116 IEEE TRANSACTIONS ON RADIATION AND PLASMA MEDICAL SCIENCES, VOL. 2, NO. 2, MARCH 2018
Selective Treatment of Pancreatic Cancer Cells
by Plasma-Activated Saline Solutions
Zhitong Chen , Li Lin, Eda Gjika, Xiaoqian Cheng, Jerome Canady, and Michael Keidar
Abstract—Atmospheric discharge between a metal pin cathode
and saline solution (SS) anode with different air gap lengths
was considered for investigating plasma self-organization patterns
at the liquid anode surface. We report the effect of plasma-
activated SSs on human pancreas adenocarcinoma cancer cells
line (BxPC-3) and human pancreatic duct epithelial normal cells
line (H6c7). The presence of reactive oxygen species and reactive
nitrogen species resulted in the anti-cancer properties of plasma-
Index Terms—Cancer therapy, cold atmospheric
plasma (CAP), saline solution (SS), self-organization
SELF-ORGANIZATION is generally referred to as a pro-
cess of spontaneous transition from a homogeneous
stable state to a regular pattern in a spatially extended
system , . Self-organization is a complex and fascinating
phenomena commonly observed in both natural and techno-
logical contexts within diverse varieties of physics, chem-
istry, and biology , . Different types of self-organization
phenomena have been reported in a wide range of plas-
mas, such as dielectric barrier discharge , high frequency
discharge , gas ﬂow stabilized discharge , , resis-
tively stabilized discharge , and discharge with liquid
electrodes –. The self-organization phenomena associ-
ated with the formation of electrode patterns are signiﬁcantly
different from these discharges, which typically occur in
the anode or cathode layer , . Self-organization pat-
terns (SOPs) of plasma include square-textures, square-lattices,
square/hexagonal superlattices, hollow-hexagonal, multiarmed
spirals, rotating-wheels patterns, etc. , . The for-
mation of these patterns depends on various parameters,
such as driving current, electrolyte conductivity, gap length,
Manuscript received August 6, 2017; revised September 2, 2017; accepted
September 28, 2017. Date of publication October 12, 2017; date of cur-
rent version March 1, 2018. This work was supported in part by the U.S.
Patent Innovations Inc. (Plasma Medicine Initiative), and in part by the
National Science Foundation under Grant 465061. (Corresponding authors:
Zhitong Chen; Michael Keidar.)
Z. Chen, L. Lin, E. Gjika, and M. Keidar are with the Department
of Mechanical and Aerospace Engineering, George Washington
University, Washington, DC 20052 USA (e-mail: email@example.com;
X. Cheng and J. Canady are with the Research Institute for Advanced
Biological and Technological Sciences, U.S. Medical innovation LLC,
Takoma Park, MD 20912 USA.
Color versions of one or more of the ﬁgures in this paper are available
online at http://ieeexplore.ieee.org.
Digital Object Identiﬁer 10.1109/TRPMS.2017.2761192
gas species, and so on –. Recently, plasma dis-
charges with the liquid electrode have been studied refer-
ring to applications ranging from water decontamination
and activation , , to nanoparticle and materials
synthesis , , and medicine . Therefore, self-
organization in plasma interacting with surfaces is interesting
not only from a fundamental point of view as intrinsic and fas-
cinating characteristics of nature, but also from practical stand-
point in current and emerging technological applications .
