Curcumin-magnesium-containing system and application thereof in improvement of tumor immune microenvironment

Abstract

The curcumin-magnesium-containing system comprises a matrix unit and an auxiliary crosslinking unit. The matrix unit is selected from at least one curable matrix of a photosensitive hydrogel, a temperature-sensitive polymer or a pH-responsive gel. And the auxiliary crosslinking unit comprises a biopolymer with an active functional group or a derivative thereof. Through verification, the system can maintain the biological activity of Cur and Mg < 2 + >, has the anti-tumor effect of remarkably enhancing microwave ablation, and provides a new strategy for adjusting the anti-tumor immune microenvironment after ablation and protecting pleural injury, so that a comprehensive treatment method is provided for tumor ablation treatment.

Description

Technical Field

[0001] This invention relates to a biomaterial, and more particularly to a gel with curcumin-magnesium as the active ingredient, for the application of improving the tumor immune microenvironment after microwave ablation. Background Technology

[0002] Lung cancer is currently the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 85%-90% of these deaths, and surgical resection is the primary means of cure. However, 25%-30% of patients are ineligible for surgical resection due to poor cardiopulmonary function, advanced age, locally advanced stage, metastatic lung cancer, or multiple primary lung cancers. Percutaneous image-guided thermal ablation (IGTA) is a minimally invasive technique suitable for patients who cannot undergo surgical resection. It can maximize the preservation of lung function while controlling local tumor growth. The NCCN guidelines recommend IGTA for these patients. IGTA includes radiofrequency ablation, cryoablation, and microwave ablation. Microwave ablation has a shorter ablation time, a larger ablation area, and is superior to the other two ablation methods for tumors >3 cm in diameter. It is also less affected by thermal sedimentation during the ablation process, making it more suitable for treating tumors adjacent to large blood vessels. However, 22%-27% of cases experience local progression after microwave ablation, seriously threatening the prognosis and survival of NSCLC patients. Therefore, a new treatment strategy is urgently needed to enhance the efficacy of microwave ablation.

[0003] Microwave ablation (WMA) can lyse cells through thermal damage, generating a large number of tumor-associated antigens (TAAs) and various damage-related molecules. These factors can activate anti-tumor immunity, but are insufficient to effectively inhibit tumor recurrence or progression. Therefore, combining microwave ablation with anti-tumor immunotherapy is a promising strategy to enhance its efficacy. Among the many immunotherapies for NSCLC patients, immune checkpoint inhibitors have shown strong efficacy and safety. Their use alone or in combination with platinum-based chemotherapy drugs can significantly prolong patient survival, but their efficacy is mainly limited by the immune cold environment of solid tumors and systemic immune-related adverse events. Therefore, there is an urgent need for strategies that can improve the immune microenvironment of solid tumors to assist microwave ablation in combination with anti-tumor immunotherapy.

[0004] With the development of nanomaterials technology, various strategies are available, including metal-polyphenol networks (MPNs). MPNs are organic-inorganic hybrid materials formed by coordinating phenolic ligands with metal ions, enabling the controlled and sustained release of the contained metal ions. Numerous studies have shown that Mg... 2+ With enhanced CD8 + It has functions such as T cell effector, increasing T cell infiltration, and reducing T cell exhaustion. Based on previous studies, the curcumin-magnesium polyphenol network has thermal stability, good biocompatibility, and the ability to alter macrophage polarization; it can slowly release Mg. 2+, give full play to Mg 2+ In addition to its functions, curcumin also imparts antioxidant, anti-inflammatory, analgesic, and cell proliferation-promoting effects.

[0005] Nevertheless, the clinical application of curcumin-magnesium, especially in improving the tumor immune microenvironment after microwave ablation, still faces many challenges, such as rapid diffusion and rapid degradation. Summary of the Invention

[0006] One object of the present invention is to provide a curcumin-magnesium system that improves the stability and bioavailability of curcumin.

[0007] Another object of the present invention is to provide a curcumin-magnesium system that improves the mechanical strength of hydrogels.

[0008] Another object of the present invention is to provide the application of a curcumin-magnesium system in the preparation of drugs or medical devices that improve the tumor immune microenvironment.

[0009] Another object of the present invention is to provide the application of a curcumin-magnesium system in the preparation of products that improve the antitumor immune microenvironment after thermal ablation.

[0010] The system of the present invention includes: Active units include metal-polyphenol materials; Matrix unit: at least one curable matrix selected from photosensitive hydrogels, temperature-sensitive polymers, or pH-responsive gels; and Assisted crosslinking unit: contains a biopolymer or its derivative with active functional groups.

[0011] In this invention, the curable matrix satisfies at least one of the following conditions: Curing occurs within 30 seconds under ultraviolet / visible light irradiation; It forms a gel within 5 minutes at body temperature; It triggers in situ cross-linking upon contact with physiological tissues.

