In-situ forming pH-responsive hydrogel for tumor immunotherapy and preparation method and application thereof

Abstract

The application discloses in-situ formed pH-responsive hydrogel for tumor immunotherapy and a preparation method and application thereof, and belongs to the technical field of biological medicine and nanomaterials. The hydrogel is formed by cross-linking aldehyde-terminated Pluronic F127 (CHO-F127-CHO) and polyvinylamine hydrochloride (PVAH) through Schiff base reaction to form a three-dimensional network structure, and is internally loaded with a blood vessel damaging agent DMXAA and type I sonodynamic nanoparticles Ti-TCPP@F127-CHO. Animal experiments show that the hydrogel combined with ultrasonic treatment realizes complete tumor regression in various tumor models, effectively inhibits postoperative recurrence and distant metastasis, and establishes long-term immune memory. The application realizes the timing synergy of blood vessel damage and type I sonodynamic therapy, provides a safe and efficient strategy for immunotherapy of hypoxic tumors, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and nanomaterials technology, specifically relating to an in-situ formed pH-responsive hydrogel for tumor immunotherapy, its preparation method, and its application. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Malignant tumors are a major disease that seriously threatens human health. Traditional tumor treatments include surgical resection, chemotherapy, and radiotherapy, but all have limitations to varying degrees. For example, surgery cannot completely remove micrometastases, and chemotherapy and radiotherapy have significant toxic side effects and are prone to drug resistance. In recent years, immunotherapy has made groundbreaking progress in the field of tumor treatment. However, the hypoxic tumor microenvironment that is prevalent in solid tumors severely inhibits the activation of the immune response, becoming one of the key bottlenecks restricting the efficacy of immunotherapy.

[0004] Panapoptosis is a newly discovered form of programmed cell death that integrates three cell death pathways: apoptosis, pyroptosis, and necroptosis. It can simultaneously activate multiple cell death signaling pathways, enhancing tumor immunogenicity and overcoming drug resistance issues associated with single cell death pathways. Reactive oxygen species (ROS) have been proven to be an effective means of inducing panapoptosis, initiating multiple cell death pathways through oxidative stress, mitochondrial dysfunction, inflammasome activation, and lipid peroxidation. However, the hypoxic tumor microenvironment severely limits ROS generation, especially for oxygen-dependent conventional photodynamic therapy or classic sonodynamic therapy, where hypoxia significantly weakens their therapeutic effects.

[0005] Sonodynamic therapy (SDT) utilizes ultrasound to activate sonosensitive agents, generating reactive oxygen species (ROS) to kill tumor cells. It offers advantages such as deep tissue penetration and non-invasiveness. Traditional SDT primarily relies on the type II pathway (energy transfer to generate singlet oxygen), which is also limited by oxygen concentration. In contrast, type I SDT generates ROS such as superoxide anion radicals and hydroxyl radicals through an oxygen-independent electron transfer process, enabling it to function efficiently under hypoxic conditions and offering a potential solution to overcome hypoxia-related therapeutic bottlenecks.

[0006] In addition, tumor angiogenesis disruptors (VDAs) such as 5,6-dimethylxanthonone-4-acetic acid (DMXAA) can selectively destroy tumor blood vessels, leading to tumor ischemia and hypoxia. Early clinical trials have shown that VDAs have manageable safety and preliminary anti-tumor efficacy.

[0007] Currently, there is no known treatment regimen that combines vascular destructive agents with type I sonodynamic therapy. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide an in-situ formed pH-responsive hydrogel for tumor immunotherapy, its preparation method, and its application.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an in-situ formed pH-responsive hydrogel for tumor immunotherapy, wherein the hydrogel is formed by cross-linking aldehyde-terminated Pluronic F127 (CHO-F127-CHO) and polyethyleneamine hydrochloride (PVAH) through a Schiff base reaction to form a three-dimensional network structure; the hydrogel is loaded with an angiogenic agent and type I sonodynamic nanoparticles; the hydrogel is formed in situ by co-extruding an aldehyde-terminated Pluronic F127 solution containing an angiogenic agent and type I sonodynamic nanoparticles with a polyethyleneamine hydrochloride solution using a dual-tube syringe.

[0010] Furthermore, this in-situ formed pH-responsive hydrogel can degrade under pH conditions of 5.0 to 6.5, first rapidly releasing a vascular disruptor, and then slowly and continuously releasing type I sonodynamic nanoparticles to achieve sequential drug release; in addition, the hydrogel can maintain structural stability under pH conditions of 7.4.

[0011] Preferably, the vascular disruptor includes monoclonal antibodies (such as Tarvacin) and small molecule VDAs, including colchicine, CA4 analogs (such as CA4P, ombrabulin, ZD6126, OXI4503, BNC105P, DX1002, and C118P) and flavonoid drugs (such as DMXAA (Vadimezan) and MN029).

[0012] Preferably, the type I acoustic dynamic nanoparticles are Ti-TCPP@F127-CHO nanoparticles obtained by modifying titanium-coordinated meso-tetra(4-carboxyphenyl)porphyrin (Ti-TCPP) with aldehyde-terminated CHO-F127-CHO groups; in the Ti-TCPP nanoparticles, titanium and TCPP are linked by coordination bonds.

[0013] Although the strategy of tumor angiogenesis disruptors may exacerbate tumor hypoxia when used alone, when combined with type I sonodynamic therapy, it can form a synergistic treatment mode of "first exacerbating hypoxia, and then efficiently killing tumors under hypoxic conditions".

[0014] This invention loads a vascular disruptor and type I sonodynamic nanoparticles into a pH-responsive injectable hydrogel to achieve time-sequential release and to induce panapoptosis of tumor cells and activate anti-tumor immunity.

[0015] Secondly, the present invention provides a method for preparing an in-situ formed pH-responsive hydrogel for tumor immunotherapy, comprising the following steps: Step 1, Preparation of Ti-TCPP nanoparticles: TiCl4·2THF was dissolved in dimethylformamide (DMF), and meso-tetra(4-carboxyphenyl)porphyrin (TCPP) was dissolved in DMF. After mixing the two, acetic acid was added, the mixture was refluxed, centrifuged, and washed to obtain Ti-TCPP nanoparticles. Step 2, Preparation of CHO-F127-CHO: Pluronic F127 was dissolved in dichloromethane, and Dess-Martin periodane reagent was added. After the reaction, the mixture was concentrated, precipitated, dialyzed, and lyophilized to obtain aldehyde-terminated Pluronic F127 (CHO-F127-CHO). Step 3, Preparation of Ti-TCPP@F127-CHO nanoparticles: The Ti-TCPP nanoparticles obtained in Step 1 and the CHO-F127-CHO obtained in Step 2 were dissolved in tetrahydrofuran (THF), ultrasonically dispersed, injected into deionized water, and the THF was evaporated to obtain Ti-TCPP@F127-CHO nanoparticles. Step 4, Preparation of PVAH: N-vinylformamide is polymerized with the polymerization initiator azobisisobutyramidine hydrochloride (AIBA), and after concentration, precipitation and drying, poly(N-vinylformamide) is obtained; it is then subjected to amide hydrolysis with NaOH solution, neutralized with HCl, dialyzed and lyophilized to obtain polyvinylamine hydrochloride (PVAH). Step 5, preparation of hydrogel: The Ti-TCPP@F127-CHO nanoparticles and vascular disruptor obtained in step 3 are dissolved in a buffer salt solution as the first precursor solution; the PVAH obtained in step 4 is dissolved in a buffer salt solution as the second precursor solution; the first and second precursor solutions are respectively loaded into the two chambers of a dual-tube syringe and co-extruded through a static mixing head to obtain an in-situ formed pH-responsive hydrogel.

