Cancer cell (mcf-7, Colo 357 and lncaP) viability on amorphous hydrogenated carbon nitride film deposited by dielectric barrier discharge plasma



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Cancer cells (MCF-7, Colo-357 and LNCaP) viability on amorphous hydrogenated carbon nitride film deposited by dielectric barrier discharge plasma
Abhijit Majumdara†, Ramesh Ummannib, Karsten Schröderc, Reinhard Waltherb, Rainer Hippler a
a Institut for Physics, Ernst-Moritz-Arndt-University Greifswald, Felix-Hausdorff-Str. 6, 17489 Greifswald, Germany

b Institute for Medical Biochemistry and Molecular biology, Klinikum, Ernst-Moritz-Arndt-University Greifswald, 17486 Greifswald, Germany

cLeibniz Institute for Plasma Science and Technology, Felix-Hausdorff-Str.2, 17489 Greifswald, Germany
Abstract

Atmospheric pressure dielectric barrier discharge (APDBD) plasma in CH4/N2 (1:1) gas mixture has been employed to deposit amorphous hydrogenated carbon nitride (aH-CNx) film. In-vitro studies with three different cancer cell lines were carried out on the coated surfaces. Preliminary biocompatibility and effect of CH4/N2 films have been investigated by measuring cell proliferation. Three different cancer cell (MCF-7, Colo-357 and LNCaP) suspensions have been exposed on the surface of aH-CNx film to investigate the effect of deposited films on viability of cells. Results from the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) proliferation assays indicated that the deposited aH-CNx film is cytotoxic to cancer cell lines. Time course cell viability assay indicated maximum cell death at 24h after seeding the cells. This effect is dependant on physico-chemical and mechanical properties of the deposited films. The deposited film has been characterized by x-ray photoelectron spectroscopy (XPS) and Fourier transformed infrared spectroscopy (FTIR). The results confirmed the presence of C-N, C≡N, C-Hx, C-O, N-O, overlapping NH and OH bands in the film.


Keywords: Cell viability, Amorphous hydrogenated Carbon-nitride film (aH-CNx), Dielectric barrier discharge (DBD) plasma, breast cancer (MCF-7), prostate cancer (LNCaP), pancreatic cancer (Colo 357) cells.

Corresponding author: majumdar@physik.uni-greifswald.de (Abhijit Majumdar)

Fax: +493834864701

I. Introduction:

Application of atmospheric pressure plasma in life sciences is a recent advancement. But there are several limitations to use plasma in biomedical applications. Over the last five years, enormous developments have been reported in biomedical plasma research and technology [1-4]. Different plasmas are being successfully used in biomedical applications such as sterilization of plastics, controlling or modifying cell surfaces, decontamination of bacteria etc. But there will be another insight to examine whether the plasma treatment give any coating or thin layer on the treated area, or not. Now a day the biocompatibility of diamond like coating (DLC) or carbon nitride film and the plasma-tissue interaction is one of the emerging issues in plasma biomedical application [1]. In this respect it would be a novel approach to study cytotoxic effect of different plasma deposited surfaces (hydrophobic, cytotoxic etc.) on mammalian cancer cells. However, the plasma generated for biological applications should be compatible and suitable for the biological environment. Atmospheric pressure (AP) plasmas have been developed for many applications such as surface modification of polymers [5-7], air purification [8-10] and sterilization [11-17]. CNx films have been prepared by magnetron sputtering [21], electron cyclotron resonance plasma-assisted vapor deposition [22 and 28], ion beam deposition [23], plasma enhanced chemical vapor deposition (PECVD) [24 and 25], dielectric barrier discharge plasma [20 and 26] and microwave plasma CVD [27 and 28]. It is generally accepted that CNx and Diamond like Coating (DLC) coatings do not induce any cytotoxity or inflammation on usual adherent cells [18] including human embryonic kidney (HEK) cells [19]. Only one contradictory result has been reported regarding cytocompatibility (with respect to HEK and PC 12 cell lines) of aH-CNx film deposited by dielectric barrier discharge plasma [20]. Therefore it is very important that the behavior of special carbon-based coatings should be discussed based on the detailed information of the coating such as atomic bond structure, composition, and/or electronic structure [18].

It happens in most of the cases that the applied plasma could develop a thin layer on the subjected tissue/cell lines. Thus before applying the CH4/N2, DBD plasma directly on the tissues or cancer cell lines, it would be important to check the impact of the thin layer on the targeted cell lines. In our previous work [20], on cytotoxic effect, it has been observed that the different mammalian cell lines are not able to survive after a certain time span on the aH-CNx film deposited by CH4/N2 DBD plasma. To proceed further in this respect we have tried to work out the same treatment/experiment on several cancer cell lines.

