Electrical Field-Assisted Gene Delivery from Polyelectrolyte Multilayers

Abstract

To sustain gene delivery and elongate transgene expression, plasmid DNA and cationic nonviral vectors can be deposited through layer-by-layer (LbL) assembly to form polyelectrolyte multilayers (PEMs). Although these macromolecules can be released for transfection purposes, their entanglement only allows partial delivery. Therefore, how to efficiently deliver immobilized genes from PEMs remains a challenge. In this study, we attempt to facilitate their delivery through the pretreatment of the external electrical field. Multilayers of polyethylenimine (PEI) and DNA were deposited onto conductive polypyrrole (PPy), which were placed in an aqueous environment to examine their release after electric field pretreatment. Only the electric field perpendicular to the substrate with constant voltage efficiently promoted the release of PEI and DNA from PEMs, and the higher potential resulted in the more releases which were enhanced with treatment time. The roughness of PEMs also increased after electric field treatment because the electrical field not only caused electrophoresis of polyelectrolytes and but also allowed electrochemical reaction on the PPy electrode. Finally, the released DNA and PEI were used for transfection. Polyplexes were successfully formed after electric field treatment, and the transfection efficiency was also improved, suggesting that this electric field pretreatment effectively assists gene delivery from PEMs and should be beneficial to regenerative medicine application.

Keywords: electrical field; gene delivery; layer-by-layer assembly; polyelectrolyte multilayer; polypyrrole.

Figure 1

The electrical field administration to PEMs. (a) Polypyrrole (PPy) was deposited on polystyrene surfaces to form a conductive substrate, which was trimmed in the dimension of 6 by 3 cm. (b) Glass rings with an inner diameter of 6 mm and a height of 7 mm were glued on PPy films. (c) For the vertically electrical field, PBS was filled in the wells without bubbles, and the stainless steel counter electrodes were placed on the top of the wells. (d) For the horizontally electrical field, electrodes were placed at two opposing sides of the PPy films.

Figure 2

The effects of electrical field modes on polyelectrolyte delivery from PEMs. (a) Schemes of three different modes of electrical field. (b) The releases of PEI and DNA during 1 h treatment of different electrical fields. (c) PEMs with 1 h treatment of electrical fields were monitored for their following delivery in aqueous environments for 48 h (n = 3; t-test compared to the results of the control group at the same time, * p < 0.05; ** p < 0.01).

Figure 2

The effects of electrical field modes on polyelectrolyte delivery from PEMs. (a) Schemes of three different modes of electrical field. (b) The releases of PEI and DNA during 1 h treatment of different electrical fields. (c) PEMs with 1 h treatment of electrical fields were monitored for their following delivery in aqueous environments for 48 h (n = 3; t-test compared to the results of the control group at the same time, * p < 0.05; ** p < 0.01).

Figure 3

The effects of the parameters of the vertically electrical field pretreatment on polyelectrolyte delivery from PEMs. (a) Ten voltage of electrical fields pretreated PEMs on PPy films with different electrical resistances for 1 h to determine the effect of electric currents. (b) Different voltages pretreated PEMs for 1 h to determine the effect of intensity of electrical fields. (c) Electrical field in 10 V pretreated PEMs for different durations to determine the effect of treatment duration. These electrical field-treated PEMs were monitored for their following delivery in aqueous environments for 48 h. (n = 3. For (b), t-test compared to the results of the 5 V pretreating group at the same time, * p < 0.05; ** p < 0.01. For (c), t-test compared to the results of the 1 h pretreated group at the same time, ** p < 0.01.).

Figure 4

Scanning electron microscopy analysis of PEMs. (a) Surfaces of PEMs before or after treating different voltages for 1 h. (b) Surfaces of PEMs in aqueous environments with or without electrical field treatment for 1 or 2 h. (scale bar = 1 µm).

Figure 5

Atomic force microscopy analysis of PEMs. (a) Surfaces of PEMs before or after treating different voltages for 1 h. (b) Surfaces of PEMs in aqueous environments with or without electrical field treatment for 1 or 2 h. Roughness averages (Ra) were also determined (n = 3, and t-test compared to the 0 V group, * p < 0.05) (c) The explanation scheme of surface roughness after vertically electrical field treatment. Entanglement of polyelectolytes caused uneven charge distribution, and these unstable regions were susceptible to electrical fields to demonstrated quick release. In addition, the thinner regions became closer to the electrode, which thus were released faster to eventually form bumps and nanoparticles on the PEM surfaces.

Figure 5

Atomic force microscopy analysis of PEMs. (a) Surfaces of PEMs before or after treating different voltages for 1 h. (b) Surfaces of PEMs in aqueous environments with or without electrical field treatment for 1 or 2 h. Roughness averages (Ra) were also determined (n = 3, and t-test compared to the 0 V group, * p < 0.05) (c) The explanation scheme of surface roughness after vertically electrical field treatment. Entanglement of polyelectolytes caused uneven charge distribution, and these unstable regions were susceptible to electrical fields to demonstrated quick release. In addition, the thinner regions became closer to the electrode, which thus were released faster to eventually form bumps and nanoparticles on the PEM surfaces.

Figure 6

The release of DNA and PEI from PEMs for transfection application. After electrical field pretreatment, PEMs were kept in PBS for 48 h and the supernatant was collected. (a) The supernatant was loaded to agarose gel for electrophoresis to determine the complexation efficiency of PEI to DNA. (b) To determine the sizes and surface charges of DNA/PEI polyplexes in supernatant, DLS analysis was performed. (c) The released polyplexes were applied to transfect NIH/3T3 cells. Because eGFP was encoded in plasmid DNA, transfected cells expressed green fluorescence. In addition, directly mixed DNA and PEI for complexation were applied as the control groups NP 1 and NP 2 to confirm whether the transfection efficiencies of DNA and PEI delivered from PEMs were similar to those prepared by the traditional complexation method. According to the results in Figure 4b, the NP 1 control group was prepared using DNA of 2.34 μg and PEI of 1.89 μg and the NP 2 control group was prepared using DNA of 3.82 μg and PEI of 3.46 μg, which were equal to those released from PEM pretreated by 0 V and 20 V, respectively. Fluorescent microscopy was performed after transfecting for 3 days, and the fluorescent images were analyzed using image software (scale bar = 200 μm; n = 3, t-test compared to the results of 0 V group, * p < 0.05; ** p < 0.01).