22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Effect of the radical species for gene transfection by discharge plasma
irradiation
Y. Ikeda1, H. Motomura1, Y. Kido1,2, S. Satoh1, 3, K. Tachibana4 and M. Jinno1
1
Department of Electrical and Electronic Engineering, Ehime University, 3 Bunkyo-cho, JP-790-8577 Matsuyama,
Ehime, Japan
2
Pearl Kogyo Co. Ltd., 3-8-13 Minami-Kagaya, Suminoe-ku, JP-559-0015 Osaka, Japan
3
Y’s Corporation, 2-31-17 Minamino, Tama-shi, JP-206-0032 Tokyo, Japan
4
Department of Electrical and Electronic Engineering, Osaka Electro-Communication University - 18-8 Hatsucho,
Neyagawa, JP-572-8530 Osaka, Japan
Abstract: The authors investigated the factors for plasma gene transfection by changing
protocols and looked at the time periods the factors become effective. The conclusion is
that nearly 10% of transfections occur during plasma irradiation and that the last 90% of
transfections occur after plasma irradiation is stopped. This second stage transfection is
caused not only by the residual chemically reactive species, but also by plasma irradiation
stress to cells and plasmids, i.e. possibly charging effect and oxidation stress.
Keywords: gene transfection, atmospheric plasma, micro plasma, radical
1. Introduction
Gene transfection is a technique of deliberately
introducing nucleic acids into cells in order to give them
specific characteristics. In practice, this can be achieved
in three different ways: chemical method, physical
method and the viral vector method. The chemical
method for gene transfection was developed first in 1973
by Graham using calcium phosphate [1]. In 1987 Felgner
developed the Lipofection method [2]. As a physical
method in 1982, Neumann developed electroporation.
With electroporation small holes are opened on the cell
membrane by applying an electric pulse to the cell
suspension and then the genes passes through those holes
[3]. Although these two methods are available only for in
vitro environments, the viral vector method is available
for both in vivo and in vitro environments, so it is
expected to be a practical method for gene therapy.
The electroporation method is the most common
technique in molecular biological and medical
experiments in laboratories. This method achieves a high
transfection rate, but it causes a lot of damage to the cells
resulting in more than half of them dying by necrosis or
apoptosis. The viral vector method provides both a high
transfection rate and a high cell survival rate. However,
the viral method has risks of pathogenicity expression or
neoplastic transformation. Therefore, the development of
a new safe and damage-free technique is in demand.
Such a technique would be expected to play an important
role in advanced medical technologies like gene therapy
and regenerative medicine through establishment of iPS
(induced pluripotent stem) cells. A technique that uses
discharge plasma irradiation was invented by Satoh, who
is one of the authors, and his group in 2002 [4] and was
reported by Ogawa in 2005 [5]. Since this technique is
free from adverse effect associated with viruses, there are
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no risks as the others mentioned above. The plasma
irradiation on genes and cells induces the transfection
process in which the genes and cells are exposed to
discharge current, that is, ions and electrons fluxes,
electric field, chemically active species such as radicals,
etc.
In the previous works [6-12], the authors have been
trying various types of plasma for gene transfection.
Recently, a small capillary copper tube with outer
diameter of 70 µm is used as a HV (high voltage)
electrode and a gas nozzle.
Consequently high
transfection rate and high cell survivability are achieved
simultaneously for plasmid. On the other hand, the
relationship between the plasma irradiation effects and the
working mechanism by the cell has not been clear yet.
Recently, the authors selectively investigate the effect of
radicals as surface interactions among radicals, plasmid
and cell membrane on transfection rate by washing out or
adding the plasmid after plasma irradiation [13]. As a
result, it is found that the transfection occurs not only
during plasma irradiation but also after plasma irradiation.
The result suggests the effects of residual radicals, the
radical-induced active species or surface charges after
plasma irradiation. In this paper, the authors more
specifically investigate the timing in which the above
species show the effect on the transfection.
2. Experimental Setup and Procedure
2.1. Experimental Setup
As the target COS7 cells, which are taken from an
African green monkey, are used. The COS7 cells are
cultured in each well. Then 0.8 µg of pCX-EGFP
plasmid diluted with Tris/EDTA (TE) buffer (0.4 µg/µl)
is used. The plasma irradiation device is shown in Fig. 1.
The high voltage (HV) electrode is made of copper, which
1
is a capillary with 70 m outer diameter and 20 µm inner
diameter. The grounded (GND) electrode is copper plate
and is placed on an XYZ-stage. A 96-well plate with
cells and plasmid is set on the GND electrode. The
distance from liquid surface of the well to the tip of the
capillary electrode was 1 mm.A custom-made HV power
supply (Pearl Kogyo) is used for discharge initiation. The
output voltage waveform is 20 kHz sinusoidal that was
pulse modulated at 25 Hz with a duty ratio of 1%. The
voltage and current waveforms are shown in Fig. 2. As
shown in this figure, a set of discharge consists of 3
groups of pulse, and the length of each group of pulse is
0.4 ms. The applied voltage between electrodes is 14 kV.
