A functionalized platelet-based drug control system and its preparation method and application

Abstract

The present invention discloses a functionalized platelet-based drug control system, its preparation method, and application, relating to the field of pharmaceutical technology. The drug control system comprises a functionalized platelet matrix, a chemotherapy drug loaded within the functionalized platelet matrix, and protein sphere nanoparticles grafted onto the surface of the functionalized platelet matrix. The present invention utilizes the advantages of platelets, such as their lack of nuclei, ability to serve as natural carriers, ability to evade macrophage phagocytosis, ability to target tumor cells, and good biosafety, to prepare a reduction-responsive functionalized platelet-based drug controlled-release system co-loaded with the chemotherapy drug DOX, the immunosuppressant Gal, and the enzyme HAase, a degrading enzyme for hyaluronic acid, a major component of ECM, to regulate combined chemotherapy and immunotherapy for tumors.

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] Malignant tumor proliferation poses a serious threat to human health. As one of the most promising cancer treatments, tumor immunotherapy can restore the body's antitumor immune response and effectively kill tumors. However, its clinical efficacy is severely limited by weak immunogenicity and a suppressive immune microenvironment. Chemotherapy combined with immunotherapy offers significant advantages in the treatment of malignant tumors, given that chemotherapeutic drugs and immunosuppressants can, respectively, induce tumor immunogenic death (ICD), enhance immunogenicity, and suppress the immune microenvironment. For example, the chemotherapeutic drug doxorubicin (DOX) can effectively induce tumor ICD, transforming "cold" tumors into "hot" tumors, enhancing immunogenicity and boosting the body's antitumor immune response. However, this strategy is limited by the risk of chemoresistance. Galunisertib, an immunosuppressant, is a TGF-β inhibitor. By inhibiting TGF-β signaling, it relieves the immunosuppressive effects of TGF-β on immune cells in the tumor microenvironment, such as T cells, macrophages, and neutrophils, thereby blocking tumor cell growth and spread. However, its clinical administration is limited by off-target effects and toxic side effects. Furthermore, DOX, which acts in the cell nucleus, must be precisely delivered to tumor cells to enhance its killing effect while avoiding damage to immune effector cells. Gal, on the other hand, must be precisely delivered to the extracellular matrix of tumor cells to effectively suppress immune tolerance. Furthermore, the unusually dense extracellular matrix (ECM) barrier is another obstacle to the effectiveness of drug delivery. Therefore, overcoming the constraints of graded drug delivery and dense ECM screening to safely and effectively deliver the chemical drug DOX and the immunosuppressant Gal into tumor cells and the extracellular matrix, respectively, is of great scientific significance and potential application prospects.

[0003] With the rapid development of nanotechnology, functionalized autologous cell drug delivery systems have received widespread attention in recent years. Platelets can be specifically recruited and activated by tumor cells through interactions between intercellular adhesion molecules, helping tumor cells evade the body's immune system. At the same time, the highly expressed integrin P-selectin on the activated platelet membrane can specifically bind to the tumor cell surface antigen CD44, facilitating targeted uptake. However, in order to safely and efficiently overcome the dense ECM barrier, improve drug bioavailability, and responsively release loaded drugs in a graded manner at the tumor lesion to activate combined chemotherapy and immunotherapy, it is necessary to perform necessary functional modifications on the platelet base to enable tumor penetration, graded release, and in vivo degradability, thereby achieving efficient tumor killing. Summary of the Invention

[0004] In response to 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, 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 be combined with tumor chemotherapy and immunotherapy to effectively kill tumors and inhibit tumor growth and metastasis. The present invention constructs a reduction-responsive functionalized platelet-based drug control system with enhanced 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 treatment.

[0005] The first object of the present invention is to provide a functionalized platelet-based drug control system, which comprises a functionalized platelet-based matrix, a chemotherapy drug loaded inside the functionalized platelet-based matrix, and protein sphere nanoparticles grafted onto the surface of the functionalized platelet-based matrix;

[0006] The protein ball nanoparticles are cross-linked into spheres by NHS-SS-NHS and HAase, and the inside of the spheres is loaded with immunosuppressants.

[0007] Preferably, the grafting rate of the protein sphere nanoparticles is 12-15%; and the loading rate of the chemotherapy drug is 4-8%.

[0008] Preferably, the protein spherical nanoparticles are prepared according to the following steps:

[0009] HAase was dissolved in PBS solution to obtain solution A; NHS-SS-NHS was dissolved in DMSO to obtain solution B. After mixing solution A and solution B, immunosuppressants were added, mixed evenly, and then centrifuged and washed to obtain protein ball nanoparticles.

[0010] 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.

[0011] Preferably, the drug control system is spherical and has a particle size of 1.0-2.0 μm; the particle size of the protein spherical nanoparticles is 100-200 nm.

[0012] 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:

[0013] Extracting the functionalized platelet matrix; loading the functionalized platelet matrix with chemotherapy drugs to obtain a functionalized platelet matrix loaded with chemotherapy drugs; preparing protein sphere nanoparticles; dissolving the protein sphere nanoparticles and a cross-linking agent in a PBS solution, reacting at 1-5°C for 2-4 hours to obtain a cross-linked product; mixing the cross-linked product with the functionalized platelet matrix loaded with chemotherapy drugs, reacting at room temperature for 1-2 hours, and centrifuging and washing the reactants to obtain a functionalized platelet-based drug control system.

[0014] Preferably, the functionalized platelet matrix loaded with chemotherapy drugs is prepared according to the following steps:

[0015] 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 in the dark at 37°C for 1 to 2 hours; the shaken product is then centrifuged and washed to obtain a functionalized platelet matrix loaded with chemotherapy drugs.

[0016] Preferably, the functionalized platelet matrix is ​​extracted according to the following steps:

[0017] After the mice were anesthetized, whole blood was extracted from the mice and placed in a centrifuge tube containing the anticoagulant EDTA. The blood was mixed and centrifuged to remove the red blood cells. The supernatant was then centrifuged again to obtain platelet-rich plasma.

[0018] After centrifuging the platelet-rich plasma, the supernatant is discarded, and the resulting precipitate is the functionalized platelet matrix.

[0019] Preferably, the cross-linking agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt.

