A nano-assembly, its preparation method and application

Abstract

The present invention provides a nano-assembly, a preparation method thereof and an application. The nano-assembly provided by the present invention has a core-shell structure, including a core composed of a procoagulant and a chemotherapeutic drug and a shell composed of a temperature-sensitive nano-polymer. Moreover, it has good temperature sensitivity and excellent gelation behavior, can be transformed into a high-strength gel, can effectively increase the strength of a blood clot, inhibit the degradation of the blood clot for a long time, is sensitive to pH, is conducive to the release of polyphosphate in blood vessels with a slightly neutral pH value, thereby triggering blood clot embolism, aggravating the anaerobic metabolism of tumor hypoxia, increasing the acidic environment at the tumor site, and further promoting the release of the chemotherapeutic drug by the acidic pH value at the tumor site, thereby improving the targeting property.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and particularly relates to a nano-assembly and its preparation method and application. Background Art

[0002] Hepatocellular carcinoma (HCC, hereinafter referred to as liver cancer) is a major disease that seriously endangers human health. It has a very high degree of malignancy, grows invasively and rapidly, is prone to recurrence after treatment, and the 5-year survival rate is less than 5%, so it is known as the "king of cancers". China is the largest country with liver cancer in the world, and the number of new cases accounts for about 55% of the global total. The mortality rate ranks second among malignant tumors, and the incidence and mortality rates are still showing a continuous upward trend. Among various treatment methods, such as ablation, resection, transplantation, and transarterial chemoembolization (TACE), etc., TACE is the preferred treatment method for intermediate and advanced HCC.

[0003] TACE can, under the guidance of imaging equipment, deliver vascular embolization materials and therapeutic drugs to the tumor target vessels in a super-selective manner through a slender catheter, playing the roles of blocking the blood flow in the tumor, slow drug release, and targeted therapy. This method aims to "starve" the tumor by depriving it of oxygen and nutrients, thereby causing ischemic injury and necrosis, and thus inhibiting tumor growth. However, the embolization material also faces a "flow-embolization" contradiction: it needs to have good fluidity to easily diffuse into the tiny tumor terminal blood vessels through the slender catheter, and at the same time, it needs to have high-strength embolization resistance to resist blood flow scouring and prevent vascular recanalization. However, currently used embolizing agents in clinical practice usually cannot completely and thoroughly embolize the rich and highly heterogeneous liver cancer terminal vascular network through physical viscosity, so there are many problems in TACE treatment, such as the tumor cannot be completely cured, is prone to recurrence, and the long-term survival rate is low, etc., which are non-radical palliative therapy problems.

[0004] In recent years, the use of thrombus spontaneously formed in the body for tumor embolization treatment has gradually attracted people's interest. Research shows that a tiny blood clot can block the nutrient supply of the entire blood vessel and cause "avalanche-like" death of thousands of cells. This treatment method of tumor vessel infarction (TVI) has the following advantages: 1) Good tumor radical cure effect: TVI not only targets tumor neovascularization, but also can efficiently block the established tumor blood vessels, and has a more superior tumor radical cure effect than angiogenesis inhibitors; 2) Quick and lasting efficacy: Research shows that tumor cells begin to die within hours after TVI treatment, and the blood clot in the tumor can exist for a long time; 3) Specific coagulation in the tumor: Different from the normal blood vessels which are usually in a non-coagulative environment, the tumor is considered to be a "wound that is difficult to heal" and has a hypersensitive procoagulant microenvironment. However, in TVI treatment, the selection of coagulation proteins, the requirements for targeting, and the inability to maintain the formed blood clot effectively for a long time are still problems that need to be solved urgently. Summary of the Invention

[0005] The object of the present invention is to provide a nano-assembly and its preparation method and application. The nano-assembly provided by the present invention has good thermosensitivity and excellent gelation behavior, can be transformed into a high-strength gel, and can effectively increase the strength of blood clots, inhibit the degradation of blood clots for a long time, is sensitive to pH, is conducive to the release of polyphosphate in blood vessels with a slightly neutral pH value, thereby triggering blood clots and embolism, aggravating the anaerobic metabolism of tumor hypoxia, increasing the acidic environment at the tumor site, and further promoting the release of chemotherapeutic drugs by the acidic pH value at the tumor site, thereby improving the targeting property.

[0006] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a nano-assembly. The nano-assembly has a core-shell structure and includes a core composed of a procoagulant and a chemotherapeutic drug and a shell composed of a thermosensitive nano-polymer. The mass ratio of the procoagulant, the chemotherapeutic drug to the thermosensitive nano-polymer in the nano-assembly is (10-75):4:80;

[0008] The thermosensitive nano-polymer is prepared by the RAFT method using N-isopropylacrylamide and diethylaminoethyl methacrylate as monomers.

[0009] Preferably, the procoagulant is polyphosphate, and the degree of polymerization of the polyphosphate is 25-250.

[0010] Preferably, the chemotherapeutic drug is nitrated cisplatin or a tetravalent platinum chemotherapeutic drug.

[0011] Preferably, the mass ratio of the procoagulant, the chemotherapeutic drug to the thermosensitive nano-polymer in the nano-assembly is 10:4:80.

[0012] Preferably, the degree of polymerization of the thermosensitive nano-polymer is 100-250.

[0013] The present invention also provides a preparation method of the nano-assembly described in the above technical solution, including the following steps: After mixing the solution of the chemotherapeutic drug and the solution of the thermosensitive nano-polymer, dropwise add the solution of the procoagulant and carry out a self-assembly reaction to obtain the nano-assembly.

[0014] Preferably, the thermosensitive nano-polymer is prepared by the RAFT method and includes the following steps:

[0015] (1) After mixing N-isopropylacrylamide and an organic solvent, perform the first liquid nitrogen freezing, the first vacuum pumping and the first argon gas introduction in sequence, and then perform the second liquid nitrogen freezing to obtain a first solid product;

[0016] (2) After mixing the first solid product obtained in step (1) with a chain transfer agent, successively perform third liquid nitrogen freezing, second vacuum pumping, and second argon introduction, and then perform fourth liquid nitrogen freezing to obtain a second solid product;

[0017] (3) After mixing the second solid product obtained in step (2) with an initiator, successively perform third vacuum pumping and third argon introduction, and then perform the first RAFT reaction (reversible addition-fragmentation chain transfer radical polymerization) to obtain a macro chain transfer agent intermediate, i.e., Macro-CTA;

[0018] (4) After mixing the Macro-CTA obtained in step (3) with a deoxygenated solution of diethylaminoethyl methacrylate, perform the second RAFT reaction to obtain a temperature-sensitive nano-polymer poly(N-isopropylacrylamide-b-diethylaminoethyl methacrylate).

[0019] Preferably, the temperature of the first RAFT reaction in step (3) is 65-75 °C, and the time of the first RAFT reaction is 6-24 h.

[0020] Preferably, after the second RAFT reaction in step (4), it further includes: successively dialyzing and freeze-drying the product of the second RAFT reaction to obtain a temperature-sensitive nano-polymer.

[0021] The present invention also provides the use of the nano-assembly described in the above technical solution or the nano-assembly prepared by the preparation method in the preparation of anti-tumor drugs.

