Shota Sasaki

Highly efficient and minimally invasive transfection using time-controlled irradiation of atmospheric-pressure plasma

To develop a highly efficient and minimally invasive gene transfection method, the cells after direct plasma irradiation under various conditions are evaluated by simultaneous analysis of transfection efficiency and cell viability. As a result, the efficiency has a maximal value at a short plasma irradiation time (3–5 s) while maintaining a very high cell viability, and the volume of irradiated cell suspension changes the time dependence of the efficiency, which could be caused by the competition between the synergetic effects of reactive oxygen species and electric field stimulation, and membrane transport such as exocytosis which is the process of excretion.

Improvement of cell membrane permeability using a cell-solution electrode for generating atmospheric-pressure plasma

The cell membrane permeability, which is strongly related to gene transfection, is improved using a cell-solution electrode for generating atmospheric-pressure plasma (APP) just above the solution. In the case of the floating cells, the cell membrane permeability is significantly improved by the cell-solution electrode APP compared with the conventional diffusion type APP, because the distance between the plasma generation area and the cell solution surface becomes short, which could reduce the radial diffusion loss of the plasma irradiated to the cell suspended solution. In the case of the adherent cells, cell membrane permeability is found to be enhanced by the shorter distance between the solution surface and the adherent cells as well as using the cell-solution electrode, which means that the short-lived reactive oxygen species generated at the solution surface are essential for the improvement of cell membrane permeability.

Calcium influx through TRP channels induced by short-lived reactive species in plasma-irradiated solution

Non-equilibrium helium atmospheric-pressure plasma (He-APP), which allows for a strong non-equilibrium chemical reaction of O2 and N2 in ambient air, uniquely produces multiple extremely reactive products, such as reactive oxygen species (ROS), in plasma-irradiated solution. We herein show that relatively short-lived unclassified reactive species (i.e., deactivated within approximately 10 min) generated by the He-APP irradiation can trigger physiologically relevant Ca2+ influx through ruthenium red- and SKF 96365-sensitive Ca2+-permeable channel(s), possibly transient receptor potential channel family member(s). Our results provide novel insight into understanding of the interactions between cells and plasmas and the mechanism by which cells detect plasma-induced chemically reactive species, in addition to facilitating development of plasma applications in medicine.

Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization

Gas-liquid interfacial atmospheric-pressure plasma jets (GLI-APPJ) are used medically for plasma-induced cell-membrane permeabilization. In an attempt to identify the dominant factors induced by GLI-APPJ responsible for enhancing cell-membrane permeability, the concentration and distribution of plasma-produced reactive species in the gas and liquid phase regions are measured. These reactive species are classified in terms of their life-span: long-lived (e.g., H2O2), short-lived (e.g., O2•−), and extremely-short-lived (e.g., •OH). The concentration of plasma-produced •OHaq in the liquid phase region decreases with an increase in solution thickness (<1 mm), and plasma-induced cell-membrane permeabilization is found to decay markedly as the thickness of the solution increases. Furthermore, the horizontally center-localized distribution of •OHaq, resulting from the center-peaked distribution of •OH in the gas phase region, corresponds with the distribution of the permeabilized cells upon APPJ irradiation, whereas the overall plasma-produced oxidizing species such as H2O2aq in solution exhibit a doughnut-shaped horizontal distribution. These results suggest that •OHaq is likely one of the dominant factors responsible for plasma-induced cell-membrane permeabilization.

Highly gate-tuneable Rashba spin-orbit interaction in a gate-all-around InAs nanowire metal-oxide-semiconductor field-effect transistor

III-V semiconductors have been intensively studied with the goal of realizing metal-oxide-semiconductor field-effect transistors (MOSFETs) with high mobility, a high on-off ratio, and low power consumption as next-generation transistors designed to replace current Si technology. Of these semiconductors, a narrow band-gap semiconductor InAs has strong Rashba spin-orbit interaction, thus making it advantageous in terms of both high field-effect transistor (FET) performance and efficient spin control. Here we report a high-performance InAs nanowire MOSFET with a gate-all-around (GAA) structure, where we simultaneously control the spin precession using the Rashba interaction. Our FET has a high on-off ratio (104~106) and a high field-effect mobility (1200 cm2/Vs) and both values are comparable to those of previously reported nanowire FETs. Simultaneously, GAA geometry combined with high- κ dielectric enables the creation of a large and uniform coaxial electric field (>107 V/m), thereby achieving highly controllable Rashba coupling (1 × 10−11 eVm within a gate-voltage swing of 1 V), i.e. an operation voltage one order of magnitude smaller than those of back-gated nanowire MOSFETs. Our demonstration of high FET performance and spin controllability offers a new way of realizing low-power consumption nanoscale spin MOSFETs.