[0020] The third object of the present invention is to provide an application of a functionalized platelet-based drug control system in the preparation of drugs for tumor chemotherapy or combined immunotherapy.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a functionalized platelet-based drug control system, its preparation method, and application. By means 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. The nanospheres can respond to the highly reducing microenvironment outside the tumor cells, rapidly degrade, and release the loaded drugs in situ, with 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 the 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 ICD of tumor cells can enhance cellular immunogenicity and immune response; on the other hand, Gal-mediated TGF-β inhibition can relieve immune tolerance. The two work together to cascade and amplify the body's anti-tumor immune response, thereby effectively killing tumors. The Pts@DOX / HANGs@Gal platelet-based drug control system prepared by this invention, through the combined action of these mechanisms, achieves personalized tumor chemotherapy and immunotherapy. This not only overcomes off-target effects and improves the safety and efficacy of delivery, but also overcomes tumor heterogeneity and broadly enhances the anti-tumor immune response in different patients.

[0023] This invention leverages the advantages of platelets, such as their lack of nuclei, ability to serve as natural carriers, ability to evade macrophage phagocytosis, ability to target tumor cells, and excellent biosafety, to develop a reduction-responsive functionalized platelet-based controlled-drug release system co-loaded with the chemotherapy drug DOX, the immunosuppressant Gal, and HAase, a degrading enzyme for hyaluronic acid, a major component of the ECM, to regulate combined chemotherapy and immunotherapy for tumors. This drug-controlled system responds to the reducing tumor microenvironment, inducing the degradation of external protein sphere nanoparticles HANGs@Gal within the extracellular matrix and the release of Gal and HAase. Simultaneously, PMP-DOX released by activated platelets delivers DOX into tumor cells, achieving precise, graded drug delivery and improving drug utilization. More importantly, this drug-controlled system-mediated combined chemotherapy and immunotherapy can effectively enhance cellular immunogenicity, suppress immune tolerance, and strengthen the body's anti-tumor immune response, thereby effectively inhibiting tumor growth, metastasis, and recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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 magnification; B is the TEM image of HANGs@Gal; C is the TEM image of Pts@DOX / HANGs@Gal and its local magnification; 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.

[0025] Figure 2 Figure 2 is a transmission electron micrograph of the product of the present invention after activation: A is a TEM image of activated Pts@DOX and a magnified TEM image of the released DOX-PMPs; B is a schematic diagram of the activation of Pts@DOX, scale bars in A: 1 μm and 200 nm.

[0026] Figure 3 These are Western-blot result graphs and quantitative statistical graphs of the products of the present invention, and SDS-PAGE electrophoresis Coomassie Brilliant Blue staining result graphs: wherein A is a Western blot result graph of Pts, Pts@DOX, Pts@DOX / HANGs, and Pts@DOX / HANGs@Gal; B is a quantitative statistical result graph of A; and C is a Coomassie Brilliant Blue staining result graph of Pts, Pts@DOX, Pts@DOX / HANGs, and Pts@DOX / HANGs@Gal.

[0027] 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.

[0028] Figure 5 2 are the drug release graphs of the products of the present invention under different conditions: A is the cumulative release graph of DOX of Pts@DOX and Pts@DOX / HANGs@Gal at pH 7.4; B is the cumulative release graph of Gal of HANGs@Gal and Pts@DOX / HANGs@Gal nanoparticles in PBS and 1 mM GSH, respectively.

[0029] Figure 6 Figure 3 is a cell activity graph of the Pts@DOX / HANGs@Gal drug control system of the present invention: Figure A is a cell activity graph of tumor cells treated with PBS, DOX, Pts@DOX, Pts@DOX / HANGs, and Pts@DOX / HANGs@Gal for 24 and 48 hours, respectively; Figure B is a cell activity graph of cells treated with Pts@DOX / HANGs@Gal for different time periods.

[0030] Figure 7 Figure 2 is an in vitro tumor penetration diagram of the Pts@DOX / HANGs@Gal drug-controlled system of the present invention: A is a CLSM image of tumor multicellular spheroids incubated with the system in the presence or absence of Thrombin for 4 h and 12 h, respectively; B is the fluorescence quantification statistics; Scale bar in A: 100 µm.

[0031] 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 after incubation with DOX, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal for 4 h and 12 h, respectively. 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.

[0032] Figure 9 CLSM images of the targeted adhesion of the products of the present invention to tumor cells: A is the CLSM images of the targeted adhesion of Pts and Pts / HANGs@Gal to tumor cells under conditions of HA pre-treatment and without HA pre-treatment, respectively; B is the fluorescence quantification statistics; Scale bar in A: 50 μm.

[0033] Figure 10 CLSM images of ICD-related proteins in tumor cells promoted by the products of the present invention: 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; cell nuclei were stained with DAPI (blue), scale bar in A: 40 µm, scale bar in B: 40 µm.

[0034] Figure 11Figure 1 shows the in vivo treatment of the functionalized platelet drug-controlled system of the present invention. Figure A shows the tumor size of tumor-bearing mice after treatment with saline, DOX, Pts@DOX, Pts@DOX / HANGs, and Pts@DOX / HANGs@Gal for different time periods. Figure B shows the relative tumor volume changes during the treatment period. Figures C and D show the mouse survival rate and weight changes.

[0035] Figure 12 Figure 1 is an immunofluorescence analysis of the functionalized platelet drug control system of the present invention: A, B, and C are immunofluorescence staining of tumor tissues for TUNEL, Ki-67, and TGF-β, respectively; scale bar: 100 μm.

[0036] Figure 13 The functional platelet drug control system of the present invention regulates the immune response of T cells, DCs and NK cells in vivo. + 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.

[0037] Figure 14 Figures 2 and 3 show the in vivo pathological analysis and biosafety testing of the functionalized platelet drug-controlled system of the present invention. Figure A shows the blood routine and liver and kidney function results of mice after drug treatment; Figure B shows the H&E staining results of major 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.

[0038] Figure 15 : This 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.

[0039] Figure 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; B is the related fluorescence quantitative statistics of A.

[0040] Figure 17 This is a schematic diagram of the Pts@DOX / HANGs@Gal drug control system of the present invention combined with chemotherapy and immunotherapy in vivo.

