Nanoparticle cross-linked hydrogel and application thereof

Abstract

The invention discloses a nanoparticle cross-linked hydrogel and an application thereof, the nanoparticle cross-linked hydrogel is prepared from end group functionalized nanoparticles and a side chain functionalized modified hydrophilic polymer material, because a thiol group and an unsaturated double bond group can be connected through a Michael addition reaction, the cross-linked hydrogel has the advantages that the cross-linked hydrogel has good biocompatibility; therefore, a thiol group and an unsaturated double-bond group are selected to form a connecting bond of the nanoparticles and a hydrophilic high-molecular material, so that hydrogel forming is realized. According to the hydrogel delivery system taking the nanoparticles as the cross-linking agent, loading and delivery of various drugs are realized through a dual loading strategy of the nanoparticles and the hydrogel.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a nanoparticle cross-linked hydrogel and application thereof. BACKGROUND

[0002] For patients with end-stage organ failure or permanent loss of organs, transplantation is a life-saving treatment option. For example, cell transplantation using hematopoietic stem cells is currently a curative method for treating highly chemotherapy-resistant fatal hematological malignancies; islet transplantation is an effective clinical treatment method for replacing beta cells to control blood glucose in patients with type 1 diabetes; skin transplantation as a form of tissue transplantation is the gold standard for surgery in the treatment of burns and is the most common choice for treating chronic non-healing wounds to prevent infection and deterioration; solid organ transplantation is still an important means for treating irreversible diseases such as liver, kidney, lung, and heart that no longer have normal functions.

[0003] According to the genetic differences between donors and recipients, transplantation is generally divided into autologous transplantation, syngeneic transplantation, allogeneic transplantation, and xenogeneic transplantation. Allogeneic transplantation refers to the transplantation of a donor who is of the same species but has different genes to a recipient. Allogeneic transplantation is the most widely used transplantation method in clinical practice due to its short treatment time, high availability, strong product standardization capability, and no need for additional invasive surgery for the recipient. However, immune system recognition is a major obstacle to allogeneic transplantation, which recognizes allogeneic transplants as foreign bodies, triggers immune rejection, and ultimately destroys the transplants. Transplant patients usually need to receive a lifelong immunosuppression regimen to suppress allogeneic transplantation rejection. Immunosuppressive agents such as rapamycin and tacrolimus inhibit T lymphocyte proliferation, stimulate the production of regulatory T (Treg) cells, and prolong the survival of transplants. Immunosuppressive regimens greatly improve the lives of transplant recipients, but repeated use of non-specific immunosuppressive agents can disrupt immune balance, leading to serious adverse reactions and complications. Long-term systemic use of immunosuppressive agents causes irreversible bone marrow suppression, causing leukopenia, granulocytopenia, and decreased platelet count, and patients affected by this develop severe infections. In addition, the immune surveillance function is destroyed, and tumor cells cannot be identified and removed in time, leading to life-threatening malignancies.

[0004] Mesenchymal stem cells (MSCs) have unique immunomodulatory properties and have been approved for the treatment of graft-versus-host disease and are increasingly being used to treat a variety of autoimmune diseases, including systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis. Immune modulatory molecules on the surface of MSCs, such as Fas ligand (FasL), programmed death ligand 1 (PD-L1), and intercellular adhesion molecule 1 (ICAM1), can induce cell death and cell cycle arrest of effector T (Teff) cells. MSCs increase the phenotype of Treg cells by interacting with the Fas death receptor and programmed cell death protein 1 (PD1) on Teff cells, and this intercellular contact effect plays a crucial role in reducing immune responses and promoting allograft tolerance. However, MSCs, as living cells with uncontrolled self-renewal and multi-directional differentiation, have a risk of tumorigenesis, and lack sustained immunomodulatory activity due to limited local retention after transplantation, aging, and eventual death.

[0005] Therefore, there is still a need to develop a local persistent immune protection system that does not cause systemic immune tolerance for allogeneic graft delivery. SUMMARY

[0006] One of the purposes of the present application is to provide a nanoparticle cross-linked hydrogel made of end-group functionalized nanoparticles and side-chain functionalized modified hydrophilic polymer materials, wherein the mass ratio of end-group functionalized nanoparticles and side-chain functionalized modified hydrophilic polymer materials is 1:1 to 10:1. The end-group functionalized nanoparticles and side-chain functionalized modified hydrophilic polymer materials are selected from one of the following combinations: (i) the end-group functionalized nanoparticles are thiol end-group functionalized nanoparticles, and the side-chain functionalized modified hydrophilic polymer materials are unsaturated double bond group modified hydrophilic polymer materials; or (ii) the end-group functionalized nanoparticles are unsaturated double bond end-group functionalized nanoparticles, and the side-chain functionalized modified hydrophilic polymer materials are thiol group modified hydrophilic polymer materials.

[0007] Further, the nanoparticles are liposome nanoparticles, polymer nanoparticles, or cell-derived nanoparticles.

[0008] Further, the hydrophilic polymer materials are selected from hyaluronic acid, chitosan, heparin, cellulose, alginic acid, dextran, amylose, amylopectin, or pectin.

[0009] Further, in combination (i), the thiol end group is a mercapto group or cysteine, and the unsaturated double bond group is an acrylate, pentenamide, maleimide, or methacrylate.

[0010] Furthermore, in combination (ii), the unsaturated double bond end group is maleimide, vinyl sulfone, acrylate, acrylamide or methacrylate, and the thiol group is mercapto or cysteine.

[0011] A second objective of this invention is to provide the application of the above-mentioned nanoparticle cross-linked hydrogel in the preparation of allogeneic transplant products.

[0012] A third objective of this invention is to provide an allogeneic transplantation product comprising the aforementioned nanoparticle cross-linked hydrogel and an allogeneic graft, wherein the end-functionalized nanoparticles are loaded with immunosuppressants.

[0013] Furthermore, the immunosuppressant is a small molecule immunosuppressant selected from tacrolimus, cyclosporine A, rapamycin, cyclophosphamide, azathioprine, methotrexate, prednisone, or dexamethasone.

[0014] Furthermore, the allogeneic graft is selected from skin cells, epithelial cells, chondrocytes, cardiomyocytes, pancreatic islet cell clusters, induced pluripotent stem cells, skin, or blood vessels.

[0015] In one embodiment of the present invention, the nanoparticles are selected from liposomes, the raw materials of which are phospholipids, cholesterol and immunosuppressants, wherein the mass ratio of cholesterol to phospholipids is 1:4 to 1:15.

[0016] Furthermore, in one embodiment of the present invention, mesenchymal stem cell membrane-derived vesicles are first coated on the surface of nanoparticles loaded with immunosuppressants and modified with functionalized end groups, and then crosslinked with functional side chains modified on a hydrophilic polymer material to form a hydrogel.