[0016] Preferably, the mass ratio of TiCl4·2THF to TCPP in step 1 is 1.5:1 to 2.5:1, more preferably 1.8:1 to 2.2:1.

[0017] Further, in step 1, the amount of acetic acid added is 180-220 μL of acetic acid per 40 mg TiCl4·2THF. The reaction time is 10-14 hours; preferably 12 hours.

[0018] Preferably, the mass ratio of Pluronic F127 to Dess-Martin periodane in step 2 is 9:1 to 11:1.

[0019] Preferably, the mass ratio of Ti-TCPP nanoparticles to CHO-F127-CHO in step 3 is 0.5:45 to 0.5:55.

[0020] Preferably, in steps 1 to 3, the organic solvent is selected from one or a combination of several of dimethylformamide (DMF), dichloromethane, tetrahydrofuran, and trichloromethane.

[0021] Further, the molar ratio of N-vinylformamide to AIBA in step 4 is 500:1 to 1000:1, preferably 545:1.

[0022] Further, the mass ratio of poly(N-vinylformamide) to NaOH in step 4 is 1~10:1~10, preferably 6:7.6.

[0023] Further, in step 5, the concentration of Ti-TCPP@F127-CHO in the first precursor solution is 50~500 μM (calculated as Ti-TCPP), the concentration of CHO-F127-CHO is 1%~20% w / v, and the concentration of DMXAA is 0.1~10 mg / mL; the concentration of PVAH in the second precursor solution is 1%~10% w / v.

[0024] Furthermore, in step 5, the volume ratio of the first precursor liquid to the second precursor liquid is 0.5~2:0.5~2, preferably 1:1.

[0025] Furthermore, the buffer salt solution mentioned in step 5 is PBS.

[0026] Thirdly, the present invention provides the application of the in-situ formed pH-responsive hydrogel in the preparation of antitumor drugs.

[0027] Furthermore, the applications include use in the preparation of drugs for inducing pan-apoptosis of tumor cells, use in the preparation of drugs for activating anti-tumor immune responses, and use in the preparation of drugs for inhibiting tumor growth, recurrence, and / or metastasis.

[0028] Furthermore, the tumors mentioned include colon cancer and melanoma.

[0029] Furthermore, the application is in conjunction with ultrasound, with an ultrasound frequency of 20~50 kHz, preferably 30 kHz; an ultrasound power of 1~5 W, preferably 2 W; and an ultrasound time of 2~20 min, preferably 10 min.

[0030] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: (1) This invention is the first to load the vascular disruptor DMXAA and type I sonodynamic nanoparticles together in a pH-responsive injectable hydrogel. By triggering the degradation of the hydrogel through the acidic microenvironment of the tumor, the sequential delivery of DMXAA is achieved first and the nanoparticles are released later, forming a synergistic treatment mode from "vascular disruption exacerbates hypoxia" to "type I sonodynamics utilizes hypoxia to efficiently produce ROS", breaking through the limitations of traditional oxygen-dependent therapy and overcoming the treatment bottleneck in hypoxic environments.

[0031] (2) The Ti-TCPP@F127-CHO nanoparticles used in this invention generate a variety of ROS such as superoxide anion radicals and hydroxyl radicals through the type I electron transfer pathway under ultrasonic irradiation. They do not depend on oxygen participation and can play an efficient role in the severely hypoxic tumor microenvironment.

[0032] (3) The ROS burst generated by this invention can efficiently induce panapoptosis in tumor cells (simultaneously activating three death pathways: apoptosis, pyroptosis and necroptosis), overcome the drug resistance problem of single cell death pathways, and induce strong immunogenic cell death (ICD), promoting the release of tumor antigens and the maturation of dendritic cells.

[0033] (4) The hydrogel of the present invention is formed by cross-linking through Schiff base bonds and has good pH responsiveness: it gradually dissociates in the acidic tumor microenvironment (pH 5.5~6.5) and remains stable under physiological pH (7.4) conditions, thus realizing the controlled release and tumor-specific delivery of drugs.

[0034] (5) The hydrogel of the present invention can be injected in situ and rapidly gelled using a dual-tube syringe. It is easy to operate, suitable for intratumoral injection, and has good prospects for clinical translation.

[0035] (6) The present invention has shown excellent anti-tumor effects in various animal tumor models (colon cancer, melanoma), and can significantly inhibit the growth of primary tumors, prevent postoperative recurrence and distant metastasis, and establish long-term anti-tumor immune memory. Attached Figure Description

[0036] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0037] Figure 1 The images show (a) the particle size distribution and (b) the transmission electron microscope image of the Ti-TCPP nanoparticles synthesized in Example 1.

[0038] Figure 2The images show (a) photoelectron spectroscopy (XPS spectrum), (b) the corresponding high-resolution Ti 2p spectrum, and (c) the UV-Vis-NIR spectra of the Ti-TCPP nanoparticles synthesized in Example 1.

[0039] Figure 3 The following are the (a) synthetic chemical formula, (b) Fourier transform infrared spectrum and (c) 1H nuclear magnetic resonance spectrum of CHO-F127-CHO in Example 1.

[0040] Figure 4 The diagram shows (a) the preparation of Ti-TCPP@F127-CHO nanoparticles in Example 1 and (b) the particle size distribution.

[0041] Figure 5 The image shows the 1H NMR spectrum of polyvinylamine hydrochloride (PVAH) in Example 1.

[0042] Figure 6 The diagram shows the formation process of DM-TC@Gel formed by the crosslinking reaction of Ti-TCPP@F127-CHO and polyethyleneamine hydrochloride in Example 1 (a) and a photograph of DM-TC@Gel (b).

[0043] Figure 7 The images show (a) a representative scanning electron microscope image of DM-TC@Gel in Example 1 and (b) the storage modulus (G') and loss modulus (G'') of DM-TC@Gel obtained by oscillation time scanning.