[0022] The nano-assembly provided by the present invention has good thermosensitivity and sol-gel phase transition behavior, and has the characteristic of shear thinning. Due to the fluidity and high dispersion stability of the nano-assembly, it can be injected through a syringe or a microcatheter to achieve drug delivery. At the same time, the formed gel complex also has good applications in clinical TAE treatment; the nano-assembly can gelify at normal human body temperature, transform into a high-strength gel, and achieve the sustained release of procoagulants and chemotherapeutic drugs. And it can effectively increase the strength of blood clots and inhibit the degradation of blood clots. The hydrophobic interface after gelation can effectively activate platelets, thereby enhancing the coagulation behavior of procoagulants; the chemotherapeutic drugs of the nano-assembly can promote the apoptosis of tumor cells and activate the ICD effect, and can improve the immune microenvironment at the tumor site and activate the immune response through the infiltration of lymphocytes and the activation of dendritic cells, thereby inhibiting the metastasis of tumors. The nano-assembly loaded with procoagulants and chemotherapeutic drugs provided by the present invention realizes the synergistic treatment of embolization-chemotherapy-immunotherapy, and has great potential for clinical liver cancer treatment. The experimental results show that the PND-Pt-PolyP nano-assembly prepared in Example 1 of the present invention has good assembly behavior and presents a core-shell structure; due to the thermosensitive characteristics of the PND-Pt-PolyP nano-assembly prepared in Example 1, it is easy to inject at room temperature, and the network structure formed after gelation can effectively slow-release drugs. At the same time, the high-strength gel can also effectively block blood vessels; the procoagulant PolyP released by the nano-assembly prepared in Example 1 can effectively promote the generation of FXII and thrombin and increase the strength and stability of fibrin; the chemotherapeutic drug Pt released by the nano-assembly prepared in Example 1 can promote the apoptosis of tumor cells and enhance the ICD effect, and induce the maturation of DCs. The nano-assembly prepared in Example 1, with the synergistic treatment mode of embolization-chemotherapy-immunotherapy, can effectively inhibit tumor growth, reduce tumor metastasis, and enhance the anti-tumor immune response at the tumor site; at the same time, it is shown in the VX2 transplanted tumor rabbit liver cancer model that the PND-Pt-PolyP nano-assembly can achieve the synergistic embolization treatment of physical embolization of thermosensitive nanopolymers and thrombus embolization induced by PolyP, and has good clinical potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic flow chart of the preparation of the PND-Pt-PolyP nano-assembly in Example 1 of the present invention;

[0024] Figure 2 It is an electron micrograph of the PND-Pt-PolyP nano-assembly prepared in Example 1 of the present invention;

[0025] Figure 3 It is a particle size and potential diagram of the PND-Pt-PolyP nano-assembly prepared in Example 1 of the present invention;

[0026] Figure 4 Drug release curve of the PND-Pt-PolyP nano-assembly prepared in Example 1 of the present invention;

[0027] Figure 5 Thermosensitive sol-gel phase transition behavior diagrams of PND-Pt-PolyP in Example 1 of the present invention, PND in Example 1, PND-Pt in Comparative Example 2, and PND-PolyP in Comparative Example 4;

[0028] Figure 6 Influence trend diagram of the PND-Pt-PolyP nano-assembly prepared in Example 1 of the present invention on FXII generation;

[0029] Figure 7 Influence trend diagram of the PND-Pt-PolyP nano-assembly prepared in Example 1 of the present invention on thrombin generation;

[0030] Figure 8 Influence trend diagram of the PND-Pt-PolyP nano-assembly prepared in Example 1 of the present invention on fibrin;

[0031] Figure 9 Scanning electron micrograph of the clot by the PND-Pt-PolyP nano-assembly prepared in Example 1 of the present invention;

[0032] Figure 10 Influence trend diagram of the PND-Pt-PolyP nano-assembly prepared in Example 1 of the present invention on apoptosis of 4T1 tumor cells;

[0033] Figure 11 ICD effect diagram of the PND-Pt-PolyP nano-assembly prepared in Example 1 of the present invention on 4T1 tumor cells;

[0034] Figure 12 Influence trend diagram of the PND-Pt-PolyP nano-assembly prepared in Example 1 of the present invention on the maturation of DC cells;

[0035] Figure 13 Gross tumor image of the 4T1 subcutaneous tumor in the sixth mouse tested in the present invention;

[0036] Figure 14 Relative tumor growth rate diagram of the 4T1 subcutaneous tumor in the sixth mouse tested in the present invention;

[0037] Figure 15 Tumor weight change diagram of the 4T1 subcutaneous tumor in the sixth mouse tested in the present invention;

[0038] Figure 16 Tumor tissue immunofluorescence image of the 4T1 subcutaneous tumor in the sixth mouse tested in the present invention;

[0039] Figure 17 It is the graph of the number of lung nodules in the mouse lung metastasis model in Test 7 of the present invention;

[0040] Figure 18 It is the graph of the lung weight in the mouse lung metastasis model in Test 7 of the present invention;

[0041] Figure 19 It is the graph of the infiltration of CD8+ cells in the para-tumor lymph nodes of the mouse lung metastasis model in Test 7 of the present invention;

[0042] Figure 20 It is the graph of the maturation of DC cells in the para-tumor lymph nodes of the mouse lung metastasis model in Test 7 of the present invention;

[0043] Figure 21 It is the DSA graph and H&E staining graph at different times of the rabbit VX2 transplanted tumor liver cancer model in Test 8 of the present invention. Detailed implementation mode

[0044] The present invention provides a nano-assembly. The nano-assembly has a core-shell structure and includes a core composed of a procoagulant and a chemotherapeutic drug and a shell composed of a thermosensitive nano-polymer. The mass ratio of the procoagulant, chemotherapeutic drug to the thermosensitive nano-polymer in the nano-assembly is (10-75):4:80;

[0045] The thermosensitive nano-polymer is prepared by the RAFT method using N-isopropylacrylamide and diethylaminoethyl methacrylate as monomers.

[0046] In the present invention, the procoagulant is preferably polyphosphate; the degree of polymerization of the polyphosphate is preferably 25-250, and more preferably 45.

[0047] In the present invention, the chemotherapeutic drug is nitrated cisplatin or tetravalent platinum chemotherapeutic drug.

[0048] In the present invention, the mass ratio of the procoagulant, chemotherapeutic drug to the thermosensitive nano-polymer in the nano-assembly is preferably (10-75):4:80, and more preferably 10:4:80. The present invention controls the mass ratio of the procoagulant, chemotherapeutic drug to the thermosensitive nano-polymer in the nano-assembly within the above range to obtain a nano-assembly with better performance.

[0049] In the present invention, the degree of polymerization of the thermosensitive nano-polymer is preferably 100-250, and more preferably 220. The present invention controls the degree of polymerization of the thermosensitive nano-polymer within the above range to have better thermosensitive sol-gel phase transition behavior.