[0017] The hydrogel delivery system of the present invention, which uses nanoparticles as crosslinking agents, achieves loading and delivery of a variety of drugs through a dual loading strategy of nanoparticles and hydrogels. Attached Figure Description

[0018] Figure 1 The particle size results and transmission electron microscope images (scale bar: 100 nm) of RAPA / NP in Example 1 are shown.

[0019] Figure 2 The results of RAPA / NP release in vitro in Example 1 are shown.

[0020] Figure 3 The image shows the gelation of RAPA-Gel after mixing SH-RAPA / NP with a-HA in Example 2.

[0021] Figure 4 The rheological measurement results of RAPA-Gel in Example 2 are shown.

[0022] Figure 5 The image shows a scanning electron microscope image of RAPA-Gel in Example 2 (scale bar: 50 μm (low magnification) and 1 μm (high magnification)).

[0023] Figure 6 The results show the PD1 expression of activated T cells immersed in hydrogel in Example 2.

[0024] Figure 7 Cryo-scanning electron microscopy image of RAPA-Gel encapsulating pancreatic islets in Example 2 (scale bar: 50 μm).

[0025] Figure 8 The glucose-stimulated insulin release and stimulation index of free islets and islets / RAPA-Gel in Example 2 are shown.

[0026] Figure 9 The percentage of dead cells in pancreatic islet / L-Gel and pancreatic islet / RAPA-Gel after 48 h of culture with activated T cells in Example 2.

[0027] Figure 10 The glucose-stimulated insulin release and stimulation index of islet / L-Gel and islet / RAPA-Gel after 48 h of culture with activated T cells in Example 2 are shown.

[0028] Figure 11 The results show the PD1 expression of activated T cells immersed in hydrogel in Example 3.

[0029] Figure 12 Fluorescence images of DiD-RAPA / NP, DiD-RAPA / NP / HA, and DiD-RAPA-Gel implanted under the renal sac in Example 4; fluorescence images of five major tissues collected on day 28 post-transplantation.

[0030] Figure 13 In Example 4, flow cytometry was used to measure Treg and CD8 levels in the islets, islet / L-Gel, and islet / RAPA-Gel, kidneys, and draining lymph nodes 7 days post-transplantation. + Teff and CD4 + Teff cell proportion.

[0031] Figure 14 The percentage of induced Treg cells in the islets, islets / L-Gel, and islets / RAPA-Gel kidneys, measured by flow cytometry 7 days post-transplantation, as described in Example 4.

[0032] Figure 15 This is the in vivo efficacy result of the immunomodulatory hydrogel against allogeneic islet rejection in Example 4.

[0033] Figure 16 The glucose tolerance test and area under the curve for normal mice, diabetic mice, and diabetic mice that survived 30 days after transplantation of pancreatic islets / RAPA-Gel in Example 4 are shown.

[0034] Figure 17 The results are from the flow cytometry determination of FasL on the MSC surface in Example 5.

[0035] Figure 18 The results show the expression of PD-L1 on the MSC surface in Example 5, both untreated and IFN-γ treated.

[0036] Figure 19 This is the in vivo efficacy result of the immunomodulatory hydrogel against allogeneic islet rejection in Example 5. Detailed Implementation

[0037] This invention designs and synthesizes a hydrogel using nanoparticles as a crosslinking agent. This hydrogel forms a synergistic drug delivery system through crosslinking of functionalized end groups modified on nanoparticles and functional side chains synthesized on hydrophilic polymers. On one hand, the nanoparticles, serving as the internal drug delivery platform of this system, have broad applicability. They can load not only hydrophilic drugs but also hydrophobic drugs, improving drug solubility and bioavailability through the diversity of nanoparticle selection; they can also effectively load protein or peptide drugs, enhancing their stability by providing protection; and they also possess the ability to load gene therapy drugs. On the other hand, the hydrogel, as an external matrix material, can stably load protein-based biomolecules, maintaining their bioactivity; effectively load hydrophilic drugs, achieving drug release through a three-dimensional network structure; and efficiently deliver grafts, providing new solutions for tissue engineering and regenerative medicine. This invention's hydrogel delivery system using nanoparticles as a crosslinking agent achieves the loading and delivery of multiple drugs through a dual loading strategy of nanoparticles and hydrogel.

[0038] Since thiol groups and unsaturated double bonds can be linked via Michael addition reactions, this invention selects thiol groups and unsaturated double bonds to form the linking bonds between nanoparticles and hydrophilic polymers. Hydrogel formation is achieved through a Michael addition reaction between end-functionalized nanoparticles and side-chain functionalized hydrophilic polymers. The end-functionalized nanoparticles and side-chain functionalized hydrophilic polymers are selected from one of the following combinations: (i) The terminal-functionalized nanoparticles are thiol-terminal-functionalized nanoparticles, and the side-chain functionalized hydrophilic polymer material is a hydrophilic polymer material modified with unsaturated double bond groups. Or (ii) the end-functionalized nanoparticles are unsaturated double-bond end-functionalized nanoparticles, and the side-chain functionalized hydrophilic polymer material is a thiol-modified hydrophilic polymer material.

[0039] Furthermore, the nanoparticles are liposome nanoparticles, polymer nanoparticles, or cell-derived nanoparticles.

[0040] Furthermore, the hydrophilic polymer material is selected from hyaluronic acid, chitosan, heparin, cellulose, alginic acid, dextran, amylose, amylopectin, or pectin.

[0041] Specifically, in combination (I), for thiol-terminated nanoparticles, block materials ABC with thiol or cysteine ​​end groups are selected to modify the nanoparticles. Here, A is a phospholipid, B is polyethylene glycol (PEG), selected from linear PEG, multi-arm PEG, dendritic PEG, and hyperbranched PEG, and C is thiol or cysteine. A can be embedded in a lipid bilayer structure to modify the nanoparticles with active end groups. For hydrophilic polymers modified with unsaturated double bond groups, the unsaturated double bond groups are selected from acrylates, pentenamides, maleimides, and methacrylates.

[0042] In combination (II), for nanoparticles functionalized with unsaturated double bond end groups, block materials ABD with maleimide, vinyl sulfone, acrylate, acrylamide, or methacrylate end groups are selected to modify the nanoparticles. Here, A is a phospholipid, B is polyethylene glycol (PEG), selected from linear PEG, multi-arm PEG, dendritic PEG, and hyperbranched PEG, and D is selected from maleimide, vinyl sulfone, acrylate, acrylamide, and methacrylate. A can be embedded in a bilayer lipid structure to modify the nanoparticles with active end groups. For hydrophilic polymers modified with thiol groups, the thiol groups are mercapto or cysteine.