Plasma interacting with the liquid generates reactive oxy-
gen species (ROS) and reactive nitrogen species (RNS) that
act as key intermediate for cancer therapy , . In this
paper, we created plasma with different SOPs to activate saline
solution (SS) and investigated the anti-tumor effects of the
plasma-activated SS on human pancreatic normal and can-
cer cells. We have recorded different self-organized patterns
formed at the liquid surface. The spectra of plasma with SOPs
were characterized by UV–visible–NIR optical emission spec-
troscopy. The concentration of hydrogen peroxide (H2O2) and
2) was measured by using a Fluorimetric Hydrogen
Peroxide Assay Kit, and the Griess reagent system, respec-
tively. The cell viability of H6c7 and BxPC-3 was measured
via Cell Counting KIT 8 assay. Typically, SS is used to treat
dehydration by injection into a vein, and it is also used to dilute
medications to be given by injection. Based on our results, one
can suggest that SOP plasma-activated SS has potential to be
utilized as an oral medicine or drug which can be injected into
II. EXPERIMENTAL SECTION
A. Discharge Setup
Fig. 1shows a schematic representation of the SOP dis-
charge setup capable of producing well-deﬁned self-organized
patterns at the surface of the liquid/plasma interface. Anode
(thin copper plate, thickness d=0.2 mm, diameter
Ø=22 mm) was placed at the bottom of a glass-made
well. The tungsten cathode of Ø =2 mm was then installed
above the SS surface. A ballast resistor (90 k) was connected
between the cathode and the direct current power supply unit
(Power Design, Model 1570 A, 1–3012 V, 40 mA). Voltage
was applied between the cathode and the liquid-immersed
anode, and a small (2–10 mm) gap between the cathode and
liquid surface accommodated a plasma formation. SS was
treated by discharge with distance of 2, 4, 6, 8, and 10 mm
air gap length to obtain plasma solutions for treating cancer
cells. 6 ml volume of SS was treated with the plasma.
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CHEN et al.: SELECTIVE TREATMENT OF PANCREATIC CANCER CELLS BY PLASMA-ACTIVATED SS 117
Fig. 1. Schematic representation of the SOP plasma discharge setup. Different
air gap distance between the cathode and surface of liquid accommodated
plasma. (dis the distance of air gap).
B. Cell Cultures
The human pancreas adenocarcinoma cancer cell line
(BxPC-3) was acquired from American Type Culture
Collection (ATCC). Cell lines were cultured in RPMI-
1640 medium (ATCC 30-2001) supplemented with 10%
(v/v) fetal bovine serum (Atlantic Biologicals) and 1% (v/v)
penicillin and streptomycin (Life Technologies). The human
pancreatic duct epithelial normal cell line (H6c7, Kerafast)
was cultured in Keratinocyte SFM (KSFM, Gibco) sup-
plemented with prequaliﬁed human recombinant epidermal
growth factor 1-53 (EGF 1-53, Gibco), bovine pituitary extract
(BPE, Gibco), and 1% (v/v) penicillin and streptomycin
(Life Technologies). Cultures were maintained at 37 ◦Cin
a humidiﬁed incubator containing 5% (v/v) CO2.
C. Evaluation of Hydrogen Peroxide (H2O2) Concentration
Fluorimetric Hydrogen Peroxide Assay Kit (Sigma-Aldrich)
was used for measuring the amount of H2O2in SS. A detailed
protocol can be found on the Sigma-Aldrich website. Brieﬂy,
we added 50 μl of standard curves samples, controls, and
experimental samples (SS treated by SOP plasma with 2, 4, 6,
8, and 10 mm air gap) to the 96-well ﬂat-bottom black plates,
and then added 50 μl of master mix (including red peroxi-
dase substrate stock, 20 units/mL peroxidase stock, and assay
buffer) to each of wells. We incubated the plates for 20 min at
room temperature protected from light on and measured ﬂuo-
rescence by Synergy H1 Hybrid Multimode Microplate Reader
at Ex/Em: 540/590 nm.
D. Evaluation of Nitrite (NO−
Nitrite level was determined by using the Griess Reagent
System, including 50 ml sulfanilamide solution, 50 ml NED
solution, and 1 ml nitrite standard, (Promega Corporation)
according to the instructions provided by the manufacturer.
Brieﬂy, we added 50 μl of standard curves samples, controls,
and experimental samples to the 96-well ﬂat-bottom plates.
Then dispense 50 μl of the sulfanilamide solution to all sam-
ples and incubate 5–10 min at room temperature. Finally,
dispense 50 μl of the NED solution to all wells and incubate at
room temperature 5–10 min. The absorbance was measured at
540 nm by Synergy H1 Hybrid Multimode Microplate Reader.