In the current study we have investigated the effect of the DBD assisted plasma deposited film (CH4/N2=1:1) on mammalian cancer cell lines to understand cytotoxicity of these films. The chemical properties/composition of the deposited film has been analyzed by photoelectron spectroscopy (XPS) and Fourier Transformed infrared spectroscopy (FTIR). Here we report how CH4/N2 plasma-deposited films influence proliferation rate of cancer cells in MTS assays.
II. Materials and methods

A. Film preparation

Three films were deposited with CH4:N2 gas mixture ratios at 3:1. 1:3 and 1:1, at a constant pressure 400 mbar with constant frequency 5.5 kHz. The time course cell viability assays has been studied on one of the promising film (CH4/N2 = 1:1) among the three deposited films (CH4/N2 = 3:1, 1:1 and 1:3). The experimental set up of the dielectric barrier discharge has been explained in detail elsewhere [29, 30 and 31]. Both the Ag electrodes are covered by dielectrics: the upper (powered) electrode is covered with aluminium oxide (ε ~ 10); the lower (grounded) electrode with a glass plate (ε ~ 3.8). Glass electrode is used as dummy substrate. Electrodes are separated by 0.15 cm gap. The upper electrode is connected to a home-built high voltage power supply, while the lower electrode is grounded. The chamber is pumped by a membrane pump down to a base pressure of about 1 mbar. Pressure inside the plasma chamber was controlled by two gas flow controllers for methane and nitrogen and by an adjustable needle valve between the chamber and the membrane pump. The high voltage power supply consists of a frequency generator delivering a sinusoidal output that is fed into an audio amplifier. The amplifier can be operated at up to 500 W. Experiments were performed at 8.8 kV (peak-to-peak) and at 5.5 kHz. The dissipated power during plasma under these conditions was of 4.5 W and plasma surface power density is 0.18 W/cm2 and 0.12 W/cm3
B. Chemical surface analysis

X-ray photoelectron spectroscopy (XPS) measurements of the aH-CNx films were performed on a multi-technique 100 mm hemispherical electron analyser (CLAM2: VG Microtech), using Mg Kα radiation (photon energy 1253.6 eV) as the excitation source and the binding energy (BE) of Au (Au 4f7/2 : 84.00 eV) as the reference. The XPS spectra were collected in a constant analyser energy mode, at a chamber pressure of 10-8 mbar and pass energy of 23.5 eV at 0.125 eV/step.

Fourier transform infrared (FTIR) transmission spectra were obtained by means of FTIR spectrometer Bruker (Vector 22). The plain sample was placed in a vacuum chamber built inside the spectrometer in order to minimize the IR signal of water vapour, CO2 content and noise. The measuring signal passed the optical way with an aperture diameter of 3 mm with spectral resolution 4 cm-1. For optimal signal-to-noise ratio 50 scans were averaged per sample spectrum and apodized by applying of the Norton Beer apodization function for Fourier transformation. Interferograms were zero-filled using a zero-filling factor of 2. The background spectrum was independently measured on a pure silicon substrate.
C. Cell culture

The Prostate (LNCaP), Breast (MCF-7), Pancreatic (Colo-357) carcinoma cell lines were purchased from DSMZ and maintained in RPMI1640 medium (Invitrogen, Germany) supplemented with 10% foetal calf serum (FCS), 100 units/ml penicillin and streptomycin, glucose, sodium pyruvate, sodium bicarbonate and HEPES. Cells were grown in an incubator at 37 °C with constant supply of 5% CO2 and split after reaching 85 to 90% confluence. Cells were regularly tested for mycoplasma contamination using the MycoAlert™ Mycoplasma Detection Kit (Cambrex).

The effect of CH4:N2 films on proliferation rate of the carcinoma cell lines LNCaP was measured by the MTS assay. This assay uses the novel tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) and the electron coupling reagent, phenazine methosulfate (PMS). MTS is chemically reduced by cells into formazan, which is soluble in tissue culture medium with maximum absorbance at 492nm. In brief, 6×104 cells in 300 µl medium were seeded in each well on deposited films using rings and allowed to grow at 37 °C/5% CO2. Then MTS stock solution was added to the cells at final 1× concentration and allowed to incubate for additional 2h at 37 °C/5% CO2. 2h after incubation, formazan production in culture medium was measured at 490 nm in an ElLISA plate reader. Cell proliferation values are the mean of three independent experiments carried out with triplicate samples each. For calculation of significance, a t-test was performed using Graph Pad Prism version 3.0.

