A gelma hydrogel composite material, and a preparation method and application thereof

Abstract

This invention discloses a GelMA hydrogel composite material, its preparation method, and its applications, belonging to the field of materials preparation technology. This invention prepares microneedle-shaped GelMA hydrogel composite materials (GelMA MNs) by grafting methacrylic anhydride functional groups onto the side chain amino groups of gelatin. Then, by loading doxorubicin and an aPD-L1 inhibitor into the GelMA MNs, a dual-drug delivery system is established. The prepared aPD-L1 / Dox@GelMA MNs significantly enhances the recognition and killing of melanoma cells by immune cells while minimizing skin damage and promoting immunogenic cell death (ICD), providing a new approach to overcome the limitations of ICIs.

Description

Technical Field

[0001] This invention belongs to the field of materials preparation technology, specifically relating to a GelMA hydrogel composite material, its preparation method, and its application. Background Technology

[0002] Cutaneous melanoma is an aggressive skin cancer that is difficult to cure even with surgical excision. This advanced disease also exhibits considerable resistance to traditional chemotherapy and single immunotherapies, including immune checkpoint inhibitors (ICIs). Its complex underlying mechanisms are attributed to the inherent microenvironment of the cold tumor, which leads to an imbalance among immune cells, non-immune cells, and the microbiome. Current systemic treatments for melanoma include surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy, with the latter two becoming the mainstays. However, in addition to potential systemic drug side effects, the in-situ drug concentration at the tumor lesion may not reach ideal levels due to microenvironmental blockade. Furthermore, infection during wound healing after surgical excision often triggers non-tumor-specific inflammatory responses. If this inflammation is automatically suppressed by the body, even if ICIs have restored tumor-specific cytotoxic T cell (CTL) activity to some extent, it can still have adverse effects.

[0003] Treatment options for cutaneous melanoma are diverse, but the tumor microenvironment is complex, rapidly changing, and often accompanied by overexpression of immune checkpoint ligands / receptors. This ultimately leads to limited immune cell infiltration and weakened cytotoxic killing, thus accelerating disease progression and making it more difficult to treat. When immune cells are overactivated, immune checkpoint-related proteins should trigger inhibitory signals to maintain stable autoimmune levels. Tumor cells, including melanoma cells, can hijack this mechanism to evade the immune system. With ongoing research into the role of immune checkpoints in tumor immunity, ICIs have been widely used in the treatment of various malignant tumors. For melanoma patients who have not responded to chemotherapy or molecularly targeted therapy, PD-1 / PD-L1 blockers can significantly prolong their survival; however, the reactivation of immune cells induced by a single ICI may not last long, and once the tumor recurs, reusing the same ICI may be ineffective or lead to complete T cell exhaustion. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a GelMA hydrogel composite material, its preparation method and application, so as to solve the technical problem of poor efficacy of existing drug treatments for melanoma.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for preparing a GelMA hydrogel composite material, comprising the following steps: dissolving collagen in an alkaline solution, then adding a photosensitizer containing an anhydride functional group, reacting at 45-55℃ for 2.5-3.5 h, then dialyzing, purifying at room temperature for 4.5-5.5 d, and freeze-drying the purified solid solution to obtain the GelMA hydrogel composite material; the collagen is gelatin; the photosensitizer containing anhydride functional group is methacrylic anhydride, glycidyl methacrylate, or acryloyl chloride; the mass ratio of collagen, alkaline solution, and photosensitizer containing anhydride functional group is 1-100:20-2000:0.1-50.

[0006] Gelatin is the main product obtained from the irreversible decomposition of collagen. This type of compound has a high affinity for protein molecules on the surface of human tissues, weak antigenicity, good biocompatibility, and biodegradability, and can be degraded and absorbed. Simultaneously, gelatin has a large molecular weight and numerous functional groups, making it easy to modify and exhibiting excellent plasticity. Methacrylic anhydride is a ketene compound containing photosensitive anhydride functional groups, which can be copolymerized through free radical polymerization to form cross-linked compounds. This invention introduces photosensitive groups by grafting the anhydride functional groups of methacrylic anhydride onto the side chain amino groups of gelatin, thereby enhancing the functionalization of gelatin. While retaining its excellent biological properties, it also possesses photosensitivity, giving GelMA hydrogel composite materials the potential for photodynamic therapy. Photodynamic therapy (PDT) relies on the activation of photosensitizers. By exciting photosensitive groups with a specific wavelength of light, singlet oxygen and other reactive oxygen species (ROS) are generated. These substances can selectively destroy melanoma cells, leading to apoptosis or necrosis. Furthermore, this process can induce an immune response against melanoma cells and alter the tumor microenvironment, reducing tumor blood supply and metastatic potential. Therefore, functionalized gelatin hydrogels, with their photosensitizing properties, demonstrate significant potential for effective melanoma treatment in PDT, offering a promising tumor therapy strategy.

