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Biosynthesis of titanium dioxide nanoparticles using a probiotic from coal fly ash effluent
Materials Research Bulletin 48 (2013) 4738–4742
Contents lists available at ScienceDirect
Materials Research Bulletin journal homepage: www.elsevier.com/locate/matresbu
Biosynthesis of titanium dioxide nanoparticles using a probiotic from coal fly ash effluent S Babitha, Purna Sai Korrapati * Biomaterials Department, CSIR – Central Leather Research Institute, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 April 2013 Received in revised form 2 July 2013 Accepted 15 August 2013 Available online 24 August 2013
The synthesis of titanium dioxide nanoparticle (TiO2 NP) has gained importance in the recent years owing to its wide range of potential biological applications. The present study demonstrates the synthesis of TiO2 NPs by a metal resistant bacterium isolated from the coal fly ash effluent. This bacterial strain was identified on the basis of morphology and 16s rDNA gene sequence [KC545833]. The physicochemical characterization of the synthesized nanoparticles is completely elucidated by energy dispersive X-ray analysis (EDAX), Fourier transform infrared spectroscopy (FTIR) and transmission and scanning electron microscopy (TEM, SEM). The crystalline nature of the nanoparticles was confirmed by X-RD pattern. Further, cell viability and haemolytic assays confirmed the biocompatible and non toxic nature of the NPs. The TiO2 NPs was found to enhance the collagen stabilization and thereby enabling the preparation of collagen based biological wound dressing. The paper essentially provides scope for an easy bioprocess for the synthesis of TiO2 NPs from the metal oxide enriched effluent sample for future biological applications. ß 2013 Elsevier Ltd. All rights reserved.
Keywords: A. Inorganic compounds A. Oxides C. Electron microscopy C. Infrared spectroscopy C. X-ray diffraction
1. Introduction Synthesis of nanoparticles and their application in various biomedical fields such as drug delivery, gene transfer, wound dressings, etc., is gaining enormous significance in the current era. Although there are conventional processes viz. Physical and Chemical methods for the synthesis of nanoparticles, these technologies have certain limitations in aspects of cost, security and time. Further, there is a growing need to develop environmentally safe nanoparticle synthesis with minimum usage of toxic chemicals. Therefore biological approaches are preferred owing to their cost effective and energy saving benefits. Microbial method is one such efficient cost effective alternative because of its low demand for energy, material and less generation of waste by product. Various micro organisms such as bacteria [1], fungi [2,3], yeast [4], etc., are known to play a crucial role in toxic remediation through reduction of metal ions. Due to the extreme environmental conditions, micro organisms adapt to a specific defense mechanism which could attribute to change the redox state of
* Corresponding author at: Biomaterials Division, CSIR – Central Leather Research Institute, TICEL Biopark, Taramani, Chennai 600113, India. Tel.: +91 44 22542490; fax: +91 44 24912150; mobile: +91 09444502584. E-mail address: [email protected] (P.S. Korrapati). 0025-5408/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.materresbull.2013.08.016
the metal ions, thus, forming the basis for nanoparticle synthesis [5]. The rich elemental composition of the fly ash effluent [6,7] paved the way for screening metal resistant bacteria which could further be used for nanoparticle synthesis. TiO2 NPs has been explored in various biomedical applications such as wound dressing [8], biosensing [9], contrast agents [10], targeted drug delivery agents [11], antiwrinkle [12] and antimicrobial [13] and anti parasitic agents [14] owing to their non-toxic and biocompatible properties. In light of these properties, TiO2 is potentially more attractive than other oxidative nanoparticles. Collagen is the most common extra cellular matrix protein with a wide range of applications from drug delivery, tissue engineering and scaffolds for wound dressing [15] owing to its excellent biocompatibility. However, native collagen is prone to higher biodegradation rate and mechanically less stable. Therefore, various crosslinkers both natural and synthetic are used to enhance the strength for in vivo applications [16,17]. Herein, we attempted to use TiO2 NPs in collagen film in order to enhance the strength of the film. The present study essentially deals with the identification and application of metal resistant bacteria for synthesizing nanoparticles. An attempt has also been made to explore the potential of these synthesized nanoparticles in strengthening the collagen films for further application as wound dressing in in vivo rat models.
