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Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma
Materials Today: Proceedings xxx (xxxx) xxx
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Materials Today: Proceedings journal homepage: www.elsevier.com/locate/matpr
Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma P. Sri Devi a,b,⇑, K.A. Vijayalakshmi c a
Research Scholar, Research and Development Centre, Bharathiar University, Coimbatore, Tamilnadu 641046, India PG Department of Physics, Vellalar College for Women, Erode, Tamilnadu 638012, India c Sri Vasavi College, Erode, Tamilnadu 638316, India b
a r t i c l e
i n f o
Article history: Received 24 April 2019 Received in revised form 21 June 2019 Accepted 30 September 2019 Available online xxxx Keywords: XRD SEM MTT assay FTIR Biocompatibility
a b s t r a c t The present work investigated the possibility of enhancing hydroxyapatite (HAp) bioactivity by substituting silver oxide. Ag2O-HAp nanoparticles were synthesized by using the wet precipitation process with exposure to DC glow discharge non thermal plasma. The phase purity, elemental composition and morphology were analyzed by XRD, FTIR, SEM and EDX. MTT assay for Silver oxide doped hydroxyapatite (Ag2O HAP) nanopowders (NPs) have been carried out for MCF-7 breast cancer cell line and the cyototoxicity effect were analysed. The biocompatibility of the synthesized silver oxide doped HAp proves it as promising candidate for upcoming biomedical applications. Ó 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Emerging Materials and Modeling.
1. Introduction Hydroxyapatite (HAp) Ca10PO4)6 (OH)2 is a calcium phosphate similar to the human hard tissues in morphology and composition with stoichiometric Ca/P ratio of 1.67, which is identical to bone apatite. HAp has challengeable features like biocompatibility, bioactivity, biodegradability and corrosion-resistance. Many metals are widely used to increase the bioactivity of hydroxyapatite (HAp) in orthopedics and dental implants. Silver Oxide(Ag2O) is widely used in various fields, especially in the biomedical field because of its excellent mechanical properties and biocompatibility [1–4]. The synthetic biomaterials such as Silver Oxide doped nano – hydroxyapatite (Ag2O – HAp) gained considerable attractiveness due to excellent biocompatibility. In recent years plasma technology acts as a novel technique for the manufacture of better materials by surface modification by plasma. It is the economical and essential surface modification of this biomaterial with growing interest in the field of biomedical engineering [5,6]. The action of the plasma promotes the formation of free radicals that can act as interlock points for polar groups. Also, depending on the type of plasma forming gas and general
⇑ Corresponding author at: Research Scholar, Research and Development Centre, Bharathiar University, Coimbatore, Tamilnadu 641046, India. E-mail address: [email protected] (P. Sri Devi).
conditions of the plasma treatment, it is possible to promote some surface etching which can induce changes in surface topography, thus having a positive effect on the enrichment of surface properties of the nanomaterials.
2. Experimental details 2.1. Materials and methods Ag2O doped HAP was synthesized by using 0.9 M of calcium nitrate tetra hydrate Ca(NO3)2.4 H2O (95% EMPLURA) and 0.6 M of di ammonium hydrogen phosphate (NH4)2 HPO4 (99% SIGMA ALDRICH) respectively, used as sources for calcium and phosphorous. Ca(NO3)2.4 H2O and (NH4)2HPO4 were dissolved in 500 ml of de-ionized water separately. 0.1 M of Silver Oxide solution was added to the phosphorous solution. The pH of each aqueous solution was maintained at 10 by the addition of ammonium hydroxide solution NH4OH (99%, SIGMA ALDRICH). A gelatinous white precipitate was produced by the drop wise addition of (NH4)2HPO4 + Ag2O solution to the vigorously stirring Ca(NO3)2.4 H2O solution for an hour. Then, the precipitate was aged for 24 h at room temperature followed by washing four times with deionized water and dried in hot air oven at 373 K for 10 h. The dried powder was then milled using a mortar and pestle and finally calcined in a silica crucible using a muffle furnace at 523 K for 2 h. The
https://doi.org/10.1016/j.matpr.2019.09.204 2214-7853/Ó 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Emerging Materials and Modeling.