[0012] In this invention, the auxiliary crosslinking unit comprises: Selected from at least one of hyaluronic acid, collagen, fibrin or their derivatives; The derivatives are modified with amino, carboxyl, thiol or aldehyde groups.

[0013] A system using a composition of gelatin-acryloyl (GelMA) and o-nitrobenzyl hyaluronic acid sodium (HANB) as a carrier, wherein the GelMA and HANB powders are constructed by strong ionic bonds, in an amount such as 2 / 1.

[0014] Another system is a hydrogel.

[0015] GelMA is biocompatible, biodegradable, non-cytotoxic, and non-immunogenic, and has been used alone as a hydrogel carrier. HANB is a photosensitive polymer material obtained by modifying hyaluronic acid (HA) with o-nitrobenzyl alcohol (NB). HA is an important component of the human extracellular matrix, possessing important physiological functions such as water retention, maintaining extracellular space, regulating osmotic pressure, lubrication, and promoting cell repair. These biomaterials, under photo-initiated free radical polymerization, such as under ultraviolet (UV) light irradiation, interact with photoinitiators to form cross-linked structures. Cross-linked hydrogels can achieve controlled release of active substances while reducing potential cytotoxicity. Furthermore, hydrogels can provide additional adhesion and stiffness, repairing thermally damaged pleura during microwave ablation, thereby avoiding complications such as pneumothorax and hemothorax.

[0016] The system provided by this invention also includes curcumin (Cur) and magnesium (Mg). 2+ Metal-polyphenol materials composed of ) are used as active ingredients.

[0017] It has been verified that Cur-Mg@GH combines Cur and Mg. 2+ The therapeutic effects of Cur-Mg@GH hydrogel are enhanced, improving the anti-tumor process of microwave ablation. The therapeutic effects of Cur-Mg@GH are optimized through the following methods: (i) Improve the stability and bioavailability of Cur; (ii) The anti-inflammatory and analgesic effects imparted by Cur after ablation; (iii) Mg 2+ Increased CD8 in the post-ablation cell reduction state + T cells / CD4 + T cell ratio; (iv) Mg 2+ It promoted the polarization of M1 macrophages in a cell-reduced state after ablation; (v) The physical barrier formed by GH hydrogel and its ability to prevent pleural damage; the multiple functions of Cur-Mg@GH have been verified in vitro and in vivo. This multifunctional Cur-Mg@GH retains the properties of Cur and Mg... 2+ The bioactivity of this study provides a new approach to promote the anti-tumor immune microenvironment after ablation and to protect against pleural damage after ablation.

[0018] The hydrogel provided by this invention also includes a PD1 monoclonal antibody to promote anti-tumor immunity after ablation.

[0019] After thermal ablation of the lung tumor, the curcumin- and magnesium-containing hydrogel (Cur-Mg@GH) of this invention is placed in the ablation area and solidified after ultraviolet irradiation. Cur-Mg@GH transforms into a solid hydrogel to continuously release Mg. 2+ And PD1 antibody.

[0020] Furthermore, Cur-Mg@GH exhibits good morphological plasticity and adhesive strength, thus improving pleural defects. The structure of the magnesium polyphenol network in Cur-Mg@GH enhances the biocompatibility and stability of Cur, and the interaction between Cur and Mg... 2+ It can promote the chemotaxis of CD8+ T cells and dendritic cells, and promote changes in CCL2 and CXCL2. The Cur-Mg@GH hydrogel successfully achieved the transition from a cold to a hot environment in the tumor, and released PD1 and Mg. 2+ The therapeutic effect on tumors has been verified in vivo.

[0021] The invented system can be made into medical devices or drugs and applied to improve the tumor immune microenvironment, especially the tumor immune microenvironment after microwave ablation. Attached Figure Description

