| Part one:Preparation and characterization of OIX＿NPsObjective:To synthesize oxygen-carried and indocyanine green/oxaliplatin-loaded nanoparticles(OIX＿NPs)and explore the basic characteristics.Methods:OIX＿NPs were prepared by modified double emulsification method,and the morphology and size of OIX＿NPs were observed by optical microscope and electron microscope;The size,polydispersity index(PDI)and zeta potential of nanoparticles were detected by Malvern zetasizer;Ultraviolet-visible(UV-Vis)spectrophotometer and high performance liquid chromatograph(HPLC)were used to determine the entrapment efficiency(EE)and loading efficiency(LE)of indocyanine green(ICG)and oxaliplatin(OXP),respectively;In addition,the optical property of OIX＿NPs was discussed through absorption spectrum and fluorescence spectrum.Results:The spherical OIX＿NPs with uniform size and good dispersion were observed under microscope;The size,PDI,and zeta potential of OIX＿NPs were 247.10±2.65nm,0.12±0.04,-14.23±1.46m V,respectively;The EE and LE of ICG were57.09±1.78%and 2.08±0.06%,the EE and LE of OXP were24.95±4.27%and 1.36±0.23%;There was no obvious shift in the absorption spectrum and fluorescence spectrum of OIX＿NPs compared with free ICG.Conclusion:In this experiment,the modified double emulsion method was successfully used to prepare oxygen-carried and indocyanine green/oxaliplatin-loaded nanoparticles(OIX＿NPs),which had good entrapment efficiency and loading efficiency.Part two:Imaging properties of OIX＿NPs in vivoObjective:To observe the aggregation of OIX＿NPs in tumor tissue and explore its ability to enhance US/PA imaging in vivo.Methods:A subcutaneous model of tumor-bearing C57BL/6mouse was established,followed by tail vein injection of OIX＿NPs(64 ug/m L of ICG).The images were captured at different time points(pre,2,4,6,12,and 24 h),and analyzed to obtain the optimal time point for the aggregation of OIX＿NPs at the tumor site.Then,the tumor-bearing mice were randomly divided into two groups to receive a caudal vein injection of free ICG or OIX＿NPs.The tumor tissues were exposed to NIR irradiation(1.5 W/cm~2,5min)at the optimal imaging time and the corresponding images were acquired.The average echo intensity and the average photoacoustic intensity were used for analysis.Results:After the injection of OIX＿NPs,the PA signal at the tumor site started to increase and peaked at 4h postinjection,and then the PA signal gradually decreased until 24 hours after injection.4 hours after injection of OIX＿NPs,the average echo intensity was about 2 times that before injection and the average photoacoustic intensity was about 5 times that before injection(p<0.05).After laser irradiation,the average echo intensity and the average photoacoustic intensity were significantly enhanced(p<0.05).However,no significant signal changes were observed in the free ICG group.Conclusion:OIX＿NPs can effectively aggregate in tumor tissue to achieve enhanced US/PA imaging.After laser irradiation,the imaging ability is further improved,laying the foundation for follow-up treatment.Part three:The in vivo therapeutic effect and mechanism of OIX＿NPs combined with photo-sonodynamicObjective:To study the therapeutic effect of OIX＿NPs combined with photo-sonodynamic on ID8 subcutaneous tumors of ovarian cancer in C57BL/6 mice,and explore its immunological mechanism.Methods:ID8 tumor-bearing mice were divided into five groups randomly(n=8):(1)saline group;(2)oxp group;(3)IX＿NPs+PSDT group;(4)OI＿NPs+PSDT group;(5)OIX＿NPs+PSDT group.The different drug formulations were intravenously injected at an oxaliplatin dose of 2mg/kg every 5days for four cycles.Four hours after injection,photo-sonodynamic therapy was performed(808 nm laser:1.5W/cm~2,5min;low-intensity ultrasound:1W/cm~2,1min).The tumor volume and body weight of each mouse were recorded.24hours after the last treatment,three mice were randomly sacrificed from each group,tumor tissue and blood samples were collected,and the remaining mice were continued to monitor daily.CRT and HMGB1 immunohistochemical staining were used to detect the induction of ICD;The serum cytokine IFN-γwas measured by ELISA;Tumor tissue necrosis was observed by HE staining;Tumor tissue apoptosis was evaluated by TUNEL assay;Angiogenesis was assessed by VEGF staining;HIF-1αwas measured by Western Blot.Results:The OIX＿NPs+PSDT group had the best therapeutic effect among all groups,the tumor growth inhibition rate was as high as 81.88±3.07%,and the mouse body weight change was the smallest.A large amount of CRT and HMGB1 expression were seen in tumor tissue in the OIX＿NPs+PSDT group,indicating the successful induction of ICD.The higher serum concentration of cytokine IFN-γwas observed in the OIX＿NPs+PSDT group,which was about 2 times that of the control group.For OIX＿NPs+PSDT group,HE staining showed the most obvious tumor cell necrosis,TUNEL assay performed the strongest positive staining,and the expression of VEGF was significantly lower than that of the other groups.In addition,the OIX＿NPs+PSDT group achieved the longest median survival time(P<0.05).Conclusion:OIX＿NPs-mediated chemotherapy combined with PSDT can successfully inhibit tumor growth,prolong the median survival time,and induce tumor ICD to enhance systemic anti-tumor immunity.Furthermore,OIX＿NPs can deliver oxygen to the tumor site and improve the tumor hypoxic microenvironment.Part four:The antitumor immune effect of OIX＿NPs combined with photo-sonodynamicObjective:To explore the abscopal effect of OIX＿NPs combined with photo-sonodynamic,and evaluate the infiltration of immune cells in tumor.Methods:The bilateral tumor model of ovarian cancer ID8 in C57BL/6 mouse was established,and then tumor-bearing mice were randomly divided into OIX＿NPs group and OIX＿NPs+PSDT group.OIX＿NPs were intravenously injected at an oxaliplatin dose of 2mg/kg every 5 days for four cycles.Four hours after injection,photo-sonodynamic therapy was performed only for primary tumours(808 nm laser:1.5W/cm~2,5min;low-intensity ultrasound:1W/cm~2,1min).The tumor volume of primary tumour and distant tumour was recorded.24 hours after treatment,three mice were randomly selected from each group for sacrifice,and their primary and distant tumor tissues were collected for CD4 and CD8immunohistochemical staining.Results:The combination of OIX＿NPs-mediated chemotherapy and PSDT could not only significantly inhibit the growth of primary tumors,but also delay the growth of distant tumors through the immune response.Moreover,the OIX＿NPs+PSDT group had higher expression of CD4 and CD8 in both primary tumour and distant tumour,indicating that our strategy could recruit more T lymphocytes to infiltrate and thereby inhibit the growth of residual and metastatic tumors(p<0.05).Conclusion:OIX＿NPs-mediated chemotherapy combined with PSDT can successfully induce anti-tumor immune effect,which can inhibit the growth of primary tumors and distant tumors through enhancing the immunogenicity of tumor cells and activating T lymphocytes in tumor tissue.It provides new ideas for the treatment of metastatic tumors. |
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