[0043] In this invention, the mass ratio of end-functionalized nanoparticles to side-chain functionalized hydrophilic polymers is 1:1 to 10:1. The inventors monitored the gelation of the mixture using a test tube inversion experiment. When the mass ratio of nanoparticles to hydrophilic polymers was below 1:1 or above 10:1, the mixture was in a flowing liquid state, indicating that hydrogel could not form at these ratios. Hydrogel formation was achieved when the mass ratio was between 1:1 and 10:1. Therefore, this invention selects a mass ratio of 1:1 to 10:1 for the end-functionalized nanoparticles and the side-chain functionalized hydrophilic polymers.

[0044] The nanoparticle-crosslinked hydrogel of this invention connects the nanoparticles and polymeric materials via a Michael addition reaction. This invention uses nanoparticles as the crosslinking agent, eliminating the need for any additional chemical initiators. The hydrogel, with nanoparticles as the crosslinking agent, is directly prepared by mixing end-functionalized nanoparticles with side-chain functionalized hydrophilic polymeric materials via a thiol-double-bond Michael addition reaction. Unlike uncontrolled systemic exposure, this hydrogel firmly immobilizes the nanoparticles, reducing the risk of systemic leakage and achieving a sustained immunosuppressive effect when implanted at predetermined target sites.

[0045] In this invention, the method for preparing the aforementioned end-functionalized nanoparticles is as follows: Immunosuppressants, phospholipids, and cholesterol are dissolved in an organic phase, and the solvent is evaporated by a nitrogen stream to form a uniform thin film at the bottom of a vial. Then, PBS is added to the vial for hydration and ultrasonic-assisted dispersion to obtain nanoparticles loaded with immunosuppressive drugs. Finally, the nanoparticles are co-incubated with materials having thiol or double bond groups at the ends, and washed with PBS through an ultrafiltration tube to obtain nanoparticles used as crosslinking agents.

[0046] In this invention, the preparation method of the above-mentioned side-chain functionalized hydrophilic polymer material is as follows: the hydrophilic polymer material is dissolved in an aqueous phase, an organic reagent with thiol or double bond groups is added to carry out the reaction, and the side-chain functionalized hydrophilic polymer material is obtained by acetone precipitation. The selected natural polymer has good hydrophilicity, biodegradability, and modifiability. It has high solubility in water, can be degraded by enzymes present in vivo, and contains hydroxyl, carboxyl, or amino groups in its structure, which can be used as a basic skeleton for hydrophobic modification. The selected thiol or double bond groups can be linked to the carboxyl or amino groups on the polymer material through amino, hydroxyl, or carboxyl groups.

[0047] The aforementioned hydrogel using nanoparticles as crosslinking agents can be used in allogeneic graft implantation devices. Allogeneic grafts are mixed with end-functionalized nanoparticles and side-chain functionalized modified hydrophilic polymers in phosphate-buffered saline (PBFS) to assemble a graft delivery hydrogel. The allogeneic grafts are selected from skin cells, epithelial cells, chondrocytes, cardiomyocytes, pancreatic islet cell clusters, induced pluripotent stem cells, skin, or blood vessels. This hydrogel can be prepared efficiently and selectively under mild conditions, thus avoiding functional impairment and decreased survival rates of the encapsulated grafts.

[0048] Specifically, in one embodiment of the present invention, nanoparticles loaded with immunosuppressants and hydrogels encapsulating allogeneic grafts form an allogeneic graft delivery system to address the technical problem of allogeneic graft damage and destruction related to immune rejection during transplantation therapy.

[0049] In this invention, immune rejection refers to the activation of Teff cells by the host's immune system after allogeneic graft transplantation by recognizing allogeneic antigens. The activated Teff cells infiltrate the transplantation area and attack the allogeneic graft, thereby causing damage and destruction of the graft. Treg cells enhance homeostasis and self-tolerance after allogeneic transplantation by inhibiting the activity of Teff cells and their subsequent effector functions.

[0050] Following allogeneic graft transplantation, the host's immune system triggers an immune response by recognizing the allogeneic antigen. Nanoparticles on the hydrogel release immunosuppressive drugs, promoting Treg cell generation, inhibiting Teff cell activity, and prolonging the survival of the allogeneic graft.

[0051] Following local implantation, the hydrogel prolongs the retention time of nanoparticles at the transplant site. The nanoparticles are uniformly and firmly fixed within the hydrogel network, minimizing the risk of systemic leakage and achieving a sustained immunomodulatory effect. The hydrogel also improves the long-term preservation of the graft, maintaining its function. After allogeneic graft transplantation, the host immune system triggers an immune response by recognizing the allogeneic antigen. Upon activation, the key immune executor, Teff cells, infiltrates the transplant site carrying the information to indiscriminately attack the allogeneic graft. The nanoparticles in the hydrogel act as carriers, continuously releasing immunosuppressive drugs. This process actively promotes the generation of Treg cells and effectively inhibits the activity of Teff cells, prolonging graft survival and maintaining its function.

[0052] Furthermore, this invention designs a process where mesenchymal stem cell membrane-derived vesicles (MMVs) are coated onto the surface of nanoparticles and modified with functionalized end groups. These are then cross-linked with functional side chains synthesized on a hydrophilic polymer to form a hydrogel. The nanoparticles release immunosuppressants to promote Treg cell generation, and the MMV-mediated Fas / FasL and PD1 / PD-L1 signaling pathways induce Teff cell death and promote Treg cell generation, respectively, inducing local rather than systemic tolerance. The fused MMV long-term expresses FasL and PD-L1 molecules, inducing Teff cell apoptosis and promoting Treg cell generation through the Fas / FasL and PD1 / PD-L1 signaling pathways. By combining nanoparticle-based immunosuppressants with MMV-based immunosuppressive proteins, a durable immune protection system is created, providing a new strategy for improving the long-term preservation and maintaining the function of grafts.

[0053] The above-mentioned method for preparing mesenchymal stem cell membrane-derived vesicles that present FasL and PD-L1 is as follows: IFN-γ stimulates the expression of PD-L1 on the MSC cell membrane, and then the cell membrane is obtained from the MSC using hypotonic and differential centrifugation methods. MMV is prepared from the MSC cell membrane by gentle sonication and repeated squeezing.

[0054] In a specific embodiment of the present invention, the above-mentioned method for preparing end-functionalized nanoparticles / MMV is as follows: nanoparticles are mixed with MMV, and MMV-coated nanoparticles NP / MMV are obtained by repeated physical extrusion. Finally, the NP / MMV is co-incubated with materials having thiol or double bond groups at the ends, and washed with PBS through an ultrafiltration tube to obtain NP / MMV as a crosslinking agent. The above steps are repeated to construct an immunomodulatory hydrogel, in which the MMV plays a crucial role in the long-term expression of FasL and PD-L1 molecules. By utilizing the two key signal transduction pathways of Fas / FasL and PD1 / PD-L1, MMV can induce apoptosis in Teff cells and further enhance the generation of Treg cells. This immunomodulatory hydrogel can improve the survival of grafts in allogeneic transplantation models without systemic immunosuppression.

