in vivo.

[0034] Fig.18 Schematic diagram of the preparation process of functionalized platelet-based drug control system. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0036] The purpose of the present invention is to achieve both safe and efficient overcoming of the dense ECM barrier, improving the bioavailability of drugs, and responsively releasing loaded drugs in stages at the tumor lesions to activate tumor chemotherapy and immunotherapy. It is necessary to perform necessary functional modifications on the platelet base so that it has the characteristics of tumor penetration, graded release and in vivo degradability, thereby achieving efficient tumor killing. Based on this, the present invention constructs a functionalized platelet-based drug control system with a reduction response that enhances tumor penetration and drug graded release characteristics, co-loads DOX and Gal, combines chemotherapy and immunotherapy strategies, effectively kills tumors and inhibits tumor growth and metastasis, and provides a scientific basis and practical reference for the construction of a functionalized platelet-based drug control system and chemotherapy and immunotherapy.

[0037] In order to achieve the above-mentioned object, the first aspect of the present invention provides a functionalized platelet-based drug control system, the drug control system comprising a functionalized platelet-based (Pts), a chemotherapy drug loaded inside the functionalized platelet-based, and protein sphere nanoparticles (HANGs@Gal) grafted on the surface of the functionalized platelet-based; The protein ball nanoparticles are cross-linked into spheres by NHS-SS-NHS and HAase, and immunosuppressants are loaded inside the spheres.

[0038] The grafting rate of the protein ball nanoparticles is 12-15%, preferably 13.82%; the loading rate of the chemotherapy drug is 4-8%, preferably 5.62%. The chemotherapy drug includes doxorubicin, carmustine, nimustine or dactinomycin D.

[0039] The protein ball nanoparticles are prepared according to the following steps: HAase was dissolved in PBS solution to obtain solution A; NHS-SS-NHS was dissolved in DMSO to obtain solution B. After solution A and solution B were mixed, immunosuppressants were added, and after mixing evenly, the mixture was centrifuged and washed to obtain protein ball nanoparticles.

[0040] The molar mass ratio of the HAase to NHS-SS-NHS is 1:10-20, preferably 1:15.

[0041] The mass ratio of the immunosuppressant to the functionalized platelet base is 0.3-0.7:1, and the preferred dosage of the immunosuppressant is 0.5 mg of the immunosuppressant for every 1 mg of platelets.

[0042] The drug control system is spherical, and the particle size is 1.0-2.0 μm; the particle size of the protein spherical nanoparticles is 100-200 nm.

[0043] In one embodiment, a functionalized platelet-based drug control system includes an internally functionalized platelet base, wherein the functionalized platelet base is loaded with a chemotherapeutic drug (DOX), and the surface of the functionalized platelet base is functionalized with grafted reduction-responsive protein sphere nanoparticles (HANGs@Gal).

[0044] The functionalized platelet base is Pts, and the functionalized platelet base is DOX-loaded platelets; The protein ball nanoparticles (HANGs@Gal) contain biological enzyme HAase and immunosuppressant Gal. Specifically, the protein ball nanoparticles HANGs@Gal are cross-linked into balls by NHS-SS-NHS and HAase, and are loaded with immunosuppressant Gal.

[0045] The protein ball nanoparticles are spherical and have a particle size of 150 nm.

[0046] The functionalized platelet matrix is ​​spherical and has a particle size of 1.7 μm.

[0047] The second aspect of the present invention provides a method for preparing a functionalized platelet-based drug control system, comprising the following steps: Extracting functionalized platelet matrix; loading the chemotherapeutic drug into the functionalized platelet matrix to obtain a functionalized platelet matrix loaded with the chemotherapeutic drug; preparing protein spherical nanoparticles; The protein ball nanoparticles and the cross-linking agent were dissolved in a PBS solution and reacted at 1-5°C for 2-4 h to obtain a cross-linked product; The cross-linked product is mixed with the functionalized platelet-based material loaded with chemotherapeutic drugs and reacted at room temperature for 1 to 2 hours. The reactants are centrifuged and washed to obtain the functionalized platelet-based drug control system.

[0048] The functionalized platelet matrix loaded with chemotherapeutic drugs is prepared according to the following steps: The chemotherapy drug is dissolved in Tyrode buffer, a platelet PBS solution is added, and then a PGE1 solution and a triethylamine solution are added to react. The reactant is shaken at 37°C in the dark for 1 to 2 hours; the shaken product is then centrifuged and washed to obtain a functionalized platelet matrix loaded with chemotherapy drugs.

[0049] Functionalized platelet matrix was extracted according to the following steps: After the mice were anesthetized, whole blood was extracted from the mice and placed in a centrifuge tube containing the anticoagulant EDTA and mixed. The blood was centrifuged and the supernatant was taken. After the red blood cells were removed, the blood was centrifuged again and the supernatant was taken to obtain platelet-rich plasma. After centrifuging the platelet-rich plasma, discard the supernatant and the resulting precipitate is the functionalized platelet matrix.

[0050] The cross-linking agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt.

[0051] In one embodiment, see Fig.18 As shown, a method for preparing a functionalized platelet-based drug control system, the specific preparation steps include: (1) extracting platelets required for core functionalized platelet matrix; (2) Preparation of platelet-based Pts@DOX loaded with chemotherapeutic drug DOX; (3) Synthesis of reduction-responsive protein spherical nanoparticles HANGs@Gal; (4) Preparation of Pts@DOX / HANGs@Gal drug control system.

[0052] In step (1), platelets required for the core functionalized platelet matrix are extracted: after anesthetizing a mouse, whole blood of the mouse is extracted and placed in a centrifuge tube containing an anticoagulant EDTA and mixed; the whole blood is centrifuged at 200 g for 20 minutes, the supernatant is taken, and red blood cells are removed; the supernatant is centrifuged again at 200 g for 20 minutes, the supernatant is taken, and platelet-rich plasma is obtained; the platelet-rich plasma is centrifuged at 800 g for 20 minutes, the supernatant is discarded, and the resulting precipitate is platelets (Pts); In step (2), the preparation of Pts@DOX: first weigh DOX and dissolve it in Tyrode buffer, then add the PBS solution of platelets; at the same time, add 1 μM PGE1 solution and triethylamine solution to the reactants to prevent platelet activation and remove the hydrochloride in DOX hydrochloride; then shake the reactants at 100 rpm in the dark at 37°C for 1 hour; then, centrifuge the resulting product at 800 g for 20 minutes at room temperature to obtain the precipitate; wash with fresh ACD solution 2-3 times to obtain Pts@DOX; In step (3), reduction-responsive protein ball nanoparticles HANGs@Gal are synthesized: HAase is weighed and dissolved in a PBS solution of pH 7.4, and NHS-SS-NHS is weighed and dissolved in a small amount of DMSO; the two solutions are mixed, and Gal is weighed and added, and the reaction is stirred at room temperature for 30 minutes; then, the resulting solution is placed in an ultrafiltration centrifuge tube and centrifuged at 14000g for 15 minutes at room temperature; the product is washed three times with PBS solution using the above method to obtain HANGs@Gal.

