GelMA hydrogel composite material as well as preparation method and application thereof

Abstract

The invention discloses a GelMA hydrogel composite material as well as a preparation method and application thereof, and belongs to the technical field of material preparation. A micro-needle-shaped GelMA hydrogel composite material (GelMA MNs) is prepared by grafting an anhydride functional group of methacrylic anhydride to a side chain amino group of gelatin, then doxorubicin and an aPD-L1 inhibitor are loaded into the GelMA MNs, a double-drug delivery system is established, and the prepared aPD-L1 / DoxatGelMA MNs can reduce skin injury to the maximum extent, and meanwhile, the aPD-L1 / DoxatGelMA MNs have a good application prospect. Recognition and killing of immune cells on melanoma cells are greatly started, immunogenic cell death (ICD) is promoted, and a new thought is provided for overcoming limitation of ICIs.

Description

Technical Field

[0001] The invention belongs to the technical field of material preparation, and specifically relates to a GelMA hydrogel composite material and a preparation method and application thereof. Background Art

[0002] Cutaneous melanoma is an aggressive skin cancer that is difficult to cure even with surgical resection. This advanced disease also shows considerable resistance to traditional chemotherapy and single immunotherapy including immune checkpoint inhibitors (ICIs). The complex underlying mechanism is attributed to the inherent microenvironment of cold tumors, which leads to an imbalance between immune cells, non-immune cells and the microbiota. Current systemic treatments for melanoma include surgery, chemotherapy, radiotherapy, immunotherapy and targeted therapy, of which the latter two have become the mainstay. However, in addition to possible systemic drug side effects, the in situ drug concentration in the tumor lesion may not reach the ideal level due to the blockage of the microenvironment, and after surgical resection, infection during the wound healing process often triggers a non-tumor-specific inflammatory response. If this inflammation is automatically suppressed by the body, it will have adverse effects even if ICIs have restored the activity of tumor-specific cytotoxic T cells (CTL) to a certain extent.

[0003] There are many treatment options for cutaneous melanoma, but the composition of the tumor microenvironment is complex, changes rapidly, and is often accompanied by overexpression of immune checkpoint ligands / receptors. This ultimately leads to limited immune cell infiltration and weakened cytotoxic killing, which accelerates disease progression and makes it more difficult to treat. When immune cells are overactivated, immune checkpoint-related proteins should trigger inhibitory signals to maintain a stable level of autoimmunity. Tumor cells, including melanoma cells, can hijack this mechanism to evade immunity. With the continuous study of the role of immune checkpoints in tumor immunity, ICIs have been widely used in the treatment of a variety of malignancies. For melanoma patients who have not responded to chemotherapy or molecular targeted therapy, PD-1 / PD-L1 blockers can significantly prolong their survival, but the reactivation of immune cells caused by a single ICI may not last for a long time. Once the tumor recurs, the use of the same ICI again may fail 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 and a preparation method and application thereof, so as to solve the technical problem that existing drugs have poor effects in treating melanoma.

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

[0006] Gelatin is the main product obtained by irreversible decomposition of collagen. This type of compound has a greater affinity for protein molecules on the surface of human tissues, weaker antigenicity, good biocompatibility and biodegradation safety, and can be degraded and absorbed; at the same time, gelatin has a larger molecular weight and more groups that are easily modified, and has excellent plasticity. Methacrylic anhydride is a ketone compound containing a photosensitive anhydride functional group, which can be copolymerized through free radical polymerization to form a cross-linked compound. The present invention increases the functional modification of gelatin by grafting the anhydride functional group of methacrylic anhydride on the side chain amino group of gelatin and introducing a photosensitive group. It has photosensitivity while retaining its excellent biological properties, and gives the GelMA hydrogel composite material the potential for photodynamic therapy. Photodynamic therapy relies on the activation of photosensitizers. Light sources of specific wavelengths excite photosensitive groups to produce singlet oxygen and other reactive oxygen species (ROS), which can selectively destroy melanoma cells, leading to apoptosis or necrosis. In addition, this process can also induce the immune system to respond to melanoma cells and change the tumor microenvironment, reducing the blood supply and metastasis of tumors. Therefore, through functionalized gelatin hydrogels, photosensitivity materials show effective therapeutic potential for melanoma in photodynamic therapy, providing a promising tumor treatment strategy.

