Experiment study on the transfection of exogenous genes promoted by ultrasound-targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal / 中华超声影像学杂志

ABSTRACT

Objective To increase the transfection of EGFP-N3 plasmids into 293T cells using ultrasound-targeted microbubbles delivery(UTMD) mediated a peptide nucleic acid (PNA) binding nuclear localization signal (NLS).Methods Antibody-targeted microbubbles were used in the experiments which can specifically recognize the SV40T antigen receptor.The SV40T antigen receptors were expressed on the membranes of 293T cells.The PNA containing the NLS were inserted in the EGFP-N3 plasmid DNA,which increased nuclear localization.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.The study was divided into five groupsContrast (group A),Common microbubble + DNA (group B),Antibody-targeted microbubbles + DNA (group C),Common microbubbles + NLS-PNA-DNA (group D),Antibody-targeted microbubbles + NLS-PNA-DNA (group E).Fluorescence microscope was used to observe the fluorescent light in each group;flow cytometry to test the transfection;RT-PCR and Western blot to detect genes' mRNA and protein expression level.Results Ultrasound and antibody-targeted microbubble delivery (UTMD) significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability(＞80％).Fluorescent microscope showed that the quantities of green fluorescence in cells were increased successfully.The transfection results of flow cytometry were 0,(9.30 ± 0.46)％,(26.46 ± 2.01)％,(29.54 ± 0.62)％,(45.72 ± 1.86)％,respectively,and the differences were statistically significant(P ＜0.05).The relative mRNA and protein expression in group E were greater than those in group C and D respectively (P ＜0.05).Conclusions UTMD combined with antibody-targeted microbubbles and a PNA binding NLS plasmid can significantly improve transfection efficiency of exogenous genes by enhancing both cytoplasmic and nuclear DNA import.