LOW-TEMPERATURE PLASMA NEEDLE FOR BIOMEDICAL TREATMENTS

Michael A. Wilson,1 Timothy R. Brubaker,2 Andrea M. Mastro,3

Michael M. Micci,4 Sven G. Biln,5 and Sean D. Knecht 6
1

Undergraduate Student, Department of Electrical Engineering; 2Ph.D. Student,

Department of Electrical Engineering; 3Professor of Microbiology and Cell Biology,
Department of Biochemistry and Molecular Biology; 4Professor of Aerospace Engineering,
Department of Aerospace Engineering; 5Assoc. Prof. of Engineering Design, Electrical
Engineering, and Aerospace Engineering, Department of Electrical Engineering; 6Research
Faculty, Applied Research Laboratory
ABSTRACT
An overview of the design methodology of a lowtemperature plasma needle and preliminary results
investigating its efficacy in treating metastatic human
breast cancer epithelial cells is reported. Low-temperature
plasma is created by flowing gas past a high-voltage
surface. This process produces ions and generates an
electrically conductive state of matter (plasma) at room
temperature. High-energy collisions of electrons with
neutral particles create reactive chemical species such as
NO, O3, H2O2 and others which have biomedical
applications. Current cancer treatment options, such as
surgery and chemotherapy, may not be sufficient to treat
metastatic cancer. Initial experiments have been conducted
in which a line of cultured metastatic human breast cancer
epithelial cells (MDA-MB-231) are exposed to the lowtemperature plasma needle. The plasma needle is lowered
into cell culture dishes and the cells are exposed to plasma
at different dosages. Cell detachment was observed for
plasma and helium-exposed cultures with a greater effect
observed for plasma-exposed cultures. This is attributed to
reactive chemical species generation in the plasma plume.
Experiments are continuing to optimize the treatment
conditions and further this research.
1. INTRODUCTION
The number of women in the U.S. with a history of breast
cancer was estimated to be more than 3.1 million as of
January 1, 2014 with an additional 232,670 women
expected to be diagnosed in 2014 [1]. Approximately 43%
of women diagnosed with breast cancer are 65 years and
older, 20% are under the age of 50, and 37% are between
50 and 65 years old [1], with an average age of 61 [1]. This
form of cancer, unless treated early, generally metastasizes
to areas such as bone, ovaries, skin, and pericardium [2].
Approximately 2030% of breast cancer victims will later
find out that their breast cancer has become metastatic and

the average survival period after this diagnosis is three

years [3]. The average 5-year survival rate for this disease
has increased from 75% between 1975 and 1977, to 90%
between 2003 and 2009 [1]. While the survival rate has
increased significantly, the methods that are currently used
to treat cancer still have limitations. Any new methods of
eradicating the areas that are affected by the breast cancer
cells without harming the adjacent healthy cells would
benefit cancer patients. This research group is working on
using low-temperature plasma as a possible therapy option
for breast cancer patients.
The term plasma was coined in 1928 by Irving
Langmuir because the substance reminded him of blood
plasma [4]. Plasma is the most common state of matter
found in the universe and finds many uses and applications
in a variety of fields. For example, the medical community
uses plasmas for disinfection and sterilization [512].
Scientists classify plasma into two main types: hightemperature (thermal) plasma, or HTP, and lowtemperature (non-thermal) plasma, or LTP. LTP remains
near room temperature making it easier to work with
compared to HTP. The generation of LTP is due to the
thermal equilibration of electron self-collisions occurring
faster than the equilibrium between electrons and ions. This
leads to high electron temperatures and very low gas
temperatures [5]. Researchers have realized that LTP can
be useful in many applications within the medical field, due
to the generation of reactive chemical species in the plasma
plume. Specifically, there have been many experiments
with plasmas in areas of cancer research and dentistry [13].
Scientists and engineers have been researching and
working to commercialize these therapies.
A dielectric barrier discharge (DBD), also known as a
barrier discharge or silent discharge, is an electrical
discharge that occurs between two electrodes that are
separated by a dielectric barrier. One of the electrodes is
connected to a high voltage (AC) power supply and the
other electrode is connected to ground. The DBD method
is beneficial because it provides a wide range of geometric

