Sertraline-loaded Liposome For Dual-modality Imaging And Photothermal Combination Therapy Against Metastatic Clear Cell Renal Cell Carcinoma

PART ? PREPARATION,BASIC PROPERTIES AND CYTOTOXICITY OF LIPOSOME CONTAINING SERTRALINE HYDROCHLORIDE AND INDOCYCLINE GREENObjective To prepare a multifunctional liposome containing sertraline hydrochloride and indocycline green(ICG),to detect its basic physicochemical properties,and to observe its cytotoxicity.Methods 1.Preparation and basic properties of liposomes : Liposome loaded sertraline hydrochloride and ICG(Ser/ICG@Lip)was prepared by a film-dispersion and hydration-sonication method.Liposome loaded sertraline hydrochloride(Ser@Lip),and liposome loaded ICG(ICG@Lip)were prepared by the same way.The morphology of Ser/ICG@Lip was observed by optical microscope,while the nanostructure of Ser/ICG@Lipwas analyzed by transmission electron microscope.The size distribution and Zeta-potential of Ser/ICG@Lip,Ser@Lip and ICG@Lip were detected by a Laser Particle Size Analyzer System(Marvin particle size instrument).The content of sertraline hydrochloride and indocycline green in the nanoparticles were determined by ultraviolet spectrophotometry.The encapsulation efficiency(EE%)and encapsulation content(EC)of sertraline hydrochloride and ICG in the prepared liposomes were calculated respectively.The particle size of Ser/ICG@Lip which was dispersed in phosphate buffer solution(PBS)was recorded at pre-determined times(0,1,3,and 7d)to observe its stability.In addition,the cumulative release rate of sertraline hydrochloride in Ser/ICG@Lip was measured by dialysis method at different PH values(PH5.5 and 7.4)at specific time(3h,6h,9h,12 h,24h,36h)using a 3500 D dialysis membrane.2.Cytotoxicity of liposomes:Cell count kit-8(CCK-8)method was used to detect the cytotoxicity of Ser/ICG@Lip liposome in vitro.Ser/ICG@Lip,Ser@Lip,ICG@Lip,free sertraline hydrochloride and free ICG were added to 96-well plates incubated by metastatic clear renal cell cancer cells(Caki-1)at different concentrations according to the corresponding concentrations,and co-incubated for 24 hours.Adding serum-free medium to a cell group as the control group.Using CCK-8 method to calculate cell activity.All concentration groups were respectively made for five replication.Results 1.Characteristics of liposomes:The new nanoparticles containing sertraline hydrochloride and ICG,Ser/ICG@Lip,were successfully prepared by a film-dispersion and hydration-sonication method.The nanoparticles were dispersed well under an optical microscope,uniform in size and round in shape.The regular spherical nanostructure of the lipsome was observed under transmission electron microscope.The particle sizes of Ser/ICG@Lip,Ser@Lip,ICG@Lip were measured with the Marvin particle size analyzer,respectively 237.33±18.61 nm,180.07±10.00 nm,and 240.17±96.32 nm.The zeta potential was-25.93±1.20 mV,-36.47±4.74 mV and-59.07±4.40 mV,respectively.The EE% and EC of sertraline hydrochloride and ICG in Ser/ICG@Lip were 50.45% and 184 ?mol/l,98% and 0.49mg/ml,respectively.The size of Ser/ICG@Lip in PBS within one-week was almost unchanged.In addition,under a constant temperature of 37?,the release of sertraline hydrochloride from Ser/ICG@Lip was promoted by the slightly acidic environment,which showed an initial burst release and a slow release in the later stage.2.Cytotoxicity of liposomes:Ser/ICG@Lip nanoparticles showed good cellular safety and reduced cytotoxicity of free sertraline hydrochloride by CCK-8 assay.Conclusion 1.Liposome containing sertraline hydrochloride and ICG(Ser/ICG@Lip)was successfully synthesized and its basic properties were tested.2.Ser/ICG@Lip basically has almost no cytotoxicity,which lays a solid foundation for the application of such nano-biological agents in diagnosis and treatment,and can reduce the systemic toxicity of free sertraline hydrochloride effectively while achieving the effect of killing tumor cells.PART ? PHOTOTHERMAL EFFECTS AND DUAL-MODE IMAGING CAPABILITIES OF LIPOSOMES CONTAINING SERTRALINE AND INDOCYCLINE GREENObjective To study the photothermal effects of liposome containing sertraline and indocycline green(ICG),as well as their near infrared fluorescence imaging and photoacoustic imaging capabilities.Methods1.In vitro photothermal effect:Ser/ICG@Lip liposome of different concentrations were irradiated at different excitation densities of 808 nm laser to evaluate its photothermal properties.Ser@Lip,ICG@Lip,free ICG,and phosphate buffer solution(PBS)were used as controls.Using an infrared thermal-imaging camera to record temperature changes and infrared thermal images.2.Fluorescence imaging and photoacoustic imaging in vitro: Xenogen IVIS Spectrum imaging system was used for near infrared fluorescence imaging and signal intensity analysis.Ser/ICG@Lip dispersions of different concentrations were placed on 96-well plates,and the corresponding ICG concentration was 0.490-0.030mg/ml.All concentration groups were respectively made for three replication.In addition,the Ser/ICG@Lip photoacoustic imaging capability was evaluated using the Vevo LAZR ultrasonic imaging system,and corresponding photoacoustic images and photoacoustic