A double-layer composite structure car-t cell in vivo delivery scaffold and application thereof

Abstract

This invention relates to a bilayer composite CAR-T cell in vivo delivery scaffold and its application. The in vivo delivery scaffold comprises an inner layer and an outer layer. The inner layer comprises a methacrylated hydrogel and δ-MnO2 nanosheets and cytokine 1 linked thereto. The outer layer comprises a composite hydrogel and cytokine 2 linked thereto. The composite hydrogel comprises hyaluronic acid and methacrylated gelatin. This bilayer scaffold achieves a synergistic system for structured immune induction and functional signal maintenance. The outer lymphoid-inducible matrix constructs a spatial framework for immune cell communication and memory formation, and delivers nutritional support to the inner layer. The inner oxygen-cytokine complex system maintains CAR-T cell activity and metabolic homeostasis. The synergy of these two layers can form a locally sustainable artificial immune microecology for immune activation and memory maintenance, providing a novel material basis for the long-term effectiveness and safety of CAR-T cell therapy in solid tumors.

Description

Technical Field

[0001] This invention relates to the field of in vivo drug delivery technology, and in particular to a bilayer composite CAR-T cell in vivo delivery scaffold and its application. Background Technology

[0002] Adoptive cell transfer (ACT) is a rapidly developing tumor immunotherapy strategy in recent years, among which chimeric antigen receptor T cell (CAR-T) therapy has achieved significant clinical efficacy in hematologic malignancies. However, the efficacy of CAR-T cell therapy in solid tumors is still limited by multiple factors. First, the tumor immune microenvironment (TIME) of solid tumors exhibits significant immunosuppressive characteristics. Studies have shown that regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) enriched in tumor tissues can inhibit CAR-T cell activity by secreting immunosuppressive factors or competitively consuming metabolic substrates; at the same time, hypoxic and acidic metabolic environments further weaken the metabolic activity and cytotoxic effects of CAR-T cells. Second, CAR-T cells have poor retention and survival capabilities in solid tumors, making it difficult to achieve sustained expansion or form immune memory within tumor tissues.

[0003] To address the aforementioned issues, existing research has attempted to improve the survival and function of CAR-T cells at tumor sites through local delivery systems. Common strategies include using hydrogels, nanocarriers, or implantable scaffolds to achieve local fixation and sustained release of CAR-T cells. However, current delivery technologies still have several shortcomings. While they can improve local cell retention or physical support, they are mostly passive sustained releases or structural fixation, lacking active, spatiotemporally controllable strategies for regulating the tumor immune microenvironment. Many adoptive immune cells, including CAR-T cells, have the potential to differentiate into memory cells, but most delivery technologies lack designs to induce lymphoid structures (TLS) formation, failing to achieve durable immune memory responses. Furthermore, while some designs incorporate oxygen release modules to alleviate hypoxia, the oxygen release mechanism and rate control are singular and usually not coupled with immune structuring or lymphatic drainage reconstruction strategies. In summary, how to construct a local CAR-T cell delivery system that can simultaneously achieve oxygen supply regulation and synergistic cytokine delivery remains a key technical problem that urgently needs to be solved in this field, leaving crucial technological gaps for improvement and invention. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a bilayer composite CAR-T cell in vivo delivery scaffold and its application. This invention achieves a synergistic effect of local immune induction, oxygen metabolism regulation, and cell function maintenance through spatial functional partitioning. The scaffold consists of an inner "oxygen supply-cytokine synergistic zone" and an outer "lymphoid structure zone," and can be applied to tumor resection cavities or local lesion areas via in situ injection or modular implantation.

[0005] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a bilayer composite CAR-T cell in vivo delivery scaffold, the in vivo delivery scaffold comprising an inner layer and an outer layer; the inner layer comprising a methacrylated hydrogel and δ-MnO2 nanosheets and cytokine 1 connected thereto; the outer layer comprising a composite hydrogel and cytokine 2 connected thereto; the composite hydrogel comprising hyaluronic acid and methacrylated gelatin.

