A hydrogel composition for antitumor immunotherapy

Abstract

This invention relates to the field of biomedical materials and provides an anti-tumor method combining magnesium-driven metal immunotherapy with iron-based magnetothermal therapy (MHT). This method synergistically exerts a vaccine-like anti-tumor effect through magnetothermal ablation and immune activation, providing a novel metal-based anti-tumor strategy. This strategy can not only effectively eliminate primary tumors but also inhibit distant lesions of immunosuppressive tumors, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more particularly to a hydrogel composition for antitumor immunotherapy. Background Technology

[0002] Compared to traditional treatments such as surgery, radiotherapy, and chemotherapy, tumor immunotherapy has become a groundbreaking clinical treatment strategy, with its core mechanism being the activation of adaptive immune responses. New immunotherapy modalities developed in recent years, especially immune checkpoint inhibitors (ICIs) and tumor vaccines, have gained widespread attention as highly promising treatment options. They induce specific anti-tumor immune responses while reducing off-target effects.

[0003] PD-1 / PD-L1 inhibitors, as representative of ICIs, have been included in the first-line treatment standards for various solid tumors, including non-small cell lung cancer, melanoma, and esophageal squamous cell carcinoma. These biologics significantly prolong overall survival and improve quality of life by promoting the formation of persistent antigen-specific immune memory.

[0004] However, for CD4 + and CD8 + Immunosuppressive tumors with low T-cell infiltration levels, particularly head and neck squamous cell carcinoma (HNSCC), which has a high global incidence, suffer from limited efficacy with PD-1 targeted therapies (such as nivolumab and pembrolizumab) due to widespread clinical resistance. Although the National Comprehensive Cancer Network (NCCN) guidelines recommend anti-EGFR monoclonal antibodies combined with ICIs for HNSCC treatment, the uncertainty of efficacy and significant adverse reactions remain pressing clinical challenges. Therefore, current immunotherapies based on bioactive molecules such as antigens, antibodies, and ICIs for these low-immunogenic tumors face the dual challenges of in vivo metabolic inactivation and low targeted delivery efficiency. This situation necessitates the development of novel immunotherapeutic systems with broad-spectrum anti-tumor effects and the ability to overcome drug resistance mechanisms, utilizing advanced biomaterial platforms.

[0005] Metallic elements, especially those essential to the human body such as sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), and zinc (Zn), play a crucial role in maintaining life activities and regulating physiological functions. Specifically, Na... + / K +Metals participate in the regulation of cell membrane potential and the maintenance of humoral homeostasis; Fe plays a central role in oxygen transport; Zn participates in the regulation of the immune system; and Ca and Mg are crucial for maintaining skeletal homeostasis and normal neuromuscular transmission. Metal-based compounds also have a long history of application in disease treatment; for example, platinum-based chemotherapy drugs are widely used due to their broad-spectrum antitumor activity, while aluminum-based adjuvants can significantly enhance the immune response efficacy of vaccines.

[0006] In recent years, with the rapid development of nanotechnology, metal-based nanomaterials, especially magnetic nanoparticles (MNPs), have shown unique advantages in the field of integrated tumor diagnosis and treatment. As an emerging biomaterial platform, MNPs not only possess excellent physicochemical properties but also enable multimodal diagnostic and therapeutic synergy, providing a new solution to overcome the bottlenecks of traditional immunotherapy.

[0007] MNP-based diagnostic and therapeutic platforms can induce magnetocaloric (MHT) effects under alternating magnetic fields (AMF), achieving targeted tumor ablation while protecting normal tissue. This technology triggers programmed cell death by activating the PKCδ-mediated PLS3 phosphorylation pathway and induces immunogenic cell death (ICD) by altering membrane permeability and disrupting subcellular structures. However, the clinical translation of magnetocaloric therapy faces bottlenecks; its effects are mainly limited to the primary lesion, and it requires combination with other therapies to produce synergistic effects. These limitations restrict the widespread application of this technology in tumor immunotherapy. Summary of the Invention

[0008] This invention constructs a synergistic therapeutic system combining magnesium-mediated metal-mediated immune regulation with iron-based nano-MHT. Through the synergistic effect of thermodynamic ablation and immune cascade activation, it achieves precise dual elimination of tumors. This metal-based anti-tumor therapeutic system not only achieves complete eradication of the primary tumor but also effectively overcomes the immunosuppressive tumor microenvironment barrier, significantly inhibiting distant metastasis and recurrence of immune-rejecting tumors, demonstrating excellent broad-spectrum anti-tumor potential.

[0009] This invention provides an injectable hydrogel composition containing magnesium microparticles and Fe3O4 magnetic nanoparticles (Mg / Fe3O4@Gel) and its delivery system, aiming to enhance anti-tumor immune responses and achieve vaccine-like immunosuppressive effects against distal and new lesions. Under the action of AMF, Mg / Fe3O4@Gel induces cell death in head and neck squamous cell carcinoma (HNSCC) cells through magnetocaloric induction, simultaneously triggering mitochondrial dysfunction and disrupting redox homeostasis, ultimately mediating a cell apoptosis rate of up to 72%. Furthermore, the bioactive components released during hydrogel degradation can efficiently neutralize the intracellular acidic microenvironment, synergistically enhancing the systemic immunotherapy effect.

[0010] Mechanistic studies have shown that lactoferrin (Ltf) plays a key mediating role in Mg / Fe3O4@Gel-induced immunogenic cell death (ICD), achieving dual inhibition of primary and distant HNSCC lesions. Furthermore, a vaccine-like immunization strategy based on Mg / Fe3O4@Gel-inactivated tumor cells, combined with adoptive transfer of CD3... + T-cell infusion can effectively activate the immune response and achieve up to 80% tumor growth inhibition in immunodeficiency models, which is significantly better than existing clinical treatments and shows great promise for anti-tumor applications.

[0011] This invention utilizes AMF to trigger a synergistic effect between metal ions and magnetothermal activity, effectively breaking through the immunosuppressive microenvironment of tumors (especially HNSCC), achieving a synergistic enhancement of in situ immune activation and systemic anti-tumor response, thereby significantly improving the therapeutic effect of tumors. This invention not only possesses excellent physicochemical stability and biocompatibility, but also combines the dual functions of magnetothermal therapy and immune regulation, providing an innovative and efficient solution for comprehensive tumor treatment.

[0012] In vitro studies, by quantifying magnetocaloric effect parameters, systematically elucidated the cascade mechanism of mitochondrial membrane potential (MMP) disintegration, reactive oxygen species (ROS) bursts, and intracellular pH (pHi) homeostasis imbalance in regulating apoptosis and ICD in HNSCC cells.

[0013] Crucially, the effectiveness of Mg / Fe3O4@Gel in in vivo in inducing ICD was verified using immunodeficient mice (BALB / c nude), immunocompetent mice (C3H), and bilateral tumor models.

[0014] To further elucidate the molecular mechanism, CRISPR / Cas9 knockout and lentivirus-mediated overexpression experiments confirmed that Ltf is a key regulator of Mg / Fe3O4@Gel-mediated ICD. Furthermore, to evaluate the vaccine potential of Mg / Fe3O4@Gel-treated tumor cells, it was injected into C3H mice to induce immune memory, and CD3+ isolated from the spleens of immunized mice was analyzed. + Adoptive transfer of T cells into NSG mice deficient in T and B cells showed that this strategy had a significant inhibitory effect on tumor progression.