[0044] Figure 8 The figures show the typical elasticity index (EI) curve, solid-liquid equilibrium (SLB) curve, and mean self-diffusion distance (MSD) curve of (a) in situ hydrogel during decomposition in buffer solutions at different pH values ​​(pH 7.4, 6.5, 5.5) in Example 1.

[0045] Figure 9 The images show (a) photographs of the in-situ hydrogel DM-TC@Gel at different time points in different pH buffer solutions and (b) the DMXAA release curve in Example 1.

[0046] Figure 10 For example 2, (a) confocal microscopy images of Ti-TCPP@F127-CHO cells at 0, 0.5, 1, and 2 h and (b) flow cytometry analysis.

[0047] Figure 11Examples 2 show (a) confocal fluorescence images of CT26 cells stained with JC-1 under different treatment conditions and (b) corresponding statistical analysis of mitochondrial membrane potential.

[0048] Figure 12 For Example 2, under normoxic and hypoxic conditions, (a) the relative cell viability of CT26 cells after co-incubation with different concentrations of TCF and ultrasound irradiation; (b, c) the relative cell viability of CT26 cells under different treatment conditions and Calcein AM / PI co-staining.

[0049] Figure 13 For example 3, under different treatment conditions (noroxic and hypoxic), (a) flow cytometry analysis of CT26 cells after co-staining with Annexin V-FITC and PI and (b) corresponding quantitative data.

[0050] Figure 14 (a, b) Confocal fluorescence images and corresponding quantitative analyses of CT26 cells co-incubated with Cleave-caspase 3 antibody under different treatment conditions in Example 3; (c) Western blot analysis of Cleave-caspase 3 in cells after different treatments under hypoxia conditions.

[0051] Figure 15 The images shown are: (a) bright-field images of CT26 cells under different treatment conditions (noroxic and hypoxic) in Example 3; and (b) confocal fluorescence images of CT26 cells after different treatments under hypoxic conditions stained with T11.

[0052] Figure 16 The images show (a) immunoblotting analysis of Cleave-caspase 1 and GSDMD-N in CT26 cells after different treatments under hypoxic conditions in Example 3, and (b) confocal fluorescence images stained with GSDMD-N antibody.

[0053] Figure 17 The release of (a) IL-1β and (b) lactate dehydrogenase in CT26 cells after different treatments in Example 3.

[0054] Figure 18 For example 3, (a) confocal fluorescence images of CT26 cells stained with YO-PRO-1 / PI after different treatments under normoxic and hypoxic conditions; (b) Western blot analysis of P-MLKL and P-RIPK1 expression in CT26 cells after different treatments under hypoxic conditions.

[0055] Figure 19The images show (a) immunofluorescence images of CRTs exposed on the surface of CT26 tumor cells under different treatment conditions in Example 4, and (b) the corresponding quantitative analysis.

[0056] Figure 20 Immunofluorescence images of HMGB1 in CT26 cells under different treatment conditions in Example 4 (a) and corresponding quantitative analysis (b).

[0057] Figure 21 The amount of ATP secreted by CT26 cells under different treatment conditions in Example 4.

[0058] Figure 22 The diagram shows (a) mature DCs treated under different hypoxic conditions in Example 4; (b) a representative flow cytometry diagram; and (c) the corresponding quantitative analysis.

[0059] Figure 23 Fluorescence imaging and corresponding quantitative fluorescence results were performed on CT26 tumor mice injected with TC NPs and DM-TC@Gel in Example 5 (a) and (b) respectively.

[0060] Figure 24 Examples 5 show (a) hypoxic immunofluorescence images of mouse tumor sections treated with different methods and (b) corresponding quantitative analyses.

[0061] Figure 25 The figures for (a) mean tumor growth curve, (b) mouse survival rate, and (c) mean weight change of CT26 tumor mice after different treatments in Example 5 are shown.

[0062] Figure 26 The following are examples from Example 5: (a) treatment regimen for CT26 tumor recurrence; (b) mean tumor growth curve of mice after re-stimulation with CT26 tumor; (c) number of central memory T cells and effector memory T cells in peripheral blood of mice; and (d) IFN-γ and TNF-α levels in serum of mice from the untreated group and the DM-TC@Gel + US treatment group.

[0063] Figure 27 The diagram shows the experimental design of (a) B16F10 melanoma-bearing mice for treatment in Example 6; and (b) immunofluorescence staining of reactive oxygen species in mouse tumors under different treatment methods.

[0064] Figure 28 The figures show (a) mean tumor growth curve, (b) survival rate, and (c) mean weight change of mice carrying B16F10 tumors under different treatments in Example 6.

[0065] Figure 29The treatment regimen for tumor recurrence in the tumor resection mouse model in Example 6 (a); and (bd) the average recurrence tumor growth curve, survival rate, and average body weight change of B16F10 tumor-bearing mice under different treatments.

[0066] Figure 30 The diagram shows (a) lung metastasis in the B16F10 tumor recurrence mouse model in Example 6; and (b) representative photographs of mouse lungs collected under different treatment methods and corresponding H&E-stained lung sections.

[0067] Figure 31 The following are flow cytometry images and corresponding quantitative results of (a) CD8+ T cells and (b) NK1.1 cells in mouse tumors under different treatments in Example 7.

[0068] Figure 32 For Example 7, (a) the secretion levels of IFN-γ, TNF-α, IL-1β and IL-6 in mouse tumors under different treatments; (b) the content of IFN-γ in mouse serum under different treatments.

[0069] Figure 33 For Example 7, (a) is the average curve of tumor growth in different groups of mice after re-exposure to CT26 tumor; (b) is a representative flow cytometry plot showing the status of central memory T cells and effector memory T cells in the peripheral blood of mice receiving different treatments; and (c) is the content of TNF-α and IFN-γ in the serum of mice in different treatment groups.