S. Babitha, P.S. Korrapati / Materials Research Bulletin 48 (2013) 4738–4742 Table 1 Physico-chemical characteristics of effluent.
Ebg ¼
Parameters
Value
Turbidity pH Total dissolved solids Total suspended solids Conductivity Total hardness as CaCo3 Total alkalinity
650 NTU 8.38 388 mg/l 3210 mg/l 610 mV/cm 177 mg/l 247 mg/l
4739
hc
l
where h = Planck’s constant (6.626 1039), c = speed of the light (3.0 108 m/s), l = cut off wavelength (382 109 m). The nanoparticles were freeze dried, powdered and used for Xray diffraction analysis using X-ray diffractometer with CuKa radiation l = 1.5405 A˚ over a wide range of Bragg angles (108 2u 808). The scanning was done at 0.028/min with the time constant of 2 s. The size of the nanoparticles was calculated using Scherrer’s equation:
2. Materials and methods 0:9l b cosu
2.1. Selection of bacterial species
d¼
The Physico-chemical parameter of the effluent was tested (Table 1). The serial dilution technique was adopted for the isolation of bacterial strains from the coal fly ash effluent enriched sediment. Later, the heavy metal resistant bacterial strains were isolated by pour plating method using nutrient agar supplemented with various concentrations of heavy metal salts. Individual pure colonies were screened for their ability to synthesize TiO2 NPs. The organism that efficiently synthesizes TiO2 NPs was then characterized completely based on biochemical characterization according to Bergy’s manual of Bacteriology [18] which includes 16S rDNA technique and G + C analysis.
where d is the mean diameter of the nanoparticle, l is the wavelength of X-ray radiation, b is the angular full width at half – maximum of the XRD peak at the diffraction angle. The dried powder was diluted with potassium bromide in the ratio of 1:100; analyzed at a resolution of 4 cm1 over 4000 to 500 cm1 in diffuse reflectance mode and the spectrum was recorded in Fourier transform infrared (FTIR) spectrophotometer. The morphology and size distribution of the nanoparticle was observed by TEM and field emission scanning electron microscope attached with EDAX to determine the elemental characterization at a specific position. For FE-SEM, a drop of the sample was placed on a stub using adhesive carbon tape with gold sputtering and observed using FEI Quanta 200 operating at a voltage of 30 kV. Sample was air dried on a carbon coated copper grid and operated at an accelerating voltage of 300 kV for observation using TEM.
2.2. Synthesis of TiO2 nanoparticles About 25 ml of the re-suspended culture of the isolate was diluted four times by adding 75 ml of sterile distilled water containing nutrients. This diluted culture was allowed to grow for another 24 h. 20 ml of 0.025 M TiO(OH)2 solution was added to the culture solution and heated on a steam bath up to 60 8C for 10– 20 min until white deposition was observed at the bottom of the flask indicating the initiation of transformation. The culture solution was then cooled and allowed to incubate at room temperature in laboratory ambience. After 12–48 h, the culture solution was observed to have distinct white precipitate deposited at the bottom of the flask. The precipitate was then annealed at 300 8C for 1 h that transforms amorphous into anatase phase and the impurities were removed. 2.3. Characterization of nanoparticles The synthesized TiO2 nanoparticles were characterized by measuring the UV absorbance at a resolution of 1 nm and the bandgap (Ebg) was calculated using
2.4. In-vitro biocompatibility assays The haemolytic assay was done for various concentrations of TiO2 NPs as described by Sai et al. [19]. Rat erythrocytes were isolated from heparinized blood by centrifugation. The cells were washed three times with 5 mM HEPES buffer containing 150 mm sodium chloride. Aliquots of 1 ml suspension containing 107 cells in microfuge tubes were incubated with different concentrations of TiO2 NPs (5–100 mg/ml) in duplicates at 37 8C for 30 min with gentle mixing. The tubes were then centrifuged and absorbance of the supernatants was measured at 540 nm. The lysis obtained with water was considered as 100%. The cytotoxic effect of TiO2 NPs was assessed by MTT (3-[4,5dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT, Sigma, USA) assay [20]. 8 103 cells (NIH/3T3-mouse embryonic fibroblasts) were seeded onto a 24 well culture dish and allowed to adhere and stabilize overnight in a CO2 incubator at 37 8C. Cells were washed with warm PBS and fresh medium containing TiO2
Fig. 1. (a) Propionibacterium jensenii and (b) observation of pleomorphic rods by Gram staining procedure at a magnification of 100.