[0006] In this invention, uniformly dispersed δ-MnO2 nanosheets in the inner layer continuously react in the tumor microenvironment or postoperative environment (where H2O2 concentration is high), decomposing and releasing O2 to significantly alleviate local hypoxia and enhance the aerobic metabolism and cytotoxic activity of CAR-T cells. To enhance sustained immune signaling, cytokine 1 is slowly released to maintain a high local concentration of cytokines while avoiding systemic toxicity. This layer functionally achieves multiple benefits: continuous oxygen production improves local tumor immunosuppression and acidification, while simultaneously promoting long-term survival and memory differentiation of CAR-T cells through cytokine 1 signaling.

[0007] The outer layer can enhance fluid exchange and metabolic supply through the vascular system, and recruit various immune cells such as dendritic cells to promote antigen presentation, while forming a local immune memory reserve area. The microporous structure of this layer allows CAR-T cells to migrate, and together with the inner CAR-T storage area, they form a "local immune factory", enabling CAR-T cells to proliferate continuously in the local area, activate and drive the systemic anti-tumor immune response.

[0008] Preferably, the cytokine 1 includes IL-15 and / or IL-21.

[0009] In this invention, IL-15 in cytokine 1 promotes the survival and proliferation of CAR-T cells; IL-21 assists CAR-T cells in resisting exhaustion, resisting terminal differentiation, and improving the killing quality; the combination can achieve long-term survival and functional maintenance of CAR-T cells in vivo.

[0010] Preferably, the delivery carrier 1 loads the cytokine 1 and is covalently linked to the methacrylated hydrogel.

[0011] Preferably, the delivery carrier 1 comprises liposomes and / or PLGA nanoparticles.

[0012] Preferably, the δ-MnO2 nanosheets are covalently linked to the methacrylic hydrogel.

[0013] Preferably, the thickness ratio of the inner layer to the outer layer is 10:(1-50). The (1-50) can be, for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50, etc.

[0014] Preferably, the concentration of δ-MnO2 nanosheets in the inner layer is 0.1%-5%. The 0.1%-5% can be, for example, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%.

[0015] Preferably, the concentration of cytokine 1 in the inner layer is 30-150 ng / mL. The 30-150 ng / mL concentration can be, for example, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, or 150 ng / mL.

[0016] Preferably, the mass ratio of hyaluronic acid to methacrylamide gelatin is 1:(3-8). The (3-8) can be, for example, 3, 4, 5, 6, 7, or 8.

[0017] Preferably, the molecular weight of the hyaluronic acid is 100-300 kDa. The 100-300 kDa range can be, for example, 100 kDa, 150 kDa, 200 kDa, 250 kDa, or 300 kDa.

[0018] Preferably, the degree of methylation of the methacrylamide gelatin is 60-90%. The 60-90% can be, for example, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0019] Preferably, the concentration of cytokine 2 in the outer layer is 150-650 ng / mL. The 150-650 ng / mL concentration can be, for example, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 350 ng / mL, 400 ng / mL, 450 ng / mL, 500 ng / mL, 550 ng / mL, 600 ng / mL, or 650 ng / mL.

[0020] Preferably, the cytokine 2 includes VEGF-C, CCL21, CXCL13, and LIGHT.

[0021] In this invention, among cytokines 2, VEGF-C promotes lymphangiogenesis; CCL21 attracts and recruits immune cells such as dendritic cells (DCs); CXCL13 recruits B cells to form lymphoid follicles; LIGHT tissues undergo lymphoid remodeling; and T cell immune co-stimulation is promoted.

[0022] The cytokine 2 used in this invention can induce the migration of lymphoendothelial cells, the formation of lymphatic vessels in the gel, promote the formation of B cell follicles, and induce the formation of structures such as high endothelial venules (HEVs), thereby realizing the structure and function of a lymphatic system.

[0023] Preferably, the delivery carrier 2 loads the cytokine 2 and is covalently linked to the composite hydrogel.

[0024] Preferably, the liposomes and / or PLGA nanoparticles are included.

[0025] In a second aspect, the present invention provides an in vivo CAR-T cell delivery system, the delivery system comprising the CAR-T cell in vivo delivery scaffold with a bilayer composite structure as described in the first aspect and CAR-T cells.