[0070] Figure 34 A schematic diagram illustrating the working mechanism of in-situ formed pH-responsive hydrogels in sequential angiogenesis and type I sonodynamic therapy-driven panapoptosis. (A) Stage 1: Tumor microenvironment modulation; (B) Stage 2: Sonodynamically driven panapoptosis. Detailed Implementation

[0071] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0072] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0073] The materials involved in this invention include: N,N-Dimethylformamide (DMF), acetic acid (AcOH), tetrahydrofuran (THF), sodium hydroxide methanol solution, and Triton X-100 were purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd. Titanium tetrachloride tetrahydrofuran complex (TiCl4·2THF) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd., China. Meso-tetra(4-carboxyphenyl)porphyrin (TCPP) was purchased from Shanghai Mairui Biochemical Technology Co., Ltd., China. DMXAA was purchased from Anhui Zesheng Technology Co., Ltd., China. Pluronic F127 (12,600 Da) was purchased from Sigma-Aldrich. Dichloromethane was purchased from Tianjin Fuyu Fine Chemical Co., Ltd., China. n-Hexane was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., China. N-Vinylformamide, Dys-Martin periodide, and 2,2-azobis(2-methylpropylimidazolium) dihydrochloride were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., China. Hydrochloric acid was purchased from Yantai Yuandong Fine Chemical Co., Ltd., China. Green singlet oxygen probe (SOSG) was purchased from Meilun Biotechnology Co., Ltd., China. 1,3-Diphenylisobenzofuran (DPBF), dihydrorhodamine 123 (DHR 123), and DCFH-DA were all purchased from Solarbio Science & Technology Co., Ltd., Beijing, China. Methylene blue (MB) was purchased from Ningbo Road Chemical Reagent Co., Ltd., Shanghai, China. Live and dead cell staining kits were purchased from Tianyu Biotechnology Co., Ltd., China. Annexin V-FITC / PI apoptosis detection kit, Calcein-AM / propidium iodide (PI), ATP detection kit, and 4',6-diamidinyl-2-phenylindole (DAPI) were all purchased from Beyotime Biotechnology Co., Ltd., China. YO-PRO-1 was purchased from Beyotime Biotechnology Co., Ltd., China. CRT and HMGB1 antibodies were purchased from Suzhou Youyilandi Biotechnology Co., Ltd., China. Hypoxyprobe™ Plus kit was purchased from Hypoxyprobe. Cleaved-Caspase 3 antibody was purchased from the China National Center for Biopharmaceutical Research. The flow cytometry antibodies were purchased from Biolegend: anti-CD3-FITC (clone 17A2), anti-CD4-APC (clone RM4-5), anti-CD8a-PE (clone S18018E), anti-CD11b-PerCP (clone M1 / 70), anti-Gr-1-AF700 (clone RB6-8C5), anti-CD45-Brilliant Violet 650 (clone 30-F11), anti-CD44-APC (clone IM7), anti-CD62L-PE / Cyanine7 (clone MEL-14), and anti-NK1.1-PE / eFluor610 (clone PK136).The enzyme-linked immunosorbent assay (ELISA) kits were purchased from Thermo Fisher Scientific: mouse tumor necrosis factor α (TNF-α, catalog number 88-7324-88), interferon γ (IFN-γ, catalog number 88-7314-88) and interleukin 12p70 (IL-12p70, catalog number 88-7121-88).

[0074] Statistical calculations: p-values ​​were calculated using Tukey's multiple comparisons one-way ANOVA; "ns" indicates no significant difference. "This indicates that P < 0.05," "This indicates that P < 0.01," This indicates that P < 0.001.

[0075] In embodiments of the present invention, a schematic diagram is provided illustrating the working mechanism of in-situ formed pH-responsive hydrogels in sequential vascular disruption and type I sonodynamic therapy-driven pan-apoptosis. For example... Figure 34 As shown in Figure (A), the preparation of DM-TC@Gel and the sequential release of DMXAA and Ti-TCPP under acidic conditions are illustrated. Furthermore, DM-TC@Gel generates a large amount of reactive oxygen species under ultrasound. After intratumoral injection, DM-TC@Gel hydrogel forms in situ. The acidic tumor microenvironment initially triggers the rapid release of DMXAA, disrupting tumor angiogenesis and exacerbating hypoxia. Figure 34 As shown in (B), the released Ti-TCPP nanoparticles are subsequently activated by ultrasound, driving oxygen-independent type I sonodynamic therapy, generating a large amount of reactive oxygen species, which efficiently induce pan-apoptosis in tumor cells. This sequential release mode can trigger strong immunogenic cell death, thereby significantly promoting tumor regression, inhibiting metastasis, and establishing durable immune memory to prevent recurrence.

[0076] In the embodiments of this invention and the accompanying drawings, Ti-TCPP@F127-CHO is abbreviated as TCF, and Ti-TCPP is abbreviated as TC.

[0077] Example 1: Synthesis and characterization of in-situ formed pH-responsive hydrogels: (1) Dissolve 40 mg TiCl4·2THF in 10 mL DMF, and dissolve 20 mg TCPP in 40 mL DMF. Mix the two solutions, add 200 μL of acetic acid, and reflux at 90°C for 12 hours with vigorous stirring. After the reaction is complete, collect the precipitate by centrifugation, wash three times with ethanol to obtain Ti-TCPP nanoparticles.

[0078] (2) The transmission electron microscopy (TEM) image of the obtained Ti-TCPP nanoparticles shows that they have a monodisperse spherical morphology with a particle size of approximately 102.7 nm. Figure 1 (ab). X-ray photoelectron spectroscopy showed that the Ti 2p peaks were located at 457.85 eV and 465.10 eV, corresponding to Ti2p3 / 2 and Ti 2p1 / 2, confirming the presence of titanium. Figure 2 a, b). The UV-Vis absorption spectrum shows the characteristic absorption peak of TCPP (416 nm), confirming the successful loading of porphyrin. Figure 2 c).

[0079] (3) Dissolve 10 g of Pluronic F127 in 200 mL of dichloromethane, add 1 g of Dess-Martin periodane reagent, stir at room temperature for 30 minutes, then heat to 40°C and react for 24 hours. After the reaction is complete, concentrate under vacuum, precipitate in ice-cold petroleum ether, dialyze the crude product against deionized water using a dialysis bag with a molecular weight cutoff of 3500 Da, and freeze-dry to obtain CHO-F127-CHO ( Figure 3 a).

[0080] (4) Fourier transform infrared spectroscopy showed that the product was at 1730 cm⁻¹ -1 The characteristic absorption peak appears at this point, corresponding to the C=O stretching vibration of the aldehyde group. Figure 3 b). The 1H NMR spectrum showed proton signal peaks for the methylene (-CH2-) and aldehyde (-CHO) groups, with an integrated area ratio of 2:1, confirming the successful introduction of the aldehyde group. Figure 3 c).

[0081] (5) Dissolve 0.5 mg of Ti-TCPP nanoparticles obtained in Example 1 and 50 mg of CHO-F127-CHO obtained in (3) in 200 μL of THF, disperse by ultrasonication, inject into 1 mL of deionized water, stir vigorously for 24 hours, evaporate the THF, and obtain Ti-TCPP@F127-CHO nanoparticles. Dynamic light scattering showed that the hydrated particle size of the obtained nanoparticles was approximately 130.9 nm, and the particle size distribution was narrow ( Figure 4 (ab).

[0082] (6) 7 mL (0.109 mol) of N-vinylformamide and 54.4 mg (0.2 mmol) of AIBA were added to a three-necked flask containing 100 mL of water and polymerized at 60°C for 4 hours under N2 protection. The reaction solution was concentrated under vacuum and precipitated in excess methanol, filtered, and dried under vacuum for 24 hours to obtain poly(N-vinylformamide).