[0022] Figure 1 Figures show the preparation and characterization results of Cur-Mg; where a is a schematic diagram of Cur-Mg synthesis, b is a field emission scanning electron microscope (FE-SEM) image of Cur-Mg, c is a magnified view of the part shown in Figure b, d is a scanning electron microscope (SEM) image of Cur-Mg, e is the elemental C mapping result of Cur-Mg, f is the elemental O mapping result of Cur-Mg, g is the elemental Mg mapping result of Cur-Mg, h is the X-ray diffraction (XRD) pattern of Cur-Mg powder, i is the elemental distribution and energy dispersive spectroscopy (EDS) analysis results of Cur-Mg, j is the absorption spectrum of Cur-Mg at various concentrations, k is the absorption intensity fitting curve of Cur-Mg at 456 nm at various concentrations, l is the optical photograph of the stability study of Cur-Mg at various concentrations, and m is the optical photograph of GH and Cur-Mg@GH. Figure 2This document describes the preparation and characterization of GH and Cur-Mg@GH hydrogels. Figure a shows a schematic diagram of the preparation process for Cur-Mg@GH hydrogels; figure b shows a schematic diagram of the mechanical testing of GH and Cur-Mg@GH hydrogels and their overlap shear test curves; figure c shows a schematic diagram of the mechanical testing of GH and Cur-Mg@GH hydrogels and their compression test curves; figure d shows the rheological analysis results of Cur-Mg@GH hydrogels (the relationship between viscosity and shear stress as a function of shear rate); figure e shows the frequency-dependent storage modulus (G′), loss modulus (G″), and complex viscosity (|η*|) results of Cur-Mg@GH hydrogels; figure f shows the degradation curves of GH and Cur-Mg@GH hydrogels over 20 days; figure g shows the cumulative release curve of Cur-Mg / aPD1@GH hydrogels in deionized water over 20 days; and figure h shows the concentrations of aPD1 at 562 nm as determined by the BCA method. The absorbance intensity fitting curve at point i is a statistical graph of the cumulative release curve of aPD1 of Cur-Mg / aPD1@GH hydrogel in deionized water for 20 days; Figure 3 The in vitro cytotoxicity assessment of Cur-Mg@GH is shown below: a) is a statistical graph of lung epithelial cell proliferation after 24 hours of co-culturing with various concentrations of Cur-Mg or normal cell culture medium; b) is a statistical graph of lung epithelial cell proliferation after 48 hours of co-culturing with various concentrations of Cur-Mg or normal cell culture medium; c) is a statistical graph of lung epithelial cell proliferation after 72 hours of co-culturing with various concentrations of Cur-Mg or normal cell culture medium; d) is a live / dead double-stained fluorescence microscopy image of lung epithelial cells and bone marrow stem cells after 48 hours of co-culturing with GH hydrogel or Cur-Mg@GH hydrogel; e) is a statistical graph of the survival rate of lung epithelial cells and bone marrow stem cells after 24 hours of co-culturing with GH hydrogel or Cur-Mg@GH hydrogel; f) is a statistical graph of the survival rate of lung epithelial cells and bone marrow stem cells after 48 hours of co-culturing with GH hydrogel or Cur-Mg@GH hydrogel. Figure 4The images show the in vivo pleural repair effects of Cur-Mg / aPD1@GH. In the images, a is an optical photograph taken at the first time point to record the tumor ablation process after induction of lung tumors in rats; b is an optical photograph taken at the second time point to record the tumor ablation process after induction of lung tumors in rats; c is an optical photograph taken at the third time point to record the tumor ablation process after induction of lung tumors in rats; d is an optical photograph taken at the fourth time point to record the tumor ablation process after induction of lung tumors in rats; e is an optical photograph taken at the fifth time point to record the tumor ablation process after induction of lung tumors in rats; f is an optical photograph taken at the sixth time point to record the tumor ablation process after induction of lung tumors in rats; g is a representative chest CT image of rats in the five experimental groups before treatment and one week after treatment (yellow dashed boxes indicate atelectasis, blue dashed boxes indicate pneumothorax); h is a statistical graph of the preoperative tumor diameter in the five experimental groups; i is a statistical graph of the incidence of atelectasis in the five groups one week after surgery; and j is a statistical graph of the incidence of pneumothorax in the five groups one week after surgery. Figure 5 The image shows the therapeutic effects of curcumin-loaded magnesium-polyphenol network hydrogel delivery of aPD1 in a rat LLC lung tumor model enhanced by microwave ablation (MWA). Figure a shows a schematic diagram of the in vivo treatment regimen in the rat LLC tumor model; figure b shows representative optical images of LLC tumors collected from rats after 35 days of treatment using various methods; figure c shows the postoperative tumor diameter in lung tissue of each experimental group after 35 days of treatment; figure d shows the difference between preoperative and postoperative tumor diameter in each group after 35 days of treatment; figure e shows the weight gain curve of rats during the 35-day treatment period in each group; figure f shows the survival rate of rats in each experimental group; figure g shows representative immunohistochemical staining fields of CCL2, CXCL12, and PCNA in tumor sections of LLC tumor-bearing rats after treatment in each group; figure h shows the quantitative analysis statistics of CCL2 expression in tumor sections; figure i shows the quantitative analysis statistics of CXCL12 expression in tumor sections; and figure j shows the quantitative analysis statistics of PCNA expression in tumor sections. Figure 6Figure 1 shows the in vivo antitumor efficacy and immune mechanism verification results of microwave ablation, curcumin-loaded magnesium-polyphenol network hydrogel, and anti-PD1 immunotherapy combined application. Specifically, a) is the flow cytometry quantitative analysis of CD4⁺ T cells (CD3⁺CD4⁺), b) is the flow cytometry quantitative analysis of CD8⁺ T cells (CD3⁺CD8⁺), c) is the flow cytometry quantitative analysis of the CD8⁺ T cell / CD4⁺ T cell ratio, d) is the flow cytometry quantitative analysis of CD103⁺ dendritic cells (DCs), e) is the flow cytometry quantitative analysis of myeloid suppressor cells (MDSCs; CD11b⁺Gr-1⁺), f) is the flow cytometry quantitative analysis of PD1 (CD279) expression, and g) shows different macrophage populations (CD68⁺CD206⁺, CD68⁺iN) in rat tumors after various treatments. Representative immunofluorescence images of OS⁺ and CD68⁺CD11c⁺ are shown. h is a quantitative analysis image of macrophage subsets (CD68⁺CD206⁺) in rat tumors after treatment with various methods. i is a quantitative analysis image of macrophage subsets (CD68⁺iNOS⁺) in rat tumors after treatment with various methods. j is a quantitative analysis image of macrophage subsets (CD68⁺CD11c⁺) in rat tumors after treatment with various methods. Detailed Implementation