[0055] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0057] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0058] Example 1 Preparation of nanoparticles In this embodiment, rapamycin (RAPA) was used as a model drug to prepare rapamycin-loaded nanoparticles (RAPA / NP).

[0059] Weigh 2 mg RAPA, 50 mg soybean lecithin and 5 mg cholesterol and dissolve them in 5 mL ethanol. Then evaporate the solvent by nitrogen gas flow for 4 h to form a uniform film at the bottom of the vial. Add 1 mL PBS to the vial for hydration and ultrasonic-assisted dispersion to obtain RAPA-loaded nanoparticles (RAPA / NP).

[0060] The particle size of RAPA / NP was determined using a particle size analyzer, and the morphology of RAPA / NP was observed using transmission electron microscopy. The results are as follows: Figure 1 As shown, the hydrated particle size of RAPA / NP is 140 nm and the shape is spherical.

[0061] The in vitro release of RAPA / NP was investigated using dialysis. 1 mL of RAPA / NP was placed in a dialysis bag, sealed, and then placed in 40 mL of release medium (PBS containing 20% ​​ethanol). The tube was then placed in a 37 °C constant-temperature shaker, and 0.5 mL samples were taken at predetermined time points, with 0.5 mL of release medium added as needed. Drug concentrations at different time points were recorded, and the cumulative drug release rate was calculated. The results are as follows: Figure 2 As shown, the cumulative release rate of RAPA / NP within 24 hours was close to 75%. In the first 6 hours, the cumulative release rate of RAPA was approximately 55%, and approximately 20% of the drug was released in the following 18 hours, demonstrating the slow release effect of the nanocarrier on RAPA. Example 2

[0062] Thiol-terminated functionalized RAPA / NP and double-bond modified polymer crosslinked hydrogels I. Preparation of RAPA / NP with thiol-terminated functionalization 100 μL of RAPA / NP prepared in Example 1 was mixed with 100 μL of distearylphosphatidylethanolamine-polyethylene glycol-thiol (DSPE-PEG-SH), disqualylphosphatidylcholine-polyethylene glycol-thiol (DEPC-PEG-SH), dioleoylphosphatidylethanolamine-polyethylene glycol-thiol (DOPE-PEG-SH), and dipalmitoylphosphatidylcholine-polyethylene glycol-thiol (DPPC-PEG-SH) at 37°C. o Incubate in a constant temperature shaker at C for 15 min, then wash twice with PBS through an ultrafiltration tube (10 K MWCO) to obtain thiol-modified RAPA / NP (SH-RAPA / NP).

[0063] 100 μL of the RAPA / NP prepared in Example 1 was mixed with 100 μL of distearylphosphatidylethanolamine-polyethylene glycol-cysteine ​​(DSPE-PEG-Cys), bisorhamnoylphosphatidylcholine-polyethylene glycol-cysteine ​​(DEPC-PEG-Cys), dioleoylphosphatidylethanolamine-polyethylene glycol-cysteine ​​(DOPE-PEG-Cys), and dipalmitoylphosphatidylcholine-polyethylene glycol-cysteine ​​(DPPC-PEG-Cys) at 37°C. o Incubate in a constant temperature shaker at C for 15 min, then wash twice with PBS through an ultrafiltration tube (10 K MWCO) to obtain cysteine-modified RAPA / NP (Cys-RAPA / NP).

[0064] 2. Polymer materials modified with double bond groups 2 g of sodium hyaluronate (HA, 90 kDA) was dissolved in 100 mL of deionized water. 1.6 mL of acrylic anhydride, 4-penten-1-amine, N-(2-aminopropyl)maleimide, and methacrylic anhydride were added and stirred until homogeneous. NaOH (5 M) was added to adjust the pH of the solution to between 8 and 9. After sealing and stirring at 4 °C for 24 h, the resulting solution was precipitated with 200 mL of acetone to obtain a cotton-like flocculent precipitate. The precipitate was washed twice with 100 mL of ethanol and then dissolved in 50 mL of deionized water. The solution was stirred overnight until clear. The solution was placed in a dialysis bag (14 kDA) and dialyzed with deionized water for 48 h. After dialysis, the liquid in the dialysis bag was collected and freeze-dried to obtain purified acrylate-modified HA (a-HA), pentenamide-modified HA (Am-HA), maleimide-modified HA (Mal-HA), and methacrylate-modified HA (Ma-HA).

[0065] 2 g of chitosan (CS, 60 kDA), heparin (Hep, 15 kDA) and sodium alginate (SA, 20 kDA) were dissolved in 100 mL of deionized water. The above steps were repeated to obtain chitosan, heparin and sodium alginate modified with double bond groups.

[0066] 3. Preparation and characterization of cross-linked hydrogels of polymer materials with thiol-terminated functionalized RAPA / NP and double bond modified groups. 100 μL of DSPE-PEG-SH modified RAPA / NP (SH-RAPA / NP), at a concentration of 30 mg / mL and pH 7.4, were added to 100 μL of a-HA, Am-HA, Mal-HA, Ma-HA, a-CS, Am-CS, Mal-CS, Ma-CS, a-Hep, Am-Hep, Mal-Hep, Ma-Hep, a-SA, Am-SA, Mal-SA, and Ma-SA solutions, respectively. After incubation at room temperature for 1 h, the gelation of the mixed solutions was determined using the inverted vial method. Hydrogel formation was considered complete if the solution did not drip from the inverted vial. The gelation results for SH-RAPA / NP and double-bond modified polymers are shown in Table 1.

[0067] Table 1. Gel formation of polymers modified with SH-RAPA / NP and double bond groups.

[0068] 100 μL of RAPA / NP modified with DEPC-PEG-SH, DOPE-PEG-SH, and DPPC-PEG-SH, and 100 mg / mL of a-HA, Am-HA, Mal-HA, Ma-HA, a-CS, Am-CS, Mal-CS, and Ma-CS, respectively, were added to RAPA / NP modified with DEPC-PEG-SH, DOPE-PEG-SH, and DPPC-PEG-SH at pH 7.4. After incubation at room temperature for 1 h, the gelation of the mixed solutions was determined using the inverted vial method. Hydrogel formation was considered complete if the solution did not drip from the inverted vial. The gelation results of RAPA / NP modified with DEPC-PEG-SH, DOPE-PEG-SH, and DPPC-PEG-SH, and the polymer materials modified with double bonds are shown in Table 2.

[0069] Table 2. Gel formation of polymers modified with SH-RAPA / NP and double bond groups.

[0070] 100 μL of DSPE-PEG-Cys modified RAPA / NP (Cys-RAPA / NP), at a concentration of 30 mg / mL and pH 7.4, were added to 100 μL of a-HA, Am-HA, Mal-HA, Ma-HA, a-CS, Am-CS, Mal-CS, Ma-CS, a-Hep, Am-Hep, Mal-Hep, Ma-Hep, a-SA, Am-SA, Mal-SA, and Ma-SA solutions, respectively. After incubation at room temperature for 1 h, the gelation of the mixed solutions was determined using the inverted vial method. Hydrogel formation was considered complete if the solution did not drip from the inverted vial. The gelation results for Cys-RAPA / NP and double-bond modified polymers are shown in Table 3.

