Ultrasound-targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal mediate transfection of exogenous genes by improving cytoplasmic and nuclear import

Abstract

The development of an efficient delivery system is critical for the successful treatment of cardiovascular diseases using non-viral gene therapies. Cytoplasmic and nuclear membrane barriers reduce delivery efficiency by impeding the transfection of foreign genes. Thus, a gene delivery system capable of transporting exogenous genes may improve gene therapy. The present study used a novel strategy involving ultrasound-targeted microbubbles and peptide nucleic acid (PNA)-binding nuclear localization signals (NLS). Ultrasound-targeted microbubble destruction (UTMD) and PNA-binding NLS were used to improve the cytoplasmic and nuclear importation of the plasmid, respectively. Experiments were performed using antibody-targeted microbubbles (AT-MCB) that specifically recognize the SV40T antigen receptor expressed on the membranes of 293T cells, resulting in the localization of ultrasound microbubbles to 293T cell membranes. Furthermore, PNA containing NLS was inserted into the enhanced green fluorescent protein (EGFP)-N3 plasmid DNA (NLS-PNA-DNA), which increased nuclear localization. The nuclear import and gene expression efficiency of the AT-MCB with PNA-binding NLS were compared with AT-MCB alone or a PNA-binding NLS. The effect of the AT-MCB containing PNA-binding NLS on transfection was investigated. The ultrasound and AT-MCB delivery significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability. The nuclear import and gene expression of combined microbubble- and PNA-transfected cells were significantly greater compared with cells that were transfected with AT-MCB or DNA with only PNA-binding NLS. The quantity of EGFP-N3 plasmids in the nuclei was increased by >5.0-fold compared with control microbubbles (CMCB) and NLS-free plasmids. The gene expression was ~1.7-fold greater compared with NLS-free plasmids and 1.3-fold greater compared with control microbubbles. In conclusion, UTMD combined with AT-MCB and a PNA-binding NLS plasmid significantly improved transfection efficiency by increasing cytoplasmic and nuclear DNA import. This method is a promising strategy for the noninvasive and effective delivery of target genes or drugs for the treatment of cardiovascular diseases.

“PFH/AGM-CBA/HSV-TK/LIPOSOME-Affibody”: Novel Targeted Nano Ultrasound Contrast Agents for Ultrasound Imaging and Inhibited the Growth of ErbB2-Overexpressing Gastric Cancer Cells

“…In recent years, nano ultrasound contrast agents (UCAs) in combination with ultrasound-targeted microbubble destruction (UTMD) technology have been developed to improve the transfection efficiency of genes in gene therapies. 12–14 Nano UCAs as gene carriers can overcome the problem of traditional microbubbles (size of 1–5 μm), which are usually limited to the vascular pool. Due to the smaller particle size, typically in the range of 380−780 nm, Nano UCAs can enter the tumor tissue directly through the gap between tumor neovascular endothelial cells.…”