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Fig. 2. (a) UV–visible, (b) X-RD and (c) FTIR spectrum of TiO2 nanoparticle.
Fig. 3. (a) TEM and (b) FE-SEM image of TiO2 nanoparticle with (c) EDAX.
NPs at various concentrations (5–100 mg/ml) was added in triplicates. after 24 h incubation with TiO2 NPs at 37 8C, the cells were treated with MTT (5 mg/ml) for 3 h. The formazan complex formed was solubilized with DMSO (dimethyl sulphoxide) and the absorption was measured at 570 nm with a reference wavelength of 630 nm, in a micro plate reader (Biorad). The percentage of viability was calculated using the optical density of the control and treated cells.
photographed and the wound margins were traced to determine the area of contraction throughout the healing period. Granulation tissue was removed every third day and the dressings were replaced till the healing was complete. The experiment was carried out after obtaining approval from the institutional animal ethics committee.
2.5. In-vivo biocompatibility study
The organism on the basis of 16S rDNA technique and the biochemical parameters was identified to be Propionibacterium jensenii [KC545833], a probiotic, high G + C rich, pleomorphic rod shaped gram-positive bacteria (Fig. 1). The G + C content of the sequence were estimated to be 57.49% using Seqool software [21].
2.5.1. Preparation of collagen – TiO2 wound dressing Nanosized titanium dioxide particles possess the property of enhancing mechanical strength. Various concentrations of TiO2 NPs (5–100 mg/ml) were dispersed in the reconstituted collagen (5 mg/ml Collagen, 0.2 M phosphate buffer, 2 M NaCl, and 1.25 N NaOH) film and assessed for their mechanical strength using Instron tensile tester at an extension rate of 5 mm/min. Dumb-bell shaped pieces of 20 mm length, 0.02 mm thickness and width of 12 mm were cut from the reconstituted collagen – TiO2 films for measuring the stress strain characteristics. The ideal concentration of TiO2 dispersed collagen film was observed to be 25 mg/ml giving a good mechanical strength of 33.37 MPa. The films were used for further studies on animal experiment. 2.5.2. Open excision wound model Female wistar rats (12 Nos) weighing 120 g were selected and their dorsal surface below the cervical region was shaved off the pelage. Uniform sized open excision type wounds of 5 cm2 were created under mild anaesthesia (intraperitoneal injection of pentobarbitone sodium (50 mg/kg). The control group (6 Nos) was treated with reconstituted collagen film while the experimental group (6 Nos) was dressed with collagen film containing TiO2 NPs and were covered with sterile cotton gauze. The wounds were
3. Results and discussion
Fig. 4. Cytotoxicity of synthesized TiO2 NPs.
S. Babitha, P.S. Korrapati / Materials Research Bulletin 48 (2013) 4738–4742
4741
Fig. 5. (a) In vivo wound healing efficiency of TiO2 NPs. (b) Percentage of wound contraction rate in control and TiO2 treated rats.
