Development of a bio-electrospray system for cell and non-viral gene delivery

Abstract

Bio-electrospray technology is a very attractive tool for preparing scaffolds and depositing desired solutions on various targets by electric force. In this study, we focused on the application of a bio-electrospray (BES) technique to spray cells on the target and to simultaneously deliver genetic constructs into the cells, called non-viral gene delivery-based bio-electrospray (NVG-BES). Using this method, we tried to harvest the electric charge produced during electrospray for the cellular internalization of cationic polymer/DNA nanoparticles as well as the delivery of living cells on the desired substrate. Furthermore, we optimized the voltage, culture medium and polymeric cationic charges for high transfection efficiency and cell viability during NVG-BES. As a result, the solutions used during the NVG-BES process played an important role in improving transfection efficiency. We determined that a voltage of 10 kV with PBS as the spraying solution showed high transfection efficiency, probably due to the facilitation of cationic polymer/DNA nanocomplexes in cellular internalization and their subsequent expression. In conclusion, NVG-BES, as a novel method, is expected to deliver genes to cells and simultaneously deliver transfected cells to any substrate or scaffold.

Fig. 1. Non-viral gene delivery-based bio-electrospray (NVG-BES) system. (a) Schematic diagram of the NVG-BES system. The NVG-BES system facilitated introduction of DNA to cells and simultaneously delivered cells to a target. In this method, a cationic polymer was used as non-viral carrier with electric force in a bio-electrospray (BES) system to electrospray living cells onto a target. (b) NVG-BES system on a clean bench. (c) The syringe pump. (d) The high voltage generator.

Fig. 2. Cell viability assay: (a) cell morphology 1 day after BES at 0 kV, 10 kV, 15 kV, and 20 kV. (b) The results of the live/dead assay 1 day after bio-electrospray at 0 kV, 10 kV, 15 kV, and 20 kV (green: live cells, red: dead cells). The case of 0 kV is the negative control (no BES). The pictures were taken at 10× magnification. (scale bars = 100 μm), (c) WST assay of cultured cells 1 day after BES from 0 kV to 20 kV. The case of 0 kV is the negative control (no BES) (n = 3, p < 0.05. Columns with different letters are significantly different according to the Duncan test). (d) WST assay of cultured cells 1 day after BES from 0 kV to 10 kV. The case of 0 kV is the negative control (no BES). (n = 3, p < 0.05. Columns with different letters are significantly different, according to the Duncan test). The results of cell viability assay after BES showed that cells were fine under conditions at 10 kV of BES. However, cell viability was rapidly decreased above 10 kV of BES.

Fig. 3. DNA expression results using NVG-BES with several solutions (a)–(d) GFP expression after NVG-BES transfection with several solutions: (a) DW, (b) PBS, (c) DMEM (w/o FBS), (d) DMEM (w/FBS). GFP expression pictures were taken at 10× magnification (scale bars = 100 μm). (e) Luciferase activity after BES with several solutions (n = 3 p < 0.05. Columns with different letters are significantly different, according to the Duncan-test). Other groups were significantly different to the DW group (p < 0.001). The use of PBS as BES solution showed higher expression of GFP than the other solutions.

Fig. 4. DNA expression results using NVG-BES at various voltages (a)–(f) GFP expression results after bio-electrospray at different voltages: (a) only DNA, (b) 0 kV, (c) 5 kV, (d) 10 kV and (e) 15 kV. The 0 kV group received only PEI + DNA (no BES), and the case labelled only DNA received only DNA. GFP expression pictures were taken at 10× magnification (scale bars = 100 μm). At 10 kV of BES, GFP was expressed more frequently than other groups. (f) Luciferase assay results after BES at different voltages (n = 3, p < 0.05. Columns with different letters are significantly different according to the Duncan test). Other groups were significantly different (p < 0.001) than the only DNA group (N.C). The groups using PEI and those applying BES had increased expression of luciferase. In particular, BES at 10 kV had the highest expression level.

Fig. 5. Comparison of viability between BES with only DNA and BES with PEI & DNA (a) image of BESed cells after one day at 0 kV, 5 kV, 10 kV and 15 kV. The pictures were taken at 10× magnification (scale bars = 100 μm). (b) WST-1 results (n = 7, p < 0.05. Columns with different letters are significantly different, according to the Duncan test). The case of 0 kV is no BES. However, cell morphology showed no difference at 10 kV of BES, and cell viability of BES with PEI decreased significantly relative to groups of BES without PEI.

Fig. 6. Proposed NVG-BES mechanism. (a) At 0 kV, normal cellular uptake occurs. (b) At 10 kV, cell membrane permeability and cellular uptake are increased by induction of electric field. As a consequence, transfection efficiency increases. (c) Above 10 kV, cell membrane disruption and cell death happen due to excessive electric field.

Fig. 7. Delivery transfected cells on the scaffold using the BES transfection method. (a) Bio-electrospray on a PCL nanofiber scaffold. (b) The PCL nanofiber scaffold. (c) The results of delivering transfected cells on a PCL nanofiber scaffold using BES. GFP expression pictures were taken at 10× magnification (scale bars = 100 μm). It showed that BES transfected living cells and delivered them to a nanofiber scaffold at the same time.