[0053] In step (4), the preparation of the Pts@DOX / HANGs@Gal drug control system: HANGs@Gal obtained in step (3) and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt (Sulfo-SMCC) are dissolved in a PBS solution with a pH of 7.4 and reacted at 4°C for 2 h; the obtained product is centrifuged at a speed of 14000g for 20 minutes in an ultrafiltration centrifuge tube to remove excess SMCC linker; the obtained product is then mixed with the Pts@DOX obtained in step (2) and reacted at room temperature for 1 hour; the reaction solution is centrifuged at a speed of 800g for 20 minutes at room temperature to remove excess HANGs@Gal; the above steps are repeated for washing three times to obtain a functionalized platelet-based drug control system, recorded as Pts@DOX / HANGs@Gal.

[0054] The third aspect of the present invention provides an application of a functionalized platelet-based drug control system in the preparation of drugs for tumor chemotherapy or combined immunotherapy.

[0055] The present invention uses gradient centrifugation to separate platelet cells from the extracted mouse whole blood, and uses mixed incubation technology to load DOX to prepare platelet-based Pts@DOX loaded with DOX. Subsequently, the reduction-responsive protein crosslinker-succinimidyl ester-SS-succinimidyl ester (NSH-SS-NHS) is cross-linked with the ECM regulator-HAase to prepare the reduction-responsive HAase nanoprotein sphere HANGs@Gal loaded with the immunosuppressant Gal, which is chemically grafted on the platelet-based surface to prepare a functionalized platelet-based drug control system. The functionalized platelet-based drug control system prepared by the present invention integrates the advantages of high drug loading, easy surface modification, good biosafety, and reduction-responsive degradability. It is easy to prepare and store, and has potential applications in bioenzyme and drug delivery and tumor treatment.

[0056] This system can safely and efficiently load the chemotherapy drug DOX and the immunosuppressant Gal, and target tumor lesions with the help of the EPR effect. Subsequently, in response to the reductive stimulation of the tumor microenvironment, the cross-linker NSH-SS-NHS bridged by the reduction-sensitive bond is broken, and the nanoprotein sphere HANGs@Gal is rapidly degraded, releasing the HAase and Gal loaded thereon. HAase, as a degrading enzyme for HA, the main component of the extracellular matrix, effectively degrades HA to make the dense ECM barrier loose, so that the drug can more easily penetrate deep into the tumor, improving the efficiency of drug delivery in vivo. More importantly, Gal is accurately delivered to the extracellular matrix of tumor cells to effectively inhibit immune tolerance. At the same time, the functionalized platelet base targets and adheres to tumor cells through the receptor-ligand binding principle, and accurately delivers DOX into tumor cells to exert its effect. On the one hand, the released DOX can not only kill tumor cells, but also improve tumor immunogenicity and recruit a large number of immune cells to invade tumor lesions. On the other hand, the released immunosuppressive Gal inhibits TGF-β signal transduction, relieves the immunosuppression of TGF-β on immune cells in the tumor microenvironment, such as T cells, macrophages, neutrophils and other cells, thereby relieving immune tolerance. The tumor chemotherapy and immunotherapy combined with the functional platelet-based drug control system not only effectively relieves the body's immune tolerance, but also efficiently enhances the body's anti-tumor immune response, synergistically improves the effect of tumor treatment, and has good biosafety and potential clinical medical prospects.

[0057] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified; the reagents and materials used are all commercially available unless otherwise specified.

[0058] Example 1 A functionalized platelet-based drug control system (Pts@DOX / HANGs@Gal) is a controlled release system prepared by using platelets loaded with the chemotherapy drug DOX as the core, functionalizing the surface of platelets with grafted reduction-responsive HANGs@Gal protein sphere nanoparticles, and co-loading Gal and HAase.

[0059] The preparation process of the functionalized platelet-based drug control system Pts@DOX / HANGs@Gal includes the following steps: (1) Preparation of platelets with the chemotherapeutic drug DOX loaded in the inner core: 11) Platelet extraction: Grab the mouse, turn its head downward, and fix it with bare hands, while using the fixed hands to gently press both sides of the neck to make the eyeballs bulge out with blood; use ophthalmic curved forceps to quickly clamp the base of the eyeball and remove the eyeball; place the EP tube containing anticoagulant under the eye socket to collect blood to obtain the whole blood of the mouse; centrifuge the whole blood of the mouse at 200g for 20 minutes, and collect the supernatant as PRP; collect PRP and centrifuge it again at 200g for 20 minutes to further remove excess red blood cells; collect the supernatant PRP and suspend it in ACD solution containing 1μM prostaglandin E1 (PGE1) and centrifuge it at 800g for 20 minutes, discard the supernatant, and the precipitate is pure platelets.

[0060] 12) Preparation of Pts@DOX: First, 5 mg of doxorubicin hydrochloride (DOX·HCL) and 2 mg of pure platelets were weighed and dissolved in Tyrode buffer containing 1 μM PGE1; secondly, in order to increase the drug loading rate, triethylamine was added to the mixture of DOX and platelets (the molar ratio was triethylamine:DOX=3:1); then, the above mixture was placed in a 37°C shaker for one hour to obtain Pts@DOX; after that, Pts@DOX was centrifuged at 800g for 20 minutes to obtain a precipitate, which was Pts@DOX. The product was washed three times with fresh ACD solution to remove free DOX; the product was collected by centrifugation and then dispersed in PBS solution for use. The obtained DOX-loaded platelets were named Pts@DOX.

[0061] (2) Surface functionalization grafting of the above core: 21) Synthesis of HANGs@Gal: First, 1.19 mg HAase was weighed and dissolved in PBS to obtain a PBS solution of HAase; secondly, 10 mg NHS-SS-NHS was weighed and added to the PBS solution of HAase; at the same time, 1 mg Gal was weighed and dissolved in about 30 μl of DMSO solution and added to the above solution; after the above mixture was stirred for reaction for 30 minutes, the reacted solution was placed in an ultrafiltration centrifuge tube (molecular retention capacity = 100 kDa), and centrifuged at 14000 g for 20 minutes to obtain a precipitate; the precipitate was resuspended in PBS to obtain the final product, protein ball nanoparticles, namely HANGs@Gal.

