Effects Of Targeted Nanoparticles That Carry Oxygen And IR780 On Epithelial Ovarian Cancer And Its Mechanism

Background:Epithelial ovarian cancer(EOC)accounts one of the most lethal malignancy diseases of woman reproductive system.Due to limited efficacy of traditional treatments,over 70%of patients experienced tumor recurrence or death within five years,more effective methods are urgently needed.Sonodynamic therapy(SDT)is a kind of non-invasive therapy,which destruct tumor cells by reactive oxygen species(ROS)produced after sonosensitizers triggered by ultrasound,focused ultrasound gathered at tumor tissues and make little injury to surrounded normal tissues.In this study,we aimed to constructed an oxygen-carrying and sonosensitizer loaded targeted nanoparticles,which can retain at tumor tissues,showed antitumor effects after activated by low intensity focused ultrasound; besides this,we further explored the potential mechanism of its antitumor effects,as to provide a new idea for ovarian cancer treatment.PART I Construction and characterization of targeted nanoparticles that carry Oxygen and IR780Objective:Folate receptor-targeted and ultrasound-responsive nanoparticles were constructed using PLGA-PEG-FA(FA:folate,PLGA:poly(lactic-co-glycolic)acid,polyethylene glycol(PEG)),reactive oxygen species(ROS)-generating sonosensitizer IR780 and oxygen-carrying material perfluorohexane(PFH),termed IRO@FA nanoparticles.And the morphology,size,targeting ability,phase-transformation and ROS production of nanoparticles were detected.Method:IRO@FA nanoparticles were constructed through a two steps emulsion method,then the morphology of IRO@FA nanoparticles was observed by transmission electron microscope(TEM);size and zeta potential were detected by Zetasizer Nano;Standard curve of IR780 was draw according to absorbance of UV-Vis and encapsulation rate was calculated;SOSG were used to detect ROS production of IRO@FA nanoparticles after triggered by ultrasound;IRO@FA nanoparticles was co-cultured with ID8 cells and the targeting ability was measured by laser confocal microscope(LSCM)and flow cytometry.Results:IRO@FA nanoparticles diffused in distilled water and displayed a dark green color;nanoparticles showed a spheric shape and uniform in size;the diameter of IRO@FA is 313.8±3.2nm and carry a negative charge;IRO@FA showed a phase-transformation and produced ROS after irradiated by ultrasound;in vitro study showed IRO@FA gathered at ID8cells and produced ROS intracellular after irradiated with ultrasound.Conclusions:we successfully prepared IRO@FA nanoparticles which were uniform in size and showed a good dispersibility in PBS,and produce ROS after trigged by ultrasound;meanwhile it showed a good targeting ability to ID8 cells,lay a foundation for subsequent experiments.PART II Antitumor effects and related immune variation of IRO@FA nanoparticles on EOCObjective:To evaluated the antitumor effects of IRO@FA nanoparticles on EOC by in vitro and in vivo study;and to explore potential mechanism through analyze immune change of tumor-bearing mice.Methods:in vitro antitumor effects of IRO@FA nanoparticles on ID8cells were detected by CCK8 and flow cytometry;the distribution of IRO@FA nanoparticles in tumor-bearing mice were observed by in vivo fluorescence imaging system,and the antitumor effects in vivo was evaluated by tumor size measurement;meanwhile the biocompatibility of IRO@FA was measured by HE staining;immune associated cytokines were detected by ELISA assay and immune cells infiltration in tumor tissues were observed by IHC;CD4~+,CD8~+T lymphocyte cells and CD80~+/CD86~+dendritic cells proportion in spleens were counted by flow cytometry.Results:IRO@FA nanoparticles showed significant antitumor effects after triggered by ultrasound;in vivo fluorescence images showed IRO@FA gathered at tumors at the first one hour and accumulated over time;and results from HE showed the structure of livers,kidneys,lungs and hearts in IRO@FA treated groups has no difference to control group;IHC results showed in SDT group the densities of CD3~+and CD8~+T cells in tumor tissues were significantly higher than others,while there is no difference in spleens;the expression level of IL-6,TNF-αand IFN-γwere upregulated in IRO@FA treated group,no similar trends were observed in serums.Conclusions:IRO@FA nanoparticles showed significant antitumor effects on ID8 cells both in vitro and in vivo,it also induced tumor immune infiltration and upregulation of cytokine expression.PART Ⅲ To explore potential mechanisms of IRO@FA on antitumor effects and immune infiltrationObjective:To study the potential mechanisms of IRO@FA nanoparticles on antitumor effects and increase of immune infiltration in EOC.Methods:the organelle structure change of ID8 cells was observed by TEM;damage associated molecular patterns(DAMPs)were determined by ELISA assay and immunofluorescence;DCs maturation were evaluated by flow cytometry.meanwhile whole protein from different treated ID8 cells were extracted,and western blot was used to analyze the expression level of cell death associated proteins.Results:cells after treated by IRO@FA nanoparticles and US showed ER edema and increase of perinuclear space which suggested ER stress,we also found the secretion of DAMPs are upregulated in IRO@FA treated groups and supernatant from which induced higher proportion of DCs maturation;proteins associated with programmed cell death were detected and results showed IRO@FA nanoparticles not only induced ID8 cells apoptosis but also pyroptosis,which may be induced by the imbalance of intracellular redox.Conclusions:IRO@FA promote tumor infiltration by inducing ID8 cells secretion of DAMPs and ICD;meanwhile,IRO@FA nanoparticles showed antitumor effects by inducing ID8 cells apoptosis and pyroptosis.