Photo-sonodynamic Synergism Oxygenate Perfluorocarbon And Indocyanine Green-Loaded Nanoparticles For Inhibition Of Ovarian Cancer SKOV3 Cells

PART ONE: PREPARATION AND PHYSICAL CHARACTERIZATION OF OXYGEN AND ICG-LOADED PERFLUOROCARBON PLGA NANOPARTICLESObjective: Preparation and characterization of oxygen and ICG-loaded perfluorocarbon PLGA nanoparticles(OPI-NPs).Methods:The OPI-NPs,OP-NPs and I-NPs were prepared by a water-in-oil-in-water emulsion(w/o/w).The morphology of nanoparticles formulations was observed by bright field microscopy and transmitting electron microscopy(TEM).The sizes,polydispersity indexes of nanoparticles were determined by dynamic light scattering instrument.The encapsulation efficiency(EE)of ICG was calculated using the ultraviolet spectrophotometer.The photoacoustic data were simultaneously collected by the Vevo LAZR imaging system and compared the effect of photoacoustic imaging between OP-NPs+ free ICG and I-NPs.Results: The morphology of OPI-NPs was round in spherical.The size was displaying a(80 nm—1000 nm)range,and the average diameter of OPI-NPs is 275.24±7.28 nm.The ICG entrapment efficiency was about 55% and ICG loading was 0.55 mg/ m L.The PA signal intensity of OP-NPs+ free ICG、 I-NPs and OPI-NPs were 0.336 ± 0.022,0.744 ± 0.029 and 1.112 ± 0.072,respectively.The PA signal intensity of OPI-NPs and I-NPs were higher than that of OP-NPs plus free ICG.The PA signal intensity of OPI-NPs was higher than that of I-NPs.(P<0.05).Conclusion: We have successfully developed an oxygenate perfluorocarbon and Indocyanine Green-Loaded nanoparticles with uniforms distribution of particle size and enhances contrast in PA and ultrasound images in response to an external optical trigger.PART TWO: APPROPRIATE PARAMETER OF LASER,ULTRALSOUND FOR OPI-NPS ACTIVATION AND ICG CONCENTRATION FOR OPI-NPS TREATMENTObjective : Screening proper laser and ultrasonic parameters for nanoparticles activation and determine the concentration of encapsulated ICG in OPI-NPs for PDT.Methods:1.The laser parameter selection: The prepared OPI-NPs was diluted 20 times by double distilled water.The laser intensity and irradiation time divide into eight groups as follows: 0.5W/cm2,30s、0.5W/cm2,60s、 1.0W/cm2,30 s 、 1.0W/cm2,60 s 、 1.5W/cm2,30 s 、 1.5W/cm2,60 s 、 2.0W/cm2,30 s and 2.0W/cm2,60 s.After treatment,the morphologic of OPI-NPs was observed by bright field microscopy.2.The ultrasound parameter selection: The prepared OPI-NPs was diluted 20 times by double distilled water and were irradiated by laser(2.0W/cm2,30s).The ultrasound intensity and irradiation time divide into eight groups as follows: 0.44W/cm2,30s、 0.44W/cm2,60s、 0.66W/cm2,30s、 0.66W/cm2,60s、 0.88W/cm2,30s、 0.88W/cm2,60s、 1.10W/cm2,30 s and 1.10W/cm2,60 s.After treatment,the morphologic of OPI-NPs was observed by bright field microscopy.3.The selection of ICG concentration of OPI-NPs: Cultured SKOV3 cells were divided into six groups,which include control,0.5μg/ml,1.0μg/ml,2.0μg/ml,4.0μg/ml,8.0μg/ml.After co-culture of SKOV3 cell and the OPI-NPs for 24 h,the ratio of proliferation inhibition was detected by CCK-8.4.Activation of OPI-NPs by laser and ultrasound: OPI-NPs was diluted 20 times by double distilled water and took the same amount of them into a tube that put in a gel and irradiated by laser(2.0W/cm2,30s)and ultrasound(0.88W/cm2,30 s and 0.88W/cm2,60s).The morphology of OPI-NPs was observed by bright field microscopy,size distribution of OPI-NPs was detected by dynamic light