[0050] The nano-assembly provided by the present invention has good thermosensitivity and excellent gelation behavior, can be transformed into a high-strength gel, and can effectively increase the strength of blood clots, inhibit the degradation of blood clots for a long time, is sensitive to pH, is conducive to the release of polyphosphate in blood vessels with a slightly neutral pH value, thereby triggering blood clot embolism, exacerbating the anaerobic metabolism of tumor hypoxia, increasing the acidic environment at the tumor site, and further promoting the release of chemotherapeutic drugs by the acidic pH value at the tumor site, thereby improving the targeting property.

[0051] The present invention also provides a preparation method of the nano-assembly described in the above technical solution, including the following steps: After mixing the solution of the chemotherapeutic drug and the solution of the thermosensitive nano-polymer, adding dropwise the solution of the blood coagulant promoter, and performing a self-assembly reaction to obtain the nano-assembly.

[0052] In the present invention, when the chemotherapeutic drug is nitro-cisplatin, the preparation method of the solution of the chemotherapeutic drug includes the following steps: Mixing cisplatin and silver nitrate according to a molar ratio of 1:2, dissolving in water, stirring overnight in the dark at room temperature, and then centrifuging to remove the precipitate of silver nitrate, and the supernatant is the solution of the chemotherapeutic drug (or nitro-cisplatin solution). In the present invention, the nitro-cisplatin solution is preferably stored in a dark and 4°C environment.

[0053] In the present invention, the thermosensitive nano-polymer is preferably prepared by the RAFT method, including the following steps:

[0054] (1) After mixing N-isopropylacrylamide and an organic solvent, successively performing the first liquid nitrogen freezing, the first vacuum pumping, and the first argon introduction, and then performing the second liquid nitrogen freezing to obtain a first solid product;

[0055] (2) After mixing the first solid product obtained in the step (1) and a chain transfer agent, successively performing the third liquid nitrogen freezing, the second vacuum pumping, and the second argon introduction, and then performing the fourth liquid nitrogen freezing to obtain a second solid product;

[0056] (3) After mixing the second solid product obtained in the step (2) and an initiator, successively performing the third vacuum pumping and the third argon introduction, and then performing the first RAFT reaction to obtain a macro-chain transfer agent intermediate Macro-CTA;

[0057] (4) After mixing the macro-chain transfer agent intermediate Macro-CTA obtained in the step (3) and the deoxygenated solution of diethylaminoethyl methacrylate, performing the second RAFT reaction to obtain the thermosensitive nano-polymer poly(N-isopropylacrylamide-b-diethylaminoethyl methacrylate).

[0058] In the present invention, N-isopropylacrylamide and an organic solvent are preferably mixed, followed by first liquid nitrogen freezing, first vacuum pumping, and first argon gas introduction, and then second liquid nitrogen freezing to obtain a first solid product.

[0059] In the present invention, the preparation of the thermosensitive nano-polymer is preferably carried out in a sealed four-necked ground reaction tube.

[0060] In the present invention, the organic solvent is preferably DMSO. In the present invention, the molar ratio of N-isopropylacrylamide to the volume of the organic solvent is preferably (15 - 25) mmol : (8 - 12) mL. In the present invention, the mixing of N-isopropylacrylamide and the organic solvent is preferably carried out under stirring conditions.

[0061] In the present invention, the time of the first liquid nitrogen freezing is preferably 3 - 8 min, more preferably 4 - 6 min. The present invention utilizes the first liquid nitrogen freezing to fully solidify the liquid and prevent the liquid from splashing during vacuum pumping.

[0062] In the present invention, the time of the first vacuum pumping is preferably 3 - 8 min, more preferably 4 - 6 min. The present invention utilizes the first vacuum pumping to fully evacuate the oxygen in the bottle and prevent the quenching of the reaction by oxygen.

[0063] In the present invention, the time of the first argon gas introduction is preferably 3 - 8 min, more preferably 4 - 6 min. The present invention utilizes the first argon gas introduction to fully fill the inert gas and maintain an anaerobic environment for the reaction. In the present invention, the thawing after liquid nitrogen freezing is carried out under water bath conditions; the temperature of the water bath is preferably 15 - 35 °C, more preferably 30 °C. The present invention thaws under water bath conditions for rapid thawing.

[0064] In the present invention, the time of the second liquid nitrogen freezing is preferably 3 - 8 min, more preferably 4 - 6 min. The present invention utilizes the second liquid nitrogen freezing to fully solidify the liquid and prevent the liquid from splashing during vacuum pumping.

[0065] After obtaining the first solid product, in the present invention, the first solid product and a chain transfer agent are preferably mixed, followed by third liquid nitrogen freezing, second vacuum pumping, and second argon gas introduction, and then fourth liquid nitrogen freezing to obtain a second solid product.

[0066] In the present invention, the chain transfer agent is preferably 4-cyano-4-(thiobenzoyl) pentanoic acid. In the present invention, the molar ratio of N-isopropylacrylamide to 4-cyano-4-(thiobenzoyl) pentanoic acid is preferably 200 : 1. The present invention controls the molar ratio of N-isopropylacrylamide to 4-cyano-4-(thiobenzoyl) pentanoic acid within the above range to synthesize a polymer with a clear structure and good thermosensitive properties.

[0067] In the present invention, the time of the third liquid nitrogen freezing is preferably 3 - 8 min, more preferably 4 - 6 min. The present invention utilizes the second liquid nitrogen freezing to fully solidify the liquid and prevent the liquid from splashing during vacuum pumping.

[0068] In the present invention, the time of the second vacuum pumping is preferably 3 - 8 min, more preferably 4 - 6 min. The present invention utilizes the second vacuum pumping to fully evacuate the oxygen in the bottle and prevent the quenching of the reaction by oxygen.

[0069] In the present invention, the time of the second argon introduction is preferably 3 - 8 min, more preferably 4 - 6 min. The present invention utilizes the second argon introduction to fully fill the inert gas and maintain an anaerobic environment for the reaction. In the present invention, the thawing after the liquid nitrogen freezing is carried out under water bath conditions; the temperature of the water bath is preferably 15 - 35 °C, more preferably 30 °C. The present invention performs thawing under water bath conditions to quickly thaw.

[0070] In the present invention, the time of the fourth liquid nitrogen freezing is preferably 5 min. The present invention utilizes the fourth liquid nitrogen freezing to fully solidify the liquid and prevent the liquid from splashing during vacuum pumping.

[0071] After obtaining the second solid product, the present invention preferably mixes the second solid product and the initiator, and then sequentially performs the third vacuum pumping and the third argon introduction, and then conducts a reversible addition-fragmentation chain transfer radical polymerization (the first RAFT reaction) to obtain a macro chain transfer agent intermediate (Macro-CTA).

[0072] In the present invention, the initiator is preferably azobisisobutyronitrile. In the present invention, the molar ratio of N-isopropylacrylamide to the initiator is preferably 2000:1. The present invention controls the molar ratio of N-isopropylacrylamide to the initiator within the above range to fully initiate the RAFT reaction.

[0073] In the present invention, the time of the third vacuum pumping is preferably 3 - 8 min, more preferably 4 - 6 min. The present invention utilizes the third vacuum pumping to fully evacuate the oxygen in the bottle and prevent the quenching of the reaction by oxygen.

