The Role And Mechanism Of Novel Photosensitive Materials And Their Induced Anti-tumor Immunity In The Treatment Of Osteosarcoma

Part 1: Low-temperature photothermal therapy mediated by Graphene oxide and its inhibition and therapeutic effects for osteosarcomaBackground: Although traditional tumor treatment methods such as surgical treatment,radiotherapy and chemotherapy can achieve the killing effect of tumors,the side effects can not be ignored,such as the metastasis and recurrence after surgery.What's more,the patient has to bear huge physical pain,financial burden and so on.These problems have prompted researchers to develop and innovate better treatment options.As an emerging tumor treatment method,photothermal therapy has received widespread attention,however,photothermal therapy has heat damage on normal tissues.The development of new photothermal materials can not only guarantee the killing effect on tumor cells,but also protect the normal tissues,which make the development of novel materials more important in the field of tumor treatment.Methods: Graphene oxide(GO)and folic acid(FA)can connect through amide bonds,the HSP90 specific inhibitor SNX2112 can connect with GO-FA through large ? bonds.Fourier infrared imaging and elemental analysis of transmission electron microscopy can confirm the characterization of materials;animal serum can be extracted to detect blood liver and kidney function and the morphology of important organs can determine the biological toxicity of the material.What's more,808 nm infrared photothermal and near-infrared thermal imaging instrument can detect the photothermal characteristics of the material;CCK8 experiment and apoptosis detection can confirm the killing effect of GFS(GO-FA-SNX2112).In vivo experiments are carried out by subcutaneous tumor formation,and small animal live imaging can detect tumor targeting ability,tumor macroscopic and immunohistochemical staining can detect the anti-tumor effects in vivo;Results: Fourier infrared imaging and elemental analysis of transmission electron microscopy confirmed that the material was prepared successfully,and the drug loading rate of SNX-2112 is about 20%;under the condition of808 nm near infrared light,the material can transfer the light energy into heat energy,which confirmed the excellent photothermal effect,what's more,the temperature can reach 43-45? stably.After 2 months of injection of the material in the tail vein,the liver and kidney function and the morphology of important organs are tested,and we confirmed that the material is injected in vivo without obvious toxic and side effects on the body.In vitro experiments confirmed that when the material is co-cultured with the osteosarcoma cell line HOS at a temperature of 43-45 ?,the tumor cells can reach about 70-80% apoptosis rate,and the subcutaneous tumor formation experiment confirmed that the material has significant targeting ability and tumor inhibition abiliy.Conclusion: The low-temperature photothermal material GO-FA-SNX2112 was successfully synthesised,which can achieve excellent tumor killing effect at relatively low temperature(43~45?).At the same time,the relatively low temperature can protect normal tissues of the body,which may have clinical application value in the future.Part 2: The Mechanism of Graphene Oxide-mediated Low-temperature Photothermal Therapy on the treatment of OsteosarcomaBackground: Adjuvant treatments for tumors,such as photothermal therapy have many problems.For example,the thermal damage to normal tissues.And the mechanism of cell death is still unclear,the body's systemic immune response after tumor cell death has not yet been investigated.These are the problems that must be solved before photothermal therapy can be applied to clinic.Exploring the physiological and pathological changes of tumor cells during low-temperature photothermal treatment,and clarifying the death mode,death mechanism and death pathway of tumor cells during low-temperature photothermal treatment are the focus of this research.Methods: Western blot and PCR experiments were used to verify the changes in tumor cell pathways.Immunofluorescence can confirm the changes in intracellular autophagy levels.In the design of the recovery experiment,the AKT pathway activator insulin and inhibitor LY294002 were used to confirm the AKT pathway in tumors.The role of cell death in the process of cell death,the autophagy activator 3-MA and inhibitor rapamycin were used to confirm the role of autophagy in the process of tumor cell death,the forward and reverse experiments were used to confirm the main pathways and key molecules in the process of low temperature photothermal.Results: Through reverse experiments,we found that insulin,the activator of AKT pathway can rescue the excessive activation of autophagy caused by low-temperature photothermal treatment,the death of tumor cells and the decrease of PDL1 expression.The inhibitor of the autophagy pathway,3-MA,can rescue the excessive activation of autophagy pathway,the death of tumor cells and the decrease of PDL1 expression caused by low-temperature photothermal treatment.In the positive rescue experiment,we found that LY294002,inhibitor of the AKT pathway,can strengthen the excessive activation of autophagy caused by low-temperature photothermal treatment,the death of tumor cells and the expression of PDL1 decrease.Rapamycin,an activator of autophagy pathway,can intensify the death of tumor cells and the decrease of PDL1 expression caused by low-temperature photothermal therapy.In addition,it was found that after co-cultivation with tumor cells after PTT,immune T cells were activated,which is confirmed