[0007] Based on the above technical solution, the present invention can be further improved as follows:

[0008] Furthermore, the mass ratio of collagen, alkaline solution, and photosensitizer containing anhydride functional groups is 4-6:80-120:2-3.

[0009] Furthermore, the alkaline solution is obtained by dissolving sodium carbonate and sodium bicarbonate in water at a mass ratio of 1:2.

[0010] Furthermore, the pH value of the alkaline solution is 9.2-10.6.

[0011] Furthermore, the freeze-drying temperature is -55 to -45°C, and the time is 46-50 hours.

[0012] The present invention also discloses a GelMA hydrogel composite material prepared by the above preparation method.

[0013] This invention also discloses the application of GelMA hydrogel composite materials in the preparation of drugs for treating skin cancer.

[0014] Based on the above technical solution, the present invention can be further improved as follows:

[0015] Furthermore, skin cancer is melanoma.

[0016] Furthermore, the specific steps of the application include: dissolving the GelMA hydrogel composite material in water, then sequentially adding a photoinitiator, doxorubicin, and aPD-L1 inhibitor, mixing thoroughly, allowing the reaction solution to stand under vacuum for 0.5-1.5 hours, then crosslinking under ultraviolet light for 3-7 minutes, and finally drying to obtain a drug for treating skin cancer; the mass ratio of GelMA hydrogel composite material, photoinitiator, doxorubicin, and aPD-L1 inhibitor is 1-100:0.01-1:0.1-10:0.1-10.

[0017] Furthermore, the photoinitiator is LAP, I2959, or VA086; the aPD-L1 inhibitor is tislelizumab, nivolumab, pentazolizumab, atezolizumab, duvalumab, or acimetab.

[0018] aPD-L1 is an inhibitor of programmed death-1 (PD-1) and its ligand (PD-L1). It can block the binding of PD-1 on the surface of T cells to PD-L1 and PD-L2 on the surface of immune cells, upregulate the growth and proliferation of T cells, enhance the recognition of tumor cells by T cells, activate their attack and killing functions, enhance the immune system's attack on tumor cells, and achieve anti-tumor effects by mobilizing the body's own immune function.

[0019] Furthermore, the drying temperature is 35-40℃, and the time is 10-14 hours.

[0020] The beneficial effects of this invention are as follows:

[0021] 1. The aPD-L1 / Dox@GelMA MNs prepared in this invention significantly enhances the recognition and killing of melanoma cells by immune cells while minimizing skin damage, and promotes immunogenic cell death (ICD). In vitro and in vivo experiments show that aPD-L1 / Dox@GelMA MNs enhances ICD by increasing Dox uptake and enhances immune activation by inhibiting PD-L1 signaling, thereby effectively inhibiting melanoma progression. Dox triggers immunogenic cell death, thereby sending signals to antigen-presenting cells to increase CRT, HMGB1, and ATP, while aPD-L1 promotes Dox uptake by melanoma cells. On the other hand, aPD-L1 and Dox synergistically enhance the proportion of CTLs and the secretion level of cytokines in melanoma tissue. The combined use of the two drugs can significantly enhance immunomodulation and significantly inhibit tumor growth, which is beneficial for reducing the systemic dosage of ICIs.