[0074] In the present invention, the time of the third argon introduction is preferably 3 - 8 min, more preferably 4 - 6 min. The present invention utilizes the third argon introduction to fully fill the inert gas and maintain an anaerobic environment for the reaction. In the present invention, the thawing after the liquid nitrogen freezing is carried out under water bath conditions; the temperature of the water bath is preferably 15 - 35 °C, more preferably 30 °C. The present invention performs thawing under water bath conditions to quickly thaw.

[0075] In the present invention, the temperature of the first RAFT reaction is preferably 65 - 75 °C, more preferably 70 °C; the time of the first RAFT reaction is preferably 6 - 24 h, more preferably 24 h. The present invention controls the temperature and time of the first RAFT reaction within the above ranges to fully react and obtain a macromolecular chain transfer agent Macro-CTA (i.e., pNIPAM).

[0076] After obtaining the intermediate, the present invention preferably mixes the intermediate with a solution of diethylaminoethyl methacrylate and then conducts a second RAFT reaction to obtain a temperature-sensitive nano-polymer poly(N-isopropylacrylamide-b-diethylaminoethyl methacrylate).

[0077] In the present invention, the solution of diethylaminoethyl methacrylate is preferably deoxygenated before use. In the present invention, the solvent used for the solution of diethylaminoethyl methacrylate is preferably DMSO. In the present invention, the molar ratio of N-isopropylacrylamide to diethylaminoethyl methacrylate is preferably (20 - 200):1. The present invention controls the molar ratio of N-isopropylacrylamide to diethylaminoethyl methacrylate within the above range to endow the polymer with both temperature-sensitive properties and surface positive charges.

[0078] In the present invention, the mixing of the intermediate and the solution of diethylaminoethyl methacrylate is preferably carried out under sealed conditions. In the present invention, the temperature of the second RAFT reaction is preferably 65 - 75 °C, more preferably 70 °C; the time of the second RAFT reaction is preferably 6 - 24 h, more preferably 24 h. The present invention controls the temperature and time of the second RAFT reaction within the above ranges to fully polymerize to obtain a temperature-sensitive nano-polymer {i.e., polymer p(NIPAM-b-DEAM) or PND}.

[0079] After the second RAFT reaction is completed, the present invention preferably subjects the product of the second RAFT reaction to dialysis and freeze-drying in sequence to obtain a temperature-sensitive nano-polymer.

[0080] In the present invention, the molecular weight cut-off of the dialysis bag used for dialysis is preferably 3000 - 10000 Da, more preferably 3500 Da; the time of dialysis is preferably 6 - 8 d. In the present invention, the freeze-drying is preferably to freeze-dry the dialysis solution obtained by dialysis at -40 °C for 48 h.

[0081] After obtaining the temperature-sensitive nano-polymer, the present invention preferably mixes the temperature-sensitive nano-polymer with ultrapure water to obtain a solution of the temperature-sensitive nano-polymer.

[0082] After obtaining the solution of the chemotherapeutic drug and the solution of the thermosensitive nano-polymer, the present invention mixes the solution of the chemotherapeutic drug and the solution of the thermosensitive nano-polymer, and then drops the solution of the coagulant to carry out a self-assembly reaction to obtain a nano-assembly.

[0083] In the present invention, the mixing of the solution of the chemotherapeutic drug and the solution of the thermosensitive nano-polymer is preferably stirred at room temperature for 45 to 55 h, more preferably 48 h. The present invention controls the mixing time within the above range to enable the full assembly of the chemotherapeutic drug and the thermosensitive nano-polymer.

[0084] In the present invention, the dropping rate is preferably 10 to 30 mL / h, more preferably 20 mL / h. The present invention controls the dropping rate within the above range to enable slow and full assembly and avoid aggregation.

[0085] In the present invention, the temperature of the self-assembly reaction is preferably room temperature; the time of the self-assembly reaction is preferably 22 to 26 h, more preferably 24 h. The present invention controls the temperature and time of the self-assembly reaction within the above range to avoid the polymer forming a sol at low temperature or a gel at high temperature, thus affecting the assembly behavior.

[0086] In the present invention, the nano-assembly is preferably stored in an environment of 4°C.

[0087] The present invention also provides the application of the nano-assembly described in the above technical solution or the nano-assembly prepared by the preparation method in the preparation of anti-tumor drugs.

[0088] In the present invention, the anti-tumor drug is preferably an anti-tumor drug for embolization-chemotherapy-immunotherapy synergy.

[0089] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

[0090] Example 1

[0091] The nano-assembly has a core-shell structure, including a core composed of a coagulant and a chemotherapeutic drug and a shell composed of a thermosensitive nano-polymer. The mass ratio of the coagulant, the chemotherapeutic drug to the thermosensitive nano-polymer in the nano-assembly is 10:4:80;

[0092] The chemotherapeutic drug is nitro-cisplatin, and the coagulant is polyphosphate with a polymerization degree of 45;

[0093] Preparation method of the nano-assembly: Weigh 300 mg of PND freeze-dried powder and dissolve it in 2.625 mL of ultrapure water. After complete dissolution, a solution of PND is obtained. Then, add 0.725 mL of a solution of nitro-cisplatin with a concentration of 21 mg / mL, and stir at room temperature for 48 h. Until the solution changes from white, clear and transparent to yellow, clear and transparent, take 900 μL of the PND-Pt solution, and dropwise add 100 μL of a solution of PolyP with a concentration of 100 mg / mL. Stir at room temperature for 24 h for self-assembly reaction to obtain the nano-assembly, denoted as PND-Pt-PolyP10, and store it in a 4 °C refrigerator;

[0094] The thermosensitive nano-polymer, i.e., PND, is prepared by the RAFT method using N-isopropylacrylamide and diethylaminoethyl methacrylate as monomers. The steps are as follows:

[0095] (1) Accurately weigh the monomer NIPAM (2.26 g, 20 mmol), add it to a tetrafluoride valve reaction tube equipped with a magnetic stir bar, accurately weigh DMSO (10 mL) and add it to the reaction tube. Stir on a constant temperature magnetic stirrer to completely dissolve NIPAM, transfer it to a liquid nitrogen bath for the first liquid nitrogen freezing for 4 min, use a vacuum pump for the first vacuum pumping for 5 min, stir in a hot water bath and conduct the first argon gas introduction for 5 min; after the first argon gas introduction is completed, conduct the second liquid nitrogen freezing to obtain a solid first solid product;

[0096] (2) Add the chain transfer agent 4-cyano-4-(thiobenzoyl) pentanoic acid (27.9 mg, 0.1 mmol) to the first solid product obtained in step (1). After stirring, conduct a cycle of the third liquid nitrogen freezing, the second vacuum pumping and the second argon gas introduction in sequence. After the second argon gas introduction is completed, conduct the fourth liquid nitrogen freezing to obtain a solid second solid product;

[0097] (3) Add the accurately weighed initiator AIBN (2.6 mg, 0.01 mmol) to the second solid product obtained in step (2), and conduct a cycle of the third vacuum pumping and the third argon gas introduction in sequence. After the reaction solution completely melts, place the reaction tube in a 70 °C oil bath for the first RAFT reaction for 24 h to obtain an intermediate;

[0098] (4) Add the deoxygenated diethylaminoethyl methacrylate solution (0.785 ml, 2 mmol) to the intermediate obtained in step (3) through a syringe, seal the syringe hole with vacuum glue, continue to place it in a 70 °C oil bath for the second RAFT reaction for 24 h. After the reaction is completed, transfer it to a dialysis bag (cut-off molecular weight 3500 Da) and dialyze for 7 d. Finally, freeze-dry the dialysate at -40 °C for 48 h to obtain the thermosensitive nano-polymer, i.e., PND 200-20 Freeze-dried powder.