[0071] Table 3. Gel formation of polymers modified with Cys-RAPA / NP and double bond groups.

[0072] The results in Tables 1, 2, and 3 show that, under pH 7.4 conditions, SH-RAPA / NP and Cys-RAPA / NP can selectively react with double-bond modified polymers (such as a-HA, Am-HA, Mal-HA, Ma-HA, a-CS, Am-CS, Mal-CS, and Ma-CS) to initiate gelation via Michael addition reaction, forming hydrogel RAPA-Gel. The results of DSPE-PEG-SH modified RAPA / NP forming hydrogel RAPA-Gel with a-HA are shown below. Figure 3 As shown.

[0073] The rheological measurements of the hydrogel (RAPA-Gel) formed by DSPE-PEG-SH modified RAPA / NP and α-HA are as follows: Figure 4 As shown, after the addition of RAPA / NP, both the G′ value (storage modulus) and the G″ value (loss modulus) increase over time. When the G′ value reaches a high point of 400 Pa, which is much higher than the G″ value, this is conclusive evidence of hydrogel formation.

[0074] The microstructure of the hydrogel (RAPA-Gel) formed by DSPE-PEG-SH modified RAPA / NP and α-HA is as follows: Figure 5 As shown, RAPA-Gel is supported by a cross-linked network in which many RAPA / NPs are visible.

[0075] 50 mg of soybean lecithin and 5 mg of cholesterol were dissolved in 5 mL of dichloromethane. The solvent was evaporated by a nitrogen stream for 4 h, forming a uniform thin film at the bottom of the vial. 1 mL of PBS was added to the vial for hydration and ultrasonic-assisted dispersion to obtain blank nanoparticles. The blank nanoparticles were incubated with DSPE-PEG-SH in a 37 ℃ constant temperature shaker for 15 min using the post-insertion method to obtain SH-modified liposomes. 100 μL of HA solution modified with 30 mg / mL, pH 7.4 and CS solution modified with acrylate groups were mixed with 100 μL of HA solution modified with acrylate groups and CS solution modified with acrylate groups at pH 7.4 to prepare control gels (L-HA-Gel and L-CS-Gel).

[0076] T cells isolated from spleen cells were treated with concanavalin A (5 μg / mL) for 24 h, and then seeded on the surfaces of HA cross-linked hydrogels (RAPA-HA-Gel), L-HA-Gel, CS cross-linked hydrogels (RAPA-CS-Gel), and L-CS-Gel, all of which were functionalized by DSPE-PEG-SH and modified with acrylate groups. The cells were then further incubated at 37°C. o C. Incubate in a 5% CO2 incubator for 48 h. Anti-PD1-FITC staining was performed on activated T cells infiltrating the hydrogel to detect PD1 expression. 2 μL of anti-PD1-FITC was added to the hydrogel and incubated for 30 min, followed by quantitative fluorescence analysis using confocal microscopy. Results are as follows: Figure 6As shown, the fluorescence signal of PD1 on the surface of T cells in RAPA-HA-Gel was significantly lower than that in L-HA-Gel, and the fluorescence signal of PD1 on the surface of T cells in RAPA-CS-Gel was also significantly lower than that in L-CS-Gel. This indicates that the RAPA released by RAPA-HA-Gel and RAPA-CS-Gel inhibits the expression of PD1 on activated T cells, which lays a solid foundation for inducing the formation of Treg cells.

[0077] 4. In vitro efficacy of RAPA cross-linked hydrogel against allogeneic islet rejection Islets were isolated from C57BL / 6 mice. At pH 7.4, 500 islet-equivalent units were mixed with 100 μL of 30 mg / mL DSPE-PEG-SH modified RAPA / NP (SH-RAPA / NP) and 100 μL of 30 mg / mL α-HA solution to prepare a hydrogel reservoir for islet delivery (islet / RAPA-Gel). Morphological characterization was performed using cryo-scanning electron microscopy. Figure 7 As shown, pancreatic islet cell clusters were successfully encapsulated in a network of interwoven hydrogels.

[0078] The effect of RAPA-Gel on pancreatic islet function was investigated using glucose-stimulated insulin release and stimulation index. Free islets and islet / RAPA-Gel mixtures were incubated in Krebs buffer at 37°C. o C. Equilibrate in a 5% CO2 environment for 1 h; then, expose to a low glucose (3 mM) solution for 1 h and collect the supernatant; subsequently, expose to a high glucose (11 mM) solution for 1 h, collect the supernatant, and use a mouse insulin ELISA kit to determine the insulin content released from the islet cell clusters in the supernatant. Figure 8 As shown, islets cultured in RAPA-Gel can respond to changes in environmental glucose levels and secrete more insulin under high glucose conditions than under low glucose conditions, thus maintaining insulin secretion function. The calculated insulin stimulation index indicates no statistically significant difference between islets / RAPA-Gel and free islets, confirming the biocompatibility of the hydrogel for islet function.

[0079] T cells isolated from BALB / C mouse spleen cells were activated using Con A and then co-incubated with islets / RAPA-Gel. At the in vitro model level, the survival rate of RAPA-Gel against allogeneic islets attacked by activated T cells was assessed using live / dead cell staining, glucose-stimulated insulin release, and stimulation index. Islet cell viability was detected using calcein AM / PI double staining. 5 μL of prepared calcein AM / PI staining solution was added and incubated for 30 min. Live / dead cell fluorescence intensity was quantitatively analyzed using confocal microscopy. The glucose-stimulated insulin release and stimulation index experiments were performed using the same methods. Figure 9 As shown, RAPA-Gel has a lower percentage of dead pancreatic islet cells. Figure 10 As shown, islets cultured in RAPA-Gel can respond to changes in environmental glucose levels, and the stimulation index of islets / RAPA-Gel is significantly higher than that of islets / L-Gel. These results confirm that under the protection of RAPA-Gel, islets can resist T-cell-mediated immune attacks and retain glucose-responsive insulin secretion function.

[0080] Example 3 Crosslinked hydrogels of polymer materials with double bond end-functionalized RAPA / NP and thiol group modification I. Preparation of RAPA / NP with double bond end groups functionalized 100 μL of RAPA / NP obtained in Example 1 was mixed with 100 μL of DSPE-PEG-Mal, DEPC-PEG-Mal, DOPE-PEG-Mal, and DPPC-PEG-Mal respectively at 37°C. o Incubate in a constant temperature shaker at C for 15 min, then wash twice with PBS through an ultrafiltration tube (10 K MWCO) to obtain maleimide-modified RAPA / NP (Mal-RAPA / NP).