[0022] 2. The GelMA hydrogel prepared by this invention also possesses excellent biocompatibility, cell reactivity, and protein hydrolysis degradation properties, providing suitable cell adhesion sites. Furthermore, the GelMA hydrogel exhibits good mechanical properties, and the 3D microscaffolds constructed from it possess tunable mechanical and chemical properties. Attached Figure Description

[0023] Figure 1 Surface morphology of aPD-L1 / Dox@GelMA MNs;

[0024] Figure 2 Microstructure diagram of aPD-L1 / Dox@GelMA MNs;

[0025] Figure 3 Hematoxylin-eosin staining of skin tissue treated with aPD-L1 / Dox@GelMAMNs;

[0026] Figure 4 Staining images of B16-F10 cells after different treatments;

[0027] Figure 5 The histogram of relative cell viability of B16-F10 cells;

[0028] Figure 6 Cellular uptake efficiency of Dox under different doses of aPD-L1;

[0029] Figure 7 The amount of ATP released from B16-F10 cells after different treatments;

[0030] Figure 8 CRT exposure of B16-F10 cells after different treatments;

[0031] Figure 9 HMGB1 release in B16-F10 cells after different treatments;

[0032] Figure 10 Tumor volume in mouse models of melanoma after treatment with different GelMAMNs

[0033] Figure 11 Survival curves of a mouse model of melanoma after treatment with different GelMAMNs;

[0034] Figure 12 The death of melanoma cells in tumors after treatment with different GelMAMNs;

[0035] Figure 13 The proliferation of melanoma cells in tumors after treatment with different GelMAMNs;

[0036] Figure 14 The proportion of CD11c+CD86+ mature DCs after treatment with different GelMAMNs;

[0037] Figure 15 Histogram of the proportion of mature CD11c+CD86+ DCs after different GelMA MNs treatments;

[0038] Figure 16 The proportion of CD8+ cytotoxic T lymphocytes after treatment with different GelMAMNs;

[0039] Figure 17 Histogram of the proportion of CD8+ cytotoxic T lymphocytes after treatment with different GelMA MNs;

[0040] Figure 18 The proportion of CD4+FoxP3+ regulatory T cells after treatment with different GelMA MNs;

[0041] Figure 19 Histogram of the proportion of CD4+FoxP3+ regulatory T cells after treatment with different GelMA MNs;

[0042] Figure 20 The production levels of immunomodulatory cytokines TNF-α(G) and IFN-γ(G) after treatment with different GelMA MNs;

[0043] Figure 21 Histograms showing the production levels of immunomodulatory cytokines TNF-α(G) and IFN-γ(G) after treatment with different GelMA MNs;

[0044] Figure 22CRT exposure in melanoma tumors after treatment with different GelMA MNs;

[0045] Figure 23 The release of HMGB1 in melanoma tumors after treatment with different GelMA MNs. Detailed Implementation

[0046] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0047] Example 1

[0048] A method for preparing a GelMA hydrogel composite material, the reaction equation of which is as follows:

[0049]

[0050] The specific steps include: dissolving 7.95g of sodium carbonate and 14.65g of sodium bicarbonate in 1L of distilled water to obtain an alkaline solution with a pH of 10; then dissolving 50g of gelatin in the alkaline solution, mixing thoroughly, and adding 10g of methacrylic anhydride. The mixture is reacted at 50°C for 3 hours, and then dialyzed using a dialysis bag with an MWCO of 1000 Da. The mixture is then purified at room temperature for 5 days, and the purified solid solution is freeze-dried at -45°C for 48 hours to obtain the GelMA hydrogel composite material (GelMA MNs).

[0051] The application of the GelMA hydrogel composite material prepared by the above method in the preparation of drugs for treating melanoma is shown in the following reaction equation:

[0052]

[0053] The specific steps include: mixing 50 mL of 15 wt% GelMA hydrogel composite material, 50 mL of 0.25 wt% LAP, 5 mg Dox, and 100 μg tislelizumab evenly, then letting the reaction solution stand under vacuum for 1 h, then crosslinking under 405 nm ultraviolet light for 5 min, and finally drying at 37 °C for 12 h to obtain a microneedle patch drug for treating melanoma (aPD-L1 / Dox@GelMA MNs).

[0054] Example 2

[0055] A method for preparing a GelMA hydrogel composite material, the reaction equation of which is as follows:

[0056]

[0057] The specific steps include: dissolving 7g of sodium carbonate and 15g of sodium bicarbonate in 1L of distilled water to obtain an alkaline solution with a pH of 9.2; then dissolving 50g of gelatin in the alkaline solution, mixing thoroughly, and adding 10g of glycidyl methacrylate. The mixture is reacted at 45°C for 3.5h, and then dialyzed using a dialysis bag with an MWCO of 1000Da. The mixture is then purified at room temperature for 4.5d, and the purified solid solution is freeze-dried at -55°C for 46h to obtain GelMA hydrogel composite material (GelMAMNs).