[0099] The preparation steps of the solution of nitro-cisplatin are as follows;

[0100] Add cisplatin (300 mg, 1 mmol) and silver nitrate (340 mg, 2 mmol) to a clean 50 mL round-bottom flask according to a molar ratio of 1:2, dissolve with water, stir overnight in the dark at room temperature, then centrifuge to remove the precipitate AgCl, and the supernatant is the nitro-cisplatin solution. Subsequently, measure the concentration by ICP-OES and store it in the dark at 4 °C;

[0101] The solution of PolyP is an aqueous solution of polyphosphate with a degree of polymerization of 45.

[0102] Figure 1 This is a schematic flow chart for preparing the nano-assembly in Example 1 of the present invention. Specifically: First, prepare the chemotherapeutic drug nitro-cisplatin and the thermosensitive nano-polymer separately, and prepare the procoagulant polyphosphate. The three are subjected to a self-assembly reaction to obtain the nano-assembly.

[0103] Example 2

[0104] Prepare the nano-assembly according to the method of Example 1. Different from Example 1, the added mass of PolyP is 75 mg, and the prepared nano-assembly is denoted as PND-Pt-PolyP75.

[0105] Comparative Example 1

[0106] Prepare nitro-cisplatin according to the method of Example 1, denoted as Pt.

[0107] Comparative Example 2

[0108] Prepare PND-Pt according to the method of Example 1. Different from Example 1, weigh 300 mg of PND freeze-dried powder and dissolve it in 2.625 mL of ultrapure water. After complete dissolution, obtain the solution of PND, and then add 0.725 mL of the solution of nitro-cisplatin with a concentration of 21 mg / mL, and stir at room temperature for 48 h until the solution changes from white clear and transparent to yellow clear and transparent to obtain PND-Pt.

[0109] Comparative Example 3

[0110] Prepare Pt-PolyP according to the method of Example 1. Different from Example 1, add 0.725 mL of the solution of nitro-cisplatin with a concentration of 21 mg / mL to 2.625 mL of ultrapure water solution, and dropwise add 100 μL of the solution of PolyP with a concentration of 100 mg / mL, and stir at room temperature for 24 h for self-assembly reaction to obtain Pt-PolyP.

[0111] Comparative Example 4

[0112] The nano-assemblies were prepared according to the method of Example 1. Different from Example 1, the added mass of PolyP was 1 mg, and the prepared nano-assemblies were denoted as PND-Pt-PolyP1.

[0113] Comparative Example 5

[0114] The nano-assemblies were prepared according to the method of Example 1. Different from Example 1, 100 μL of a PolyP solution with a concentration of 100 mg / mL was added dropwise to 900 μL of a PND solution (88.9 mg / mL), and self-assembly reaction was carried out by stirring at room temperature for 24 h, denoted as PND-PolyP.

[0115] Test 1. Characterization of PND-Pt-PolyP10

[0116] The PND-Pt-PolyP10 nano-assemblies prepared in Example 1 and the assemblies prepared in the comparative example were diluted to 1 mg / mL according to the polymer concentration, observed by electron microscopy, the particle size and potential were measured by a dynamic light scattering particle size analyzer, and elemental Mapping was carried out. The electron micrographs are as Figure 2 shown, where Figure 2 in the first row, a is Pt-Poly, b is PND-Pt, c is PND-Pt-PolyP1, and d is PND-Pt-PolyP10, Figure 2 the second row is the elemental Mapping diagram (indicating the positions of different elements in the nano-assemblies), and the particle size and potential analysis diagrams are as Figure 3 shown.

[0117] It can be seen from Figure 2 that in the absence of polymer stabilization, nitro-cisplatin and PolyP showed an aggregated state. After the addition of PND for stabilization, with different addition amounts of PolyP, the morphology of the prepared nanomaterials gradually changed from a dispersed spherical structure to a uniform core-shell structure. The results of the elemental Mapping diagram showed that both Pt and PolyP were at the core position in the nano-assemblies, while the polymer formed a stable outer shell.

[0118] It can be seen from Figure 3It can be seen that without polymer stabilization, Pt and PolyP aggregate to form aggregates with a particle size of 4000 nm, and the potential is about -30 mV. The PND-Pt prepared in Comparative Example 1 is reduced to about -180 nm. Due to the action of cationic nanogels, the potential is about 15 mV. With the addition of polyphosphate PolyP, the potential gradually decreases. At the same time, when the addition amount of PolyP is 1, the charge of the assembly is neutral, the particles are unstable and aggregate, and the particle size increases to 400 nm. When PolyP is continuously added from 15 to 75, the particles tend to be stable, and the particle size is maintained at about 180 nm. Due to the addition of negatively charged polyphosphate PolyP, the potential gradually increases from -15 mV to about -22 mV. Therefore, the PND-Pt-PolyP10 prepared in Example 1 has good stability, forms a core-shell structure, and can effectively load Pt and PolyP into the core, which is beneficial to the stability and slow release of drugs.

[0119] Test 2. Drug release behavior of the PND-Pt-PolyP10 nanoassembly prepared in Example 1

[0120] Measure the drug release behavior of the PND-Pt-PolyP10 nanoassembly prepared in Example 1 under different conditions. The specific method is as follows:

[0121] (1) Prepare the release medium: By different ratios of sodium dihydrogen phosphate and disodium hydrogen phosphate, prepare PBS release buffers with pH values of 7.4 and 6.5 respectively.

[0122] (2) Take 200 μL of the prepared PND-Pt-PolyP10 nanoassembly solution in Example 1 and place it in a dialysis bag with a molecular weight cut-off of 3500 Da. Seal it with a clip. Set 3 parallels for each group. After gelation at 37 °C, then immerse the dialysis bag in a 50 mL centrifuge tube containing 30 mL of release solution that has been stabilized at 37 °C, and shake it in a shaker. The temperature of the shaker is 37 °C and the rotation speed is 180 rpm.

[0123] (3) At predetermined time points (0.5, 1, 2, 4, 8, 12, 24, 48, 72 h), take 1 mL of the release solution and supplement it with 1 mL of blank release solution. The released Pt and P contents in the released solution are detected by ICP-OES to obtain the drug release curve of the PND-Pt-PolyP10 nanoassembly prepared in Example 1, as shown in Figure 4 .

