Preparation And Imaging Investigation Of Dual-Targeted Poly(Lactic-Co-Glycolic Acid)Ultrasound Contrast Agent Carrying Anti-HER2 And VEGFR2 Antibodies

Purpose: To prepare the dual-targeted PLGA ultrasound contrast agent carrying anti-HER2 antibody and anti-VEGFR2 antibody,and explored its targeting ability and the effects ultrasound imaging in vitro and in vivo.Material and methods: The anti-HER2 antibody and anti-VEGFR2 antibody were conjugated to the PLGA NPs in the carbodiimide method to prepare the dual-targeted ultrasound contrast agent.The searching target ability of dual-targeted contrast agent in vitro was assessed by the laser scanning confocal microscope(LSCM)and the flow cytometry instrument detects its rate of combination with targeted cells.Prepare four kinds of nano-polymer contrast agent: PLGA NPs UCAs(MBC),carrying anti-HER2 antibody PLGA NPs UCAs(MBH),carrying anti-VEGFR2 antibody PLGA NPs UCAs(MBV),carrying anti-HER2 antibody and anti-VEGFR2 antibody dual targeted PLGA NPs UCAs(MBD),and investigate the imaging ability in vitro at 22 MHz and 40 MHz ultrasound in agarose gel.Established nude subcutaneous xenograft model of breast cancer and they were randomly divided into four groups--MBC,MBH,MBV,MBD.Obtain the 22 MHz ultrasound images of the tumor or muscle without injection contrast agents,before blasted and substraction.Inject the agents with 0.04 ml dose in the tail vein.PLGA NPs UCAs could penetrate into the tissue across blood vessels in the tumor area and could imaging.MBD had better targeting ability and can specifically enhancing tumor blood vessels and tissue.The results of target imaging of MBD were better than other PLGA NPs UCAs.Immunofluorescence staining showed that the expression of HER2 and VEGFR2 in breast tumors of nude mice.Results: The dual-targeted PLGA NPs with anti-HER2 antibody and anti-VEGFR2 antibody was firmly attached to the targeted cells,and the binding rates were all over 70%.Under 22 MHz,1 mg/ml and 40 MHz contrast agents ultrasonic imaging effect was good in vitro.The green stripe dot number of MBD was significantly more than other groups and the distribution was more uniform in the tumor area.MBD had the highest average signal strength(137.0?5.57)dB.Immunofluorescence staining showed that the expression of HER2 and VEGFR2 in breast tumors of nude mice.Conclusions: It was successfully to prepare the PLGA dual-targeted ultrasound contrast agent with anti-HER2 antibody and anti-VEGFR2 antibody.It had good targeting ability and the effects ultrasound imaging in vitro and in vivo.