Study On The Role And Mechanism Of Temperature-responsive Hydrogels Co-loaded With Radiotherapy Microparticles And Mn²⁺ In Anti-tumor Immunity

Immunosuppressive tumor microenvironment is the primary cause of tumor immune escape,and breaking immunosuppressive tumor microenvironment plays an important role in anti-tumor immunity.Previous studies of our team have shown that microvesicles（RT-MPs）produced by radiation inhibit tumor growth by promoting tumor cell immunogenic death and remodeling macrophage function.However,the limited immune activation efficiency weakens the long-term control of RT-MPs on tumor.As one of the necessary metal elements,manganese(Mn²⁺)has a strong activation effect on c GAS-STING signaling pathway,and acts as an immune adjuvant to activate antigen presenting cells（APCs）to enhance adaptive immune response.However,the metabolism of Mn²⁺in vivo is fast,so it needs to be administered repeatedly,and it can not produce specific killing to tumor cells directly.In order to solve the problem of limited immune system activation by RT-MPs and the problem of fast metabolism and non-specific activation of immune system caused by Mn²⁺as a strong adjuvant,our team successfully loaded RT-MPs and Mn Cl₂ with poly（amino acid）temperature responsive hydrogel as carrier,and realized the in vivo sustained release effect of both.RT-MPs significantly enhanced the expression of costimulatory molecules on the surface of APCs by increasing the stimulation of c GAS-STING signal pathway of Mn Cl₂ to APCs.Meanwhile,it induced the production of a large number of proinflammatory cytokines and the infiltration of effector T cells,thus reactivating the immunosuppressive tumor microenvironment and inhibiting the growth of tumor.Furthermore,the combination of PD-1 antibody significantly increased the anti-tumor efficacy of RT-MPs and Mn Cl₂,promoted the formation of immune memory and inhibited tumor recurrence.The main achievements and conclusions of this subject are summarized as follows:1)By detecting the phase transition of different concentrations of polyamino acid materials to confirm the appropriate material concentration（6wt%）.Through enzyme degradation experiments,magnetic resonance detection confirmed that the hydrogel materials can effectively load RT-MPs and Mn Cl₂,and play the role of drug release in vivo.2)Western blotting,ELISA and flow cytometry showed that RT-MPs and Mn Cl₂could synergistically increase the activation of c GAS-STING signal pathway,increase the expression of type I IFN downstream of STING,and promote the activation of APCs in cooperation with Mn Cl₂ through increasing the expression of costimulatory molecules CD80 and CD86.3)Subcutaneous transplanted tumor model showed that the thermosensitive hydrogel（RT-MPs@Mn Cl₂ gel）loaded with RT-MPs and Mn Cl₂ could significantly inhibit the growth of tumor in mice after intratumoral injection.Flow cytometry confirmed that RT-MPs released into tumor microenvironment was mainly absorbed by antigen presenting cells and cooperated with Mn Cl₂ to promote the activation of DC cells in draining lymph nodes,breaking the tumor microenvironment of immunosuppression and activating systemic anti-tumor immune response.4)Through the model of subcutaneous transplanted tumor,it was proved that RT-MPs@Mn Cl₂ gel combined with PD-1 antibody could significantly reduce tumor burden,and improve the survival in mice.Flow cytometry and flow multifactor quantitative detection showed that combined with PD-1 antibody significantly increased the activation of CTLs,promoted the infiltration of more immune cells,increased the expression of cytokines and chemokines,transformed"cold tumor"into"hot tumor",increased the proportion of effective memory T cells,and inhibited tumor recurrence.In vivo intravital imaging was used to confirm that when the cured mice were re-inoculated with tumor,the infiltration and movement of T cells in the tumor area increased,and then inhibited the growth of re-inoculated tumor.