| Part one:Preparation and characterization of folate-targeted and oxygen/indocyanine green loaded lipid nanoparticlesObjective:To synthesize folate-targeted and oxygen/indocyanine green loaded lipid nanoparticles?FA-OINPs?and detect its characteristics.Methods:FA-OINPs were synthesized by a modified two-step emulsion method.The morphologic characterization of FA-OINPs were observed by optical microscopy.The EE?entrapment efficiency?and LE?loading efficiency?of ICG in FA-OINPs were measured by an ultraviolet–visible?UV–Vis?spectrophotometer.The Malvern Zetasizer Nano ZS unit?Malvern Instrument,UK?was used to determine the size distribution and the zeta potential of FA-OINPs.The optical property and fluorescence stability of FA-OINPs were detected by UV-Vis spectrophotometer and fluorescence spectrometer,respectively.Results:The FA-OINPs were dispersed separately with a uniform spherical structure and homogeneously distribution.The average diameter and zeta potential of FA-OINPs were?301.08±11.12?nm and?-23.40±1.51?mv,respectively.The EE of ICG encapsulated in FA-OINPs was?95.67±0.46?%and the LE was?2.79±0.01?%.Besides,compared with the free ICG,the absorption spectra and fluorescence spectra of FA-OINPs showed no obvious wavelength excursion,while the fluorescence stability increased significantly?P<0.01?.Conclusion:FA-OINPs were successfully prepared by a modified two-step emulsion method,which was expected to realize a new mode of diagnosis and therapy integration in ovarian cancer.Part two:The dual-mode imaging characteristics of FA-OINPs in vitro and in vivoObjective:To investigate the dual-mode imaging characteristics of FA-OINPs as a contrast agent in vitro and in vivo.Methods:An agar gel phantom with several holes was used to study the imaging effect in vitro and holes in the agar gel phantom were divided into three groups:?a?PBS control,?b?oxygen loaded lipid nanoparticles?ONPs?and?c?folate-targeted and oxygen/indocyanine green loaded lipid nanoparticles?FA-OINPs?,in which the group c was divided into 8?g/ml,16?g/ml,32?g/ml and 64?g/ml according to the different concentrations of ICG.Then an 808 nm laser was used to irradiate the various groups at1.5w/cm2 for 5min.Before and after irradiation,the samples were scanned by the ultrasonic diagnostic instrument for ultrasound imaging[brightness modulation?B-Mode?and contrast-enhanced ultrasound?CEUS?].Similarly,photoacoustic imaging of the different groups before and after irradiation were assessed by the Vevo LAZR PA imaging system.The results were quantificationally analyzed by using the corresponding image processing software.In vivo,the SKOV3 tumor-bearing mice were randomly divided into two groups:?a?OINPs group and?b?FA-OINPs group,which were injected the corresponding nanoparticles with the same concentration through tail-vein,and the PA images of tumor at 0,3,6,12,24 and 48h post injection were obtained by the Vevo LAZR PA imaging system.Another SKOV3 tumor-bearing mice were classified into three groups?saline,OINPs and FA-OINPs?and intravenously injected the corresponding nanoparticles or saline.The photoacoustic imaging containing before injection,after injection and after laser irradiation?1.5 w/cm2 for 5min after 6h of post-injection?were gained using the Vevo LAZR PA imaging system.Quantitative statistics methods were the same as in vitro.Results:In vitro,compared with the other two groups,the dual-mode imaging effects of laser irradiated FA-OINPs group was significantly higher than that before irradiation?P<0.01?.Meanwhile,it was evidently to notice that the B-mode,CEUS and PA signal intensity enhanced linearly with ICG concentrations after laser irradiation,which satisfied y=1.07x+23.51?R2=0.92?,y=0.41x-0.59?R2=0.99?and y=0.0080x+0.21?R2=0.93?,respectively.In vivo,the photoacoustic signal intensity in tumor site of FA-OINPs group peaked at 6 h post injection.While the OINPs group reached maximal PA signal intensity in tumor region at 12h post injection and the signal intensity