Please cite this article as: P. Sri Devi and K. A. Vijayalakshmi, Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.09.204
2
P. Sri Devi, K.A. Vijayalakshmi / Materials Today: Proceedings xxx (xxxx) xxx
synthesized samples are treated in DC glow discharge in air plasma atmosphere for three different exposure times 5 min, 10 min and 15 min [7–10]. 3. Results and discussion 3.1. X-ray diffraction analysis (XRD) The XRD analysis of Ag2O doped HAp has been carried out for untreated and for 5 min, 10 min, 15 min plasma treated at 400 V DC voltage. The X – ray patterns of untreated and plasma treated Ag2O doped HAp for three different times was illustrated in Fig. 1 (a–d). The diffraction peaks of all the samples shows that the reflections with high intensity peaks at (2 1 1), (3 0 0), (0 0 2) and (0 0 4) miller planes which reveals the hexagonal structure with
reference to JCPDS card no. #09-0432 for hydroxyapatite. Nearly identical peaks were observed without any apparent phase change in the XRD pattern of Ag2O doped HAp, before and after plasma treatment [11,12]. There is no severe fluctuation due to variation of treatment time of air plasma. The average crystallite size were calculated using the Debye Scherrer’s formula is 26.55 nm, 16.43 nm, 8.15 nm and 11.91 nm for untreated and 5 min,10 min , 15 min plasma treated Ag2O doped HAp nano powders. 3.2. Scanning electron microscope (SEM) analysis The morphologies of the untreated and plasma treated Ag2O doped HAp for three different times was illustrated in Fig. 2. The surface morphology of Ag2O doped HAp was examined by using scanning electron microscope. It was observed that the particles exhibit nearly platelet shape and the particles agglomerated due to vanderwaals force of attraction [13–15]. There is an improvement in the morphology due to surface etching of air plasma atmosphere. 3.3. Energy dispersive X-ray (EDAX) analysis EDAX pattern of the untreated and plasma treated Ag2O doped HAp are illustrated in Fig. 3. In the elemental composition, the contents Ag, O, P and Ca presence confirms the elements present in Ag2O doped HAp [16–18]. No other impurity peaks and elements are present except a small variation in oxygen content. 3.4. Fourier transform infrared (FTIR) spectral analysis
Fig. 1. X-ray diffraction patterns of untreated and plasma treated Ag2O doped HAp.
The FTIR spectra in the functional regions (2000–400 cm 1) of untreated, plasma treated Ag2O doped HAp nanopowders are illustrated in Fig. 4(a) untreated and (b–d) 5 min, 10 min, 15 min plasma treated TiO2 doped HAp the FTIR band observed at 3448.27 cm 1 was assigned to the presence of hydroxyl group. The peak observed at 566.59 cm 1 indicates the presence of PO34 (t4) [19–21]. The band observed at 1072.91 cm 1 confirmed to
Fig. 2. SEM images of untreated (a) and plasma treated (b - d) Ag2O doped HAp.
Please cite this article as: P. Sri Devi and K. A. Vijayalakshmi, Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.09.204
P. Sri Devi, K.A. Vijayalakshmi / Materials Today: Proceedings xxx (xxxx) xxx
3
Fig. 3. EDAX spectrum of untreated (a) and plasma treated (b - d) Ag2O doped HAp.
phosphate stretching vibration. For peak observed at 1096 cm 1 indicates the presence of PO3(t3). The band observed at 4 3432.84 cm 1 examined the presence of hydroxyl group. The bending mode of FTIR spectra show bands of phosphate groups by stretching vibration at about 1034.82 cm 1 and 1039.64 cm 1. Broad band about 3400–3600 cm 1 can be related to water or hydrated layer along with HAp [22,23]. The presence of (CO2– 3 ) groups is likely due to sample preparation and in the air plasma 3atmosphere and incorporation of formed (CO2– 3 ) into PO4 sites. The variation of plasma exposure time led to gradual increase of
Fig. 4. FTIR spectrum of untreated (a) and plasma treated (b - d) Ag2O doped HAp.
