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Objective To prepare a targeted ultrasound contrast agent (UCA) carrying gene and cell-penetrating peptide (CPP),and to evaluate their targeting affinity for hypoxia human umbilical vein endothelial cell (HUVEC) and gene transfection in vitro.Methods The targeted UCA carrying gene and CPP was prepared by mechanical vibration and carbodiimide techniques.The apprearance, concentration and diameter of the UCA were measured.Binding force of DNA and protective effect on DNA of targeted UCA carrying gene and CPP were detected by agarose gel electrophoresis.The gene and CPP distribution on the UCA were observed using confocallaser scanning microscopy (CLSM).The encapsulation efficiencies of the gene and CPP carried in UCA were investigated using fluorospectrophotometry.The connection of anti-P-selectin antibody with UCA was detected by immunofluorescent assay, and targeted UCA conjugation with hypoxia HUVEC was observed by light microscope and flow cytometry (FCM).In gene transfection experiments in vitro,the expression of enhanced green fluorescent protein (EGFP) was observed by fluorescence microscopy and gene transfection efficiency was detected by FCM.Results The average diameter of targeted UCA carrying gene and CPP was (2.15±-0.36)μm and the concentration was (1.58±0.23)×107/ml.Strong binding force of targeted UCA carrying gene and CPP with DNA was shown by agarose gel electrophoresis and DNA on the UCA was protected from nuclease hydrolysis.The results of CLSM showed that gene and CPP were distributed on the shell of UCA.The gene encapsulation efficiency was 28%, and the CPP encapsulation efficiency was 25%.Red fluorescence was observed on the surface of targeted UCA using immunofluorescent microscopy.The conjugation of the targeted UCA with hypoxia HUVEC was firm,while the conjugation in control group was negative.The result of FCM showed that 73.80% of the cells surfaces were combined with the targeted UCA,while 1.39% of the cells surfaces were combined with the UCA in the non-targeted group.24h after transfection, EGFP expression was observed by fluorescence microscopy both in targeted and non-targeted UCA groups.The result of FCM showed that gene transfection efficiency in targeted group was higher than that in non-targeted group.Conclusion The targeted UCA carrying gene and CPP was prepared successfully.It had a strong target binding capacity to hypoxia HUVEC and could enhance gene transfection, which maybe a novel strategy for targeted gene therapy.