[0083] (7) 6.0 g of poly(N-vinylformamide) and 7.6 g of NaOH were dissolved in 60 mL of deionized water and hydrolyzed at 60°C for 10 hours under N2 protection to carry out deformylation. The reaction solution was neutralized with concentrated HCl, dialyzed against deionized water for 48 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, and lyophilized to obtain PVAH. The chemical structure of PVAH was confirmed by 1H NMR spectroscopy. Figure 5 ).

[0084] (8) The Ti-TCPP@F127-CHO nanoparticles (200 μM based on Ti-TCPP) obtained in Example 3 and DMXAA (3 mg / mL) were dissolved in PBS (pH 7.4) to obtain the first precursor solution (containing 5% w / v CHO-F127-CHO). The PVAH obtained in (7) was dissolved in PBS (pH 7.4) at a concentration of 3% w / v to serve as the second precursor solution.

[0085] (9) Equal volumes of the first and second precursor solutions are respectively loaded into the two chambers of a dual-tube syringe and co-extruded through a static mixing head to obtain an in-situ pH-responsive hydrogel (DM-TC@Gel). Figure 6 (ab). The resulting hydrogel was a pale yellow transparent gel, and scanning electron microscopy showed that it had a three-dimensional porous network structure. Figure 7 a). Rheological tests showed that the storage modulus (G') was greater than the loss modulus (G''), confirming the formation of a stable hydrogel. Figure 7 b).

[0086] (10) The hydrogel obtained in Example 5 was placed in buffer solutions at pH 7.4, 6.5, and 5.5, respectively, and its dissociation behavior was monitored using an optical microrheometer. The results showed that at pH 5.5, the elastic index of the hydrogel decreased rapidly within 48 hours, and the solid-liquid equilibrium value changed from 0-0.5 (elastic solid) to 0.5-1 (viscoelastic fluid), indicating that the hydrogel was completely dissociated. At pH 6.5, the dissociation rate slowed down significantly. At pH 7.4, both the elastic index and the solid-liquid equilibrium value remained stable, confirming the structural integrity of the hydrogel. Figure 8 (c).

[0087] (11) The hydrogel obtained in Example 5 was sealed in a dialysis membrane (molecular weight cutoff of 1 kDa) and immersed in buffer solutions at pH 7.4, 6.5, and 5.5, respectively. Samples were taken at different time points, and the release amount of DMXAA was determined by high performance liquid chromatography. The results showed that the release of DMXAA was strongly pH-dependent. Under pH 5.5 conditions, the cumulative release rate reached 92.1% within 72 hours; under pH 6.5 conditions, it was 76.7%; and under pH 7.4 conditions, it was only 25.3%. This indicates that the hydrogel of the present invention has excellent acid-triggered drug release characteristics. Figure 9 (ab).

[0088] Example 2: Study on the in vitro enhancement of type I sonodynamic killing of tumor cells by pH-responsive hydrogels: (1) Cell uptake experiment To evaluate the uptake efficiency of Ti-TCPP@F127-CHO nanoparticles by CT26 tumor cells, the nanoparticles were incubated with CT26 cells for 0, 0.5, 1, and 2 hours. Cell uptake was then detected using flow cytometry and laser confocal microscopy. The results of laser confocal microscopy observations are shown below. Figure 10 The results showed that as the incubation time increased from 0 hours to 2 hours, the intensity of the red fluorescence of Ti-TCPP in CT26 cells gradually increased, indicating that Ti-TCPP@F127-CHO nanoparticles could be effectively taken up by tumor cells in a time-dependent manner. Flow cytometry quantitative analysis further confirmed this trend. Figure 10 (b) The average fluorescence intensity after 2 hours of incubation was approximately 3.2 times that after 0.5 hours of incubation, and the difference was statistically significant (P<0.01). This result indicates that Ti-TCPP@F127-CHO nanoparticles have good cellular uptake efficiency, providing a material basis for subsequent sonodynamic therapy.

[0089] (2) Detection of mitochondrial membrane potential Subsequently, the present invention performed mitochondrial membrane potential detection, and the JC-1 staining results are as follows: Figure 11 As shown in Figures ab, cells in the PBS control group and the US (ultrasound)-only group exhibited strong red fluorescence (J-aggregates) and a high red / green fluorescence ratio, indicating normal mitochondrial membrane potential. The Ti-TCPP@F127-CHO group (without ultrasound) showed a slightly lower red / green ratio, suggesting that the nanoparticles themselves have some effect on mitochondria, but to a limited extent. In the Ti-TCPP@F127-CHO+US (ultrasound) group, under normoxic conditions, red fluorescence was significantly weakened while green fluorescence was significantly enhanced, with the red / green ratio decreasing by approximately 75% compared to the control group, indicating a significant decrease in mitochondrial membrane potential and severe mitochondrial damage. Notably, under hypoxic conditions, a similar decrease in mitochondrial membrane potential was observed in the Ti-TCPP@F127-CHO+US (ultrasound) group, with no significant difference in red / green ratio compared to normoxic conditions (P>0.05). These results indicate that Ti-TCPP@F127-CHO-mediated type I sonodynamic therapy can cause severe mitochondrial dysfunction under both normoxic and hypoxic conditions.

[0090] (3) In vitro sonodynamic cytotoxicity of Ti-TCPP@F127-CHO Subsequently, the cytotoxicity of TCF under ultrasound irradiation was detected by MTT assay. The results showed that cytotoxicity increased in a concentration-dependent manner, and the cytotoxicity was comparable under normoxic and hypoxic conditions. At the highest concentration (50 μM), cell viability decreased to 29.5% under normoxic conditions and 30.2% under hypoxic conditions. Finally, Calcein-AM / PI staining was performed, which further confirmed ultrasound-triggered cell death. In summary, these results indicate that TCF can serve as an effective type I sonosensitive agent for killing hypoxic cancer cells. Figure 12 (c).

[0091] Example 3: Verification of the mechanism of pH-responsive hydrogel combined with type I sonodynamic induction of pan-apoptosis in vitro: (1) Investigation on the in vitro induction of apoptosis by Ti-TCPP@F127-CHO This invention then investigated the potential mechanism of Ti-TCPP@F127-CHO-mediated type I sonodynamic cell death. Flow cytometry analysis using annexin V / PI staining showed that, compared to the PBS, TCF, and US (ultrasound) groups, TCF + US (30 kHz, 3 W, 2 min) effectively induced apoptosis in CT26 cells under both normoxic and hypoxic conditions, with apoptosis rates reaching 56.5% and 44.2%, respectively. Figure 13 (ab).