[0124] From Figure 4It can be seen that for the PND-Pt-PolyP10 nano-assemblies prepared in Example 1, at different pH values, Pt and PolyP exhibit different release behaviors. Pt is released under acidic conditions, while PolyP is slowly released in an environment with a pH of 6.5 and is released faster in a neutral environment with a pH of 7.4. The cumulative drug release amounts of the two reached 80% (Pt, pH 6.5) and 65% (PolyP, pH 7.4), respectively. The advantage of this is that when the nano-assemblies are injected into tumor blood vessels, the neutral pH value of the blood vessels is conducive to the release of PolyP, thereby triggering thrombus embolization, exacerbating the anaerobic metabolism of tumor hypoxia, increasing the acidic environment at the tumor site, and further promoting the release of Pt, thus exerting the chemotherapeutic anti-tumor treatment effect. That is to say, the PND-Pt-PolyP10 nano-assemblies prepared in Example 1 have pH sensitivity to the release of Pt and PolyP, improving their targeting.

[0125] Test 3: Sol-gel phase transition behavior of the PND-Pt-PolyP10 nano-assemblies prepared in Example 1

[0126] The PND-Pt-PolyP10 nano-assemblies in Example 1, the PND prepared in Example 1, the PND-Pt prepared in Comparative Example 2, and the PND-PolyP prepared in Comparative Example 5 were configured into solutions with a concentration of 80 mg / mL, and their thermosensitive sol-gel phase transition behaviors were characterized by a high-level rotational rheometer to obtain a thermosensitive sol-gel phase transition behavior diagram, as Figure 5 shown. It can be Figure 5 seen that due to the thermosensitive properties of the cationic thermosensitive nano-polymer, the PND-Pt-PolyP10 nano-assemblies, PND, PND-Pt, and PND-PolyP all have good gelation behaviors, and the gelation temperatures are all around 35°C. As the temperature increases, the elastic modulus of the nano-assembly material increases from 10 Pa at room temperature to 1000 Pa after gelation (8%, PND-Pt-PolyP10), indicating that the PND-Pt-PolyP10 nano-assemblies in Example 1 have excellent gelation behaviors, which can not only maintain the long-term release of drugs but also effectively block the blood vessels at the tumor site. At the same time, it has good shear thinning characteristics, and as the shear rate increases, the shear viscosity decreases significantly, making it easy to inject.

[0127] Test 4: In vitro procoagulant study of the PND-Pt-PolyP10 nano-assemblies

[0128] The PND-Pt-PolyP10 nanoassemblies in Example 1 were mixed with plasma, and the chromogenic substrates of factor XII (S-2302) and thrombin (S-2238) were added respectively. The absorbance at a wavelength of 405 nm was detected by an enzyme-labeled instrument to explore the influence trends of the material on the generation of FXII, thrombin, and fibrin. The graphs are respectively as Figure 6 , 7 and shown in Figure 8.

[0129] It can be seen from Figures 6 - 8 that the PND-Pt-PolyP10 nanoassemblies prepared in Example 1 can significantly promote the generation of FXII ( Figure 6 ) and thrombin ( Figure 7 ), can increase the turbidity of fibrin, and improve the stability of the fibrin structure ( Figure 8 );

[0130] At the same time, after mixing the PND-Pt-PolyP10 nanoassemblies with whole blood, it was placed at 37 °C for 10 min, then quickly frozen in liquid nitrogen, and freeze-dried to observe the influence of the material on the blood clot structure. The scanning electron micrograph of the blood clot of the PND-Pt-PolyP10 nanoassemblies prepared in Example 1 is as Figure 9 shown in Figure. It can be seen from Figure 9 that the nanoassemblies prepared in Example 1 exhibit a porous structure of the gel, which can facilitate the slow release of drugs. The pure blood clot shows a large number of red blood cells, with a complete and dispersed morphology. After the two are combined, it can be seen that the gel pores are filled with aggregated red blood cells, and the overall structure is denser, indicating that the gel formed by the PND-Pt-PolyP10 nanoassemblies prepared in Example 1 can stabilize the structure of the blood clot, facilitate the long-term maintenance of the blood clot, and inhibit the degradation of the blood clot.

[0131] Test Five: In Vitro Immune Activation of PND-Pt-PolyP10 Nanoassemblies

[0132] To investigate the effect of the PND-Pt-PolyP10 nanoassemblies prepared in Example 1 on the apoptosis of tumor cells, the specific steps are as follows: The 4T1 cells in good growth state were digested and collected, and then seeded into 24-well plates at 70,000 cells per well, and incubated overnight in an incubator to allow them to adhere. Solutions of Pt (prepared in Comparative Example 1), PND-Pt (prepared in Comparative Example 2), and PND-Pt-PolyP10 (prepared in Example 1) were prepared according to a Pt concentration of 20 μg / mL. The old medium in the 24-well plates was aspirated, and 1 mL of the above-prepared Pt, PND-Pt, and PND-Pt-PolyP10 solutions was added, with 3 wells in each group. The blank group was added with drug-free complete medium. After 24 h, staining was performed according to the apoptosis kit, and a flow cytometer was used to observe the proportion of cells in different apoptotic stages, and a trend graph of the effect on the apoptosis of 4T1 tumor cells was obtained as Figure 10 shown. It can be seen from Figure 10 this that the apoptosis is mainly due to the chemotherapeutic effect of Pt, and the groups containing Pt all have good ability to induce apoptosis of tumor cells. Among them, the PND-Pt-PolyP10 nanoassemblies can effectively promote the early and late apoptosis of tumor cells.

[0133] To investigate the effect of the PND-Pt-PolyP10 nanoassemblies prepared in Example 1 on the immunogenic cell death (ICD effect) of tumor cells, the specific steps are as follows: The 4T1 cells in good growth state were digested and collected, and then seeded into 24-well plates at 70,000 cells per well, and incubated overnight in an incubator to allow them to adhere. Solutions of Pt (prepared in Comparative Example 1), PND-Pt (prepared in Comparative Example 2), and PND-Pt-PolyP10 (prepared in Example 1) were prepared according to a Pt concentration of 20 μg / mL. The old medium in the 24-well plates was aspirated, and 1 mL of the prepared Pt, PND-Pt, and PND-Pt-PolyP10 solutions (where the concentration of each component was PND: 400 μg / mL, Pt: 20 μg / mL, PolyP: 50 μg / mL) was added, with 3 wells in each group. The blank group was added with drug-free complete medium. After 24 h, the expressions of CRT and HMGB1 were measured by flow cytometry, and the amount of ATP secreted into the supernatant was measured by an ATP kit to obtain the ICD effect graph of the nanoassemblies prepared in Example 1 on 4T1 tumor cells. Figure 11 The results show that the groups containing Pt can all induce the release of HMGB1 well, increase the expression of CRT, enhance the secretion of ATP, and enhance the ICD effect caused by the material, thereby activating the immune system for tumor immunotherapy.