[0007] On the basis of the above technical solution, the present invention can also be improved as follows:

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

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

[0010] Further, the pH value of the alkali solution is 9.2-10.6.

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

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

[0013] The invention also discloses the application of the GelMA hydrogel composite material in preparing a drug for treating skin cancer.

[0014] On the basis of the above technical solution, the present invention can also be improved as follows:

[0015] Further, the skin cancer is melanoma.

[0016] Furthermore, the specific steps of the application include: dissolving the GelMA hydrogel composite material in water, then adding a photoinitiator, doxorubicin and aPD-L1 inhibitor in sequence, mixing evenly, allowing the reaction solution to stand under vacuum for 0.5-1.5 hours, then cross-linking under ultraviolet light for 3-7 minutes, and finally drying to obtain a drug for treating skin cancer; the mass ratio of the 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, panlizumab, atezolizumab, durvalumab or avelumab.

[0018] aPD-L1 is an inhibitor of programmed cell death protein-1 (PD-1) and its ligand (PD-L1), which can block the binding of PD-1 on the surface of T cells with PD-L1 and PD-L2 on the surface of immune cells, upregulate the growth and proliferation of T cells, enhance T cells' recognition of tumor 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°C and the drying time is 10-14h.

[0020] The beneficial effects of the present invention are:

[0021] 1. The aPD-L1 / Dox@GelMA MNs prepared by the present invention greatly activated the recognition and killing of melanoma cells by immune cells while minimizing skin damage, and promoted immunogenic cell death (ICD). In vitro and in vivo experiments showed that aPD-L1 / Dox@GelMA MNs enhanced immunogenic cell death (ICD) by enhancing the uptake capacity of Dox, and enhanced immune activation by inhibiting PD-L1 signals, thereby effectively inhibiting melanoma progression. Dox triggered immunogenic cell death, thereby sending CRT, HMGB1 and ATP increase signals to antigen presenting cells, while aPD-L1 promoted the uptake of Dox by melanoma cells. On the other hand, aPD-L1 and Dox synergistically enhanced the proportion of CTLs in melanoma tissues and their cytokine secretion levels. The combined use of the two drugs can significantly enhance the immunomodulatory effect and significantly inhibit tumor growth, which is conducive to reducing the systemic administration dose of ICIs.

[0022] 2. The GelMA hydrogel prepared by the present invention also has excellent biocompatibility, cell response characteristics and proteolytic degradation, and can provide suitable cell adhesion sites. In addition, the GelMA hydrogel has good mechanical properties, and the 3D microscaffold constructed by it has adjustable mechanical and chemical properties. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 2 The microstructure of aPD-L1 / Dox@GelMA MNs;

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

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

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

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

[0029] Figure 7 is the ATP release of B16-F10 cells after different treatments;

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

[0031] Fig. 9 HMGB1 release of B16-F10 cells after different treatments;

[0032] Fig.10 Tumor volume of melanoma mouse model after treatment with different GelMAMNs

[0033] Fig.11 Survival curves of melanoma mouse models after treatment with different GelMAMNs;

[0034] Fig.12 The death of melanoma cells in the tumor after treatment with different GelMAMNs;

[0035] Fig.13 The proliferation of melanoma cells in the tumor after treatment with different GelMAMNs;

[0036] Fig.14 is the proportion of CD11c+CD86+ mature DCs after treatment with different GelMAMNs;

[0037] Fig.15 The histograms are the proportions of CD11c+CD86+ mature DCs after treatment with different GelMA MNs;

