Single Cellular and Endoscopic Micro-plasma Cancer Therapy
Jae Young Kim1,3, John Ballato2,3, Paul Foy2,3, Thomas Hawkins2,3, Yanzhang Wei4, Jinhua Li4,
and Sung-O Kim1,3
1
Holcombe Department of Electrical and Computer Engineering, Clemson University, Clemson, USA
2
School of Material Science and Engineering, Clemson University, Clemson, USA
3
Center for Optical Materials Science and Engineering Technologies (COMSET), Clemson University, USA
4
Department of Biological Sciences, Clemson University Biomedical Institute, Clemson University, USA
Abstract: A highly flexible micro-plasma jet device made of hollow glass optical
fiber having an inner diameter of 15 µm is reported. Even at this small dimension
and given the extremely low gas flow rate, the generated micro-plasma jet is
shown to induce apoptosis of cultured tumor cells in a dose-dependent manner.
Murine melanoma tumor cells were found to be more sensitive to plasma
treatments than murine fibroblast cells at constant plasma dose conditions. This
work enables new directed cancer therapies based on highly flexible microplasma device and offers the micro-plasma cancer endoscopy as a physical
cancer therapeutic method.
Keywords: Atmospheric pressure plasma, Cancer therapy, Micro-plasma jets,
Plasma medicine
1. Introduction
The use of atmospheric pressure plasmas in
cancer therapies is becoming attractive since
plasma contain short-lived free radicals,
including reactive oxygen species (ROS), that
can induce apoptosis in tumor cells [1-5]. For
the precise treatment of tumor cells with
plasmas, it would be preferred to have a small
plasma device that can treat tumor cells at the
single-cellular-level. Particularly, the flexible
micro-plasma jet device can be utilized in vivo
for targeting individual tumor cells or other cells
in need of plasma treatment in an area where
only certain specific cells are to be treated [6, 7].
A hollow-core optical fiber as a conduit for a
delivery of atmospheric pressure plasma has
several advantages, such as an excellent
flexibility, very small inner diameter
commensurate with cellular scales, mechanical
and chemical strong properties, and very low
production cost. For example, such a flexible
micrometer-scale optical fiber-based plasma jet
device may permit the treatment of colorectal
cancers without laparotomy.
2. Experiments
2.1. Fabrication of hollow-core optical fiber
based plasma jet device
The hollow-core optical fiber employed had
an inner diameter of 15 µm, an outer diameter
of 60 µm, and an additional protective plastic
coating such that the full diameter of the fiber
was 125 µm. The small scale of the glass optical
fiber makes the plasma delivery system highly
flexible and able to precisely treat a small
collection of tumors. A single electrode, made
by coating with a silver paste, was placed 1 mm
from the end of the fiber. High purity helium
gas (99.998%) was used as the carrier gas due to
its inertness, lower voltage needed to induce a
plasma and more reactive species than other
candidate gasses such as argon [8]. The helium
Plasma plume
He flow
Silver electrode
100 mm
Figure 1. Dimensional comparison between murine
B16F0 tumor cells and hollow-core optical fiber
with 15 µm ID.
gas flow rate was held constant at around 10
standard cubic centimeters per minute (sccm),
which is extremely low when compared to other
plasma jet devices based on millimeter-sized
tubes. Such a reduced gas flow rates is
additionally advantageous as it can limit damage
by dehydration in sensitive cells and tissue
samples [9]. The voltage and current waveforms
from the single electrode were measured using a
high voltage probe (Tektronix P6015A) and a
current probe (Pearson 4100) to monitor the
input electrical energy. In the driving circuit, an
inverter was used to amplify a low primary
voltage to a high secondary voltage. In order to
verify the diverse reactive species generated by
the helium plasma plume in the ambient air, the
emission spectra of atmospheric pressure microplasma jet were monitored by the fiber optic
spectrometer (Ocean Optics, USB-4000UVVIS) in which the distance between the end of
the device and the spectrometer was about 2 mm.
2.2. Cell preparation and cell treatments by
micro-plasma jet
Murine melanoma B16F0 tumor cells (ATCC
CRL-6322) and murine fibroblast C L.7
cells(ATCC TIB-80) were seeded in wells of
High voltage
Figure 2. Plasma plume from the hollow-core
optical fiber into ambient air.
24-well plates (2×105 cell/well) and cultured in
DMEM (Gibco BRL, Grand Island, NY),
supplemented with 10 % FBS (Hyclone, Logan,
UT) and 50 µg/ml gentamicin (Gibco BRL) at
37˚C in a humidified atmosphere of 5 % CO2
for 24 hours. Prior to plasma treatment, the cells
at approximately 90% confluence were washed
twice with PBS and treated with plasmas. After
plasma treatments, the wells were immediately
filled with cell culture medium and incubated
for 24 hours. After harvesting and washing
twice with PBS, the cells were analyzed for
apoptosis using the Annexin V-FITC Apoptosis
Detection Kit (eBiosciences) and a FACS
Calibur (BD Biosciences, San Jose, CA).
3. Results and discussion
3.1. Plasma plumes from micro-plasma jet
device
Figure 1 shows a comparison of the dimensions
of murine B16F0 melanoma tumor cells (ATCC
CRL-6322) to the hollow-core glass optical
fiber showing that the inner diameter of the
hollow-core optical fiber is as small as a single
B16F0 tumor cell. Therefore, the plasma jet
device employing this flexible yet robust
hollow-core glass optical fiber is capable of
treating a small collection of tumor cells. When
5
He
Intensity [a.u.]