E. Measurement of Cell Viability
The cells were plated in 96-well ﬂat-bottom microplates at
a density of 3000 cells per well in 70 μL of complete cul-
ture medium. Cells were incubated for 24 h to ensure proper
cell adherence and stability. Conﬂuence of each well was
conﬁrmed to be at ∼40%. 30 μl of RPMI, SS, and plasma-
activated SSs were added to the corresponding cells. Cells
were further incubated at 37 ◦C for 24 and 48 h. The viability
of the pancreas normal and cancer cells was measured with
Cell Counting Kit 8 assay (Dojindo Molecular Technologies,
MD, USA). The original culture medium was aspirated and
10 μL of CCK 8 reagent was added per well. The plates were
incubated for 3 h at 37 ◦C. The absorbance was measured at
450 nm by Synergy H1 Hybrid Multimode Microplate Reader.
We normalized data according to control group (RPMI for
BxPC-3 and KSFM for H6c7). We calculated the mean and
standard deviation independently.
F. Optical Emission Spectra Measurement
UV–visible–NIR, a range of wavelength 200–850 nm, was
investigated on SOP plasma to detect various RNS and ROS
(nitrogen [N2], nitric oxide [–NO], nitrogen cation [N+],
atomic oxygen [O], and hydroxyl radical [–OH]). The spec-
trometer and the detection probe were purchased from Stellar
Net Inc. The optical probe was placed at a distance of 2 cm
in front of the plasma beam. Integration time of the collecting
data was set to 100 ms.
G. Statistical Analysis
All results were presented as mean ±standard deviation
plotted using origin 8. Student’s t-test was applied to check
the statistical signiﬁcance (*p<0.05, **p<0.01, ***p<0.001).
A. Current–Voltage Characteristics of Discharge
Fig. 2shows the current–voltage characteristics of the dis-
charge with air gap at distance of 2–10 mm. With gap
increasing, the discharge current decreases while discharge
voltage increases. Similar features of the discharge voltage
increasing with air gap length are found in the case of elec-
trolyte anode/cathode discharge , . The SOP appears
at the plasma-liquid interface and the discharge is stabilized
when the air gap length is about 6 mm. At 2 mm gap, the
discharge voltage is low while discharge current is high, and
the discharge pattern represents a single ﬁlament. As the air
gap length increases from 2 to 4 mm, the anode spot changes
to a double ring-like structure. At air gap length of 8 mm, var-
ious types of SOPs are formed above the liquid media surface
as shown in Fig. 2. When the air gap is within the range of
2–8 mm, the plasma discharge is stable. When the air gap is
118 IEEE TRANSACTIONS ON RADIATION AND PLASMA MEDICAL SCIENCES, VOL. 2, NO. 2, MARCH 2018
Fig. 2. Current–voltage dependence for different air gap lengths with optical
photographs of the self-organized interface patterns.
Fig. 3. Optical emission spectrum by the SOP plasma discharge above SS
with different air gap length taken using UV–visible–NIR in the 200–850 nm
wavelength range. (a) 2 mm, (b) 4 mm, (c) 6 mm, (d) 8 mm, and (e) 10 mm.
10 mm, however, the plasma discharge becomes unstable. If
the air gap is larger than 10 mm, the plasma discharge cannot
be sustained anymore.
B. Optical Spectrum of SOP Plasma
We have measured spectra of plasma from the plasma-liquid
interface. Typical optical emission spectra are shown in Fig. 3.
One can see that with air gap length increasing, the emis-
sion intensity decreases. The identiﬁcation of the emission
bands was performed according to Pearse and Gaydon .
Fig. 4. H2O2and NO−
2concentrations in SS treated by plasma with
SOP plasma with different air gap length (each air gap length treated by
SOP plasma for 40 s): (a) H2O2 concentration and (b) NO−
Student’s t-test was performed, and the signiﬁcance compared to the 2 mm
is indicated as *p<0.05, **p<0.01, and ***p<0.001. (n=3).
Small gaps (2 and 4 mm) lead to high intensity of spec-
tra. From 6 to 10 mm, the cathode was far to anode
resulting in intensity decreasing. In the 250–300 nm wave-
length range, weak emission bands (258, 267, and 284) were
detected as NO lines . Species at wavelengths of 337 and
358 nm were deﬁned as N2C3uor NO β3g(denoted
as N2/NO), because both species have possible optical emis-
sion at these wavelengths . The emission bands between
300 and 500 nm have still not been clearly identiﬁed in .