[0058] The application of the GelMA hydrogel composite material prepared by the above method in the preparation of drugs for treating melanoma is shown in the following reaction equation:

[0059]

[0060] The specific steps include: mixing 50 mL of 15 wt% GelMA hydrogel composite material, 50 mL of 0.25 wt% I2959, 5 mg Dox, and 100 μg nivolumab evenly, then letting the reaction solution stand under vacuum for 0.5 h, then crosslinking under 405 nm ultraviolet light for 7 min, and finally drying at 35 °C for 14 h to obtain a microneedle patch drug for treating melanoma (aPD-L1 / Dox@GelMA MNs).

[0061] Example 3

[0062] A method for preparing a GelMA hydrogel composite material, the reaction equation of which is as follows:

[0063]

[0064] The specific steps include: dissolving 8.5g of sodium carbonate and 14g of sodium bicarbonate in 1L of distilled water to obtain an alkaline solution with a pH of 10.6; then dissolving 50g of gelatin in the alkaline solution, mixing thoroughly, and adding 10g of methacrylic anhydride. The reaction is carried out at 55°C for 2.5h, and then dialyzing the reaction solution using a dialysis bag with an MWCO of 1000Da. The solution is then purified at room temperature for 5.5 days, and the purified solid solution is freeze-dried at -45°C for 50h to obtain the GelMA hydrogel composite material (GelMA MNs).

[0065] The application of the GelMA hydrogel composite material prepared by the above method in the preparation of drugs for treating melanoma is shown in the following reaction equation:

[0066]

[0067] The specific steps include: mixing 50 mL of 15 wt% GelMA hydrogel composite material, 50 mL of 0.25 wt% VA086, 5 mg Dox, and 100 μg pentazocine antibody evenly, then letting the reaction solution stand under vacuum for 1.5 h, then crosslinking under 405 nm ultraviolet light for 3 min, and finally drying at 40 °C for 10 h to obtain a microneedle patch drug for treating melanoma (aPD-L1 / Dox@GelMA MNs).

[0068] Comparative Example 1

[0069] The difference between this comparative example and Example 1 is that the aPD-L1 inhibitor is omitted, while the rest of the preparation process is the same as in Example 1, to obtain Dox@GelMA MNs.

[0070] Comparative Example 2

[0071] The difference between this comparative example and Example 1 is that Dox is omitted, while the rest of the preparation process is the same as in Example 1, to obtain aPD-L1@GelMA MNs.

[0072] Experimental Example 1: Structural Characterization

[0073] The surface morphology of aPD-L1 / Dox@GelMA MNs prepared in Example 1 was observed using SEM, as shown below. Figure 1 As shown, the surface of aPD-L1 / Dox@GelMA MNs exhibits a neatly arranged tapered feature. The microstructure is as follows... Figure 2 As shown, the sponge-like appearance and surface roughness of aPD-L1 / Dox@GelMA MNs reflect the preservation of the porous structure of the GelMA hydrogel composite and the successful encapsulation of aPD-L1 and Dox, respectively. With an average pore size of 10 μm, it is suitable for drug delivery and potential tissue penetration. Figure 3 As shown, HE staining revealed a large number of inflammatory cells, including neutrophils, lymphocytes, and monocytes, indicating that aPD-L1 / Dox@GelMA MNs can effectively induce an inflammatory response in the skin, presumably due to the synergistic effect of aPD-L1 and Dox.

[0074] Experimental Example 2: In vitro anti-melanoma effect

[0075] To evaluate the anti-melanoma efficacy of GelMA MNs, cytotoxicity, cell viability, and drug uptake efficiency were assessed using a live / dead cell staining assay. B16-F10 cells incubated with four different MN formulations (GelMA MNs and aPD-L1 / Dox@GelMA MNs prepared in Example 1, Dox@GelMA MNs prepared in Comparative Example 1, and aPD-L1@GelMAMNs prepared in Comparative Example 2) were double-stained with calcein-AM and EthD-1. Live cells emitted green fluorescence, while dead cells emitted red fluorescence.