[0092] Cleaved caspase-3 is an important enzyme involved in the apoptosis process and a key executor of programmed cell death. This invention uses Western blotting and immunofluorescence staining to verify the expression of cleave caspase-3 in tumor cells treated with TCF + US (ultrasound). The results showed that under both normoxic and hypoxic conditions, TCF + US (ultrasound) treatment significantly upregulated cleave-caspase-3 expression in tumor cells, providing crucial evidence that this method induces apoptosis in tumor cells. Figure 14 (c).

[0093] (2) Investigation on the in vitro pyroptosis induced by Ti-TCPP@F127-CHO Furthermore, this invention also observed the morphological changes exhibited by CT26 cells treated with TCF combined with ultrasound irradiation. Bright-field images of the treated tumor cells were taken using an inverted microscope. The results showed significant membrane swelling and blistering in the TCF combined with ultrasound irradiation groups (noroxic and hypoxic), as indicated by the red arrows, demonstrating that this treatment effectively induced the secretion of pyroptosis bodies by tumor cells. Subsequently, this invention further confirmed through 4-(dimethylamino)benzene-1,3-diol (T11) membrane integrity staining that TCF + US irradiation significantly increases membrane permeability under hypoxic conditions, inducing pyroptosis. Figure 15 (ab).

[0094] Subsequently, this invention investigated the expression of pyroptosis-related proteins. Immunoblotting and immunofluorescence analyses showed upregulation of N-GSDMD and Cleave-caspase 1 expression in the TCF + US (ultrasound) group, confirming Caspase 1 activation. GSDMD was subsequently cleaved into N-GSDMD. Immunofluorescence analysis also showed significant upregulation of GSDMD-N in the TCF + US irradiation group under hypoxic conditions. Figure 16 (ab).

[0095] Meanwhile, after different treatments of tumor cells, the secretion levels of IL-1β and lactate dehydrogenase were detected in the corresponding culture supernatants. The results showed that the secretion levels of these enzymes were significantly increased after TCF + US treatment. Figure 17 (ab). In summary, these results indicate that TCF-mediated type I sonodynamic therapy can effectively trigger pyroptosis.

[0096] (3) Investigation on the in vitro induction of necrosis and apoptosis by Ti-TCPP@F127-CHO Mounting evidence suggests that reactive oxygen species-induced mitochondrial damage is accompanied by necroptosis, characterized by RIPK3-mediated MLKL phosphorylation. To verify the ability of TCF-mediated type I sonodynamic therapy to induce necroptosis, this invention employed YO-PRO-1 / PI double staining. The nuclei of apoptotic cells were preferentially stained with YO-PRO-1 (green), while the nuclei of necroptotic cells were stained with both YO-PRO-1 and PI, producing overlapping yellow fluorescence. Figure 18 As shown in Figures ab, cells treated with TCF + US irradiation exhibited significant yellow fluorescence under both normoxic and hypoxic conditions, indicating that necrosis and apoptosis occurred. Furthermore, Western blot analysis confirmed the upregulation of P-MLKL and P-RIPK1, further validating the induction of necrosis and apoptosis.

[0097] In summary, these results indicate that three cell death modes, including apoptosis, pyroptosis, and necrotic apoptosis, can be observed in tumor cells, suggesting that TCF-mediated type I sonodynamic therapy can induce panapoptosis in cells.

[0098] Example 4: pH-responsive hydrogel combined with type I sonodynamically induced panapoptosis to drive immunogenic cell death and DC cell maturation: (1) Given TCF's strong pan-apoptotic induction ability, this invention further investigated its ability to trigger immunogenic cell death by assessing the secretion of damage-related molecular patterns in cells, including calreticulin (CRT), adenosine triphosphate (ATP), and high-mobility group box 1 (HMGB1). Confocal immunofluorescence imaging showed that under normoxic and hypoxic conditions, TCF + US treatment induced a significant increase in CRT green fluorescence, indicating enhanced CRT expression on the cell surface. Figure 19 (ab).

[0099] (2) Meanwhile, HMGB1 is a Toll-like receptor agonist that plays a key role in activating dendritic cell (DC) maturation. This invention further investigated its activity using confocal immunofluorescence imaging. The results showed that, compared to other groups, after TCF + US treatment, significant translocation from the nucleus to the cytoplasm was observed under both normoxic and hypoxic conditions. Figure 20 (ab).

[0100] (3) In addition, the present invention used an ATP detection kit to detect the level of extracellular ATP after different treatments. The trend of the detection results was the same as above. Under both oxygen conditions, the ATP level in the TCF + US (ultrasound) group was significantly higher than that in the US and TCF groups. These results collectively indicate that TCF + US-induced pan-apoptosis can effectively promote immunogenic cell death ( Figure 21 ).

[0101] (4) Dendritic cells, as key mediators of immune activation, can be stimulated by damage-related molecular patterns released during immunogenic cell death. Mature dendritic cells can further activate T cells by releasing inflammatory factors, thereby initiating a strong immune response. To investigate the ability of TCF + US-induced pan-apoptosis to promote dendritic cell maturation, this invention analyzed the expression of dendritic cell maturation markers CD80 and CD86. Under hypoxic conditions, after TCF + US treatment, the population of CD80+CD86+ positive dendritic cells increased significantly, and the dendritic cell maturation rate was approximately 55.3%, about 2.2 times that of the control group. Figure 22 (c).

[0102] All these results confirm that TCF+US-induced panapoptosis can drive immunogenic cell death and promote efficient dendritic cell maturation under hypoxic conditions mimicking tumors, highlighting its potential as a powerful strategy for activating anti-tumor immunity.

[0103] Example 5: In vivo retention and therapeutic effects of pH-responsive hydrogel combined with type I sonodynamics in a CT26 colon cancer model: (1) Intratumoral hypoxia severely limits the efficacy of oxygen-dependent treatments, while type I sonodynamic therapy offers a promising solution by circumventing this limitation. Given the excellent panapoptotic induction and potent cytotoxicity of TCF under ultrasound irradiation, this invention subsequently established a subcutaneous CT26 tumor model in mice to evaluate its therapeutic effect on hypoxic tumors. First, this invention evaluated the in situ gelation and retention behavior of injectable DM-TC@Gel within tumors by monitoring TCPP fluorescence using a three-dimensional small animal imaging system (IVIS). Figure 23 As shown in Figures ab, compared with free TC nanoparticles, DM-TC@Gel has a significantly longer retention time in tumors. 63.3% of the fluorescence signal remained on day 4 after injection, indicating the formation of in-situ gelation and the ability of this hydrogel to achieve long-term intratumoral retention.

[0104] (2) DM-TC@Gel dissociates in the slightly acidic tumor microenvironment, leading to the release of DMXAA, which damages tumor microvessels and exacerbates hypoxia in the tumor microenvironment. This invention utilizes immunofluorescence staining of the tumor site to detect hypoxia. The staining results show that 24 hours after intratumoral injection of DM-TC@Gel, tumor hypoxia significantly increases, with the most significant effect observed in the DM-TC@Gel + US (ultrasound) group. Figure 24 (ab). This enhancement may be attributed to the combination of DMXAA-induced vascular occlusion and oxygen consumption during sonodynamic therapy, which synergistically promotes intratumoral hypoxia.

