22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Analysis of energetic oxygen species effect on in vitro biological responses of RF
plasma-treated polymer
F. Rezaei1 and B. Shokri1,2
1
Laser & Plasma Research Institute, Shahid Beheshti University G.C., Evin, Tehran, 1983963113, Iran
2
Physics Department, Shahid Beheshti University G.C., Evin, Tehran, Iran
Abstract: The low pressure RF (13.56 MHz) plasma generated from high purity oxygen
gas was used to impart biological characteristics to Polymethylmethacrylate (PMMA).
PMMA samples were modified in constant pressure and voltage. The plasma process was
monitored using optical emission spectroscopy (OES) to study the oxygen species which
were generated in the plasma. Water contact angle goniometry showed that the plasma
process was followed by a decrease of contact angle. Samples were characterized for their
cell viability and biocompatibility by MTT assay using MEF cells. Moreover, antibacterial
activity of samples utilizing the method of plate counting against Escherichia Coli was
evaluated. Based on the obtained results, cell viability and antibacterial performance of
samples were statistically studied in terms of water contact angle and intensity of most
predominant oxygen species.
Keywords: oxygen species, OES, antibacterial activity, MEF cells, goniometry
1. Introduction
Polymethylmethacrylate (PMMA), because of its
desirable volume properties, is one of the most
extensively used polymeric biomaterials in different
biomedical applications. One of the disadvantages of
polymeric biomaterials is the general lack of the desirable
surface properties, such as biocompatibility and
bioactivity, in the majority of cases [1, 2]. Surface
properties of biomaterials, including wettability,
morphology and chemistry are of great importance in
determining their biological responses and long term
performance. Therefore, the main goal in designing the
biomaterials is to ensure that they exhibit appropriate
surface properties as well as desired physical and
mechanical characteristics [3].
Therefore, surface
modification of polymers for biomedical applications is
required.
Plasma-based processing is one of the common
methods for modifying surface characteristics without
affecting bulky properties [4, 5]. It is thus possible to
have desired volume properties of the biomaterial and at
the same time also improve its surface properties to be
able to function properly in the biological environment.
Plasma can be used for regulation of cells and bacteria
attachment processes as a source of biologically active
agents, like reactive species, charged particles and
ultraviolet light. It is quite important to understand how
these agents affect the surface of biomaterials to optimize
plasma parameters. It is also critical to identify plasma
components which have different effects on cells and
bacteria.
In the present study, we investigated the effect of most
predominant oxygen species intensities on the cellular and
antibacterial responses of PMMA samples. It is stated
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that use of energetic oxygen species in plasma treatment
of the polymer leads to enhancement of biocompatibility
and antibacterial characteristic of PMMA films.
2. Experimental
2.1 Surface Modification Process
In present study, PMMA films were used as samples.
Samples were ultrasonically cleaned in deionized water
and ethanol successively. Oxygen (99.99% purity) was
used as carrier gas. PMMA samples were modified via
low pressure RF frequency of 13.56 MHz plasma in a
parallel plate capacitively coupled reactor, described
elsewhere [5]. The pressure and RF power were kept
constant at 40 mTorr and 60 W, respectively. Samples
were treated for 2 min at various gas flow rates
(10 - 120 sccm). To ensure the reproducibility of the
results, each experiment was done at least three times.
Therefore, the results obtained by taking the average of
analysed data.
2.2 Optical Emission Spectroscopy (OES)
OES was employed for monitoring the reactive oxygen
plasma. OES measurements are very sensitive to change
in the high-energy fraction of the electron distribution
function (EDF), which is responsible for production of
radicals and ions in reactive plasmas [6]. Coinciding with
treatments the optical spectra were detected in the
wavelength range from 200 to 1100 nm by an optical
spectrometer (AvaSpec-3648).
2.3 Contact Angle Goniometry
A simple technique to measure the reactive level of a
surface is contact angle measurement. Contact angles
were determined by goniometry with Sessile Drop
1
2.5 In Vitro Bacterial Adhesion Assay
The antibacterial performance of plasma treated
samples was assessed using a modified plate-counting
method against Escherichia coli ATCC 25922 (Gram
negative). This assay has been described in detail
previously [5].
2.6 Statistical Analysis
The statistical analysis was performed using OriginPro
8 SR0 software (v8.0724, OriginLab Corporation, USA).
One way analysis of variance (ANOVA) was used to
compare mean values.
3. Results and Discussion
The samples were exposed to oxygen plasma at
different gas flow rates between 10 to 120 sccm. The
optical spectra were measured continuously during the
plasma treatment, as explained earlier. A typical optical
spectrum of oxygen plasma is shown in Fig. 1.
Table 1. Predominant transition lines observed in optical
emission spectra.
Oxygen species
𝑂
𝑂
𝑂2+
𝑂2+
Gas flow: 60 sccm
O (777 nm)
Intensity (a.u.)