CO-2 3 vibration intensity. The OH vibrations are well-defined at 3569.79 cm 1 (stretching) and 632.17 cm 1 (bending). 3.5. In vitro cytotoxic analysis MTT Cell Proliferation and Viability Assay is a safe, sensitive, in vitro assay for the measurement of cell proliferation. Cells are cultured in flat-bottomed, 96-well tissue culture plates and the suspension of the samples. The cells are treated as per experimental design and incubation times are optimized for each cell type and system. The tetrazolium compound MTT (3-[4,5-dimethylthia zol-2-yl]-2,5-diphenyltetrazolium bromide) is added to the wells and the cells are incubated. MTT is reduced by metabolically active cells to insoluble purple formazan dye crystals. Detergent is then added to the wells, solubilizing the crystals so the absorbance can be read using a spectrophotometer. Samples are read directly in the wells. The optimal wavelength for absorbance is 570 nm, the data is analyzed by plotting concentration of extracts versus absorbance, allowing quantization of changes in cell proliferation. The rate of tetrazolium reduction is proportional to the rate of cell proliferation [24,25]. Silver Oxide doped Hydroxyapatite was screened for their cytotoxicity against human MCF-7 Breast cancer cells lines at different concentrations (6 mg/mL 85 mg/mL), to determine the mean percent (%) cell viability by MTT assay. All cytotoxic activity was assessed at 24 hrs time duration. The suspension of samples demonstrated antiproliferative activities on the growth of MCF-7 Breast cancer cell lines. Morphological alteration of (MCF-7) cell line upon exposure using Silver oxide doped Hydroxyapatite suspension was observed under phase contrast microscope. The Trypan blue exclusion method was utilized to predict percentage of cell viability on cytostatic effects. The plasma treated sample at 85 mg/mL showed better antiproliferative effect. The cells indicated the most promi-
Please cite this article as: P. Sri Devi and K. A. Vijayalakshmi, Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.09.204
4
P. Sri Devi, K.A. Vijayalakshmi / Materials Today: Proceedings xxx (xxxx) xxx
Fig. 5. Images of MTT assay for MCF-7 cancer cell line (a) control, (b–d) Untreated and (e–g) Plasma treated Ag2O doped HAp.
nent effects after exposure to before and after plasma treated suspension of the samples was shown in Fig. 5(a) control, (b–d) Untreated and (e–g) Plasma treated Ag2O doped HAp. The microscopic observations revealed the study the samples to be having outstanding effect on treated cells. The number of dead cells increased correspondingly with concentration increment of the suspension treatment. At highest concentration (85 mg/mL) the cells became rounder, shrunken and showed signs of detachment from
Fig. 6. MTT Assay for MCF-7 Cells IC50 (6 mg/mL
85 mg/mL).
the surface of the wells denoting cell death. MTT results have shown that the above stated concentrations of Ag2O doped HAp could significantly induce cytotoxicity in the MCF-7 cells in a dose-dependent manner at IC50 was shown in Fig. 6. 3.6. Antibacterial activity The Ag2O doped HAp untreated and plasma treated samples were dissolved in DMSO and tested for antimicrobial activity by well diffusion method. Liquid Mueller Hinton agar media and the Petri plates were sterilized by autoclaving at 121 °C for about 30 min at 15 lbs pressure. Under aseptic conditions in the laminar airflow chamber, about 20 ml of the agar medium was dispensed into each Petri plate to yield a uniform depth of 4 mm. After solidification of the media, 18 hrs culture of Gram positive microorganisms such as Bacillus cereus(MTCC 430), Staphylococcus aureus (MTCC 3160), Gram negative microorganisms such as E.coli (MTCC 1698) and Pseudomonas aeruginosa (MTCC424) obtained from IMTECH, Chandigarh were swabbed on the surface of the agar plates. Well was prepared by using cork borer followed with loading of 50 ml & 100 ml of each sample to the distinct well with DMSO as negative control and Vancomycin (30mcg/disc) as positive control [26]. At the lower the concentration of Ag2O doped HAp 2, 4 lg/ml shows less effect when compared with higher concentration i.e at 50 ml & 100 ml lg/ml. The sample loaded plates were then incubated at 37 °C for 24 h to observe the zone of inhibition. From the Table 1 it is observed that the due to surface modification due to plasma treatment, plasma treated Ag2O doped HAp samples shows better antibacterial activity when compared with untreated Ag2O doped HAp sample.