[0015] Introducing Mg and Fe3O4 into the hydrogel significantly enhances the antitumor efficacy of this vaccine-like metal-based platform and broadens its application prospects in the field of broad-spectrum immunotherapy for immunosuppressive tumors.

[0016] Figure 1The mechanism of action of magnesium-enhanced magnetothermal immunotherapy was elucidated: this strategy aims to induce a vaccine-like antitumor response against primary and distant tumors. Under AMF irradiation, Mg / Fe3O4@Gel generates a local magnetothermal effect, inducing SCC VII cell death. During this process, mitochondrial function is impaired and redox balance is disrupted, ultimately leading to apoptosis and ICD. It is worth emphasizing that CD33 from immunocompetent C3H mice... + Following translocation, T cells were able to significantly inhibit tumor progression in immunodeficient NSG mice, further demonstrating that this strategy can achieve effective systemic immune activation.

[0017] Therefore, the present invention provides a hydrogel composition characterized by comprising magnesium microparticles and Fe3O4 magnetic nanoparticles.

[0018] The composition as described above is characterized in that the diameter of the magnesium microparticles is 5-500 μm, preferably 10-200 μm, 20-100 μm, 30-70 μm, or 50-55 μm; and the diameter of the Fe3O4 magnetic nanoparticles is 10-1000 nm, preferably 20-500 nm, 30-250 nm, 40-200 nm, 50-150 nm, 60-120 nm, 70-110 nm, or 90-100 nm.

[0019] The composition described above is characterized in that it further comprises a hydrogel matrix material, preferably including: hyaluronic acid, chitosan, alginate, dextran, sodium carboxymethyl cellulose, polylactic acid, polyglycolic acid, or polylactic acid-polyglycolic acid copolymer (PLGA); preferably, the hydrogel matrix material is chitosan, more preferably amino-containing chitosan (chitosan-NH2).

[0020] The composition described above may optionally include a crosslinking agent.

[0021] The composition described above is characterized in that: the composition comprises a crosslinking agent, which is glutaraldehyde, epichlorohydrin, divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE), dicyclohexylcarbodiimide (DCC), boric acid, polyethyleneimine, tetra-arm polyethylene glycol, and more preferably dibenzaldehyde polyethylene glycol (DF-PEG-DF), dialdehyde polyethylene glycol (OHC-PEG-CHO), dimercapto polyethylene glycol (HS-PEG-SH), or dicarboxylated polyethylene glycol (COOH-PEG-COOH).

[0022] The composition described above is characterized in that each 1 mL of the composition contains 5-100 mg of magnesium microparticles (preferably 10-80 mg or 20-50 mg) and 2-100 mg of Fe3O4 magnetic nanoparticles (preferably 5-80 mg or 10-50 mg).

[0023] The present invention also provides the hydrogel composition as described above, characterized in that its preparation method is as follows:

[0024] The hydrogel matrix material is prepared by mixing magnesium microparticles and Fe3O4 magnetic nanoparticles with a hydrogel matrix material; preferably, the hydrogel matrix material is cross-linked by a cross-linking agent.

[0025] Preferably, magnesium microparticles and Fe3O4 magnetic nanoparticles are mixed with chitosan-NH2 to form an intermediate solution, which is then cross-linked with a cross-linking agent to form a hydrogel;

[0026] More preferably, the preparation method of the Fe3O4 magnetic nanoparticles is: ultrasonic precipitation, co-precipitation, high-temperature decomposition, microemulsion, oxidation, or sol-gel method.

[0027] The preferred method for preparing the Fe3O4 magnetic nanoparticles is to use Fe... 3+ Fe3O4 magnetic nanoparticles were prepared by precipitation of salt under alkaline conditions; preferably, the Fe... 3+ The salt is FeCl3, and the preferred alkaline substance is ammonia, sodium hydroxide solution or sodium acetate; the preferred preparation method also includes a surfactant, which is preferably a nonionic surfactant, cationic surfactant or anionic surfactant; the preferred surfactant is sodium dodecylbenzenesulfonate (SDBS).

[0028] The present invention also provides a method for preparing the above composition, characterized in that:

[0029] The hydrogel matrix material is prepared by mixing magnesium microparticles and Fe3O4 magnetic nanoparticles with a hydrogel matrix material; preferably, the hydrogel matrix material is cross-linked by a cross-linking agent.

[0030] Preferably, magnesium microparticles and Fe3O4 magnetic nanoparticles are mixed with chitosan-NH2 to form an intermediate solution, which is then cross-linked with a cross-linking agent to form a hydrogel;

[0031] More preferably, the preparation method of the Fe3O4 magnetic nanoparticles is as follows:

[0032] More preferably, the preparation method of the Fe3O4 magnetic nanoparticles is: ultrasonic precipitation, co-precipitation, high-temperature decomposition, microemulsion, oxidation, or sol-gel method.

[0033] The preferred method for preparing the Fe3O4 magnetic nanoparticles is to use Fe... 3+ Fe3O4 magnetic nanoparticles were prepared by precipitation of salt under alkaline conditions; preferably, the Fe... 3+ The salt is FeCl3, and the preferred alkaline substance is ammonia, sodium hydroxide solution or sodium acetate; the preferred preparation method also includes a surfactant, which is preferably a nonionic surfactant, cationic surfactant or anionic surfactant; the preferred surfactant is sodium dodecylbenzenesulfonate (SDBS).

[0034] More preferably, the preparation method is as follows: ethylene glycol and diethylene glycol are mixed evenly, ferric chloride, sodium acetate and sodium dodecylbenzenesulfonate (SDBS) are added, the mixture is stirred and mixed evenly, and then heated at a high temperature (e.g., 200°C) to obtain Fe3O4 magnetic nanoparticles.

[0035] Mg particles and Fe3O4 MNPs were uniformly mixed in a chitosan-NH2 aqueous solution to form solution A. DF-PEG-DF was dissolved in the chitosan-NH2 aqueous solution to form solution B. Solutions A and B were mixed and stirred at room temperature to allow them to crosslink and form a composite hydrogel.

[0036] Preferably, the volume ratio of solutions A and B is 1:1.

[0037] The present invention also provides pharmaceutical use of the composition, characterized in that it is used for the prevention and / or treatment of tumor diseases; preferably, in combination with magnetothermal therapy for antitumor immunotherapy.

