Application Of Novel Polymeric Nanomedicines In The Immunotherapy Of Osteosarcoma

Background:Immunotherapy,an emerging treatment that kills cancer cells by activating the body’s immune system,has been widely used in the treatment of many cancers.However,only patients with inflammatory tumors that are enriched in tumor-infiltrating lymphocytes can benefit from these treatments.Treating immune"cold"tumors that lack cytotoxic T-cell infiltration and fail T-cell priming remains a great challenge.Currently,one promising strategy is to convert immunosuppressive"cold"tumors into immunologically active"hot"tumors by triggering immunogenic cell death（ICD）,thereby improving the efficacy of immunotherapy.Polymeric nanodrugs,as a novel drug delivery system,have been widely studied and applied in cancer therapy.In addition to the advantages of improving the bioavailability of drugs,controlling the release rate of drugs,and reducing the side effects of drugs,polymeric nanodrugs can also enhance the effect of immunotherapy by activating the immune system,enhancing antigen delivery and stimulating immune cell activity.From the perspective of osteosarcoma immunotherapy,this thesis designs polymeric nanodrugs with amplified ICD effect and promoted anti-tumor immunity,hoping to provide new solution ideas for osteosarcoma treatment.（1）Amplification of Mitochondrial Oxidative Stress-Induced ICD for Osteosarcoma ImmunotherapyObjective:To test the physicochemical properties of NP-I-CA-TPP and elucidate the mechanism of action of NP-I-CA-TPP in amplifying mitochondrial oxidative stress,inducing ICD effect and triggering anti-tumor immunity.Methods:We first synthesized an amphiphilic polymer P^I-CAthat can consume glutathione（GSH）to amplify ROS oxidative stress damage.P^I-CAis characterized by a pendant iodide（I）that can deplete GSH through iodine-thiol click chemistry and cinnamaldehyde（CA）that can generate ROS.We then self-assembled P^I-CAwith the mitochondrial targeting polymer P^TPPto form a mitochondrial oxidative stress amplifier NP-I-CA-TPP.（1）The synthesized polymers were characterized by ¹H NMR,¹³C NMR and ³¹P NMR.The morphology of nanomedicine was characterized by transmission electron microscopy（TME）and dynamic light scattering（DLS）.DTNB method and high performance liquid chromatography（HPLC）were used to characterize the ability of nanomedicines to consume GSH and respond to CA release.（2）Mitochondrial fluorescent probes were used to detect the ability of nanomedicines to target mitochondria;ROS kit,Mito SOX kit and GSH/GSSG kit were used to detect the ability of nanomedicines to amplify mitochondrial oxidative stress.（3）In vitro antitumor activity of nanomedicines was detected by MTT method,Annexin V-FITC apoptosis assay kit,cell viability assay kit and mitochondrial membrane potential assay kit;the ability of nanomedicines to induce ICD was assessed by immunofluorescence,ELISA,flow cytometry and other methods.（4）The animal model of osteosarcoma mice was constructed,and the in vivo distribution and tumor site enrichment of nanomedicine were detected by small animal live imaging technique;the in vivo tumor suppression effect of nanomedicine was observed by recording the tumor volume and survival period of mice after treatment in different groups;the immune cell infiltration in tumor-draining lymph nodes,spleen and tumor tissues of tumor-bearing mice were analyzed by flow cytometry to detect the anti-tumor immune effect induced by nanomedicine.Results:（1）A mitochondria-targeting nanoparticle（NP-I-CA-TPP）with the function of amplifying mitochondrial oxidative stress was successfully designed and synthesized.（2）NP-I-CA-TPP can specifically target mitochondria after being taken up by osteosarcoma cells,and then endogenous mitochondrial ROS can induce CA release and stimulate mitochondrial ROS production.At the same time,iodide depletes mitochondrial GSH through iodine-thiol click chemistry.Elevated ROS and GSH depletion amplify oxidative stress in mitochondria,directly killing tumor cells while also inducing ICD activation of antitumor immune responses.