Double-layer hydrogel scaffolds, methods of making and uses thereof

Abstract

The application belongs to the technical field of tumor combined immunotherapy, and particularly relates to a double-layer hydrogel support and a preparation method and application thereof. In view of the problem that the existing drugs do not combine immunotherapy and cell therapy, the application provides a double-layer hydrogel support DLS, the outer layer of which is methacrylated gelatin GelMA, and the inner layer of which is methacrylated gelatin PR. The double-layer hydrogel support can simultaneously load gene drugs and immune cells, which not only guarantees the delivery capacity of the nano-carrier and the stability of the nucleic acid, but also maintains the activity and phenotype of the immune cells, so as to realize the simultaneous treatment of solid tumors by the gene drugs and the immune cells, kill tumor cells more efficiently and conveniently, and realize precise treatment of tumors.

Description

TECHNICAL FIELD

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

[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 treatment methods such as surgery, chemotherapy, and radiotherapy, tumor immunotherapy does not directly attack cancer cells, but rather activates or enhances the immune system to recognize and eliminate cancer cells. Tumor immunotherapy has the advantages of strong targeting, strong persistence, and high effectiveness, but due to the complex pathogenesis of tumors and the characteristics of multi-step development, the immune response to tumors is mainly composed of multiple tumor immune cycles. Therefore, the effect of single intervention cancer immunotherapy is usually not satisfactory, and there is an urgent need to develop combined immunotherapy strategies.

[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 target cells in the human body, thereby achieving the purpose of treating diseases. Non-viral vectors improve drug penetration, retention, and pharmacokinetic characteristics, providing a broad prospect for improving immunotherapy efficiency and becoming a leading candidate for anticancer drugs.

[0004] Adoptive cell therapy (ACT) is an immunotherapy method that collects immune active cells from the patient's body, amplifies, genetically engineers, and functionally identifies them in vitro, and then reinfuses them into the patient's body to exert an anti-tumor effect. ACT can regulate and increase the immune function of tumor patients, effectively overcoming the tumor immune escape mechanism. However, ACT is sensitive to the barrier of immunosuppressive tumor microenvironment and poor tumor infiltration, which hinders its application in solid tumor treatment.

[0005] However, gene therapy and ACT have their own advantages and disadvantages in tumor treatment, but 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 antigen recognition and processing, and has the effect of long-term immune infiltration. Therefore, it is expected to combine the two technologies together, cooperate with each other, and produce multiple synergistic immunotherapy effects. However, there is no precedent for combining tumor gene therapy and cell therapy together. Generally, the drugs for gene therapy work through local injection or intravenous infusion. The cell therapy product is generally returned through intravenous infusion. The drug delivery mode of such injection liquid has the advantages of rapid action and quick effect. However, in the treatment of solid tumors, there are problems of drug leakage and poor persistence. It causes poor compliance, unstable blood drug concentration, and risk of acute cytokine storm. Moreover, simple injection administration is not conducive to the mutual cooperation of the mechanisms of the two. Therefore, 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 containing a large amount of water with a three-dimensional network structure formed by a simple reaction of one or more monomers. The physical properties of hydrogel are very beneficial to drug delivery, and are mainly used in the delivery of small molecule drugs, protein drugs, radiotherapy drugs, and cell therapy drugs. It can achieve sustained release of the loaded drugs. Through a suitable 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 stimuli. Therefore, it is expected to use hydrogel to combine gene therapy and ACT for treatment, however, how to use hydrogel to combine the two, how to ensure the biological activity of genes and cells, especially to maintain the delivery ability of nanocarriers and the stability of nucleic acids, while also maintaining the activity and phenotype of immune cells, is still a problem to be solved in the field. SUMMARY

[0007] The technical problem to be solved by the present application is that the existing lack of a suitable carrier for combining gene therapy and cell therapy cannot simultaneously deliver gene therapy drugs and cell therapy drugs.

[0008] The technical solution of the present application to solve the above technical problem is to provide a double-layer hydrogel scaffold DLS, the outer layer is methacrylated gelatin GelMA, and the inner layer is methacrylated gelatin PR.

[0009] The concentration of the methacrylated gelatin GelMA and the methacrylated gelatin PR in the double-layer hydrogel scaffold DLS is 5-10%. When the concentration of the methacrylated gelatin is 5-10%, the drug can be loaded by forming a gel, and since the outer layer needs to be degraded faster, a lower cross-linking degree and concentration can be selected. The application confirms through tests that the hydrogel concentration of the methacrylated 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 internal and external immune environments for a long time, so a higher cross-linking degree and concentration are selected. The hydrogel concentration of the methacrylated gelatin PR in the inner layer is 6%, and the cross-linking degree is 60%.