[0105] (3) Subsequently, BALB / c mice with CT26 tumors were divided into five treatment groups: PBS (G1), DM@Gel (G2), Gel + US (G3), TC@Gel + US (G4), and DM-TC@Gel + US (G5). On day 0, the corresponding hydrogel was injected into the tumor, followed by sonication (30 kHz, 3 W, 10 min). The tumor growth curves showed that, compared with untreated mice, irradiation with DM@Gel or US alone had no significant inhibitory effect on tumor growth. TC@Gel, however, showed a moderate inhibitory effect on tumor growth. Notably, this invention found that DM-TC@Gel + US treatment completely eradicated the tumor and significantly prolonged the survival time of these tumor-bearing mice. No significant changes in body weight were observed after any of the treatments throughout the treatment period, indicating high biocompatibility. Figure 25 (c).

[0106] (4) Given the significant efficacy of DM-TC@Gel + US treatment, this invention further investigated its long-term immune memory effect in limiting tumor recurrence. Forty days after the first round of treatment, healthy mice and mice cured by DM-TC@Gel + US treatment were challenged again with CT26 tumor cells. Rapid tumor growth was observed in healthy mice, but mice treated with DM-TC@Gel + US showed significant tumor growth inhibition. To elucidate the potential mechanisms contributing to the superior tumor suppression effect, blood samples were collected from mice on day 47 post-treatment, and the proportion of CD8+ effector memory T cells was analyzed. The results showed that DM-TC@Gel + US treatment significantly increased the number of central memory T cells and effector memory T cells in the blood. Furthermore, elevated levels of IFN-γ and TNF-α were detected in the serum of treated mice, further promoting the establishment of anti-tumor immune memory. Figure 26 (ad).

[0107] Therefore, these results indicate that DM-TC@Gel releases DMXAA under acidic tumor conditions to disrupt tumor angiogenesis, while ultrasound-triggered type I sonodynamic therapy induces panapoptosis, which can synergistically inhibit tumor growth and generate long-term immune memory to prevent recurrence.

[0108] Example 6: In vivo therapeutic effect of pH-responsive hydrogel combined with type I sonodynamics in a B16F10 melanoma model: (1) Given the good therapeutic effect of DM-TC@Gel + US in the CT26 tumor model, this invention further evaluates its efficacy by establishing a B16F10 melanoma mouse tumor model. Mice with B16F10 tumors were randomly divided into five groups, the same as the CT26 tumor model, and treated as described above. Immunofluorescence staining of tumor tissue showed that DM-TC@Gel + US treatment effectively increased the level of intratumoral reactive oxygen species (ROS). Figure 27 (ab).

[0109] (2) Subsequently, the tumor size and body weight of mice in the different treatment groups were monitored. The monitoring results showed that the tumors of mice treated with DM-TC@Gel + US were significantly suppressed, and complete tumor eradication was observed in four out of five mice. The survival rate of these mice was also significantly improved compared to other groups. Furthermore, no significant changes in body weight were observed in any treatment group during the monitoring period. Figure 28 (c).

[0110] (3) Subsequently, a postoperative tumor resection model (99% tumor resection) was established to evaluate the ability of DM-TC@Gel + US treatment to inhibit residual tumor recurrence. Tumor growth curves showed that tumors in mice that underwent surgical resection without further treatment recurred rapidly. The Gel + US and TC@Gel + US (ultrasound) groups showed only limited tumor recurrence inhibition, with the TC@Gel + US (ultrasound) group showing moderate efficacy. In contrast, the DM-TC@Gel + US (ultrasound) group demonstrated significant efficacy in preventing tumor recurrence, with no visible tumors observed in any of the five treated mice. Figure 29 (ad).

[0111] (4) Subsequently, the lung metastases of these recurrent tumors were further evaluated. In the PBS and DM@Gel groups, clearly visible black nodules were observed in the lungs, while the Gel + US (ultrasound) group showed smaller but diffusely distributed tumors. Some metastatic lesions remained in the TC@Gel + US (ultrasound) group. In contrast, no visible tumor nodules were observed in the DM-TC@Gel + US (ultrasound) group. H&E staining of lung sections confirmed these observations, and compared with all other groups, the DM-TC@Gel + US (ultrasound) group showed a significant reduction in lung metastases. Figure 30 (ab). In summary, these results indicate that DM-TC@Gel + US treatment effectively inhibits tumor growth, recurrence, and metastasis.

[0112] Example 7: Evaluation of the immune mechanism of pH-responsive hydrogel combined with type I sonodynamics in a B16F10 melanoma model: Furthermore, this invention carefully investigated the potential immunological mechanism of combined therapy involving DMXAA-induced angiogenesis and DM-TC@Gel + US-triggered pan-apoptosis. B16F10 melanoma mice (n ≥ 4 per group) were treated as described above, and on day 6, tumors were excised to prepare single-cell suspensions for immunological analysis. Immune cell populations, including CD8+ T cells and NK cells, were assessed by flow cytometry. Flow cytometry analysis showed that DM-TC@Gel + US treatment significantly increased the proportion of intratumoral CD3+ CD8+ T cells to 45.2% (compared to 23.9% in the control group) and the proportion of NK1.1 cells to 8.28% (compared to 1.82% in the control group). These results indicate enhanced cytotoxic activity. Figure 31 (ab).

[0113] Furthermore, this invention also measured the levels of intratumoral cytokines, including IFN-γ, TNF-α, IL-6, and IL-1β, as well as the serum level of IFN-γ. The results showed that DM-TC@Gel + US treatment significantly increased the production of pro-inflammatory factors in both the tumor and serum. Figure 32 (ab). These results indicate that the combination of angiogenesis disruption and panapoptosis effectively promotes anti-tumor immunity by activating cytotoxic T cells, NK cells, and pro-inflammatory cytokine responses, thereby contributing to robust adaptive immune activation.

[0114] Then, to further verify the immune memory effect induced by DM-TC@Gel + US treatment, the present invention re-inoculated cured mice with B16F10 melanoma cells on day 40 after DM-TC@Gel + US treatment. Compared with the control group, tumor growth in DM-TC@Gel + US cured mice was significantly inhibited, demonstrating the induction of a durable anti-tumor immune memory response. Simultaneously, DM-TC@Gel + US treatment significantly increased the number of memory T cells, including central memory T cells and effector memory T cells. Furthermore, the levels of IFN-γ and TNF-α in the serum of treated mice were elevated, further supporting the establishment of durable anti-tumor immune memory. Figure 33 (c).

