Double-layer hydrogel stent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of tumor combined immunotherapy, and particularly relates to a double-layer hydrogel stent as well as a preparation method and application thereof. In order to solve the problem that no medicine combining immunotherapy and cell therapy exists in the prior art, the double-layer hydrogel stent DLS is provided, the outer layer is methacrylated gelatin GelMA, and the inner layer is methacrylated gelatin PR. The double-layer hydrogel scaffold can load gene drugs and immune cells at the same time, the delivery capacity of the nano-carrier and the stability of nucleic acid are guaranteed, the activity and phenotype of the immune cells are kept, then the gene drugs and the immune cells can treat solid tumors at the same time, tumor cells can be killed more efficiently and conveniently, and the application prospect is wide. And precise treatment of tumors is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor combined immunotherapy, and in particular relates to a double-layer hydrogel scaffold and a preparation method and application thereof. Background Art

[0002] Tumor immunotherapy, also known as cancer immunotherapy, is an innovative treatment method that uses the body's own immune system to fight cancer. Unlike traditional treatments such as surgery, chemotherapy, and radiotherapy, tumor immunotherapy does not directly attack cancer cells, but instead identifies and eliminates cancer cells by activating or enhancing the immune system. Tumor immunotherapy has the advantages of strong targeting, strong durability, and high effectiveness. However, due to the complex pathogenesis of tumors and their multi-step development characteristics, the immune response to tumors is mainly composed of multiple tumor immune cycles. Therefore, the effect of cancer immunotherapy with a single intervention measure is usually unsatisfactory, and there is an urgent need to develop a combined immunotherapy strategy.

[0003] Gene therapy is a new generation of precision therapy that corrects diseases caused by genetic defects or abnormalities by introducing therapeutic exogenous genes into human target cells, thereby achieving the purpose of treating diseases. Non-viral vectors improve the permeability, retention and pharmacokinetic characteristics of drugs, providing broad prospects for improving the efficiency of immunotherapy and becoming a leading anti-cancer drug candidate.

[0004] Adoptive cell therapy (ACT) is an immunotherapy method that collects immune-competent cells from patients, amplifies them in vitro, genetically engineered them, and functionally identifies them, and then infuses them back into the patient to exert anti-tumor effects. ACT can regulate and increase the immune function of tumor patients and effectively overcome tumor immune escape mechanisms. However, ACT is very sensitive to the barriers of the immunosuppressive tumor microenvironment and poor tumor infiltration, which hinders its application in the treatment of solid tumors.

[0005] However, gene therapy and ACT have their own advantages and disadvantages in tumor treatment, but they also have certain complementarity in working mechanism. For gene therapy, non-viral delivery system can ignore the barrier effect of tumor microenvironment and directly act on tumor cells through targeting. On the other hand, cell therapy is good at recognizing and processing antigens, which has the effect of long-term immune infiltration. Therefore, it is expected that these two technologies can be combined and cooperate with each other to produce multiple synergistic immunotherapy effects. However, there is no precedent for combining tumor gene therapy and cell therapy. Generally speaking, gene therapy drugs work through local injection or intravenous infusion. Cell therapy products are generally reinfused intravenously. This type of drug delivery method in the form of injection has the advantages of rapid action and fast onset. However, there are problems of drug leakage and poor sustainability in the treatment of solid tumors. It causes risks such as poor compliance, unstable blood drug concentration, and acute cytokine storm. In addition, simple injection administration is not conducive to the mutual coordination of the two mechanisms of action. To this end, the development of a sustained-release delivery carrier that can effectively combine the two is expected to solve the above problems.

