AVS 63rd International Symposium & Exhibition
    Plasma Processing for Biomedical Applications Focus Topic Tuesday Sessions
       Session PB+BI+PS-TuM

Paper PB+BI+PS-TuM11
Generation of Reactive Species in Medium Irradiated Laser-Induced-Plasmas

Tuesday, November 8, 2016, 11:20 am, Room 101A

Session: Plasma Processing of Biological/Biomimetic Surfaces
Presenter: Yukihiro Kurokawa, Nagoya University, Japan
Authors: K. Kurokawa, Nagoya University, Japan
N. Kurake, Nagoya University, Japan
K. Takeda, Nagoya University, Japan
K. Ishikawa, Nagoya University, Japan
H. Hashizume, Nagoya University, Japan
H. Tanaka, Nagoya University, Japan
H. Kondo, Nagoya University, Japan
M. Sekine, Nagoya University, Japan
M. Hori, Nagoya University, Japan
Correspondent: Click to Email
The non-equilibrium atmospheric pressure plasma (NEAPP) was irradiated to the cell culture medium as liquid. The antitumor effect, showing the selective killing effect for cancer cells without killing normal human cells, was reported [1,2]. This effect are considered to be caused by large amounts of reactive nitrogen and oxygen species (RONS) generated by the plasma. However, chemical reactions during transport of plasma in ambient to the liquid surface is complicated; therefore we have applied the laser-induced plasma.

Previously, we reported that the high ratio of NO2−/ H2O2, even in low H2O2 contained in the plasma activated medium [3]. However, relations of reactive species concentrations with antitumor effects have not been fully elucidated. Here, we focus on the concentrations of reactive species generated in culture media by the laser-induced plasma.

A Nd:YAG laser and harmonic generators (Quanta Ray Pro 230, Spectra Physics) provided the pulsed-laser light with a wavelength of 266 nm, a frequency of 30 Hz, a power at sample surface of 25 mW. The light was focused on the gas-liquid interface of ultrapure water or Dulbecco’s Modified eagle Medium (DMEM; cat. no. 5796; Sigma) by using plano-convex lens, made of synthetic quartz. 2 mL of the liquid was typically irradiated for 5 min. This is called as LPAM. Just after irradiation, H2O2 and NO2− concentrations were measured by using absorption that was measured by ultraviolet-visible near infrared spectrometer (V-650, JASCO). Moreover, HeLa cells were incubated in the LPAM and cell survival was measured after 24 h incubation. For analysis of killing mechanism, activated caspase3/7 as apoptosis marker (CellEvent Caspase-3/7) was measured after fluorescent staining by a fluorescent microscope.

The LPAM generated effectively H2O2 causing by photo-dissociation of water, hydroxyl radicals (･OH) works a precursor of H2O2 with the reaction of ･OH + ･OH →H2O2. Survival of HeLa cells in the LPAM was dependent on dilution of the LPAM and standard DMEM. We prepared the diluted LPAM for a half of killing of HeLa cells. After the cultivation for 24 h in the diluted LPAM, the caspase-3/7 activity of dead cells as apoptosis death was observed clearly. Notably, the cell-death was almost inhibited by catalase.

We will discuss on the generation mechanism of active species and the mechanism of antitumor effect of the LPAM with comparison of the PAM.

This work was partly supported by MEXT KAKENHI on Innovative Areas Grant no. 24108002.

[1] H. Tanaka et al., Plasma Medicine, 3, 265 (2013); [2] F. Utsumi et al., PLoS ONE, 4, e81576 (2013); [3] N. Kurake et al., Arch. Biochem. Biophys. (2016) doi:10.1016/j.abb.2016.01.011