scattering instrument,the tube image was observed by diagnosis ultrasound before and after treatment.5.To investigate the effection of laser and ultrasound on the proliferation of SKOV3 ovarian cancer cells: SKOV3 cells were divided into four groups,which include control,laser,ultrasound,laser and ultrasound.24 h after different treatment,the cell proliferation was detected by CCK-8.Results: 1.The OPI-NPs can occur phase transition when were irradiated by laser(1.5W/cm2)and the rate of phase transition was increased by extending the irradiation time.But the rate of phase transition has no obvious increased by extending the irradiation time when the OPI-NPs were irradiated by laser(2.0W/cm2).2.The OPI-NPs of phase transition have no obvious decreased when were irradiated by ultrasound(0.44W/cm2 and 0.66W/cm2).The OPI-NPs of phase transition can be effective smashed by ultrasound irradiation(0.88W/cm2 and 1.10W/cm2)and have no difference between irradiation 30 s and irradiation 60 s.3.The cell proliferation can be effective inhibited when the concentrations of encapsulated ICG in OPI-NPs was 8μg/ml.The cell proliferation haven’t been effected when the concentrations of encapsulated ICG in OPI-NPs equal to or less than 4μg/ml.4.The ultrasound image was enhanced and the particle size of OPI-NPs was increase from 275.24 nm to 1533.65 nm after laser irradiation.The ultrasound imaging attenuate and the average particle size decrease to 1060.56 nm and 771.38 nm after laser(2.0W/cm2,30s)and ultrasound irradiation for 30 s and 60 s,respectively.5.The selected laser(2.0W/cm2,30s)and ultrasound(0.88W/cm2,60s)parameter have no obvious effect on SKOV3 ovarian cancer cell proliferation.Conclusion: The OPI-NPs can successful transform nanoparticle into microbubble by laser(2.0W/cm2,30s)irradiation and the microbubble can be destructed by ultrasound irradiation(0.88W/cm2,60s).The selected laser(2.0W/cm2,30s)and ultrasound(0.88W/cm2,60s)parameter have no obvious effect on SKOV3 ovarian cancer cell proliferation.The cell proliferation haven’t been effected when the concentrations of encapsulated ICG in OPI-NPs equal to or less than 4μg/ml.The selected laser(2.0W/cm2,30s)and ultrasound(0.88W/cm2,60s)parameter and the concentration of encapsulated ICG in OPI-NPs will be applied to the follow-up studies.PART THREE: PHOTO-SONODYNAMIC SYNERGISM OXYGENATE PERFLUOROCARBON AND INDOCYANINE GREEN-LOADED NANOPARTICLES FOR INHIBITION OF OVARIAN CANCER SKOV3 CELLSObjective: To study the effect of photo-sonodynamic synergism oxygenate perfluorocarbon and indocyanine green-loaded nanoparticles on the proliferation of ovarian cancer SKOV3 cells and its mechanism.Methods: The effect of photo-sonodynamic synergism OPI-NPs on the proliferation of SKOV3 cells: Cultured SKOV3 cells were divided into six groups,which include ICG+L+U,I-NPs +L+U,PI-NPs +L+U,OPI-NPs +L+U,OPI-NPs +L and OPI-NPs +U.After different treatment,the ratio of proliferation inhibition was detected by CCK-8.2.The effect of different concentration of encapsulated ICG in OPI-NPs on the proliferation of SKOV3 cells: Cultured SKOV3 cells were divided into five groups,which include 0.25μg/ml,0.5μg/ml,1.0μg/ml,2.0μg/ml and 4.0μg/ml.After co-culture for 24 h with SKOV3 cell,exposed with laser(2.0W/cm2,30s)and ultrasound(0.88W/cm2,60s),the ratio of proliferation inhibition was detected by CCK-8 24 h after treatment.3.Detection of singlet oxygen in free cell system solution : 0.1ml samples and 0.02 ml of 50 m M SOSG were