Please cite this article as: P. Sri Devi and K. A. Vijayalakshmi, Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.09.204
5
P. Sri Devi, K.A. Vijayalakshmi / Materials Today: Proceedings xxx (xxxx) xxx Table 1 Zone of Inhibition for Silver oxide doped HAp. Microorganisms
Bacillus cereus Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa
Zone of Inhibition of Silver oxide doped HAp in Diameter (mm) Control (100 ml)
Untreated 50 ml
Nil Nil Nil Nil
25 23 26 21
5 Min Plasma Treated
10 Min Plasma Treated
100 ml
50 ml
100 ml
50 ml
100 ml
50 ml
100 ml
26 21 30 21
26 23 28 21
28 24 30 22
28 25 28 27
28 24 31 24
28 28 29 28
29 30 32 29
4. Conclusion In this study the facile sol gel aided with DC glow discharge plasma technique HAp has been successfully prepared Ag2O-HAp nanoparticles by varying the of different plasma exposure times. The analysis of XRD, SEM, EDAX, FTIR and cytotoxicity of silver doped HAp samples are characterized by different analytical techniques for bone graft substitute. From the above investigation it is evident that the silver oxide HAp has appreciable cytotoxicity activity. This makes it a precise material for bone substitution in the biomedical field. The results shows that Siver oxide doped Hydroxyapatite was screened for their cytotoxicity against human MCF-7 Breast cancer cells lines at different concentrations. The untreated and plasma treated samples demonstrated antiproliferative activities on the growth of MCF-7 Breast cancer cell lines. References [1] M.O. Li, X.F. Xiao, R. Liu, C. Chen, L. Huang, Structural characterization of zincsubstituted hydroxyapatite prepared by hydrothermal method, J. Mater. Sci. – Mater. Med. 19 (2008) 797–803. [2] S. Kim, H.S. Ryu, H. Shin, H.S. Jung, K.S. Hong, In situ observation of hydroxyapatite nanocrystal formation from amorphous calcium phosphate in calcium-rich solutions, Mater. Chem. Phys. 91 (2005) 500–506. [3] D.S. Seo, J.K. Lee, Dissolution of human teeth-derived hydroxyapatite, Ann. Biomed. Eng. 36 (2008) 132–140. [4] K.A. Vijayalakshmi, M. Mekala, C.P. Yoganand, K. Navaneetha Pandiyaraj, Studies on modification of surface properties in polycarbonate (PC) film induced by DC glow discharge plasma, Int. J. Poly Sci. 11 (2011) 1–7. [5] E. Schepers, M. de Clercq, P. Ducheyne, R. Kempeneers, Bioactive glass particulate material as filler for bone lesions, J. Oral. Rehab. 18 (1991) 439– 452. [6] K.N. Pandiyaraj, V. Selvarajan, R.R. Deshmukh, C. Gao, Appl. Surf. Sci. 255 (7) (2009) 3965–3971. [7] S.F. Hulbert, L.L. Hench, D. Forbers, L.S. Bowman, History of bioceramics, Ceram. Int. 8 (1982) 131–140. [8] B. Gross, B. Grycz, K. Miklossy, Plasma Technology, Iliffe Books, London, 1969. [9] K.P. Sanosh, A. Min Cheolchu, Balakrishnan, T.N. Kim, Seong Jai Cho, Preparation and characterization of Nano hydroxyapatite powder using sol gel technique, Bull Mater. Sci. 32 (5) (2009) 465–470. [10] L. Mohan, D. Durgalakshmi, M. Geetha, T.S.N. Sankara Narayanan, R. Asokamani, Electrophoretic deposition of nanocomposite (Hap + TiO2) on titanium alloy for biomedical applications, Ceram. Int. 38 (2012) 3435–3443.
15 Min Plasma Treated
Std. Antibiotic (Vancomycin) 30mcg/disc
27 32 28 18
[11] I.S. Kim, P.N. Kumta, Sol-gel synthesis and characterization of nanostructured hydroxyapatite powder, Mater. Sci. Eng. B 111 (2004) 232–236. [12] L.Y. Huang, K.W. Xu, J. Lu, A study of the process and kinetics of electrochemical deposition and the hydrothermal synthesis of hydroxyapatite coatings, J. Mater. Sci.-Mater. Med. 11 (2000) 667–673. [13] F.Z. Mezahi, H. Oudadesse, A. Harabi, Effect of ZrO2, TiO2, and Al2O3 additions on process and kinetics of bonelike apatite formation on sintered natural hydroxyapatite surfaces, Int. J. Appl. Ceram. Technol. 9 (3) (2012) 529–540. [14] D.M. Liu, T. Troczynski, W.J. Tseng, Water-based sol gel synthesis of hydroxyapatite: process development, Biomaterials 22 (2001) 1721–1730. [15] Salima Ziani, Samira Meski, Hafit Khireddine, Characterization of magnesiumdoped hydroxyapatite prepared by sol-gel process, Int. J. Appl. Ceram. Technol. 11 (2013) 1–9. [16] C. Deepa, A. Nishara Begam, S. Aravindan, Preparation and antimicrobial observations of Zinc doped nanohydroxyapatite, Nanosystems: Phys. Chem. Math. 4 (3) (2013) 370–377. [17] F. Miyaji, Y. Kono, Y. Suyama, Formation and structure of zinc-substituted calcium hydroxyapatite, Mater. Res. Bull. 40 (2005) 209–220. [18] A. Ito, M. Otsuka, H. Kawamura, Masako Ikeuchi, Hajime Ohgushi, Yu Sogo, Noboru Ichinose, Zinc-containing tricalcium phosphate and related materials for promoting bone formation, Curr. Appl. Phys. 5 (2005) 402–406. [19] L. Li, H. Pan, J. Tao, X. Xu, C. Mao, X. Gu, et al., Repair of enamel by using hydroxyapatite nanoparticles as the building blocks, J. Mater. Chem. 18 (34) (2008) 4079–4084. [20] K. Niespodziana, K. Jurczyk, J. Jakubowicz, M. Jurczyk, Fabrication and properties of titanium-hydroxyapatite nanocomposites, Mater. Chem. Phys. 123 (2010) 160–165. [21] P. Anitha, Haresh M. Pandya, Comprehensive review of preparation methodologies of nanohydroxyapatite, J. Environ. Nanotechnol. 