Search

Inefficient gene delivery poses a challenge for non-viral gene therapy. Cytoplasmic and nuclear membrane barriers are responsible for the inefficiency as they restrict the import of exogenous genes. The present study aimed to improve the transfection efficiency using a novel gene delivery system, which consisted of two components: ultrasound microbubbles and the nuclear factor κB (NFκB) binding motif. Ultrasound-targeted microbubble destruction (UTMD) was used to enhance the cytoplasmic import of plasmids and the NFκB binding motif was added to promote the nuclear intake of the plasmid from the cytoplasm. In the present study, human umbilical vein endothelial cells were transfected using UTMD with two different Cy3-labeled plasmids, phSDF-1α and phSDF-1α-NFκB. phSDF-1α-NFκB was constructed by inserting a specific DNA targeting sequence (five optimal repeats of the binding motif for the inducible transcription factor NFκB) into phSDF-1α. The nuclear import and gene expression efficiency of phSDF-1α-NFκB were compared with those of phSDF-1α to investigate the effect of the NFκB binding motif on transfection. The results showed that UTMD significantly increased the cytoplasmic intake of pDNA and maintained high cell viability. The nuclear import and gene expression of phSDF-1α-NFκB-transfected cells were significantly higher than those transfected with phSDF-1α. Compared with the NFκB-free plasmids, the quantity of NFκB plasmids in the nucleus increased 6.5-fold and the expression of SDF-1α was 4.4-fold greater. These results suggest that UTMD combined with NFκB binding motif significantly improve transfection efficiency by enhancing the cytoplasmic and nuclear import of exogenous plasmid DNA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]