[0038] As described above, the tumors include: breast cancer, lung cancer, head and neck cancer, gastric cancer, cervical cancer, melanoma, prostate cancer, ovarian cancer, squamous cell carcinoma, and glioma; preferably, the tumors include head and neck squamous cell carcinoma and thyroid cancer; more preferably, the tumors are resistant, and more preferably, the tumors are immune-rejecting (cold tumors). Attached Figure Description

[0039] Figure 1 Schematic diagram of the mechanism of action of magnesium-enhanced magnetothermal immunotherapy;

[0040] Figure 2 Morphological characterization of Gel, Mg@Gel, Fe3O4@Gel and Mg / Fe3O4@Gel;

[0041] Figure 3 Synthesis and physicochemical characterization of Mg / Fe3O4@Gel;

[0042] Figure 4 Mg / Fe3O4@Gel activates SCC VII cell death in vitro;

[0043] Figure 5 Quantitative analysis of SCC VII cell death triggered by Mg / Fe3O4@Gel;

[0044] Figure 6 In vivo antitumor therapeutic effect of Mg / Fe3O4@Gel under AMF irradiation on orthotopic SCC VII tumor-bearing C3H mice;

[0045] Figure 7 Evaluation of the in vivo antitumor activity and biocompatibility of Mg / Fe3O4@Gel;

[0046] Figure 8 Transcriptome analysis reveals the antitumor molecular mechanism of Mg / Fe3O4@Gel under AMF irradiation;

[0047] Figure 9 Correlation analysis based on weighted gene co-expression network analysis (WGCNA);

[0048] Figure 10 Analysis of Mg / Fe3O4@Gel remodeling the tumor immune microenvironment (TIME) and its correlation with Ltf;

[0049] Figure 11 Correlation analysis of LTF;

[0050] Figure 12 Mg / Fe3O4@Gel drives antitumor efficacy by activating ICD via Ltf;

[0051] Figure 13 Establishment and evaluation of Ltf-modified SCC VII cells and corresponding xenograft tumor models in BALB / c nude mice. Detailed Implementation

[0052] The present invention will be further described below with reference to the embodiments.

[0053] Experimental materials and instruments used in this invention:

[0054] Mg / Fe3O4@Gel: Mg particles were prepared from Shanghai Naio Nanotechnology Co., Ltd. (Shanghai, China).

[0055] Fe3O4 MNPs were prepared according to the method described in Example 1 S1.

[0056] Chitosan-NH2 and DF-PEG-DF were purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan).

[0057] Microscopic morphology observation: Scanning electron microscope (SEM) S-4800 (Hitachi, Japan) and transmission electron microscope (TEM) HT7700 (Hitachi, Japan).

[0058] Elemental content analysis: Mg / Fe3O4@Gel was analyzed by SEM-EDS.

[0059] Aperture measurement: ImageJ software.

[0060] Rheological property testing: AR1000 rheometer (TA Instruments, Inc., USA). Test conditions: 0.01–10% strain step cycling at room temperature and 10 rad / s ω; 1–100 rad / s frequency step cycling at room temperature and 1% strain; and 0–37°C temperature step cycling at 10 rad / s ω and 1% strain. The dynamic changes of storage modulus (G') and loss modulus (G'') were recorded and analyzed.

[0061] Injection molding performance testing: Universal testing machine (Suzhou Qiantong Instruments, China).

[0062] Magnetothermal performance testing: Magnetic induction heating device (Shenzhen Shuangping Electric Technology Co., Ltd., China), AMF field strength 20kA / m.

[0063] Example 1 Synthesis and Characterization of Mg / Fe3O4@Gel

[0064] S1: Preparation of Fe3O4 MNPs

[0065] 1) Accurately measure 10 mL of ethylene glycol (EG) solution, then add 30 mL of diethylene glycol (DEG), place in a magnetic stirrer (800 rpm), and stir continuously for 15 min until homogeneous and transparent. Weigh 1.35 g of ferric chloride hexahydrate (FeCl3⋅6H2O), 2.325 g of sodium acetate (NaAc), and 1.40 g of sodium dodecylbenzenesulfonate (SDBS) sequentially, adding them to the mixed solvent system in batches at 5-minute intervals. Maintain a constant temperature of 25±2℃ and stir continuously for 5 h to obtain a dark green precursor solution.

[0066] 2) Transfer the precursor solution to a 50 mL PTFE-lined high-pressure reactor, ensuring the filling degree does not exceed 80% of the volume. Heat to 200°C using a programmable temperature-controlled vacuum drying oven and maintain this temperature for 12 hours. Maintain system pressure balance during the reaction, avoiding drastic pressure fluctuations. After the reaction, turn off the heating and allow the reactor to cool naturally to below 40°C to obtain a dark green suspension.

[0067] 3) Open the reactor and transfer the dark green suspension to a centrifuge tube. Centrifuge at 12,000 rpm for 10 min. Discard the supernatant and wash the product three times each with anhydrous ethanol and deionized water. Then, dry the product in a vacuum drying oven at 60°C for 12 h to obtain a black nanopowder product.

[0068] 4) The morphology, structure, and particle size distribution of Fe3O4 MNPs were characterized by transmission electron microscopy.

[0069] S2: Preparation of Mg / Fe3O4@Gel

[0070] This step involves removing Mg particles (50-55 μm in diameter). Figure 2 A, 2B), Fe3O4 magnetic nanoparticles (MNPs, diameter 90-100 nm, Figure 2 C, 2D) are mixed with chitosan-hydrogel to obtain good injectability and significant magnetothermal properties.

[0071] Mg / Fe3O4@Gel was prepared entirely in a glove box filled with nitrogen (N2).

[0072] Specific steps: Mg particles (20 mg) and Fe3O4 MNPs (10 mg) are uniformly mixed in 500 μL of chitosan-NH2 aqueous solution to form solution A. DF-PEG-DF is uniformly dissolved in 500 μL of chitosan-NH2 aqueous solution to form solution B. Solutions A and B are mixed at a 1:1 volume ratio and stirred at room temperature to allow cross-linking and form a composite hydrogel. Figure 3 A, 3B).

[0073] The same method was used to prepare control group pure hydrogel (Gel), Mg-loaded hydrogel (Mg@Gel) and Fe3O4-loaded hydrogel (Fe3O4@Gel).

[0074] Scanning electron microscopy / energy dispersive X-ray spectroscopy (SEM-EDS) confirmed that Mg and Fe elements were uniformly dispersed throughout the Mg / Fe3O4@Gel microstructure, indicating a uniform metal distribution within the hydrogel. Figure 3 C, 3D). The elemental composition of Mg / Fe3O4@Gel was also quantified as 47.5% carbon (C), 40.5% oxygen (O), 8.5% Mg, and 3.5% Fe, demonstrating consistent stability across different production batches. Figure 3 E).

[0075] The pure hydrogel (Gel) prepared above Figure 2 E), Mg-supported hydrogel (Mg@Gel), Figure 2 F) and Fe3O4 supported hydrogel (Fe3O4@Gel) Figure 2 As a control group, we compared its surface pore size with that of Mg / Fe3O4@Gel. Figure 2 H). It is worth noting that the surface porosity of Mg / Fe3O4@Gel is significantly larger than that of Gel and Fe3O4@Gel, and comparable to that of Mg@Gel ( Figure 2 I).