[0081] Repeat the above steps, mixing 100 μL of the RAPA / NP obtained in Example 1 with 100 μL of phospholipid-polyethylene glycol-vinyl sulfone, phospholipid-polyethylene glycol-acrylate, phospholipid-polyethylene glycol-acrylamide, and phospholipid-polyethylene glycol-methacrylate at 37°C. o Incubate in a constant temperature shaker at C for 15 min, then wash twice with PBS through an ultrafiltration tube (10 K MWCO) to obtain vinyl sulfone-modified RAPA / NP (Vs-RAPA / NP), acrylate-modified RAPA / NP (Ac-RAPA / NP), acrylamide-modified RAPA / NP (Am-RAPA / NP), and methacrylate-modified RAPA / NP (Ma-RAPA / NP).

[0082] II. Thiol-modified polymers 2 g of sodium hyaluronate (HA, 90 kDA) was dissolved in 100 mL of deionized water, and then hydrochloric acid was added to adjust the pH of the solution to be between 5 and 6. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 4 g) and N-hydroxysuccinimide (NHS, 2 g) were added to the solution and stirred for 30 min. Then, 2 g of cysteine, thioglycolic acid, and thiopropanol were added, respectively. The reaction was carried out under stirring for 6 h and stored in the dark under nitrogen atmosphere. The solution was placed in a dialysis bag (14 kDA) and dialyzed with deionized water for 48 h. After dialysis, the liquid in the dialysis bag was collected and freeze-dried to obtain purified cysteine-modified HA (Cys-HA), thioglycolic acid-modified HA (TgSH-HA), and thiopropanol-modified HA (TpSH-HA).

[0083] 2 g of chitosan (CS, 60 kDA), heparin (Hep, 15 kDA) and sodium alginate (SA, 20 kDA) were dissolved in 100 mL of deionized water. The above steps were repeated to obtain thiol-modified chitosan, heparin and sodium alginate.

[0084] III. Preparation and Characterization of Crosslinked Hydrogels of Polymer Materials with Double Bond End-Group Functionalized RAPA / NP and Thiol Group Modification 100 μL of each of the following hydrogel types (Cys-HA, TgSH-HA, TpSH-HA, Cys-CS, TgSH-CS, TpSH-CS, Cys-Hep, TgSH-Hep, TpSH-Hep, Cys-SA, TgSH-SA, and TpSH-SA) were added to 100 μL of DSPE-PEG-Mal modified RAPA / NP (Mal-RAPA / NP) at 30 mg / mL and pH 7.4. After incubation at room temperature for 1 h, the gelation of the mixed solutions was determined using the inverted vial method. Hydrogel formation was considered complete if the solution did not drip from the inverted vial. The gelation results for Mal-RAPA / NP and thiol-modified polymers are shown in Table 4.

[0085] Table 4. Gel formation of polymers modified with Mal-RAPA / NP and thiol groups.

[0086] As shown in Table 4, under pH 7.4 conditions, Mal-RAPA / NP can selectively react with thiol-modified polymers (such as Cys-HA, TgSH-HA, TpSH-HA, Cys-CS, TgSH-CS, and TpSH-CS) to initiate gelation through Michael addition reaction to form hydrogel RAPA-Gel.

[0087] 40 mg of soybean lecithin and 5 mg of cholesterol were dissolved in 5 mL of dichloromethane, and the solvent was evaporated by a nitrogen stream for 4 h to form a uniform film at the bottom of the vial. 1 mL of PBS was added to the vial for hydration and ultrasonic-assisted dispersion to obtain blank nanoparticles. The blank nanoparticles were incubated with DSPE-PEG-Mal in a 37 ℃ constant temperature shaker for 15 min using the post-insertion method to obtain Mal-modified liposomes. 100 μL of Mal-modified liposomes (30 mg / mL, pH 7.4) was mixed with 100 μL of cysteine-modified HA solution (30 mg / mL, pH 7.4) and cysteine-modified CS solution (30 mg / mL, pH 7.4) to prepare control gels (L-HA-Gel and L-CS-Gel).

[0088] T cells isolated from spleen cells were treated with concanavalin A (5 μg / mL) for 24 h, and then seeded on the surfaces of HA cross-linked hydrogels (RAPA-HA-Gel), L-HA-Gel, CS cross-linked hydrogels (RAPA-CS-Gel), and L-CS-Gel, all of which were functionalized with DSPE-PEG-Mal and modified with RAPA / NP and cysteine ​​groups. The cells were then further incubated at 37°C. o C. Incubate in a 5% CO2 incubator for 48 h. Anti-PD1-FITC staining was performed on activated T cells infiltrating the hydrogel to detect PD1 expression. 2 μL of anti-PD1-FITC was added to the hydrogel and incubated for 30 min, followed by quantitative fluorescence analysis using confocal microscopy. Results are as follows: Figure 11 As shown, the fluorescence signal of PD1 on the surface of T cells in RAPA-HA-Gel was significantly lower than that in L-HA-Gel, and the fluorescence signal of PD1 on the surface of T cells in RAPA-CS-Gel was also significantly lower than that in L-CS-Gel. This indicates that the RAPA released by RAPA-HA-Gel and RAPA-CS-Gel inhibits the expression of PD1 on activated T cells, which lays a solid foundation for inducing the formation of Treg cells.

[0089] Example 4 In vivo pharmacodynamics of immunomodulatory hydrogels I. Local preservation ability of RAPA / NP cross-linked hydrogel after in vivo implantation DiD-RAPA / NP was obtained by fluorescently labeling DSPE-PEG-SH modified RAPA / NP (SH-RAPA / NP) with the cell membrane fluorescent dye DiD. 100 μL of SH-modified DiD-RAPA / NP solution (30 mg / mL, pH 7.4) was mixed with 100 μL of α-HA solution (30 mg / mL, pH 7.4) and stabilized at room temperature for 1 h to obtain DiD-labeled RAPA-Gel (DiD-RAPA-Gel). 100 μL of SH-modified DiD-RAPA / NP solution (30 mg / mL, pH 7.4) was mixed with 100 μL of HA solution (30 mg / mL, pH 7.4) and stabilized at room temperature for 1 h to obtain a mixture of DiD-labeled RAPA / NP and HA (DiD-RAPA / NP / HA). DiD-RAPA / NP, DiD-RAPA / NP / HA, and DiD-RAPA-Gel were implanted under the renal sac of BALB / C mice. In vivo imaging was used to detect changes in DiD fluorescence signal and evaluate the local retention level of RAPA-Gel. Results are as follows: Figure 12 As shown, the DiD fluorescence signal in the DiD-RAPA / NP and DiD-RAPA / NP / HA groups almost completely disappeared after 14 days. In stark contrast, the DiD fluorescence signal persisted until 28 days in the kidneys of mice with locally implanted DiD-RAPA-Gel. 28 days post-implantation, the kidneys and other normal major tissues of the mice were removed for in vitro DiD fluorescence imaging. Strong DiD signals were observed in the kidneys of mice in the DiD-RAPA-Gel group, while no signals were detected in other tissues. No DiD fluorescence signals were found in mice treated with DiD-RAPA / NP and DiD-RAPA / NP / HA, either at the transplant site or in major organs. Therefore, the cross-linked hydrogel prolonged the retention time of RAPA / NP in the transplantation area while minimizing the risk of its systemic effects.