Fig. 1.
device.
Schematic diagram of the plasma irradiation
Applied Voltage /kV
15
10
5
0
-5
-10
Discharge Current /mA
-15
60
40
20
0
-20
Frequency of AC voltage: 20 kHz
Pulse Frequency: 25 Hz
Applying Time of pulse: 0.4 ms(Duty 1%)
-40
-60
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Time /s
a) Total period (3 groups of pulses)
Applied Voltage /kV
15
Frequency of AC voltage: 20 kHz
Pulse Frequency: 25 Hz
Applying Time of pulse: 0.4 ms(Duty 1%)
10
5
0
-5
-10
Discharge Current /mA
-15
60
40
20
0
-20
-40
-60
-0.5
0.0
b) One group pulse
0.5
1.0
1.5
2.0
2.5
Time /ms
Fig. 2. Voltage and current waveforms.
2
3.0
2.2. Plasma Treatment Procedure
In order to investigate the timing of transfection and
effective factors, the samples are treated in seven different
protocols as shown in Fig. 3. The Protocol 0 is just a
“Control”, that is, without plasma irradiation.
The
Protocol 1 is the standard treatment. In the Protocol 2,
there is no effective factor after plasma treatment (without
After Effect) by washing out with PBS. The Protocol 3 is
the treatment in which the plasmid is added after plasma
irradiation and washing out in Protocol 2. In the
Protocols 4 and 5, the plasmid does not exist during
plasma irradiation (After Effect Only). The plasmid is
added just after the plasma irradiation (Protocol 5) or is
added after the plasma irradiation and the washing out
(Protocol 4). In the Protocol 6, there is no plasma
irradiation on the cells, just like the Control protocol.
Instead, the GFP treated by plasma irradiation is poured.
The detailed processes in each protocol are as follows. The
COS7 cells are incubated at 37°C under 5% carbon dioxide
in D-MEM medium in Process (a). Before plasma
treatment, the medium is removed in Process (b). Then the
sample is washed by PBS (phosphate buffered saline, 0.1
M, pH 7.4) in Process (b). After the Process (b), in the
Protocols 0, 1, 2 and 3, GFP plasmid diluted with TE
buffer (0.4 µg/µl) is poured into each well in Process (d1).
Then in the Protocols 1, 2, 3 and 4, each well is treated by
the plasma irradiation in Process (e). After the Process (e),
in the Protocol 1 the medium is poured into each well in
Process (h). In the Protocols 2 and 3, after the Process (e),
GFP plasmid and TE buffer are removed from each well in
Process (f), and each well is washed by PBS and the PBS is
removed, finally the medium is poured into each well in
Process (h). After the Process (c) in the Protocols 4, 5
and 6, 4 µl of TE buffer without plasmid is poured into
each well in Process (d2). In the Protocols 4 and 5,
plasma treatment is done in Process (e). Following the
plasma treatment in the Protocols 4 and 5, the medium is
poured with the plasmid into each well in Process (f). In
the Protocol 6, after the Process (d2), GFP plasmid treated
by the plasma irradiation is poured into the well in Process
(g2). The medium is poured into each well in Process (h).
In each Protocol, after the Process (f) or (i), the samples
are incubated for 24 hours, then they are observed by
fluorescence microscope. The effect of plasma treatment is
investigated in comparison between Protocols 0 and 1.
By washing out with PBS immediately after plasma
treatment, the direct effect of a plasma is investigated in
comparison between Protocols 1 and 2. The interaction
between plasma-treated COS7 and untreated GFP plasmid
is investigated in comparison between Protocols 2 and 3.
The effect of treated GFP plasmid dropped at the process
(d1) is investigated in comparison between Protocols 3
and 4. By washing out with PBS after plasma treatment
(e), the indirect effect of the plasma irradiation to untreated
GFP plasmid is investigated in comparison between
Protocols 4 and 5. The only effect of plasma-treated GFP
plasmid is investigated in comparison between Protocols 6
and 1.
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Fig. 3. Detailed procedure of seven Protocols.
Fig. 4. Bright field and fluorescent microscope images of the plasma treated COS7 cells with GFP plasmid.