30000
20000
10000
O2+ (559 nm)
O2+ (525 nm)
O (844 nm)
5000
0
200 300 400 500 600 700 800 900 1000 1100
Wavelength (nm)
Fig. 1. Typical optical spectrum of oxygen plasma.
2
559
525
-
9800
15600
O2+(559 nm)
O(844 nm)
15500
9700
15400
9600
15300
9500
15200
9400
15100
9300
15000
9200
14900
9100
14800
9000
14700
15
30
45
60
75
90
105 120
Gas flow rate (sccm)
Fig. 2. Variation of emission intensities of the oxygen
species in function of gas flow rate.
25000
15000
Transition line
3𝑝5 𝑃 → 3𝑠 5 𝑆
3𝑝3 𝑃 → 3𝑠 3 𝑆
According to Fig. 2, it is generally accepted that the
intensity of both atomic oxygen (844 nm) and oxygen
molecular ion (559 nm) decreases with increasing gas
flow rate. This is more likely due to increasing of
recombination of active species with increasing gas flow.
Oxygen is an electronegative gas and so decreases the
number of electrons by attachment under formation of
negative 𝑂− and 𝑂2− ions [8]. Therefore, by considering
the mechanisms [1] and [2], less free electrons in plasma
environment leads to less production of these oxygen
species.
0
35000
Wavelength
777
844
Intensity at 844 nm (a.u.)
2.4 In Vitro MTT Cell Viability Assay
Cell viability of samples was examined using MTT
assay after 48 h the mouse embryonic fibroblast (MEF)
cell seeding. The MTT method is based on the fact that
living cells are capable of reducing less colored
tetrazolium salts into intensely colored formazan
derivatives. After 48 h, the culture medium was aspirated
off and samples were rinsed twice with PBS to remove
unattached cells. The remaining cells attached to samples
were detached by trypsin-EDTA. Subsequently, MTT
solution (0.5 mg/ml) was added to each well and cells
were then incubated at 37 °C in a humidified 5% CO 2
atmosphere for 4 h, afterwards, the remaining solution
was removed and DMSO was added to wells to dissolve
the formazan crystals. Cell viability was evaluated by
measuring absorbance (optical density) at 570 nm using a
plate reader (ELISA Plate Reader). Obtained optical
density (OD) for each well is proportional to the number
of living cells [7].
The major spectral features are corresponding to
oxygen atom lines at 777 and 844 nm and the 𝑂2+ ion lines
at 559 and 525 nm. These transition lines are given in
Table 1.
Intensity variations of the 844 and 559 nm emission
bands as a function of gas flow rate were studied and are
presented in Fig. 2.
Intensity at 559 nm (a.u.)
technique. The measurements were performed using
3.0 𝜇𝜇 distilled water in triplicate.
Results of contact angle goniometry are listed in
Table 2. The decrease in contact angle with reducing gas
flow rate indicates that the surface is becoming
hydrophilic. This can be explained by the formation of
more atomic 𝑂 (844 nm) and 𝑂2+ ion (559 nm) as a result
of plasma treatment and their dispersion on the surface
[9], which is in complete compliance with Fig. 2.
The effect of intensity of these two emission lines on
cell viability of samples is evaluated. Viability of MEF
cells that were exposed to samples was calculated using
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Table 2. Variation of droplet contact angle as a function
of gas flow rate.
Contact angle (°)
22.6±0.1°
17.0±0.8°
36.2±1.2°
41.5±0.5°
46.1±0.6°
50.7±0.6°
With reference to Fig. 3, it is revealed that two stages of
cell viability changes are identified. Firstly, the cell
viability increases with a rapid rising of 𝑂2+ ion (559 nm)
and atomic 𝑂 (844 nm) intensities. In the second stage,
the viability continues to increase with a more slightly
increasing of the 𝑂2+ and 𝑂 intensities.
9800
15600
9600
15400
9500
15300
9400
15200
9300
9200
15100
559 nm: R2=0.650
844 nm: R2=0.866
15000
9100
9000
50
52
54
56
58
60
62
64
66
68
70
Viability (%)
Fig. 3. Viability variations of PMMA samples via
emission intensity of O at 844 nm and O+
2 at 559 nm.
Statistical analysis showed that the relationship between
cell viability (𝑉) and intensity of these oxygen species (𝐼)
can be expressed by exponential model. For the 𝑂2+ at
559 nm and the atomic 𝑂 at 844 nm equations [4] and [5]
were obtained, respectively.
Presence of the reactive oxygen species may improve
the reactive level of the surface [10]. When the highly
reactive species reach the sample surface, they break
weak bonds stabilized with radicals, promoting the
formation of polar and oxygen-containing groups at the
surface and opening up the chemical sites to future
bonding [11].