[0115] In summary, this invention develops an in-situ formed, pH-responsive injectable hydrogel capable of sequentially delivering the angiogenic agent DMXAA and type I sonodynamic nanoparticles (DM-TC@Gel), providing a robust strategy for hypoxic tumor immunotherapy. The DM-TC@Gel system utilizes the acidic tumor environment to trigger in-situ degradation of the hydrogel, first releasing DMXAA to induce vascular occlusion and exacerbate tumor hypoxia, followed by ultrasound-activated nanoparticles generating reactive oxygen species (ROS), thereby efficiently inducing pan-apoptosis in tumor cells. This combined strategy not only drives immunogenic cell death and dendritic cell maturation but also achieves significant tumor regression, inhibits metastasis, and establishes durable anti-tumor immune memory in various hypoxic tumor models. Overall, this study demonstrates the potential of combining angiogenesis with pan-apoptotic sonodynamic therapy as a novel therapeutic strategy, achieving highly efficient hypoxic tumor treatment by leveraging anti-tumor immune responses.

[0116] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An in-situ formed pH-responsive hydrogel for tumor immunotherapy, characterized in that, The hydrogel is a three-dimensional network structure formed by cross-linking aldehyde-terminated Pluronic F127 and polyethyleneamine hydrochloride through a Schiff base reaction; the hydrogel is loaded with a vascular disruptor and type I sonodynamic nanoparticles; the hydrogel is formed in situ by co-extruding an aldehyde-terminated Pluronic F127 solution containing the vascular disruptor and type I sonodynamic nanoparticles with a polyethyleneamine hydrochloride solution using a dual-tube syringe.

2. The in-situ formed pH-responsive hydrogel for tumor immunotherapy according to claim 1, characterized in that, The vascular disruptor includes monoclonal antibodies and small molecule VDAs, including colchicine, CA4 analogs, and flavonoids; the CA4 analogs include CA4P, ombrabulin, ZD6126, OXI4503, BNC105P, DX1002, and C118P; the flavonoids include DMXAA and MN029; DMXAA is preferred.

3. The in-situ formed pH-responsive hydrogel according to claim 1, characterized in that, The type I acoustic dynamic nanoparticles are Ti-TCPP@F127-CHO nanoparticles obtained by modifying titanium-coordinated meso-tetra(4-carboxyphenyl)porphyrin with aldehyde-terminated CHO-F127-CHO groups; in the titanium-coordinated meso-tetra(4-carboxyphenyl)porphyrin nanoparticles, titanium and meso-tetra(4-carboxyphenyl)porphyrin are linked by coordination bonds.

4. A method for preparing in-situ pH-responsive hydrogels for tumor immunotherapy, characterized in that, Includes the following steps: Step 1, Preparation of Ti-TCPP nanoparticles: TiCl4·2THF was dissolved in dimethylformamide, and meso-tetra(4-carboxyphenyl)porphyrin was dissolved in an organic solvent. After mixing the two, acetic acid was added, the mixture was refluxed, centrifuged, and washed to obtain Ti-TCPP nanoparticles. Step 2, Preparation of CHO-F127-CHO: Pluronic F127 was dissolved in an organic solvent, and Dess-Martin periodoyl alkane reagent was added. After the reaction, the mixture was concentrated, precipitated, dialyzed, and lyophilized to obtain aldehyde-terminated Pluronic F127. Step 3, Preparation of Ti-TCPP@F127-CHO nanoparticles: The titanium-coordinated meso-tetra(4-carboxyphenyl)porphyrin nanoparticles obtained in Step 1 and the aldehyde-terminated Pluronic F127 obtained in Step 2 were dissolved in an organic solvent, ultrasonically dispersed, and then injected into deionized water. The THF was evaporated to obtain Ti-TCPP@F127-CHO nanoparticles. Step 4, Preparation of PVAH: N-vinylformamide is polymerized with a polymerization initiator, and then concentrated, precipitated, and dried to obtain poly(N-vinylformamide); The product was subjected to amide hydrolysis with NaOH solution, neutralized with HCl, dialyzed, and lyophilized to obtain polyvinylamine hydrochloride. Step 5, preparation of hydrogel: The Ti-TCPP@F127-CHO nanoparticles and vascular disruptor obtained in step 3 are dissolved in a buffer salt solution as the first precursor solution; the PVAH obtained in step 4 is dissolved in a buffer salt solution as the second precursor solution; the first and second precursor solutions are respectively loaded into the two chambers of a dual-tube syringe and co-extruded through a static mixing head to obtain an in-situ formed pH-responsive hydrogel.

5. The preparation method according to claim 4, characterized in that, The mass ratio of TiCl4·2THF to TCPP in step 1 is 1.5:1 to 2.5:1, preferably 1.8:1 to 2.2:1; Alternatively, the amount of acetic acid added in step 1 is 180~220 μL of acetic acid per 40 mg TiCl4·2THF; the reaction time is 10~14 hours.

6. The preparation method according to claim 4, characterized in that, The mass ratio of Pluronic F127 to Dess-Martin periodane in step 2 is 9:1 to 11:

1.

7. The preparation method according to claim 4, characterized in that, In step 3, the mass ratio of titanium-coordinated meso-tetra(4-carboxyphenyl)porphyrin nanoparticles to aldehyde-terminated Pluronic F127 is 0.5:45 to 0.5:

55.

8. The preparation method according to claim 4, characterized in that, The molar ratio of N-vinylformamide to polymerization initiator AIBA in step (4) is 500~1000:1, preferably 545:1; Further, the mass ratio of poly(N-vinylformamide) to NaOH in step (4) is 1~10:1~10, preferably 6:7.

6.

9. The preparation method according to claim 5, characterized in that, In step (5), the concentration of Ti-TCPP@F127-CHO in the first precursor solution is 50~500 μM, the concentration of aldehyde-terminated Pluronic F127 is 1%~20% w / v based on Ti-TCPP, and the concentration of DMXAA is 0.1~10 mg / mL; the concentration of PVAH in the second precursor solution is 1%~10% w / v. Furthermore, in step (5), the volume ratio of the first precursor liquid to the second precursor liquid is 0.5~2:0.5~2, preferably 1:

1.

10. The application of the in-situ formed pH-responsive hydrogel according to claim 1 in the preparation of antitumor drugs; characterized in that, The applications include the use in the preparation of drugs for inducing panapoptosis of tumor cells, the use in the preparation of drugs for activating antitumor immune responses, and the use in the preparation of drugs for inhibiting tumor growth, recurrence and / or metastasis. Furthermore, the tumors include colon cancer and melanoma; Furthermore, the application is in conjunction with ultrasound, with an ultrasound frequency of 20~50 kHz, preferably 30 kHz; an ultrasound power of 1~5 W, preferably 2 W; and an ultrasound time of 2~20 min, preferably 10 min.

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