Immunological Effects Of Drug-Loaded Phase-Transition Nanoparticles Combined With Photodynamic And Sonodynamic Therapy On Ovarian Cancer

Part one:Physicochemical property and dual-model imaging of Oxaliplatin/Indocyanine green loaded phase-transition nanoparticlesObjective:Preparation of Oxaliplatin/Indocyanine green loaded phase-transition nanoparticles(OI_NPs)and detection of its physicochemical property and efficacy of dual-model imaging in vitro.Methods:1.OI_NPs were fabricated by modified double emulsion method.The appearance and morphology of OI_NPs were observed by transmission electron microscopy and scanning electron microscopy.The size distribution,polydispersity index and zeta potential of OI_NPs were measured by Malvern zetasizer.The optical property and stability of ICG loaded in OI_NPs and the encapsulation efficiency and drug loading of ICG were detected by ultraviolet-visible(UV-Vis)spectrophotometer.High performance liquid chromatography(HPLC)was performed to analyze the encapsulation efficiency and drug loading of OXP.2.Ultrasound imaging and PA imaging in vitro were performed by using an agar-gel model with voids.PBS,free ICG,Blank NPs and OI_NPs solutions were put into the different voids.Before or after 1.5W/cm~2 808nm laser irradiation for 2minutes,the B-mode and contrast-enhanced ultrasound(CEUS)images of each solution were captured by ultrasonic diagnostic instrument.Photoacoustic signals of the samples were gained and analyzed using the VEVO LASR PA imaging system.Finally,the corresponding image processing software was used to quantitatively analyze the captured images.Results:1.The OI_NPs have smooth surface and spherical shape.The average diameter,polydispersity index and zeta potential of OI_NPs were264.50±11.53nm,0.14±0.05 and-15.50±2.78mV respectively.The encapsulation efficiency and drug loading of ICG were 55.90±2.45%and2.03±0.09%respectively.The encapsulation efficiency and drug loading of OXP 26.02±0.72%and 1.42±0.04%.Compared with free ICG,ICG loaded in OI_NPs had no obvious wavelength shift in absorption and fluorescence spectra,importantly,with better optical stability.2.In vitro,after 808nm irradiation,the ultrasound imaging(B-mode and Contrast-enhanced mode)and photoacoustic imaging of OI_NPs were enhanced,and significantly enhanced when compared with other groups.Conclusion:We successfully fabricated OXP and ICG loaded phase-transition nanoparticles by modified double emulsion method.OI_NPs can be act as a contrast agent for ultrasound imaging and photoacoustic imaging,which contribute to the integration of diagnosis and treatment on ovarian cancer.Part two:The therapeutic effect of Oxaliplatin/Indocyanine green loaded phase-transition nanoparticles in combination with photodynamic therapy and sonodynamic therapy on ovarian cancer cellsObjective:To detect the therapeutic effect of Oxaliplatin and Indocyanine green loaded phase-transition nanoparticles in combination with photodynamic therapy and sonodynamic therapy on ovarian cancer cellsMethods:The ID8 cells were divided into the following 7 groups:Control,PSDT,I_NPs,I_NPs+PSDT,free OXP,OI_NPs,OI_NPs+PSDT.And ID8 cells were treated according to above groups.For another incubation 24h,the cell viability was detected by MTT assay.Annexin V-FITC/PI double staining was used to detect the apoptosis of ID8 cells and analyzed by flow cytometry.Results:The cell viability of free OXP,OI_NPs,I_NPs+PSDT and OI_NPs+PSDT were 80.94±1.04%,75.72±5.44%,59.36±5.77%,33.31±5.27%respectively.The cell viability of the OI_NPs+PSDT was lower than that of the other groups(P<0.001).The apoptotic trend was in accordance with MTT assay.The apoptotic rate of OI_NPs+PSDT was67.01±4.60%,which was significantly enhanced(P<0.001).Conclusion:With laser and ultrasound irradiation,OI_NPs in combination of the photochemical,sonochemical and chemotherapeutic effect of OXP,enhance the anti-tumor effect of chemotherapy and photo-sonodynamic therapy,inhibit cell proliferation and promote apoptosis.Part three:Immunological effects of Oxaliplatin and Indocyanine green loaded phase-transition nanoparticles in combination with photo-sonodynamic therapyObjective:To investigate the immunological effects of Oxaliplatin and Indocyanine green loaded phase-transition nanoparticles in combined with photo-sonodynamic therapyMethods:ID8 cells were treated with following groups:Control,PSDT,I_NPs,I_NPs+PSDT,free OXP,OI_NPs,OI_NPs+PSDT.Following a further incubation of 4 h,the translocation of calreticulin(CRT)were detected by flow cytometry.After incubation for 24h,the release of high mobility group box 1(HMGB1)and the secretion of adenosine triphosphate(ATP)were each investigated by western blot and luminometric assay.After scavenging the generation of reactive oxygen species with N-acetyl-L-cysteine(NAC),the CRT translocation was detected by flow cytometry.Tumor vaccination were divided into the following 6 groups:Control,PSDT,I_NPs+PSDT,free OXP,OI_NPs,OI_NPs+PSDT.After corresponding treatment,6-8 week old female C57BL/6 mice were inoculated subcutaneously on the lower left shoulder with different pre-treated 3x10~6 ID8 cells.7 days later,these mice were re-challenged by injecting 1 x 10~6 live ID8 cells on the lower right shoulder.The development of the tumor of the lower right shoulder was monitored for 60days and the tumor-free rate was recorded.Lactate dehydrogenase(LDH)assay was used to detect the specific lysis of spleen lymphocytes in vaccinated immunocompetent mice.Results:The translocation of CRT,ATP secretion and HMGB1 release in the free OXP,OI_NPs,I_NPs+PSDT and OI_NPs+PSDT were increased with varying degrees,while the OI_NPs+PSDT increased significantly higher than that of other groups.We use ROS inhibitor NAC to clear up the generation of cellular ROS in above four groups.The CRT translocation of all the groups were decreased with varying degrees,especially I_NPs+PSDT which was decreased from 43.44±0.50%to 2.56±0.57%.The C57BL/6 mice were vaccinated with OI_NPs+PSDT treated ID8 cells which showed72.73%of tumor-free mice after 60 days observation to reject the living ID8 cells attack and the results were higher than other groups(P<0.05).The cytotoxicity of spleen lymphocytes of the immunocompetent mouse immunization with free OXP,OI_NPs,I_NPs+PSDT,OI_NPs+PSDT respectively were enhanced with the increase of the effector/target ratio,especially in OI_NPs+PSDT.When the ratio of effector and target ratio100:1,the cytotoxicity of spleen lymphocytes of OI_NPs+PSDT group was43.58±4.61%,which was significantly higher than other groups(P<0.01).Conclusion:OI_NPs with near-infrared laser and low-intensity ultrasound irradiation enhance the ability of OXP and photo-sonodynamic to induce cancer cells immunogenic death,resulting in release or exposure of DAMPs enhanced.Its improved immunogenicity may be related to the production of reactive oxygen species.Vaccination in mice can produce long-term anti-tumor vaccination effects,which may be in relation with the host enhanced anti-tumor immune response.