（3）NP-I-CA-TPP showed excellent tumor suppressive effects in in vivo experiments and could activate anti-tumor immunity.In addition,NP-I-CA-TPP was able to synergize with immune checkpoint inhibitor（αPD-L1）to induce systemic antitumor immune effects in a bilateral subcutaneous model of osteosarcoma.Conclusion:NP-I-CA-TPP amplifies ICD effects by disrupting mitochondrial redox homeostasis and amplifying ICD effects,thereby activating antitumor immune responses.（2）Remodeling immunosuppressive TME amplification ICD for photodynamic immunotherapy of osteosarcomaObjective:To construct photodynamic immunotherapeutic nanomedicine（NP-PDT@Reg）based on degradable conjugated polymer combined with multi-targeted complex kinase inhibitor regorafenib（Reg）and systematically investigate its self-assembly properties and intracellular release mechanism;to explore the inhibitory effect of NP-PDT@Reg on osteosarcoma cell lines and its ability to reprogram macrophages;to study the distribution pattern of NP-PDT@Reg in organisms and explore its inhibitory effect on osteosarcoma and amplify the effect of anti-tumor immunity.Methods:Firstly,we designed and synthesized a near-infrared-II degradable polymer(PSP^Bodipy)with a large amount of tumor microenvironment-responsive soft segments,which contains disulfide bonds and Bodipy units in the main chain of the soft segment polymer.PSP^Bodipycan generate ROS under 808 nm light irradiation for photodynamic therapy and can also degrade under ROS triggering to improve biocompatibility.Subsequently,we synthesized an oxidatively responsive polymer containing diselenide bonds（P1）for encapsulating PSP^Bodipyand the multi-target tyrosine kinase inhibitor,Regorafenib（Reg）,to form a biodegradable photodynamic immunotherapy nanoparticle（NP-PDT@Reg）.（1）The synthesized polymers were characterized by ¹H NMR and¹³C NMR.The nanomedicines were characterized by TME,DLS,ultraviolet absorption spectroscopy,fluorescence emission spectroscopy and singlet oxygen indicator fluorescent probe（DPBF）.（2）MTT assay,Annexin V-FITC apoptosis assay kit and cell viability assay kit were used to detect the anti-tumor activity of nanomedicine in vitro.（3）Immunofluorescence,ELISA and flow cytometry were used to evaluate the ability of nanomedicine to induce macrophage reprogramming and ICD.（4）The animal model of osteosarcoma mice was constructed,and the in vivo distribution and tumor site enrichment of nanomedicines were detected by small animal in vivo imaging technology.The anti-tumor effect of nanomedicine in vivo was observed by recording the tumor volume and survival time of mice treated with different groups.（5）The ability of nanomedicines to induce vascular normalization was detected by immunofluorescence staining of tumor tissues;the ability of nanomedicines to induce TAMs reprogramming was detected by immunofluorescence staining of tumor tissues and flow cytometry.（6）The infiltration of immune cells in tumor draining lymph nodes,spleen and tumor tissues of tumor-bearing mice after immunosuppressive microenvironment remodeling was analyzed by immunofluorescence staining and flow cytometry.Results:（1）Photodynamic immunotherapy nanomedicine（NP-PDT@Reg）was successfully designed and synthesized.（2）NP-PDT@Reg produces cytotoxic ROS under 808 nm laser irradiation to directly kill cancer cells while simultaneously inducing ICD to activate anti-tumor immune response.Meanwhile,NP-PDT@Reg was able to induce macrophages to polarize from the tumor-promoting M2phenotype to the tumor-killing M1 phenotype.（3）In in vivo experiments,NP-PDT@Reg enhanced the efficacy of photodynamic therapy by alleviating tumor hypoxia dilemma through vascular normalization.Meanwhile,the relief of tumor hypoxia and TAMs reprogramming reversed immunosuppressive TME and amplified the effect of photodynamic immunotherapy.Conclusion:NP-PDT@Reg amplifies the effect of photodynamic immunotherapy for osteosarcoma by reprogramming immunosuppressive TME.70 Figures,0 Tables,384 References...