Optimization of a Cold Atmospheric Plasma Treatment to Selectively

Affect the Viability of Skin Cancer Cells

Sara Pereira

, Paulo António Ribeiro

and Susana Sério

CEFITEC, Departamento de Física, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa,

2829-516, Caparica, Portugal

Keywords: Cold Plasmas, Skin Cells, Plasma Jet Device, Hydrogen Peroxide, Plasma Medicine.

Abstract: Cold atmospheric plasmas (CAPs) are a specific type of non-thermal plasmas mainly composed by reactive

oxygen and nitrogen species (ROS and RNS, respectively), UV radiation and charged particles. In the last

years, liquids treated by CAPs (indirect CAPs treatments) have attracted a significant interest in oncology due

to its ability to kill cancer cells with an effectiveness similar to direct irradiation of the cells by cold plasmas.

It is important to point out that indirect treatments have the advantage of avoiding the effects of UV radiation

and the electrical fields present in plasmas being their effects mainly dependent on the ROS and RNS

produced in the liquid phase. To better understand the mechanisms behind the interaction between CAPs,

treated liquids and cells, it was engineered a plasma jet device and studied the vulnerability of different cell

lines to the culture medium previously exposed to CAPs. For that, it was analysed the concentration of H

produced during the treatments by means of colorimetric assays and evaluated the influence of using different

working parameters such as volume of medium and gap. According to the obtained results it could be observed

that the cancer cell line (Met-1) in study is more sensitive to the liquids treated by CAPs than the non-cancer

one (HGF-1), which in the particular case of this jet, seems to be mainly related with the concentration of

RNS species produced in the liquids during the plasma exposure since the concentration of the H

produced

is very low.

1 INTRODUCTION

Cold atmospheric plasmas (CAPs) are a near-room

temperature plasma, generally produced in laboratory

conditions, by applying an external source of energy

to a neutral gas up to a critical point, at which

electrons dissociate from atoms (Fridman, Chirokov,

and Gutsol 2005; Weltmann and Von Woedtke 2017).

The resulting ionized gas will be mainly composed by

a mixture of reactive oxygen and nitrogen species

(ROS and RNS), UV, visible and infrared light,

electromagnetic fields, electrons and ions (Pipa et al.

2012; Reuter et al. 2009, 2012). It is important to note

that, since these plasmas are laboratory-generated,

their properties, such as their energy and charged

particles density, will be dependent on the features of

the used set-up: applied power and its type, and also

of the feeding gas (Bekeschus et al. 2013; Weltmann

and Von Woedtke 2017).

https://orcid.org/0000-0002-8682-5462

https://orcid.org/0000-0001-9665-7610

https://orcid.org/0000-0002-8086-7792

Since, under in vivo conditions, cells and tissues

are surrounded by a liquid environment, during the

past decade, liquids treated by CAPs (indirect plasma

treatments) have attracted attention in clinical plasma

medicine (Jablonowski and von Woedtke 2015). So

far, a significant number of studies, have shown

similar effectiveness between treated liquids and

direct irradiation of cells (Keidar et al. 2013; Liedtke

et al. 2017; Nakamura et al. 2017; Tanaka et al. 2011;

Wende et al. 2014; Yan et al. 2014, 2017), over a wide

range of cancer cell lines, including melanomas and

carcinomas (Pereira et al. 2019; Yan et al. 2015).

Additionally, indirect treatments have the advantage

of avoiding the effects of UV radiation and of the

electromagnetic fields present in plasmas, being their

effects mainly related with the ROS and RNS species

produced in the liquid phase. Although, the explana-

tion in detail of which species are active in plasma-

treated liquids remains a challenge. Some general

Pereira, S., Ribeiro, P. and Sério, S.

Optimization of a Cold Atmospheric Plasma Treatment to Selectively Affect the Viability of Skin Cancer Cells.

DOI: 10.5220/0009373802010208

In Proceedings of the 8th International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS 2020), pages 201-208

ISBN: 978-989-758-401-5; ISSN: 2184-4364

201

conclusions have been taken about the anti-cancer

mechanisms of CAPs. For example, it was shown that

the increase in ROS species will cause damages to the

antioxidant system and consequently lead to DNA

double-strand breaks (Adachi et al. 2015; Sauer,

Wartenberg, and Hescheler 2001). Another conclu-

sion is that the CAPs effects will result in cellular

apoptosis or necrosis in a dose-dependent way.