[0038] Fig.16 is the proportion of CD8+ cytotoxic T lymphocytes after treatment with different GelMAMNs;

[0039] Fig.17 The histograms are the proportions of CD8+ cytotoxic T lymphocytes after treatment with different GelMA MNs;

[0040] Fig.18 is the proportion of CD4+FoxP3+ regulatory T cells after treatment with different GelMA MNs;

[0041] Fig.19 The histograms are the proportions of CD4+FoxP3+ regulatory T cells after treatment with different GelMA MNs;

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

[0043] Fig.21 The histograms are the production levels of immunoregulatory cytokines TNF-α (G) and IFN-γ (G) after treatment with different GelMA MNs;

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

[0045] Fig.23 HMGB1 release in melanoma tumors after treatment with different GelMA MNs. DETAILED DESCRIPTION

[0046] The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

[0047] Example 1

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

[0049]

[0050] Specifically, the method includes the following steps: dissolving 7.95 g of sodium carbonate and 14.65 g of sodium bicarbonate in 1 L of distilled water to obtain an alkaline solution with a pH value of 10; then dissolving 50 g of gelatin in the alkaline solution, adding 10 g of methacrylic anhydride after mixing evenly, reacting at 50° C. for 3 h, then dialyzing the reaction solution using a dialysis bag with a MWCO of 1000 Da, and then purifying at room temperature for 5 days, and freeze-drying the purified solid solution at -45° C. for 48 h to obtain a GelMA hydrogel composite material (GelMA MNs).

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

[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 allowing the reaction solution to stand under vacuum for 1 hour, then cross-linking under 405 nm ultraviolet light for 5 minutes, and finally drying at 37 ° C for 12 hours to obtain a microneedle patch drug (aPD-L1 / Dox@GelMA MNs) for the treatment of melanoma.

[0054] Example 2

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

[0056]

[0057] Specifically, the method includes the following steps: dissolving 7 g of sodium carbonate and 15 g of sodium bicarbonate in 1 L of distilled water to obtain an alkaline solution with a pH value of 9.2; then dissolving 50 g of gelatin in the alkaline solution, mixing well, adding 10 g of glycidyl methacrylate, reacting at 45°C for 3.5 h, and then dialyzing the reaction solution using a dialysis bag with a MWCO of 1000 Da, followed by purification at room temperature for 4.5 d, and freeze-drying the purified solid solution at -55°C for 46 h to obtain a GelMA hydrogel composite material (GelMAMNs).

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

[0059]

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

[0061] Example 3

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

[0063]

[0064] Specifically, the method includes the following steps: dissolving 8.5 g of sodium carbonate and 14 g of sodium bicarbonate in 1 L of distilled water to obtain an alkaline solution with a pH value of 10.6; then dissolving 50 g of gelatin in the alkaline solution, adding 10 g of methacrylic anhydride after mixing evenly, reacting at 55° C. for 2.5 h, then dialyzing the reaction solution using a dialysis bag with a MWCO of 1000 Da, and then purifying at room temperature for 5.5 d, and freeze-drying the purified solid solution at -45° C. for 50 h to obtain a GelMA hydrogel composite material (GelMA MNs).

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

[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 of panlizumab, then letting the reaction solution stand under vacuum for 1.5 h, then cross-linking under 405 nm ultraviolet light for 3 min, and finally drying at 40 ° C for 10 h to obtain a microneedle patch drug (aPD-L1 / Dox@GelMA MNs) for the treatment of melanoma.