[0076] To characterize the rheological properties of Mg / Fe3O4@Gel, we evaluated its elastic solid behavior and shear thinning characteristics by monitoring the changes in its storage modulus (G') and loss modulus (G'') with time, angular frequency (ω), and strain. The results showed that the gelation time of Mg / Fe3O4@Gel was approximately 200 s, and G' approached 1,280 Pa at 25 °C. Figure 3 F(i)). Within the test frequency range of 1-100 Hz, G' is always greater than G(i). Figure 3 F (ii)). When ω is fixed at 1 rad / s, the G' and G'' curves intersect at approximately 2% strain, marking the start of the sol-gel transition of Mg / Fe3O4@Gel ( Figure 3 F (iii)). Furthermore, compression tests showed that Mg / Fe3O4@Gel had the highest compressive strength among all groups ( Figure 3 G).

[0077] To characterize the magnetism of Fe3O4MNPs, we used a vibrating sample magnetometer to evaluate the intrinsic magnetization of Mg / Fe3O4@Gel. The magnetization curves showed that both Fe3O4@Gel and Mg / Fe3O4@Gel exhibited high saturation magnetization (Ms), with little difference between them, while the magnetization of Gel and Mg@Gel was almost negligible (~0 emu / g). Figure 3 H). Subsequently, using an AMF generator to induce the magnetocaloric effect, after heating for 300 s, the temperatures of Fe3O4@Gel and Mg / Fe3O4@Gel significantly increased to 28℃ and 30℃, respectively, while the temperature changes of Gel and Mg@Gel were negligible. Figure 3 I). Furthermore, thermal infrared images of Mg / Fe3O4@Gel after 5 min of AMF irradiation further confirmed its excellent magnetocaloric properties. Figure 3 J). Cyclic heating curves of Mg / Fe3O4@Gel under 5 AMF cycles ( Figure 3 K) shows that the temperature peak variation was controlled within ±0.5℃ throughout the entire magnetothermal cycle.

[0078] Through AMF irradiation, heating and cooling can be precisely and repeatably controlled. Figure 3 K).

[0079] Figure 2 Figure 1 shows the morphological characterization of Gel, Mg@Gel, Fe3O4@Gel, and Mg / Fe3O4@Gel. Figure A is a SEM image of Mg particles. Figure B shows the particle size distribution of Mg particles; scale bar: 30 μm. Figure C is a TEM image of Fe3O4 MNPs; Figure D shows the particle size distribution of Fe3O4 MNPs; scale bar: 100 nm. Figures E and H are representative SEM images of Gel, Mg@Gel, Fe3O4@Gel, and Mg / Fe3O4@Gel, respectively; scale bars: E: 100 μm, F: 200 μm, G: 100 μm, H: 200 μm. Figure I shows the surface pore size diagram of Gel, Mg@Gel, Fe3O4@Gel, and Mg / Fe3O4@Gel.

[0080] Figure 3 Figure 1 shows the synthesis and physicochemical characterization of Mg / Fe3O4@Gel. Figure A is a schematic diagram of the preparation of Mg / Fe3O4@Gel (drawn by bioRender.com). Figure B shows the result of the sol-gel transition of Mg / Fe3O4@Gel induced by DF-PEG-DF crosslinking. Figure C is a SEM image of Mg / Fe3O4@Gel; scale bar: 600 μm. Figure D is an EDS elemental distribution map of C, O, Mg, and Fe in Mg / Fe3O4@Gel; scale bar: 600 μm. Figure E shows the percentage of each element in Mg / Fe3O4@Gel. Figure F shows the changes in storage modulus (G') and loss modulus (G'') of Mg / Fe3O4@Gel with time (i), angular frequency (ii), and strain (iii). Figure G is a characterization diagram of the mechanical strength of Gel, Mg@Gel, Fe3O4@Gel, and Mg / Fe3O4@Gel. Figure H shows the curves of saturation magnetization (Ms) as a function of magnetic field strength for each of the above gel groups. Figure I shows the temperature change curves under AMF. Figure J shows the dynamic infrared thermograph of Mg / Fe3O4@Gel under AMF. Figure K shows the AMF thermal cycling curve of Mg / Fe3O4@Gel.

[0081] The excellent injectability and controllable electromagnetic heating properties of Mg / Fe3O4@Gel indicate that this material has good application prospects in the field of anti-tumor therapy.

[0082] Example 2: Mg / Fe3O4@Gel-mediated magnetothermal therapy (MHT) combined with metal therapy induces apoptosis and immunogenic cell death (ICD).

[0083] The in vitro antitumor effect of Mg / Fe3O4@Gel was evaluated using SCC VII cells. Flow cytometry analysis showed that, compared with the other five groups (blank, gel, Mg@Gel, Mg@Gel+AMF, Fe3O4@Gel+AMF), the apoptosis rate of SCC VII cells treated with Mg / Fe3O4@Gel combined with AMF was significantly increased, reaching 72%. Figure 4 A, Figure 5 A). TUNEL assays further confirmed that apoptosis was significantly enhanced in the Mg / Fe3O4@Gel+AMF group ( Figure 4 B, Figure 5 B).

[0084] Furthermore, there was no significant difference in apoptosis rates between the Mg@Gel and Mg@Gel+AMF groups. Figure 5 (A, 5B) indicates that AMF irradiation did not enhance Mg-induced apoptosis. In contrast, the apoptosis rate of cells in the Mg / Fe3O4@Gel+AMF treatment group was significantly higher than that in the Mg@Gel group and the Fe3O4@Gel+AMF group ( Figure 5 (A, 5B) indicates a significant synergistic effect between MHT and Mg metal therapy.

[0085] Mitochondrial membrane potential (MMP) instability, oxidative stress, and intracellular pH (pHi) dysregulation can all activate apoptosis. This study further investigates the synergistic mechanism between MHT and Mg metal therapy. JC-1 assays of MMP showed that the Mg / Fe3O4@Gel+AMF group exhibited the most significant changes in MMP in SCC VII cells. Figure 4 C, Figure 5 C). The DCFH-DA probe was used to detect ROS levels, and the results showed that the Mg / Fe3O4@Gel+AMF treatment group induced the most significant ROS accumulation. Figure 4 D, Figure 5 D), suggesting that the synergistic oxidative burst was caused by the combination of Mg metal therapy and MHT. pHi measurements using the BCECF-AM probe showed that, compared to the control group, the SCC VII cells in the Mg / Fe3O4@Gel+AMF treatment group exhibited the largest pHi variation. Figure 4 E, Figure 5 E). In summary, Mg / Fe3O4@Gel induces apoptosis by disrupting the intracellular environment of cancer cells, thus exhibiting good anti-tumor effects in vitro.

[0086] Changes in the intracellular environment during apoptosis (mitochondrial damage, disruption of redox homeostasis, and pHi dysregulation) are important factors inducing intracellular cognitive decline (ICD). Therefore, this experiment further investigated the induction of ICD in cells treated with Mg / Fe3O4@Gel+AMF. Immunofluorescence staining was used to detect calreticulin (CRT) and high-mobility group box 1 (HMGB1). The results showed that the Mg / Fe3O4@Gel+AMF group had the highest accumulation of CRT on the cell membrane and the most significant loss of intracellular HMGB1. Figure 4 F, Figure 5 F, Figure 5 G).

[0087] Since HMGB1 is secreted back into the extracellular space during ICD, the HMGB1 content in the culture medium of each group was detected by ELISA. The results showed that the abundance of HMGB1 in the Mg / Fe3O4@Gel+AMF group was the highest, which was 4 times that of the blank group. Figure 4 G). These results indicate that Fe3O4-mediated MHT combined with Mg metal therapy is more effective in inducing ICD than single therapy.