[0026] Preferably, the ratio of CAR-T cells to in vivo delivery scaffold is (1-5) × 10⁻⁶. 6 cells: 1 scaffold. The number of cells (1-5) can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

[0027] Preferably, the CAR-T cells are distributed in the inner layer of the in vivo delivery scaffold.

[0028] Thirdly, the present invention provides a method for preparing the CAR-T cell in vivo delivery system according to the second aspect, the method comprising: (1) Hyaluronic acid, methacrylamide gelatin and photoinitiator are mixed, and delivery carrier 1 loaded with cytokine 1 is added. After photocuring, the outer layer is obtained. (2) The inner layer is prepared by photocuring δ-MnO2 nanosheets, methacrylated hydrogel, delivery carrier 2 loaded with cytokine 2 and photoinitiator; (3) CAR-T cells are mixed with the inner layer, and then the outer layer is photocrosslinked with the mixed inner layer to obtain the product.

[0029] Preferably, the photoinitiator in steps (1) and (2) is each independently one or a combination of at least two of lithium phenyl-2,4,6-trimethylbenzoyl phosphate, eosin Y, Bengal rose red, or riboflavin.

[0030] Preferably, in step (1), the photocuring time is 30-60 s, and the light wavelength is 390-550 nm. The 30-60 s can be, for example, 30 s, 35 s, 40 s, 45 s, 50 s, 55 s, or 60 s. The 390-550 nm can be, for example, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, or 550 nm.

[0031] Preferably, in step (2), the photocuring time is 30-60 s, and the light wavelength is 390-550 nm. The 30-60 s can be, for example, 30 s, 35 s, 40 s, 45 s, 50 s, 55 s, or 60 s. The 390-550 nm can be, for example, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, or 550 nm.

[0032] Preferably, in step (3), the light intensity of the photocrosslinking is 10-100 mW / cm. 2 The 10-100 mW / cm 2 For example, it could be 10 mW / cm 2 20 mW / cm 2 30 mW / cm 2 40 mW / cm 2 50 mW / cm 2 60 mW / cm 2 70 mW / cm 2 80 mW / cm 2 90 mW / cm 2 Or 100 mW / cm 2 wait.

[0033] Fourthly, the present invention provides an application of the preparation method of the CAR-T cell in vivo delivery scaffold with the bilayer composite structure described in the first aspect, the CAR-T cell in vivo delivery system described in the second aspect, or the CAR-T cell in vivo delivery system described in the third aspect in the preparation of targeted drugs.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The bilayer composite CAR-T cell in vivo delivery scaffold prepared in this invention can achieve multiple functions in one step, including oxygen supply, nutritional support, local function maintenance, and memory signal enhancement, overcoming the problem that a single module cannot simultaneously address the three major bottlenecks of metabolism, structure, and signaling. It significantly improves the local survival and efficacy of CAR-T cells, and is expected to significantly increase the local CAR-T cell survival rate, intracellular expansion, and cytotoxic activity at the same infusion dose, while reducing the risk of functional exhaustion and depletion marker expression.

[0035] 2. In this invention, the lymphoid structures induced by the outer layer and the sustained factor support of the inner layer jointly increase the probability of forming persistent memory cells locally, thereby enhancing long-term protection against relapse and metastasis and promoting long-term immune memory and systemic protection. The lymphatic vessels and HEV formation promoted by the outer layer facilitate hydrodynamics and cell communication, which is conducive to antigen transport and the presentation of dendritic cells to lymphoid structures, thus enhancing the initiation and spread of the immune response at the histological level.

[0036] 3. The structure prepared in this invention locally anchors and slowly releases cytokines and stimulating signals, reducing peripheral circulation exposure and the risk of systemic cytokine release syndrome, decreasing systemic toxicity, and increasing the safety window. By adjusting the amount of MnO2, factor loading, release rate, and outer chemotactic molecular spectrum, personalized combination schemes can be developed according to different tumor types and postoperative conditions, giving the structure controllability and customizable development potential.

[0037] 4. The modular process of the injectable or implantable stent prepared by this invention facilitates clinical translation. The design takes into account both injection feasibility and implantation stability, and meets the clinical application scenarios of postoperative filling or local repeated drug administration. Attached Figure Description

[0038] Figure 1 This is a graph showing the changes in oxygen concentration.