3. Results and Discussion
As shown in Fig. 5, the standard plasma irradiation
protocol (Protocol 1) shows 12.2% transfection rate,
whereas only 0.1% transfection without plasma
irradiation (Protocol 0). It is confirmed that the plasma
treatment is effective to gene transfection into cell. By
washing the plasma irradiated sample out (Protocol 2),
the transfection rated drastically dropped to 1.3%. This is
only 1/10 of that of Protocol 1. From this result, the
direct effect of the plasma irradiation (e.g., electric field,
discharge current, etc.) for the gene transfection is
estimated to be 1/10 of all the effects induced by plasma
irradiation. On the other hand, the indirect effects (after
effect) of the plasma irradiation (chemically reactive
species, electric charge and bio-chemical reactions)
occupies 9/10 of all the effects induced by plasma
irradiation. By adding GPF plasmids again after washing
out (Protocol 3), the transfection rate increases double
(2.3%) compared to that of Protocol 2. One possible
reason for this enhancement is that the stress by electric
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charge or chemically reactive species on the cells has an
“after effect”, which induces transfection of the GFP
plasmids that are dropped after washing out. However, if
GFP plasmids are not dropped before plasma irradiation
(Protocol 4), the transfection rate increases double again
(4.5%) compared to that of Protocol 3. A possible
reason of this enhancement is that existence of GFP
plasmids decreases the stress to the cells during plasma
irradiation. Another possible reason is that residual
chemical species exist even after washing out process and
their amount is not stable. If the washing out process is
omitted (Protocol 5), the transfection rate dropped to
0.6%. Besides the instability of washing out process
mentioned above, there is a possibility that washing out
process itself by PBS solution has some effect such as
endocytosis enhancement. Finally, to pour plasmairradiated GFP plasmids solution to the cell (Protocol 6)
does not result in the transfection (0.5%). From the
comparison between Protocols 1, 2, 5 and 6, the “after
effect” is effective only when both GFP plasmids and
3
cells are irradiated by plasma. Therefore, the “after
effect” is caused not only by the residual chemically
reactive species, but also by plasma irradiation stress to
cells and plasmids (possibly charging effect or oxidation
stress).
[6]
[7]
[8]
[9]
[10]
Fig. 5. Transfection Rate Rt2 for each Protocol.
[11]
4. Conclusion
The authors investigated the factors for plasma gene
transfection by changing Protocols and looked at the
periods the factors become effective. The conclusion is
that nearly 1/10 of transfections occur during plasma
irradiation and that the last 9/10 of transfections occur
after plasma irradiation is stopped. This second stage
transfection is caused not only by the residual chemically
reactive species, but also by plasma irradiation stress to
cells and plasmids, i.e., possibly charging effect or
oxidation stress. As a next step, quantitative investigation
of charging effect and assignment of residual chemically
reactive species is going on.
[12]
[13]
T. Okihiro, K. Ikeda, J. Matsuda, H. Motomura,
M. Jinno, K. Tachibana, S. Satoh and N. Saeki. in:
Ext. Abst. 72nd Autumn Meeting of JSPS. 31P-ZD6 (in Japanese) (2011)
T. Okihiro, J. Matsuda, K. Ikeda, H. Motomura,
M. Jinno, K. Tachibana, S. Satoh and N. Saeki. in:
Ext. Abst. 59th Spring Meeting of JSPS. 16P-B8-19
(in Japanese) (2012)
H. Motomura, J. Matsuda, K. Ikeda, T. Okihiro,
M. Jinno, K. Tachibana, S. Satoh and N. Saeki. in:
Proc. 4th Int. Conf. Plasma Medicine. (Orléans,
France) 76 (2012)
T. Okihiro, J. Matsuda, K. Ikeda, H. Motomura,
M. Jinno, K. Tachibana, S. Satoh and N. Saeki. in:
Proc. 4th Int. Conf. Plasma Medicine. (Orléans,
France) 74 (2012)
M. Jinno, T. Okihiro, H. Murakami, T. Yamasaki,
S. Shibakawa, H. Motomura, K. Tachibana, S. Satoh
and N. Saeki. in: Ext. Abst. 73rd Autumn Meeting of
JSPS. 12P-E1-10 (in Japanese) (2012)
M. Jinno, H. Motomura, S. Satoh, Y. Kido and
K. Tachibana. in: Abst. Int. Workshop on Control of
Fluctuation of Plasma Processing.
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Japan) 3B-WS-11 (2014)
M. Jinno, H. Motomura, Y. Ikeda, S. Satoh, Y. Kido
and K. Tachibana. J. Eur. Phys. Lett. (submitted)
(2015)
M. Jinno, Y. Ikeda, H. Motomura, Y. Kido,
K. Tachibana and S. Satoh. J. Photopolymer Sci.
Technol., 27, 399 (2014)
5. Acknowledgements
This work was partly supported by the Grant-in-Aid
(25108509) from JSPS and a grant from Ehime
University. The plasmids are provided by the INCS
Shigenobu of Ehime University.
6. References
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