Oxygen-containing functional groups like hydroxyl and
carboxylic groups help to improve the surface
biocompatibility and cellular response, and also hydroxyl
groups contribute to cell colonization [12]. Moreover,
carboxylic functions contribute to bond proteins to the
surface, so the cells do not perceive the surface as a
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9800
15540
9700
15480
9600
15420
9500
15360
9400
15300
9300
15240
9200
559 nm: R2=0.574
844 nm: R2=0.924
15180
15120
9100
9000
60
63
66
69
72
75
78
81
84
Antibacterial activity (%)
Intensity at 844 nm (a.u.)
Intensity at 559 nm (a.u.)
9700
15500
15600
Intensity at 844 nm (a.u.)
Gas flow rate (sccm)
10
20
30
60
90
120
foreign material, which is corresponding to the
enhancement of biocompatibility [13]. Therefore, with
increasing the intensity of the energetic oxygen species,
biocompatibility and cellular attachment enhances.
Variation of antibacterial behaviour of samples in term
of the emission intensities of these energetic oxygen
species is presented in Fig. 4. Antibacterial activity of the
modified samples was calculated using equation [6],
where 𝑀 and 𝐶 are mean number of bacteria on the
modified and control samples, respectively.
Intensity at 559 nm (a.u.)
equation [3], where 𝑂𝑂𝑠 and 𝑂𝑂𝑐 are the average optical
density of sample group and control group, respectively.
The results are plotted in Fig. 3.
Fig. 4. Antibacterial activity of PMMA as a function of
emission intensity of O at 844 nm and O+
2 at 559 nm.
As can be seen, with increasing both intensities,
samples show more appropriate antibacterial behaviour.
Statistical analysis indicated that antibacterial activity (𝐵)
of samples varies linearly in term of the intensity of the
oxygen species (𝐼). Equations [7] and [8] were obtained
for the 𝑂2+ at 559 nm and the atomic 𝑂 at 844 nm,
respectively.
It is well accepted that cell walls of most bacterial
strains are negatively charged [14]. As mentioned earlier,
energetic oxygen species are responsible for surface
activation and formation more oxygen-containing
functional groups at the surface. Some of these groups
are negatively charged like carboxylic group. With
increasing concentration of these groups, the surface
becomes more hydrophilic and negatively charged.
Thereupon because of the strong repulsive forces,
increasing the intensities of the oxygen species enhances
the antibacterial efficiency.
Also, in this work changes in cell viability and
antibacterial activity of PMMA samples as a function of
contact angle values is studied (Fig. 5.).
As expected, according to the earlier discussion in this
paper, it is clearly observed that higher polymer surface
hydrophilicity (corresponding to lower contact angles) is
positively affecting the biological responses of PMMA
samples and enhances cell adhesion and antibacterial
efficiency.
3
70
84
68
78
Viability (%)
64
75
62
72
60
69
58
66
56
63
54
Viability (%)
Bactericidal activity (%)
52
Antibacterial activity (%)
81
66
60
50
57
15
20
25
30
35
40
45
50
55
o
Contact angle ( )
Fig. 5. Changes in cell viability and antibacterial activity
via contact angles.
4. Conclusion
In this paper, the role of atomic oxygen (844 nm) and
𝑂2+ ion (559 nm) intensities in cellular viability and
antibacterial activity of PMMA surface using reactive
oxygen plasma was investigated. The plasma was
characterized by optical emission spectroscopy. Obtained
results demonstrated that presence of the energetic
oxygen species leads to significant enhancement of MEF
cells adhesion to the surface of PMMA samples and also
better antibacterial performance of the surface after the
plasma treatment. Oxygen-containing functional groups,
which were introduced to the surface via low pressure RF
plasma treatment, play important role in determining final
biological efficiency and responses of the surface.
Statistical study revealed that cell viability varies
exponentially in terms of both atomic oxygen and oxygen
molecular ion intensities, while antibacterial activity
varies linearly.
Moreover, water contact angle
goniometry demonstrated that enhancing the surface
hydrophilicity cause an increase in both cellular
attachment and antibacterial activity.
4
Equations
[1] 𝑒 + 𝑂2 → 𝑒 + 𝑂∗ + 𝑂
[2] 𝑒 + 𝑂2 → 2𝑒 + 𝑂2+
𝑂𝑂
[3] 𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 (%) = 1 − ��1 − 𝑠 � × 100�
𝑂𝑂𝑐
[4] 𝐼559 = 15459.5 �1 − 𝑒𝑒𝑒�−0.8(𝑉 − 47.9)��
[5] 𝐼844 = 9681.3 − 2.4 exp(−0.3𝑉)
𝑀
[6] 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (%) = 1 − � × 100�
𝐶
[7] 𝐼559 = 14415.4 + 13.2 𝐵
[8] 𝐼844 = 7488.2 + 27 𝐵
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