[0088] The CCK-8 assay was used to comprehensively evaluate the antitumor effect of Mg / Fe3O4@Gel on SCC VII cells under AMF treatment and its biocompatibility with normal MC3T3-E1 cells. Figure 4 As shown in Figure H, both the Fe3O4@Gel+AMF group and the Mg@Gel group exhibited moderate cytotoxicity against SCCVII cells, which was attributed to the metal therapy effects of Fe3O4+AMF-mediated MHT and Mg, respectively. When MHT was combined with metal therapy (Mg / Fe3O4@Gel+AMF group), the viability of SCCVII cells decreased significantly, to only about 19% of that in the control group, indicating that Mg-enhanced MHT has a strong cytotoxic effect. Meanwhile, the control group, Gel group, Mg@Gel group, Fe3O4@Gel group, and Mg / Fe3O4@Gel group had little effect on normal mouse MC3T3-E1 cells. Among them, the cell viability of the Mg-containing hydrogel group (Mg@Gel) was slightly increased, while the cell survival rate of the other groups remained at around 100%, showing a slight promoting effect on normal cell proliferation. Figure 4 I). In summary, Mg / Fe3O4@Gel not only exhibits good biocompatibility with normal cells, but also demonstrates potent tumor-killing ability under AMF irradiation.

[0089] Figure 4Figure 1 shows the in vitro activation of SCC VII cell death by Mg / Fe3O4@Gel. Figures A and B show the flow cytometry results and TUNEL assay results of SCC VII cell apoptosis, respectively; scale bar: 100 μm. Figure C shows the JC-1 staining results of MMP changes in SCC VII cells (JC-1 monomers: green, JC-1 aggregates: red); scale bar: 100 μm. Figures D and E show the flow cytometry results of intracellular reactive oxygen species (ROS) and pHi levels in SCC VII cells, respectively. Figure F shows the immunofluorescence staining of CRT and HMGB1 expression in SCC VII cells; scale bar: 100 μm. Figure G shows the ELISA results of HMGB1 levels in cell culture supernatant. Figure H evaluates the antitumor effect of SCC VII cancer cells using the CCK-8 assay. Figure I evaluates the biocompatibility of MC3T3-E1 normal cells. *: p < 0.05, ***: p < 0.001, ns: p > 0.05.

[0090] Figure 5 This is a quantitative analysis of Mg / Fe3O4@Gel-triggered SCC VII cell death. Figures A and B show the quantitative analysis of SCC VII cell apoptosis induced by Annexin V / PI staining and TUNEL assay, respectively. Figure C shows the quantitative analysis of SCC VII cells labeled with JC-1 dye. Figure D shows the quantitative analysis of mean fluorescence intensity (MFI) of SCC VII cells using the DCFH-DA probe. Figure E shows the quantitative analysis of MFI of SCC VII cells using the BCECF-AM probe. Figures F and G show the quantitative analysis of CRT and HMGB1 expression in SCC VII cells, respectively. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

[0091] Example 3: Mg / Fe3O4@Gel-mediated in vivo antitumor therapy

[0092] Based on its proven biocompatibility and in vitro ICD-inducing ability, we further evaluated the in vivo antitumor effect of Mg / Fe3O4@Gel+AMF and its relationship with TIME remodeling. In situ SCC VII tumor models were established in immunodeficient BALB / c nude mice and immunocompetent C3H mice for related experiments.

[0093] Model establishment: On day -7 (D-7), SCC VII cells were subcutaneously injected into the skull region of mice, and visible in situ tumors formed one week later.

[0094] On day 0 (D-0), Mg@Gel, Fe3O4@Gel, and Mg / Fe3O4@Gel gels were injected into the tumor in situ, respectively. Figure 6 A, Figure 7 A), PBS was injected as a blank control. Simultaneously, mice treated with the above three hydrogels were irradiated with AMF to induce a magnetocaloric effect. Data showed that the tumor area temperature was significantly increased in the Fe3O4@Gel+AMF group and the Mg / Fe3O4@Gel+AMF group, while the temperature increase in other groups was negligible, consistent with in vitro experimental results. Figure 6 B, 6C, Figure 7 B. Figure 7 C).

[0095] The efficacy was evaluated on day 12 (D12). Results showed that in both immunodeficient BALB / c nude mouse and immunocompetent C3H mouse models, the Mg / Fe3O4@Gel+AMF group had the smallest tumor volume and weight, and the highest tumor growth inhibition rate. Figure 6 D, 6E, Figure 7 D, 7E). As expected, the Mg / Fe3O4@Gel+AMF group mice had the longest median survival time (>60 days), while mice in other groups died within 60 days due to tumor burden. Figure 6 D (iv)).

[0096] Histological analysis showed that, compared with other groups, the Mg / Fe3O4@Gel+AMF group had relatively normal mitotic patterns in in situ tumor cells, the lowest cell proliferation level (low Ki-67 positivity rate), and the highest apoptosis rate (high TUNEL positivity rate). This further demonstrates its significant in vivo antitumor effect. Figure 6 F). In summary, based on the in vitro experimental results, Mg@Gel and Fe3O4@Gel+AMF both showed certain inhibitory effects on in situ SCC VII tumors in vivo, while the blank control group and the Gel group did not show significant anti-tumor effects, which fully demonstrates that MHT and magnesium-based metal therapy have significant synergistic anti-tumor effects in vivo.

[0097] Notably, after injection of Mg / Fe3O4@Gel, the in situ tumor suppression effect of SCC VII in immunocompetent C3H mice was significantly better than that in immunodeficient BALB / c nude mice. In C3H mice, after 12 days of treatment with Mg / Fe3O4@Gel + AMF, the tumor volume was significantly reduced from baseline (D0) (D12). Figure 6 D (i)). In immunodeficient mice, however, the tumor continued to grow ( Figure 7 D (i)). Analysis of tumor growth inhibition rate further confirmed the difference between the two groups ( Figure 6 D (iii), Figure 7D(iii)). The above results indicate that the immune system plays a key role in the in vivo antitumor activity of Mg / Fe3O4@Gel hydrogel.

[0098] A systematic evaluation of the biosafety of Mg / Fe3O4@Gel hydrogel was conducted. Given the potential release of metal ions during hydrolysis of metal-hydrogel composites, this study used a tumor-bearing C3H mouse model to measure the levels of magnesium (Mg) and iron (Fe) in blood and major organs (heart, liver, spleen, lung, and kidney) on days 4, 8, and 12 post-injection. The results showed that all indicators remained within the normal physiological range, indicating no significant impact on magnesium or iron homeostasis in vivo. Figure 6 G). Furthermore, no abnormalities were found in blood biochemical indicators or the histological structure of major organs. Figure 6 G, Figure 7 No histological abnormalities or fluctuations in physiological parameters were observed in F-7H, further verifying that Mg / Fe3O4@Gel has good biocompatibility in vivo.