[0134] To investigate the effect of PND-Pt-PolyP10 nanoassemblies on the maturation of dendritic cells (DCs), the specific steps are as follows: Well-grown 4T1 cells and DC2.4 cells were digested and collected, and then seeded into 24-well plates at 70,000 cells per dish and incubated overnight in an incubator to allow them to adhere. For the 4T1 cell plate, solutions of Pt (prepared in Comparative Example 1), PND-Pt (prepared in Comparative Example 2), and PND-Pt-PolyP10 (prepared in Example 1) were prepared at a Pt concentration of 20 μg / mL. The old medium in the 24-well plates was aspirated, and 1 mL of the prepared Pt, PND-Pt, and PND-Pt-PolyP10 solutions (with component concentrations of PND: 400 μg / mL, Pt: 20 μg / mL, and PolyP: 50 μg / mL) was added. There were 3 wells in each group, and the blank group was added with drug-free complete medium. After 12 h, the drug-containing medium was aspirated. The treated 4T1 cells were digested and collected, resuspended in 1 mL of medium, and used to replace the old medium of DC2.4, and they were co-cultured for 24 h. After the cells were digested and collected, a flow cytometer was used to analyze the maturation of DC cells, and a trend chart of the effect on DC cell maturation was obtained. The results are as Figure 12 shown. As Figure 12 can be seen, the groups containing Pt all have the ability to promote DC maturation. The main reason is that Pt promotes tumor cell apoptosis and produces the ICD effect. Among them, the PND-Pt-PolyP10 nanoassemblies can significantly increase the maturation of DC cells, thereby increasing the presentation of tumor-associated antigens and enhancing the immune response against tumors.

[0135] Test 6: Antitumor effect of the PND-Pt-PolyP10 nanoassemblies prepared in Example 1

[0136] Female BALB / c mice, six weeks old, with a body weight between 16 - 19 g, were purchased. After one week of adaptive feeding in the laboratory animal house, the hair around the right hind limb of the mice was shaved clean. 4T1 cells were cultured. When the cell number was sufficient and in the logarithmic phase, the cells were digested, centrifuged, and collected. After washing once with PBS, they were resuspended in PBS again to prepare a cell suspension of 10 7 / mL and placed on ice for use. 100 μL of the cell suspension was subcutaneously injected above the right hind limb of each mouse using a syringe. After the injection, the mice were continued to be fed. The tumor volume calculation formula is: V = (L × W^2) / 2, where V represents the tumor volume, L represents the major axis of the tumor, and W represents the minor axis. When the tumor grew to 100 mm 3Around, the mice were randomly divided into 5 groups with 5 mice in each group, and 50 μL of drugs were administered into the tumors respectively: (1) normal saline; (2) nitro-cisplatin; (3) thermosensitive nanopolymer PND; (4) PND-Pt; (5) PND-Pt-PolyP10; among which the concentration of PND in each group was 80 mg / mL, the concentration of Pt was 4 mg / mL, and the concentration of PolyP was 10 mg / mL. During the experiment, the body weight and tumor volume of the mice were measured every two days. On the 14th day after drug administration, the mice were sacrificed. After the mice were sacrificed, the tumors were dissected, and the excised tumors were weighed and photographed. The tumors were fixed with 4% paraformaldehyde, embedded in paraffin and sectioned, and subjected to HE staining, Ki67 and TUNEL immunofluorescence staining. The results are as Figure 6 shown. Due to the strong side effects of Pt, the free nitro-cisplatin group was basically dead on the third day, while the PND-Pt-PolyP nano-assembly significantly inhibited the side effects of Pt and was able to significantly inhibit the growth of tumors. The relative tumor growth rate graph of the 4T1 subcutaneous tumor in mice is as Figure 14 , the tumor weight change graph of the 4T1 subcutaneous tumor in mice is as Figure 15 , and the immunofluorescence graph of the tumor tissue of the 4T1 subcutaneous tumor in mice is as Figure 16 .

[0137] Through immunofluorescence staining of tumor tissues, the results are as Figure 16 shown, indicating that the PND-Pt-PolyP10 nano-assembly can significantly increase the apoptosis of tumor cells. At the same time, the release of PolyP can trigger the coagulation cascade reaction of tumor blood vessels, induce platelet aggregation, and the long-term embolization result leads to the destruction of blood vessels in tumor tissues, indicating that the PND-Pt-PolyP10 nano-assembly has the best anti-tumor effect.

[0138] Test Seven: Anti-tumor metastasis effect of PND-Pt-PolyP10 nano-assembly

[0139] Female BALB / c mice, six weeks old, with body weight between 16 - 19 g, were purchased. After being adaptively fed in the laboratory animal house for one week, the hair around the right hind limb of the mice was shaved clean. 4T1 cells were cultured. When the number of cells was sufficient and in the logarithmic phase, the cells were digested, centrifuged and collected. After washing once with PBS, the cells were resuspended with PBS again and prepared into a cell suspension of 10 7 / mL and placed on ice for use. Using a syringe, 100 μL of the cell suspension was subcutaneously injected above the right hind limb of each mouse. After the injection, the mice were continued to be fed. The tumor volume calculation formula is: V = (L×W^2) / 2, where V represents the tumor volume, L represents the long diameter of the tumor, and W represents the short diameter. When the tumor grew to 100 mm 3Around, mice were randomly divided into 5 groups with 5 mice in each group, and intratumoral administration was carried out respectively: (1) normal saline; (2) nitro-cisplatin; (3) thermosensitive nanopolymer PND; (4) PND-Pt; (5) PND-Pt-PolyP10; among which the concentration of PND in each group was 80 mg / mL, the concentration of Pt was 4 mg / mL, and the concentration of PolyP was 10 mg / mL. At the same time, 4T1 cells (100 μL, 10 6 / mL) were injected intravenously. After 21 days, the mice were sacrificed, the lung tissues were dissected, the lung nodules and weights were recorded, the para-tumor lymph nodes were dissected, and the infiltration of CD8 + and the maturation of DC cells were observed by flow cytometry, and the number of lung nodules in the mouse lung metastasis model was obtained as shown in Figure 17 , the lung weight diagram of the mouse lung metastasis model was as shown in Figure 18 , and the infiltration of CD8 + cells in the para-tumor lymph nodes of the mouse lung metastasis model was as shown in Figure 19 , and the maturation of DC cells in the para-tumor lymph nodes of the mouse lung metastasis model was as shown in Figure 20 ;

[0140] It can be seen from Figures 17 - 20 that after treatment with the PND-Pt-PolyP10 nanoassembly, the number of lung nodules in mice was significantly reduced, and the metastasis of tumors was inhibited ( Figure 17 and 18 ), at the same time, the infiltration of CD8 + cells in the para-tumor lymph nodes increased, and the maturation of DC cells increased. The above results indicate that the PND-Pt-PolyP10 nanoassembly prepared in Example 1 can effectively trigger the immune response of tumors, enhance the infiltration of lymphocytes and the maturation of DC cells, thereby inhibiting the metastasis of tumor cells.

