Evaluation of plasmid DNA stability against ultrasonic shear stress and its in vitro delivery efficiency using ionic liquid [Bmim][PF6]

Abstract

The hydrophobic ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate [Bmim][PF₆] forms nanostructures with negatively charged plasmid DNA through electrostatic interactions. The formation of plasmid DNA/IL nanostructures was confirmed by measuring the zeta potential of plasmid DNA as well as plasmid DNA/IL nanostructures. The zeta potential of the nanostructures was positive, although plasmid DNA is negatively charged. The positive zeta potential is due to the complexation between plasmid DNA and positively charged ionic liquid [Bmim][PF₆]. The ability of ionic liquid [Bmim][PF₆] to protect plasmid DNA against ultrasonic shear stress was also investigated using an agarose gel electrophoretic assay and showed that ionic liquid stabilizes plasmid DNA against ultrasonication. The plasmid DNA and plasmid DNA/IL nanostructures were subjected to ultrasonic shear stress for different time periods and the biological functionality of pristine plasmid DNA (i.e., expression of the eGFP gene) as well as the self-assembled nanostructures was investigated in vitro using three different cell lines, COS7, HEK293 and HeLa. Ionic liquid [Bmim][PF₆] protected the plasmid DNA against ultrasonic shear stress and also enhanced gene transfection efficiency in vitro. Furthermore, the cytotoxicity of ionic liquid [Bmim][PF₆] was assayed in vitro using all three cell lines and the toxicity was very low. Therefore, the ionic liquid [Bmim][PF₆] stabilizes plasmid DNA against ultrasonic shear stress and also enhances its in vitro delivery efficiency.

Fig. 1. The stability of plasmid DNA, and plasmid DNA/IL nanocomplexes against ultrasonic shear stress was investigated using agarose gel electrophoresis assay. Both the plasmid DNA and plasmid DNA/IL nanocomplexes were subjected to ultrasonic shear stress for different time points. The plasmid DNA was exposed to ultrasonic shear stress for 0, 10, 20, 30, and 40 minutes. However, the plasmid DNA/IL nanocomplexes were exposed to ultrasonic shear stress for 0, 10, 20, 30, 40, 60, 90, and 120 minutes.

Fig. 2. Delivery of native plasmid DNA (pDNA), and pDNA exposed to ultrasonic shear stress for different time periods to COS7 cells. (a) pDNA; (b) pDNA was complexed with lipofectamine (LA). (c) pDNA was subjected to 10 min ultrasonic shear stress and complexed with LA; (d) pDNA was subjected to 20 min ultrasonic shear stress and complexed with LA; (e) pDNA was subjected to 30 min ultrasonic shear stress and complexed with LA; (f) pDNA was subjected to 40 min ultrasonic shear stress and complexed with LA; scale bar: 100 μm.

Fig. 3. Delivery of pDNA/IL nanocomplexes, and plasmid DNA/IL nanocomplexes subjected to ultrasonic shear stress for different time periods to COS7 cells. (a) pDNA/IL nanocomplexes; (b) pDNA/IL nanocomplexes were complexed with 2 μL lipofectamine (LA). (c) pDNA/IL nanocomplexes were subjected to 10 min ultrasonic shear stress and complexed with LA; (d) pDNA/IL nanocomplexes were subjected to 20 min ultrasonic shear stress and complexed with LA; (e) pDNA/IL nanocomplexes were subjected to 30 min ultrasonic shear stress and complexed with LA; (f) pDNA/IL nanocomplexes were subjected to 40 min ultrasonic shear stress and complexed with LA; (g) pDNA/IL nanocomplexes were subjected to 60 min ultrasonic shear stress and complexed with LA; (h) pDNA/IL nanocomplexes were subjected to 90 min ultrasonic shear stress and complexed with LA; (i) pDNA/IL nanocomplexes were subjected to 120 min ultrasonic shear stress and complexed with LA; scale bar: 100 μm.

Fig. 4. Delivery of plasmid DNA (pDNA), and pDNA/IL nanocomplexes to HEK293 cells. (a) Only pDNA; (b) pDNA was complexed with lipofectamine (LA); (c) pDNA/IL nanocomplexes complexed with LA. Scale bar: 100 μm.

Fig. 5. In vitro cytotoxicity assay for hydrophobic ionic liquid (IL) [Bmim][PF₆] to different cell lines. (a) COS7 cells; (b) HEK293 cells; (c) HeLa cells.