Moreover, H

and NO produced in treated liquids

are proposed to be key molecules to preferably kill

cancer cells instead of non-cancerous ones

(Bekeschus et al. 2014; Jablonowski and von

Woedtke 2015).

In the present work, the indirect treatment of Met-

1 cells (Squamous Cell Carcinoma keratinocytes),

which represents 24% of all skin cancers, was

performed. Squamous Cell Carcinoma represents 20-

30% of the reported cases of non-melanoma skin

cancers and its incidence is increasing over the world

(Graham and Tuchayi 2016; Waldman and Schmults

2019). To understand the mechanisms behind the

interaction between CAPs, treated liquids, and

eukaryotic cells, it was engineered a plasma jet

device, which will be described in this paper, and

study the vulnerability of Met-1 and HGF-1 cells

(human fibroblasts) to indirect CAPs treatment. It

was also investigated if CAPs anti-cancer capacity

was dependent on the parameters that were chosen to

perform the plasma treatments. For that, different

volumes of the medium, distances from the jet to the

liquid to be treated and times of plasma exposure

were tested. The vulnerability of Met-1 cells to CAPs

treatment was then compared to one of the HGF-1

cells (human fibroblasts). In addition, the

concentration of H

produced in the treated liquid

was measured and the vulnerability of Met-1 and

HGF-1 cell lines to H

rich DMEM w/o sodium

pyruvate was studied, to determine if H

is the only

reactive species responsible for the CAPs effects.

2 MATERIALS AND METHODS

2.1 Experimental Set-up

The CAP jet device used in this research was

designed and constructed in our laboratory (Plasmas

and Applications laboratory, CEFITEC, Physics

Department, FCT/UNL, Portugal). It consists of a

hand-held principal unit composed by a borosilicate

capillary with an outer diameter of 6.93 mm and an

inner diameter of 3.76 mm, with two metal electrodes,

a custom made DC power supply (2.5 mA, 20 kV),

and a gas supply unit (Pereira et al 2019). The

Figure 1: Tested electrodes: (a) copper ring, (b) titanium

ring, and (c) copper wire.

electrode on the inside of the capillary is a stainless-

steel needle with a diameter of 2 mm. For the outer

electrode (connected to the high voltage) three

different hypotheses were tested: a copper ring, a

titanium ring and one made from an enameled copper

wire, Figures 1 (a), (b) and (c), respectively. In order

to choose the electrode that allowed a more stable

plasma, spectroscopic analysis of the plasma plume

obtained using each one of the referred electrodes was

performed. According to the obtained results, it was

decided to develop the jet using the third

configuration (Figure 1 (c)). The copper wire coil

used has a 1 mm of thickness and a height of 7 mm,

corresponding to seven turns. Once the optimal

configuration in terms of stability was determined,

the device was coated in order to facilitate safe

handling and system automation.

2.2 Optical Emission Spectroscopy

OES was performed using an optic fiber (FC-UV600-

2, Avantes) coupled to a spectrometer (SPEC STD,

Sarspec’s). The size of the spectrometric quartz glass

lens covered the whole length of the effluent and was

kept at approximately 5 mm perpendicular to the

plasma plume. The instrument has a resolution of 1.7

mm and emission intensities on the range 180-1100

nm were recorded.

2.3 Cell Lines and Cell Culture

Human Squamous Cell Carcinoma (Met-1) cells were

purchased from Ximbio (153539, London) while

human fibroblasts obtained from a gingival biopsy

(HGF-1) were purchased from American Type

Culture Collection (ATCC

CRL-2014

Barcelona). Both cell lines were cultured in 75 cm

flasks (Corning

, 4314640) with complete DMEM

[(Dulbecco’s Modified Eagle’s Medium, Sigma,

D5030), supplemented with 1.0 g/L D-glucose

(Gibco, 15023-021), 3.7 g/L sodium bicarbonate

(Sigma-Aldrich, S5761), 1% GlutaMAX

(L-

Biophotonics 2020 - Special Session on Biomedical Optics

202

alanyl-L-glutamine dipeptide, Life Technologies,

35050-038), 1% sodium pyruvate (Gibco, 11360039),

penicillin (100U/ml) and streptomycin (100 µg/mL)

(Invitrogen, 15140122), 10% FBS (Fetal Bovine

Serum, Invitrogen, 10270106)]. When reaching

confluence, the cells had to be transferred into new

flasks by an enzymatic method. Met-1 and HGF-1

cells are adherent cell lines that have to be maintained

under 37°C in a humidified atmosphere of 5% CO

(standard conditions).