The UV–visible spectrum showed a well defined surface plasmon resonance peak (Fig. 2a). The cut off wavelength of TiO2 nanoparticles was observed at 382 nm which was subsequently used for calculating the band gap (Ebg) using the formula. The band gap value of TiO2 was found to be 3.247 eV which confirms the excitation absorption peak correspond to anatase form of nano TiO2. The crystal phase of the TiO2 was confirmed using XRD (Fig. 2b). The line broadening of the diffraction peaks indicated that the synthesized materials were in nanometer range. The diffraction patterns showed that the anatase phase (a = 3.782 A˚, JCPDS no: 84-1286) was formed, and the crystallite size was calculated using Scherrer’s formula. The average particle size was found to be 65 nm. The identified 2u peaks at 25.378, 37.818, 47.988, 62.548, 74.838 matching the planes 101, 004, 200, 204 and 215 confirmed the characteristics of biologically synthesized TiO2 NPs crystallites. FTIR spectrum of the TiO2 NPs (Fig. 2c) revealed the purity owing to the observation of bands at 1235, 1643 and 1068 cm1 which were assigned to the bending vibrations of primary and secondary amines and carboxylic groups respectively. The band observed at 2924 cm1 confirms the bound carboxylic groups with reference to Ti–O–Ti in accordance to Hardy et al. [22]. The presence of this carboxylic group probably attributes to the nucleation site for nanoparticle formation owing to the ability of co-ordination with metal ions. A broad peak at 3411 cm1, the other peaks at 1549 cm1, 1408 cm1 correspond to the O–H stretching of alcoholic groups, C5 5C ring stretching and bending vibration of CH2 in the lipids and proteins respectively. The amide linkage between the bacterial proteins and the TiO2 during the reaction was confirmed by the presence of a peak at 1235 cm1. The uniform shape and size of the nanoparticle and their polydispersed nature is depicted in the (Fig. 3a) ranging between 10 and 80 nm. This data is in line with XRD studies. The difference in size observed amongst the synthesized nanoparticles could be attributed to variation in time scales during the process of bacterial synthesis. The FE-SEM studies clearly showed that the nanoparticles were smooth and spherical in shape and ranged in size from 15 to 80 nm (Fig. 3b). The EDAX spectrum recorded in the spot profile mode from one of the densely populated TiO2 nanoparticle area with the binding energy region of 0–20 keV is shown in (Fig. 3c). The peak
from the spectrum revealed the presence of Ti and O at 4.5 and 0.5 keV. The atomic percentage of Ti and O was 54.73 and 45.27 respectively. The most prominent signals were corresponding to that of titanium while the lesser owing chlorine, oxygen and ka atoms were probably due to X-ray emission from proteins and enzymes present in the cell wall of the biomass. No haemolysis or cell death with reference to NIH/3T3 cell lines was observed with TiO2 NPs even at a concentration of 100 mg/ml (Fig. 4). This clearly demonstrates the non toxic nature of the nanoparticles. Further, the in vivo wound healing experiment in wistar rat model showed that the synthesized TiO2 nanoparticles have an ability to enhance wound healing. The rats treated with collagen film containing TiO2 NPs nanoparticles (25 mg/ml) showed a complete healing in 14 days, while the control group containing only collagen film healed in 18 days (Fig. 5). The enhanced wound closure rate observed in the experimental group might be attributed to the sustained release of Ti ions from the TiO2 NPs incorporated wound dressing. 4. Conclusion Eco friendly synthesis of TiO2 NPs using a highly efficient Propionibacterium species isolated from the coal fly ash effluent is illustrated. This process of synthesis resulted in a well dispersed uniform sized anatase form of nanoparticles that are highly stable, biocompatible and cost effective thereby offering several advantages over conventional methods. The present study provides scope for the synthesis of non toxic nanoparticles with enhanced wound healing activity. Acknowledgements The authors thank the Director, CSIR – CLRI for his constant support. The first author gratefully acknowledges the DST – INSPIRE, New Delhi for the fellowship (IF110583). References [1] B. Nair, T. Pradeep, Cryst. Growth Des. 2 (2002) 293–298. [2] G. Rajakumar, A. Abdul Rahuman, S. Mohana Roopan, V. Gopiesh Khanna, G. Elango, C. Kamaraj, Spectrochim. Acta Part A 91 (2012) 23–29.