[0023] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0024] The main materials used in the following embodiments of the present invention are sourced from the following sources: GelMA (NG-M250) and HANB (NG-M400) were purchased from Shanghai Linggeer Medical Technology Co., Ltd., China. Lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) was supplied by Suzhou Engineering Life Industrialization Co., Ltd., China. Curcumin (Cur, 98% purity) was obtained from Maclean Biochemical Technology Co., Ltd., China. High-glucose DuPont modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco, Inc. (Carlsbad, California, USA). Human bronchial epithelial cell line (Beas2B) and bone marrow mesenchymal stem cells (BMSCs) were cultured in high-glucose DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37°C and 5% CO2.

[0025] The specific experimental methods used in the following embodiments of the present invention are described below: 1) Preparation of curcumin-magnesium (Cur-Mg) The Cur-Mg complex was synthesized via a solvothermal method. Briefly, 8 g of curcumin (Cur) was dispersed in 500 mL of deionized water under magnetic stirring for 10 minutes, followed by pH adjustment to 12 using 10 M NaOH. Next, 10 g of magnesium chloride (MgCl2) was added to the mixture, and stirring was continued for another 10 minutes. The solution was heated at 120°C for 24 hours, and the resulting reddish-brown precipitate was collected by centrifugation (10,000 rpm, 15 minutes, 4°C) and washed twice each with ethanol and deionized water.

[0026] 2) Preparation of hydrogels The GH hydrogel was prepared as follows: 10 mL of 5 wt% GelMA and 10 mL of 2.5 wt% HANB were mixed, and 0.05 g of photoinitiator LAP was added. After irradiation with ultraviolet light (365 nm, 30 seconds), 10 mg of Cur-Mg powder was dispersed into the GH solution under ultrasonic treatment, and then Cur-Mg@GH hydrogel was obtained by ultraviolet crosslinking. For Cur-Mg / aPD1@GH hydrogel, before ultraviolet irradiation, 10 mg of anti-PD1 antibody (aPD1) was added to the Cur-Mg@GH solution, and the mixture was shaken for 4 hours to ensure homogeneity.

[0027] 3) Characterization of Cur-Mg Cur-Mg was dissolved in PBS to prepare solutions with concentrations of 0, 15.625, 31.25, 62.5, 125, and 250 μg / mL. Absorbance spectra were measured using a SpectraMAX iD3 microplate reader (Molecular Devices, USA) to identify characteristic peaks and establish standard curves.

[0028] 4) Mechanical property testing Compressive strength was evaluated using a GT-TCS-2000 universal testing machine. For the lap shear test, the hydrogel was adhered to a glass slide (contact area 5 cm²) and a tensile load was applied. All tests were repeated three times at room temperature.

[0029] 5) Rheological analysis Dynamic viscoelastic properties were measured using a Thermo HAAKE MARS 60 rheometer (25 mm parallel plate, 25°C, 1% constant strain). Viscosity was determined in rotational mode (shear rate 0.01–100 s⁻¹), while oscillation frequency sweeps (10–0.1 Hz) were used to determine storage modulus (G'), loss modulus (G''), and complex viscosity.

[0030] 6) Release kinetics 2.5 ml of Cur-Mg@GH hydrogel was immersed in 1 ml of deionized water, and the supernatant was collected at predetermined time points and analyzed at 456 nm to quantify the release of Cur-Mg.

[0031] 7) Degradation research The hydrogel was incubated in PBS (37°C, 5% CO2) and lyophilized at specified time points. The degradation rate was calculated using the formula: (W0 - W t ) / W0 ×100%, where W0 and W t These represent the initial weight and the weight at the time point, respectively.

[0032] 8) CCK-8 Detection Beas2B cells and BMSCs (5000 cells / well) were co-cultured with hydrogel extracts (prepared by soaking 125-1000 μg / ml Cur-Mg@GH in complete medium for 3 days) for 24-72 hours. Cell viability was assessed using 10% CCK-8 reagent, and absorbance was measured at 450 nm.

[0033] 9) Live / Dead dyeing Cells (2×10) 4 The sample was co-cultured with hydrogel for 72 hours, then stained with calcein-AM / PI (China Yisheng Biotechnology), and imaged using a fluorescence microscope.

[0034] 10) Animal experiments Female SD rats (6-8 weeks old) were purchased from Hunan Borui New Life Science Co., Ltd. All experimental protocols were approved by the International Association of Laboratory Animal Ethics (IACUC) of Shanghai Jiao Tong University.

