Objective Development of efficient delivery systems is critical to non-viral gene therapy which has great potential for cardiovascular diseases.Cytoplasmic and nuclear membrane barriers are the crucial factor for the inefficiency which hamper the import of foreign genes.Designating a special gene delivery system to transport exogenous genes can solve this barrier efficiently,which consists of ultrasound-targeted microbubbles and PNA (Peptide Nucleic Acid) binding NLS (Nuclear Localization Signal).Methods Ultrasound-targeted microbubble destruction (UTMD) and the PNA binding NLS were utilized to improve the cytoplasmic import of plasmids and the nuclear intake of the plasmid from the cytoplasm respectively.We conducted experiments using antibody-targeted microbubbles that specifically recognize the antigen receptor--SV40T in the membranes of 293t cells,thereby,the ultrasound microbubbles were gathered around 293t cell membrane.What's more, we inserted PNA with nuclear localization signal (NLS) in the EGFP-N3 plasmid DNA, which can localize the cell nucleus.Under optimize ultrasounds parameters,the nuclear import and gene expression efficiency of antibody-microbubble with PNA binding NLS were compared with those of antibody-targeted microbubble or PNA binding NLS to investigate the effect of antibody-targeted microbubble with PNA binding NLS on transfection.Results The results revealed that ultrasound and antibody-targeted microbubble delivery (UTMD) significantly enhanced the cytoplasmic intake of exogenous genes and still maintained high cell viability.In the group with PNA binding NLS plasmids, the nuclear import and gene expression of transfected cells were significantly higher than those transfected with common plasmids.Compared with the common microbubble and NLS-free plasmids,the quantity of pEGFP-N3 plasmids in the nucleus increased at least 3.2-fold and the expression of the genes was 2.0-fold greater.Conclusion These results demonstrate that UTMD combined with antibody-targeted microbubble and PNA binding NLS DNA significantly improve transfection efficiency by enhancing the cytoplasmic and nuclear import of exogenous plasmid DNA.