[0006] Hydrogel is a polymer system with a three-dimensional network structure containing a large amount of water formed by a simple reaction of one or more monomers. The physical properties of hydrogel are very conducive to drug delivery. It is mainly used in the delivery research of small molecule drugs, protein drugs, radiotherapy drugs, and cell therapy drugs, and can achieve the sustained release of encapsulated drugs. Through the appropriate release mechanism, hydrogel can maintain a high local drug concentration for a long time. The release mechanism can be based on diffusion, swelling, chemical or other environmental stimulation. Therefore, it is expected that hydrogels can be used to combine gene therapy and ACT for treatment. However, how to use hydrogels to combine the two, how to ensure the biological activity of genes and cells, especially while maintaining the delivery ability of nanocarriers and the stability of nucleic acids, the activity and phenotype of immune cells should also be maintained, which is still a problem to be solved in this field. Summary of the invention

[0007] The technical problem to be solved by the present invention is that there is a lack of suitable carriers for combining gene therapy with cell therapy, and gene therapy drugs and cell therapy drugs cannot be delivered simultaneously.

[0008] The technical solution of the present invention to solve the above technical problems is: providing a double-layer hydrogel scaffold DLS, the outer layer of which is methacrylated gelatin GelMA, and the inner layer of which is methacrylated gelatin PR.

[0009] Among them, in the above-mentioned double-layer hydrogel scaffold DLS, the concentration of the methacrylylated gelatin GelMA and the methacrylylated gelatin PR is 5-10%. When the concentration of methacrylylated gelatin is 5-10%, it can form a gel to load drugs. Since the outer layer needs to degrade faster, a lower cross-linking degree and concentration can be selected. The present application has confirmed through experiments that the hydrogel concentration of the methacrylylated gelatin GelMA in the outer layer is 5% and the cross-linking degree is 30%. The inner layer needs to store cells and accommodate the exchange of the internal and external immune environments for a long time, so a higher cross-linking degree and concentration are selected. The hydrogel concentration of the methacrylylated gelatin PR in the inner layer is 6%, and the cross-linking degree is 60%.

[0010] When the methacryloyl gelatin is used, since the inner layer needs to prepare a sparse and porous hydrogel, it needs to be stirred at a constant temperature of 35° C. for 30 minutes, and the outer layer GelMA can be used directly.

[0011] Wherein, in the above-mentioned double-layer hydrogel scaffold DLS, the scaffold is spherical and has a diameter of 3 to 10 mm.

[0012] Furthermore, the inner layer diameter of the stent is 2 to 7 mm, and the outer layer thickness is 1 to 5 mm.

[0013] The present invention also provides a method for preparing the above-mentioned double-layer hydrogel scaffold DLS, comprising the following steps:

[0014] a. Drop the inner porous PR hydrogel into mineral oil and solidify it by irradiation with 405nm wavelength blue light to form the inner layer ILS of the stent;

[0015] b. Ordinary GelMA hydrogel was then added to cover the ILS, and then cured for the second time by irradiation with 405nm wavelength blue light to form the outer layer OLS of the scaffold. After washing, a double-layer hydrogel scaffold DLS was obtained.

[0016] Wherein, in the preparation method of the double-layer hydrogel scaffold DLS, the volume ratio of the PR hydrogel to the GelMA hydrogel is 1-10:5-20.

[0017] Wherein, in the preparation method of the above-mentioned double-layer hydrogel scaffold DLS, the blue light irradiation time is 30 to 60 seconds.

[0018] The present invention also provides a use of the double-layer hydrogel scaffold DLS for loading drugs for treating tumors.

[0019] Among them, in the above-mentioned uses, the drug includes at least one of gene drugs, immune cells, small molecule drugs, stem cells, monoclonal antibodies, cytokines, tumor vaccines, polypeptide drugs or radioactive therapeutic drugs.

[0020] Furthermore, in the above use, the gene drug is loaded on the outer layer of the double-layer hydrogel scaffold DLS, and the immune cells are loaded on the inner layer of the double-layer hydrogel scaffold DLS.