was weaker than that of FA-OINPs.It was suggested that FA-OINPs can assemble more rapidly than OINPs at the tumor site.After laser irradiation,the ultrasound and photoacoustic signals of the OINPs group increased by 6.25%and 16.39%,respectively;while the FA-OINPs group increased by 13.17%and 20.32%,respectively.Conclusion:FA-OINPs can be used as a contrast agent for ultrasonic/photoacoustic dual-mode imaging,and their imaging effects were significantly enhanced after laser irradiation.At the same time,compared with non-target nanoparticles,FA-OINPs can aggregation to the tumor region more rapidly,which may provide the experimental evidence for the new model of diagnosis and therapy integration in ovarian cancer.Part three:Study of the targeting performance of FA-OINPs and the therapeutic effect of photo-sonodynamic mediated FA-OINPs on SKOV3 ovarian cancer cells Section oneThe targeting performance of FA-OINPs in ovarian cancer cellsObjective:To discuss the targeting performance of FA-OINPs in SKOV3 ovarian cancer cells.Methods:The SKOV3 cells in logarithmic growth phase were randomly divided into three groups:?a?OINPs group,?b?FA-OINPs group and?c?FA-OINPs+free folic acid block group.And excessive free folic acid was used to block FRs in advance in group?c?.The A549 cells were randomly classified into two groups:?a?OINPs group and?b?FA-OINPs group;DiI-labeled FA-OINPs or OINPs were added into homologous groups respectively and co-incubated for 30min.Finally the confocal laser scanning microscopy was used to observe the targeted binding affinity of nanoparticles in different groups respectively.Results:In SKOV3 cells,the FA-OINPs group had a significantly stronger fluorescence signal than OINPs group,which showed that FA-OINPs can active targeting to the SKOV3 cells and be internalized.While after blocked by folic acid,the fluorescence signal decreased observably,which showed that the active targeting of FA-OINPs was mediated by folic acid.Meanwhile,compared with the A549 cell?low folate receptor expressing?,FA-OINPs were more effectively be internalized by the SKOV3cells?high FRs expressing?.These results further suggested that the specific uptake of FA-OINPs was mediated by folic acid.Conclusion:FA-OINPs can specifically identify the SKOV3 cells,which were known to overexpressing FRs,and performed excellent target ability.Section twoThe therapeutic effect of photo-sonodynamic mediated FA-OINPs on SKOV3 ovarian cancer cellsObjective:To evaluate the effect and related mechanism of photo-sonodynamic mediated FA-OINPs on SKOV3 ovarian cancer cells.Methods:The SKOV3 cells were randomly divided into 7 groups:?a?control,?b?laser+ultrasound alone?L.U.alone?,?c?free ICG+L.U.,?d?ONPs+L.U.,?e?OINPs+L.U.,?f?FA-OINPs+L.U.,?g?FA-OINPs+L.U.+N-acetyl-L-cysteine?FA-OINPs+L.U.+LNAC?,after corresponding treatments,the reactive oxygen species assay kit was used to detect the generation of reactive oxygen species?ROS?in the cells.Next,the SKOV3cells were randomly classified into 6 groups,which were the same as above?a?-?f?,and were given various treatments.Finally,the cell viability was evaluated by the MTT assay and flow cytometry analysis was used to analyze the apoptosis of cells with an Annexin V-FITC/PI double staining.Results:Except for the L.U.alone group,there was an increase in the generation of ROS among all the other treatment groups.And the production of ROS in the FA-OINPs+L.U.group increased most significantly,being?179.46±22.46?%.It was significantly higher than that in the other treatment groups?P<0.01?.At the same time,the cell proliferation inhibition rate and apoptosis rate in FA-OINPs group were?68.94±4.83?%and?75.51±0.88?