[0099] Figure 6 This study describes the in vivo antitumor therapeutic effect of Mg / Fe3O4@Gel on orthotopic SCC VII tumor-bearing C3H mice under AMF irradiation. Figure A is a schematic diagram of the treatment process (created using bioRender.com). Figure B shows the temperature changes at the tumor sites in each group. Figure C is an infrared thermographic image of tumor-bearing C3H mice treated with Mg / Fe3O4@Gel+AMF. Figure D shows the tumor volume (i), body weight (ii), growth inhibition rate (iii), and survival rate (iv) of tumor-bearing C3H mice during treatment (n = 5). Figure E is a photograph of the tumor in orthotopic SCC VII tumor-bearing C3H mice; scale bar: 1 cm. Figure F shows H&E, Ki67, and TUNEL staining of orthotopic SCC VII tumors in C3H mice; scale bar: 50 μm. Figure G shows the Mg and Fe content, complete blood cell count, and biochemical indicators in major organs of the Mg / Fe3O4@Gel+AMF group. ***: p < 0.001.

[0100] Figure 7This section assesses the in vivo antitumor activity and biocompatibility of Mg / Fe3O4@Gel. Figure A is a schematic diagram of the treatment process in BALB / c nude mice (created using bioRender.com). Figure B shows the temperature change curves at the tumor sites in each group. Figure C is an infrared thermographic image of tumor-bearing BALB / c nude mice treated with Mg / Fe3O4@Gel+AMF. Figure D shows the tumor volume (i), body weight (ii), and growth inhibition rate (iii) (n = 5) of tumor-bearing C3H mice during treatment. Figure E is a photograph of the tumor in orthotopic SCC VII tumor-bearing BALB / c nude mice; scale bar: 1 cm. Figure F shows representative HE-stained images of the heart, liver, spleen, lung, and kidney of C3H mice in each group; scale bar: 100 μm. Figures G and H show the levels of Mg and Fe in the blood of C3H mice after 4, 8, and 12 days of Mg / Fe3O4@Gel+AMF treatment, respectively. *: p < 0.05, **: p < 0.001, ns: p > 0.05.

[0101] Example 4: Identification of related module genes for the antitumor effect of Mg / Fe3O4@Gel

[0102] To clarify the regulatory mechanism of the antitumor effect of Mg / Fe3O4@Gel, the transcriptome characteristics of SCC VII tumors in C3H mice before treatment (blank group) and after treatment with Mg@Gel, Fe3O4@Gel+AMF, and Mg / Fe3O4@Gel+AMF were analyzed. RNA-seq analysis showed that the hydrogels in each group could remodel the transcriptome expression pattern of SCC VII tumors. Figure 8 A).

[0103] Specifically, compared with the control group, the Mg@Gel treatment group had 442 upregulated genes and 63 downregulated genes; while the Mg / Fe3O4@Gel+AMF group had 418 upregulated genes and 62 downregulated genes. Figure 8 B,8C). Based on gene set enrichment analysis (GSEA) and GO enrichment analysis, differentially expressed genes upregulated in the Mg@Gel group were mainly enriched in the interleukin signaling pathway and significantly enriched in immune-related biological processes such as inflammation and immune response. Figure 8 D, 8E). Similarly, the upregulated genes in the Mg / Fe3O4@Gel+AMF group were also significantly enriched in biological processes such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation pathways, chemotaxis, inflammatory responses, cellular responses to tumor necrosis factor (TNF), and immune responses. Figure 8 These results demonstrate at the molecular level the crucial role of immunity in the antitumor effects of Mg / Fe3O4@Gel.

[0104] Weighted co-expression network (WGCNA) analysis was performed using RNA-seq data from the blank group, Mg@Gel, Fe3O4@Gel+AMF, and Mg / Fe3O4@Gel+AMF groups. Considering both scale independence and average connectivity, a power exponent β=6 (scale-free R²) was selected. 2 = 0.85) as the soft threshold ( Figure 9 A). Under the conditions of a merging threshold height of 0.25 and a minimum module gene count of 30, a total of 16 gene co-expression modules were obtained, and they were distinguished by different colors ( Figure 8 H, Figure 9 B). Subsequently, Pearson correlation analysis was used to analyze the relationship between each module and the treatment group. Among the 16 identified modules, the "greenyellow" module, containing 692 genes, showed the highest correlation with the Mg / Fe3O4@Gel+AMF group (r = 0.79, p < 0.01). Figure 8 Therefore, this module was identified as a key module for subsequent research on its functions and mechanisms.

[0105] To further elucidate the biological functions of the "greenyellow" module genes, the inventors performed GO functional enrichment analysis and KEGG pathway enrichment analysis on the genes contained in this module. GO enrichment results showed that this module was significantly enriched in multiple biological processes closely related to tumor progression, specifically including oxidative stress, immune response, hypoxia response, and IL-1-mediated cellular response (Figure 8J). KEGG pathway analysis showed that the genes in this module are mainly involved in typical tumor-related pathways such as apoptosis, oxidative phosphorylation, ferroptosis, and the p53 signaling pathway (see Figure 9C). Notably, the GO enrichment results for "immune response" and "IL-1-mediated cellular response" clearly revealed that Mg / Fe3O4@Gel can effectively activate the immune response in the SCC VII tumor microenvironment. In vitro and in vivo experiments further confirmed that the enrichment of this module in the regulatory pathways of oxidative stress and immune activation is highly consistent with the mechanism of ICD induction. In conclusion, the "greenyellow" module gene can be identified as a key regulatory module gene for the anti-tumor effect of Mg / Fe3O4@Gel.

[0106] Figure 8Transcriptome analysis revealed the antitumor molecular mechanism of Mg / Fe3O4@Gel under AMF irradiation. Figure A shows the distribution of differentially expressed genes (DEGs) among different groups. Figure B is a heatmap of DEGs in the Mg@Gel group and the control group. Figure C is a heatmap of DEGs in the Mg / Fe3O4@Gel+AMF group and the control group. Figure D, based on Reactome database GSEA analysis, shows that the upregulated DEGs in the Mg@Gel group were significantly enriched in the interleukin signaling pathway compared to the control group. Figure E shows the functional enrichment analysis results of the upregulated DEGs in the Mg@Gel group compared to the control group. Figure F, based on Reactome database GSEA analysis, shows that the upregulated DEGs in the Mg / Fe3O4@Gel+AMF group were significantly enriched in ROS and RNS generation compared to the control group. Figure G shows the functional enrichment analysis results of the upregulated DEGs in the Mg@Gel group compared to the control group. Figure H is a topological overlap matrix diagram showing the gene network expressed in all four groups. Figure I shows the correlation analysis between module genes and groups, and the heatmap shows the correlation r and p values. Figure J shows the GO pathway enrichment analysis results of the "greenyellow" module gene.

[0107] Figure 9 This is a correlation analysis based on weighted gene co-expression network analysis (WGCNA). Figure A shows the determination of soft thresholding (β=6) and scale-free topology. Figure B is a clustering dendrogram of each group of expressed genes based on topological overlap dissimilarity, with module colors assigned. Figure C shows the KEGG pathway enrichment analysis results for genes in the "greenyellow" module.

[0108] Example 5: Mg / Fe3O4@Gel remodels the immune microenvironment and is positively correlated with Ltf expression.