[0041] Figure 18 Schematic diagram of the preparation process of functionalized platelet-based drug control system. DETAILED DESCRIPTION

[0042] 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 with reference to specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0043] The purpose of the present invention is to achieve both safe and efficient overcoming of the dense ECM barrier, improving drug bioavailability, and responsively releasing loaded drugs in a graded manner at the tumor lesion to activate combined chemotherapy and immunotherapy for tumors. It is necessary to perform necessary functional modifications on the platelet base to enable it to have the characteristics of tumor penetration, graded release, and in vivo degradability, thereby achieving efficient tumor killing. Based on this, the present invention constructs a reduction-responsive functionalized platelet-based drug control system with enhanced tumor penetration and graded drug release properties, co-loads DOX and Gal, and combines chemotherapy and immunotherapy strategies to effectively kill tumors and inhibit tumor growth and metastasis, providing a scientific basis and practical reference for the construction of functionalized platelet-based drug control systems and combined chemotherapy and immunotherapy.

[0044] To achieve the above objectives, the present invention provides a functionalized platelet-based drug control system in a first aspect, comprising a functionalized platelet-based matrix (Pts), a chemotherapy drug loaded inside the functionalized platelet-based matrix, and protein sphere nanoparticles (HANGs@Gal) grafted onto the surface of the functionalized platelet-based matrix.

[0045] The protein ball nanoparticles are cross-linked into spheres by NHS-SS-NHS and HAase, and the inside of the spheres is loaded with immunosuppressants.

[0046] The grafting rate of the protein sphere nanoparticles is 12-15%, preferably 13.82%, and the loading rate of the chemotherapy drug is 4-8%, preferably 5.62%. The chemotherapy drug includes doxorubicin, carmustine, nimustine, or dactinomycin D.

[0047] The protein sphere nanoparticles are prepared according to the following steps:

[0048] HAase was dissolved in PBS solution to obtain solution A; NHS-SS-NHS was dissolved in DMSO to obtain solution B. After mixing solution A and solution B, immunosuppressants were added, mixed evenly, and then centrifuged and washed to obtain protein ball nanoparticles.

[0049] The molar mass ratio of the HAase to NHS-SS-NHS is 1:10-20, preferably 1:15.

[0050] The mass ratio of the immunosuppressant to the functionalized platelet base is 0.3-0.7:1. The preferred dosage of the immunosuppressant is 0.5 mg of immunosuppressant per 1 mg of platelets.

[0051] The drug control system is spherical and has a particle size of 1.0-2.0 μm; the particle size of the protein spherical nanoparticles is 100-200 nm.

[0052] In one embodiment, a functionalized platelet-based drug control system includes an internal functionalized platelet base, wherein the functionalized platelet base is loaded with a chemotherapy drug (DOX), and the surface of the functionalized platelet base is functionalized with grafted reduction-responsive protein sphere nanoparticles (HANGs@Gal).

[0053] The functionalized platelet base is Pts, and the functionalized platelet base is DOX-loaded platelets;

[0054] The protein sphere nanoparticles (HANGs@Gal) contain the biological enzyme HAase and the immunosuppressant Gal. Specifically, the protein sphere nanoparticles HANGs@Gal are cross-linked by NHS-SS-NHS and HAase to form spheres, and are loaded with the immunosuppressant Gal.

[0055] The protein ball nanoparticles are spherical and have a particle size of 150 nm.

[0056] The functionalized platelet matrix is ​​spherical and has a particle size of 1.7 μm.

[0057] A second aspect of the present invention provides a method for preparing a functionalized platelet-based drug control system, comprising the following steps:

[0058] Extraction of functionalized platelet matrix;

[0059] loading chemotherapy drugs into the functionalized platelet matrix to obtain a functionalized platelet matrix loaded with chemotherapy drugs;

[0060] Preparation of protein spherical nanoparticles;

[0061] The protein ball nanoparticles and cross-linking agent were dissolved in PBS solution and reacted at 1-5°C for 2-4 h to obtain a cross-linked product;

[0062] The cross-linked product is mixed with the functionalized platelet-based matrix loaded with chemotherapy 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.

[0063] The functionalized platelet matrix loaded with chemotherapy drugs is prepared according to the following steps:

[0064] 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 in the dark at 37°C for 1 to 2 hours; the shaken product is then centrifuged and washed to obtain a functionalized platelet matrix loaded with chemotherapy drugs.

[0065] Functionalized platelet matrix is ​​extracted according to the following steps:

[0066] After the mice were anesthetized, whole blood was extracted from the mice and placed in a centrifuge tube containing the anticoagulant EDTA. The blood was mixed and centrifuged to remove the red blood cells. The supernatant was then centrifuged again to obtain platelet-rich plasma.

[0067] After centrifuging the platelet-rich plasma, the supernatant is discarded, and the resulting precipitate is the functionalized platelet matrix.

[0068] The cross-linking agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt.

[0069] In one embodiment, see Figure 18 As shown, a method for preparing a functionalized platelet-based drug control system comprises the following specific steps:

[0070] (1) Extracting platelets required for core functionalized platelet matrix;

[0071] (2) Preparation of platelet-based Pts@DOX loaded with chemotherapy drug DOX;

[0072] (3) Synthesis of reduction-responsive protein spherical nanoparticles HANGs@Gal;

[0073] (4) Preparation of Pts@DOX / HANGs@Gal drug control system.

[0074] 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 the anticoagulant EDTA and mixed; the whole blood is centrifuged at 200g for 20 minutes, the supernatant is collected, and red blood cells are removed; the supernatant is centrifuged again at 200g for 20 minutes, the supernatant is collected, and platelet-rich plasma is obtained; the platelet-rich plasma is centrifuged at 800g for 20 minutes, the supernatant is discarded, and the resulting precipitate is platelets (Pts);

[0075] In step (2), the preparation of Pts@DOX: first weigh DOX and dissolve it in Tyrode buffer, then add the platelet PBS solution; at the same time, a 1 μM PGE1 solution and a triethylamine solution are added to the reaction to prevent platelet activation and remove the hydrochloride in the hydrochloric acid DOX; then the reaction is shaken at 100 rpm at 37°C for 1 hour in the dark; then, the resulting product is centrifuged at 800 g for 20 minutes at room temperature to obtain the precipitate; and washed 2-3 times with fresh ACD solution to obtain Pts@DOX;

[0076] In step (3), reduction-responsive protein ball nanoparticles HANGs@Gal were synthesized: HAase was weighed and dissolved in a pH 7.4 PBS solution, and NHS-SS-NHS was weighed and dissolved in a small amount of DMSO; the two solutions were mixed, and Gal was weighed and added at the same time, and the reaction was stirred at room temperature for 30 minutes; then, the resulting solution was placed in an ultrafiltration centrifuge tube and centrifuged at 14,000 g for 15 minutes at room temperature; the product was washed three times with PBS solution using the above method to obtain HANGs@Gal.

