Study on Control of Macromolecular Drug Transfer toEpithelial Cells Using Non-Invasive Microplasma
Ryo Yokoyama1, Sadia Afrin Rimi2, Ahmad Guji Yahaya2, Marius Blajan3, Jaroslav Kristof3, Kazuo Shimizu1,2,3
1Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, 432 8561, Japan
2Graduate School of Science and Technology, Department of Optoelectronics and Nanostructure Science, Shizuoka University, Hamamatsu, 432 8561, Japan
3Organization for Innovation and Social Collaboration, Shizuoka University, Hamamatsu, 432 8561, Japan

In recent years, drug introduction using Atmospheric pressure and low-temperature plasma has been studied as a method for introducing polymer drugs into epithelial cells.[1][2]In this study, with the miniaturization of the device in mind, we investigated using a film-type electrode that can irradiate a wide area using air at atmospheric pressure, unlike theconventional plasma jet. In this presentation, as a preliminary step to examine the introduction of drugs into normal cells, we report on the effects of plasma irradiation on normal
cells in the upper intestine using fluorescent dye.

Experimental method

Experimental conditions
Cell name
Cell characteristics
Culture form
Culture medium
Fluorescent dye
Medium volume
Carrier gas
Air Flow
Treatment timeFrequency
VoltageDistance

RCB0993 : IEC6
Rat small intestinal epithelial cell line
Adherent cells
DMEM (low glucose) + 5% FBS + 4μg/ml Insulin+Penicillin-Streptomycin Solution 100mg/ml
5μM YOYO-1, 5μM FD-4, 0.1 mM FD-1503ml
Dry Air
2 L/min
0 - 120 s
10 kHz940 V0-p
5 mm

Direct plasma irradiation(DPI)

Plasma-activated medium(PAM)

A method of irradiating theculture solution with the cells
adhered to the culture container.

A method of dropping a culture
solution that has been irradiatedwith plasma in advance into aculture vessel.

1hours(YOYO-1)
3hours(FD4,FD150)

24hours
Sowing(a) Surface(b) Cross section
Electrodes used in the experiment

Medium exchange
Plasma irradiation

Fluorescenceobservation

Applied voltage and current

Nitrogen active species produced in the liquid

Results

NO radical is highly reactive and forms NO2⁻ ,NO3⁻ in water. After that, it reacts with
H2O2 to produce peroxynitrite (ONOO⁻), which has high oxidizing power. [3]
Nitrogen active species produced in the liquid were quantitatively evaluated using anion chromatograph. An increase in the concentration of NO2⁻ and NO3⁻ in the liquid
was observed according to the plasma irradiation time.

Fluorescence intensity of YOYO-1

Fluorescent image of YOYO-1(Scale bar is 50 μm)

In the case of YOYO-1, there is no difference in fluorescence intensity
between direct irradiation(DPI) and Plasma-activated medium(PAM).
When the plasma was irradiated for 120 seconds, the fluorescence intensity
was about twice that of Cont.

Ion chromatograph measurement results

Amount of NO2⁻ and NO3⁻ generated

Oxygen active species produced in the liquid
OH radical improves cell membrane permeability and forms cell membranepores by oxidizing membrane lipids.[4]OH radical produced in the liquid was quantitatively evaluated using sodium
terephthalate (NaTA). An increase in the concentration of OH radicals in theliquid was observed according to the plasma irradiation time.

Fluorescence intensity of FD4 and FD150

Fluorescent image of FD4(Scale bars in a,b,c are 50 μm, and in d is 30 μm )In FD4, the fluorescence intensity increases with the irradiation time,and direct irradiation tends to be higher than indirect irradiation.
It is considered that FD150 was not introduced into the cells because
the fluorescence intensity did not change with the irradiation time.

Molecular structure and reaction
mechanism of NaTA and HTA

Amount of OH radicals generated

Conclusions• Up to 4 kDa drug uptake was observed in the cells by plasma irradiation. On the other hand, no uptake of FD150 was observed.
• Since the fluorescence intensity increases with the irradiation time, it is considered that the uptake of the drug is promoted by plasma irradiation.
• An increase in the amount of NO2-, NO3- and OH radicals produced were observed depending on the irradiation time.
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[2] T. Kaneko, S. Sasaki, Y. Hokari, S. Horiuchi, R. Honda , K. Makoto, “Improvement of cell membrane permeability using a cell-solution electrode for generating atmospheric-pressure plasma,” Science & Technology ofMaterials, Interfaces, and Processing, 2015.
[3]W. H. Koppenol, “THE CHEMISTRY OF PEROXYNITRITE, A BIOLOGICAL TOXIN,” Química Nova. 21 (3): 326–331, 1998.
[4]A. Zerrouki, M. Yousfi, A. Rhallabi, H. Motomura , M. Jinno, “Monte Carlo Poration Model of Cell Membranes for Application to Plasma Gene Transfection,” Plasma Process. Polym. 2016, 13, 633–648, 2016