%,respectively,which showed an obviously highest apoptosis and proliferation inhibition rate than those of the rest treatment groups?P<0.01?.Conclusion:Photo-sonodynamic mediated FA-OINPs could promote the apoptosis of SKOV3 ovarian cells,inhibit cell proliferation and reduce the cell viability.The mechanism may be connected with the markedly enhanced generation of intracellular reactive oxygen species and the cavitation effect mediated by photo-sonodynamic.Part four:The Therapeutic Effect of Photo-Sonodynamic Mediated Folate-targeted and Oxygen/Indocyanine green Loaded Lipid Nanoparticles on Ovarian Cancer XenograftsSection onePhotothermal effects of FA-OINPs induced by laser irradiationObjective:To demonstrated the photothermal effects of FA-OINPs induced by laser irradiation in vitro and in vivo.Methods:The 48-well plates were used for the study in vitro and the nude mice with subcutaneous tumor of SKOV3 ovarian cancer were used in vivo study.There were four groups in the experiment:?a?saline;?b?free ICG;?c?OINPs;?d?FA-OINPs,then irradiated by 808nm laser at 1.5 w/cm2 for 5min.Meanwhile,an infrared?IR?thermal imaging system was used to record the images and temperature during irradiation.Results:In vitro,after 808 nm laser irradiation,the temperature of the saline,free ICG,OINPs and FA-OINPs groups were 26.8°C,41.3°C,45.6°C,and 47°C,respectively.Compared with the saline group,the temperature of the other three groups increased significantly,especially the OINPs and the FA-OINPs groups exceeded 43°C,which could cause irreversible damage to the tumor.These results revealed that the laser-mediated nanoparticle has photothermal effects.In vivo,the temperature of the saline,free ICG,OINPs and FA-OINPs groups were 36.4°C,37.1°C,40.1°C,and 44.8°C,respectively.Compared with the other three groups,FA-OINPs have the most significant photothermal effects.Conclusion:In the presence of laser irradiation,the photothermal effects of FA-OINPs could be induced efficiently.It was expected to provide a new strategy for the combined treatment of tumor.Section twoTreatment of ovarian cancer xenografts by photo-sonodynamic mediated FA-OINPsObjective:To demonstrated therapeutic efficacy of photo-sonodynamic mediated FA-OINPs for ovarian cancer xenografts.Methods:The tumor-bearing nude mice were randomly divided into five groups:?a?saline,?b?free ICG+L.U.,?c?ONPs+L.U.,?d?OINPs+L.U.and?e?FA-OINPs+L.U.,eight mice in each group.Each group was randomly sacrificed three nude mice to obtain the tumor and major organs on the 15th day after the end of treatment.Hematoxylin and eosin?HE?staining was used to observe the biosafety of nanoparticles for the major organs,terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay?TUNEL assay?was implemented to evaluate the tumor cell apoptosis index,VEGF and CD34 were used to label the tumor microvascular density and positive CD68 cells was used to indicate the tumor-associated macrophages?TEMs?,which were also high-expression of folate receptors?FRs?.The rest mice of each group were monitored daily for survival time.Results:No noticeable organic damage of the major organs were observed among all the groups.The apoptosis rate in the FA-OINPs+L.U.group was?69.47±4.49?%,which was significantly higher than that of the other treatments?P<0.05?.The integral optical density?IOD?of CD68-positive cells in FA-OINPs+L.U.group was significantly lower than that in the other groups?P<0.05?.Compared with the control group,all the treatment groups except free ICG+L.U.group displayed lower expression of VEGF and MVD?P<0.05?,and the FA-OINPs+L.U.group had the most significant decrease?P<0.05?.The relevant median survival times in group?a?to group?e?were 28,29,32,39and 50 days,respectively.Conclusion:FA-OINPs combined with photo-sonodynamic synergistically could improve the lethality and inhibition effect of tumor cells of drug-loaded nano-system through active targeting performance.And simultaneously extend the median survival time of the subcutaneous ovarian tumor xenograft model.The mechanism may be in connection with the improvement of hypoxic microenvironment,the increase of reactive oxygen species,the cavitation and mechanical effect of photo-sonodynamic mediated nanoparticles as well as the photothermal effects. |
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