[0021] The beneficial effects of the present invention are:

[0022] The present invention prepares a double-layer hydrogel scaffold DLS for the first time, wherein the outer layer is methacrylated gelatin GelMA, and the inner layer is methacrylated gelatin PR. By reasonably setting the concentration and ratio of the inner and outer hydrogels, double-layer hydrogels of different sizes can be prepared, and different drugs can be loaded in the double-layer hydrogels, thereby achieving the effect of releasing different drugs at different times. The double-layer hydrogel scaffold DLS of the present invention can be used to load gene drugs and immune cells at the same time, which can ensure the delivery ability of nanocarriers and the stability of nucleic acids, and can also maintain the activity and phenotype of immune cells, thereby achieving the simultaneous treatment of solid tumors by gene drugs and immune cells, which can kill tumor cells more efficiently and conveniently, and achieve precise treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shown is a DLS cross-sectional view of a double-layer spherical stent;

[0024] Figure 2 The figure shows the characterization results of the double-layer spherical stent DLS, where Figure a shows the stent being clamped with surgical tweezers, Figure b shows the adhesion characteristics of DLS, Figure c shows the compression and recovery characteristics of the stent, and Figure d shows the DLS structure and morphology under a scanning electron microscope;

[0025] Figure 3 The figure shows the degradation of the double-layer spherical scaffold DLS by collagenase, wherein Figure a shows the degradation of the double-layer spherical scaffold DLS at different collagenase concentrations, and Figure b shows the degradation of the double-layer spherical scaffold DLS at different time periods;

[0026] Figure 4 The figure shows the effect of the DMP-Bim complex loaded on the outer layer of the double-layer spherical scaffold DLS on tumor cells. Figure a shows the result of mRNA release, Figure b shows the result of transfection of CT26 cells by the released mRNA, Figure c shows the result of flow cytometry evaluation of the transfection efficiency of the daily supernatant, and Figures d and e show the results of tumor inhibition by the released mRNA;

[0027] Figure 5 The results are shown for T cells loaded in the inner layer of the double-layer spherical scaffold DLS. Figure a shows the results of primary T cells cultured in PR or GM hydrogels, Figure b shows the effect of T cells cultured in ILS on nonspecific activation of T cells, Figure c shows the proliferation of T cells cultured under different conditions, and Figure d shows the release of T cells loaded in the inner layer of the scaffold.

[0028] Figure 6 Shown is the chronological release of two payloads from DLS; Figure a is the in vitro release evaluation results of the Jurkat T cell line stably expressing BimmRNA and mCherry labeled with U12, and Figure b is the in vivo evaluation of the release of gene complexes and T cells from the GD-920 scaffold. DETAILED DESCRIPTION

[0029] The present invention develops a double-layer spherical scaffold (DLS) based on photocurable hydrogel, named GD-920 scaffold. The DMP-Bim complex loaded on the outer layer of the scaffold can continuously release and transfect CT26 cells, and the expression of Bim mRNA can induce tumor cell apoptosis, antigen exposure and immunogenic cell death. The inner layer of the scaffold is loaded with primary T cells, which can interact with recruited DCs and immune activators in situ. The GD-920 scaffold releases gene and cell loads in a controllable and orderly manner, achieving a sustained TME activation effect. The scaffold only needs to be implanted once in situ to effectively inhibit tumor growth, and its effect is far better than that of the control scaffold containing a single payload. This GD-920 scaffold that co-delivers genes and cells has excellent potential in cancer immunotherapy as a strategy combining gene therapy and adoptive cell therapy.

[0030] The photocurable hydrogels PR and GelMA used in the present invention are synthesized by the reaction of methacrylic anhydride and gelatin, wherein the hydroxyl group and the amino group in the gelatin react with methacrylic anhydride respectively, and cross-link under the catalysis of the photosensitive initiator phenyl-2,4,6-trimethylbenzoylphosphonate lithium (LAP) to form a photosensitive hydrogel, wherein the cross-linking degree of GelMA is 30%, and the cross-linking degree of PR is 60%. PR and GelMA are photosensitive hydrogels with the ability of "rapidly transforming from sol to gel", and the addition of mRNA does not affect this property; PR and GelMA have a three-dimensional network structure, can stably load drugs, and are biodegradable and safer photosensitive hydrogels.