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[3] T. Ayon, R. Ramesh, W. Wei-Ning, B. Pratim, J.C. Tarafdar, Adv. Sci. – Eng. Med. 5 (2013) 943–947. [4] A.K. Jha, K. Prasad, A.R. Kulkarni, Colloids Surf. B: Biointerface 71 (2009) 226–229. [5] Y. Nangia, N. Wangoo, N. Goyal, G. Shekawat, C. Raman Suri, Micro. Cell Fact. 8 (2009) 39–46. [6] S. Dhadse, P. Kumari, L.J. Bhagia, Environmental Impact and Risk Assessment Division, National Environmental Engineering Research Institute (NEERI). 2008. [7] K.K. Shivpuri, B. Lokeshappa, D.A. Kulkarni, A.K. Dikshit, J. Environ. Eng. 1 (2011) 21–27. [8] D. Archana, Joydeep Dutta, P.K. Dutta, Int. J. Biol. Macromol. 57 (2013) 193–203. [9] A.E. Ansary, L.M. Faddah, Nano Sci. Appl. 3 (2010) 65–76. [10] R.W. Cheyne, Tim A.D. Smith, L. Trembleau, A.C. Mclaughlin, Nano Res. Lett. 6 (2011) 423–428. [11] H. Zhang, C. Wang, B. Chen, X. Wang, Int. J. Nanomed. 7 (2012) 235–242. [12] Z. Senic, S. Bauk, M.V. Todorovic, N. Pajic, A. Samolov, D. Rajic, Sci. Technol. Rev. 61 (2011) 63–72. [13] P. Dhandapani, S. Maruthamuthu, G. Rajagopal, J. Photochem. Photobiol. 110 (2012) 43–49.
[14] K. Velayutham, A. Abdul Rahuman, G. Rajakumar, T. Santhoshkumar, S. Marimuthu, C. Jayaseelan, A. Bagavan, A. Vishnu Kirthi, C. Kamaraj, A. Abduz Zahir, G. Elango, Parasitol. Res. 111 (2012) 2329–2337. [15] M.E. Nimni, D. Cheung, B. Strates, M. Kodama, K. Sheikh, J. Biomed. Mater. Res. 21 (1987) 741–771. [16] L. Castaneda, J. Valle, N. Yang, S. Pluskat, K. Slowinska, Bio. Mac. Mol. 9 (2008) 3383–3388. [17] C. Gaidu, M. Giurginca, T. Dragomir, A. Petica, W. Chen, J. Optoelectron. Adv. Mater. 12 (2010) 2157–2163. [18] D.H. Bergey, Bergey’s Manual of Systematic Bacteriology, Williams & Wilkins Company, Baltimore, MD, 1984. [19] K.P. Sai, M.V. Jagannadham, M. Vairamani, N.P. Raju, A.S. Devi, R. Nagaraj, J. Biol. Chem. 276 (2001) 2701–2707. [20] T. Mosmann, J. Immunol. Meth. 55 (1983) 55–63. [21] W.M. Seqool, A sequence analysis tool for signal search, pattern recognition and sequence statistics 2011; Version 3.0: http://biossc.de/seqool. [22] A. Hardy, J. D’Haen, M.K. Van Bael, J. Mullens, J. Sol-Gel Sci. Technol. 44 (2007) 65–74.