[0035] 11) Tumor Model Lewis lung cancer (LLC) cells (5 × 10⁻⁶) 6 Administer 50 μl PBS via injection into the left lung parenchyma of anesthetized rats (3% sodium pentobarbital, 30 mg / kg). Postoperative care includes antibiotic prophylaxis (penicillin, 2 × 10⁻⁶ mg / kg). 5 U / kg / day, for 3 consecutive days).

[0036] 12) Treatment group Tumor-bearing SD rats were randomly divided into five groups (n=3): G1: control group, G2: microwave ablation (WMA) group, G3: HY hydrogel + WMA group, G4: Cur-Mg@GH hydrogel + microwave group, and G5: Cur-Mg / aPD1@GH hydrogel + microwave group. WMA treatment was performed using an MTC-3C microwave therapy device (Nanjing Vision Medical, China) (Figure S4). Based on cytotoxicity assay results, the hydrogel was injected into the wound at a concentration of 500 μg / ml Cur-Mg@GH. The average weight of the rats was approximately 200 g. Each rat received 1 mg aPD1 (10 mg / kg), Cur-Mg (10 mg / kg, based on aPD1, Gd / M to aPD1 mass ratio 1:1), and 2 ml GH hydrogel. Weight was monitored every two days. Mice were sacrificed on day 35, and tumors were collected for immunohistochemical, immunofluorescence, and flow cytometry analysis.

[0037] 13) CT imaging On the first day after surgery, a chest CT scan was performed using a SCENARIA View 16-slice spiral CT system (Hitachi, Japan).

[0038] 14) Flow cytometry Tumor single-cell suspensions were prepared by enzymatic digestion (collagenase IV / DNase / hyaluronidase) and stained with fluorescent antibodies against CD3, CD4, CD8, CD11b, Gr-1, CD11c, CD103, and PD1. Data were acquired using a CytoFLEX flow cytometer (Beckman) and analyzed using FlowJo software.

[0039] 15) Histological analysis Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 μm), and subjected to H&E and immunohistochemical staining (CCL2, CXCL12, PCNA) or immunofluorescence staining (CD68 / CD206 / iNOS / CD11c). Images were acquired using a digital slide scanner and a Leica SP5 microscope.

[0040] 16) Statistical Analysis Data are expressed as mean ± standard deviation. Comparisons were performed using unpaired t-tests or one-way ANOVA combined with Dunnett's multiple comparison test (SPSS 26.0). Significance was defined as p < 0.05, no significance in ns, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Graphs were generated using GraphPad Prism, and image analysis was performed using ImageJ (NIH). Example 1: Preparation and Characterization of Cur-Mg Cur-Mg is synthesized via a hydrothermal method. It is a porous material with magnesium ions as bonding sites and Cur as a framework (Figure 1a). Scanning electron microscopy (SEM) images show that Cur-Mg has a spherical structure with a diameter of 5-10 micrometers (Figure 1b and 1c). Figure 1 c). To verify the synthesis and composition of Cur-Mg, elemental mapping was performed, and the results showed that carbon (C), magnesium (Mg), and oxygen (O) were uniformly distributed in Cur-Mg (Figure 1d). Figure 1 e Figure 1 f、 Figure 1 g and Figure 1 h). The X-ray diffraction (XRD) pattern of Cur-Mg powder shows obvious and characteristic peaks ( Figure 1 i), indicating the successful synthesis of Cur-Mg. To investigate the relationship between different concentrations of Cur-Mg and the absorption peak, spectral analysis was performed using concentrations of 62.5 µm / ml, 125 µm / ml, 250 µm / ml, and 500 µm / ml. It was observed that all concentrations of Cur-Mg exhibited a characteristic absorption peak at 456 nm. Figure 1 j and Figure 1 The absorption peak of Cur-Mg shifted compared to Cur at 428 nm, due to the chelation between the carbonyl group of Cur and the metal ion. Images of the appearance at 125 µm / ml, 250 µm / ml, and 500 µm / ml are shown below. Figure 1 l. Example 2: Synthesis and Characterization of Cur-Mg@GH Hydrogel Figure 2 A demonstrates the process for preparing Cur-Mg / GH composite hydrogel blocks. Fluorescence images and 3D reconstructions of CT images of the Cur-Mg / GH composite hydrogel blocks are shown below. Figure 2 As shown in Figure a, the white GH hydrogel and the orange Cur-Mg@GH hydrogel are as follows: Figure 1As shown in the figure. The respiratory movements of the thoracic cavity are mainly manifested as the expansion and compression of the thoracic cage. To characterize the adhesion and compressibility of the hydrogels, shear and compression tests were conducted on Gelma, HANB, GH, and Cur-Mg@GH. The results showed that the combination of GH and Cur-Mg significantly improved the mechanical properties of Gelma and HANB. Compared with single hydrogels, GH's shear strength and resistance to displacement were between those of Gelma and HANB, with its shear strength being twice that of HANB and its resistance to displacement being nearly three times that of single Gelma hydrogel. Figure 2 a). The toughness of GH hydrogel is between that of Gelma and HANB, and the compressive strength of GH is nearly twice that of HANB alone. Figure 2 b and Figure 2 c). To investigate the rheological properties of Cur-Mg@GH hydrogel, the viscosity and shear stress of Cur-Mg@GH hydrogel under varying shear rates were measured. Modulus and composite viscosity were also tested (Figure 2d and 2d). Figure 2 e). The results show that the Cur-Mg@GH hydrogel has good viscoelasticity.