[0010] The methacrylated gelatin is used in the above manner. Since the inner layer needs to prepare a sparse porous hydrogel, it needs to be stirred at 35℃ for 30 minutes, and the outer layer GelMA can be directly used.

[0011] In the double-layer hydrogel scaffold DLS, the scaffold is spherical, and the diameter is 3-10mm.

[0012] Further, the diameter of the inner layer of the scaffold is 2-7mm, and the thickness of the outer layer is 1-5mm.

[0013] The application also provides a preparation method of the double-layer hydrogel scaffold DLS, comprising the following steps:

[0014] a. The inner layer of the porous PR hydrogel is dropped into mineral oil, and is cured by irradiation of 405nm wavelength blue light to form the inner layer ILS of the scaffold;

[0015] b. The ILS is coated with ordinary GelMA hydrogel, and is cured for the second time by irradiation of 405nm wavelength blue light to form the outer layer OLS of the scaffold, and the double-layer hydrogel scaffold DLS is obtained after washing.

[0016] 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] In the preparation method of the double-layer hydrogel scaffold DLS, the irradiation time of the blue light is 30-60 seconds.

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

[0019] In the use, the drug includes at least one of a gene drug, an immune cell, a small molecule drug, a stem cell, a monoclonal antibody, a cytokine, a tumor vaccine, a polypeptide drug, or a radiotherapy drug.

[0020] Further, 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 application are:

[0022] The present application first prepares 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. By reasonably setting the concentration and ratio of the inner and outer layers of the hydrogel, double-layer hydrogels of different sizes can be prepared, and different drugs can be loaded in the double-layer hydrogel, thereby achieving the effect of releasing different drugs at different times. The double-layer hydrogel scaffold DLS of the present application can be used to simultaneously load gene drugs and immune cells, can ensure the delivery capacity of the nanocarrier and the stability of the nucleic acid, and can also maintain the activity and phenotype of the immune cells, thereby realizing the simultaneous treatment of solid tumors by gene drugs and immune cells, and can more efficiently and conveniently kill tumor cells, and realize precise treatment of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The figure shows a cross-sectional view of the double-layer spherical scaffold DLS;

[0024] Figure 2 The figure shows the characterization results of the double-layer spherical scaffold DLS, wherein figure a shows the scaffold clamped using surgical forceps, figure b shows the adhesion properties of the DLS, figure c shows the compression and recovery properties of the scaffold, and figure d shows the structure and morphology of the DLS 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 under 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 double-layer spherical scaffold DLS loaded with the DMP-Bim complex on tumor cells, figure a shows the release results of mRNA, figure b shows the results of transfecting CT26 cells with the released mRNA, figure c shows the results of flow cytometry evaluation of the transfection efficiency of the supernatant every day, and figures d and e show the results of tumor inhibition by the released mRNA;

[0027] Figure 5 The figure shows the results of loading T cells in the inner layer of the double-layer spherical scaffold DLS, figure a shows the results of culturing primary T cells in PR or GM hydrogel, respectively, figure b shows the effect of culturing T cells in ILS on the non-specific activation of T cells, figure c shows the results of the proliferation of T cells cultured under different conditions, and figure d shows the release results of T cells loaded in the inner layer of the scaffold;

[0028] Figure 6 The release of two payloads from DLS in chronological order is shown; Figure a is the release in vitro of U12 labeled BimmRNA and mCherry stable expression Jurkat T cell line, and Figure b is the release of GD-920 scaffold releasing gene complex and T cells in vivo. DETAILED DESCRIPTION

[0029] The present application develops a double-layer spherical scaffold (DLS) based on photocured hydrogel, named GD-920 scaffold. The DMP-Bim complex loaded in the outer layer of the scaffold can be released 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, and the T cells can interact with recruited DCs and immune activators in situ. The GD-920 scaffold releases gene and cell load in a controllable and orderly manner, achieving sustained TME activation effect. The scaffold only needs to be implanted once in situ, and can effectively inhibit tumor growth, which is much better than the control scaffold containing a single payload. This GD-920 scaffold for co-delivery of genes and cells has excellent potential as a strategy combining gene therapy and adoptive cell therapy in cancer immunotherapy.

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

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

[0032] The specific embodiments of the present application will be further explained and described by the following examples, but it does not mean that the protection scope of the present application is limited in the range described in the examples.