Contents lists available at ScienceDirect
Materials Research Bulletin journal homepage: www.elsevier.com/locate/matresbu
Biosynthesis of titanium dioxide nanoparticles using a probiotic from coal fly ash effluent S Babitha, Purna Sai Korrapati * Biomaterials Department, CSIR – Central Leather Research Institute, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 April 2013 Received in revised form 2 July 2013 Accepted 15 August 2013 Available online 24 August 2013
The synthesis of titanium dioxide nanoparticle (TiO2 NP) has gained importance in the recent years owing to its wide range of potential biological applications. The present study demonstrates the synthesis of TiO2 NPs by a metal resistant bacterium isolated from the coal fly ash effluent. This bacterial strain was identified on the basis of morphology and 16s rDNA gene sequence [KC545833]. The physicochemical characterization of the synthesized nanoparticles is completely elucidated by energy dispersive X-ray analysis (EDAX), Fourier transform infrared spectroscopy (FTIR) and transmission and scanning electron microscopy (TEM, SEM). The crystalline nature of the nanoparticles was confirmed by X-RD pattern. Further, cell viability and haemolytic assays confirmed the biocompatible and non toxic nature of the NPs. The TiO2 NPs was found to enhance the collagen stabilization and thereby enabling the preparation of collagen based biological wound dressing. The paper essentially provides scope for an easy bioprocess for the synthesis of TiO2 NPs from the metal oxide enriched effluent sample for future biological applications. ß 2013 Elsevier Ltd. All rights reserved.
Keywords: A. Inorganic compounds A. Oxides C. Electron microscopy C. Infrared spectroscopy C. X-ray diffraction
1. Introduction Synthesis of nanoparticles and their application in various biomedical fields such as drug delivery, gene transfer, wound dressings, etc., is gaining enormous significance in the current era. Although there are conventional processes viz. Physical and Chemical methods for the synthesis of nanoparticles, these technologies have certain limitations in aspects of cost, security and time. Further, there is a growing need to develop environmentally safe nanoparticle synthesis with minimum usage of toxic chemicals. Therefore biological approaches are preferred owing to their cost effective and energy saving benefits. Microbial method is one such efficient cost effective alternative because of its low demand for energy, material and less generation of waste by product. Various micro organisms such as bacteria [1], fungi [2,3], yeast [4], etc., are known to play a crucial role in toxic remediation through reduction of metal ions. Due to the extreme environmental conditions, micro organisms adapt to a specific defense mechanism which could attribute to change the redox state of
* Corresponding author at: Biomaterials Division, CSIR – Central Leather Research Institute, TICEL Biopark, Taramani, Chennai 600113, India. Tel.: +91 44 22542490; fax: +91 44 24912150; mobile: +91 09444502584. E-mail address: [email protected] (P.S. Korrapati). 0025-5408/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.materresbull.2013.08.016
the metal ions, thus, forming the basis for nanoparticle synthesis [5]. The rich elemental composition of the fly ash effluent [6,7] paved the way for screening metal resistant bacteria which could further be used for nanoparticle synthesis. TiO2 NPs has been explored in various biomedical applications such as wound dressing [8], biosensing [9], contrast agents [10], targeted drug delivery agents [11], antiwrinkle [12] and antimicrobial [13] and anti parasitic agents [14] owing to their non-toxic and biocompatible properties. In light of these properties, TiO2 is potentially more attractive than other oxidative nanoparticles. Collagen is the most common extra cellular matrix protein with a wide range of applications from drug delivery, tissue engineering and scaffolds for wound dressing [15] owing to its excellent biocompatibility. However, native collagen is prone to higher biodegradation rate and mechanically less stable. Therefore, various crosslinkers both natural and synthetic are used to enhance the strength for in vivo applications [16,17]. Herein, we attempted to use TiO2 NPs in collagen film in order to enhance the strength of the film. The present study essentially deals with the identification and application of metal resistant bacteria for synthesizing nanoparticles. An attempt has also been made to explore the potential of these synthesized nanoparticles in strengthening the collagen films for further application as wound dressing in in vivo rat models.
S. Babitha, P.S. Korrapati / Materials Research Bulletin 48 (2013) 4738–4742 Table 1 Physico-chemical characteristics of effluent.
