Clarification of electrical current importance in plasma gene transfection by equivalent circuit analysis.

We have been developing a method of plasma gene transfection that uses microdischarge plasma (MDP) and is highly efficient, minimally invasive, and safe. Using this technique, electrical factors (such as the electrical current and electric field created through processing discharge plasma) and the chemical factors of active species and other substances focusing on radicals are supplied to the cells and then collectively work to introduce nucleic acids in the cell. In this paper, we focus on the electrical factors to identify whether the electric field or electrical current is the major factor acting on the cells. More specifically, we built a spatial distribution model that uses an electrical network to represent the buffer solution and cells separately, as a substitute for the previously reported uniform medium model (based on the finite element method), calculated the voltage and electrical current acting on cells, and examined their intensity. Although equivalent circuit models of single cells are widely used, this study was a novel attempt to build a model wherein adherent cells distributed in two dimensions were represented as a group of equivalent cell circuits and analyzed as an electrical network that included a buffer solution and a 96-well plate. Using this model, we could demonstrate the feasibility of applying equivalent circuit network analysis to calculate electrical factors using fewer components than those required for the finite element method, with regard to electrical processing systems targeting organisms. The results obtained through this equivalent circuit network analysis revealed for the first time that the distribution of voltage and current applied to a cellular membrane matched the spatial distribution of experimentally determined gene transfection efficiency and that the electrical current is the major factor contributing to introduction.

Investigation of plasma induced electrical and chemical factors and their contribution processes to plasma gene transfection.

This study has been done to know what kind of factors in plasmas and processes on cells induce plasma gene transfection. We evaluated the contribution weight of three groups of the effects and processes, i.e. electrical, chemical and biochemical ones, inducing gene transfection. First, the laser produced plasma (LPP) was employed to estimate the contribution of the chemical factors. Second, liposomes were fabricated and employed to evaluate the effects of plasma irradiation on membrane under the condition without biochemical reaction. Third, the clathrin-dependent endocytosis, one of the biochemical processes was suppressed. It becomes clear that chemical factors (radicals and reactive oxygen/nitrogen species) do not work by itself alone and electrical factors (electrical current, charge and field) are essential to plasma gene transfection. It turned out the clathrin-dependent endocytosis is the process of the transfection against the 60% in all the transfected cells. The endocytosis and electrical poration are dominant in plasma gene transfection, and neither permeation through ion channels nor chemical poration is dominant processes. The simultaneous achievement of high transfection efficiency and high cell survivability is attributed to the optimization of the contribution weight among three groups of processes by controlling the weight of electrical and chemical factors.