[0077] In step (4), the Pts@DOX / HANGs@Gal drug control system was prepared as follows: HANGs@Gal obtained in step (3) and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (Sulfo-SMCC) were dissolved in a PBS solution with a pH of 7.4 and reacted at 4°C for 2 h; the resulting product was centrifuged at 14,000 g for 20 min in an ultrafiltration centrifuge tube to remove excess SMCC linker; the resulting product was then mixed with the Pts@DOX obtained in step (2) and reacted at room temperature for 1 h; the reaction solution was centrifuged at 800 g for 20 min at room temperature to remove excess HANGs@Gal; the above steps were repeated and washed three times to obtain a functionalized platelet-based drug control system, which was recorded as Pts@DOX / HANGs@Gal.

[0078] 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.

[0079] The present invention uses gradient centrifugation to separate platelets from extracted mouse whole blood. Platelets are then loaded with DOX using a mixed incubation technique to prepare DOX-loaded platelet-based Pts@DOX. Subsequently, the reduction-responsive protein crosslinker, succinimidyl ester-SS-succinimidyl ester (NSH-SS-NHS), is cross-linked with the ECM modulator, HAase, to prepare reduction-responsive HAase nanoparticles (HANGs@Gal) loaded with the immunosuppressant Gal. These HAase nanoparticles are then chemically grafted onto the platelet-based surface to create a functionalized platelet-based drug control system. The functionalized platelet-based drug control system prepared in this invention combines 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 enzyme and drug delivery and tumor therapy.

[0080] This system can safely and efficiently load the chemotherapy drug DOX and the immunosuppressant Gal, targeting 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 that bridges the reduction-sensitive bonds is broken, rapidly degrading the nanoprotein spheres HANGs@Gal, releasing the HAase and Gal loaded therein. HAase, as a degrading enzyme for HA, the main component of the extracellular matrix, effectively degrades HA, making the dense ECM barrier loose, thereby making it easier for drugs to penetrate deep into the tumor and improving the efficiency of drug delivery in the body. More importantly, Gal is accurately delivered to the extracellular matrix of tumor cells to effectively suppress immune tolerance. At the same time, the functionalized platelet base targets and adheres to tumor cells through the receptor-ligand binding principle, accurately delivering DOX into tumor cells to exert its effect. On the one hand, the released DOX can not only kill tumor cells, but also enhance 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 signaling, relieving TGF-β's immunosuppression of immune cells in the tumor microenvironment, such as T cells, macrophages, and neutrophils, thereby relieving immune tolerance. Combined chemotherapy and immunotherapy for tumors regulated by a functionalized platelet-based drug control system not only effectively relieves immune tolerance in the body but also effectively enhances the body's anti-tumor immune response, synergistically improving tumor treatment efficacy. It has excellent biosafety and potential clinical applications.

[0081] 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.

[0082] Example 1

[0083] 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 and grafting reduction-responsive HANGs@Gal protein sphere nanoparticles on the surface, and co-loading Gal and HAase.

[0084] The preparation process of the functionalized platelet-based drug control system Pts@DOX / HANGs@Gal includes the following steps:

[0085] (1) Preparation of platelets loaded with the chemotherapy drug DOX:

[0086] 11) Platelet extraction:

[0087] Grab the mouse, turn its head downward, and fix it with your bare hands. At the same time, use the fixed hands to gently press the sides of the neck to make the eyeballs become congested and bulge out. 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 whole blood from the mouse. Centrifuge the whole blood of the mouse at 200g for 20 minutes, and collect the supernatant as PRP. Collect the 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.

[0088] 12) Preparation of Pts@DOX:

[0089] First, 5 mg of doxorubicin hydrochloride (DOX·HCL) and 2 mg of pure platelets were weighed and dissolved in Tyrode's buffer containing 1 μM PGE1. To increase drug loading efficiency, triethylamine was added to the DOX / platelet mixture (molar ratio of triethylamine:DOX = 3:1). The mixture was then incubated in a shaker at 37°C for one hour to obtain Pts@DOX. The Pts@DOX was then centrifuged at 800 g for 20 minutes to obtain a precipitate, the Pts@DOX. The product was washed three times with fresh ACD solution to remove free DOX. The product was collected by centrifugation and dispersed in PBS for later use. The resulting DOX-loaded platelets were designated Pts@DOX.

[0090] (2) Surface functionalization grafting of the above core:

[0091] 21) Synthesis of HANGs@Gal:

[0092] First, 1.19 mg of HAase was weighed and dissolved in PBS to obtain a HAase PBS solution. Second, 10 mg of NHS-SS-NHS was weighed and added to the HAase PBS solution. Simultaneously, 1 mg of Gal was weighed, dissolved in approximately 30 μL of DMSO solution, and added to the above solution. After the mixture was stirred for 30 minutes, the reacted solution was placed in an ultrafiltration centrifuge tube (molecular cutoff = 100 kDa) and centrifuged at 14,000 g for 20 minutes to obtain a precipitate. The precipitate was resuspended in PBS to obtain the final product, protein sphere nanoparticles, namely HANGs@Gal.

[0093] 22) Modification of Sulfo-SMCC:

[0094] Weigh 1.19 mg of Sulfo-SMCC and add it to the reaction mixture. Stir and continue the reaction for 30 minutes. The resulting solution is then placed in an ultrafiltration centrifuge tube (molecular cutoff = 100 kDa) and centrifuged at 14,000 g for 20 minutes to obtain a precipitate. The precipitate is resuspended in PBS and washed three times with PBS following the above centrifugation method to remove excess NHS-SS-NHS and Sulfo-SMCC. The resulting cross-linked product is resuspended in PBS, resulting in Sulfo-SMCC-modified HANGs@Gal.

