Ultrasound-Targeted Microbubbles Combined with a Peptide Nucleic Acid Binding Nuclear Localization Signal Mediates the Transfection of Exogenous Genes by Improving both Cytoplasmic and Nuclear Import

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The development of an efficient delivery systems is critical for non-viral gene therapies used to treat cardiovascular diseases. The cytoplasmic and nuclear membrane barriers reduce delivery efficiency by impeding the import of foreign genes. Thus, a gene delivery system capable of transporting exogenous genes could improve gene therapies. We have employed a novel strategy involving ultrasound-targeted microbubbles and a PNA (Peptide Nucleic Acid) binding NLS (Nuclear Localization Signal). Ultrasound-targeted microbubble delivery (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 SV40T antigen receptor expressed on the membranes of 293t cells so that ultrasound microbubbles could be gathered around the 293t cell membranes. Furthermore, we inserted the PNA containing the nuclear localization signal (NLS) in the egfp-n3 plasmid DNA, which increased nuclear localization. The nuclear import and gene expression efficiency of the antibody-microbubbles with the PNA binding NLS were then compared with antibody-targeted microbubbles alone or a PNA binding NLS. We specifically investigated the effect of the antibody-targeted microbubbles containing the PNA binding NLS on transfection. The results indicate that ultrasound and antibody-targeted microbubble delivery (UTMD) significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability. We found the nuclear import and gene expression of combined microbubble and PNA transfected cells were significantly higher than cells transfected with antibody-targeted microbubbles or DNA with only the PNA binding NLS.The quantity of pEGFP-N3 plasmids present in the nucleus increased by more than 3.2-fold relative to the control mircrobubbles and NLS-free plasmids.The data indicate that the gene expression was 2.0-fold greater. These results demonstrate that UTMD combined with antibody-targeted microbubbles and a PNA binding NLS plasmid significantly improved transfection efficiency by enhancing both cytoplasmic and nuclear DNA import. It's a very promising method for noninvasive and effective delivering target genes or drugs to the targeted to treat cardiovascular diseases.