signal intensity were collected.Ser/ICG@Lip dispersion at1.250mg/ml obtained the optimal imaging wavelength at the wavelength of680-970 nm.At the optimal imaging wavelength,the photoacoustic images and photoacoustic signal values of Ser/ICG@Lip suspension with different concentrations(1.250-0.039mg/ml)were obtained.Results1.In vitro photothermal effect:Ser/ICG@Lip of 1.25mf/ml achieved the highest temperature irridiated by 808 nm laser at power density of 2w/cm~2,up to 80?.Both free ICG and ICG@Lip had obvious photothermal effect,and the photothermal conversion effects of the two liposomes co-loaded with ICG was higher than that of free ICG,while PBS and Ser@lip had no obvious temperature change under the same conditions.2.In vitro dual mode imaging capability:Ser/ICG@Lip showed a trend of fluorescence signal decreasing with the increase of concentration in near infrared fluorescence imaging,but the relationship was not linear.Accordingly,Ser/ICG@Lip has the maximum absorbance and the optimal photoacoustic image at 825 nm.Under this wavelength,the photoacoustic signal of Ser/ICG@Lip was enhanced in a concentration-dependent manner.Conclusion Ser/ICG@Lip has good photothermal effect and can be used for near-infrared fluorescence and photoacoustic imaging.PART ? TARGETING OF LIPOSOME CONTAINING SERTRALINE AND INDOCYCLINE GREEN AND ITS CHEMO-PHOTOTHERMAL COMBINATION THERAPY AGAINST METASTATIC CLEAR CELL RENAL CELL CARCINOMAObjective To observe the targeting of Ser/ICG@Lip liposomes in vitro and to anatomize the chemo-photothermal associated combined killing effect of the nanoparticle on metastatic clear cell renal cell carcinoma.Methods1.In vitro cell uptake: The uptake of Ser/ICG@Lip by metastatic clear cell renal cell carcinoma(Caki-1)was observed by confocal laser scanning microscopy(CLSM)at the set time(0.5,1,2,3,4h).Quantifying and analyzing the fluorescence intensity of region of interest(ROI)was also done.2.In Vitro chemo-photothermal treatment of Caki-1 cells2.1 CCK-8 method was used to evaluate the killing effect of nanoparticle on target cells:Caki-1 cells were planted in 96-well plates,incubated with Ser/ICG@Lip at different concentrations for 4 hours,and then treated with808 nm laser at different power densities.Another 12 hours of co-incubation,using CCK-8 method to calculate cell activity.The control group was treated with serum-free medium,and each treatment group was made for three replication.To compare the effects of photothermal therapy,chemotherapy and chemo-photothermal combination therapy,seven groups were set:serum-free medium group,laser only group,Ser/ICG@Lip Only group,Ser/ICG@Lip+laser group,ICG@Lip only group,ICG@Lip+laser group and free sertraline hydrechloride group.All the above groups were treated with different ways and incubated for 4hours respectively.Three groups(laser only group,Ser/ICG@Lip+laser group,ICG@Lip+laser group)were treated with 808 nm laser for 5mins at a power density of 2w/cm~2.After another 12 h incubation,cell activity in each group was determined by CCK-8 assay.All treatment groups were made for three replication respectively.2.2 CLSM was used to observe the killing effect of nanoparticle on target cells: The caki-1 cells were grown in a laser confocal cell culture dish,and were grouped and treated as described before.After an additional12 hours of incubation,the cells were co-stained with Calcein acetoxymethyl ester(Calcein-AM)and Propidium iodide(PI),and the fluorescence images were observed and collected by CLSM.Results1.In vitro cell uptake:The uptake of Ser/ICG@Lip by Caki-1cells was time-dependent.A little uptake of liposome was observed at 0.5h and peaked at 4h.The fluorescence intensity of 4h and 3h showed no statistical difference,but it was still1.2-fold higher than that of the latter.2.In Vitro chemo-photothermal treatment of Caki-1 cells:When the ICG concentration in Ser/ICG@Lip was 0.49mg/ml,the cell survival rate remained extremely low with the increase of laser power density,while under irradiation by 808 nm laser at a power density of 2w/cm~2,the cell survival rate significantly decreased with the increase of ICG concentration in Ser/ICG@Lip.In experiments to explore the therapeutic effects of chemo-photothermal combination therapy,we found that when the concentration of Ser/ICG@Lip was 1.25mg/ml,cell survival was significantly reduced in three groups,Ser/ICG@Lip+laser group(as chemo-photothermal combination therapy group),ICG@Lip+laser group(as photothermal therapy group)and free sertraline hydrochloride group(as chemotherapy group).When Ser/ICG@Lip concentration was lower than0.625mg/ml,the chemo-photothermal combination therapy group' cell survival rate was obviously lower than the single treatment groups,while the difference between the photothermal treatment group and chemotherapy group was not significant.Three groups(i.e.laser only,Ser/ICG@Lip only and ICG@Lip only)had almost no lethal effect on tumor cells.Almost the same results were observed by CLSM.Conclusion The Ser/ICG@Lip can be rapidly and effectively uptaked by Caki-1 cells,and the liposome can produce chemo-photothermal combined killing effect on cells under the irradiation of 808 nm laser.