However, we anticipated that OH was present at 309 nm, the
wavelength of 375 nm could be indicative of N+
atomic oxygen (O) was denoted at the wavelength of 777 nm.
Atomic oxygen (ground/excited states) was believed to have
a signiﬁcant effect on cells and therefore a broad biomedical
application . The dominant species of the spectra in this
paper were NO or N2 lines (258, 267, 337, and 357 nm), OH
(309 nm), N+
2(391 nm), and O (777 nm).
C. H2O2and NO−
Plasma species penetrate through the plasma-liquid
interface, and can produce chemically reactive species in the
SS. Complex chemistry is associated with plasma produced
species in liquid . These reactions lead to the formation
of short- and long-lived species. H2O2and NO−
long-lived species in the plasma-activated SS and will be focus
of this paper. The air gap length dependencies of the H2O2
2concentrations in the plasma-activated SS with gap
distance as a parameter are shown in Fig. 4. The concentration
of H2O2increases with an up to 4 mm air gap then decreased
up to 8 mm before increasing again at 10 mm as shown in
Fig. 4(a). The concentration of NO−
2increases with air gap
from 2 to 8 mm, then decreases at 10 mm.
D. Cell Viability of H6c7 and BxPC-3
To investigate the potential of plasma-activated SS, we
treated BxPC-3 human pancreas cancer cells and H6c7 human
normal cells with them. RPMI, KSFM, and untreated SS were
used as controls. Fig. 5shows the cell viability of BxPC-
3 human pancreas cancer cells and H6c7 human pancreas nor-
mal cells exposed to RPMI/KSFM, SS, and plasma-activated
SSs for 24 h and 48 h. We can see that plasma-activated SSs
CHEN et al.: SELECTIVE TREATMENT OF PANCREATIC CANCER CELLS BY PLASMA-ACTIVATED SS 119
Fig. 5. Effects of seven media: RPMI/KSFM, SS, and ﬁve plasma-activated
media (SS activated by plasma with SOP at 2, 4, 6, 8, and 10 mm distance
for 40 s’ treatment) on viability of the BxPC-3 human pancreas cancer cells
and the H6c7 human pancreas normal cells after (a) 24 h and (b) 48 h incu-
bation, respectively. The percentages of surviving cells for each cell line were
calculated relative to controls (RPMI/KSFM).
have stronger effect on the cancer cells than that on the nor-
mal cells. For BxPC-3 cancer cells, when incubated for 24 h
and 48 h, cell viability decreased ﬁrst, then increased with
air gap length increasing. The minimum cell viability appears
at 4 mm air gap. For H6c7 normal cells, when incubated for
24 h and 48 h, plasma with SOP at 6 mm air gap has the most
signiﬁcant effect of plasma-activated SSs.
We have previously reported that under certain conditions
cold atmospheric plasma can be directly applied to cancer
cells without inﬂuencing healthy tissues –. At the
same time plasma-activated media have been reported to have
a cytotoxic effect in oncology. Unique processes at the self-
organized interfaces could be the key to the production of
novel, highly efﬁcient tumor-inhibiting media. SOP pattern-
ing at the interfaces drastically widens the spectrum of the
involved structural, physical, and chemical processes, and thus
opens new horizons to tackling tumors of various origins and
locations. In this paper, SSs were treated by plasma with vari-
ous SOPs to be applied to human pancreatic cancer and normal
cells. Discharge was formed between pin and liquid electrode
and resulted in SOP formation which depended on discharge
gap as shown in Fig. 2. Transport of ROS/RNS across the
plasma/liquid interface is affected by SOP. As such modiﬁca-
tion of SS by discharge is affected and controlled by SOP at
the plasma-liquid interface. Typical optical emission spectra of
such plasmas at different air gap were shown in Fig. 3indi-
cating that plasma at each air gap length contains ROS and
RNS in the gas phase. ROS and RNS were also formed in
plasma-activated SS. RNS are well known to induce cell death
via DNA double-strand breaks and apoptosis, where ROS are
capable of inducing the apoptosis and necrosis , . Our
results in Fig. 4show that the H2O2concentration is highest
at 4 mm air gap distance while NO−
2concentration is highest
at 8 mm air gap distance. Possible reactions illustrating the
routes of H2O2and NO−
2formation in liquid and plasma have
been reported in our previous articles , , . From
Fig. 2we can see that plasma average discharge power is
growing with increasing air gap, which results in the tem-
perature of plasma-activated SSs going up (except 10 mm).