[0031] The photosensitive hydrogel double-layer spherical scaffold (DLS) prepared by the present invention is a biodegradable carrier and a relatively safe delivery carrier. The prepared GD-920 scaffold has a gene complex loaded on the outer layer and primary T cells loaded on the inner layer, providing a new idea for the cancer treatment strategy combining gene therapy and adoptive cell therapy.

[0032] The specific implementation modes of the present invention will be further explained below through examples, but it is not intended to limit the protection scope of the present invention to the scope described in the examples.

[0033] Example 1 Preparation of double-layer spherical scaffold DLS

[0034] The preparation process of double-layer spherical scaffold DLS is as follows: First, 50 μL of porous GelMA (PR) solution was dropped into mineral oil and photocured to form the inner layer of the scaffold (ILS). Then 100 μL of ordinary GelMA (GM) solution was added to coat the ILS, and the outer layer of the scaffold (OLS) was formed by secondary photocuring. The scaffold was washed three times with PBS to remove the external mineral oil, thereby obtaining a DLS with uniform morphology. The results are shown in Figure 2. Figure 1 As shown, the diameter of the ILS layer is about 5mm, and the diameter of the entire stent is about 7mm. The thickness of the OLS layer is evenly distributed at about 1mm. After being cut in half from the middle, the DLS is a three-dimensional (3D) sphere rather than a flat ellipse, which can achieve a better sequential release of loaded drugs through uniform degradation. DLS can be prepared in different sizes to meet different application scenarios. The two layers of hydrogel are evenly distributed, and the two payloads can be released through orderly biodegradation.

[0035] Example 2 Characterization of double-layer spherical scaffold DLS

[0036] The characterization results of double-layer spherical scaffold DLS are shown in Figure 2 .like Figure 2 As shown in a, the stent can be clamped using surgical forceps without severe deformation. Figure 2 b Demonstrates the adhesive properties of DLS, which helps it to stay fixed at the implantation site and not be affected by movement. Figure 2 c shows the compression properties of the scaffold. After the pressure is removed, the DLS still maintains a spherical structure, proving that the scaffold has favorable properties of long-term biointegration at the implantation site, enabling it to release drugs in situ and respond to the physiological environment. The two-layer structure and morphology (e.g. Figure 2 d). The pore size distribution of ILS is around 100 μm, which is larger than that of OLS (30 μm). The interface between the OLS layer and the ILS layer can be clearly observed under scanning electron microscopy (white dashed line). These results indicate that DLS can maintain its three-dimensional shape and porous morphological structure, which is conducive to drug loading and uniform release.

[0037] Example 3 Degradation of double-layer spherical scaffold DLS

[0038] The in vitro degradation of DLS was evaluated after 24 h of exposure to different concentrations of collagenase. Figure 3As shown in a, with the increase of collagenase concentration, the outer layer or inner layer of the scaffold was degraded in an orderly manner. In addition, the relative fluorescence intensity of the degraded scaffolds under the action of different concentrations of collagenase in the supernatant was also detected. The OLS signal (detected by blue fluorescent dye staining) gradually increased and reached a maximum value at a collagenase concentration of 1 mg, indicating that the OLS was completely degraded. In contrast, the fluorescence signal of the ILS (stained with red fluorescent dye) could be detected at a collagenase concentration of 1 mg and then increased rapidly, indicating that the ILS layer began to degrade after the OLS layer was completely degraded, indicating that the DLS was degraded sequentially in vitro. To evaluate the degradation in vivo, the stained DLS (ILS layer stained with red ink and OLS layer stained with blue ink) was implanted subcutaneously and its volume was measured every day. Figure 3 b shows that on day 4, the blue OLS layer was slightly degraded, and the remaining OLS remained spherical with a diameter of nearly 8 mm. On day 12, the OLS layer was almost completely degraded, and the diameter of the ILS was still 5 mm. On day 15, the DLS was completely degraded in vivo. These results indicate that DLS can be degraded sequentially in vivo within 15 days. In summary, spherical DLS was successfully prepared and exhibited stable physicochemical properties, implantability, and continuous biodegradability.