[0039] Figure 2 This is a diagram of cytokine release.

[0040] Figure 3 This is a diagram of cell composition.

[0041] Figure 4 This image shows the effectiveness of the CAR-T cell in vivo delivery system.

[0042] Figure 5 This is a graph showing the effect of inhibiting tumor recurrence. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0044] Example 1 This embodiment describes the preparation of an in vivo CAR-T cell delivery system. (1) Preparation of the lymphoid induction layer (outer layer) Hyaluronic acid (HA, molecular weight 200 kDa) was dissolved in sterile PBS to prepare a 2% (w / v) solution; methacrylamide gelatin (GelMA, methylation degree 80%) was dissolved in PBS to prepare a 10% (w / v) solution, and photoinitiator LAP (0.25% w / v) was added. The two solutions were mixed at a volume ratio of 1:1 to form a composite precursor solution. To impart immunoinducing function, liposomes co-assembled with DSPE-PEG-NH2 were used to co-load nanoparticles (particle size 200 nm) with VEGF-C (100 ng / mL), CCL21 (100 ng / mL), CXCL13 (200 ng / mL), and LIGHT (20 ng / mL) via a thin-film method. The carboxyl groups on GelMA were activated by EDC / NHS, and then reacted with the amino groups on the nanoparticles to form amide bonds, thus covalently linking the nanoparticles to GelMA. The resulting composite solution was cured by irradiation with 405 nm light for 40 s to form a porous gel layer with a pore size of 100 μm. Residual reactants were removed by washing with PBS. This layer can promote the recruitment of dendritic cells (DCs), lymphoendothelial cells, and high endothelial venules (HEVs), providing local immune microstructure and material delivery channels to support subsequent CAR-T reactivation.

[0045] (2) Preparation of the oxygen supply-cytokine synergistic layer (inner layer) KMnO4 (final concentration 0.1 M) was added to a GelMA (10% w / v) solution, stirred until homogeneous, and then ascorbic acid (0.05 M) was added dropwise for 30 min to reduce and form MnO2 nanosheets (particle size 30 nm). To construct an immune-activating environment, SPE-PEG-NH2 co-assembled liposomes were used as carriers to prepare IL-15 (50 ng / mL) and IL-21 (30 ng / mL) via a thin-film method. EDC / NHS activated the carboxyl groups on GelMA, which then reacted with the amino groups on the nanoparticles to form amide bonds, achieving covalent linkage between the nanoparticles and GelMA. LAP (0.25%) was added to the mixed solution, and crosslinking was performed under 405 nm light for 30-60 s to form a gel with a pore size of 100 μm and a mechanical modulus of 1.5 kPa.

[0046] (3) Preparation of CAR-T cell in vivo delivery system The oxygen-cytokine synergistic inner layer was placed at the bottom of the mold, and a lymphoid-inducing layer precursor solution was dropped onto the surface and cured by irradiation with 405 nm light for 40 s, allowing the two layers to interpenetrate at the interface to form a stable whole with a thickness of 2 mm and a thickness ratio of 1:1 between the inner and outer layers. Before implantation, CD3 / CD28 activated CAR-T cells (CEA-targeted CAR-T cells) (3×10⁻⁶) were implanted. 6 CAR-T cells / scaffolds are mixed into the inner GelMA network (CAR-T cells are mixed in before the inner layer is cured by light) to maintain uniform distribution and viability.

[0047] Example 2 This embodiment verifies the oxygen production capacity of the delivery stent. The prepared bilayer hydrogel scaffold from Example 1 was cut into circular pieces (50 mg in weight) with a diameter of 8 mm and a thickness of 2 mm. These pieces were placed in a 50 mL Erlenmeyer flask, and 10 mL of 0.5 M phosphate-buffered saline (PBS, pH 7.4) was added. The temperature was equilibrated to 37°C. A low-concentration hydrogen peroxide solution was continuously and slowly added using a syringe to simulate a tumor or postoperative environment. Dissolved oxygen (DO) was measured online. An electrode (dissolved oxygen electrode / dissolved oxygen meter) was inserted into the center of the solution, and DO changes were recorded every minute. The results are as follows: Figure 1 As shown, the oxygen concentration is significantly increased and continuously released.