[0095] (3) Preparation of Pts@DOX / HANGs@Gal particles

[0096] The products Pts@DOX and Sulfo-SMCC-modified HANGs@Gal obtained from (1) and (2) were added to Tyrode buffer containing 1 μM PGE1 and reacted in the dark for 1 hour. After the reaction, the product was centrifuged at 800 g for 20 minutes, and the precipitate was washed three times according to the above centrifugation method. Finally, the precipitate was resuspended in physiological saline containing 1 μM PGE1 to obtain the final product, a functionalized platelet-based drug control system, namely Pts@DOX / HANGs@Gal.

[0097] The products obtained in each step of Example 1 were systematically studied for their morphology, composition and biological activity using modern nano-analysis techniques such as TEM, SEM, Western-blot, Coomassie Brilliant Blue staining, DLS and Zeta potential analyzer, CD, and ELISA. The results are as follows:

[0098] 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 1As 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 onto HANGs@Gal. It is also spherical with a size of about 1.7 μm. The magnified image shows that nanoparticles are clearly grafted onto the edge of Pts. Figure 1 Figure 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. These results confirm that Pts@DOX / HANGs@Gal of appropriate sizes has been successfully synthesized.

[0099] 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 activated Pts@DOX changes its morphology to an irregular shape, and spherical secretions of DOX-PMPs are released. These results demonstrate that the activation and release properties of Pts are not affected after modification, and further confirm the successful synthesis of Pts@DOX.

[0100] The present invention used Western-blot and Coomassie Brilliant Blue staining to analyze the protein structures of Pts, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal obtained in each step of Example 1. Figure 3 As shown in Figure A, the expression of Pts' characteristic membrane proteins CD41, CD61 and P-Selectin were highly consistent in the Western-blot results; 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 3As shown in Figure C, SDS-PAGE staining revealed no significant differences in the expression of intrinsic Pts proteins among the Pts, Pts@DOX, Pts@DOX / HANGs, and Pts@DOX / HANGs@Gal groups. These results demonstrate that various modifications to Pts maintain the original structure of platelets and do not affect their original function.

[0101] The present invention uses UV spectrophotometry, CCK-8, CD and ELISA to analyze the biological activities of Pts, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal obtained in each step of Example 1. Figure 4 As shown in Figure A, under the condition of no reducing stimulation, the DOX content in Pts@DOX / HANGs@Gal did not decrease significantly after 48 h, 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 of Pts still showed good cell viability after 24 hours, proving that the above modification process has almost no 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, HAase released from Pts@DOX / HANGs@Gal and free HAase exhibited good HAase bioactivity as determined by enzyme-linked immunosorbent assay (ELISA). These results further demonstrate that the native bioactivities of HAase and Pts remain intact after various functional modifications of platelets, laying a solid foundation for further functional validation.

[0102] Experimental Example 2

[0103] The functionalized platelet-based drug control system Pts@DOX / HANGs@Gal provided in Example 1 has reduction-responsive drug release characteristics.

[0104] The present invention uses GSH to simulate a reductive tumor microenvironment and examines the drug release characteristics of this system. The specific results are as follows:

[0105] 1) Release characteristics of DOX

[0106] 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.

[0107] 2) Pts@DOX / HANGs@Gal controlled release system restores responsive drug release properties

[0108] The reduction-responsive drug release characteristics of the Pts@DOX / HANGs@Gal controlled release system were investigated by UV-Vis. After incubating 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 under GSH stimulation was quantified. After 48 hours of incubation under physiological conditions (PBS), the amount of Gal released from HANGs@Gal and Pts@DOX / HANGs@Gal was negligible (less than 28%), demonstrating the excellent stability of the Pts@DOX / HANGs@Gal controlled-release system. Compared to the control group, when the experimental group was co-incubated with 1 mM GSH, over 80% of Gal was released from the HANGs@Gal and Pts@DOX / HANGs@Gal controlled-release systems, further confirming the reduction-sensitive drug release characteristics of the Pts@DOX / HANGs@Gal system. These results demonstrate that the Pts@DOX / HANGs@Gal controlled-release system exhibits excellent and expected drug-controlled release characteristics.

[0109] Experimental Example 3

[0110] In vitro biological evaluation of the functionalized platelet-based drug control system Pts@DOX / HANGs@Gal provided in Example 1.

[0111] (1) Cell activity

[0112] Cell culture: The present invention used highly metastatic mouse melanoma cells (B16F10) provided by the Institute of Cell Biology, Chinese Academy of Sciences. B16F10 cells were cultured in DMEM high-glucose medium supplemented with 10% FBS and 1% bispecific antibiotics (100 μg / mL penicillin and 100 μg / mL streptomycin) in a 37°C, 5% CO2 incubator.

[0113] In order to evaluate the in vitro tumor cell cytotoxicity of the Pts@DOX / HANGs@Gal controlled release system, the present invention first used the CCK-8 method to detect the effects of PBS, free DOX, Pts@DOX, Pts@DOX / HANGs and Pts@DOX / HANGs@Gal on the activity of B16F10 cells. 4 cells / cm 2 B16F10 cells were seeded into 24-well plates at a density of 10 μg / mL. When the cell confluency reached approximately 60-70%, they were co-cultured with the above drugs for 24 and 48 hours, respectively. The cells were washed with PBS, and then a mixture of 200 μL of fresh culture medium and 20 μL of CCK8 was added. The reaction was continued at 37°C for 1.5-2 hours. The reaction solution in the well plate was then transferred to a 96-well plate. The absorbance of the solution at a wavelength of 450 nm was measured using a microplate reader to calculate the cell viability. The results are shown in Figure 2. 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 severe cytotoxicity. This is because Pts accurately targets tumor cells, improves the uptake of drugs 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 1. 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 was 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.