[0039] Example 4 mRNA drug loading and release

[0040] In order to achieve the sustained and effective tumor suppression function of the outer layer, the DMP-Bim complex was mixed with GM hydrogel and dropped into mineral oil, and the therapeutic DMP-Bim@OLS was formed after cross-linking. To evaluate the long-term release and protection ability of mRNA, DMP-Bim@OLS (loaded with 7 μg Bim) was incubated in DMEM medium containing collagenase at 37°C for 1 week. The supernatant was collected every day to quantify the release of mRNA. Figure 4 a shows the cumulative release rate of mRNA over these days. Rapid release of mRNA was detected in the first 2 days, followed by a relatively slow and stable release rate over the next 5 days; 100% released mRNA was detected on the 7th day. These results indicate that DMP-Bim@OLS can release Bim in a controllable manner and protect it from degradation in vitro for 7 days, demonstrating that DMP-Bim@OLS has a lasting tumor inhibitory effect.

[0041] To further evaluate the biological activity of the released complex, the DMP-EGFP complex was loaded into the GM scaffold and cultured in DMEM medium containing collagenase at 37°C for 7 days, and the supernatant was collected every day. Figure 4 As shown in b, the supernatants from day 7 all showed that EGFP mRNA could efficiently transfect CT26 cells without obvious cytotoxicity.

[0042] In addition, the transfection efficiency of the daily supernatants was assessed by flow cytometry. Figure 4As shown in Figure c, the DMP-EGFP complex released from OLS maintained a fairly high transfection capacity within 7 days, with an average transfection efficiency ranging from 11.57% to 33.06%. It is worth noting that a high transfection efficiency was still detected in the last two days, further demonstrating the sustained protection and long-term transfection capacity of the DMP-EGFP complex. These results indicate that the mRNA loaded with OLS has a long-lasting and stable biological activity in vitro.

[0043] We also evaluated the tumor suppressive effect of DMP-Bim@OLS in vitro. The supernatant of DMP-Bim@OLS (4 μg Bim) pretreated with collagenase was collected from treated CT26 cells. Figure 4 As shown in Figures 4d and 4e, more red-stained dead cells were observed in the DMP-Bim@OLS-treated group compared with the other groups by live / dead staining. The cell morphology of the DMP-Bim@OLS group showed atrophy and destruction, while there was no significant change in the cell morphology of the OLS group and the DMP@OLS group. These results indicate that the DMP-Bim complex can be released by DMP-Bim@OLS while biodegrading and maintains the transfection ability of CT26 cells. The transfected Bim mRNA can be expressed in CT26 cells and play a tumor-killing role. Therefore, our results demonstrate that DMP-Bim@OLS has long-term mRNA protection ability, stable controllable release ability, and persistent tumor cell killing effect.

[0044] Example 5 T cell culture

[0045] To investigate the feasibility of T cell deposition in ILS, we evaluated the cell culture properties of different hydrogels. First, lymphocytes were extracted from the lymph nodes of BALB / c mice and then stimulated with anti-mouse CD3 / CD28 antibodies for 24 h. A high proportion of CD3-positive lymphocytes (95.19%) was detected in the stimulated lymphocytes, indicating that primary T cells were successfully collected.

[0046] Then, the collected T cells were labeled with CFSE and cultured in PR or GM hydrogels. Figure 5 As shown in a, the proliferation rate of T cells cultured in PR, GM or culture medium was similar on the second day, all 40%. From day 4 to 6, the proliferation rate of T cells cultured in PR hydrogels reached 68%, which was higher than that of T cells cultured in GM (49%, P<0.01). The proliferation rate of T cells cultured in culture medium reached 77%, which was higher than that of the PR group (P<0.05) and the GM culture group (P<0.0001). These results show that the proliferation function of T cells can be maintained in both PR and GM hydrogels, but the proliferation efficiency obtained in culture medium is higher. Compared with GM, PR has a stronger adaptability to T cell culture and proliferation and is suitable for the composition of ILS.