2.4 Plasma Treatment Conditions

The protocol used for the both cell lines in study was

identical. Met-1 and HGF-1 cells were seeded in a 96-

well plate (83.3924, Sarstedt) with a confluence of

3.5  10



cells/mL and incubated overnight under

standard conditions. A specific volume of complete

DMEM without sodium pyruvate were plasma-

treated in wells of a 12-well plate (83.3921.005,

Sarstedt), for different times. To perform the

treatments, the plasma jet was placed above the upper

edge of the well containing the liquid to be treated and

it rested at the chosen position until the end of the

treatment. The treated liquid was mixed by an argon

flux of 3 standard liters per minute (slm) controlled

by a flowmeter (Dynamal Argon 0-15 L/min, Air

Liquid). 100 or 150 µL of the treated culture medium

were then immediately transferred to the previously

cultured cells in sextuplicate. The same volume of

untreated medium was used as a positive control in

sextupllicate, in all the performed experiments.

Before this, the medium used to culture the cells

overnight was discarded and the cells were washed

with Phosphate Buffer Saline (PBS). The cells were

incubated for 48 hours under standard conditions and

only then the cellular viability was assessed by the

resazurin assay. On the present study, it was decided

to use DMEM without sodium pyruvate, since the last

could act as a scavenger for hydrogen peroxide

(Pereira et al. 2019; Wende et al. 2015).

2.5 pH and Temperature

Measurements

2 mL of cell culture medium were treated as

previously described. Immediately after the

treatment, the pH of the treated solution was

measured using a ph-meter. The temperature of the

treated solution was determined using a multimeter

associated with a k type thermocouple (RS, Portugal).

2.6 Hydrogen Peroxide Determination

in Complete DMEM

The H

concentration of the treated liquid was

analyzed using a Fluorimetric Hydrogen Peroxide

Assay Kit (Sigma-Aldrich, MAK165). After the

plasma treatment, 50 µL/well of the treated liquid

were transferred in triplicate to different wells of a

black 96 well flat-bottom plate. As control, 50 µL of

DMEM without sodium pyruvate were also

transferred to a well on the same plate in triplicate. 50

µL of a previously prepared master mix (assay

reaction solution) was added to all the wells in study

(samples, standards, and controls). The plate was then

incubated at room temperature for 30 minutes

protected from light and the fluorescence intensity

was measured at λ

excitation

=540 nm and λ

emission

=590

nm using a fluorescence plate reader (Tecan infinite

200). Final concentrations were calculated using a

standard curve.

2.7 Effects of H

Rich Medium

Met-1 and HGF-1 cells were seeded in a 96-well plate

with a cell confluence of 3.5   10



cells/mL and

cultured in an incubator overnight under standard

conditions. Then, different solutions of H

rich

medium were prepared. The H

solutions were

prepared by mixing a 30% w/w H

solution (Sigma)

into the complete DMEM without sodium pyruvate.

The prepared H

rich medium was then transferred

to the previously cultured cells in sextuplicate. Before

this step, the medium that has been used to culture the

cells overnight was discarded and the cells were

washed with PBS. After that, the cells were incubated

for 48 hours under standard conditions, and only then

the cellular viability was assessed by the resazurin

assay. Positive control of cells in untreated DMEM

without sodium pyruvate and without H

was used

in all the realized experiments.

2.8 Cell Viability: Resazurin Assay

The resazurin assay is based on the ability of the

dehydrogenase enzyme, present in metabolically

active cells, to reduce the resazurin (7-Hydroxy-3H-

phenoxazin-3-one 10-oxide) blue dye into a pink

colored and highly red fluorescent resorufin (3H-

phenoxazin-3-one) product. The amount of resorufin

produced is directly proportional to the mitochondrial

enzyme activity, i.e. to the number of viable present

cells (Anoopkumar-Dukie et al. 2005; Riss et al.

2004), which can be easily quantified using a

Optimization of a Cold Atmospheric Plasma Treatment to Selectively Affect the Viability of Skin Cancer Cells

203

Figure 2: Optical emission spectrum of the plasma plume

for three different electrodes, obtained using an Argon flow

of 3 slm, where the black spectrum represents the electrode

of copper wire, the red the titanium ring and the blue the

copper ring.

microplate reader. To assess cell viability, the culture

medium in the wells was discarded and 100 µL of

resazurin solution (0.04% resazurin, Life

Technologies, USA) were added to all the wells in

study. After an adequate time of incubation in the

dark at standard conditions, the absorbance was

measured in a microplate reader, using a wavelength

of 570 nm and a reference standard of 600 nm.