N2
4
3
2
1
He
N2
He
N2+
N2
He
OH
He
O
N2+
0
300
400
500
600
700
800
Wavelength [nm]
Figure 3. Optical emission spectra of atmospheric
pressure micro-plasma jet.
a sinusoidal voltage, with a peak value of 10 kV
and a frequency of 32 kHz, is applied to the
single electrode, the resultant plasma plume has
the length of approximately 5 mm as shown in
Figure 2. Since the plasma plume in the ambient
air is quite narrow (15 µm) but long (5 mm), the
micro-plasma device is sufficiently small
enough to apply the direct treatments of a small
collection of tumor cells. Figure 3 depicts the
optical emission spectra of the plasma plume
over the range from 250 to 800 nm further
verifying that excited species of OH, N2, N2+,
He, and O exist in the plasma plume. The clear
presence of nitrogen and oxygen species in the
optical emission spectra of Figure 3 indicates
that many gaseous species from the air
participate along with the helium in the plasma
process.
3.2. Apoptotic analysis of micro-plasma
treatments using Annexin V-FITC
The micro-plasma jet was applied directly to
either cultured murine tumor cells or cultured
murine fibroblasts to study the specific
influence of the plasma treatment. Figure 4
shows the results of apoptotic studies on the
murine melanoma B16F0 tumor cells and the
murine fibroblast CL.7 cells at doses of 0 (no
treatment), 2, 5, 10 and 20 seconds, respectively.
When the micro-plasma was applied to the cells,
the applied voltage was 8.5 kV with a frequency
of 32 kHz and He gas flow rate was fixed at 10
sccm. In each assay well, a circle motion of 6
spots were treated, with the distance between
the plasma jet device and the cells was fixed to
5 mm. The upper graphs in Figure 4 represent
the apoptotic analysis using the Annexin VFITC in murine melanoma B16F0 tumor cells
and the lower graphs represent the analysis in
murine fibroblast CL.7 cells. As can be seen, the
micro-plasma jet induced the cultured murine
cells to undergo apoptosis in a dose-dependent
manner. In the graphs, the necrotic cells are
Annexin V negative and PI positive (top-left
sector), apoptotic dead cells are both PI and
Annexin V positive (top-right sector), and
apoptotic cells are Annexin V positive and PI
negative (lower-right sector), respectively. The
percentage of the cells in the region among the
total cells is represented in the graphs. Under
these experimental conditions, the plasma
treatments mainly affect apoptotic cells (lowerright sector, purple). Despite the small sized
plasma plume, these conditions yielded
measureable results for the induction of
apoptosis. The proportion of apoptotic live and
apoptotic dead the B16F0 tumor cells treated
with plasma is higher than that of fibroblast
CL.7 cells at all plasma dosages. The plasma
treatment induced more apoptosis in B16F0
tumor cells than in CL.7 cells. Note that when
the dose is increased to 20 seconds, the plasma
treatment seems to induce equal levels of
apoptosis in B16F0 tumor cells and in the CL.7
fibroblast cells, indicating that the differential
effect of the micro-plasma treatment on
different cells disappears when the dose
increases to a certain level.
No treatment
2 seconds x6
10
3
3.09%
10
1.42%
3
0
10
1
2
10 10
3
10
10
10
0.70%
7.45%
0
10
10
1.36%
3
1
2
10 10
3
10
3
0
10
1
2
10 10
3
10
10
10
0
10
1
2
10 10
3
10
10
2
10 10
3
4.73%
10
2
10 10
3
10
10
1
2
10 10
3
10
4
10
3
0.90%
6.87%
10
2
10
CL.7
1
10
0
4 10
10
10
5.19%
1
1
0
4
1.73%
2
0
10
10
10
24.12%
0
4
3
10
0
10
4
10
1
10
10
3.84%
0
4
15.73%
0
10
2.15%
B16
1
10
4
0.84%
1
10
2.84%
0
10
10
2
1
10
10 10
3.07%
2
0
4 10
3
10
10
1
2
10
2
2
10
1
2.42%
10
10
7.61%
0
10
10
2.08%
3
10
1
4
0.98%
10
3.97%
2
0
4 10
10
3.60%
10
10
10
10
3
10
1
4
4
10
3.46%
2
0
4
3.27%
10
10
3.35%
0
10
10
3
20 seconds x6
4
4
10
1
10
3
10
2.72%
10
1
10
4.17%
2
2
10
10 seconds x6
4
10
10
PI
5 seconds x6
4
4
10
10.99%
0
10
1
2
10 10
3
10
0
4 10
10
21.32%
0
10
1
2
10 10
3
10
4
10
Annexin V-FITC
Figure 4. Apoptotic analysis using the Annexin V-FITC in murine melanoma B16F0 and fibroblast CL.7 cells by
plasma treatments.
4. Conclusions
A flexible and single cell-level micro-plasma
cancer endoscope based on a 15 µm hollow-core
glass optical fiber has been proposed and tumor
cell apoptotic analysis supports its potential use
in targeted cancer therapies. Despite the small
inner diameter and very low gas flow rate, the
generated plasma jet was able to induce
apoptosis of the cultured tumor cells. The results
demonstrate that the 15 µm-sized hollow-core
optical fiber micro-plasma jet induced apoptosis
in both cultured murine melanoma tumor cells
and fibroblast cells in a dose-dependent manner.
The murine melanoma tumor cells were found
to be more sensitive to plasma treatment than
murine fibroblast cells under specific plasma
dose conditions. This work offers a potential
application of micro-plasma cancer endoscopy
for physical cancer therapeutic methods.
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