Injectable Chitosan-based Thermosensitive Hydrogel Enhanced Mild Photothermal-immune Therapy

Photothermal-immune therapy is based on photothermal materials which can transfer light energy into thermal energy to kill tumor cells and therefrom induce anti-tumor immune response.It has attracted much attention in recent years because it has some advantages including good specificity,non-invasive,low toxicity,and effective killing the tumor cells and causing systemic anti-tumor immune response.But overhigh temperature will damage the surrounding normal tissues.Although mild photothermal therapy(MPTT)can effectively reduce the damage to peripheral tissues due to low temperature,the therapeutic effect is also weakened,which is difficult to inhibit the growth of“edge”tumor.Therefore,multiple hyperthermia and the introduction of(indoleamine2,3-dioxygenase)IDO inhibitors at tumor sites can not only enhance the drug delivery in cells,improve the tumor immunosuppressive microenvironment,improve the anti-tumor efficacy,but also ensure the safety to the greatest extent.In addition,the tumor antigens induced by photothermal therapy are easily cleared from the body,which reduces its immunogenicity.In order to improve the therapeutic effect and immune response of MPTT,chitosan based injectable thermosensitive hydrogel was used to capture autoantigen released from MPTT,to prolong the time of antigen stimulation,and combined with IDO inhibitor 1-methyl-D-tryptophan(1MT)to further enhance its anti-tumor immune response.The main research contents and results were shown as follows:1.Poly(N-isopropylacrylamide-co-chitosan)(PNC)thermosensitive nanogel were prepared by radical polymerization cross-linked with N,N′-methylene-bis-acrylamide.Its structure was confirmed by FTIR and~1H NMR.The results of dynamic light scattering showed that the average hydrated diameter of nanogels was about 250 nm at room temperature,the volume phase transition temperature(VPTT)was ca.32°C.The PNC nanogel dispersion(80 mg/m L)was easy to be injected as a sol at room temperature,and became a gel above 34°C.After coincubation with protein including tumor antigens for 12h,the size of the PNC nanogels was increased from 91.4 nm to 4274 nm,and the zeta potential was changed from positive to negative,and the mass of capture protein was 400μg/mg.It indicates PNC nanogels have good temperature sensitivity and protein adsorption properties.In addition,the PNC bulk gel can degrade in PBS.2.Polydopamine nanoparticles(PDA NPs)were prepared by the oxided self-polymerization of dopamine.And 1MT and PDA NPs were mixed with the PNC nanogel dispersion to form PDA/PNC and 1MT-PDA/PNC dispersion,respectively.The concentration of 1MT,PDA NPs and PNC nanogels were set as 1.8 mg/m L,200μg/m L and80 mg/m L,respectively.The results showed there was no obvious effect of the addition of PDA NPs and 1MT on the sol-gel phase transition behavior of PNC nanogel dispersion.These dispersions possessed the properties with shear thinning behavior,protein adsorption capacity and antigen capture ability.Under the irradiation of near infrared(NIR)laser(808nm,1.0 W/cm~2),the temperature of PDA/PNC dispersion increased by 20°C rapidly,and has good photothermal stability,which is suitable for the application in tumor hyperthermia therapy.The in vitro drug release behavior of 1MT-PDA/PNC gel was studied using dialysis method.It was found that the cumulative release of 1MT was about 70%at 72 h,and the release of 1MT was accelerated by NIR irradiation.3.The results of coincubation of PNC dispersions with L929 cells and HUVEC suggested that PNC had good biosafety.Using 4T1 breast cancer cell as a tumor cell model,it was found that the cell viability in PDA/PNC and 1MT-PDA/PNC treated group decreased significantly under NIRlaser irradiation.By detecting the exposure of calreticulin and the release of high-mobility group box 1,it was found that 1MT-PDA/PNC based MPTT could induce the immunogenic cell death of 4T1 cells.The results of flow cytometry showed that antigen-capturing gels could promote the maturation of bone marrow derived dendritic cells in vitro.4.The mild photothermal effect and immune response induced by1MT-PDA/PNC-based MPTT in vivo were studied using 4T1 tumor bearing mice model.Under 808 nm laser irradiation,the temperature of tumor site could be maintained at 42～45°C.Flow cytometry and ELISA kit were used to detect immune cells and related cytokines.It was found that the amount of the matured DCs in lymph nodes and the infiltration of CD8~+effector T cells into tumor sites were significantly enhanced.While the amount of regulatory T cells in tumor sites was reduced,resulting in improved tumor immunosuppressive microenvironment.In addition,the levels of interferon-γand tumor necrosis factor-αin the blood in the group of 1MT-PDA/PNC-based MPTT were significantly increased,indicating that systemic anti-tumor immune response was improved.