as indicated. Each well is circular with a diameter of 15.6

mm and holds a maximum volume of 3.4 mL
3. EXPERIMENTAL DESIGN

Figure 1. Schematic of an LTP dielectric barrier

discharge. The HV power supply is connected to the
syringe which is encased within the dielectric barrier
(PEEK). The gas flows through the back and out of the
tip of the syringe as plasma.
configuration options. With respect to biomedical
applications, configurations can include quartz plates
covering one or both electrodes; high-voltage insulated
wires plasma stamp; plasma stick; plasma comb; and
plasma roller [14], among many others. Rather than
generating a plasma plume within small discharge gaps,
plasma jets generate plasma in the open air (see Figure 1).
Due to the applications on living tissue, the current is kept
in the milliamp range to prevent electric shock. In terms of
the grounding electrode, an external or internal ground
configuration may be used. The external configuration is
required for plasma jets using noble gases. An internal
configuration can be used for air-plasma jets in which the
grounding electrode is within a metal cap. The high energy
collisions between the gases and neutral molecules create
the reactive species of interest for biomedical treatments.
This paper details the design of preliminary LTP
experiments with emphasis on breast cancer research. The
remainder of this paper is structured as follows. Section 2
introduces information about the cancer cell line used in the
experiments. Section 3 explains how the LTP needle was
designed and discusses the experiments and the methods
used in testing the plasma on breast cancer cultures. Section
4 discusses the results. Section 5 provides concluding
remarks and plans for future research.

The goal of the experiments is to investigate the effects of

LTP on metastatic BCs. A design methodology for the LTP
source was established such that it could eventually suit the
needs of doctors working with this equipment in a clinical
setting.
The syringe system includes two electrodes to generate
the plasma. The syringe itselfa 22-gauge needle that is 2
long serves as the high-voltage electrode. Helium flows
axially through the syringe (note: this configuration is
slightly different that that shown in Figure 1, but is
conceptually the same). The syringe is sheathed with PEEK
(polyether ether ketone) plastic tubing, which serves as the
dielectric barrier around the needle. PEEK has a high
dielectric strength and is used as a barrier to prevent arc
production. The overall diameter of the device is equivalent
to a 16-gauge needle.
The ground electrode is placed approximately 1/8
from the tip of the syringe. The placement of the ground is
important for generating plasma, as it can affect the electric
field that is formed on the needle. This electric field is
necessary for the formation of plasma because it accelerates
electrons, resulting in high energy collisions with neutral
particles (atoms, molecules) that are already flowing
axially. These collisions result in atomic excitation or
molecular dissociation processes which result in reactive
chemical species in the plasma plume. The operation of this
setup is demonstrated in Figure 2, which shows the
dielectric barrier discharge interface on a saline sample.
Since the device is exposed to very high voltage, it is
essential to make sure that the high voltage and ground
were encased safely in a polycarbonate clear tubing (1.5
inner diameter, 1.75 outer diameter). This arrangement
allows the operator to hold onto the syringe without being

2. CELL CULTURE
For these experiments human metastatic breast cancer cells
(MDA-MB-231, ATCC-HTB 26 presumptive equivalent)
that were genetically engineered to produce green
fluorescent protein (GFP) were utilized. Hereafter these are
referred to as BCs. Derived from a pleural effusion [15],
MDA-MB-23GFP cells are known to invade the murine
skeleton [16].
Before use, the cells were grown to confluency in
Dulbeccos Modified Eagles Medium (DMEM) with 5%
fetal bovine serum and 1 essential amino acids in 24- or
48-well standard tissue culture plates made of polystyrene

Figure 2. The interface between the LTP syringe

interacting with a saline sample. The visible plasma jet
has a diameter similar to the syringe.

exposed to the high voltage. It is also preferable to have

optical access to the device which is why clear
polycarbonate tubing was used. Holes were drilled within
the tubing to thread the voltage line through the device to
its respective input location.
An optical table is used to hold the syringe in place and
allows precise motion in the x, y, and z directions. This
optical table is able to hold the syringe at one spot in the
cell culture wells. As mentioned in Section 2, the cultures
were placed in 24- and 48-well cell culture plates. The
plasma jet is lowered into the cell culture wells to a height
at which the jet is visibly in contact with the surface of the
buffer solution in the wells.
Helium is used for plasma production during the
experiments. Additional gases will be mixed with the
helium as dopants in future experiments to increase reactive
species generation. The helium flow rate is controlled via a
mass flow controller (MFC) which is calibrated for gases at
70 F, 14.7 psia. The flow rate can be controlled over the
range of 15-775 mL/min.
The electric field is generated by the high voltage
applied to the metal syringe. An Agilent 33220A Function
Generator generates a 5-kHz, 2-Vpp sine wave which is
amplified 1000 to 2 kV by a Trek 10-40 amplifier.
Although the voltage amplifier has current-limiting

Figure 3. Full LTP setup. Helium is introduced to the

system using a mass flow controller. The voltage and
ground inputs are connected to the power supply and
threaded through the tube to their respective input
locations. The optical table is used for support and
measurement purposes.