[0062] 22) Modification of Sulfo-SMCC: Weigh 1.19 mg Sulfo-SMCC and add it to the above reaction system, continue stirring and reacting for 30 minutes; then place the solution after the reaction in an ultrafiltration centrifuge tube (molecular cutoff = 100 kDa), centrifuge at 14000g for 20 minutes to obtain a precipitate; resuspend the precipitate in PBS, and wash the product with PBS three times according to the above centrifugation method to remove excess NHS-SS-NHS and Sulfo-SMCC. The final cross-linked product is resuspended in PBS solution, which is Sulfo-SMCC-modified HANGs@Gal.

[0063] (3) Preparation of Pts@DOX / HANGs@Gal particles The products Pts@DOX and Sulfo-SMCC-modified HANGs@Gal obtained by (1) and (2) were added to Tyrode buffer containing 1 μM PGE1 and reacted for 1 hour in the dark. After the reaction, the product was centrifuged at 800 g for 20 minutes, and the precipitate was washed three times by the above centrifugation method. Finally, the precipitate was resuspended in physiological saline containing 1 μM PGE1. The final product, a functionalized platelet-based drug control system, was obtained, namely Pts@DOX / HANGs@Gal.

[0064] The products obtained in each step of Example 1 were systematically studied for their morphology, composition and biological activity using modern nano-testing and analysis techniques such as TEM, SEM, Western-blot, Coomassie Brilliant Blue staining, DLS and Zeta potential analyzer, CD, Elisa, etc. The results are as follows: The present invention characterized the Pts@DOX, HANGs@Gal and Pts@DOX / HANGs@Gal obtained in the examples by transmission electron microscopy, scanning electron microscopy and dynamic light scattering. Figure 1 As shown in Figure A, Pts@DOX has a spherical structure with a size of about 1.6 μm, and as shown in its enlarged image, the edges of Pts@DOX are clear without small particles grafted. Figure 1 Figure B is the transmission electron microscopy result of HANGs@Gal. HANGs@Gal is spherical and its size is about 150nm. Figure 1 Figure C is a transmission electron micrograph of Pts@DOX / HANGs@Gal obtained after Pts@DOX was grafted with HANGs@Gal. It is also spherical with a size of about 1.7 μm. The enlarged image shows that nanoparticles are obviously grafted on the edge of Pts. Figure 1Figure D is a pseudo-color SEM image of Pts@DOX and Pts@DOX / HANGs@Gal, where purple is Pts and yellow is HANGs@Gal. As shown in the result figure, compared with Pts@DOX, Pts@DOX / HANGs@Gal has obvious spherical nanoparticles grafted on the platelet surface, indicating the successful grafting of HANGs@Gal. Figure 1 As shown in Figure E, the dynamic light scattering size results of Pts@DOX, HANGs@Gal and Pts@DOX / HANGs@Gal also correspond to their transmission electron microscopy results. The above results confirm that Pts@DOX / HANGs@Gal of appropriate size has been successfully synthesized.

[0065] The present invention verifies the activation performance of Pts@DOX by scanning electron microscopy. After platelets are activated, their morphology changes, and the particles in the platelets fuse with the plasma membrane, allowing them to be released from the open duct system. Figure 2 As shown in A and B, the morphology of activated Pts@DOX changed to an irregular shape, and spherical secretions of DOX-PMPs were released. The above results proved that the activation and release properties of Pts were not affected after modification, and also further confirmed the successful synthesis of Pts@DOX.

[0066] The protein structures of Pts, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal obtained in each step of Example 1 were analyzed by Western-blot and Coomassie Brilliant Blue staining. Figure 3 As shown in Figure A, the expression of Pts' characteristic membrane proteins CD41, CD61 and P-Selectin in Western-blot results are highly consistent; Figure 3 As shown in Figure B, ImageJ software was used to quantify the results, and there was no significant difference in the expression of characteristic proteins; Figure 3 As shown in Figure C, the results of SDS-PAGE staining show that there is no significant difference in the expression of intrinsic proteins of Pts in the Pts, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal groups. The above results collectively indicate that the various modifications of Pts can still maintain the original structure of platelets and do not affect the original function of platelets.

[0067] The present invention uses UV spectrophotometry, CCK-8, CD and Elisa to analyze the biological activities of each part of Pts, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal obtained in each step of Example 1. Figure 4As shown in Figure A, under the condition of no reducing stimulation, after 48 h, the DOX content in Pts@DOX / HANGs@Gal still did not show a significant decrease, which proved that DOX had a good retention ability in the Pts@DOX / HANGs@Gal system for a long time. Figure 4 The results of Figure B show that the Pts@DOX / HANGs@Gal obtained after functional modification still showed good cell viability after 24 hours, proving that the above modification process had little effect on the cell viability of Pts; Figure 4 As shown in Figure C, the HAase released from Pts@DOX / HANGs@Gal and the free HAase showed almost the same circular dichroism spectra, indicating that the secondary structure of HAase in Pts@DOX / HANGs@Gal was well preserved; Figure 4 As shown in Figure D, the HAase released from Pts@DOX / HANGs@Gal and the free HAase showed good HAase bioactivity by enzyme-linked immunosorbent assay (ELISA). These results once again prove that the natural bioactivity of HAase and Pts is not affected after various functional modifications of platelets, laying a good foundation for further functional verification.

[0068] Experimental Example 2 The functionalized platelet-based drug control system Pts@DOX / HANGs@Gal provided in Example 1 has reduction-responsive drug release characteristics.

[0069] The present invention uses GSH to simulate a reductive tumor microenvironment and detects the characteristics of drug release in this system. The specific results are as follows: 1) Release characteristics of DOX The release characteristics of DOX contained in Pts@DOX and Pts@DOX / HANGs@Gal in vivo were investigated by UV-Vis spectroscopy (e.g. Figure 5 As shown in Figure A in the figure, the release of DOX within 48 hours was investigated using a PBS solution containing 4% DMSO to simulate the in vivo environment. The results showed that more than 80% of DOX was released from Pts@DOX and Pts@DOX / HANGs@Gal within 48 hours, confirming the feasibility of drug release within cells.