[0090] II. Immunosuppressive Effect of RAPA / NP Crosslinked Hydrogel in Allogeneic Islet Transplantation Model A mouse allogeneic islet transplantation model was established by intraperitoneal injection of streptozotocin (220 mg / kg) into BALB / C mice. Islets from C57BL / 6 donor mice were transplanted into the renal capsule of diabetic BALB / C recipient mice. The immunosuppressive potential of immunoprotective hydrogels in this model was investigated, with three groups tested: islet group, islet / L-Gel group, and islet / RAPA-Gel group. The islet / L-Gel group was prepared by mixing 500 islet equivalents of islets with 100 μL of 30 mg / mL DSPE-PEG-SH modified liposomes and 100 μL of 30 mg / mL α-HA solution at pH 7.4. Islets equivalent to 500 islets were mixed with 100 μL of 30 mg / mL DSPE-PEG-SH modified RAPA / NP and 100 μL of 30 mg / mL α-HA solution at pH 7.4 to prepare the islet / RAPA-Gel group. Grafts and draining lymph nodes were collected 7 days after allogeneic islet transplantation, and flow cytometry was used to analyze the immune cell population and the proportion of induced Treg cells. Results are as follows... Figure 13 As shown, RAPA-Gel significantly increased the proportion of Treg cells in the kidney and draining lymph nodes, thereby reducing the accumulation of Teff cells in the kidney and draining lymph nodes. Results are as follows... Figure 14 As shown, mice treated with islet / RAPA-Gel had a significantly higher proportion of induced Treg cells in the transplanted region.

[0091] III. In vivo efficacy of RAPA / NP cross-linked hydrogel against allogeneic islet rejection A mouse allogeneic islet transplantation model was established by intraperitoneal injection of streptozotocin (220 mg / kg) into BALB / c mice. Mice with two consecutive blood glucose levels exceeding 350 mg / dL were identified as diabetic mice. Islets, islets / L-Gel, and islets / RAPA-Gel obtained from C57BL / 6 mice were transplanted subrenally into diabetic BALB / c mice using the same method, with a single dose of 500 islet equivalents / mouse. Islet allogeneic transplantation rejection was defined as two consecutive blood glucose levels exceeding 200 mg / dL. Results are as follows: Figure 15 As shown, all grafts in mice receiving islet allogeneic transplants exhibited acute rejection during the two-week observation period. L-Gel did not alleviate the immune rejection of allogeneic islets. In stark contrast, graft survival was significantly prolonged in mice receiving islet / RAPA-Gel transplants.

[0092] Pancreatic islet function in successfully transplanted mice was characterized using a glucose tolerance test. Mice were fasted for 12 hours prior to the experiment, but allowed free access to water. Mice were weighed and their weight recorded. Diabetic mice treated with islet / RAPA-Gel, normal mice, and untreated diabetic mice were administered a glucose solution (2 g / kg) intraperitoneally. Blood glucose levels were measured at predetermined time points using a portable glucometer. Normal and untreated diabetic mice served as controls. Results are as follows: Figure 16 As shown, mice transplanted with pancreatic islets / RAPA-Gel exhibited a similar glucose clearance pattern to normal mice, and the area under the curve calculation results showed no significant difference.

[0093] Example 5 Biomimetic nanovesicle cross-linked immunomodulatory hydrogel in vivo drug efficacy I. Preparation of biomimetic nanovesicle cross-linked immunomodulatory hydrogels Healthy female BALB / c mice (20 g) were euthanized, and the bilateral femurs were dissected and separated, removing attached muscles and periosteum. Pre-chilled PBS solution (approximately 4 °C) was aspirated into a syringe and used to flush the bone marrow cavity three times until the bone shaft turned white. Bone marrow cell suspension was collected. The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 300 × g for 5 min. The supernatant was discarded, and the bottom cell clusters were resuspended in 5 mL of MEM α complete culture medium. The cell suspension was seeded into 25 cm... 2 Cells were cultured in culture flasks at 37 °C in a 5% CO2 incubator. After 5 days, unattached cells were removed. Fresh MEM α complete medium was added every 3 days thereafter. When cells reached 90% or higher confluence in the culture flasks, they were digested and passaged. Cells in the logarithmic growth phase of passage 3 were collected, the old medium was removed, and the cells were washed three times with PBS. 1 mL of trypsin was added to digest the cells for 5 min, and 2 mL of fresh MEM α complete medium was added to stop the digestion. The cells were centrifuged at 300 × g for 5 min, and the supernatant was discarded to obtain MSCs.

[0094] Extraction of Red Blood Cells (RBCs) and Platelets (PLTs). Blood was collected from the orbital sinus of healthy BALB / c mice, 10 drops from each mouse, for a total of 10 mL of heparinized blood. The blood was centrifuged at 100 × g for 20 min, and the supernatant rich in PLTs was collected. The lower layer of RBC particles was washed three times with pre-chilled PBS (approximately 4 °C) for further experiments. 4 mL of the supernatant plasma was added to 4 mL of Tris-HCl buffer (pH 6.5) containing disodium EDTA (5 mM) and prostaglandin E1 (1 μM). The mixture was centrifuged at 800 × g for 20 min to obtain stationary PLTs. The stationary PLTs were washed twice with pre-chilled PBS and incubated with 2 mL of thrombin (10 μM) at room temperature for 10 min. The activation process was terminated by adding 2 mL of paraformaldehyde (2%), and the platelets were washed twice with PBS to obtain activated platelets (aPLTs). The expression of FasL in MSCs, RBCs, PLTs, and aPLTs was determined by flow cytometry. Results are as follows: Figure 17 As shown, FasL expression was low on RBCs and PLTs, and thrombin-induced activation increased FasL expression on PLTs, while FasL expression was significantly higher on the surface of MSCs.

[0095] MSCs were inoculated at a depth of 25 cm. 2 Placed in a culture flask at 37°C o Cells were cultured in a 5% CO2 incubator for 24 h. Cells were then incubated with exogenous IFN-γ at a final concentration (1 ng / mL) for 12 h to stimulate upregulation of PD-L1 on the surface of MSCs. PD-L1 expression on MSCs was analyzed by flow cytometry, and the results are as follows: Figure 18 As shown, PD-L1 expression was significantly increased in MSCs treated with IFN-γ compared to untreated MSCs.

