Study On In Vitro Imaging Of Targeting Fluorescent Nanoparticles And Their Mediated Photodynamics In Rat Choroidal Endothelial Cells

PART ? PREPARATION AND CHARACTERIZATIONS OF IR780 LOADED VEGFR2 TARGETING NANOPARTICLESObjective To prepare a novel IR780 loaded targeting VEGFR2nanoparticles(VEGFR2/IR780/PLGA).investigate their general properties,encapsulation rate of IR780 in nanoparticles and the conjugation rate between nanoparticles and VEGFR2 antibodies.Methods The non-targeting nanoparticles loaded with IR780(IR780/PLGA)were prepared by ultrasonic emulsification method.The anti-VEGFR2 antibodies was coupled with nanoparticle by carbodiimide method,finally the(VEGFR2/IR780/PLGA-NPs)were prepared.Optical microscope,laser confocal microscope,scanning electron microscope and transmission electron microscope were used to observe and detect the morphology and structure of nanoparticles.Particle size and potential weredetected by Malvern particle size analyzer.The absorption spectra of nanoparticles were detected by multifunctional enzyme marker.The encapsulation rate of IR780 in nanoparticles was detected by High Performance Liquid Chromatography(HPLC).The connection between the nanoparticles and VEGFR2 antibody was observed by laser confocal microscope;The conjugation rate between the nanoparticles and VEGFR2 antibody was detected by Flow cytometry.Results IR780 loaded targeting VEGFR2 nanoparticles(VEGFR2/IR780/PLGA-NPs)were successfully prepared.The nanoparticles were spherical and uniform in size under different microscopes.The mean particle size of the nanoparticles detected by the Malvern particle size and zeta potential analyzer was(273.23±31.55)nm.The average zeta potential was(-8.85±1.60)mV.The absorption spectrum of the nanoparticles detected by the multifunctional enzyme marker showed the maximum absorption peak at 780 nm,and the results showed that the IR780 has been successfully loaded in PLGA nanoparticles,the encapsulation rate of IR780 was(87.37±2.44)%.Laser confocal microscopy showed that VEGFR2 antibody was successfully connect to nanoparticles,And The conjugation rate between the nanoparticles and VEGFR2 antibody detected by flow cytometry was(83.60±4.79)%.Conclusion(VEGFR2/IR780/PLGA-NPs)was successfully prepared with uniform particle size,good dispersibility,high drug loading and high conjugation rate between the nanoparticles and VEGFR2 antibody.PART ? IN VITRO FLUORESCENCE IMAGING CAPABILITY AND PHOTODYNAMIC PERFORMANCE DETECTION OF IR780 LOADED VEGFR2 TARGETING NANOPARTICLESObjective To detect in vitro fluorescence imaging and photodynamic properties of(VEGFR2/IR780/PLGA-NPs).Methods Fetal bovine serum was used to prepare(VEGFR2/IR780/PLGA-NPs)with concentrations of(62.5,125,250,375,500?g/m L),the in vitro fluorescence imaging capability of(VEGFR2/IR780/PLGA-NPs)was detected by using IVIS fluorescent imaging system,The signal intensity was measured by the system software,the relationship between the intensity of the fluorescence signal and the concentration of nanoparticles was analyzed to evaluate its fluorescence imaging ability in vitro.Nanoparticles with concentrations of(62.5,125,187.5,250,375,500?g/m L)were irradiated by 808 nm laser(density50J/cm2,power 600 m W/cm2,time 83s),The singlet oxygen sensor green reagent(SOSG)was used to detect the reactive oxygen species yield of nanoparticles,and the relationship between the reactive oxygen species yield and the concentration of nanoparticles was analyzed photodynamic performance in vitro.Results Fluorescence imaging results showed that the fluorescence signal intensity increased as the concentration of(VEGFR2/IR780/PLGA-NPs)increased,it shows that nanoparticles have good fluorescence imaging ability.The SOSG showed that a large number of reactive oxygen species were generated after laser irradiation,and the production of reactive oxygen species gradually increased with the increase of the concentration of(VEGFR2/IR780/PLGA-NPs),so the nanoparticles had good photodynamic performance.Conclusion(VEGFR2/IR780/PLGA-NPs)have good fluorescence imaging capability and photodynamic performance in vitro.PART ? IN VITRO TARGETING AND PHOTODYNAMIC INDUCED APOPTOSIS OF RAT CHOROID VASCULAR ENDOTHELIAL CELLS WITH IR780 LOADED VEGFR2 TARGETING NANOPARTICLESObjective To culture rat choroid vascular endothelial cells(RCVECs)and detect the cytotoxicity of(VEGFR2/IR780/PLGA-NPs).To detect the targeting ability and the photodynamic therapy capability.Methods RCVECs cells were cultured,and The medium was used to prepare(VEGFR2/IR780/PLGA-NPs)with concentrations of(50,125,250,375,500?g/m L),and the cytotoxicity of nanoparticles at different concentrations was detected by cck-8 method.The nanoparticles were divided into targeted group,untargeted group,antibody blocked group and the control group,the targeting ability of the nanoparticles in vitro was evaluated by laser confocal and flow cytometry.The nanoparticles were divided into the targeted laser group,the untargeted laser group,the pure laser group and the control group.the proportion of photodynamic induced apoptosis in RCVECs was detected by Flow cytometry.Results RCVECs were successfully cultured.CCK-8 test showed that the survival rate of RCVECs was(91.78±1.79)%,(91.37±1.23)%,(89.25±1.96)%,(88.89±2.04)% and(88.74±2.30)%,There was no statistical difference between each group(P>0.05),indicated that the nanoparticles had no obvious cytotoxicity.The confocal laser showed that the number of nanoparticles around RCVECs in the targeted group was significantly higher than that in the untargeted group and the antibody blocked group.The connection rate between nanoparticles and cells in the targeted group detected by flow cytometry was(58.13±1.64)%,which was significantly higher than that in the untargeted group(20.64±1.89)% and the antibody blocking group(15.12±1.16)%,and the difference was statistically significant(P<0.05).The proportion of RCVECs apoptosis detected by flow cytometry in the targeted laser group was(82.99±0.98)%,which was significantly higher than that in the untargeted laser group(30.75±1.81)% the pure laser group(7.05±0.96)%,and the difference was statistically significant(P<0.05).Therefore,targeted nanoparticles have better PDT effect.Conclusion(VEGFR2/IR780/PLGA-NPs)have no obvious cytotoxicity to RCVECs,And It has good targeting ability,excellent photodynamic therapy effect in vitro.