[0070] 2) Pts@DOX / HANGs@Gal controlled release system restores responsive drug controlled release characteristics The reduction-responsive drug release characteristics of the Pts@DOX / HANGs@Gal controlled release system were investigated by UV-Vis. After incubating the HANGs@Gal and Pts@DOX / HANGs@Gal controlled release systems with different concentrations of GSH, the characteristic peak intensity of Gal showed a significant enhancement, such as Figure 5 As shown in Figure B, the cumulative drug release level under GSH stimulation was quantified and statistically analyzed. After 48 h of incubation under physiological conditions (PBS), the Gal release from HANGs@Gal and Pts@DOX / HANGs@Gal was negligible (less than 28%), indicating that the Pts@DOX / HANGs@Gal controlled release system had good stability. Compared with the control group, when the experimental group was co-incubated with 1mM GSH, more than 80% of Gal was released from the HANGs@Gal and Pts@DOX / HANGs@Gal controlled release systems, which once again confirmed that the Pts@DOX / HANGs@Gal system had reduction-sensitive drug release characteristics. The above results show that the Pts@DOX / HANGs@Gal controlled release system has good and expected drug controlled release characteristics.

[0071] Experimental Example 3 In vitro biological evaluation of the functionalized platelet-based drug control system Pts@DOX / HANGs@Gal provided in Example 1.

[0072] (1) Cell activity Cell culture: The present invention uses mouse melanoma highly metastatic cells (B16F10) provided by the Institute of Cell Biology, Chinese Academy of Sciences. B16F10 cells were cultured in DMEM high-glucose medium containing 10% FBS and 1% double antibody (100 μg / mL penicillin and 100 μg / mL streptomycin) in a cell incubator at 37°C and 5% CO2.

[0073] In order to evaluate the in vitro tumor cell cytotoxicity of the Pts@DOX / HANGs@Gal controlled release system, the present invention firstly used the CCK-8 method to detect the effects of PBS, free DOX, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal treatment on the activity of B16F10 cells. 4 cells / cm 2The cells were inoculated into a 24-well plate at a density of 100 μg / mL. When the cell confluence reached about 60-70%, B16F10 cells were co-cultured with the above drugs for 24 h and 48 h, respectively. The cells were washed with PBS, and then 200 μL of fresh culture medium and 20 μL of CCK8 mixed solution were added. The reaction was continued at 37 °C for 1.5-2 h, and then the reaction solution in the well plate was transferred to a 96-well plate. The absorbance of the solution at a wavelength of 450 nm was detected by an ELISA reader to calculate the cell activity. The results are shown in Figure 6 As shown in Figure A, regardless of the incubation time, the control group showed higher cell activity; free DOX caused slight cell damage; while the Pts@DOX group, Pts@DOX / HANGs group and Pts@DOX / HANGs@Gal group caused more serious cytotoxicity. This is because Pts accurately targets tumor cells, improves the drug uptake by tumor cells, and leads to higher cytotoxicity; this result confirms that the Pts@DOX / HANGs@Gal controlled release system has a good in vitro tumor killing effect. Secondly, the present invention uses the CCK-8 method to detect the effect of different doses of Pts@DOX / HANGs@Gal treatment on the activity of B16F10 cells. The results are shown in Figure 6 As shown in Figure B, with the increase of the dosage, the relative activity of tumor cells showed a downward trend, but when the dosage increased to 10 μg / mL, the cell activity no longer decreased significantly. This result confirmed that the Pts@DOX / HANGs@Gal controlled release system has a good in vitro tumor killing effect and shows a dose-dependent trend within a certain range.

[0074] (2) In vitro tumor penetration and cell uptake: First, a 3D multicellular spheroid (MCS) based on B16F10 cells, i.e., a tumor sphere model, was constructed. 80 μL of 1.5% hot agarose solution was added to a 96-well plate. After it cooled and solidified into a gel, it was sterilized by ultraviolet irradiation overnight. B16F10 cells were then incubated at 2 × 10 3 The initial density of cells / well was inoculated in the above-mentioned well plate, and tumor spheres were formed after 6 days of co-culture. Subsequently, the FITC-labeled Pts@DOX / HANGs@Gal controlled release system was co-incubated with the tumor spheres, and CLSM monitored the MCSs penetration of the controlled release system under the conditions of pre-addition of Thrombin stimulation and without the addition of Thrombin, and investigated the enhanced tumor penetration behavior of the controlled release system after platelet activation. Figure 7As shown in Figure A, without the addition of Thrombin stimulation, after 4 h of incubation, only a very small amount of green fluorescence-labeled HAase was dispersed around the outer boundary of MCSs, and only a very small amount of DOX was taken up into the cells; even if the incubation time was extended to 12 h, it was still consistent with the above situation, the fluorescence intensity of FITC and DOX was slightly enhanced, and the green fluorescence-labeled HAase was still dispersed around the outer boundary of MCSs, and more DOX was internalized into the cells. Under the condition of Thrombin stimulation, after 4 h of incubation, the green fluorescence-labeled HAase was widely distributed in the outer boundary area of ​​​​MCSs, and most of the red fluorescence DOX was internalized into the cells and the fluorescence intensity was stronger than that of the group without Thrombin addition; after 12 h of incubation, the entire MCS sphere dispersed strong red fluorescence, and the green fluorescence was still on the periphery of MCSs, but the fluorescence intensity was significantly enhanced compared with 4 h and the group without Thrombin addition, indicating that more HANGs@Gal disintegrated and released HAase. The profile analysis further confirmed this experimental result (such as Figure 7 The above results together prove that the Pts@DOX / HANGs@Gal controlled release system has a good graded delivery effect.

[0075] In order to further investigate the cellular uptake efficiency, CLSM and FCM were used to monitor the endocytosis level of B16F10 cells to the Pts@DOX / HANGs@Gal controlled release system in real time. 5 cells / cm 2 B16F10 cells were seeded in confocal culture dishes. When the cell confluence reached 60-70%, the cells were treated with free DOX, Pts@DOX, CY3-labeled Pts@DOX / HANGs and CY3-labeled Pts@DOX / HANGs@Gal and incubated for 4 h and 12 h. The nucleus and skeleton of the cells were then stained and analyzed, and the endocytosis efficiency was quantitatively detected by flow cytometry. Figure 8 As shown in Figures A and B, compared with the free DOX group, the concentration and intensity of DOX endocytosed by B16F10 in the Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal groups were significantly higher than those in the free DOX group; this is consistent with the results of FCM quantitative analysis. This is because Pts precisely targets tumor cells, allowing DOX to be more efficiently taken up by tumor cells, further proving the excellent drug delivery effect of Pts@DOX / HANGs@Gal.