[0041] Furthermore, this embodiment investigated the degradability of the hydrogels and found that due to the addition of Cur-Mg, the linkage of Cur-Mg@GH was more stable, thus its stability was superior to GH. The degradation rate of both hydrogels began to slow down on day 10. On day 20, the degradation rates of Cur-Mg@PP and PP hydrogels were 40% and 70%, respectively. Figure 2 f). The release characteristics of Cur-Mg in Cur-Mg@GH hydrogel were studied. The results showed that the release concentration of Cur-Mg peaked between day 5 and day 10, stabilized until day 15, and then reached a second peak, after which it tended to stabilize at a stable level. Figure 2 The release characteristics of PD1 in Cur-Mg@GH hydrogel were investigated. The results showed that the release concentration of PD1 peaked between days 5 and 10 and then remained stable. Figure 2 h and Figure 2 i). Therefore, the application of Cur-Mg / PD1@GH enables the slow release and continuous administration of Cur-Mg and PD1.

[0042] Subsequently, the repair effect of Cur-Mg@GH hydrogel on pleural rupture was verified. A rat lung was harvested, and an incision was made in the surface of the left lung to connect it to a ventilator. The lung was submerged below the water surface, and the ventilator was activated to inflate the lungs; bubbles were observed emerging (e.g., ...). Figure 2 j and Figure 2 k), after coating the broken surface with Cur-Mg@GH hydrogel and curing it under light, bubbles no longer emerged (e.g. Figure 2 l、 Figure 2 m and Figure 2 (n) indicates that the hydrogel, after solidification, can repair pleural ruptures and treat lung leaks. Example 3: In vitro biocompatibility of Cur-Mg@GH To investigate the cytotoxicity of Cur-Mg@GH, Cur-Mg@GH extracts at various concentrations were obtained after soaking in DMEM for 3 days and used in in vitro CCK-8 assays. Lung epithelial cells were co-cultured with Cur-Mg@GH extract for 24 hours. When the Cur-Mg solution concentration reached 1000 μg mL⁻¹, no significant cytotoxicity was observed in the lung epithelial cells (Figure 3a), and the optimal proliferation concentration was 125 µg / mL. No significant toxicity was observed after co-culturing for 48 and 72 hours. Figure 3 b and Figure 3 c), and as time progressed, there was no significant difference in the proliferative effect of different concentrations. Therefore, combined with the aforementioned concentration of 500 µg / ml, the absorption wavelength reached its peak (e.g., Figure 1 (j) Unless otherwise stated, a 500 µg / ml Cur-Mg solution was used in subsequent experiments. Similar results were obtained from the same experiments performed on human embryonic stem cells (BMSCs).

[0043] Further testing of the biocompatibility of GH and Cur-Mg@GH was conducted by co-culturing lung epithelial cells and human embryonic stem cells with GH and Cur-Mg@GH hydrogels for 24 and 48 hours, followed by live / dead cell double staining, yielding similar results. Figure 3 d、 Figure 3 e and Figure 3 f). Example 4: In vivo pleural repair effect of Cur-Mg@GH hydrogel The efficacy of Cur-Mg@GH in repairing pleural effusion and treating air leakage in an in vitro model of lung pleural injury was demonstrated, but its in vivo pleural repair effect requires further confirmation. An orthotopic tumor model was constructed in the left lung of SD rats. Figure 4 a) Rats with successfully constructed pulmonary orthotopic tumors were divided into 5 groups: G1: control group; G2: microwave ablation group; G3: GH hydrogel group; G4: Cur-Mg@GH hydrogel group; G5: Cur-Mg / PD1@GH hydrogel group. Microwave ablation was performed after incising the skin, muscle layer, and pleura. Figure 4 (b) It can be seen that the ablated tumor appears scorched yellow and shrunken. Figure 4 c) Apply the corresponding hydrogel to the surface of the tumor after ablation in groups G3, G4, and G5, and then irradiate with ultraviolet light. Figure 4 d). The cured hydrogel covers the surgical area in a gel-like state. Figure 4 e), suture the skin ( Figure 4f).

