| Background:Small molecule chemotherapeutic drugs can produce marked toxic and side effects.With the cross-fertilization of nanotechnology and modern medicine,nanomedicines are increasingly used in tumor treatment.Recently,immunotherapy and photodynamic therapy(PDT)have brought new hope to cancer patients.Therefore,our group has developed several nanomedicines in the early stage.However,the current cancer therapies still face many challenges,including the existence of multiple physiological-physical barriers before reaching the final target and the construction of immunosuppressive tumor microenvironment by immunonegative regulatory molecules.In this study,two new multifunctional nanomedicines(named NPER/BO-PDTand NPPDT@CXB)were selected from the perspective of enhancing immunotherapy of osteosarcoma,and further investigate their mechnanisms against osteosarcoma.Objective:To test the physicochemical properties of these two nanomedicines(NPER/BO-PDTand NPPDT@CXB),and to elucidate the mechanisms of NPER/BO-PDT breaking the step-reduction delivery dilemma(SRDD)of drugs and NPPDT@CXB relieving the immunosuppressive state of tumor tissues for achieving efficient anti-osteosarcoma immunotherapeutic effects,providing new therapeutic strategies for the treatment of osteosarcoma.Methods:(1)Introducing endoplasmic reticulum(ER)targeting ligand and bone targeting ligand into amphiphilic polymers,the targeting polymers were synthesized respectively.Subsequently,the targeting polymers were mixed to prepare the dual targeting nanomedicine NPER/BO-PDT;(2)A novel BODIPY molecule containing double hydroxyl groups was synthesized.Subsequently,the reative oxygen species(ROS)sensitive degradable polymer was synthesized by condensation reactions.Furthermore,celecoxib(CXB)was encapsulated with the polymers to form nanomedicine NPPDT@CXB that can neutralize pro-tumorigenesis inflammation in tumor tissues;(3)The nanomedicines were identified and quantified by transmission electron microscopy,dynamic light scattering,ultraviolet absorbtion spectroscopy and inductively coupled plasma mass spectrometry,etc.The in vitro anti-tumor activity of nanomedicines was analyzed by MTT,Annexin V-FITC/PI double staining,live/death cells staining and clone formation assay,etc.The anti-tumor mechanisms were explored by various experimental methods,such as laser confocal co-localization,immunofluorescence,flow cytometry,Western-blot,ELISA assay and metabolomics analysis,etc.The effects of nanomedicines on enhancing immune response in vitro were evaluated by cell co-culture technology.Subsequently,the orthotopic osteosarcoma mouse model,unilateral osteosarcoma subcutaneous model,bilateral osteosarcoma subcutaneous model and osteosarcoma patient-derived tumor xenograft model were constructed.Finally,the in vivo distribution and tumor site enrichment of nanomedicines were detected by the live imaging technique.The anti-tumor effects were observed by recording the tumor volumes and survival time of mice.The levels of cytokines in peripheral serum and tumor tissues of mice were evaluated by ELISA,and the infiltration of related immunity cells in tumor draining lymph nodes(TDLNs),spleen and tumor tissues of mice were detected by flow cytometry to analyze the effects of nanomedices on enhancing immune response and the related mechanisms.Results:(1)The new polymeric nanomedicine NPER/BO-PDT is stable in the aqueous phase,capable of generating ROS and undergoing responsive degradation upon light irradiation,and can target delivery to bone tumor tissues and the endoplasmic reticulum;(2)NPER/BO-PDT entered tumor cells through endocytosis and further targeted the ER,which can generate ROS in situ of ER under light irradiation,disrupt cytoplasmic Ca2+homeostasis,trigger sustained endoplasmic reticulum stress(ERS),enhance immunogenic cell death(ICD),and effectively inhibit osteosarcoma cell growth.In addition,the near-infared-Ⅱregion(NIR-Ⅱ)fluorescence imaging of tumor-bearing mice revealed that NPER/BO-PDT can targeted accumulate in osteosarcoma tissues.Under light irradiation,the NPER/BO-PDTcan kill tumor cells directly while enhancing the infiltration of CD8+T cells and inducing the maturation of dendritic cells(DCs)in tumor-bearing mice,achieving highly effective anti-tumor photodynamic-immunotherapy;(3)Meanwhile,the nano-delivery system with photodynamic effect,controllable release of CXB and biodegradability(NPPDT@CXB),a nanomedicine with emitting long wavelength fluorescence and ROS-responsive degradation,was successfully designed and synthesized;(4)NPPDT@CXB were effective intaken by osteosarcoma cells,can produce ROS under light irradiation,and release CXB in response to ROS,which in turn inhibit the expression of cyclooxygenase 2(COX-2)and the synthesis of prostaglandin E2(PGE2),and can promote DNA damage,reduce the expression of CD44 and programmed cell death ligand 1(PD-L1)in the cells,which effectively inhibit the growth of osteosarcoma cells.Meanwhile,NPPDT@CXB can induce intense ICD effects in osteosarcoma cells under light irradiation,further promoting the maturation of DCs and the polarization of macrophages to M1 type,enhancing the anti-tumor immune response,which were further verified by metabolomic analysis;(5)Additionally,the fluorescence imagings of tumor-bearing mice reveal that NPPDT@CXB can emit NIR-Ⅱfluorescence for bioimaging and can targeting accumulate in osteosarcoma tissues after itravenously injected into mice.Under light irradiation,NPPDT@CXB is able to induce intense ICD effects and inhibite pro-tumorigenesis inflammation(reduce the activity of COX-2/PGE2 pathway)to remodel the tumor immunosuppressive microenvironment,thereby triggering a strong anti-tumor immune response and effectively inhibiting the growth of primary and distant osteosarcoma.Conclusions:(1)The targeting delivery nanomedicine NPER/BO-PDTsuccessfully constructed in this study has good biocompatibility and can generate ROS efficiently after light irradiation;(2)NPER/BO-PDT can enhance the anti-tumor immunity by breaking the SRDD of drugs,triggering sustained ERS and intense ICD effects,and activate the body’s anti-tumor immunity while directly killing osteosarcoma cells to achieve efficient anti-tumor photodynamic-immunotherapy;(3)The successfully designed and synthesized multifunctional light-responsive nanomedicine NPPDT@CXB has good biocompatibility,biodegradability and NIR-Ⅱfluorescence imaging,and can controllable release of CXB,thus reducing the activity of COX-2/PGE2pathway;(4)NPPDT@CXB can generate ROS efficiently after light irradiation,which directly kill osteosarcoma cells and activate the body’s anti-tumor immunity simultaneously,the controlled release CXB can further remodel the immunosuppressive tumor microenvironment by inhibiting pro-tumorigenesis inflammation,achieving efficient anti-tumor photodynamic-immunotherapy. |