[0076] (3) In vitro tumor cell targeting: In order to study the in vitro tumor targeting effect of the Pts@DOX / HANGs@Gal delivery system, B16-F10 cells were used to verify the targeting effect of Pts@DOX / HANGs@Gal on them. 5 cells / cm 2 The density of cells was inoculated into the confocal dish. When the cell confluence reached about 60-70%, HA solution was added to the control group in advance to block the CD44 receptor. The experimental group did not receive any treatment. B16-F10 cells were co-cultured with Dil-labeled Pts and Pts / HANGs@Gal for 6 h, and then the cells were fixed, the cell nuclei were stained, and CLSM images were collected. The results are shown in Fig. 9 As shown in Figure A, in the experimental group that was not blocked by HA, Dil-labeled Pts and Pts / HANGs@Gal were distributed and targeted on the surface of B16-F10 cells, proving that Pts and Pts-based delivery systems have good adhesion and targeting to tumor cells; and after blocking the CD44 receptors on the surface of tumor cells with HA solution in advance, the Pts and Pts / HANGs@Gal targeted distribution around them were significantly reduced, proving that the targeting of the Pts@DOX / HANGs@Gal delivery system is based on the interaction between CD44 receptors and P-selectin. After quantitative analysis of the corresponding fluorescence (such as Fig. 9 The above results show that the functional modification of platelets did not affect their targeting effect on tumor cells, which further proves the excellent targeting ability of Pts@DOX / HANGs@Gal delivery system on tumor cells.

[0077] (4) Pts@DOX / HANGs@Gal enhances tumor cell ICD The chemotherapy drug DOX can enhance the ICD of tumor cells. In order to study the in vitro enhancement effect of the Pts@DOX / HANGs@Gal controlled release system on the ICD of tumor cells, the expression levels of ICD-related proteins CRT and HMGB1 in tumor cells were investigated by CLSM. B16F10 cells seeded in confocal dishes were treated with PBS, DOX, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal for 24 h, and then immunofluorescence staining of tumor cell-related proteins was performed, and the cell nuclei were labeled with DAPI. Fig.10As shown in Figure A, compared with the PBS group and the free DOX group, the green fluorescence of tumor cells in the Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal groups was significantly enhanced after treatment, indicating that the expression of CRT proteins on the cell surface increased. These CRT proteins can bind to receptors on the surface of some innate immune cells and enhance the immune system's response to these cells. High mobility group protein B1 (HMGB1) is a highly conserved nuclear protein, such as Fig.10 As shown in Figure B, the nuclear green fluorescence of the free DOX group was slightly lower than that of the PBS group, while the green fluorescence of the tumor cell nuclei in the Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal groups was significantly reduced after treatment, proving that the HMGB1 protein migrated outside the cells, mediated stronger immunogenic death and enhanced anti-tumor immunogenicity. The above results jointly prove that the precise delivery of DOX by the Pts@DOX / HANGs@Gal drug control system effectively enhances the immunogenic death of tumor cells and enhances their immunogenicity, and also further proves the good targeting ability of Pts@DOX / HANGs@Gal for tumor cells and higher drug delivery efficiency.

[0078] Experimental Example 4 In vivo anti-tumor evaluation of the functionalized platelet-based drug control system Pts@DOX / HANGs@Gal provided in Example 1.

[0079] (1) Construction of tumor-bearing mouse model: All animal experiments in the present invention were conducted in accordance with the relevant provisions of the Experimental Animal Care and Use Committee Work Manual. Female C57BL / 6 mice weighing about 20 g, aged 5 to 6 weeks, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., China. The present invention used B16F10 cells in the exponential growth phase to prepare 100 μL of cell suspension, which was subcutaneously inoculated into the right groin of each mouse to establish a tumor-bearing mouse model. The health status and behavior of the mice were observed every day, and the weight and tumor volume of the tumor-bearing mice were recorded every 2 days. The tumor volume (V) was calculated according to the following formula: V = L×S 2 / 2 (L, long diameter of the tumor; S, short diameter of the tumor). (2) Drug intervention: The successfully constructed tumor-bearing mice were randomly divided into five groups according to the principle of weight, and intravenous injection of normal saline, DOX, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal was given twice a week for 20 days. After the end of the administration, the mice were euthanized. The heart, liver, spleen, lung, kidney and tumor of the mice were collected and histopathological analysis was performed by H&E, TUNEL and Ki-67 staining.

[0080] After the mice were killed, the main organs and tumor tissues were collected. Fig.11 As shown in Figure A, the tumor size in the free DOX group was slightly smaller than that in the saline group, and the tumor showed a relatively rapid growth trend during the administration period, indicating that the inhibitory effect of free DOX on tumor growth was relatively limited; the Pts@DOX group and the Pts@DOX / HANGs group showed slight and moderate inhibitory effects on tumor growth, respectively. Among all the treatment groups, the Pts@DOX / HANGs@Gal group induced the most significant tumor growth inhibition, and the B16-F10 malignant tumor almost disappeared after 18 days of treatment, indicating that the Pts@DOX / HANGs@Gal-mediated tumor chemotherapy and immunotherapy combined therapy can effectively kill tumors. Fig.11 The tumor volume statistics in Figure B confirmed the same tumor growth inhibition trend. In addition, compared with other treatment groups, the Pts@DOX / HANGs@Gal group not only significantly prolonged the survival time of tumor-bearing mice (50-day survival rate was 67%), Fig.11 C, which was significantly higher than that of other treatment groups), and the weight of tumor-bearing mice did not decrease during the administration period (e.g. Fig.11 (as shown in Figure D), which indicates that the Pts@DOX / HANGs@Gal controlled release system has good biosafety and excellent anti-tumor effect.

[0081] (3) Histopathological analysis: The results of histopathological analysis are as follows: Fig.12 As shown in Figure 3, compared with the control group, the free DOX group induced mild tumor tissue apoptosis, while the Pts@DOX group and the Pts@DOX / HANGs group induced moderate and higher degrees of tumor tissue apoptosis; the Pts@DOX / HANGs@Gal group induced the most severe tumor tissue apoptosis, which was manifested by a large number of magenta fluorescent spots marking DNA damage in the TUNEL map (such as Fig.12 In addition, the results of Ki-67 immunofluorescence staining showed that (as shown in Figure A) Fig.12 As shown in Figure B), compared with the free DOX group, the Pts@DOX group more effectively downregulated the expression of Ki-67 in tumor cells, and the expression of Ki-67 in the Pts@DOX / HANGs group further decreased, among which the Pts@DOX / HANGs@Gal group showed the weakest expression of Ki-67. The results of TGF-β immunofluorescence staining showed (as shown in Figure Fig.12 As shown in Figure C, compared with the saline group, free DOX group, Pts@DOX group and Pts@DOX / HANGs group, the fluorescence intensity of TGF-β in the tissue of the Pts@DOX / HANGs@Gal group was significantly reduced after treatment, and the expression of TGF-β was more effectively inhibited. The above results once again confirmed that the Pts@DOX / HANGs@Gal drug control system has a good anti-tumor effect.