[0076] The results are as follows Figure 4 As shown, compared with the blank control group (incubated without MN preparation), cells incubated with unloaded GelMA MNs exhibited a significant green fluorescent cell population, confirming that the cytotoxicity of the GelMA hydrogel composite material itself is negligible. Furthermore, a small number of red fluorescent cells were observed in both the Dox@GelMA MNs and aPD-L1@GelMA MNs treatment groups, indicating limited cytotoxicity; the red fluorescence in the Dox / aPD-L1@GelMA MNs treatment group was significantly increased.

[0077] like Figure 5 As shown, Dox / aPD-L1@GelMAMNs exhibited 2-3 times higher inhibitory activity against tumor cell viability than single-drug loaded MNs formulations (Dox@GelMA MNs and aPD-L1@GelMA MNs), and 5 times higher than unloaded GelMA MNs and the control group.

[0078] In the absence of immune cells, the chemotherapy drug Dox may account for a larger proportion of the cytotoxic effects in cultured tumor cells. Therefore, the effect of different doses (1, 5, and 10 μM) of aPD-L1 on Dox uptake efficiency in cells was investigated. The results are as follows: Figure 6 As shown, aPD-L1 can enhance Dox uptake to varying degrees (1 μM aPD-L1 showed the strongest enhancement effect) and promote Dox translocation into tumor cell nuclei. These results indicate that Dox / aPD-L1@GelMAMNs have significant anti-tumor effects in vitro, and that there is a positive synergistic effect between aPD-L1 and Dox.

[0079] Experimental Example 3a: PD-L1 enhances Dox-induced ICD.

[0080] A comprehensive in vitro assay was performed to reveal whether Dox / aPD-L1@GelMAMNs can utilize the immune system to combat melanoma. The assay focused on the complex interactions between Dox and aPD-L1 in the context of immunogenic cell death (ICD) by measuring hallmarks of ICD such as ATP release, calreticulin (CRT) exposure, and high-mobility group box 1 (HMGB1) release.

[0081] The results are as follows Figure 7 As shown, co-administration of Dox / aPD-L1@GelMA MNs significantly increased ATP release (p<0.001) and enhanced immune recognition. Furthermore, melanoma cells treated with Dox / aPD-L1@GelMA MNs exhibited enhanced externalization of CRTs on their cell surface. Figure 8 ), accompanied by a significant release of HMGB1 from the cell nucleus ( Figure 9 The enhanced cytotoxicity of Dox / aPD-L1@GelMA MNs is attributed not only to the drug's direct cytotoxic effects but also to the induction of an immune response through ICD unleash.

[0082] Experiment Example 4: In vivo anti-melanoma effect

[0083] The concentration is 2.0 × 10 6100 μL of B16-F10 cells were subcutaneously injected into the back of C57BL / 6 mice. After 7 days of growth, the mice were treated with 1.0 mL of MNs formulations (GelMA MNs and aPD-L1 / Dox@GelMA MNs prepared in Example 1, Dox@GelMA MNs prepared in Comparative Example 1, and aPD-L1@GelMA MNs prepared in Comparative Example 2). Patches containing the above MNs formulations were penetrated into the area near the B16 melanoma and fixed to the skin with a sealing film. The MNs were fixed to the tumor, and the patch was changed every 4 days. On day 12, the melanoma tumor was excised, and immunofluorescence staining and flow cytometry analysis were performed. The flow cytometry analysis procedure was as follows: Tumor tissue was surgically removed from mice 12 days after tumor cell inoculation. The tissue was washed with PBS, cut into small pieces, and filtered through a 100 μm cell filter. The filtrate from each tumor was centrifuged at 500 × g for 5 min. The cell pellet was then washed twice with PBS and gently resuspended in staining buffer (PBS containing 2% FBS) for subsequent surface staining. Finally, the cells were stained with fluorescently labeled surface antigen antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD11c, and anti-CD86) at 4 °C for 30 min. The expression of transcription factor Foxp3 was detected using a Foxp3 / transcription factor staining buffer kit (eBioscience, 00-5523-00).

[0084] The results are as follows Figure 10 As shown, compared with the PBS treatment group, GelMA MNs treatment group, Dox@GelMA MNs treatment group, and aPD-L1@GelMA MNs treatment group, the PD-L1 / Dox@GelMA MNs treatment group showed significantly reduced tumor volume (p<0.05), while aPD-L1 / Dox@GelMA MNs showed a remarkable improvement in survival rate within the extended trajectory (p<0.001). Figure 11 ).