III. Results and Discussion:

A. Chemical Properties:

X-ray photo electron spectroscopy (XPS)

Full scale XPS spectra of the deposited films are shown in figure 1, with BE from 100 eV to 1160 eV for the film CH4: N2= 1:1, supporting the presence of C 1s, N 1s and O 1s bands in the deposited polymer film. Photoelectron and Auger electron are shown in one full scale XPS spectrum. After fitting the photoelectron peak (which is also the XPS peak area) area by Gaussian deconvolution method [26], it helps to determine the percent amount of elements present in the film. The observed chemical shift have been calibrated by peak fitting program [26]. With the increase of nitrogen concentration, the C-1s peak broadens and also becomes more asymmetric in nature which indicate that the nitrogen is involved in chemical bonds with carbon in three possible chemical states: C-N, C=N, C≡N and C-O bonds [26]. The best Gaussian fits to the XPS lines resulted in to four different peaks for the C--1s line and three peaks for the N--1s line. From Figure 2 (a), the C--1s spectrum (CH4:N2 ~ 1:1) exhibits peaks at 284.84 eV, 285.87 eV, 287.02 eV and 288.12 eV, which are attributed to C=C, C=N, C-N or C≡N and C-O bonds, respectively [20, 23, 25, 26, 31]. Figure 2 (b) shows, the N—1s spectrum (CH4:N2 ~ 1:1) peaks at 399.22 eV, 400.23 eV and 401.13 eV which are attributed to C-N or C≡N, C=N and N-O bonds, respectively [20, 26]. In some cases, a peak appears at 286.4 eV and is attributed to the nitrile group (C≡N) [23, 31]. The energy of the sp2 C-N peak falls within 285.5 - 285.9 eV and that of the sp3 C-N peak is assigned in the range 287.0 eV to 287.8 eV. Due to these wide spectral ranges a clear understanding of the differences between C-N and C≡N bonds [23, 31] in the analysis of C 1s XPS spectra is quite difficult. It can be deduced from figure 3, those carbon-nitrogen bonds (C=N and C-N) irrespectively of the nitrogen content of the CH4: N2 gas mixture at the expense of carbon-carbon and carbon-oxygen bonds.



Fourier transforms infrared spectroscopy (FTIR)

FTIR absorption measures the vibrational, stretching, symmetric/antisymmetric bands configuration (range 4000-400 cm-1) present in the deposited polymer film. The aH-CNx films have been analyzed by FTIR technique. Typical IR transmission spectra are shown in figure 3, within the range from 4000 cm-1 to 400 cm-1. Spectroscopic properties of the polymerized films deposited at different mixture concentrations of the reactive gase CH4:N2 are presented in figure 3 [23]. The band between 3395.83 cm-1 is attributed to stretching vibrations of NH and OH functionally groups [26]. However, the separation of the overlapped bands is not possible due to intermolecular interactions as H-bridges, which are very intensive in this region and cause the broadening of the bands. The second interval (3000 cm-1 - 2810 cm-1) is the characteristic for CH2 and CH3 groups [26]. The intensity of the bands is low (absorption up to 1%) and is related with the concentration of methane in the gas discharge. The broad absorption single peak at 2174 cm-1 is attributed to C≡N triple bond stretching vibration (so called nitrile group). The broad band at 1645.46 cm-1 is attributed to C=C and C=N stretching mode [27]. The absorption band observed in the interval 1350 cm-1 - 1480 cm-1 corresponds to the C-N single bond stretch [27]. The absorption peak at 1155.51 cm-1 is referred as C-O stretching mode [29]. The broad absorption from zone 820 cm-1 to 450 cm-1 corresponds to band [29]. From figure 3, we see that the bands corresponding to the intervals to 1350-1480 cm-1, 1645-1665 cm-1 and 2260-2290 cm-1 indicate that the carbon and nitrogen atoms in the CNx film are linked as C-N, C=N and C≡N bonds, respectively.

B. Biological response

Figure 4 shows the rate of proliferation of cancer cells after an incubation period of 24h on the aH-CNx. MTS assay results showed a significantly decreased proliferation rate of all three carcinoma cell lines. The cells seeded on to glass surface without deposition represent respective controls and the formazon formation is set at 100%. The proliferation rate of the cells on coated glass was 60% lower than their corresponding controls. The cell adhesion and proliferation at the biomaterial surface is determined by physical, chemical and mechanical characteristics. It has been shown that cells are highly sensitive to surface morphology and this interaction affects several cellular functions (cell shape and predominant type, migration, adhesion and tissue organization) [32]. Therefore we deposited CH4/N2 films at different ratios and tested them for their cytotoxic effects. The thickness of the coating depends upon the ratio of gases and current used in the DBD assisted plasma deposition. Fig. 5 shows the cell proliferation rate of cancer cells grown on films deposited with different CH4/N2 ratios. The results indicate that the films deposited with 1:1 ratio using CH4/N2 are more efficient to induce cell death in cancer cells. On the other hand we would like to point that the DBD assisted plasma deposition with 3:1 or 1:3 produced dark yellow colour films. These films produce brown colour in culture medium after seeding the cells and it has nearly the same absorption maxima as in current assay used to measure cell proliferation. Our intention is to examine this aspect in a more controlled manner in the near future. Fig.6 shows the result of the proliferation assay carried out after 12 h, 1, 2 and 3 days on deposited films or sterile glass seeded with cells. The initial number of cells (50000) is represented by the control and is set as 100%. The plot shows that the time dependant cytotoxic effect on cells. Only after 24h the proliferation rate of cancer cells is significantly less than then controls.