[0114] (2) In vitro tumor penetration and cellular uptake: First, a 3D multicellular spheroid (MCS) based on B16F10 cells was constructed, i.e., a tumor sphere model. 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 UV irradiation overnight. B16F10 cells were then plated at a density of 2 × 10 3The initial density of cells / well was seeded in the above-mentioned well plate, and after 6 days of co-culture, tumor spheres were formed. Subsequently, the FITC-labeled Pts@DOX / HANGs@Gal controlled release system was co-incubated with the tumor spheres, and CLSM was used to monitor the penetration of the controlled release system into MCSs with and without the addition of Thrombin, to investigate the enhanced tumor penetration behavior of the controlled release system after platelet activation. Figure 7 As shown in Figure A, without the addition of Thrombin stimulation, after 4 h of incubation, only a very small amount of green fluorescent labeled HAase was dispersed around the outer boundary of MCSs, and only a very small amount of DOX was taken up into the cell; 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 fluorescent labeled HAase was still dispersed around the outer boundary of MCSs, while more DOX was internalized into the cell. Under the condition of Thrombin stimulation, after 4 h of incubation, the green fluorescent labeled HAase was widely distributed in the outer boundary area of ​​​​MCSs, and most of the red fluorescent DOX was internalized into the cell, and the fluorescence intensity was stronger than that of the group without Thrombin addition; after 12 h of incubation, the entire MCS sphere was dispersed with strong red fluorescence, and the green fluorescence was still on the periphery of MCSs, but the fluorescence intensity was significantly enhanced compared with the 4 h time 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 demonstrate that the Pts@DOX / HANGs@Gal controlled-release system has a good graded delivery effect.

[0115] In order to further investigate the cellular uptake efficiency, CLSM and FCM were used to monitor the endocytosis level of B16F10 cells in response to the Pts@DOX / HANGs@Gal controlled release system in real time. 5 cells / cm 2 B16F10 cells were seeded in confocal microplates. 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 and 12 hours. The nuclei and skeletons of the cells were then stained and analyzed, and the endocytosis efficiency was quantitatively detected by flow cytometry. Figure 8As shown in Figures A and B, compared with the free DOX group, the concentration and intensity of DOX endocytosed by B16F10 cells 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 quantification. This is because Pts precisely targets tumor cells, enabling more efficient DOX uptake, further demonstrating the excellent drug delivery efficacy of Pts@DOX / HANGs@Gal.

[0116] (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. 5 cells / cm 2 The cells were seeded into confocal microplates at a density of 100 μg / mL. 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, fixed, and the cell nuclei were stained. CLSM images were collected. The results are shown in Figure 2. Figure 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 properties 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 Figure 9 (As shown in Figure B), the results are consistent with those in Figure A. These results demonstrate that functional modification of platelets does not affect their targeting effect on tumor cells, further demonstrating the excellent targeting ability of the Pts@DOX / HANGs@Gal delivery system for tumor cells.

[0117] (4) Pts@DOX / HANGs@Gal enhances tumor cell ICD

[0118] The chemotherapy drug DOX can enhance the ICD of tumor cells. To investigate the in vitro enhancing effect of the Pts@DOX / HANGs@Gal controlled release system on tumor cell ICD, CLSM was used to investigate the expression levels of ICD-related proteins CRT and HMGB1 in tumor cells. B16F10 cells seeded on confocal microscopy 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, with cell nuclei labeled with DAPI. Figure 10 As shown in Figure A, compared with the PBS group and 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 Figure 10 As shown in Figure B, the nuclear green fluorescence of the free DOX group was slightly reduced compared to the PBS group, while the green fluorescence of the tumor cell nuclei was significantly reduced after treatment with Pts@DOX, Pts@DOX / HANGs, and Pts@DOX / HANGs@Gal. This demonstrates that HMGB1 protein migrates outside the cell, mediating stronger immunogenic cell death and enhancing anti-tumor immunogenicity. These results demonstrate that the precise delivery of DOX by the Pts@DOX / HANGs@Gal drug control system effectively enhances the immunogenic cell death and enhances its immunogenicity. This also further demonstrates the excellent tumor cell targeting ability and higher drug delivery efficiency of Pts@DOX / HANGs@Gal.

[0119] Experimental Example 4

[0120] In vivo anti-tumor evaluation of the functionalized platelet-based drug control system Pts@DOX / HANGs@Gal provided in Example 1.

[0121] (1) Construction of tumor-bearing mouse model: All animal experiments in the present invention were conducted in accordance with the relevant provisions of the Laboratory Animal Care and Use Committee Work Manual. Female C57BL / 6 mice weighing approximately 20 g and aged 5 to 6 weeks were purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., China. In the present invention, 100 μL of cell suspension was prepared using B16F10 cells in the exponential growth phase. The suspension 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).

[0122] (2) Drug Intervention: The successfully constructed tumor-bearing mice were randomly divided into five groups based on weight and intravenously injected with saline, DOX, Pts@DOX, Pts@DOX / HANGs, and Pts@DOX / HANGs@Gal twice a week for 20 days. After the end of the drug administration, the mice were euthanized. The hearts, livers, spleens, lungs, kidneys, and tumors of the mice were collected for histopathological analysis using H&E, TUNEL, and Ki-67 staining.

[0123] After the mice were killed, the main organs and tumor tissues were collected. Figure 11 As shown in Figure A, the tumor in the free DOX group was slightly smaller than that in the normal 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 mild 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 combination therapy can effectively kill tumors. Figure 11 The tumor volume statistics in Figure B confirmed the same trend of tumor growth inhibition. 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%), Figure 11 C, which was significantly higher than that of the other treatment groups), and the weight of tumor-bearing mice did not decrease during the administration period (e.g. Figure 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.

[0124] (3) Histopathological analysis: The results of histopathological analysis are as follows: Figure 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 Figure 12 In addition, the results of Ki-67 immunofluorescence staining showed that (as shown in Figure A) Figure 12As 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 Figure 12 As shown in Figure C, compared with the saline, free DOX, Pts@DOX, and Pts@DOX / HANGs groups, the TGF-β fluorescence intensity in the Pts@DOX / HANGs@Gal group was significantly reduced after treatment, indicating more effective inhibition of TGF-β expression. These results further demonstrate the excellent anti-tumor effect of the Pts@DOX / HANGs@Gal drug control system.

[0125] (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 combined therapy mediated by the Pts@DOX / HANGs@Gal drug control system. The specific experimental process is as follows: After drug treatment, the mice are euthanized, and the tumor tissues are collected and lysed in a 37°C shaker 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, washed with a washing solution, and resuspended in a PBS solution. Subsequently, the cells are stained with Live / Dead dye in the dark for 20 min.