mixed in black 96-well plates and were divided into eight groups,which include control,PBS+L+U,ICG+L+U,I-NPs +L+U,PI-NPs +L+U,OPI-NPs +L+U,OPI-NPs +L and OPI-NPs +U;20 mins after different treatment,the oxidized SOSG was quantified by measuring the fluorescence intensity using a multifunctional microplate reader.4.Detection of singlet oxygen in free cell system solution with different concentration of encapsulated ICG in OPI-NPs synergism laser and ultrasound: 0.1ml samples and 0.02 ml of 50 m M SOSG were mixed in black 96-well plates and were divided into five groups,which include 0.25μg/ml,0.5μg/ml,1.0μg/ml,2.0μg/ml and 4.0μg/ml.20 mins after laser(2.0W/cm2,30s)and ultrasound(0.88W/cm2,60s)treatment,the oxidized SOSG was quantified by measuring the fluorescence intensity using a multifunctional microplate reader.5.Detection of intracellular singlet oxygen : Cultured SKOV3 cells were co-culture with 2μl DCFH-DA(10m M)for 20 mins and then divided into eight groups,which include control,PBS+L+U,ICG+L+U,I-NPs +L+U,PI-NPs +L+U,OPI-NPs +L+U,OPI-NPs +L and OPI-NPs +U.The fluorescence emission spectrums of DCF were immediately captured on a fluorescence microscope and measured the fluorescence intensity using a Flow Cytometry 20 mins after different treatment.Results:1.The cell proliferation inhibition rate of PI-NPs +L+U,OPI-NPs +L+U and OPI-NPs +L were(30.2 ±1.291)%,(77.24 ±0.4634)% and(59.06 ±2.003)%,respectively,which were higher than other treatment groups(P<0.05).The cell proliferation inhibition rate of OPI-NPs +L+U and OPI-NPs +L were higher than that of PI-NPs +L+U(P<0.05);The cell proliferation inhibition rate of OPI-NPs +L+U was higher than that of OPI-NPs +L(P<0.05).2.The cell proliferation inhibition rate was increased with the concentration of encapsulated ICG in OPI-NPs.The cell proliferation inhibition rate was maximum(72.04 ±5.273)% when concentration of encapsulated ICG in OPI-NPs reached 4μg/ml,which was significantly higher than the other concentration(P<0.05).3.The fluorescence intensity of singlet oxygen in free cell system solution of PI-NPs +L+U,OPI-NPs +L+U and OPI-NPs +L were 185.3±19.46,440.1±11.47 and 355.6±31.15,respectively.Which were significantly higher than the other treatment groups(P<0.05).The fluorescence intensity of OPI-NPs +L+U and OPI-NPs +L were higher than that of PI-NPs +L+U(P<0.05);The singlet oxygen intensity of OPI-NPs +L+U was higher than that of OPI-NPs +L(P<0.05).4.The fluorescence intensity of singlet oxygen in free cell system solution was increased with the concentration of encapsulated ICG in OPI-NPs.The fluorescence intensity of singlet oxygen was maximum(440.1 ±11.47)when concentration of encapsulated ICG in OPI-NPs reached 4μg/ml,which was significantly higher than the other concentration(P<0.05).5.The intracellular fluorescence intensity of singlet oxygen of PI-NPs +L+U,OPI-NPs +L+U and OPI-NPs +L were 1935±53.14,2939±300.2 and 2935±295.3,respectively,which were significantly higher than the other treatment groups(P<0.05).The intracellular fluorescence singlet oxygen intensity of OPI-NPs +L+U and OPI-NPs +L were higher than that of PI-NPs +L+U(P<0.05).But the intracellular fluorescence of singlet oxygen have no obvious difference between OPI-NPs +L+U and OPI-NPs +L(P>0.05).Conclusion: Our data provide evidence that laser and ultrasound combined with OPI-NPs inhibited proliferation of SKOV3 ovarian cancer cells.The mechanism may be related to improve extracellular and intracellular ROS production.