by the increased expression of activated surface markers CD69 and the decresed expression of inhibitory surface markers PD1,TIM-3 and LAG-3.Conclusion: The killing effect of this material on tumors comes from two aspects.One is the excessive activation of autophagy of the cell itself,which becomes a direct death factor.The other is the change in the expression of immune checkpoint molecules on the cell surface,which induces an increase in autoimmune response.The cold immune condition of the tumor microenvironment is improved,thereby promoting the killing effect of the tumor.Part 3.The immunological effects of graphene oxide-mediated low-temperature photothermal therapyBackground: Low-temperature photothermal therapy is a non-invasive treatment program that can kill and ablate tumors quickly with relatively few side effects.However,the body's systemic immunity has not been explored during the process of low-temperature photothermal therapy.In-depth exploration of the body's immune response have great significance to the clinical application of low-temperature photothermal therapy.Therefore,exploring the changes in the body's immune system in graphene oxide-mediated low-temperature photothermal therapy is the focus of this research.Methods: The tumor-associated macrophages after photothermal treatment were co-cultured with tumor cells.Migration test,scratch test and PCR detection of migration-related genes were used to verify the migration ability of tumor cells,and CCK8 test was used to detect the proliferation ability of tumor cells.The tumor-associated macrophages and tumor cells after photothermal treatment are injected into nude mice in a certain proportion,and the effect of tumor-associated macrophages can be detected by subcutaneous tumor formation experiments.For tumor-associated macrophages,the polarization direction is detected by PCR and flow cytometry after low-temperature photothermal treatment.Results: After low-temperature photothermal treatment at 43-45?,the M2 polarization of tumor-associated macrophages was inhibited,and the supernatant could inhibit the proliferation of tumor cells.These were confirmed by the CCK8 proliferation experiment.Macrophage supernatant can also inhibit tumor cell migration,which is confirmed by scratch test,transwell test and PCR results of migration-related genes.In vivo,tumor-associated macrophages treated with low temperature can inhibit the growth of tumor tissues in vivo.Conclusion: Low-temperature photothermal therapy can inhibit tumor cell proliferation and migration by inhibiting the M2 polarization of tumor-associated macrophages.Part 4.Genetically modified Escherichia coli loaded with p DA/Ce6 to achieve an enhanced photodynamic and photothermal combined therapy strategy for osteosarcomaBackground: Although simple low-temperature photothermal therapy can achieve good tumor killing and inhibiting effects,it is still not enough to replace traditional treatment methods.This is related to the insufficient killing effect of a single treatment method.Preliminary experimental studies have confirmed that photothermal therapy can stimulate the body's anti-tumor immunity,therefore,taking full advantage of the body's immune activation,combining photothermal therapy with photodynamic therapy and immunotherapy may achieve excellent tumor treatment effects.Making full use of the body's anti-tumor immunity and developing a combined treatment strategy of photothermal/photodynamic therapy/immunotherapy is the focus of this research.Method: Because the positive charge of dopamine particles and the negetive charge of Escherichia coli,they can attract each other to form a new type of biological material.The material synthesis process is confirmed by scanning electron microscopy,transmission electron microscopy imaging and the zeta potential change during the material synthesis process.The base coating experiment confirms the biological activity of the bacteria.The immunoblotting electrophoresis of the target protein experiment confirms that E.coli has been successfully genetically modified and has functions.The CCK8 experiment and apoptosis test confirm the anti-tumor properties of the material.The in vivo experiment is carried out by subcutaneous tumor formation.Small animals imaging is used to detect tumor targeting ability,and the effect of materials on tumors in vivo is detected by observation of tumor gross morphology and immunohistochemical staining.Results: In the combined treatment strategy,the morphology of scanning electron microscopy confirmed that the p DA/Ce6 nanoparticles were uniformly loaded on the surface of E.coli,forming a new type of biologically active material.The antibiotic agarose solid was used to confirm the successful transfer of plasmid in E.coli.The culture of E.coli in the medium confirmed that the loading of p DA/Ce6 nanoparticles does not affect the proliferation and targeting ability of E.coli.E.coli has the function of loading p DA/Ce6 nanoparticles,targeting the hypoxic area of the tumor,and expressing human catalase.This combined treatment strategy of photothermal therapy/photodynamic therapy/biological therapy can achieve good tumor suppression effects.It is an extension and development of previous photothermal therapy and is expected to be applied in clinical in the future.Conclusion: A new combination therapy strategy of photothermal therapy/enhanced photodynamic therapy/biological therapy has been developed through genetically modified Escherichia coli,which can achieve powerful tumor killing and inhibitory effects.This new tumor phototherapy and combination therapy have broad potential for clinical application.