Since H2O2is thermodynamically unstable, its rate of decom-
position increases with rising temperature . It should be
pointed out that the plasma discharge becomes unstable at
the air gap length of about 10 mm. The discharge has to be
reignited. As such, the discharge instability at 10 mm gap
might lead to a low concentration of nitrite. Fig 5 shows that
plasma-activated SS affects cancer and normal pancreatic cells
in a selective manner. Gain-of-function mutations in onco-
genes and loss-of-function mutation in tumor suppressor genes
drive malignant transformation, which results in cell deregula-
tion that is frequently associated with enhanced cellular stress
including oxidative, replicative, metabolic, and proteotoxic
stress, and DNA damage . Adaptation to this stress phe-
notype is required for cancer cells to survive. Consequently,
cancer cells may become dependent upon nononcogenes that
do not ordinarily perform such a vital function in normal cells.
Thus, SOP plasma-activated SSs might affect these nononco-
gene dependencies in the context of a transformed genotype
and might result in a synthetic lethal interaction and the selec-
tive death of BxPC-3 cancer cell compared to H6c7 normal
cell. Plasma with SOP activating SSs have more effect on
cancer cells. The trend of pancreatic normal and cancer cells
can be attributed to the trend of ROS and RNS concentra-
tion with different air gap distances. On the other hand, H2O2
reacts with NO−
2to form peroxynitrite OONO−and H2O.
ONOO−is a powerful oxidant and nitrating agent that is
known to be more damaging to cancer cells . Therefore,
a synergistic effect of ROS and RNS is suspected to play a key
role in the apoptosis of the plasma solutions. For BxPC-3 can-
cer cells, intracellular ROS-mediated up-regulation of DR5 can
leads to apoptosis (procaspase-8 is a direct downstream tar-
get of DR5) . On the other hand, intracellular generation
of ROS induces increasing protein expression of Bax, dis-
ruption of the mitochondrial membrane potential and release
of cytochrome c and AIF into the cytosol resulting in to the
activation of caspase9/3 cascade . Therefore, plasma with
SOP-induced intracellular generation of ROS induced apop-
tosis in BcPC-3 cancer cells might be orchestrated by the
synergistic effects of both extrinsic and intrinsic pathways.
The results indicate the cytotoxicity of plasma-activated SS
is speciﬁc to pancreatic adenocarcinoma cancer cells. The
plasma-activated SS at 4 mm air gap distance had the most sig-
niﬁcant affect in inducing cell death in pancreatic cancer cells.
This is related to certain amounts of ROS and RNS generated
by double ring-like structure plasma with SOPs.
The presented ﬁndings demonstrate that self-organized pat-
tern plasma-activated SSs applied to both cancerous and
normal pancreatic cells exhibit selective manners. The air
gap at a distance between 2 and 10 mm results into vari-
ous shapes of SOP on SS anode. A synergistic effect of RNS
and ROS present in the plasma solution is suspected to play
a key role in the cell death. The SOP plasma-activated SS at
4 and 6 mm air gap distance between the pin electrode and
the solution had the most signiﬁcant effect in inducing cell
death in both pancreatic cancer and normal cells, respectively.
120 IEEE TRANSACTIONS ON RADIATION AND PLASMA MEDICAL SCIENCES, VOL. 2, NO. 2, MARCH 2018
The SOP plasma-activated SSs have more serious effect on
BxPC-3 human pancreatic adenocarcinoma cancer cells than
H6c7 human pancreatic epithelial normal cells. These results
suggest that it might be possible to use SOP plasma-activated
SSs with anti-tumor effect for clinical applications.
The authors would like to thank the cell lines used in this
paper were kindly provided by U.S. Patent Innovations LLC.
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