3 (1) (2014) 101–121. [22] Zhang Li, Li Yubae, Yang Aiping, Preparation and in vitro investigation of chitosoan/nano hdroxyapatite composite used as bone substituted materials, J. Mater. Sci: Mat.Med. 16 (3) (2005) 213–219. [23] Kateryna Bazaka, Mohan V. Jacob, Russell J. Crawford, Elena P. Ivanova, Plasma-assisted surface modification of organic biopolymers to prevent bacterial attachment, Act. Biomat. 7 (2011) 2015–2028. [24] C.M. Mardziah, I. Sopyan, S. Ramesh, Strontium-doped hydroxyapatite nanopowder via sol-gel method: effect of strontium concentration and calcination temperature on phase behavior, Trends Biomater. Artif. Organs 23 (2009) 105–113. [25] Y. Huang, M. Hao, X. Nian, H. Qiao, X. Zhang, X. Zhang, G. Song, J. Guo, X. Pang, H. Zhang, Strontium and copper co-substituted hydroxyapatite-based coatings with improved antibacterial activity and cytocompatibility fabricated by electrodeposition, Ceram. Int. 42 (2016) 11876–11888. [26] A. Akhavan, N. Sheikh, F. Khoylou, F. Naimian, E. Ataeivarjovi, Synthesis of antimicrobial silver-hydroxyapatite nanocomposite by gamma irradiation, Rad. Physics and Chemistry 98 (2014) 46–50.
Please cite this article as: P. Sri Devi and K. A. Vijayalakshmi, Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.09.204
Contents lists available at ScienceDirect
Materials Today: Proceedings journal homepage: www.elsevier.com/locate/matpr
Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma P. Sri Devi a,b,⇑, K.A. Vijayalakshmi c a
Research Scholar, Research and Development Centre, Bharathiar University, Coimbatore, Tamilnadu 641046, India PG Department of Physics, Vellalar College for Women, Erode, Tamilnadu 638012, India c Sri Vasavi College, Erode, Tamilnadu 638316, India b
a r t i c l e
i n f o
Article history: Received 24 April 2019 Received in revised form 21 June 2019 Accepted 30 September 2019 Available online xxxx Keywords: XRD SEM MTT assay FTIR Biocompatibility
a b s t r a c t The present work investigated the possibility of enhancing hydroxyapatite (HAp) bioactivity by substituting silver oxide. Ag2O-HAp nanoparticles were synthesized by using the wet precipitation process with exposure to DC glow discharge non thermal plasma. The phase purity, elemental composition and morphology were analyzed by XRD, FTIR, SEM and EDX. MTT assay for Silver oxide doped hydroxyapatite (Ag2O HAP) nanopowders (NPs) have been carried out for MCF-7 breast cancer cell line and the cyototoxicity effect were analysed. The biocompatibility of the synthesized silver oxide doped HAp proves it as promising candidate for upcoming biomedical applications. Ó 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Emerging Materials and Modeling.
1. Introduction Hydroxyapatite (HAp) Ca10PO4)6 (OH)2 is a calcium phosphate similar to the human hard tissues in morphology and composition with stoichiometric Ca/P ratio of 1.67, which is identical to bone apatite. HAp has challengeable features like biocompatibility, bioactivity, biodegradability and corrosion-resistance. Many metals are widely used to increase the bioactivity of hydroxyapatite (HAp) in orthopedics and dental implants. Silver Oxide(Ag2O) is widely used in various fields, especially in the biomedical field because of its excellent mechanical properties and biocompatibility [1–4]. The synthetic biomaterials such as Silver Oxide doped nano – hydroxyapatite (Ag2O – HAp) gained considerable attractiveness due to excellent biocompatibility. In recent years plasma technology acts as a novel technique for the manufacture of better materials by surface modification by plasma. It is the economical and essential surface modification of this biomaterial with growing interest in the field of biomedical engineering [5,6]. The action of the plasma promotes the formation of free radicals that can act as interlock points for polar groups. Also, depending on the type of plasma forming gas and general
⇑ Corresponding author at: Research Scholar, Research and Development Centre, Bharathiar University, Coimbatore, Tamilnadu 641046, India. E-mail address: [email protected] (P. Sri Devi).
conditions of the plasma treatment, it is possible to promote some surface etching which can induce changes in surface topography, thus having a positive effect on the enrichment of surface properties of the nanomaterials.