[0096] Take 175 cm 2MSCs pretreated with IFN-γ were placed in culture flasks, the old culture medium was removed, and the cells were washed three times with PBS. 5 mL of trypsin was added to digest the cells for 5 min, and 10 mL of fresh MEM α complete culture medium was added to terminate the digestion. The cells were centrifuged at 300 × g for 5 min, the supernatant was discarded, and the MSC pellet was collected after washing three times with PBS and placed in 15 mL sample tubes. The MSCs were placed in 5 mL of Tris buffer (30 mM) containing mannitol (225 mM), sucrose (75 mM), bovine serum albumin (0.5%), EDTA (0.5 mM), and a protease inhibitor (1×) and lysed overnight at 4 °C. The cell suspension was sonicated at 20% intensity for 3 min using a cell disruptor, centrifuged at 20,000 × g for 20 min, and the supernatant was collected. The resulting supernatant was centrifuged at 120,000 × g for 50 min at 4 °C to obtain cell membrane particles. The cell membrane precipitate was resuspended to 1 mL, and the cell suspension was treated with sonication at 10% intensity for 3 min using a cell disruptor. The MMV was obtained by squeezing the filter through a 100 nm polycarbonate membrane filter 21 times.

[0097] The RAPA / NP solution and MMV solution were mixed evenly at a mass ratio of 1:5. The mixture was then extruded 21 times through a 200 nm polycarbonate membrane filter using an extruder to obtain MMV-coated NPs, i.e., biomimetic nanovesicles (RAPA / NP / MMV).

[0098] Add 20 μL of RAPA / NP / MMV solution to each well of a 96-well plate, and add 200 μL of BCA working solution to each well. Incubate at 37 °C with a shaker for 30 min, and measure the absorbance at 562 nm using a microplate reader. The protein concentration of MMV was quantified to 150 mg / mL using a BCA protein assay kit.

[0099] RAPA / NP / MMV and DSPE-PEG-SH were incubated in a constant temperature shaker at 37 ℃ for 15 min using the post-insertion method to obtain SH-modified RAPA / NP / MMV. An immunomodulatory hydrogel (RAPA / MMV-Gel) was prepared by mixing 100 μL of SH-modified RAPA / NP / MMV with 100 mg / mL of 30 mg / mL protein at pH 7.4 and 100 μL of α-HA solution with 30 mg / mL protein at pH 7.4.

[0100] II. In vivo efficacy of biomimetic nanovesicle cross-linked immunomodulatory hydrogels against allogeneic islet rejection The steps in Example 4 were repeated to establish a BALB / c diabetes model, and islets, islet / RAPA-Gel, and islet / RAPA / MMV-Gel from C57BL / 6 mice were transplanted at a single dose of 500 islet equivalents per mouse. Islets were mixed with 100 μL of DSPE-PEG-SH modified RAPA / NP and 100 μL of α-HA solution at pH 7.4 to prepare islet / RAPA-Gel. Islets were mixed with 100 μL of DSPE-PEG-SH modified RAPA / NP / MMV and 100 μL of α-HA solution at pH 7.4 to prepare islet / RAPA / MMV-Gel. Islets were mixed with 100 μL of DSPE-PEG-SH modified RAPA / NP / MMV and 100 mg / mL of α-HA solution at pH 7.4 to prepare islet / RAPA / MMV-Gel. Islet allogeneic transplant rejection was defined as two consecutive blood glucose levels exceeding 200 mg / dL. Results are as follows. Figure 19 As shown, all grafts from mice receiving islet allogeneic transplants exhibited acute rejection during the 30-day observation period. In stark contrast, 60% of grafts from mice receiving islet / RAPA-Gel transplants survived for 30 days. Furthermore, in mice treated with islet / RAPA / MMV-Gel, 80% of grafts survived for up to 30 days. This suggests that the interaction between the immunosuppressant-loaded NP and MMV enhances the immunosuppressive effect and prolongs the survival of the allogeneic graft.

Claims

1. Nanoparticle crosslinked hydrogel, characterized in that, The nanogel is made of end-group functionalized nanoparticles and side-chain functionalized modified hydrophilic polymer materials, wherein the mass ratio of the end-group functionalized nanoparticles and the side-chain functionalized modified hydrophilic polymer materials is 1:1-10:

1. The end-group functionalized nanoparticles and the side-chain functionalized modified hydrophilic polymer materials are selected from one of the following combinations: (i) the end-group functionalized nanoparticles are thiol end-group functionalized nanoparticles, and the side-chain functionalized modified hydrophilic polymer materials are unsaturated double bond group modified hydrophilic polymer materials; or (ii) the end-group functionalized nanoparticles are unsaturated double bond end-group functionalized nanoparticles, and the side-chain functionalized modified hydrophilic polymer materials are thiol group modified hydrophilic polymer materials.

2. The nanoparticle crosslinked hydrogel of claim 1, wherein, The nanoparticles in the end-group functionalized nanoparticles are selected from liposome nanoparticles, polymer nanoparticles, and cell-derived nanoparticles.

3. The nanoparticle crosslinked hydrogel of claim 1, wherein, The hydrophilic polymer materials in the side-chain functionalized modified hydrophilic polymer materials are selected from hyaluronic acid, chitosan, heparin, cellulose, alginic acid, dextran, amylose, amylopectin, and pectin.

4. The nanoparticle crosslinked hydrogel of claim 1, wherein, In combination (i), the thiol end group is a mercapto group or cysteine, and the unsaturated double bond group is an acrylate, pentenamide, maleimide, or methacrylate.

5. The nanoparticle crosslinked hydrogel of claim 1, wherein, In combination (ii), the unsaturated double bond end group is a maleimide, vinyl sulfone, acrylate, acrylamide, or methacrylate, and the thiol group is a mercapto group or cysteine.

6. Use of the nanogel of any one of claims 1-5 in the preparation of an allogeneic transplantation product.

7. An allogeneic transplant product, characterized in that, The nanogel of any one of claims 1-5 and an allogeneic transplant, wherein the end-group functionalized nanoparticles carry an immunosuppressant.

8. The allograft product of claim 7, wherein, The immunosuppressant is a small molecule immunosuppressant selected from tacrolimus, cyclosporin A, rapamycin, cyclophosphamide, azathioprine, methotrexate, prednisone, or dexamethasone.

9. The allograft product of claim 7, wherein, The allogeneic transplant is selected from skin cells, epithelial cells, chondrocytes, cardiomyocytes, islet cell clusters, induced pluripotent stem cells, skin, or blood vessels.

10. An allogeneic transplant product, characterized in that, The nanogel is made of nanogels coated with mesenchymal stem cell membrane-derived vesicles, hydrophilic polymer materials, and allogeneic transplants, wherein the nanogels coated with mesenchymal stem cell membrane-derived vesicles and the hydrophilic polymer materials are connected through functionalized end groups on the surface of the mesenchymal stem cell membrane-derived vesicles and functionalized side chains on the hydrophilic polymer materials.

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