In cell culture proliferating cells needs cell to cell contacts. Usually when cells are directly treated with plasma, the growing cells will detach from each other [20, 33 and 34]. Further the detached cells will enter into cell death [20, 33-35]. This disruption of cell to cell contacts is dependant on the cultivation conditions. Therefore we have checked for time dependant effect of plasma deposited films on cell proliferation. In order to investigate the cytotoxicity of the aH-CNx films we studied the changes in the rate of proliferation of prostate, pancreatic and breast cancer cells seeded on to the plasma coated glass slides by MTS assay. The assay measures dehydrogenase enzyme activity found in metabolically active cells. This method gave us direct information about the cytotoxicity of the deposited material. Results also indicate that the cytotoxic effect of the films to pancreatic cancer cells (figure 6 (c)) is minimal compared to prostate (a) and breast (b) cancer cells. However, values are significant when compared with Student’s t-test at p<0.05. Both the spectroscopic (XPS and FTIR) results exhibit the common feature to the presence of nitrile (-C≡N) and NH group in the deposited aH-CNx film. The presence of nitrile and NH group could exhibit the toxicity towards cell lines. The deposited aH-CNx film is adhesive and not soluble in water and has slightly acidic pH observed over pH indicator strips. In the contact angle measurement (which is not discussed here) we observed that the adhesive property is changing with time (~ after 15 minutes). And also the interesting thing is that the film is not soluble in those precursors. Still there are some arguments regarding this behavior. aH-CNx films seem to have a tendency to impair the protein-based cell adhesion mechanism and to stop the cell growth process. Strong homo- and hetero-nuclear dipolar coupling properties of aH-CNx film could may break the peptide configuration and induce apoptotic signals [20]. At present stage, it is difficult to explain clearly from the biological, physical, and chemical point of view about the actual mechanism of the cells death and growth on such an amorphous-H-CNx coated substrate. Such surfaces could be very interesting for the suppression of unwanted cell growth with help of biological implants on biomedical devices inside the human body. To support this assumption, a further study of those films in cell culture is necessary.

IV. Conclusion

Cytotoxic effect of the amorphous hydrogenated carbon nitride (aH-CNx) film was studied in-vitro with three different adherent cancer cell lines (MCF-7, Colo-357 and LNCaP). Cytotoxic effect was deduced from the fact that the treated cells do not initially die but stop growth and die en masse after 24 h by the following treatment, where the untreated cells continue to grow and proliferate. Chemical properties of the films confirmed the presence of C-N, C≡N, C-Hx, C-O, N-O, overlapping NH and OH bands in the film. Deeper understanding of films-cell interaction and of the specific biochemistry is needed to answer how and why the plasma assisted deposited films promotes cytotoxic behavior towards mammalian cells. Further studies on these films (biological applications) might be worth pursuing for the development of novel biomedical implantation materials to control cellular processes.
Acknowledgement
Part of this work was supported by the Deutsche Forschungemeinschaft (DFG) through Sonderforschungsbereich SFB/TR24 Fundamental of Complex Plasmas and by the Federal Ministry of Education and Research (BMBF) through Verbundprojekt “Campus PlasmaMed”. Authors are also thankful to Prof. Winfried Hinrichs, Dr. Rajesh Kumar Singh and Dr. Gottfried J. Palm, Institute for Biochemistry, Greifswald, Germany, for helpful discussions.

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Figure legends

Figure 1. Full scale XPS spectrum of the deposited aH-CNx film by CH4/N2 = 1:1 DBD plasma (f=5.5 kHz and P = 400 mbar).
Figure 2. Typical XPS spectrum of C 1s and N 1s of aH-CNx film deposited by CH4/N2=1:1 gas mixture DBD plasma.
Figure 3. FTIR absorption spectra of aH-CNx film deposited by CH4/N2 = 1:1 mixture DBD plasma.

Figure 4. Rate of proliferation of cancer cells after an incubation period of 24h on the aH-CNx (CH4/N2 = 1:1) gray filled bars represents cell lines where as white bars represents their respective controls .

Figure 5. The cell proliferation rate of cancer cells grown on aH-CNx films deposited with different CH4/N2 ratios (CH4/N2 = 3:1, 1:3 and 1:1).
Figure 6. The result of the proliferation assay carried out 12, 24, 36 and 48h after seeding the cells on deposited aH-CNx film (selected film with ratio, CH4/N2 = 1:1) or sterile glass slide as control.

Figure





Figure 4








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