[0047] We then evaluated whether loading T cells within ILS would alter the biological functions of T cells. Briefly, T cells were placed into the inner layer of DLS to form T@ILS. First, to determine whether ILS culture would induce nonspecific activation of T cells, the expression of T cell activation markers CD69 and CD25 was detected. Figure 5 As shown in b, there was no significant difference in the expression of CD69 and CD25 between T cells cultured in ILS and culture medium. This proves that ILS does not induce nonspecific activation of T cells. Then, the phenotype of T cells cultured under these two conditions was detected by measuring the proportion of helper T cells (CD4+) or CTL (cytotoxic T cells, CD8+). In addition, no significant difference was found in the ratio of CD8+ / CD4+T cells between T cells (0.32 times) and T@ILS (0.28 times), indicating that ILS culture does not change the phenotype of T cells.

[0048] Mouse lymphocytes are divided into naive T cells (Tn, CD44-CD62L+) and memory T cells (Tm, CD44+). According to our results ( Figure 5 b), T cells in both groups were mostly composed of Tm. In addition, compared with T cells cultured in medium, the proportion of effector memory T cells (Tem, CD44+CD62L-) in Tm cultured in ILS was slightly higher, while the proportion of central memory T cells (Tcm, CD44+CD62L+) was lower. These results indicate that culturing in ILS has little effect on the normal biological function of T cells, so this method of storing and releasing T cells is feasible.

[0049] According to our design, GD-920 contains an inner layer of T@ILS and an outer layer of DMP-Bim@OLS. Our goal was to determine whether the proliferation of T cells would be affected by the payload in the outer layer. Therefore, we evaluated the proliferation of T cells cultured under different conditions. T cells were labeled with CFSE and then cultured in culture medium, DLS inner layer (T@ILS), DMP-loaded DLS (DMP@OLS-T@ILS), or GD-920 scaffolds. After five days, the CFSE signal ( Figure 5 c). The proliferation rate of T cells in each group was similar (greater than 95%) compared with day 0. This result indicates that the DMP-Bim complex loaded on the outer layer has little effect on the survival and proliferation of T cells.

[0050] We then evaluated the release of T cells loaded onto the inner layer of the scaffold. 6T cells were loaded onto ILS and cultured in medium containing collagenase. Supernatants were collected and quantified daily to count T cells released from ILS. Figure 5 As shown in (d), T cells were first detected in the supernatant on day 4, and their released number reached a maximum on day 6. Then, T cells were released at a constant rate until day 8. These results indicate that T cells can be deposited in ILS and gradually released with the help of collagenase. The release of T cells does not occur immediately but proceeds in a controlled and orderly manner as they are loaded into the inner layer of the scaffold.

[0051] Example 6 mRNA and T cells release

[0052] In order to observe the chronological release of the two payloads from DLS, a U12-labeled Jurkat T cell line stably expressing Bim mRNA and mCherry was synthesized and constructed. The two compounds were loaded on ILS or OLS, respectively, to form a labeled GD-920 scaffold. It was then cultured in medium containing collagenase and the release of its payload was monitored once a day. The results are shown in Figure 2. Figure 6 As shown in Figure 1, the boundary between the outer layer (white) and the inner layer (yellow) of the scaffold can be clearly observed on day 1. With the digestion of collagenase on days 2 and 3, the boundary of the outer layer became blurred, but the boundary of the inner layer could be clearly observed. This indicates that the outer layer of the scaffold gradually degraded with the release of U12-labeled mRNA, and the cells were still stored in the inner layer of the scaffold. In addition, on day 4, the outer layer of the scaffold was completely degraded, while the boundary of the inner layer was still clear. Then, on days 5 and 6, due to the rapid release of Jurkat T cells, the inner layer of the scaffold had no clear boundary. These results indicate the time-sequential degradation process of DLS, and the time-sequential release of mRNA loaded on the outer layer and T cells loaded on the inner layer in vitro with the biodegradation of the scaffold.