[0109] In-depth analysis of the 692 gene characteristics of the "greenyellow" module was conducted to screen key genes closely related to the antitumor effect of Mg / Fe3O4@Gel. Scatter plot analysis showed a significant linear positive correlation between module membership (MM) and gene significance (GS) (r = 0.62, p < 0.01), suggesting that these genes play an important role in the antitumor mechanism. Figure 10 A). To further screen potential targets, the top 200 genes by MM and GS values ​​were selected as candidate genes. Subsequently, Venn diagram intersection analysis was performed on these 200 candidate genes and the top 200 upregulated DEGs in the Mg@Gel group and the Mg / Fe3O4@Gel+AMF group. Figure 10B). The results showed that five genes were found in the intersection of the three groups: Ltf, Camp, Ngp, Slc4a1, and CD177. Further analysis indicated that the expression levels of these five key genes gradually increased from the blank group, the Mg@Gel group, to the Mg / Fe3O4@Gel+AMF group. Figure 11 A).

[0110] Given that Mg / Fe3O4@Gel may exert its anti-tumor effect through an immune-mediated mechanism, this study selected the Ltf gene as a starting point for in-depth analysis. Immunohistochemical and Western blotting results showed that the expression level of Ltf gradually increased from the blank group, the Mg@Gel group to the Mg / Fe3O4@Gel+AMF group. Figure 10 C Figure 11 B). Analysis using the GEPIA2 database revealed that Ltf expression in adjacent normal tissues was significantly higher than that in tumor tissues. Figure 11 C). Analysis of immune cell infiltration using the CIBERSORT algorithm and RNA-seq data revealed that the expression level of Ltf in orthotopic SCC VII tumors was related to CD8 expression. + T cell activation is positively correlated with ( Figure 10 D). Further survival analysis using the GEPIA2 database showed high expression of Ltf and CD8. + HNSCC patients with higher T-cell infiltration levels had better survival rates than patients with both low and low T-cell infiltration levels. Figure 11 D).

[0111] The tumor immune microenvironment (TIME) remodeling of in situ SCC VII tumors in each group was studied. The results showed that after injection of Mg@Gel and Mg / Fe3O4@Gel, CD4+ in the tumor tissue was significantly reduced. + CD8 + and CD69 + (Activate CD8) + T cell infiltration was significantly increased. Figure 10 E, Figure 11 E, Figure 11 F). Notably, Mg / Fe3O4@Gel-induced CD8 + CD4 + and CD69 + T cell infiltration was significantly higher than in other groups.

[0112] Furthermore, immunostimulatory cytokines (IFN-γ and TNF-α) are important indicators for assessing antitumor immune efficiency. Studies have found that the levels of IFN-γ and TNF-α in tumor tissue and serum of the Mg / Fe3O4@Gel+AMF group were higher than those in the Mg@Gel group and the blank control group. Figure 10 (F-10I). In summary, Mg / Fe3O4@Gel may enhance anti-tumor immune responses by upregulating Ltf expression and reshaping the immune microenvironment of in situ SCCVII tumors.

[0113] Figure 10 This section presents an analysis of the remodeling of the tumor immune microenvironment (TIME) by Mg / Fe3O4@Gel and its correlation with Ltf. Figure A is a scatter plot of module membership (MM) and gene significance (GS) in the "greenyellow" module. Figure B is a Venn diagram showing the screening and identification process of candidate regulatory genes. Figure C is an immunohistochemical gradient analysis of Ltf protein expression levels in the blank group, Mg@Gel group, and Mg / Fe3O4@Gel+AMF group; scale bar: 30 μm. Figure D shows the correlation between five candidate regulatory genes and dendritic cell (DC) activation, natural killer cell (NK) activation, and CD8+. + Correlation analysis of T cell activation. Figure E shows the flow cytometry assessment of CD4 in orthotopic SCC VII tumors. + CD8 + Tumor-infiltrating T cells and CD8 + CD69 in T cells + The expression of IFN-γ. Figures F and G show the quantitative analysis of IFN-γ concentration in serum and tumor tissue in different groups (n = 5). Figures H and I show the quantitative analysis of TNF-α concentration in serum and tumor tissue in different groups (n = 5). **: p < 0.01, ***: p < 0.001.

[0114] Figure 11 This section presents a correlation analysis of Ltf. Figure A shows the expression heatmap of five candidate genes in the control group, Mg@Gel group, and Mg / Fe3O4@Gel+AMF group. Figure B shows the expression level of Ltf protein in the control group, Mg@Gel group, and Mg / Fe3O4@Gel+AMF group as detected by Western blotting. Figure C shows the difference in Ltf expression between HNSCC tumors and corresponding normal tissues analyzed using the GEPIA2 database. Figure D shows the high and low co-expression of Ltf and CD8. + Survival analysis of HNSCC patients with T cells. Figure E shows T cells (CD4+) in SCC VII tumors. + / CD8 + Flow cytometry results of CD4 infiltration and CD69+ expression. Figure F shows the CD4 infiltration and expression results for each group. + CD8 + and CD69 + Immunofluorescence images of T cell infiltration; scale bar: 50 μm. *: p < 0.05.

[0115] Example 6: Mg / Fe3O4@Gel drives antitumor effect via ltf-mediated ICD

[0116] TIME regulation is an effective strategy for triggering ICD. The foregoing examples have demonstrated that Mg / Fe3O4@Gel can induce ICD in SCC VII cells in vitro. To further verify the ability of Mg / Fe3O4@Gel to induce ICD in orthotopic SCC VII tumors in vivo, this example uses a bilateral orthotopic SCC VII tumor model for evaluation.

[0117] The model was constructed as follows: On day -7 (D-7), SCC VII cells were inoculated into the skull region of C3H mice (to form a primary tumor); on day -3 (D-3), SCC VII cells were inoculated into the distal left side of the mice (to form a distal tumor). On day 0 (D-0), the primary tumor was injected with Mg / Fe3O4@Gel followed by AMF treatment. Figure 12 A). Two weeks later (D14), Mg / Fe3O4@Gel showed significant inhibitory effects on both primary and distal SCC VII tumors, with tumor volumes of approximately 14.5 mm. 3 and 215 mm 3 ( Figure 12 B). On day 14 (D14), the quality of both primary and distal tumors was significantly lower than that of the control group ( Figure 12 C, 12D). Results from the bilateral tumor model further confirmed that Mg / Fe3O4@Gel can simultaneously inhibit the growth of primary and distal SCC VII tumors, suggesting that it exerts its anti-tumor effect by stimulating ICD.