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

[0034] The preparation process of the double-layered spherical scaffold DLS is as follows: first, 50 μL of the porous GelMA (PR) solution is dropped into mineral oil, and the inner layer of the scaffold (ILS) is formed by light curing. Then, 100 μL of the common GelMA (GM) solution is added to coat the ILS, and the outer layer of the scaffold (OLS) is formed by secondary light curing. After washing with PBS for three times to remove the external mineral oil, the DLS with uniform morphology is obtained. The results are shown in Figure 1 As shown in FIG. 8A, the diameter of the ILS layer is about 5 mm, and the diameter of the whole scaffold is about 7 mm. The thickness of the OLS layer is uniformly distributed at about 1 mm. After being cut in half from the middle, the DLS is in a three-dimensional (3D) spherical shape instead of a flat elliptical shape, which can achieve better sequential release of the loaded drugs through uniform degradation. The DLS can be prepared in different sizes to meet different application scenarios. The two layers of hydrogel are uniformly distributed, and the two kinds of payloads can be released through ordered biodegradation.

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

[0036] The characterization results of the double-layered spherical scaffold DLS are shown in Figure 2 As shown in FIG. 8A, the diameter of the ILS layer is about 5 mm, and the diameter of the whole scaffold is about 7 mm. The thickness of the OLS layer is uniformly distributed at about 1 mm. After being cut in half from the middle, the DLS is in a three-dimensional (3D) spherical shape instead of a flat elliptical shape, which can achieve better sequential release of the loaded drugs through uniform degradation. The DLS can be prepared in different sizes to meet different application scenarios. The two layers of hydrogel are uniformly distributed, and the two kinds of payloads can be released through ordered biodegradation. Figure 2 a shows that the scaffold can be clamped using surgical forceps without serious deformation. Figure 2 b shows the adhesion properties of the DLS, which helps it to be fixed at the implantation site and not affected by movement. Figure 2 c shows the compression properties of the scaffold. After the pressure is removed, the DLS still maintains the spherical structure, which proves that the scaffold has the advantageous property of long-term biological fusion at the implantation site, enabling it to release drugs in situ and respond to the physiological environment. The two-layer structure and morphology are observed by scanning electron microscopy (SEM), as shown in Figure 2 d. The pore size of the ILS is about 100 μm, which is larger than that of the OLS (30 μm). The interface between the OLS layer and the ILS layer (white dashed line) can be clearly observed under the scanning electron microscope. These results show that the DLS can maintain its three-dimensional shape and porous morphological structure, which is beneficial for drug loading and uniform release.

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

[0038] The in vitro degradation of the DLS is evaluated after 24 hours of action of different concentrations of collagenase. As shown in Figure 3As shown in Figure 2a, with the increase of collagenase concentration, the outer layer or inner layer of the scaffold was sequentially degraded. In addition, the relative fluorescence intensity of the degradation of the scaffold under different concentrations of collagenase was also detected. The OLS signal (detected by staining with blue fluorescent dye) gradually increased and reached a maximum 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, which indicated that the DLS was sequentially degraded in vitro. In order to evaluate the in vivo degradation, the dyed DLS (ILS layer dyed with red ink and OLS layer dyed with blue ink) was implanted subcutaneously, and its volume was measured every day. Figure 3 b shows that on the 4th day, the blue OLS layer was slightly degraded, and the remaining OLS still maintained a spherical shape with a diameter close to 8 mm. On the 12th day, the OLS layer was almost completely degraded, and the diameter of the ILS was still 5 mm. On the 15th day, the DLS was completely degraded in vivo. These results indicate that the DLS can be sequentially degraded in vivo within 15 days. In summary, the spherical DLS was successfully prepared and exhibited stable physicochemical properties, implantable properties, and continuous biodegradation properties.

[0039] Example 4 mRNA drug loading and release

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

[0041] In order to further evaluate the biological activity of the released complex, the DMP-EGFP complex was loaded into the GM scaffold and incubated in DMEM medium containing collagenase at 37°C for 7 days, and the supernatant was collected every day. As shown in Figure 3a, the supernatant collected on the 7th day showed that the EGFP mRNA could efficiently transfect CT26 cells, and there was no obvious cytotoxicity. Figure 4 b shows that the supernatant collected on the 7th day showed that the EGFP mRNA could efficiently transfect CT26 cells, and there was no obvious cytotoxicity.

