Construction Of Injectable Multifunctional Hydrogels And Their Effects And Mechanisms In Cancer Immunotherapy

As an emerging tumor treatment,cancer immunotherapy has attracted much attention in the last decade.Compared with traditional chemotherapy,radiotherapy,and surgical treatment,immunotherapy has many advantages: high safety,minor damage to normal tissues,and immune memory to cope with tumor metastasis and recurrence.Represented by immune checkpoint inhibitors and tumor vaccines,various immunotherapeutic strategies bring hope to relapsed and metastatic patients.However,in practical applications,these approaches often suffer from an insufficient response and poor prognostic effects,which is mainly caused by the following aspects:(1)Effector T cells(Teffs)often do not receive enough tumor-related antigens(TAAs)information,resulting in their limited activation and unable to perform the killing function effectively;(2)Most cancers are "cold" tumors with poor lymphocyte infiltration,low tumor mutation antigen and resistance to immune checkpoint inhibitors(ICBs),in which lots of immunosuppressive factors,such as regulatory T cells(Treg),bone marrow-derived suppressive cells(MDSC),transforming growth factor β(TGF-β),will exhaust Teffs,causing failure of the anti-tumor immune response.Therefore,enhancing the killing function of effector T cells while reducing the inhibitory factors in tumors is expected to improve the antitumor immunotherapy effect.With the development of material science and biomedical technology,cancer immunotherapy meets new opportunities.As a biological material with excellent performance,hydrogels have made significant progress in drug delivery,biosensing,medical imaging,tissue engineering,and other aspects.Harnessing hydrogels for cancer immunotherapy are also one of the emerging therapeutic strategies in recent years.As delivery vehicles,hydrogels have the following advantages:(1)As a carrier,hydrogels can load not only small molecular drugs but also a variety of immunomodulators,such as antibodies,tumor antigens,cytokines,and adjuvants,which could work together to enhance the immune response rate;(2)Hydrogels based on natural polysaccharides or natural polyamino acids always have excellent biosafety,which can be gradually degraded in the body and have minor side effects on normal tissues and organs;(3)Hydrogel systems protect active immunomodulators from degradation in vivo,such as antibodies,cytokines,etc.and control the sustained release rate of active substances and consequently enhance the immune stimulation of the vaccine.(4)Some biomaterials themselves have an adjuvant effect.Therefore,through reasonable design,hydrogels with adjuvant effect can be formed to amplify the anti-tumor immune effect.Besides,hydrogels also have many advantages such as flexible design,controllable response performance,and controllable release rate,providing new opportunities for solving the problems mentioned above in tumor immunotherapy.To this end,this thesis takes the advantages of hydrogels to solve the two common problems in cancer immunotherapy.Firstly,to solve the problem that T cells cannot effectively perform the adaptive immune response due to the limited antigen presentation,a spontaneous injectable hydrogel(NOCC-Cp G/OX-M,Ncom Gel)based on natural polysaccharides for cancer immunotherapy is reported with multiple stimuli capability to amplify innate immune response and harness innate immunity to launch and maintain robust adaptive immune response.Specifically,Cp G is first modified in N,O-carboxymethyl chitosan(NOCC)to form one of the gel skeletons.Cp G is an agonist of Toll-like receptor 9(TLR9),promoting dendritic cells(DC cells)activation.OX-M is partially oxidized mannan,which can be bound to the Mannose receptor(MR)on the surface of DC cells and macrophages to promote the maturation of DC cells and the activation and polarization of macrophages.Therefore,the hydrogel formed by the cross-linking of NOCC-Cp G and OX-M has multiple stimulating effects.The Ncom gel vaccine is developed by thoroughly mixing with Ncom gel and the model antigen-OVA.After two-point immunization subcutaneous injection into the inguinal area of mice,through the synergistic effect of Cp G and mannose in the Ncom gel,the primary APCs such as DC cells and macrophages are first efficiently activated.By uptake and processing antigen,mature APCs then present the antigen to T cells and activate effector T cells to reach the tumor site.Effector T cells then directly kill tumors by secreting IFN-γ,granzyme B,and other cytokines to perform adaptive immune responses.Meanwhile,APCs maturation favors the formation of antigen-specific humoral immunity,which could direct phagocytosis and kill tumor-associated antigens through ADCC,ADCP.Finally,the multiple effects caused by Ncom gel increase the level of anti-tumor immune cells,such as CD8+ T cells,DCs,M1 type macrophages,while decreasing the pro-tumor immune cells,such as Treg cells,M2 type macrophages,and MDSCs,which finally enhance the anti-tumor killing ability.After the Ncom gel vaccine is used in the mouse melanoma model,it has a significant inhibitory