[0085] Apoptotic cells in each group of tumors were detected by TUNEL staining, and the results are as follows: Figure 12 The proportion of TUNEL-positive (FITC) cells increased in the aPD-L1 / Dox@GelMA MNs group, with strong fluorescence indicating the presence of extensive DNA fragmentation in the tumor cells. In contrast, histopathological analysis of serial tumor tissue sections showed ( Figure 13 Ki-67 staining and HE staining confirmed a significant reduction in cell proliferation.

[0086] By detecting the infiltration of immune cells within tumors, we can understand the activated immune response. For example... Figure 14 and Figure 15 As shown, both Dox@GelMA MNs and aPD-L1@GelMAMNs significantly increased the proportions of mature DCs (mDCs) of CD11c+ and CD86+ (22.7% and 25.9%, respectively, p<0.001) and CD8+ cytotoxic T lymphocytes (CTLs) (21.2% and 23.2%, respectively, p<0.05 and 0.01); Figure 16 and Figure 17 As shown, aPD-L1 / Dox@GelMA MNs resulted in the highest proportions of mDCs and CTLs (39.7% and 44.3%, respectively, p<0.01).

[0087] In addition, such as Figure 18 and Figure 19 As shown, the number of CD4+FoxP3+ regulatory T cells (Tregs) was slightly reduced in the single-drug loaded MN formulations (Dox@GelMA MNs and aPD-L1@GelMAMNs) (11.3% and 11.6%, respectively, p<0.01), while the number of CD4+FoxP3+ regulatory T cells was significantly reduced in the aPD-L1 / Dox@GelMA MNs group (4.43%, p<0.01). In the aPD-L1 / Dox@GelMA MNs treatment group, the production levels of immunomodulatory cytokines TNF-α and IFN-γ in the tumor microenvironment were significantly higher than those in the control group and the unloaded GelMAMNs group (more than 6-fold and 4-fold, respectively), and 1.5-2 times higher than those in the Dox@GelMAMNs group and the aPD-L1@GelMA MNs group. Figure 20 and Figure 21 ).

[0088] like Figure 22 and Figure 23 As shown, the trends of CRT and HMGB1 in the xenograft model were consistent with those in in vitro experiments. These results indicate that GelMAMNs loaded with immunotherapeutic agents and chemotherapeutic agents enhance the efficacy of immunochemotherapy, providing a new approach to overcome the limitations of ICIs and demonstrating excellent potential for clinical application.

Claims

1. The application of a microneedle-shaped patch drug in the preparation of a drug for treating skin cancer, characterized in that, The hydrogel composite material was dissolved in water, followed by the sequential addition of a photoinitiator, doxorubicin, and aPD-1 inhibitor. After thorough mixing, the reaction solution was allowed to stand under vacuum for 0.5-1.5 hours, then crosslinked under ultraviolet light for 3-7 minutes, and finally dried to obtain a microneedle-like patch drug. The mass ratio of the hydrogel composite material, photoinitiator, doxorubicin, and aPD-1 inhibitor was 7.5:0.125:5:0.

1. The aPD-1 inhibitor was tislelizumab, nivolumab, or pentazolizumab. The preparation method of the hydrogel composite material includes the following steps: dissolving collagen in an alkaline solution, then adding a photosensitizer containing anhydride functional groups, reacting at 45-55℃ for 2.5-3.5 h, then dialyzing, purifying at room temperature for 4.5-5.5 days, and freeze-drying the purified solid solution to obtain the hydrogel composite material; wherein the collagen is gelatin; the photosensitizer containing anhydride functional groups is methacrylic anhydride or glycidyl methacrylate; the mass ratio of collagen, alkaline solution and photosensitizer containing anhydride functional groups is 4-6:80-120:2-3; the alkaline solution is obtained by dissolving sodium carbonate and sodium bicarbonate in water at a mass ratio of 1:2; and the skin cancer is melanoma.

2. The application according to claim 1, characterized in that, The freeze-drying temperature is -55~-45℃, and the time is 46-50h.

3. The application according to claim 1, characterized in that, The photoinitiator is LAP, I2959, or VA086.

4. The application according to claim 1, characterized in that, The drying temperature is 35-40℃, and the time is 10-14 hours.

Images