[0068] Comparative Example 1

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

[0070] Comparative Example 2

[0071] The difference between this comparative example and Example 1 is that Dox is omitted, and the rest of the preparation process is the same as that of Example 1 to prepare 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 by SEM. Figure 1 As shown in Figure 2, the surface of aPD-L1 / Dox@GelMA MNs showed neatly arranged cone-shaped features. Figure 2 As shown in Figure 3, the sponge-like appearance and surface roughness of aPD-L1 / Dox@GelMA MNs reflect the retention 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, which is suitable for drug delivery and potential tissue penetration. Figure 3 As shown, HE staining indicated a large number of inflammatory cells, including neutrophils, lymphocytes, and monocytes, indicating that aPD-L1 / Dox@GelMA MNs can effectively induce 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] In order to evaluate the anti-melanoma efficacy of GelMA MNs, the cytotoxicity, cell viability and drug uptake efficiency were evaluated by live / dead cell staining experiments. B16-F10 cells incubated with four different MNs preparations (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, and live cells emitted green fluorescence and dead cells emitted red fluorescence.

[0076] The results are as follows Figure 4 As shown in the figure, compared with the blank control group (incubated without adding MN preparation), the cells incubated with GelMA MNs without drug loading showed a significant green fluorescent cell population, confirming that the cytotoxicity of the GelMA hydrogel composite itself was negligible. In addition, a small number of red fluorescent cells were observed in both the Dox@GelMA MNs-treated group and the aPD-L1@GelMA MNs-treated group, indicating that their cytotoxic effect was limited, and the red fluorescence of the Dox / aPD-L1@GelMA MNs-treated group increased significantly.

[0077] like Figure 5 As shown, the inhibitory ability of Dox / aPD-L1@GelMA MNs on tumor cell viability was 2-3 times higher than that of single drug-loaded MNs preparations (Dox@GelMA MNs and aPD-L1@GelMA MNs), and 5 times higher than that of un-drug-loaded GelMA MNs and the control group.

[0078] In the absence of immune cells, the chemotherapy drug Dox may account for a greater proportion of the cytotoxic effect of tumor cells cultured in vitro. Therefore, the effect of different doses (1, 5, and 10 μM) of aPD-L1 on the cellular uptake efficiency of Dox was detected. Figure 6 As shown in the figure, aPD-L1 can enhance the uptake of Dox to varying degrees (1 μM aPD-L1 has the strongest enhancement effect) and promote the transfer of Dox into the nucleus of tumor cells. These results indicate that Dox / aPD-L1@GelMAMNs has a significant anti-tumor effect in vitro and there is a positive synergistic effect between aPD-L1 and Dox.

[0079] Experimental Example 3aPD-L1 enhances the ability of Dox to induce ICD

[0080] Comprehensive in vitro testing was performed to reveal whether Dox / aPD-L1@GelMAMNs can harness the immune system to fight melanoma. The testing focused on the complex interaction between Dox and aPD-L1 in the context of immunogenic cell death (ICD), by measuring hallmark indicators 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 in Figure 2, co-administration of Dox / aPD-L1@GelMA MNs significantly increased ATP release (p<0.001) and enhanced immune recognition. In addition, melanoma cells treated with Dox / aPD-L1@GelMA MNs showed enhanced externalization of CRT on the cell surface ( Figure 8 ), accompanied by a significant release of nuclear HMGB1 ( Fig. 9 ). The enhanced cytotoxicity of Dox / aPD-L1@GelMA MNs was not only attributed to the direct cytocidal effect of the drug, but also to the unleashing of immune responses by inducing ICD.

[0082] Experimental Example 4 In vivo anti-melanoma effect

[0083] The concentration was 2.0×10 6100 μL of B16-F10 cells / mL were subcutaneously injected into the back of C57BL / 6 mice. After the mice grew for 7 days, 1.0 mL of MNs preparation (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) was used to treat the mice. The patch containing the above MNs preparation was penetrated into the area near the B16 melanoma and fixed on the skin with a sealing film. MNs were fixed on the tumor, and the patch was replaced every 4 days. The melanoma tumor was removed on the 12th day, and immunofluorescence staining and flow cytometry analysis were performed. The flow cytometry analysis process is as follows: tumor tissue was surgically removed from mice 12 days after tumor cell inoculation, the tissue was rinsed with PBS, cut into small pieces, and filtered through a 100 μm cell strainer, and the filtrate of 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 the Foxp3 / transcription factor staining buffer set (eBioscience, 00-5523-00).