[0053] We evaluated the release of gene complexes and T cells from GD-920 scaffolds in mice bearing CT26 subcutaneous tumors. Briefly, cy5.5-labeled DMPs and DiR-stained T cells were added to GD-920 scaffolds and implanted near the tumor. Figure 6As shown in b, a strong cy5.5 signal was detected in the tumor area from day 3 to day 5, and the signal slowly disappeared by day 15. At the same time, the DiR signal was strongly detected from day 3 to day 13. These results indicate that the release and action of DMP occurred from day 3 to day 5 after implantation, while the release and tumor infiltration of T cells lasted longer. The staining results of frozen sections also support this conclusion. Before implantation, biotin-labeled DMP and CFSE-stained T cells were incorporated into the GD-920 scaffolds to track their distribution in the tissue.

[0054] It can be seen that the present invention prepares a double-layer hydrogel scaffold that can simultaneously load gene drugs and immune cells. After loading the drugs, the release of the drugs can be controlled in sequence, thereby combining gene therapy and immunotherapy, providing a new possibility for the treatment of tumors and having great practical value.

Claims

1. A double-layer hydrogel scaffold DLS, characterized in that: The outer layer is methacrylated gelatin GelMA, and the inner layer is methacrylated gelatin PR.

2. The double-layer hydrogel scaffold DLS according to claim 1, characterized in that: The concentrations of the methacryloyl gelatin GelMA and methacryloyl gelatin PR are 5-10%; preferably, the hydrogel concentration of the methacryloyl gelatin GelMA is 5%, and the crosslinking degree is 30%; the hydrogel concentration of the methacryloyl gelatin PR is 6%, and the crosslinking degree is 60%.

3. The double-layer hydrogel scaffold DLS according to claim 1, characterized in that: The bracket is spherical and has a diameter of 2 to 10 mm.

4. The double-layer hydrogel scaffold DLS according to claim 1, characterized in that: The inner layer diameter of the stent is 2-7 mm, and the outer layer thickness is 1-5 mm.

5. The method for preparing the double-layer hydrogel scaffold DLS according to any one of claims 1 to 4, characterized in that: The following steps are involved: a. Drop the inner porous PR hydrogel into mineral oil and solidify it by irradiation with 405nm wavelength blue light to form the inner layer ILS of the stent; b. Ordinary GelMA hydrogel was then added to cover the ILS, and then cured for the second time by irradiation with 405nm wavelength blue light to form the outer layer OLS of the scaffold. After washing, a double-layer hydrogel scaffold DLS was obtained.

6. The method for preparing the double-layer hydrogel scaffold DLS according to claim 5, characterized in that: The volume ratio of the PR hydrogel to the GelMA hydrogel is 1-10:5-20.

7. The method for preparing the double-layer hydrogel scaffold DLS according to claim 5, characterized in that: The blue light irradiation time is 30 to 60 seconds.

8. Use of the double-layer hydrogel scaffold DLS according to any one of claims 1 to 4, characterized in that: Used to load drugs for treating tumors.

9. The use of the double-layer hydrogel scaffold DLS according to claim 8, characterized in that: The drug includes at least one of gene drugs, immune cells, small molecule drugs, stem cells, monoclonal antibodies, cytokines, tumor vaccines, polypeptide drugs or radioactive therapeutic drugs.

10. The use of the double-layer hydrogel scaffold DLS according to claim 8, characterized in that: The gene medicine is loaded on the outer layer of the double-layer hydrogel scaffold DLS, and the immune cells are loaded on the inner layer of the double-layer hydrogel scaffold DLS.

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