capability, a secondary current limiter is included via a 500k resistor stack. Increasing the voltage or mass flow rate
of the helium results in an increasing length and visible
intensity of the plasma plume. Figure 3 shows the setup
used for these experiments.
4. RESULTS
Over several days, data were collected to determine how
the cells responded to treatment with LTP. Cell cultures
were observed with microscopy pre- and post-exposure.
For the first group of experiments, it was observed that
there was an equal amount of cell detachment from both the
plasma and helium exposure. This result suggests that there
was a physical effect of the flow velocity on the cell culture
medium. Specifically, it is likely that the kinetic energy of
the flowing helium was sufficient to damage the cultured
cells under these conditions. It was necessary to limit this
effect in the subsequent experiments in order to isolate
potential effects of reactive chemistry.
For the second group of experiments, the cells were
exposed to helium and plasma for 90 seconds with 200-L
of PBS (phosphate buffered saline). PBS provides a
physical buffer to the cell culture and also prevents the cells
from drying out and dying. This buffer was then
immediately removed after the treatment and cell culture
medium was re-introduced. No effect was observed for
both helium- and plasma-exposed cells indicating that jet
kinetic energy did not penetrate the PBS, which further
supports the conclusions from the first group of
experiments. However, the diffusion of reactive chemical
species was also likely prevented in this case.
For the third group of experiments, the growth medium
was removed from the cells and 200 L of PBS was added
to two sets of wells and 100 L of PBS to one set. The
plasma was then applied for 120 seconds to the 200- and
100-L samples. The other 200-L sample was also
exposed for 180 seconds. After exposure, the samples were
left alone for 5 minutes before the PBS was removed and
the growth medium re-introduced. The same procedure was
followed for the pure-helium exposure to the cells as a
control. The voltage was 2.5 kVpp at a frequency of 5 kHz.
It was observed that the cell cultures had large circular
holes in the areas that were exposed to both the plasma and
the helium. Critically, the holes left from the pure helium
were smaller in area.
The final group of experiments utilized 50 L of PBS
in each of six total wells. Three cell culture wells were
exposed to the helium and three wells were exposed to the
plasma. Two of the six wells were exposed for 60 seconds,
whereas the other four were exposed for 180 seconds. The
mass flow rate was at its maximum of 775 mL/min. After
the experiments, it was observed that the plasma exposed
cultures had voids in the cell culture that were two to three
times the diameter of the voids in the helium-exposed
cultures, as shown in Figure 4. This result indicates an

(a)

(b)

(c)

(d)

Figure 4. Images of metastatic breast cancer culture MDA-MB-231 post treatment. Images are equivalent in scale. Each
culture was exposed to 3-minute dosages. (a) Helium-exposed culture, (b) Plasma-exposed culture, (c) Helium-exposed
culture, (d) Plasma-exposed culture. The voids in cell culture are larger for plasma-exposed culture.
additional mechanism of cell destruction is active in the
low-temperature plasma beyond the kinetic energy of the
jet molecules. The hypothesis is that reactive oxygen
species generated in the plasma plume are responsible for
the increased destructive effects due to increased oxidative
stress on the cells. These reactive species can diffuse
through the surrounding air and the PBS buffer, resulting in
a larger area of cell destruction compared to the helium jet.
Further experimentation is planned to quantify the
generation of reactive chemical species and evaluate this
hypothesis.

Further investigation is needed to determine why the

pure helium exposure can result in cell destruction.
Different gases will also be used in future experiments as
well as longer treatment times to the cultures themselves.
Larger diameter wells and a greater volume of PBS will
also be used. Further experiments will be executed in the
near future to investigate the role that low-temperature
plasma, and subsequent enhanced reactive chemistry in the
plasma plume, has in successfully treating cancer cells
without harming neighboring healthy tissue by expanding
upon the results from these preliminary tests.
Acknowledgments

5. CONCLUSIONS
In summary, a preliminary investigation of the effects of a
low-temperature plasma needle on metastatic breast cancer
cultures was undertaken. The results indicate that the
helium jet and the plasma jet both result in cell destruction
under certain conditions. Cell destruction was observed to
occur for a larger area in the case of the plasma jet
indicating an additional mechanism of cell destruction
beyond the kinetic energy of the jet molecules. The
generation of reactive oxygen species in the plasma plume
and the subsequent oxidative stress on the cells is the
proposed method of action. The results provide a solid basis
for continuing investigations regarding the mechanisms
and efficacy of low-temperature plasma on metastatic
breast cancer.

The authors wish to thank D. Sosnoski, for helping prepare

the cell cultures. The BC line (MDA-MB-231, ATCC-HTB
26 presumptive equivalent) was initially provided by Dr. D.
Welch, University of Alabama at Birmingham.
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