2. Experimental details 2.1. Materials and methods Ag2O doped HAP was synthesized by using 0.9 M of calcium nitrate tetra hydrate Ca(NO3)2.4 H2O (95% EMPLURA) and 0.6 M of di ammonium hydrogen phosphate (NH4)2 HPO4 (99% SIGMA ALDRICH) respectively, used as sources for calcium and phosphorous. Ca(NO3)2.4 H2O and (NH4)2HPO4 were dissolved in 500 ml of de-ionized water separately. 0.1 M of Silver Oxide solution was added to the phosphorous solution. The pH of each aqueous solution was maintained at 10 by the addition of ammonium hydroxide solution NH4OH (99%, SIGMA ALDRICH). A gelatinous white precipitate was produced by the drop wise addition of (NH4)2HPO4 + Ag2O solution to the vigorously stirring Ca(NO3)2.4 H2O solution for an hour. Then, the precipitate was aged for 24 h at room temperature followed by washing four times with deionized water and dried in hot air oven at 373 K for 10 h. The dried powder was then milled using a mortar and pestle and finally calcined in a silica crucible using a muffle furnace at 523 K for 2 h. The
https://doi.org/10.1016/j.matpr.2019.09.204 2214-7853/Ó 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Emerging Materials and Modeling.
Please cite this article as: P. Sri Devi and K. A. Vijayalakshmi, Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.09.204
2
P. Sri Devi, K.A. Vijayalakshmi / Materials Today: Proceedings xxx (xxxx) xxx
synthesized samples are treated in DC glow discharge in air plasma atmosphere for three different exposure times 5 min, 10 min and 15 min [7–10]. 3. Results and discussion 3.1. X-ray diffraction analysis (XRD) The XRD analysis of Ag2O doped HAp has been carried out for untreated and for 5 min, 10 min, 15 min plasma treated at 400 V DC voltage. The X – ray patterns of untreated and plasma treated Ag2O doped HAp for three different times was illustrated in Fig. 1 (a–d). The diffraction peaks of all the samples shows that the reflections with high intensity peaks at (2 1 1), (3 0 0), (0 0 2) and (0 0 4) miller planes which reveals the hexagonal structure with
reference to JCPDS card no. #09-0432 for hydroxyapatite. Nearly identical peaks were observed without any apparent phase change in the XRD pattern of Ag2O doped HAp, before and after plasma treatment [11,12]. There is no severe fluctuation due to variation of treatment time of air plasma. The average crystallite size were calculated using the Debye Scherrer’s formula is 26.55 nm, 16.43 nm, 8.15 nm and 11.91 nm for untreated and 5 min,10 min , 15 min plasma treated Ag2O doped HAp nano powders. 3.2. Scanning electron microscope (SEM) analysis The morphologies of the untreated and plasma treated Ag2O doped HAp for three different times was illustrated in Fig. 2. The surface morphology of Ag2O doped HAp was examined by using scanning electron microscope. It was observed that the particles exhibit nearly platelet shape and the particles agglomerated due to vanderwaals force of attraction [13–15]. There is an improvement in the morphology due to surface etching of air plasma atmosphere. 3.3. Energy dispersive X-ray (EDAX) analysis EDAX pattern of the untreated and plasma treated Ag2O doped HAp are illustrated in Fig. 3. In the elemental composition, the contents Ag, O, P and Ca presence confirms the elements present in Ag2O doped HAp [16–18]. No other impurity peaks and elements are present except a small variation in oxygen content. 3.4. Fourier transform infrared (FTIR) spectral analysis
Fig. 1. X-ray diffraction patterns of untreated and plasma treated Ag2O doped HAp.
The FTIR spectra in the functional regions (2000–400 cm 1) of untreated, plasma treated Ag2O doped HAp nanopowders are illustrated in Fig. 4(a) untreated and (b–d) 5 min, 10 min, 15 min plasma treated TiO2 doped HAp the FTIR band observed at 3448.27 cm 1 was assigned to the presence of hydroxyl group. The peak observed at 566.59 cm 1 indicates the presence of PO34 (t4) [19–21]. The band observed at 1072.91 cm 1 confirmed to
Fig. 2. SEM images of untreated (a) and plasma treated (b - d) Ag2O doped HAp.
