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

1Undergraduate 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 low-

temperature 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 low-

temperature 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: high-

temperature (thermal) plasma, or HTP, and low-

temperature (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

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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.

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

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

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

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.

Figure 2. The interface between the LTP syringe

interacting with a saline sample. The visible plasma jet

has a diameter similar to the syringe.

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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

capability, a secondary current limiter is included via a 500-

k 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

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.

4

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.

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.

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

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.

REFERENCES

[1] American Cancer Society. Cancer Treatment and

Survivorship Facts & Figures 20142015. Atlanta: American Cancer Society; 2014.

[2] G. diSibio and S. French, Metastatic Patterns of Cancers: Results from a Large Autopsy Study, Arch. Pathol. Lab. Med., vol. 132, 2007.

(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.

5

[3] J. OShaughnessy, Extending Survival with Chemotherapy in Metastatic Breast Cancer, The Oncologist, vol. 10, no. 3, pp. 2029, 2005.

[4] W. Thornhill and D. Talbott, The Electric Universe.

Portland, OR: Mikamar Pub., 2007.

[5] M. Wang, B. Holmes, X. Cheng, W. Zhu, M. Keidar and L.

Zhang, Cold Atmospheric Plasma for Selectively Ablating Metastatic Breast Cancer Cells, PLoS ONE, vol. 8, no. 9, p. e73741, 2013.

[6] R. Walk, J. Snyder, P. Srinivasan, J. Kirsch, S. Diaz, F.

Blanco, A. Shashurin, M. Keidar, and A. Sandler, Cold atmospheric plasma for the ablative treatment of

neuroblastoma, Journal of Pediatric Surgery, vol. 48, no. 1, pp. 6773, 2013.

[7] M. Keidar, A. Shashurin, O. Volotskova, M. Ann Stepp, P.

Srinivasan, A. Sandler and B. Trink, Cold atmospheric plasma in cancer therapy, Physics of Plasmas, vol. 20, no. 5, p. 057101, 2013.

[8] R. Guerrero-Preston, T. Ogawa, M. Uemura, G.

Shumulinsky, B. Valle, F. Pirini, R. Ravi, D. Sidransky, M.

Keidar and B. Trink, Cold atmospheric plasma treatment selectively targets head and neck squamous cell carcinoma

cells, Int. J. Mol. Med., 2014.

[9] A. Hirst, F. Frame, N. Maitland and D. OConnell, Low Temperature Plasma: A Novel Focal Therapy for Localized

Prostate Cancer? BioMed Research International, vol. 2014, pp. 115, 2014.

[10] O. Volotskova, T. Hawley, M. Stepp and M. Keidar,

Targeting the cancer cell cycle by cold atmospheric plasma, Scientific Reports, vol. 2, 2012.

[11] X. Cheng, J. Sherman, W. Murphy, E. Ratovitski, J. Canady

and M. Keidar, The Effect of Tuning Cold Plasma Composition on Glioblastoma Cell Viability, PLoS ONE, vol. 9, no. 5, p. e98652, 2014.

[12] S. Mirpour, H. Ghomi, S. Piroozmand, M. Nikkhah, S.

Tavassoli and S. Azad, The Selective Characterization of Nonthermal Atmospheric Pressure Plasma Jet on Treatment

of Human Breast Cancer and Normal Cells, IEEE Transactions on Plasma Science, vol. 42, no. 2, pp. 315322, 2014.

[13] C. Hoffmann, C. Berganza and J. Zhang, Cold Atmospheric Plasma: methods of production and application in dentistry

and oncology, Med Gas Res, vol. 3, no. 1, p. 21, 2013.

[14] Y. Kim, S. Jin, G. Han, G. Kwon, J. Choi, E. Choi, H.

Uhm and G. Cho, 'Plasma Apparatuses for Biomedical

Applications', IEEE Transactions on Plasma Science, pp. 1-

1, 2015.

[15] R. Cailleau, M. Oliv and Q. Cruciger, Long-term human breast carcinoma cell lines of metastatic origin: Preliminary

characterization, In Vitro, vol. 14, no. 11, pp. 911915, 1978.

[16] D. Rusciano, D. Welch and M. Burger, In vivo cancer metastasis assays, in Cancer metastasis: experimental approaches. Amsterdam: Elsevier, 2000, pp. 207242.