[0048] Example 3 This embodiment performs cytokine release detection. To verify the long-term sustained-release performance of cytokines in the bilayer GelMA / HA–MnO2 composite scaffold of this invention, IL-15 and CCL21 were selected as representative immunomodulatory factors for evaluation. Using the bilayer GelMA / HA–MnO2 composite scaffold prepared in Example 1, the scaffold was placed in PBS buffer and incubated at 37°C. Cytokine levels in the supernatant were measured at 1, 6, 12, 24, 48, 96, and 168 h, and the release curve was quantitatively analyzed using ELISA. The results are as follows: Figure 2 The results showed that each cytokine was released at a rate of approximately 20–35% within the first 48 hours, with a cumulative release rate of 70–85% by day 7.

[0049] Example 4 This embodiment verifies the function of an in vivo gel system. To verify the in vivo immune structure formation and anti-tumor function of the bilayer composite scaffold of this invention, GPC3 was selected to target CAR-T cells and a mouse model of in situ recurrence of liver cancer after surgery was established.

[0050] The CAR-T cell in vivo delivery system prepared using the method in Example 1 was used to subcutaneously inoculate C57BL / 6 mice with Hepa1-6-GPC3 cells (1×10⁻⁶). 6 (Each scaffold is individually loaded with CAR-T cells). Local resection is performed after the tumor reaches a diameter of 8–10 mm, and the prepared double-layered scaffold is immediately implanted into the tumor resection cavity post-operatively. Each scaffold is loaded with 2 × 10⁶ CAR-T cells. 6 The control group consisted of mice receiving CAR-T therapy with a blank scaffold (without MnO2 and cytokines). On day 14 post-surgery, some mice were sacrificed, and tissue samples were collected for flow cytometry analysis. Results are as follows... Figure 3 and Figure 4 The results showed that B cells (CD19) were visible at the site of the double-layer scaffold implantation on day 14. + ), dendritic cells (CD11c + Lymphoendothelial cells (LYVE1) + ) and high endothelial microvenous cells (CD31) + CAR-T cell aggregation was observed; the survival rate of CAR-T cells in the scaffold group reached 78%, significantly higher than that in the control group (27%); the expression levels of IFN-γ and Granzyme-B were increased by approximately 2.5 times, indicating good functional maintenance. Further analysis of long-term follow-up data at 60 days post-surgery showed that CD44 could be detected in the spleen of the double-layer scaffold group. + CD62L + The persistence of memory T cells (45%) indicates that the scaffold can induce the formation of systemic immune memory.

[0051] Example 5 This embodiment tests the anti-tumor effect. To evaluate the antitumor efficacy and survival benefit of CAR-T cell delivery via a bilayer scaffold system, GPC3-targeted CAR-T cells were selected, and a mouse model of in situ recurrence of liver cancer after surgery was established. C57BL / 6 mice were subcutaneously inoculated with Hepa1-6-GPC3 cells (1×10⁻⁶). 6 (Each tumor is individually sampled and implanted). Once the tumor diameter reaches 8–10 mm, local resection is performed, leaving approximately 1% of the tissue to construct a recurrence model. Immediately post-surgery, a double-layered scaffold prepared using the method described in Example 1 is implanted into the tumor resection cavity. Each scaffold is loaded with approximately 2 × 10⁶ CAR-T cells. 6 The control group consisted of patients receiving CAR-T therapy with a blank scaffold (without MnO2 and cytokines). Tumor development and growth were monitored every 2 days, and the specific results are as follows: Figure 5 As shown, the tumor recurrence inhibition efficiency of the double-layer stent group reached over 90%, while that of the control group was only 50%.

[0052] In summary, the bilayer scaffold of this invention achieves a synergistic system for structured immune induction and functional signal maintenance. The outer lymphoid-inducible matrix constructs a spatial framework for immune cell communication and memory formation, and delivers nutritional support to the inner layer; the inner oxygen-cytokine complex system maintains CAR-T cell activity and metabolic homeostasis. The synergy of these two layers can locally form an artificial immune microecology capable of sustainable immune activation and memory maintenance, providing a novel material basis for the long-term effectiveness and safety of CAR-T cell therapy in solid tumors.