2.9 Statistical Analysis

All data are expressed as mean ± standard deviation

(SD) of at least three independent experiments with

six replications. The statistical significance of the

differences was evaluated using the Student t-test and

it was recognized as * for p0.05, ** for p0.01 and

*** for p0.005.

3 RESULTS

3.1 OES Obtained for the Different

Tested Electrodes

To verify if the plasma produced using the three

different electrodes (copper ring, titanium ring, and

copper wire) have the same composition, the optical

emission spectrum of the discharge was acquired for

all of them. Observing Figure 2, despite some small

differences in terms of relative intensities, the

produced species seems not be dependent on the outer

electrode used. The obtained emission spectra show

an emission peak belonging to OH

radical in the UV-

B region at 308 nm and some peaks between 330 and

400 nm representing the nitrogen (N

) emission in the

UV-A range. Since neither oxygen nor nitrogen are

Figure 3: Plasma effects are dependent on the used gap.

present in the working gas used, the appearance of

these emission bands can be attributed to interactions

between the generated plasma and the surrounding

ambient air (Hoentsch et al. 2014). According to

Lukes and Locke 2005, the OH

radical in the gas

phase discharge appears as a consequence of the

electron impact of H

O molecules in the water vapor

above and near the liquid surface. The near infrared

(700-900 nm) emission peaks mainly represent

excited molecules of Argon.

3.2 Effect of the Plasma Treatment

Conditions on the Cell Viability

In order to understand the relationship between

plasma treatment and cell viability, the plasma

treatment conditions, namelly, the distance between

the jet and the liquid to be treated (gap), the treated

volume, and the time of exposition to plasma were

changed. First, to understand how the chosen gap can

affect cell viability, using a cell concentration of

3.5  10



cells/mL and treatment time of 2 minutes,

the used gap was varied from 2 mm to 7 mm, and to

9 mm. The results, shown in Figure 3, indicate that

the viability of Met-1 cells increases as the distance

from the jet increases. This means that proximity of

the jet enhances its anti-tumor capacity, and for this

Figure 4: Influence of use different volumes during plasma

treatment.

2 mm 7 mm 9 mm

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

***

Relative cell viability (a.u.

)

Gap (mm)

***

Biophotonics 2020 - Special Session on Biomedical Optics

204

Figure 5: Plasma treatments are time dependent.

reason the gap used to carry out the treatments must

be carefully chosen.

Likewise, to see if the effects of plasma treatment

are volume dependent, different volumes of DMEM

w/o sodium pyruvate were treated under the same

conditions. After the treatment 150µL of the treated

medium were transferred to the previously cultured

Met-1 cells, and cell viability was measured 48 hours

after plasma treatment. Three different volumes of

medium were plasma treated: 2, 3 and 4 mL. By the

analysis, of the Figure 4, it is observed that the killing

capacity of CAPs treated medium seems to decrease

as the total volume of treated medium increase.

According to literature, these results can be explained

by the dilution of the reactive species formed during

the plasma exposure in the treated medium(Wende et

al. 2015, 2016; Yan et al. 2015).

Moreover, using the same volume and gap, the

plasma treatment was performed for different times

of exposure. In this case, the assays were performed

for Met-1 and also HGF-1, in order to investigate if

cancerous, and non-cancerous cells have a similar

response to CAPs treatment. According to the

obtained results, Figure 5, Met-1 cells seems to be

much more sensitive to plasma treatment than HGF-

1 cells, whose viability remains almost unaffected.

For Met-1 cells, and a treatment time of 2 minutes,

the viability was already reduced to less than 50%

relative to the control, while for HGF-1 no significant

differences were found between the different tested

times. Here, it can be concluded that cancerous cells

are much more sensitive to CAPs treatment than non-

cancerous cells, and CAPs treatment is time

dependent. Accordingly, to the literature (Chauvin et

al. 2017; Wende et al. 2016), this occurs due to the

increased concentration of short and long reactive

species in the medium as the time of plasma exposure

increase (section 3.2.2).

Figure 6: pH measurements of plasma treated culture

medium. Results of 3 independent experiments, expressed

in terms of mean±SD.

