Preparation Of HER2-targeted, Phase-shift Polymer Ultrasound Contrast Agent And In Vitro Experiment

PART I REPARATION OF HER2-TARGETED ANDPHASE-SHIFT POLYMER ULTRASOUND CONTRAST AGENTSAND STUDYING IT’S CHARACTERISTICSObjective To prepare a novel extravascular HER2-targetedpoly(1actic-co-glycolic acid, PLGA)-COOH ultrasound contrast agent withliquid perfluorocarbons: PFH-PLGA-tra, and to observe it’s generalproperties and it’s effects on ultrasound imaging in vitro.Methods Polymeric nanocapsules of liquid perfluorocarbons wereprepared by the single emulsion technique and conjugated withtrastuzumab monoclonal antibody by EDC/NHS. The generalcharacteristics were observed. The conjugation was demonstrated byimmunofluorescence and fluorescence-activated cell sorting (FACS) invitro.The encapsulation of liquid fluorocarbons was detected by heating invitro. The effects on ultrasound imaging were observed in vitro.Results The mean diameter of agents was (261±28) nm. The averageZeta potential was (-2.92±0.83) mv. The targeted nanocapsules were positive in immunofluorescence. In the targeted study. The rate of antibodybinding was as high as98.07％. Droplet-to-bubble transition experiment invitro showed that there were a lot of nano/microbubbles appeared innanocapsules formulations at80℃, while there were no nano/microbubblesformed by heating in control groups at80℃. The agents presented a goodultrasound imaging in vitro.Conclusions The HER2-targeted polymeric nanocapsules of liquidperfluorocarbons are successfully prepared, which may become a novelextravascular targeted ultrasound contrast agents. PART Ⅱ TARGETING STUDY AND EFFECT OFHER2-TARGETED POLYMER ULTRASOUND CONTRASTAGENT ON HUMAN BREAST CANCER CELLS IN VITROObjective To prepare a kind of HER2-targeted and phase-shift polymerultrasound contrast agent, and to investigate its targeting performance invitro and it’s effect in inhibiting cell proliferation of humanHER2-positive breast cancer cells.Methods To prepare HER2-targeted and phase-shift polymer ultrasoundcontrast agent by single emulsion and carbodiimide technique. To evaluate its targeting performance by being incubated with human HER2-positivebreast cancer cells lines(MCF-7) in vitro and compared with non-targetedand free trastuzumab intervention group. Inhibition ratios and apoptosisrate to human HER2-positive breast cancer cells(MCF-7) were assayed bythemethod of MTT and flow cytometry respectively.Results The HER2-targeted and phase-shift polymer ultrasound contrastagent were prepared successfully. The targeting study in vitro showed that anumber of HER2-targeted nanocapsules conjugated to MCF-7cells tightly,the connection rate was as high as (98.51±1.32)%, which was higher thannon-targeted group and the free trastuzumab intervention group. Theinhibition ratios on treatment of HER2-targeted nanocapsules group weresignificantly higher than the non-targeted group and the control grouprespectively (p<0.05). Flow cytometry showed that apoptosis rate oftargeted contrast agent group[(38.67±1.44)%] on MCF-7cells wassignificantly higher than non-targeted contrast agent group [(6.91±0.52)%]and blank control group [(3.20±0.21)%] respectively, the differences werestatistically significant (P <0.05).Conclusions The HER2-targeted and phase-shift polymer ultrasoundcontrast agent can targeted to human HER2-positive breast cancer cellsspecifically and effectively in vitro and also has the ability to inhibite cellproliferation of human HER2-positive breast cancer cells. It will hopefullybe an ideal molecular probe for the targeted imaging and treatment of HER2-positive carcinoma.