[0118] To verify the mediating role of Ltf in Mg / Fe3O4@Gel-induced ICD and antitumor processes, Ltf was knocked out in SCC VII cells using CRISPR / Cas9 technology (Ltf-KO), and Ltf was overexpressed via a lentiviral system (Ltf-OE). Figure 13 A,13B). Tumor allogeneic xenograft models of Ltf-KO, Ltf-OE, and their control groups were established in BALB / c nude mice and C3H mice, respectively (named Ltf-KO group, Ltf-OE group, and control group, respectively). Figure 12 E, Figure 13 C). In BALB / c nude mice, there was no significant difference in tumor volume among the groups (D-14, Figure 13 D-13F). In C3H mice, the tumor volume in the Ltf-KO group was significantly larger than that in the control group, while the tumor volume in the Ltf-OE group was significantly smaller than that in the control group (D-13F). Figure 12F-12H). This further confirmed the immunomodulatory role of Ltf in tumor suppression. Notably, previous studies have found that the Ltf-induced tumor suppression effect was eliminated in mice with depleted CD3⁺ cells. Furthermore, when treated with Mg / Fe3O4@Gel, both the Ltf-OE group and the wild-type group showed significantly better tumor suppression than the Ltf-KO group, with the Ltf-OE group showing superior efficacy compared to the wild-type group. Figure 12 I). In summary, the results further validate that ICD activation participates in the in vivo antitumor effect of Mg / Fe3O4@Gel, and clarify that Ltf plays a key mediating role in this process.

[0119] Figure 12 This diagram illustrates the antitumor efficacy of Mg / Fe3O4@Gel through Ltf activation of the ICD. Figure A is a schematic diagram of a bilateral orthotopic tumor model in C3H mice treated with Mg / Fe3O4@Gel (created by bioRender.com). Figure B shows the growth curves of primary and distal tumors in C3H mice after treatment with Mg / Fe3O4@Gel+AMF. Figures C and D are representative images and corresponding volumes and weights of primary and distal SCC VII tumors in the Mg / Fe3O4@Gel+AMF group and the control group, respectively (n = 5); scale bar: 1 cm. Figure E is a schematic diagram of a tumor xenograft model established in C3H mice using Ltf-OE and Ltf-KO SCC VII cells (created using bioRender.com). Figure F shows the growth curves of tumors in C3H mice using Ltf-OE, Ltf-KO, and the control group. Figures G and H are tumor images in the Ltf-OE, Ltf-KO, and control groups of C3H mice, respectively; scale bar: 1 cm. Figure I shows images (i) and volumes (ii) of different groups of in situ SCC VII tumors treated with Mg / Fe3O4@Gel+AMF (n = 5); scale bar: 1 cm; ***: p < 0.001.

[0120] Figure 13 This section describes the establishment and evaluation of Ltf-modified SCC VII cells and corresponding tumor xenograft models in BALB / c nude mice. Figures A and B are schematic diagrams of the construction and Western blotting validation of Ltf knockout (Ltf-KO) and Ltf overexpression (Ltf-OE) SCC VII cells, respectively (created using bioRender.com). Figure C is a schematic diagram of the BALB / c nude mouse tumor xenograft model (created using bioRender.com). Figures D and F show images (D) and growth curves (E, F) of tumors in the BALB / c nude mouse Ltf-OE group, Ltf-KO group, and control group; scale bar: 50 μm; ns: p > 0.05.

[0121] The above research was approved by the Laboratory Animal Research Ethics Committee of Peking University School of Medicine.

[0122] This invention provides a Mg / Fe3O4@Gel hydrogel combining magnetic nanoparticles and metal elements, offering a highly efficient approach for synergistic enhancement of tumor immunotherapy through magnetothermal therapy (MHT) and metal therapy. Under alternating magnetic field (AMF) irradiation, Mg / Fe3O4@Gel triggers TIME remodeling, inducing MMP instability, ROS generation, and pHi dysregulation, ultimately leading to cell death. This synergistic effect effectively activates TIME regulation and systemic immune response, inducing Ltf-mediated ICD, thereby inhibiting the progression of primary and distant tumor lesions. In a mouse model of this invention, ICD induction experiments confirmed that inoculation of tumor cells treated with Mg / Fe3O4@Gel+AMF induced protective immunity against HNSCC re-challenge. Simultaneously, CD3+ isolated from donor mice... + T, after metastasis, can significantly inhibit tumor growth in immunodeficient hosts. Complete experimental data further validated that under AMF irradiation, Mg / Fe3O4@Gel, through the Ltf-induced ICD mechanism, has significant anti-tumor efficacy in immunosuppressive HNSCC and can effectively inhibit the progression of primary and distant lesions.

[0123] The present invention has the following advantages over the prior art:

[0124] 1. Simple Synthesis and Excellent Biocompatibility: The synthesis process of the Mg / Fe3O4@Gel described in this invention is simple, and the raw materials used have high biocompatibility, avoiding complex preparation processes and facilitating large-scale production and clinical application. 2. Synergistic Effect and Superior Efficacy: This invention expands the application of injectable metal-based hydrogels. Through the synergistic effect of magnetothermal ablation and immune activation, it achieves highly efficient inhibition of primary tumors and immunosuppressive tumors, showing good clinical translational prospects in the field of magnetothermal therapy / metal therapy-enhanced immunotherapy. 3. Broad-Spectrum Application Potential: The composition described in this invention contains multiple metal elements, endowing it with broad anti-tumor immunotherapy capabilities, providing a universal treatment solution for expanding from HNSCC to various immunosuppressive tumors. 4. Dual Function of Treatment and Distant Lesion Protection: Based on the unique mechanism of action of Mg / Fe3O4@Gel, it can not only be used for direct tumor treatment but also has the potential to treat distant lesions of tumors. The above description is a general description of the invention. Depending on the circumstances or actual needs, variations in form and equivalent substitutions may be made. Although specific terminology is used herein, it is intended for descriptive purposes and not for limitation. Those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A hydrogel composition for antitumor immunotherapy, characterized in that: The composition contains magnesium microparticles, Fe3O4 magnetic nanoparticles, amino-containing chitosan, and crosslinking agent dibenzaldehyde polyethylene glycol, wherein the diameter of the magnesium microparticles is 50-55 μm and the diameter of the Fe3O4 magnetic nanoparticles is 90-100 nm; each 1 mL of the composition contains 20-50 mg of magnesium microparticles and 10-50 mg of Fe3O4 magnetic nanoparticles. The magnesium microparticles and Fe3O4 magnetic nanoparticles are mixed with amino-containing chitosan to form an intermediate solution, which is then cross-linked with a cross-linking agent to form a hydrogel.

2. A method for preparing the hydrogel composition as described in claim 1, characterized in that: The mixture of magnesium microparticles and Fe3O4 magnetic nanoparticles with a hydrogel matrix material was used to obtain the product. Hydrogel matrix materials are cross-linked using cross-linking agents.

3. The method as described in claim 2, characterized in that: Magnesium microparticles and Fe3O4 magnetic nanoparticles are mixed with amino-containing chitosan to form an intermediate solution, which is then cross-linked with a cross-linking agent to form a hydrogel. The Fe3O4 magnetic nanoparticles are composed of Fe 3+ The Fe3O4 magnetic nanoparticles are prepared by precipitation of salt under alkaline conditions; a surfactant is added during the preparation of the Fe3O4 magnetic nanoparticles.

4. The use of the composition according to claim 1 in the preparation of a pharmaceutical product, characterized in that... The drug is used to treat a tumor, specifically a squamous cell carcinoma of the head and neck.

5. The application as described in claim 4, characterized in that: The application is in combination with magnetothermal therapy for anti-tumor immunotherapy.

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