[0082] (3) In vivo immune response detection: The present invention uses FCM to quantitatively analyze the proportion of various immune cells infiltrating the tumor site to verify the in vivo anti-tumor immune response effect of the chemotherapy-immunotherapy mediated by the Pts@DOX / HANGs@Gal drug control system. The specific experimental process is as follows: After drug administration, the mice are euthanized, and the tumor tissues are collected and lysed in a shaker at 37°C using a lysis solution consisting of 0.2% collagenase IV, 0.1% hyaluronidase V, 0.002% DNase I and DMEM culture medium for 1 h. The lysed solution is then passed through a 75 μM filter to separate monodispersed lymphocytes, collected by centrifugation, and washed with a washing solution, and the cells are resuspended in a PBS solution. Next, the cells are stained with Live / Dead dye in the dark for 20 min.

[0083] 1) For tumor infiltrating CD4 + and CD8 + T cell detection: The above collected cells were incubated with anti-CD3-APC / Cy7, anti-CD4-FITC and anti-CD8- PerCP-Cy5.5 at 4°C for 30 min, and then FCM was used to detect tumor infiltrating CD4 + and CD8 + T cell levels; 2) Detection of tumor-infiltrating mature DC cells: The above collected cells were co-stained with anti-CD11b-FITC, anti-CD80-APC and anti-CD86-PE for 30 min, and DC cells (CD11b + CD80 + CD86 + ) level; 3) Detection of tumor-infiltrating NK cells: The above collected cells were co-stained with anti-CD3-APC / Cy7 and anti-CD49b-PE / Cy7 for 30 min, and NK cells (CD3 - CD49b + ) level; the results are as follows: 1) T cell immune response: The immunosuppressant Gal can relieve the inhibition of TGF-β on immune active cells by inhibiting TGF-β receptors, thereby enhancing tumor infiltration of CD8 + Cytotoxic T cells and CD4 + The activity of helper T cells, and thus the level of tumor-infiltrating CD8+ cytotoxic T cells and CD4+ helper T cells, can most directly reflect the anti-tumor immune response. Therefore, the present invention firstly uses FCM to quantitatively characterize the CD8 +Toxic T cells, CD4 + The levels of helper T cells. As shown in Fig.13 Figure A in + , the proportion of tumor-infiltrating CD8 cytotoxic T cells (CTLs) in the control group was low; the number of CTLs in the free DOX group increased moderately; while the trend in the Pts@DOX group was significantly higher than that in the free DOX group, thanks to the excellent drug delivery efficiency after Pts precisely targeted tumor cells; and the trend in the Pts@DOX / HANGs group was higher than that in the Pts@DOX group because, while the Pts@DOX / HANGs group had excellent drug delivery efficiency after Pts precisely targeted tumor cells, the HAase released after the degradation of HANGs loosened the dense ECM, facilitating the recruitment and infiltration of a large number of tumor-infiltrating CD8 + and contributing to the restoration of the activity of effector T cells. Additionally, among all treatment groups, Pts@DOX / HANGs@Gal showed the highest CTL infiltration ratio, indicating that the Pts@DOX / HANGs@Gal controlled-release system could effectively activate the immune response, enhance the anti-tumor immune response, and inhibit immune tolerance. This is because, on the basis of the precise delivery of the chemotherapeutic drug DOX by Pts and the action of the extracellular matrix main component degrading enzyme HAase, the immunosuppressant Gal was introduced, further enhancing the anti-tumor immune response of the body.

[0084] 2) Immune responses of DC cells and NK cells: The present invention further reveals the anti-tumor immune response enhancement mechanism of the Pts@DOX / HANGs@Gal drug control system by examining the maturation degree of DC cells. As shown in Fig.13 Figure B in Fig.13 , both the free DOX and the experimental groups loaded with DOX (Pts@DOX, Pts@DOX / HANGs, and Pts@DOX / HANGs@Gal) promoted the maturation of DC cells to varying degrees, and the order of their maturation degrees was: free DOX < Pts@DOX < Pts@DOX / HANGs < Pts@DOX / HANGs@Gal, indicating that the Pts@DOX / HANGs@Gal drug control system successfully promoted the maturation of DC cells. Additionally, the Pts@DOX / HANGs@Gal drug control system also successfully recruited tumor-infiltrating NK cells (as shown in + Figure C in +Intratumoral recruitment of T cells; in addition, the Pts@DOX / HANGs@Gal drug control system relieves immune tolerance by inhibiting TGF-β receptors, while HAase degrades the extracellular matrix to establish a highway for the recruitment of immune cells, further enhancing the body's anti-tumor immune response.

[0085] (4) Biosafety analysis: First, the blood glucose levels of mice were tested before and after intravenous injection of the Pts@DOX / HANGs@Gal drug control system. Fig.14 As shown in Figure A, compared with the control group, all treatment groups did not induce a sustained decrease in peripheral blood glucose, indicating that the controlled release system has good blood safety. Secondly, we tested the levels of liver function indicators aspartate aminotransferase AST and alanine aminotransferase ALT in the blood after injection of different drugs according to the kit to evaluate liver function or liver damage. The results are shown in Figure 1. Fig.14 As shown in Figure A, the concentrations of the two enzymes did not change significantly, and there was no significant difference from the control group, which indicates that the Pts@DOX / HANGs@Gal drug control system has good biosafety for liver tissue. In addition, the H&E analysis results of major organs showed that all treatment groups using Pts as a carrier did not cause damage to the body's organs (such as Fig.14 This once again confirms that the Pts@DOX / HANGs@Gal drug control system has good biosafety in vivo.

