Preparation Of EGFRv?-targeted High Molecular Polymer Nanoparticles With Liquid Perfluorocarbon And Evaluate Its Properties And Targeting Ability

Objective: To prepare an epidermal growth factor receptor variant ?(EGFRv?)targeted high molecular polymer nanoparticles with liquid perfluorocarbon,and evaluate its physical properties and targeting ability in vitro.PART ONE VALIDATED THE SPECIFICITY OF EGFRV? MONOCLONAL ANTIBODY 4G1Methods: Cultivation of glioma cells F98EGFR(EGFR+/ EGFRv?-)and F98npEGFRv?(EGFR-/EGFRv?+),using cell protein for western blot experiment to verify the EFGRv ? specificity of antibody 4G1.Using F98 EGFR and F98 npEGFRv? cells for flow cytometry and immunofluorescence experiment to verify the EFGRv? specificity of antibody 4G1.Prepared 131I-4G1 by chloramine T,to explore the best time of stability combination between antibody 4G1 and cell lines.Results: 4G1 combined with EGFRv? positive protein in western blot bands,while not combined with EGFR protein.The combination rate of 4G1 and F98 npEGFRv? cell was(90.84 ± 0.56)%,that of F98 EGFR cell was only(2.90 ± 0.07)%.The nucleus of F98 np EGFRv? cell stained blue,and its cell membrane presented green fluorescence,while F98 EGFR cells only stained blue in nucleus,its cell membrane with nothing.131I-4G1 were prepared by chloramine T method,which binding rate with F98 npEGFRv? cell in 30 min,60 min,120 min,240 min and 480 min were(39.45 ± 0.06)%,(49.19 ± 0.48)%,(69.64 ± 0.19)%,(59.19 ± 0.63)% and(55.79 ± 0.69)%,and its binding rate at the same time points with the F98 EGFR cell respectively(2.91 ± 0.21)%,(2.97 ± 0.75)%,(2.95 ± 0.65)%,(2.96 ± 0.43)% and(3.02 ± 0.50)%.Conclusion: Antibody 4G1 can only specificity identify EGFRv? protein.131I-4G1 can't combine with EGFR positive cells,and that can combined with EGFRv? positive cells.After interaction of 120 min between 131I-4G1 and cells,its binding rate reached the best.PART TWO PREPARATION OF EGFRV?-TARGETED HIGH MOLECULAR POLYMER NANOPARTICLES NANOPARTICLES AND EVALUATE ITS PROPERTIESMethods: The polymer liquid perfluorocarbon nanoparticles(P-NPs)were prepared by a double emulsion solvent evaporation method.The P-NPs were conjugated with 4G1 by EDC/NHS to prepare a targeted ultrasound contrast agent(P-NPs-4G1).The properties and distribution of P-NPs-4G1 were observed by light microscope and scanning electron microscope,and its distribution of size and potential were determined by laser particle size instrument.Further,its binding rate was detected by FCM.The ultrasound imaging was observed by ultrasonic diagnostic apparatus,after irradiation by low intensity focused ultrasound(LIFU).The targeting ability of P-NPs-4G1 was evaluated by FCM and confocal microscope in vitro.Tumor in nude mice model were detected targeted ultrasound imaging ability of P-NPs-4G1 in vivo.Results: The prepared P-NPs-4G1 polymer nanoparticles have the appearance of milky white suspension,which are spherical and uniform in size under the light microscope,and spherical or spherical like under the electron microscope.The P-NPs-4G1 have the average size(563.8 ± 60.3)nm and potential(-32.4 ± 3.37)mV.The binding rate of P-NPs-4G1 reached up to(97.37 ± 1.57)%,which was detected by FCM.After irradiation by LIFU in vitro,the P-NPs-4G1 can be used to ultrasonic imaging.P-NPs-4G1 can bind to EGFRv? positive cells(F98npEGFRv?),while can't bind to EGFRv? negative cells(F98EGFR).Nude mice in tumor model,injected P-NPs-4G1 via tail vein,after irradiation by LIFU,had no ideal results of ultrasonic imaging in vivo.Conclusion: P-NPs-4G1 prepared in this study had stability characters,after irradiation by LIFU,the P-NPs-4G1 with liquid-gas phase transition after ultrasonic or thermal effect,can be used for ultrasonic imaging.In vitro,the P-NPs-4G1 also can specificity bind to EGFRv ? positive tumor cells.