3.3 Study of the Liquid Phase

3.3.1 pH and Temperature Measurements

It is known that plasma treatment may alter the pH of

the treated culture medium and consequently affect

the cell viability. For that reason, it was measured the

pH of the plasma-treated culture medium before and

after the treatments. As shown in Figure 6, a slight

increase in the pH could be observed as the time of

plasma exposure increase. These increase in pH can

be explained by the degassing effect of the carbonate

buffer sodium bicarbonate, present in the treated

medium (DMEM w/o sodium pyruvate)

(Bundscherer et al. 2013, Rumbach et al 2015)

Despite, these small differences are not significant in

a statically point, however biologically they can

contribute to increasing the alkaline stress,

contributing to apoptosis induction.

In terms of temperature, the longest plasma

exposure of 270 seconds yielded a temperature of

31°C. Since the cellular tolerance threshold without

thermal damage is around 40°C, it can be concluded

that this increase is not enough to cause cellular

damage on its own. This indicates that the results

obtained after the cellular assays are not a

consequence of the thermal effects caused by the

plasma treatments, but of the oxidative stress caused

due to the presence of some reactive species, such as

hydroxyl radicals, that are formed by plasma

treatment of liquids.

3.3.2 Effectiveness of H

Produced in

Caps and H

O2 Rich Medium

Some authors claim that H

is the main reactive

species responsible for the selective effects of CAPs,

due to its stability and role in a variety of multiple

cellular pathways (Liedtke et al, 2017). In order to

0 60 180 270

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

pH values

Time of plasma exposure (s)

Optimization of a Cold Atmospheric Plasma Treatment to Selectively Affect the Viability of Skin Cancer Cells

205

investigate this fact, it was measured the

concentration of H

produced during the CAPs

treatment and studied the response of MET-1 and

HGF-1 cells to different concentrations of H

rich

medium. As shown in Figure 7, Met-1 cells are much

more sensitive to H

rich medium than HGF-1 cells

(IC

=11.2µM and IC

=184µM, respectively with

corresponding to the half maximal inhibitory

concentration), whose viability seems to be almost

unaffected by the presence of the H

in the tested

concentrations. However, it is important to note that

the real concentration of H

rich medium is

different than the one calculated for the preparation

of the solutions (being lower) since H

reacts with

the proteins present in the medium, for example via

Fenton reaction (Yan et al. 2015).

These results are very different from the ones

observed in treatments with CAPs medium on these

cell lines. In this last case, it was calculated an IC

only 2.69µM of H

to Met-1 cells, corresponding

to a treatment between 1 and 2 minutes. These

differences demonstrate that H

is not the only

reactive species responsible for the anti-cancer effects

of CAPs treated medium.

Figure 7: Vulnerability of H

rich medium on HGF-1 and

Met-1 cells. Results are present as mean±SD, and the

significance compared to the last bar (concentration of

0.29µM) is indicated as * p<0.05, ** p<0.01 and

***p<0.005.

4 CONCLUSIONS

The aim of this work was to understand the principles

behind the use of CAPs treated liquids in cancer

treatment, specifically on Squamous Cell Carcinoma.

For that, several parameters were studied in order to

try to optimize the anti-cancer capacity of a custom-

made Argon plasma jet device. The results presented

in this paper, show that the effectiveness of CAPs

treatment is time, volume and distance dependent.

Specifically, a shorter volume of medium and a closer

distance from the jet device to the liquid to be treated,

increase the effectiveness of the treatment. Regarding

time, this must be carefully chosen, since longer

treatments will produce high concentrations of

reactive species and consequently both cancer and

non-cancer cells may suffer irreversible damage. To

prove this anti-cancer capacity, the vulnerability of a

cancerous cell line, Met-1, was compared to the one

of a non-cancerous cell line, HGF-1, for treatments

performed in the same conditions. Despite a

significant reduction in Met-1 viability was

registered, no significant reduction in non-cancerous

HGF-1 cells was observed. To exclude possible

thermal damage and acidification of the medium,

changes on temperature and pH were monitored. In

addition, it was observed that cells respond in a

different way to the H

produced in the medium

during the treatment and to H

rich medium. This

indicates that H

is an active specie contributing to

the anti-cancer ability of CAPs treated medium but is

not the only one.

ACKNOWLEDGEMENTS

This work as supported by Fundação para a Ciência e

Tecnologia (FCT), within Radiation Biology and

Biophysics Doctoral Training Programme (Rabbit,

PD/00193/2012), through the scholarship grant

number PD/BD/114444/2016 (S. Pereira), the project

UID/Multi/04378/2013 (UCIBIO) and the project

UID/FIS/00068/2019 (CEFITEC). The authors

acknowledge Professor Doutor Jorge Carvalho Silva

from Physics Department, FCT/UNL for the use of

TELab-Tissue Engineering Laboratory facilities

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