[0042] In addition, the transfection efficiency of the supernatant every day was also evaluated by flow cytometry. As shown in Figure 3c, the transfection efficiency of the supernatant collected on the 7th day was 70%, which was higher than that of the commercial transfection reagent Lipofectamine 3000 (about 50%). Figure 4c, the DMP-EGFP complex released from OLS maintained a fairly high transfection ability within 7 days, with an average transfection efficiency from 11.57% to 33.06%. Notably, a higher transfection efficiency was still detected in the last two days, further demonstrating the sustained protection ability and long-term transfection ability of DMP-EGFP complex. These results indicate that the mRNA loaded OLS has a persistent and stable biological activity in vitro.

[0043] We also evaluated the tumor inhibition effect of DMP-Bim@OLS in vitro. The supernatant of DMP-Bim@OLS (4 pg Bim) pre-treated with collagenase was collected from treated CT26 cells. As shown in Figure 4 d and 4e, more red-stained dead cells were observed in the DMP-Bim@OLS treatment group compared with other groups, as detected by live / dead staining. The cell morphology of the DMP-Bim@OLS group showed atrophy and destruction, while the cell morphology of the OLS group and the DMP@OLS group showed no obvious changes. These results indicate that the DMP-Bim complex can be released from DMP-Bim@OLS while biodegrading, and maintain the transfection ability of CT26 cells. The transfected Bim mRNA can be expressed in CT26 cells and exert a tumor-killing effect. Therefore, our research results demonstrate that DMP-Bim@OLS has long-term mRNA protection ability, stable controlled 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 characteristics 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 hours. The CD3 positive proportion of stimulated lymphocytes was found to be very high (up to 95.19%), indicating the success of primary T cell collection.

[0046] Then, the collected T cells were labeled with CFSE and cultured in PR or GM hydrogel, respectively. As shown in Figure 5 a, the proliferation rates of T cells cultured in PR, GM or medium were similar on day 2, all at 40%. From day 4 to day 6, the proliferation rate of T cells cultured in PR hydrogel 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 medium reached 77%, which was higher than that of PR group (P<0.05) and GM culture group (P<0.0001). These results indicate that T cell proliferation function can be maintained in both PR and GM hydrogels, but the proliferation efficiency is higher in medium culture. Compared with GM, PR is more suitable for T cell culture and proliferation, and is suitable for the composition of ILS.

[0047] We then evaluated whether loading T cells inside ILS would change the biological function of T cells. Briefly, T cells were put inside the inner layer of DLS, thus forming T@ILS. First, to determine whether ILS culture would induce non-specific activation of T cells, the expression of T cell activation markers CD69 and CD25 was examined. As shown in Fig. Figure 5 b, there was no significant difference in the expression of CD69 and CD25 between T cells cultured in ILS and in medium. This demonstrated that ILS did not induce non-specific activation of T cells. Then, the phenotype of T cells cultured under these two conditions was examined by determining the proportion of helper T cells (CD4+) or CTL (cytotoxic T cells, CD8+). Moreover, there was no significant difference in the proportion of CD8+ / CD4+ T cells between T cells (0.32-fold) and T@ILS (0.28-fold), which indicated that ILS culture did not change the phenotype of T cells.

[0048] Mouse lymphocytes were divided into naive T cells (Tn, CD44-CD62L+) and memory T cells (Tm, CD44+). According to our results (Fig. Figure 5 b), T cells in both groups were mostly composed of Tm. Moreover, the proportion of effector memory T cells (Tem, CD44+CD62L-) was slightly higher, while the proportion of central memory T cells (Tcm, CD44+CD62L+) was lower in Tm cultured in ILS than in medium. These results indicated that culture in ILS had little effect on the normal biological function of T cells, thus making this method of storing and releasing T cells feasible.

[0049] According to our design, GD-920 contains inner layer T@ILS and outer layer DMP-Bim@OLS. Our aim was to determine whether the proliferation of T cells would be affected by the outer layer payload. Therefore, we evaluated the proliferation of T cells cultured under different conditions. T cells were labeled with CFSE and then cultured in medium, inner layer of DLS (T@ILS), DMP-loaded DLS (DMP@OLS-T@ILS), or GD-920 scaffold. Five days later, CFSE signals (Fig. Figure 5 c) were examined. The proliferation rate of T cells in each group was similar (more than 95%) compared to day 0. This result indicated that the outer layer-loaded DMP-Bim complex had little effect on the survival and proliferation of T cells.