[0044] The mice underwent chest CT scans 7 days post-surgery. Figure 4 g) To observe the in vivo pleural repair effect, it was observed that there was no statistically significant difference in the preoperative tumor diameter. Figure 4 h), postoperative atelectasis was observed in three mice in groups G2 and G3, while only one mouse in groups G4 and G5 developed atelectasis. The incidence of atelectasis was significantly lower than that in the ablation group and the GH hydrogel group. Figure 4 g and Figure 4 i). Subsequently, the occurrence of pneumothorax due to pleural rupture was observed. All three mice in group G2 developed pneumothorax, while no pneumothorax occurred in groups G3, G4, and G5. Figure 4 g and Figure 4 j). Example 5: Effect of Cur-Mg@GH hydrogel on promoting burn wound healing in vivo. After validating the in vitro and in vivo pleural repair results of Cur-Mg@GH, further research is needed on the in vivo antitumor effect of microwave ablation of Cur-Mg / PD1@GH, such as... Figure 3 As shown in figure a, a mouse model of an in situ tumor in the left lung was constructed.

[0045] First, tumor cells (1×10⁶) were injected into the left lower lung of mice to form primary tumors. When the diameter of the primary tumor reached 7 mm (12 days), the mice were divided into 5 groups (G1: control group; G2: MWA group; G3: GH hydrogel group; G4: Cur-Mg@GH hydrogel group; G5: Cur-Mg / aPD1@GH hydrogel group). On day 22, the tumors were treated with MWA (microwave heating to 60°C and maintaining 60°C for 3 minutes). After MWA treatment, 500 µL GH, Cur-Mg@GH (500 µg / mL), and Cur-Mg / PD1@GH (500 µg / mL) were injected into the tumor in the surgical area. The weight of the rats was measured every 2 days after surgery, and the tumor growth and detection indicators were measured after 35 days. Figure 5 a). MWA alone can inhibit tumor growth, while the addition of GH, Cur-Mg@GH, or Cur-Mg / PD1@GH shows a better ability to inhibit tumor growth. Figure 5 b and Figure 5 c), further statistical analysis of preoperative and postoperative tumor diameters confirmed the above conclusions. Figure 5 d). During the 35-day observation period, two rats died in the blank control group (days 9 and 21), one rat died in the second group (day 6), and there were no deaths in groups three through five. Figure 5e). Tumor and ablation therapy are painful experiences, and pain relief is very important in ablation therapy. Numerous studies have demonstrated that Cur has analgesic properties (44). Tumor, pain, and inflammation caused by ablation can affect the diet and metabolism of mice, thereby affecting their weight. Therefore, the weight changes of mice after treatment were recorded. The results showed that on the second day after treatment, the weight of rats in the control group decreased by nearly 6% compared with that before incision, the weight of rats in the ablation group and GH group decreased by nearly 8% compared with that before treatment, while the weight of rats in the Cur-Mg@GH and Cur-Mg / PD1@GH groups decreased by nearly 5% compared with that before treatment. Over the next 35 days, the weight of mice in all four groups continued to increase, with the Cur-Mg / PD1@GH group showing the fastest weight gain. The weight gain of the other four groups of rats gradually became more consistent ( Figure 5 g). Studies have shown that ablation can alter the tumor's immune microenvironment and change the secretion of immune factors; therefore, immunohistochemical staining was performed on five groups of rats after 35 days. This study used CCL2, CXCL12, and PCNA markers ( Figure 5 g). Quantitative results of CCL2 showed that, compared with the control group, the ablation group decreased by 46.22 times, while Cur-Mg@GH and Cur-Mg / PD1@GH increased by 3.95 times and 2.55 times, respectively. Figure 5 h).

[0046] Quantitative results for CXCL12 showed that, compared with the control group, the ablation group decreased by 3.69 times, while the Cur-Mg@GH and Cur-Mg / PD1@GH groups showed no significant rebound. Figure 5 i). PCNA quantitative results showed that the ablation group decreased by 4.56 times compared with the control group, while Cur-Mg@GH and Cur-Mg / PD1@GH did not show a significant rebound (i). Figure 5 j). Example 6: Cur-Mg@GH hydrogel induces polarization transition of M1 macrophages in vivo In the alteration of the tumor immune microenvironment, the proportions of various related immune cells are crucial, particularly CD4. + / CD8 + Therefore, on day 35, flow cytometry was performed on lung tumor tissues from the control group, WMA group, GH group, Cur-Mg@GH group, and Cur-Mg / PD1@GH group. The results are as follows: Figure 6 As shown in the figure, G1: control group; G2: microwave ablation group; G3: GH hydrogel group; G4: Cur-Mg@GH hydrogel group; G5: Cur-Mg / aPD1@GH hydrogel group; n = 3.