[0126] 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 the tumor-infiltrating CD4 + and CD8 + T cell levels;

[0127] 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;

[0128] 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:

[0129] 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 first used FCM to quantitatively characterize the CD8 + Cytotoxic T cells, CD4 + The level of helper T cells. Figure 13 As shown in Figure A, tumor-infiltrating CD8 + The proportion of cytotoxic T cells (CTLs) was low; the number of CTLs in the free DOX group increased moderately; 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 even higher than that in the Pts@DOX group, because while Pts precisely targeted tumor cells with excellent drug delivery efficiency, the HAase released after HANGs degradation made the dense ECM loose, which was conducive to a large number of tumor-infiltrating CD8 + The recruitment and infiltration of effector T cells helps to restore the activity of effector T cells. In addition, among all treatment groups, Pts@DOX / HANGs@Gal showed the highest CTL infiltration ratio, indicating that the Pts@DOX / HANGs@Gal controlled-release system can 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 chemotherapy drug DOX and the enzyme HAase, the main component of the extracellular matrix, by Pts, the immunosuppressant Gal is also introduced, further enhancing the body's anti-tumor immune response.

[0130] 2) Immune response of DC cells and NK cells: This paper further reveals the mechanism of anti-tumor immune response enhanced by the Pts@DOX / HANGs@Gal drug control system by examining the maturity of DC cells. Figure 13As shown in Figure B of [reference], the free DOX and the experimental groups loaded with DOX (Pts@DOX, Pts@DOX / HANGs, and Pts@DOX / HANGs@Gal) all promoted the maturation of DC cells to varying degrees, and the order of their maturity 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. In addition, the Pts@DOX / HANGs@Gal drug control system also successfully recruited tumor infiltrating NK cells (as shown in Figure 13 Figure C of [reference]). This is because the Pts@DOX / HANGs@Gal drug control system can enhance tumor immunogenicity through DOX-mediated tumor damage, promote the exposure of tumor associated antigen TAA, and enhance the recruitment of cytotoxic CD8 + T cells and helper CD4 + T cells into the tumor; in addition, the Pts@DOX / HANGs@Gal drug control system inhibits the TGF-β receptor,解除免疫耐受, and HAase degrades the extracellular matrix to establish a highway for the recruitment of immune cells, further enhancing the anti-tumor immune response of the body.

[0131] (4)Bio-safety analysis: First, the blood glucose levels of mice before and after intravenous injection of the Pts@DOX / HANGs@Gal drug control system were detected. The results are shown in Figure 14 Figure A of [reference]. Compared with the control group, none of the treatment groups induced a continuous decrease in peripheral blood glucose, indicating that the controlled release system has good blood safety. Second, we evaluated liver function or liver injury by detecting the levels of aspartate aminotransferase AST and alanine aminotransferase ALT, the liver function indicators in the blood after injection of different drugs according to the kit. The results are shown in Figure 14 Figure A of [reference]. The concentrations of the two enzymes did not change significantly and there was no significant difference from the control group, indicating that the Pts@DOX / HANGs@Gal drug control system has good bio-safety for liver tissue. In addition, the results of H&E analysis of the main organs showed that all treatment groups with Pts as the carrier did not cause damage to the body organs (as shown in Figure 14 Figure B of [reference]), which again confirmed that the Pts@DOX / HANGs@Gal drug control system has good bio-safety in vivo.

[0132] (5)Anti-tumor metastasis: In this invention, a lung metastasis model was constructed to further investigate the anti-tumor metastasis effect of the Pts@DOX / HANGs@Gal drug control system. 100 μL containing 1×10 6 It should be noted that there is an unclear expression "解除免疫耐受" in the original text, which may need to be further clarified in the context to ensure more accurate translation.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 counted 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 Figure 2. Figure 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 different (e.g. Figure 15 (Figure B) This demonstrates that the Pts@DOX / HANGs@Gal drug-controlled system significantly and effectively inhibits tumor metastasis. In contrast, mice in the other treatment groups exhibited varying degrees of metastatic nodules in their lung tissue. This result demonstrates that the Pts@DOX / HANGs@Gal system has a significant anti-tumor metastasis effect.

[0133] (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, mice were divided into two groups and injected with free DOX and Pts@DOX / HANGs@Gal via the tail vein. Six hours after injection, the mice were euthanized, and the main organs (heart, liver, spleen, lung, and kidney) and tumors of the mice in each group were collected and placed in culture dishes. The drug distribution was observed based on the DOX fluorescence signal using a small animal in vivo imaging system. Figure 16 As shown in Figure A, the free DOX group accumulated the most drug in the liver, followed by the kidney, with less drug accumulation in the tumor site. However, the Pts@DOX / HANGs@Gal group accumulated less drug in the liver and kidney tissues, with a large amount of drug accumulated in the tumor site. The fluorescence quantitative analysis results also correspond to this result (e.g. Figure 16 The above results demonstrate that Pts@DOX / HANGs@Gal has a good tumor targeting distribution effect in vivo and once again demonstrates that Pts@DOX / HANGs@Gal has good biosafety for major tissues and organs.

[0134] Figure 17 Schematic diagram of the Pts@DOX / HANGs@Gal drug control system provided in Example 1 combined with chemotherapy and immunotherapy in vivo. Figure 17As 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 platelet surface degrade in response to GSH and release the immunosuppressant Gal and matrix degrading enzyme HAase to the outside of the tumor cells, which are used to relieve the extracellular inhibitory immune microenvironment and degrade the dense extracellular matrix, respectively, paving the way for drug penetration and infiltration of immune effector cells; DOX is delivered into the tumor cells through membrane fusion and platelet secretion of DOX-PMPs, acting on the tumor cell nucleus, causing tumor cell ICD and enhancing its immunogenicity.