[0050] We then evaluated the release of T cells loaded in the inner layer of the scaffold. Briefly, 3x10 6T cells were loaded on ILS and cultured in collagenase-containing media. Supernatants were collected and quantified daily, and T cells released from ILS were counted. As shown in Figure 5 d, T cells were first detected in the supernatant on day 4, and the amount of release reached a maximum on day 6. Then, T cells were released at a constant rate until day 8. These results show 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 in a controlled, orderly manner, as they are loaded in the inner layer of the scaffold.

[0051] Example 6 Sequential release of mRNA and T cells

[0052] To observe the sequential release of the two payloads from DLS, U12-tagged Bim mRNA and mCherry-stable-expressing Jurkat T cell line were synthesized and constructed. These two compounds were loaded on ILS or OLS, respectively, to form a tagged GD-920 scaffold. Then it was cultured in collagenase-containing media, and the release of its payloads was monitored daily. The results are shown in Figure 6 a, 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 day 2 and day 3, the boundary of the outer layer becomes blurred, but the boundary of the inner layer can be clearly observed. This indicates that the outer layer of the scaffold is gradually degraded with the release of U12-tagged mRNA, and the cells are still stored in the inner layer of the scaffold. In addition, on day 4, the outer layer of the scaffold is completely degraded, while the boundary of the inner layer is still clear. Then, on day 5 and day 6, the inner layer of the scaffold has no clear boundary due to the rapid release of Jurkat T cells. These results show the sequential degradation process of DLS, and the sequential release of mRNA loaded in the outer layer and T cells loaded in 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 vivo in mice carrying CT26 subcutaneous tumors. Briefly, cy5.5-labeled DMP and DiR-stained T cells were added to the GD-920 scaffold, and it was implanted near the tumor. The results are shown in Figure 6As shown in Figure b, from day 3 to day 5, strong cy5.5 signal was detected in tumor area, and the signal disappeared slowly until day 15. Meanwhile, from day 3 to day 13, strong DiR signal was detected. These results showed that the release and effect of DMP occurred from day 3 to day 5 after implantation, while the release and tumor infiltration of T cells lasted for a longer time. The staining results of frozen sections also supported this conclusion. Before implantation, biotin-labeled DMP and CFSE-stained T cells were incorporated into GD-920 scaffolds to track their distribution in tissues.

[0054] It can be seen that the present application prepares a double-layer hydrogel scaffold which can simultaneously load gene drugs and immune cells, and after loading drugs, the release of the drugs can be controlled, and the combination of gene therapy and immunotherapy is provided for the treatment of tumors, which provides a new possibility for the treatment of tumors and has good practical value.

Claims

1. A double-layered hydrogel scaffold (DLS) characterized in that: The outer layer is methacrylated gelatin GelMA, and the inner layer is methacrylated gelatin PR; the concentration of the methacrylated gelatin GelMA and the methacrylated gelatin PR is 5-10%; the cross-linking degree of the methacrylated gelatin GelMA is 30%, and the cross-linking degree of the methacrylated gelatin PR is 60%; the outer layer of the double-layer hydrogel scaffold DLS is loaded with a gene drug, and the inner layer of the double-layer hydrogel scaffold DLS is loaded with immune cells.

2. The double-layer hydrogel scaffold (DLS) according to claim 1, wherein: The hydrogel concentration of the methacrylated gelatin GelMA is 5%, and the cross-linking degree is 30%; the hydrogel concentration of the methacrylated gelatin PR is 6%, and the cross-linking degree is 60%.

3. The double-layer hydrogel scaffold (DLS) according to claim 1, wherein: The scaffold is spherical, and the diameter is 2-10 mm.

4. The dual-layer hydrogel scaffold (DLS) of claim 1, wherein: The diameter of the inner layer of the scaffold is 2-7 mm, and the thickness of the outer layer is 1-5 mm.

5. Process for the preparation of a double-layer hydrogel scaffold DLS according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: a. The inner layer of the porous PR hydrogel is dropped into mineral oil, and cured by 405 nm wavelength blue light irradiation to form the inner layer ILS of the scaffold; b. The ILS is coated with common GelMA hydrogel, and the second curing is performed by 405 nm wavelength blue light irradiation to form the outer layer OLS of the scaffold, and the double-layer hydrogel scaffold DLS is obtained after washing.

6. The method of claim 5, wherein the method further comprises: The volume ratio of the PR hydrogel to the GelMA hydrogel is 1-10:5-20.

7. The method of claim 5, wherein the DLS is prepared by: The blue light irradiation time is 30-60 seconds.

8. Use of the double-layer hydrogel scaffold DLS in the preparation of a drug for treating tumors according to any one of claims 1-4.

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