Characterization of plasma-induced cell membrane permeabilization: focus on OH radical distribution

Non-equilibrium atmospheric-pressure plasma (APP) is used medically for plasma-induced cell permeabilization. However, how plasma irradiation specifically triggers permeabilization remains unclear. In an attempt to identify the dominant factor(s), the distribution of plasma-produced reactive species was investigated, primarily focusing on OH radicals. A stronger plasma discharge, which produced more OH radicals in the gas phase, also produced more OH radicals in the liquid phase (OHaq), enhancing the cell membrane permeability. In addition, plasma irradiation-induced enhancement of cell membrane permeability decreased markedly with increased solution thickness (<1 mm), and the plasma-produced OHaq decayed in solution (diffusion length on the order of several hundred micrometers). Furthermore, the horizontally center-localized distribution of OHaq corresponded with the distribution of the permeabilized cells by plasma irradiation, while the overall plasma-produced oxidizing species in solution (detected by iodine-starch reaction) exhibited a doughnut-shaped horizontal distribution. These results suggest that OHaq, among the plasma-produced oxidizing species, represents the dominant factor in plasma-induced cell permeabilization. These results enhance the current understanding of the mechanism of APP as a cell-permeabilization tool.

Roles of charged particles and reactive species on cell membrane permeabilization induced by atmospheric-pressure plasma irradiation

As factors that influence cell membrane permeabilization during direct and indirect atmospheric-pressure plasma irradiation, charged particle influx, superoxide anion radicals (O2 −), and hydrogen peroxide (H2O2) in plasma-irradiated solution were evaluated. These are the three strong candidate factors and might multiply contribute to cell membrane permeabilization. In particular, a shorter plasma diffusion distance leads to the enhancement of the direct effects such as charged particle influx and further increase cell membrane permeability. In addition, O2 − dissipates over time (a life span of the order of minutes) in plasma-irradiated water, and the deactivation of a plasma-irradiated solution in term of cell membrane permeabilization occurs in a life span of the same order. These results could promote the understanding of the mechanism of plasma-induced cell membrane permeabilization.

Cold atmospheric plasma enhances osteoblast differentiation

This study was designed to assess the effects of cold atmospheric plasma on osteoblastic differentiation in pre-osteoblastic MC3T3-E1 cells. Plasma was irradiated directly to a culture medium containing plated cells for 5 s or 10 s. Alkaline phosphatase (ALP) activity assay and alizarin red staining were applied to assess osteoblastic differentiation. The plasma-generated radicals were detected directly using an electron spin resonance-spin trapping technique. Results show that plasma irradiation under specific conditions increased ALP activity and enhanced mineralization, and demonstrated that the yield of radicals was increased in an irradiation-time-dependent manner. Appropriate plasma irradiation stimulated the osteoblastic differentiation of the cells. This process offers the potential of promoting bone regeneration.

Apoptotic effects on cultured cells of atmospheric-pressure plasma produced using various gases

This study investigated the effects of low-temperature atmospheric-pressure plasma on various cells such as rat fibroblastic Rat-1 cell line, rat neuroblastoma-like PC12 cell line, and rat macrophage-like NR8383 cell line. The plasma was irradiated directly to a culture medium containing plated cells for 0?20 s. The applied voltage, excitation frequency, and argon or helium gas flow were, respectively, 3?6 kV, 10 kHz, and 3 L/min. Cell viability and apoptotic activity were evaluated using annexin-V/propidium iodide staining. Results showed that the low-temperature atmospheric-pressure plasma irradiation promoted cell death in a discharge-voltage-dependent and irradiation-time-dependent manner. Furthermore, different effects are produced depending on the cell type. Moreover, entirely different mechanisms might be responsible for the induction of apoptosis in cells by helium and argon plasma.

Atmospheric-pressure plasma-induced cellular responses in human colorectal adenocarcinoma Caco-2 cells: A study of comprehensive quantitative proteomics

[Purpose] The non-equilibrium atmospheric pressure plasma alters various cellular functions such as the membrane transport1 and the intracellular signaling2. While the plasma has recently emerged as a tool in cancer therapy, the mechanisms remain to be determined in comprehensive and quantitative manners. The purpose of this study was thus to investigate the plasma-induced cellular responses using the SWATHTM-based comprehensive quantitative proteomics. [Method] Membrane protein fractions and whole cell lysates were prepared from human colorectal adenocarcinoma Caco-2 cells, which were incubated with the plasma-irradiated physiological buffer or the normal buffer as control. The trypsin-digested peptides were comprehensively quantified with liquid chromatography-mass spectrometry (LC-MS/MS)-based SWATH™ method. The phosphorylated peptides were concentrated by the hydroxy acid-modified metal oxide chromatography followed by dephosphorization. Spectral peptide ion database of various proteomes was const ructed to identify the proteins in the SWATHTM analysis. The peak area intensities of each identified peptide were compared between the plasma-treated and non-treated group. [Results and Discussion] The expression levels of 1468 peptides in the membrane proteins of the plasma-treated Caco-2 cells were significantly changed by over 1.2-fold or less than 0.8-fold (p<;0.05). The identified proteins include transport proteins, receptors of macromolecules, and endocytosis-related proteins. T he abundance of phosphorylated 253 peptides was altered in the whole cell lys a t e s of the plasma-treated Caco-2 cells. The levels of phosphorylated peptides were decreased in the epithelial adherens signaling-related proteins. These changes in the phosphorylation profiles were distinguished from those in hydrogen peroxide-treated Caco-2 cells. The results support the belief that the atmospheric-pressure plasma would affect the membrane transport and intracellular signaling by changing the protein expression profiles in the plasma membrane and the phosphorylation levels of intracellular signaling-related proteins.