[0135] Figure 18 This is the preparation process of the Pts@DOX / HANGs@Gal drug control system provided in Example 1. As shown in the figure, first, whole blood is extracted from mice, and platelet-rich plasma is obtained through multiple centrifugation. Pure mouse platelets are then centrifuged again to obtain DOX-loaded platelets, namely Pts@DOX. Simultaneously, HAase and NHS-SS-NHS bridging reactions are simultaneously reacted with the immunosuppressant Gal to produce Gal-loaded protein nanospheres, namely HANGs@Gal. Finally, the protein nanospheres are grafted onto the platelet surface using Sulfo-SMCC, resulting in the final product, the platelet-based drug control system Pts@DOX / HANGs@Gal.

[0136] Example 5

[0137] A functionalized platelet-based drug control system (Pts@Car / HANGs@Gal) uses platelets loaded with the chemotherapy drug carmustine as a core, with reduction-responsive HANGs@Gal protein sphere nanoparticles functionalized on their surface and co-loaded with Gal and HAase to create a controlled-release system. The preparation process is the same as in Example 1.

[0138] Example 6

[0139] A functionalized platelet-based drug control system (Pts@Nim / HANGs@Gal) uses platelets loaded with the chemotherapy drug Nimustine as a core, with reduction-responsive HANGs@Gal protein sphere nanoparticles functionalized on their surface and co-loaded with Gal and HAase to create a controlled-release system. The preparation process is the same as in Example 1.

[0140] Example 7

[0141] A functionalized platelet-based drug control system (Pts@Dac / HANGs@Gal) uses platelets loaded with the chemotherapy drug Dactinomycin D as a core, with reduction-responsive HANGs@Gal protein sphere nanoparticles functionalized on their surface and co-loaded with Gal and HAase to create a controlled-release system. The preparation process is the same as in Example 1.

[0142] In summary, the present invention utilizes the advantages of platelets, such as their lack of nuclei, ability to serve as natural carriers, ability to evade macrophage phagocytosis, ability to target tumor cells, and 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 degrading enzyme HAase, a major component of the ECM, to regulate combined chemotherapy and immunotherapy for tumors. This nanosystem can respond to the reducing tumor microenvironment and induce the regulation of combined chemotherapy and immunotherapy for tumors. This drug control system can respond to the reducing tumor microenvironment, inducing the degradation of external protein sphere nanoparticles HANGs@Gal in the extracellular matrix and releasing Gal and HAase. Simultaneously, PMP-DOX released by activated platelets is used to deliver DOX into tumor cells, achieving precise, graded drug delivery and improving drug utilization. Furthermore, the present invention has the ability to enhance tumor immune responses: on the one hand, the chemotherapy drug DOX can induce ICD in tumor cells and enhance their immunogenicity; on the other hand, HAase is released into the extracellular matrix, degrading HA, the main component of the extracellular matrix, loosening the dense ECM and paving a highway for immune cell infiltration; and finally, the immunosuppressant Gal, by inhibiting TGF-β signaling, relieves TGF-β from immunosuppressive effects on immune cells in the tumor microenvironment, such as T cells, macrophages, and neutrophils, thereby enhancing anti-tumor immune responses. The combination of these three effectively enhances the body's anti-tumor immune response, inhibits tumor growth and metastasis, and achieves better therapeutic effects.

[0143] This application solves the following problems:

[0144] (1) Solve the problem of low bioavailability such as easy inactivation of biological enzymes 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 a restricted reaction space, while maintaining high-fidelity enzyme activity and improving their bioavailability.

[0145] (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 role in killing tumors; HAase mainly acts 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 proposed to construct a functionalized platelet-based Pts@DOX / HANGs@Gal drug-control system. The chemotherapeutic drug DOX is loaded internally on platelets, and reduction-responsive, degradable protein nanospheres HANGs@Gal are grafted externally. The immunosuppressant Gal and the enzyme HAase are co-loaded to create a functionalized platelet-based Pts@DOX / HANGs@Gal drug-control system. On the one hand, this system responds to reductive stimuli from the tumor microenvironment, cleaving the bridging molecules NHS-SS-NHS in the grafted protein nanospheres. This then degrades the protein nanospheres in the extracellular matrix, releasing Gal and HAase, thereby improving drug utilization efficiency. On the other hand, after activation, platelets adhere to tumor cells and precisely deliver DOX into the tumor cells via DOX-PMPs. In summary, this system overcomes numerous physiological barriers, successfully achieving hierarchical drug delivery and achieving more efficient drug utilization.

[0146] (3) Solve the problem of unsatisfactory tumor immunotherapy effects 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.

[0147] The present invention describes preferred embodiments and their effects. However, those skilled in the art, once informed of the basic inventive concept, may make additional changes and modifications to these embodiments. Therefore, it is intended that the appended claims be interpreted to include the preferred embodiments as well as all changes and modifications that fall within the scope of the invention.

[0148] While 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 these embodiments without departing from the principles and spirit of the invention, and that the scope of the 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 matrix, a chemotherapy drug loaded inside the functionalized platelet matrix, and protein sphere nanoparticles grafted onto the surface of the functionalized platelet matrix; The protein sphere nanoparticles are cross-linked into spheres by NHS-SS-NHS and HAase, and the spheres are loaded with immunosuppressants; The chemotherapy drug is doxorubicin; Sulfo-SMCC was used to graft protein nanospheres onto the platelet surface; The immunosuppressant is Galunisertib; The functionalized platelet matrix loaded with chemotherapy 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 in the dark at 37°C for 1 to 2 hours; the shaken product is then centrifuged and washed to obtain a functionalized platelet matrix loaded with chemotherapy drugs.

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 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 mixing solution A and solution B, immunosuppressants were added, mixed evenly, and then 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 has a particle size of 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: Extraction of functionalized platelet matrix; loading chemotherapy drugs into the functionalized platelet matrix to obtain a functionalized platelet matrix loaded with chemotherapy drugs; Preparation of protein spherical nanoparticles; The protein ball nanoparticles and the crosslinking agent were dissolved in PBS solution and reacted at 1-5°C for 2-4 hours to obtain a crosslinked product; The cross-linked product is mixed with the functionalized platelet-based matrix loaded with chemotherapy 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: 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. The blood was mixed and centrifuged to remove the red blood cells. The supernatant was then centrifuged again to obtain platelet-rich plasma. After centrifuging the platelet-rich plasma, the supernatant is discarded, and the resulting precipitate is the functionalized platelet matrix.

8. 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 sulfosuccinimide ester sodium salt.

9. 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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