S13-5
Experimental study of Plasma-Liquid Interaction
プラズマ照射液相の実験的解析
Kenji Ishikawa, Naoyuki Kurake, Hiromasa Tanaka, Hiroshi Hashizume,
Kae Nakamura†, Hiroaki Kajiyama†, Fumitaka Kikkawa†, Masaaki Mizuno†,
Takashi Kondo‡, Keigo Takeda, Hiroki Kondo, Makoto Sekine, and Masaru Hori
石川健治, 倉家尚之、田中宏昌、橋爪博司、中村香江†、梶山広明†、
吉川史隆†、水野正明†、近藤隆‡、竹田圭吾、近藤博基、関根誠、堀勝Graduate school of Engineering, Nagoya University
Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
名古屋大学大学院工学系研究科 〒464-8603 名古屋市千種区不老町†Graduate school of Medicine, Nagoya University
65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan†
名古屋大学大学院医学系研究科 〒464-8603 名古屋市昭和区鶴舞65‡Toyama University2630 Sugitani, Toyama 930-0194, Japan‡
富山大学 〒930-0194 富山市杉谷2630
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1. Introduction
Cancer therapy by indirect plasma-activatedmedium (PAM) exposure has been reported someeffects on anti-proliferative activity against
cancer cells such as carcinoma and glioma. [1-3]Although the atmospheric pressure plasma (APP)
consists of various active particles such as
charged species, neutral species, radicals,
energetic photons and so on. Questions how
reactive species generated inside the PAM needs
to answer.
In this study, we focused on clarification ofgeneration of reactive species such as radicals inthePAM.
We
have
detectedelectron-spin-resonance (ESR) signals originating
generation of radicals in the PAM. From analysisof kinetic behavior of the ESR signals, we will
discuss about anti-proliferation effects on thebasis of experimental results for chemical
modification on the PAM.
2. Experimental
3 ml of cell culture medium (D-MEM+FBS+P/S)
in a microplate with 6 well was irradiated by theAPP for 3 min. First, HO* radical having highly
chemical reactivity was trapped by using
spin-trapping agent of 5,5-dimetyl-1-pyrroline-N-oxide (DMPO). Radical densities were analyzed
by referencing signal arisen from stable radical;
2,2,6,6-tetramethyl-4-hydroxy piperidine 1-oxyl(TEMPOL).
After the plasma treatment of medium, ESR
signals from OH adduct were observed clearly andits concentration was about 11.7 μM. This signal
decayed exponentially with reaching the initialvalue of 1.0 μM for 10 min. The amount of OHadducts and temporal behaviors on the PAM wereapparently different compared with plasma
activated water activated identically by the APP.
Therefore, the set of reactive species involving
HO* at least in the PAM may contribute on theeffect of anti-proliferation of cancer cells.
3. Results and discussion
After the plasma treatment of the culture medium
for 1 min, ESR signals from OH adduct wereobserved clearly as shown in Figure 1.
Quantitatively, concentration of the OH adduct wasestimated about 11.7 μM. The observation of OH
adduct in water treated plasma was agreed withprevious publications [4,5].
The temporal behaviors of signal intensity fromthe OH adduct were observed as shown in Figure 2.
For the culture medium, the signals were rapidly

4. Summary
We studied about generation mechanism ofactive species in the cell culture medium byexposing reactive species such as O atoms. The
relatively stable species generated in the plasma
activated medium (PAM). The concentration of
H2O2 increased linearly with elapsing of plasma
exposure periods with 15.7 μM for 1 min. On thebasis of experimental evidence of the generated
chemical species, we will discuss the effect of free
radicals generated in liquids by the treatments ofplasma, related on the biological response under
exposure of the plasma activated medium.

1DMPO-OH

0-10

DMPO-H isalso appeared

ESR intensity (arb. unit.)

ESR intensity (arb. unit.)

103480 3500 3520 3540 3560
Magnetic field (G)DMPO-OHDMPO-H0-1

3480 3500 3520 3540 3560
Magnetic field (G)Fig.1. ESR spectra of pure water (left) and culture
medium (right) treated plasma for 1 min.

Intensity of DMPO-OH (arb. unit.)

10 2

pure water

10 1
D-MEM

10 010 -10102030

Elapsed time after plasma exposure (min)Fig.2. Temporal behavior of signal intensity ofDMPO-OH adduct.
50
H2O2 concentration (µM)

decayed exponentially with reaching the initialvalue of 1.0 μM for 10 min. The amount of OH
adducts and temporal behaviors on the PAM wereapparently different compared with plasma
activated water activated identically by the APP.
For water, the generated HO· radical recombined
to form hydrogen peroxide (H2O2). Indeed, asshown in Figure 2, the decay of OH adduct wasapproximately 870s and agreed well in literature
value [6]. This indicates that HO·+HO·→H2O2
reaction dominated. On the other hand, Figure 3
shows H2O2 concentration in PAM as a function ofplasma exposure time. The H2O2 increased linearlywith increasing the exposure period. It isnoteworthy that absolute concentration of H2O2 isrelatively small of 1/10 compared with other reports[7-10]. This result of low H2O2 might be originatedfrom treatment with contactless of plasma onliquids. Atomic O and N atoms rather than charged
species might be effective generate the HO· radical
as a source of H2O2. Experimentally HO radical
generation in culture medium was detected,
however no NO radical was observed. Therefore,
the set of reactive species involving HO· at least in
the PAM may contribute on the effect ofanti-proliferation of cancer cells.

403020100012345

Plasma exposure period (min)Fig.3. Hydrogen peroxide concentration of PAM

Acknowledgments
This work was partly supported by aGrant-in-Aid for Scientific Research on Innovative
Areas, “Plasma Medical Innovation” (No.
24108002) from the Ministry of Education, Culture,Sports, Science and Technology of Japan. We
would like to thank Profs. Y. Okazaki, S. Toyokuni,and S. Maruyama for fruitful discussions.
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