effect on the tumor.Blood biochemical tests and the organ pathological H&E staining of the treated mice are then analyzed.It proves that the Ncom gel vaccine has no noticeable side effects.These results demonstrate that innate immunity was amplified after Ncom Gel vaccine administration.The effector response of adaptive immunity was further enhanced,which reduces the dependence of T cells mediated cytotoxicity,in part.Our work confirms that it could be more effective to design a vaccine with a multiple stimuli capability.The strategy opens a new sight for multilayered cancer immunotherapy.What’s more,our research also implies the potential application of injectable hydrogel as a platform for tumor vaccine.Next,aiming to solve the problem of the inadequate response of "cold" tumor immunotherapy,based on the previous research of the hydrogel system,an engineered gel system R848&a-OX40@Gel with ROS response is designed for "cold" tumor immunotherapy,which can inhibit the immunosuppressive factors in the tumor microenvironment to clear "the roadblocks" for effector T cells and directly promote the proliferation and activation of effector T cells by providing stimulus signals to "accelerate" effector T cells.Resiquimod(R848)is a dual agonist for Toll-like receptors 7 and 8(TLR7/8),promoting the activation and maturation of APCs,thereby enabling the presentation of TAAs and enhancing the anti-tumor immune response.What’s more,R848 can regulate immunosuppressive cells.By transforming MDSCs into APC and polarizing tumor-associated macrophage(TAMs)from the inflammatory M2 phenotype into pro-inflammatory M1 phenotype,R848 reduces these inhibitory effects cells,which can be applied for clearing "roadblocks" for Teffs.OX40 belongs to the tumor necrosis factor(TNF)superfamily,augments T-cell expansion and cytokine secretion.Specifically,OX40 signaling activation could elicit Teffs activation and inhibit Tregs function,leading to an anti-tumor immune response.Notably,OX40 agonist treatment,such as anti-OX40(a OX40),could bypass the need for DCs maturation and directly active Teffs,which provides a co-stimulatory signal to step on the "accelerator" for T cells.Moreover,accumulating evidence indicates that highly abundant reactive oxygen species(ROS)in the tumor site contribute to tumor progression and metastasis and are closely associated with the immunosuppressive tumor microenvironment.Reducing ROS level favors restoring the tumor microenvironment.After local injection into tumors with abundant ROS,the engineered gel system R848&a-OX40@Gel could be gradually degraded.R848,firstly released from the gel system,can reduce the inhibitory effect of MDSCs and TAMs,consequently reverse the immunosuppressive tumor microenvironment for clearing "roadblocks" for CTLs.Then,a OX40 subsequently transports into TME and provides an acceleration to elicit T cells activation and expansion even in the absence of TAAs.Teffs function is recovered for direct killing tumor cells and producing neoantigen.The neoantigen with R848 excites APCs and further enhances the infiltration of lymphocytes.Cascade-amplification effects of the combination strategy emphasize T cells activation and expansion,result in boosting the immune response of "cold." When the engineered hydrogel system R848&a-OX40@Gel is used in a typical "cold" tumor,the mouse triple-negative breast cancer model has a significant inhibitory effect on in situ tumors.Ulteriorly,it is used in the recurrence and distant metastasis model of triple-negative breast cancer in mice,and it is found that the tumor metastasis and recurrence inhibition effect were evident.The survival time of mice was prolonged after R848&a-OX40@Gel treatment.Finally,to explore the broad applicability of the engineered hydrogel system,mouse melanoma and colorectal cancer models are established and treated with R848&a-OX40@Gel.As expected,the growth of the tumor is inhibited.Meanwhile,the mice weight and other survival conditions were observed and found no noticeable toxic and side effects in R848&a-OX40@Gel.The design and application of the engineered hydrogel system R848&a-OX40@Gel demonstrate a unique immunotherapy strategy with clearing "roadblocks" and stepping the "accelerator" for T cells.Its design concept also reflects the concept of "immune normalization" in cancer immunotherapy while provides a novel platform for "cold" immunotherapy.This promising program also enriches "in situ tumor vaccine" strategies and expands the application of the hydrogel delivery system.In this thesis,starting from the common problems of tumor immunotherapy,two hydrogel systems are designed to enhance the presentation efficiency of APC and reduce the "cold" tumor-suppressive immune microenvironment,thereby improving T cell-mediated adaptive immune response while raising the response rate to "cold" tumor immunotherapy.The thesis also harnesses the delivery advantages of hydrogels and subsequently synergizes with immunomodulators to finally achieve an anti-tumor immune response.By reasonably designed,such injectable multifunctional hydrogels will provide a new delivery platform for tumor immunotherapy.