[0086] (5) Anti-tumor metastasis: The present invention constructs a lung metastasis model to further investigate the anti-tumor metastasis effect of the Pts@DOX / HANGs@Gal drug control system. 100 μL of 1×10 6 PBS solution of B16F10 tumor cells. After 7 days, the mice were randomly divided into 5 groups and treated with different medications. After the 10th day, the medication was terminated and the mice were euthanized. The lung tissues of the mice were collected, washed with PBS and photographed with a camera. The number of lung metastatic nodules was calculated to evaluate the anti-tumor metastasis efficiency of the controlled release system. The lung tissues of the mice were then preserved in 10% formalin solution and analyzed by H&E staining. The results are shown in Fig.15 As shown in Figure A, it can be observed that among all treatment groups, there are only a few tumor metastatic nodules in the lung tissue of the Pts@DOX / HANGs@Gal group, and the quantitative statistics are statistically significant (e.g. Fig.15 (Figure B). This indicates that the Pts@DOX / HANGs@Gal drug control system significantly and efficiently inhibits tumor metastasis. In contrast, in other drug treatment groups, there were tumor metastatic nodules in the lung tissues of mice to varying degrees. This result shows that the Pts@DOX / HANGs@Gal system has a significant anti-tumor metastasis effect.

[0087] (6) Drug distribution in vivo: The present invention constructs a mouse B16-F10 melanoma subcutaneous tumor model. When the tumor volume reaches 50 mm 3 At the same time, the mice were divided into two groups and injected with free DOX and Pts@DOX / HANGs@Gal through the tail vein. Six hours after the injection, the mice were euthanized, and the main organs (heart, liver, spleen, lung and kidney) and tumors of the mice in each group were dissected and collected into culture dishes. The drug distribution was observed based on the DOX fluorescence signal using a small animal in vivo imaging system. The results are shown in Figure 2. Fig.16 As shown in Figure A, the free DOX group had the largest drug accumulation in the liver, followed by the kidney, and less drug accumulation in the tumor site, while the Pts@DOX / HANGs@Gal group had less drug accumulation in the liver and kidney tissues, and a large amount of drug accumulated in the tumor site. The fluorescence quantitative analysis results also correspond to this result (such as Fig.16 The above results together prove that Pts@DOX / HANGs@Gal has a good tumor targeting distribution effect in vivo, and once again show that Pts@DOX / HANGs@Gal has good biosafety for major tissues and organs.

[0088] Fig.17 Schematic diagram of the Pts@DOX / HANGs@Gal drug control system provided in Example 1 combined with chemotherapy and immunotherapy in vivo. Fig.17 As shown in the figure, the Pts@DOX / HANGs@Gal drug control system penetrates from blood vessels into tumor tissues through the EPR effect, and targets and adheres to tumor cells based on the principle of receptor-ligand interaction through the P-selectin receptor on the surface of activated platelets and the CD44 receptor on the surface of tumor cells. At the same time, the protein nanospheres HANGs@Gal grafted on the surface of platelets degrade in response to GSH and release the immunosuppressant Gal and matrix degrading enzyme HAase to the outside of tumor cells, which are used to relieve the extracellular inhibitory immune microenvironment and degrade the dense extracellular matrix, respectively, opening up a highway for drug penetration and infiltration of immune effector cells; DOX is delivered into tumor cells through membrane fusion and platelet secretion of DOX-PMPs to act on the nucleus of tumor cells, causing tumor cell ICD and enhancing its immunogenicity.

[0089] Fig.18This is the preparation process of the Pts@DOX / HANGs@Gal drug control system provided in Example 1. As shown in the figure, first, extract whole blood from mice, obtain platelet-rich plasma after multiple centrifugation, obtain pure platelets from mice after centrifugation again, and obtain DOX-loaded platelets after co-incubation with platelets and DOX, namely Pts@DOX; at the same time, HAase and NHS-SS-NHS bridging reaction are added with immunosuppressant Gal to react together, and obtain Gal-loaded protein nanospheres, namely HANGs@Gal; finally, Sulfo-SMCC is used to graft protein nanospheres onto the surface of platelets, and the final product, platelet-based drug control system Pts@DOX / HANGs@Gal, is obtained.

[0090] Example 5 A functionalized platelet-based drug control system (Pts@Car / HANGs@Gal) is a controlled release system prepared by using platelets loaded with the chemotherapy drug Carmustine as the core, functionalizing and grafting reduction-responsive HANGs@Gal protein sphere nanoparticles on the surface, and co-loading Gal and HAase. The preparation process is the same as that of Example 1.

[0091] Example 6 A functionalized platelet-based drug control system (Pts@Nim / HANGs@Gal) is a controlled release system prepared by using platelets loaded with the chemotherapy drug Nimustine as the core, functionalizing and grafting reduction-responsive HANGs@Gal protein sphere nanoparticles on the surface, and co-loading Gal and HAase. The preparation process is the same as that of Example 1.

[0092] Example 7 A functionalized platelet-based drug control system (Pts@Dac / HANGs@Gal) is a controlled release system prepared by using platelets loaded with the chemotherapy drug Dactinomycin D as the core, functionalizing and grafting reduction-responsive HANGs@Gal protein sphere nanoparticles on the surface, and co-loading Gal and HAase. The preparation process is the same as that of Example 1.

[0093] In summary, the present invention utilizes the advantages of platelets that have no cell nucleus and can be used as natural carriers, can escape macrophage phagocytosis, can target tumor cells, and have good biosafety to prepare a reduction-responsive functionalized platelet-based drug controlled release system that co-loads the chemotherapy drug DOX, the immunosuppressant Gal, and the ECM main component hyaluronic acid degrading enzyme HAase to regulate tumor chemotherapy and immunotherapy. The nanosystem can respond to the reductive tumor microenvironment and induce the regulation of tumor chemotherapy and immunotherapy. The drug control system can respond to the reductive tumor microenvironment, induce the external protein sphere nanoparticles HANGs@Gal to degrade in the extracellular matrix and release Gal and HAase, and at the same time use the PMP-DOX released by activated platelets to deliver DOX into the tumor cells, achieve precise graded drug delivery, and improve drug utilization. In addition, the present invention has the performance of enhancing tumor immune response: on the one hand, the chemotherapy drug DOX can induce tumor cell ICD and enhance the immunogenicity of tumor cells; on the other hand, HAase is released into the extracellular matrix, which can degrade the main component of the extracellular matrix HA, making the dense ECM loose, and opening a highway for the infiltration of immune cells; in addition, the immunosuppressant Gal inhibits TGF-β signal transduction, relieves TGF-β from immunosuppression of immune cells in the tumor microenvironment, such as T cells, macrophages, neutrophils and other cells, and enhances anti-tumor immune response. The combination of the three effectively enhances the body's anti-tumor immune response, inhibits tumor growth and metastasis, and achieves better therapeutic effects.