[0047] This embodiment uses CD4. + T cells (CD3) + CD4+ ), CD8+ T cells (CD3) + CD8 + DC cell markers (CD11C) + CD103 + Tumor-associated macrophage markers (CD11C) + Gr1 + () Figure 6 af). CD4 + T cell results showed that, compared to the control group, the number of T cells increased in the WMA group and decreased in the GH group, Cur-Mg@GH group, and Cur-Mg / PD1@GH group. Figure 6 a). CD8 + T cell quantification results showed an upward trend in all five groups ( Figure 6 b). CD8 + / CD4 + Quantitative results of cell percentage showed no significant change in the WMA group compared to the control group, while the GH group, Cur-Mg@GH, and Cur-Mg / PD1@GH treatments significantly reduced CD8+ cell percentage. + / CD4 + Increased by 2.07 times, 2.63 times, and 3.33 times ( Figure 6 c). Quantitative results of DC cells showed an upward trend in all five groups ( Figure 6 d). Quantitative results of tumor-associated macrophages showed a decreasing trend in all five groups ( Figure 6 e), PD-L1 quantification results showed no significant changes in the 5 groups ( Figure 6 f), which demonstrates that Cur-Mg@GH improves the efficacy of microwave ablation by influencing the tumor's immune microenvironment. To further investigate its mechanism, we performed immunofluorescence staining on tumor tissues from the five groups on day 35. This study used macrophage markers (CD68, green), M1 macrophage markers (iNOS, red), M1 macrophage markers (CD11C, red), and M2 macrophage markers (CD206, red) (f). Figure 6 g,h). CD206 + / CD68 + Quantitative results of cell percentage showed no significant difference between the WMA group and the GH group compared with the control group, while Cur-Mg@GH and Cur-Mg / PD1@GH treatments reduced M2 polarization by 7.90-fold and 8.21-fold, respectively. Figure 6 h). iNOS + / CD68 +Quantitative results of cell percentage showed no significant difference between the WMA group and the GH group compared with the control group, while Cur-Mg@GH and Cur-Mg / PD1@GH treatments increased M1 polarization by 4.06-fold and 5.49-fold, respectively. Figure 6 i). CD11C + / CD68 + Quantitative results of cell percentage showed that, compared with the control group, the WMA group and GH group decreased by 5.85 times, while Cur-Mg@GH and Cur-Mg / PD1@GH treatments increased M1 polarization by 3.06 times and 3.42 times, respectively. Figure 6 (j) These results indicate that, over a longer period of 35 days, ablation damages tumor cells while reducing the infiltration of immune cells, and Cur-Mg@GH can promote macrophage M1 polarization under ablation conditions, thereby increasing CD8+. + / CD4 + The proportion of PD1 enhances the efficacy of PD1 immunotherapy. In summary, the Cur-Mg / aPD1@GH provided in this embodiment integrates the therapeutic effects of curcumin (Cur), magnesium ions (Mg²⁺), anti-PD1 (aPD1), and gelatin hydrogel (GH) to enhance the antitumor efficacy of microwave ablation. The therapeutic performance of Cur-Mg@GH is optimized through multiple mechanisms: (i) improved stability and bioavailability of curcumin; (ii) anti-inflammatory and analgesic effects of curcumin during the recovery period after ablation; (iii) Mg²⁺ mediates an increase in the CD8⁺ T cell / CD4⁺ T cell ratio under conditions of lymphopenia after ablation; (iv) Mg²⁺ promotes macrophage polarization towards the M1 phenotype after ablation; and (v) the GH hydrogel forms a physical barrier to prevent pleural damage. The multifunctional properties of Cur-Mg@GH have been systematically validated in vitro and in vivo. This compound formulation, while maintaining the bioactivity of Cur and Mg²⁺, provides a new strategy for regulating the anti-tumor immune microenvironment and protecting against pleural damage after ablation, thus offering a comprehensive treatment approach for tumor ablation therapy.

Claims

1. A system containing curcumin-magnesium, characterized in that... include: Matrix unit: at least one curable matrix selected from photosensitive hydrogels, temperature-sensitive polymers, or pH-responsive gels; and Assisted crosslinking unit: contains a biopolymer or its derivative with active functional groups.

2. The system according to claim 1, characterized in that... The active unit is a metal-polyphenol formed by curcumin and magnesium.

3. The system according to claim 1, characterized in that... Curable substrates must meet at least one of the following conditions: Curing occurs within 30 seconds under ultraviolet / visible light irradiation; It forms a gel within 5 minutes at body temperature; It triggers in situ cross-linking upon contact with physiological tissues.

4. The system according to claim 1, characterized in that... The auxiliary crosslinking unit includes: Selected from at least one of hyaluronic acid, collagen, fibrin or their derivatives; The derivatives are modified with amino, carboxyl, thiol or aldehyde groups.

5. The system according to claim 1, characterized in that... Including GelMA.

6. The system according to claim 1, characterized in that... Including sodium hyaluronate with o-nitrobenzyl alcohol.

7. The system according to claim 1, characterized in that... It also includes PD1 monoclonal antibodies.

8. The application of the system according to claim 1 in the manufacture of medical devices.

9. The application of the system according to claim 1 in the preparation of drugs that improve the tumor immune microenvironment.

10. The application of the system according to claim 1 in the preparation of products that improve the tumor immune microenvironment after microwave ablation.

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