Please cite this article as: P. Sri Devi and K. A. Vijayalakshmi, Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.09.204
P. Sri Devi, K.A. Vijayalakshmi / Materials Today: Proceedings xxx (xxxx) xxx
3
Fig. 3. EDAX spectrum of untreated (a) and plasma treated (b - d) Ag2O doped HAp.
phosphate stretching vibration. For peak observed at 1096 cm 1 indicates the presence of PO3(t3). The band observed at 4 3432.84 cm 1 examined the presence of hydroxyl group. The bending mode of FTIR spectra show bands of phosphate groups by stretching vibration at about 1034.82 cm 1 and 1039.64 cm 1. Broad band about 3400–3600 cm 1 can be related to water or hydrated layer along with HAp [22,23]. The presence of (CO2– 3 ) groups is likely due to sample preparation and in the air plasma 3atmosphere and incorporation of formed (CO2– 3 ) into PO4 sites. The variation of plasma exposure time led to gradual increase of
Fig. 4. FTIR spectrum of untreated (a) and plasma treated (b - d) Ag2O doped HAp.
CO-2 3 vibration intensity. The OH vibrations are well-defined at 3569.79 cm 1 (stretching) and 632.17 cm 1 (bending). 3.5. In vitro cytotoxic analysis MTT Cell Proliferation and Viability Assay is a safe, sensitive, in vitro assay for the measurement of cell proliferation. Cells are cultured in flat-bottomed, 96-well tissue culture plates and the suspension of the samples. The cells are treated as per experimental design and incubation times are optimized for each cell type and system. The tetrazolium compound MTT (3-[4,5-dimethylthia zol-2-yl]-2,5-diphenyltetrazolium bromide) is added to the wells and the cells are incubated. MTT is reduced by metabolically active cells to insoluble purple formazan dye crystals. Detergent is then added to the wells, solubilizing the crystals so the absorbance can be read using a spectrophotometer. Samples are read directly in the wells. The optimal wavelength for absorbance is 570 nm, the data is analyzed by plotting concentration of extracts versus absorbance, allowing quantization of changes in cell proliferation. The rate of tetrazolium reduction is proportional to the rate of cell proliferation [24,25]. Silver Oxide doped Hydroxyapatite was screened for their cytotoxicity against human MCF-7 Breast cancer cells lines at different concentrations (6 mg/mL 85 mg/mL), to determine the mean percent (%) cell viability by MTT assay. All cytotoxic activity was assessed at 24 hrs time duration. The suspension of samples demonstrated antiproliferative activities on the growth of MCF-7 Breast cancer cell lines. Morphological alteration of (MCF-7) cell line upon exposure using Silver oxide doped Hydroxyapatite suspension was observed under phase contrast microscope. The Trypan blue exclusion method was utilized to predict percentage of cell viability on cytostatic effects. The plasma treated sample at 85 mg/mL showed better antiproliferative effect. The cells indicated the most promi-
Please cite this article as: P. Sri Devi and K. A. Vijayalakshmi, Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.09.204
4
P. Sri Devi, K.A. Vijayalakshmi / Materials Today: Proceedings xxx (xxxx) xxx
Fig. 5. Images of MTT assay for MCF-7 cancer cell line (a) control, (b–d) Untreated and (e–g) Plasma treated Ag2O doped HAp.
nent effects after exposure to before and after plasma treated suspension of the samples was shown in Fig. 5(a) control, (b–d) Untreated and (e–g) Plasma treated Ag2O doped HAp. The microscopic observations revealed the study the samples to be having outstanding effect on treated cells. The number of dead cells increased correspondingly with concentration increment of the suspension treatment. At highest concentration (85 mg/mL) the cells became rounder, shrunken and showed signs of detachment from
Fig. 6. MTT Assay for MCF-7 Cells IC50 (6 mg/mL
85 mg/mL).
the surface of the wells denoting cell death. MTT results have shown that the above stated concentrations of Ag2O doped HAp could significantly induce cytotoxicity in the MCF-7 cells in a dose-dependent manner at IC50 was shown in Fig. 6. 3.6. Antibacterial activity The Ag2O doped HAp untreated and plasma treated samples were dissolved in DMSO and tested for antimicrobial activity by well diffusion method. Liquid Mueller Hinton agar media and the Petri plates were sterilized by autoclaving at 121 °C for about 30 min at 15 lbs pressure. Under aseptic conditions in the laminar airflow chamber, about 20 ml of the agar medium was dispensed into each Petri plate to yield a uniform depth of 4 mm. After solidification of the media, 18 hrs culture of Gram positive microorganisms such as Bacillus cereus(MTCC 430), Staphylococcus aureus (MTCC 3160), Gram negative microorganisms such as E.coli (MTCC 1698) and Pseudomonas aeruginosa (MTCC424) obtained from IMTECH, Chandigarh were swabbed on the surface of the agar plates. Well was prepared by using cork borer followed with loading of 50 ml & 100 ml of each sample to the distinct well with DMSO as negative control and Vancomycin (30mcg/disc) as positive control [26]. At the lower the concentration of Ag2O doped HAp 2, 4 lg/ml shows less effect when compared with higher concentration i.e at 50 ml & 100 ml lg/ml. The sample loaded plates were then incubated at 37 °C for 24 h to observe the zone of inhibition. From the Table 1 it is observed that the due to surface modification due to plasma treatment, plasma treated Ag2O doped HAp samples shows better antibacterial activity when compared with untreated Ag2O doped HAp sample.