[0094] This application solves the following problems: (1) Solve the problem of low bioavailability of biological enzymes due to easy inactivation during in vivo delivery: Based on the advantages of platelets themselves, a functionalized platelet-based drug control system is fitted to efficiently load biological enzymes, giving the enzymes restricted reaction space, while maintaining high-fidelity enzyme activity and improving their bioavailability.

[0095] (2) Solve the problems of therapeutic drugs not being accurately delivered to their reaction sites and low utilization efficiency in the body: DOX mainly acts on the nucleus of tumor cells and should be delivered to the inside of tumor cells to better play a tumor-killing role; HAase’s main function is to degrade HA, the main component of the extracellular matrix, and should be released in the extracellular matrix to better play its role; similarly, Gal should be delivered to the extracellular matrix to inhibit TGF-β signal transduction, relieve TGF-β’s immunosuppression of immune cells in the tumor microenvironment, and enhance the anti-tumor immune response. Therefore, we intend to construct a functional platelet-based Pts@DOX / HANGs@Gal drug control system, load the chemotherapy drug DOX inside the platelets, graft the reduction-responsive degradable protein nanospheres HANGs@Gal on the outside, and co-load the immunosuppressant Gal and the enzyme HAase, so as to construct a functional platelet-based Pts@DOX / HANGs@Gal drug control system; on the one hand, the system responds to the reductive stimulation of the tumor microenvironment, breaks the bridging molecule NHS-SS-NHS in the externally grafted protein nanospheres, and then degrades the protein nanospheres in the extracellular matrix and releases Gal and HAase, thereby improving the drug utilization efficiency. On the other hand, after the platelets are activated, they target and adhere to tumor cells, and accurately deliver DOX into the tumor cells in the form of DOX-PMPs. In summary, this system overcomes many physiological barriers, successfully realizes the hierarchical delivery of drugs, and achieves more efficient drug utilization.

[0096] (3) Solve the problem of unsatisfactory tumor immunotherapy effect caused by immune tolerance: The chemotherapy drug DOX can induce tumor immunogenic death, enhance its immunogenicity, recruit a large number of immune cells to invade tumor lesions, improve tumor immunogenicity and enhance the body's immune response; HAase is a degrading enzyme of HA, the main component of the extracellular matrix, which can degrade the extracellular matrix and make the dense ECM loose. On the one hand, loose ECM is more conducive to drug delivery, and more importantly, loose ECM is more conducive to the recruitment and infiltration of immune effector cells, achieving a stronger anti-tumor immune response; the immunosuppressant Gal can inhibit TGF-β signal transduction, relieve TGF-β from the immunosuppression of immune cells in the tumor microenvironment, such as T cells, macrophages, neutrophils, etc., block the growth and spread of tumor cells, and enhance the body's anti-tumor immune response. Therefore, this study plans to construct a functional platelet-based drug control system to safely and efficiently deliver DOX and Gal to tumor lesions, achieve chemotherapy and immunotherapy, inhibit the body's immune tolerance, and enhance the body's anti-tumor immune response, thereby improving the effect of tumor immunotherapy.

[0097] The present invention describes preferred embodiments and their effects. However, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0098] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A functionalized platelet-based drug control system, characterized in that: The drug control system includes a functionalized platelet base, a chemotherapy drug loaded inside the functionalized platelet base, and protein sphere nanoparticles grafted on the surface of the functionalized platelet base; The protein ball nanoparticles are cross-linked into spheres by NHS-SS-NHS and HAase, and immunosuppressants are loaded inside the spheres.

2. The functionalized platelet-based drug control system according to claim 1, characterized in that: The grafting rate of the protein ball nanoparticles is 12-15%; the loading rate of the chemotherapy drug is 4-8%.

3. The functionalized platelet-based drug control system according to claim 1, characterized in that: The protein ball nanoparticles are prepared according to the following steps: HAase was dissolved in PBS solution to obtain solution A; NHS-SS-NHS was dissolved in DMSO to obtain solution B. After solution A and solution B were mixed, immunosuppressants were added, and after mixing evenly, the mixture was centrifuged and washed to obtain protein ball nanoparticles.

4. The functionalized platelet-based drug control system according to claim 3, characterized in that: The molar mass ratio of the HAase to NHS-SS-NHS is 1:10-20; the mass ratio of the immunosuppressant to the functionalized platelet base is 0.3-0.7:

1.

5. The functionalized platelet-based drug control system according to claim 1, characterized in that: The drug control system is spherical, and the particle size is 1.0-2.0 μm; the particle size of the protein spherical nanoparticles is 100-200 nm.

6. A method for preparing the functionalized platelet-based drug control system according to any one of claims 1 to 5, characterized in that: The following steps are involved: Extracting functionalized platelet matrix; loading the chemotherapeutic drug into the functionalized platelet matrix to obtain a functionalized platelet matrix loaded with the chemotherapeutic drug; preparing protein spherical nanoparticles; The protein ball nanoparticles and the cross-linking agent are dissolved in a PBS solution and reacted at 1-5°C for 2-4 hours to obtain a cross-linked product; The cross-linked product is mixed with the functionalized platelet-based material loaded with chemotherapeutic drugs and reacted at room temperature for 1 to 2 hours. The reactants are centrifuged and washed to obtain the functionalized platelet-based drug control system.

7. The method for preparing the functionalized platelet-based drug control system according to claim 6, characterized in that: The functionalized platelet matrix loaded with chemotherapeutic drugs is prepared according to the following steps: The chemotherapy drug is dissolved in Tyrode buffer, a platelet PBS solution is added, and then a PGE1 solution and a triethylamine solution are added to react. The reactants are shaken at 37°C in the dark for 1 to 2 hours. The shaken product is then centrifuged and washed to obtain a functionalized platelet matrix loaded with chemotherapy drugs.

8. The method for preparing the functionalized platelet-based drug control system according to claim 6, characterized in that: Functionalized platelet matrix was extracted according to the following steps: After the mice were anesthetized, whole blood was extracted from the mice and placed in a centrifuge tube containing the anticoagulant EDTA and mixed. The blood was centrifuged and the supernatant was taken. After the red blood cells were removed, the blood was centrifuged again and the supernatant was taken to obtain platelet-rich plasma. After centrifuging the platelet-rich plasma, discard the supernatant and the resulting precipitate is the functionalized platelet matrix.

9. The method for preparing the functionalized platelet-based drug control system according to claim 6, characterized in that: The cross-linking agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt.

10. Use of the functionalized platelet-based drug control system according to any one of claims 1 to 5 in the preparation of drugs for tumor chemotherapy or combined immunotherapy.

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