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.

Figure 1.

Antibody binding to microbubbles. Arrows indicate the fluorescein isothiocyanate-conjugated antibody bound to microbubbles in the microbubble suspension (magnification, ×100).

Figure 2.

Enhanced green fluorescent protein-N3 plasmid construction. Lane 1, plasmid DNA; lane 2, plasmid DNA + PNA; lane 3, marker; lane 4, purified plasmid DNA + PNA. PNA, peptide nucleic acid.

Figure 3.

Flow cytometric analysis and a graph of the results following gene transfection in the three groups (group 1, 2 and 3). Histograms reveal the expression of EGFP by cells from: (A) Group 1, control; (B) group 1, CMCB + DNA; (C) group 1, AT-MCB + DNA; (D) group 2, control; (E) group 2, CMCB + DNA; (F) group 2, CMCB + NLS-PNA-DNA; (G) group 3, AT-MCB + DNA; (H) group 3, CMCB + NLS-PNA-DNA; and (I) group 3, AT-MCB + NLS-PNA-DNA. EGFP, enhanced green fluorescent protein; CMCB, ordinary microbubbles; AT-MCB, antibody targeted microbubbles; NLS, nuclear localization signal; PNA, peptide nucleic acid.

Figure 4.

Fluorescence of cells following gene transfection in the three groups (group 1, 2 and 3). Images reveal the expression of enhanced green fluorescent protein by cells from: (A) Group 1, control; (B) group 1, CMCB + DNA; (C) group 1, AT-MCB + DNA; (D) group 2, control; (E) group 2, CMCB + DNA; (F) group 2, CMCB + NLS-PNA-DNA; (G) group 3, AT-MCB + DNA; (H) group 3, CMCB + NLS-PNA-DNA; and (I) group 3, AT-MCB + NLS-PNA-DNA. EGFP, enhanced green fluorescent protein; CMCB, ordinary microbubbles; AT-MCB, antibody targeted microbubbles; NLS, nuclear localization signal; PNA, peptide nucleic acid (magnification, ×60).

Figure 5.

mRNA expression levels of EGFP-N3, as assessed using reverse transcription-polymerase chain reaction. Data are expressed as the mean ± standard deviation. EGFP, enhanced green fluorescent protein; CMCB, ordinary microbubbles; AT-MCB, antibody targeted microbubbles; NLS, nuclear localization signal; PNA, peptide nucleic acid.

Figure 6.

Protein expression levels of EGFP-N3, as assessed by western blot analysis. Data are expressed as the mean ± standard deviation. EGFP, enhanced green fluorescent protein; CMCB, ordinary microbubbles; AT-MCB, antibody targeted microbubbles; NLS, nuclear localization signal; PNA, peptide nucleic acid.

Figure 7.

Subcellular localization of the plasmids was observed using fluorescence microscopy. 293T cells were transfected with EGFP-N3 and were subsequently stained with DAPI, a fluorochrome that combines with DNA in the cell nucleus. (A-C) 293T cells were transfected with NLS-free EGFP-N3 plasmid DNA by UTMD. The cell nuclei contained no EGFP-N3. (D-F) 293T cells were transfected with NLS-PNA-EGFP-N3 plasmid DNA by UTMD. EGFP-N3 was present in the cell nuclei. EGFP-N3 plasmids appear green; DAPI appears blue. The boxed images present a typical single cell (magnification, ×100). EGFP, enhanced green fluorescent protein; NLS, nuclear localization signal; UTMD, ultrasound-targeted microbubble destruction; PNA, peptide nucleic acid.