[0053] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A bilayer composite CAR-T cell in vivo delivery scaffold, characterized in that, The in vivo delivery stent comprises an inner layer and an outer layer; The inner layer comprises a methacrylated hydrogel and its linked δ-MnO2 nanosheets and cytokine 1; The outer layer includes a composite hydrogel and a linked cytokine 2; The composite hydrogel comprises hyaluronic acid and methacrylamide gelatin.

2. The CAR-T cell in vivo delivery scaffold with a bilayer composite structure according to claim 1, characterized in that, The cytokine 1 includes IL-15 and / or IL-21; Preferably, the delivery carrier 1 loads the cytokine 1 and is covalently linked to the methacrylated hydrogel; Preferably, the delivery carrier 1 comprises liposomes and / or PLGA nanoparticles; Preferably, the δ-MnO2 nanosheets are covalently linked to the methacrylic hydrogel; Preferably, the thickness ratio of the inner layer to the outer layer is 10:(1-50).

3. The CAR-T cell in vivo delivery scaffold with a bilayer composite structure according to claim 1 or 2, characterized in that, The concentration of δ-MnO2 nanosheets in the inner layer is 0.1%-5%; Preferably, the concentration of cytokine 1 in the inner layer is 30-150 ng / mL.

4. The CAR-T cell in vivo delivery scaffold with a bilayer composite structure according to any one of claims 1-3, characterized in that, The mass ratio of the hyaluronic acid to the methacrylamide gelatin is 1:(3-8); Preferably, the molecular weight of the hyaluronic acid is 100-300 kDa; Preferably, the degree of methylation of the methacrylamide gelatin is 60-90%.

5. The CAR-T cell in vivo delivery scaffold with a bilayer composite structure according to any one of claims 1-4, characterized in that, The concentration of cytokine 2 in the outer layer is 150-650 ng / mL; Preferably, the cytokine 2 includes any one or a combination of at least two of VEGF-C, CCL21, CXCL13, or LIGHT; Preferably, the delivery carrier 2 loads the cytokine 2 and is covalently linked to the composite hydrogel; Preferably, the delivery carrier 2 comprises liposomes and / or PLGA nanoparticles.

6. A CAR-T cell in vivo delivery system, characterized in that, The delivery system comprises a bilayer composite CAR-T cell in vivo delivery scaffold and CAR-T cells as described in any one of claims 1-5.

7. The CAR-T cell in vivo delivery system according to claim 6, characterized in that, The ratio of CAR-T cells to in vivo delivery scaffold is (1-5) × 10⁻⁶. 6 cells: 1 scaffold; Preferably, the CAR-T cells are distributed in the inner layer of the in vivo delivery scaffold.

8. A method for preparing the CAR-T cell in vivo delivery system according to claim 6 or 7, characterized in that, The preparation method includes: (1) Hyaluronic acid, methacrylamide gelatin and photoinitiator are mixed, and delivery carrier 1 loaded with cytokine 1 is added. After photocuring, the outer layer is obtained. (2) The inner layer is prepared by photocuring δ-MnO2 nanosheets, methacrylated hydrogel, delivery carrier 2 loaded with cytokine 2 and photoinitiator; (3) CAR-T cells are mixed with the inner layer, and then the outer layer is photocrosslinked with the mixed inner layer to obtain the product.

9. The method for preparing the CAR-T cell in vivo delivery system according to claim 8, characterized in that, The photoinitiator in steps (1) and (2) is each independently one or a combination of at least two of lithium phenyl-2,4,6-trimethylbenzoyl phosphate, eosin Y, Bengal rose red, or riboflavin; Preferably, in step (1), the photocuring time is 30-60 s and the light wavelength is 390-550 nm; Preferably, in step (2), the photocuring time is 30-60 s and the light wavelength is 390-550 nm; Preferably, in step (3), the light intensity of the photocrosslinking is 10-100 mW / cm. 2 .

10. The application of the preparation method of the bilayer composite CAR-T cell in vivo delivery scaffold according to any one of claims 1-5, the CAR-T cell in vivo delivery system according to claim 6 or 7, or the CAR-T cell in vivo delivery system according to claim 8 or 9 in the preparation of targeted drugs.

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