Preparation And In Vitro Study On VEGFR2-targeted PA/US Dual-mode Contrast Agent

PART I PREPARATION AND CHARACTERIZATION OF VEGFR2-TARGETED PA/US DUAL-MODE CONTRAST AGENTObjective To prepare a VEGFR2-targeted photoacoustic/ultrasound dual-mode contrast agent, investigate it’s general properties and optical droplet vaporization effect in vitro.Methods Targeted agent loaded with India ink and perfluorohexane were prepared by multi-emulsion technology and conjugated with VEGFR2 monoclonal antibody by EDC/NHS. Its physicochemical properties were observed, and its conjugation rate of antibody was detected by LSCM and FCM. After irradiated by laser, its optical droplet vaporization were observed by optical microscope and its photoacoustic signal during the vaporization was detected by Vevo? LAZR photoacoustic system.Results The Vi-PFH-PLGA were spherical, with average particle size of(565.1±15.6)nm and potential of(-15.8±3.21)m V, respectively. The conjugation of nanoparticles with antibody was demonstrated by indirect immunofluoreseence assay and its binding rate reached up to 99.72%. After irradiated by laser, the phase-transition could be observed by the optical microscope. And the amplitude of the photoacoustic signal detected by the photoacoustic system increased rapidly to a high level,then gradually declined to a steady level as respect to irradiation exposure time.Conclusions The VEGFR2-targeted PA/US dual-mode contrast agent was prepared successfully. It has uniform size, high connection rate of antibody, excellent photoacoustic and optical droplet vaporization properties, which provide a good experimental base for further targeted research and PA/US imaging.PART II VEGFR2-TARGETED PHOTOACOUSTIC /ULTRASOUND DUAL-MODE CONTRAST AGENTSFOR PA/US DUAL-MODE IMAGING AND TARGETING STUDYObjective To observe the targeting and dual-mode imaging capability of VEGFR2-targeted photoacoustic/ultrasound dual-mode contrast agent in vitro. Methods The VEGFR2 targeted PA/US dual-mode contrast agent labeled with Di I were prepared. Its targeting capability to HUVEC were assessed by LSCM, with non-targeted group and free antibody intervention group acted as controls. After irradiated by laser for 2min,its PA/US imaging were observed, and compared with ink group and PFH-PLGA group.Results Under LSCM scope, there was a number of Vi-PFH-PLGA conjugated and crowded around the HUVEC tightly; while no specific binding was found in the non-targeted and free antibody intervention group. After the pulsed laser irradiation, strong PA and US signals were detected, and its US signal increased significantly compared with before irradiation(P <0.05).Conclusions The VEGFR2-targeted PA/US dual-mode contrast agent could targeted to HUVEC specifically and enhanced PA/US imaging significantly, which provides a good foundation for deeper research work.