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

[0085] The apoptotic cells in each group of tumors were detected by TUNEL staining. Fig.12 The proportion of TUNEL-positive (FITC) cells in the aPD-L1 / Dox@GelMA MNs group increased, and its strong fluorescence signal indicated the presence of a large amount of DNA fragmentation in tumor cells. In contrast, histopathological analysis of serial tumor tissue sections showed that ( Fig.13 ), Ki-67 staining and HE staining confirmed that cell proliferation was significantly reduced.

[0086] By detecting the infiltration of immune cells in tumors, we can understand the immune response activated by them. Fig.14 and Fig.15 As shown in the figure, both Dox@GelMA MNs and aPD-L1@GelMA MNs could significantly increase the proportions of CD11c+, CD86+ mature DCs (mDCs) (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); Fig.16 and Fig.17 As shown, aPD-L1 / Dox@GelMA MNs increased the proportions of mDCs and CTLs to the highest levels (39.7% and 44.3%, respectively, p<0.01).

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

[0088] like Fig. 22 and Fig.23 As shown, in the xenograft model, the change trends of CRT and HMGB1 were consistent with the in vitro experiments. These results indicate that GelMAMNs loaded with immunotherapeutic agents and chemotherapeutic agents enhance the effect of immunochemotherapy, provide new ideas for overcoming the limitations of ICIs, and have excellent clinical application potential.

Claims

1. A method for preparing a GelMA hydrogel composite material, characterized in that: The following steps are involved: The collagen is dissolved in an alkali solution, and then a photosensitizer containing an anhydride functional group is added, and the reaction is carried out at 45-55° C. for 2.5-3.5 hours, and then dialyzed and purified at room temperature for 4.5-5.5 days. The purified solid solution is freeze-dried to obtain a GelMA hydrogel composite material; the collagen is gelatin; the photosensitizer containing anhydride functional groups is methacrylic anhydride, glycidyl methacrylate or acryloyl chloride; the mass ratio of the collagen, the alkali solution and the photosensitizer containing anhydride functional groups is 1-100:20-2000:0.1-50.

2. The method for preparing the GelMA hydrogel composite material according to claim 1, characterized in that: The mass ratio of the collagen, the alkali solution and the photosensitizer containing anhydride functional groups is 4-6:80-120:2-3.

3. The method for preparing the GelMA hydrogel composite material according to claim 1, characterized in that: The alkali solution is obtained by dissolving sodium carbonate and sodium bicarbonate in water in a mass ratio of 1:

2.

4. The method for preparing the GelMA hydrogel composite material according to claim 1, characterized in that: The freeze-drying temperature is -55 to -45°C and the time is 46 to 50 hours.

5. A GelMA hydrogel composite material, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the GelMA hydrogel composite material according to claim 5 in preparing a drug for treating skin cancer.

7. The use according to claim 6, characterized in that: The skin cancer is melanoma.

8. The use according to claim 6, characterized in that: The specific steps include: dissolving the GelMA hydrogel composite material in water, then adding a photoinitiator, doxorubicin and aPD-L1 inhibitor in sequence, mixing evenly, allowing the reaction solution to stand under vacuum for 0.5-1.5 hours, then cross-linking under ultraviolet light for 3-7 minutes, and finally drying to obtain a drug for treating skin cancer; the mass ratio of the GelMA hydrogel composite material, photoinitiator, doxorubicin and aPD-L1 inhibitor is 1-100: 0.01-1: 0.1-10: 0.1-10.

9. The use according to claim 8, characterized in that: The photoinitiator is LAP, I2959 or VA086; the aPD-L1 inhibitor is tilelizumab, nivolumab, panlizumab, atezolizumab, durvalumab or avelumab.

10. The use according to claim 8, characterized in that: The drying temperature is 35-40°C and the drying time is 10-14h.

Images