[0141] Test VIII. Evaluation of the in vivo embolization effect of the PND-Pt-PolyP10 nanoassembly

[0142] New Zealand white rabbits. After the experimental rabbits with VX2 tumors implanted in the hind legs were anesthetized with 10% chloral hydrate (2.5 ml / mg), the skin was prepared and routinely disinfected. Then, the tumor tissue in the shape of fish meat with good activity was taken, rinsed with sterile phosphate buffered saline (PBS), and then cut into pieces with a size of 1 mm 3Left and right tumor masses were placed in Hanks' solution for later use. After weighing the experimental rabbits that had fasted and abstained from water for 12 hours, an intraperitoneal injection was performed at a dose of 1.4 - 1.7 ml / kg, with appropriate adjustments as needed. After routine skin preparation, the fully anesthetized experimental rabbits were fixed on a self-made rabbit board, placed on a sterile operating table, disinfected and covered with drapes. A longitudinal incision about 3 cm was made along the midline of the lower abdomen under the xiphoid process of the rabbit to fully expose the liver. An incision was made on the left lateral lobe of the liver (near the falciform ligament of the liver) with sterile ophthalmic forceps, and the VX2 tumor mass was implanted, and a 1-cm-sized absorbent gelatin sponge strip cut in advance was used to tamp it. After the incision was completely hemostatic, the liver was returned to the abdominal cavity, and then gentamicin (20,000 - 40,000 IU) was injected into the wound. After suturing the wound, ampicillin (10,000 - 20,000 IU) was intramuscularly injected for three consecutive days after the operation. Seventeen days later, after routine disinfection and covering with drapes, the rabbits with successful catheterization were placed on the table. Under the guidance of a 4F Cobra catheter and guide wire through the vascular sheath, the catheter was inserted into the main trunk of the rabbit celiac artery. The guide wire was withdrawn, and an appropriate amount of heparinized saline and iohexol contrast agent (300 mg I / ml) mixture was injected. After confirmation by "smoking", an appropriate amount of heparinized saline was injected to flush the catheter, and a high-pressure syringe was connected. Celiac arteriography was performed at a flow rate of 1 ml / s, a total volume of 3 ml, and a pressure of 200 Kpa. Then, through microcatheter superselection, the catheter tip was placed in the tumor-feeding blood vessel. After angiography confirmation at a flow rate of 0.5 ml / s, a total volume of 2 ml, and a pressure of 200 Kpa, the PND-Pt-PolyP nanocomposite was injected to obtain DSA images and H&E staining images of the rabbit VX2 transplanted liver cancer model at different times, as Figure 21 shown. As Figure 21 can be seen, before injecting the PND-Pt-PolyP10 nanocomposite, the tumor blood vessels showed a spherical structure and multi-polar branches under DSA ( Figure 21 a). After embolizing the tumor with PND-Pt-PolyP10, the tumor blood vessels could be blocked within 10 minutes and could not be visualized under DSA ( Figure 21 b). A good embolization effect was still presented four hours later ( Figure 21 c). After 4 hours, the tumor was dissected for H&E staining ( Figure 21 d). The results showed that there was a large amount of red blood cell aggregation in the tumor blood vessels and tumor tissues. This was because the released PolyP could effectively trigger the coagulation cascade reaction of tumor blood vessels, resulting in the formation of thrombus. At the same time, due to the physical barrier effect of the gel, the long-term embolization of the blood clot could be maintained, achieving a better liver cancer treatment effect.

[0143] In summary, the PND-Pt-PolyP10 nanoassemblies prepared in Example 1 have good assembly behavior and exhibit a core-shell structure; due to the thermosensitive properties of the PND-Pt-PolyP10 nanoassemblies prepared in Example 1, they are easy to inject at room temperature, and the network structure formed after gelation can effectively achieve sustained release of drugs. At the same time, the high-strength gel can also effectively embolize blood vessels; the procoagulant PolyP released by the nanoassemblies prepared in Example 1 can effectively promote the generation of FXII and thrombin, increasing the strength and stability of fibrin; the chemotherapeutic drug Pt released by the nanoassemblies prepared in Example 1 can promote the apoptosis of tumor cells and enhance the ICD effect, inducing the maturation of DCs. The nanoassemblies prepared in Example 1, with the synergistic treatment mode of embolization-chemotherapy-immunity, can effectively inhibit tumor growth, reduce tumor metastasis, and enhance the anti-tumor immune response at the tumor site; at the same time, it is shown in the VX2 transplanted tumor rabbit liver cancer model that the PND-Pt-PolyP10 nanoassemblies can achieve the synergistic embolization treatment of physical embolization of thermosensitive nanopolymers and thrombus embolization induced by PolyP, and have good clinical potential.

[0144] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A nano-assembly, characterized in that, The nano-assembly has a core-shell structure, including a core composed of a procoagulant and a chemotherapeutic drug, and a shell composed of a thermosensitive nano-polymer. The mass ratio of the procoagulant, chemotherapeutic drug and thermosensitive nano-polymer in the nano-assembly is (10-75):4:80; The procoagulant is polyphosphate, and the degree of polymerization of the polyphosphate is 25-250; The chemotherapeutic drug is nitrated cisplatin or a tetravalent platinum chemotherapeutic drug; The thermosensitive nano-polymer is prepared by the RAFT method using N-isopropylacrylamide and diethylaminoethyl methacrylate as monomers.

2. The nano-assembly according to claim 1, characterized in that, The mass ratio of the procoagulant, chemotherapeutic drug and thermosensitive nano-polymer is 10:4:

80.

3. The nano-assembly according to claim 1, characterized in that, The degree of polymerization of the thermosensitive nano-polymer is 100-250.

4. The preparation method of the nano-assembly according to any one of claims 1-3, comprising the following steps: after mixing the solution of the chemotherapeutic drug and the solution of the thermosensitive nano-polymer, dropping the solution of the procoagulant, and performing a self-assembly reaction to obtain the nano-assembly.

5. The preparation method according to claim 4, wherein The thermosensitive nano-polymer is prepared by the RAFT method, including the following steps: (1) After mixing N-isopropylacrylamide and an organic solvent, successively performing the first liquid nitrogen freezing, the first vacuum pumping and the first argon gas introduction, and then performing the second liquid nitrogen freezing to obtain a first solid product; (2) After mixing the first solid product obtained in the step (1) and a chain transfer agent, successively performing the third liquid nitrogen freezing, the second vacuum pumping and the second argon gas introduction, and then performing the fourth liquid nitrogen freezing to obtain a second solid product; (3) After mixing the second solid product obtained in the step (2) and an initiator, successively performing the third vacuum pumping and the third argon gas introduction, and then performing the first RAFT reaction to obtain a macromolecular chain transfer agent intermediate Macro-CTA; (4) After mixing the macromolecular chain transfer agent intermediate Macro-CTA obtained in the step (3) and the deoxygenated solution of diethylaminoethyl methacrylate, performing the second RAFT reaction to obtain the thermosensitive nano-polymer poly(N-isopropylacrylamide-b-diethylaminoethyl methacrylate).

6. The preparation method according to claim 5, wherein The temperature of the first RAFT reaction in the step (3) is 65-75 °C, and the time of the first RAFT reaction is 6-24 h.

7. The preparation method according to claim 5, wherein After the second RAFT reaction in the step (4), it further includes: dialyzing and freeze-drying the product of the second RAFT reaction to obtain the thermosensitive nano-polymer.

8. The application of the nano-assembly according to any one of claims 1-3 or the nano-assembly prepared by the preparation method according to any one of claims 4-7 in the preparation of anti-tumor drugs.

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