Please cite this article as: P. Sri Devi and K. A. Vijayalakshmi, Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.09.204
5
P. Sri Devi, K.A. Vijayalakshmi / Materials Today: Proceedings xxx (xxxx) xxx Table 1 Zone of Inhibition for Silver oxide doped HAp. Microorganisms
Bacillus cereus Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa
Zone of Inhibition of Silver oxide doped HAp in Diameter (mm) Control (100 ml)
Untreated 50 ml
Nil Nil Nil Nil
25 23 26 21
5 Min Plasma Treated
10 Min Plasma Treated
100 ml
50 ml
100 ml
50 ml
100 ml
50 ml
100 ml
26 21 30 21
26 23 28 21
28 24 30 22
28 25 28 27
28 24 31 24
28 28 29 28
29 30 32 29
4. Conclusion In this study the facile sol gel aided with DC glow discharge plasma technique HAp has been successfully prepared Ag2O-HAp nanoparticles by varying the of different plasma exposure times. The analysis of XRD, SEM, EDAX, FTIR and cytotoxicity of silver doped HAp samples are characterized by different analytical techniques for bone graft substitute. From the above investigation it is evident that the silver oxide HAp has appreciable cytotoxicity activity. This makes it a precise material for bone substitution in the biomedical field. The results shows that Siver oxide doped Hydroxyapatite was screened for their cytotoxicity against human MCF-7 Breast cancer cells lines at different concentrations. The untreated and plasma treated samples demonstrated antiproliferative activities on the growth of MCF-7 Breast cancer cell lines. References [1] M.O. Li, X.F. Xiao, R. Liu, C. Chen, L. Huang, Structural characterization of zincsubstituted hydroxyapatite prepared by hydrothermal method, J. Mater. Sci. – Mater. Med. 19 (2008) 797–803. [2] S. Kim, H.S. Ryu, H. Shin, H.S. Jung, K.S. Hong, In situ observation of hydroxyapatite nanocrystal formation from amorphous calcium phosphate in calcium-rich solutions, Mater. Chem. Phys. 91 (2005) 500–506. [3] D.S. Seo, J.K. Lee, Dissolution of human teeth-derived hydroxyapatite, Ann. Biomed. Eng. 36 (2008) 132–140. [4] K.A. Vijayalakshmi, M. Mekala, C.P. Yoganand, K. Navaneetha Pandiyaraj, Studies on modification of surface properties in polycarbonate (PC) film induced by DC glow discharge plasma, Int. J. Poly Sci. 11 (2011) 1–7. [5] E. Schepers, M. de Clercq, P. Ducheyne, R. Kempeneers, Bioactive glass particulate material as filler for bone lesions, J. Oral. Rehab. 18 (1991) 439– 452. [6] K.N. Pandiyaraj, V. Selvarajan, R.R. Deshmukh, C. Gao, Appl. Surf. Sci. 255 (7) (2009) 3965–3971. [7] S.F. Hulbert, L.L. Hench, D. Forbers, L.S. Bowman, History of bioceramics, Ceram. Int. 8 (1982) 131–140. [8] B. Gross, B. Grycz, K. Miklossy, Plasma Technology, Iliffe Books, London, 1969. [9] K.P. Sanosh, A. Min Cheolchu, Balakrishnan, T.N. Kim, Seong Jai Cho, Preparation and characterization of Nano hydroxyapatite powder using sol gel technique, Bull Mater. Sci. 32 (5) (2009) 465–470. [10] L. Mohan, D. Durgalakshmi, M. Geetha, T.S.N. Sankara Narayanan, R. Asokamani, Electrophoretic deposition of nanocomposite (Hap + TiO2) on titanium alloy for biomedical applications, Ceram. Int. 38 (2012) 3435–3443.
15 Min Plasma Treated
Std. Antibiotic (Vancomycin) 30mcg/disc
27 32 28 18
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Please cite this article as: P. Sri Devi and K. A. Vijayalakshmi, Analysis of antibacterial activity and cytotoxicity of silver oxide doped hydroxyapatite exposed to DC glow discharge plasma, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2019.09.204