The feasibility of a targeted ultrasound contrast agent carrying genes and cell-penetrating peptides to hypoxic HUVEC

Abstract

Objective To prepare an anti-P-selectin targeted ultrasound contrast agent carrying genes and cell-penetrating peptides (CPP) and to investigate its feasibility of delivery to hypoxic human umbilical vein endothelial cells (HUVEC).Methods Anti-P-selectin targeted ultrasound contrast agent carrying a green fluorescent protein gene (pEGFP-N1) and CPP was prepared by mechanical vibration and carbodiimide techniques.The appearance,distribution,concentration and diameter of the ultrasound contrast agent were measured.The gene and CPP distribution on the agent was investigated using confocal laser scanning microscopy (CLSM).The efficiency of the ultrasound contrast agent to carry the gene and CPP was investigated by fluorospectrophotometry.HUVEC were cultured in vitro and hypoxic HUVEC were prepared using hydrogen peroxide (H2O2 ).Hypoxic HUVEC were randomly assigned targeted ultrasound contrast agents and non-targeted ultrasound contrast agents for transfection.The transfection effect of green fluorescent protein in the two groups was observed using fluorescence microscopy and flow cytometry.T-test and linear correlation analysis were used for statistical analysis.Results The average diameter of anti-P-selectin targeted ultrasound contrast agents carrying gene and CPP was (2.15 ±0.36) μm and the concentration was ( 1.58 ± 0.23) × 107/ml.The results of CLSM showed that gene and CPP were distributed on the shell of the agent.The gene encapsulation efficiency was 28％ (y =0.932x - 0.09,r =0.993,P ＜ 0.05 ),and the CPP encapsulation efficiency was 25％ (y =5.875x -0.81,r =0.987,P ＜0.05).EGFP expression was observed using fluorescence microscopy in targeted ultrasound contrast agents and non-targeted ultrasound contrast agents.The average transfection efficiencies of targeted ultrasound contrast agents and nontargeted ultrasound contrast agents were ( 18.74 ± 0.47 ) ％ and ( 15.34 ± 0.22) ％ after 24 h ( t =10.923,P ＜ 0.001 ).Conclusions The in vitro studies showed that the targeted ultrasound contrast agent could significantly enhance the transfection efficiency in hypoxic HUVEC.This may be a new idea for gene therapy. Key words: Contrast media; Genes; Cell-penetrating peptide; Transfection; Endothelial cells; Ultrasonography