Ebg ¼
Parameters
Value
Turbidity pH Total dissolved solids Total suspended solids Conductivity Total hardness as CaCo3 Total alkalinity
650 NTU 8.38 388 mg/l 3210 mg/l 610 mV/cm 177 mg/l 247 mg/l
4739
hc
l
where h = Planck’s constant (6.626 1039), c = speed of the light (3.0 108 m/s), l = cut off wavelength (382 109 m). The nanoparticles were freeze dried, powdered and used for Xray diffraction analysis using X-ray diffractometer with CuKa radiation l = 1.5405 A˚ over a wide range of Bragg angles (108 2u 808). The scanning was done at 0.028/min with the time constant of 2 s. The size of the nanoparticles was calculated using Scherrer’s equation:
2. Materials and methods 0:9l b cosu
2.1. Selection of bacterial species
d¼
The Physico-chemical parameter of the effluent was tested (Table 1). The serial dilution technique was adopted for the isolation of bacterial strains from the coal fly ash effluent enriched sediment. Later, the heavy metal resistant bacterial strains were isolated by pour plating method using nutrient agar supplemented with various concentrations of heavy metal salts. Individual pure colonies were screened for their ability to synthesize TiO2 NPs. The organism that efficiently synthesizes TiO2 NPs was then characterized completely based on biochemical characterization according to Bergy’s manual of Bacteriology [18] which includes 16S rDNA technique and G + C analysis.
where d is the mean diameter of the nanoparticle, l is the wavelength of X-ray radiation, b is the angular full width at half – maximum of the XRD peak at the diffraction angle. The dried powder was diluted with potassium bromide in the ratio of 1:100; analyzed at a resolution of 4 cm1 over 4000 to 500 cm1 in diffuse reflectance mode and the spectrum was recorded in Fourier transform infrared (FTIR) spectrophotometer. The morphology and size distribution of the nanoparticle was observed by TEM and field emission scanning electron microscope attached with EDAX to determine the elemental characterization at a specific position. For FE-SEM, a drop of the sample was placed on a stub using adhesive carbon tape with gold sputtering and observed using FEI Quanta 200 operating at a voltage of 30 kV. Sample was air dried on a carbon coated copper grid and operated at an accelerating voltage of 300 kV for observation using TEM.
2.2. Synthesis of TiO2 nanoparticles About 25 ml of the re-suspended culture of the isolate was diluted four times by adding 75 ml of sterile distilled water containing nutrients. This diluted culture was allowed to grow for another 24 h. 20 ml of 0.025 M TiO(OH)2 solution was added to the culture solution and heated on a steam bath up to 60 8C for 10– 20 min until white deposition was observed at the bottom of the flask indicating the initiation of transformation. The culture solution was then cooled and allowed to incubate at room temperature in laboratory ambience. After 12–48 h, the culture solution was observed to have distinct white precipitate deposited at the bottom of the flask. The precipitate was then annealed at 300 8C for 1 h that transforms amorphous into anatase phase and the impurities were removed. 2.3. Characterization of nanoparticles The synthesized TiO2 nanoparticles were characterized by measuring the UV absorbance at a resolution of 1 nm and the bandgap (Ebg) was calculated using
2.4. In-vitro biocompatibility assays The haemolytic assay was done for various concentrations of TiO2 NPs as described by Sai et al. [19]. Rat erythrocytes were isolated from heparinized blood by centrifugation. The cells were washed three times with 5 mM HEPES buffer containing 150 mm sodium chloride. Aliquots of 1 ml suspension containing 107 cells in microfuge tubes were incubated with different concentrations of TiO2 NPs (5–100 mg/ml) in duplicates at 37 8C for 30 min with gentle mixing. The tubes were then centrifuged and absorbance of the supernatants was measured at 540 nm. The lysis obtained with water was considered as 100%. The cytotoxic effect of TiO2 NPs was assessed by MTT (3-[4,5dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT, Sigma, USA) assay [20]. 8 103 cells (NIH/3T3-mouse embryonic fibroblasts) were seeded onto a 24 well culture dish and allowed to adhere and stabilize overnight in a CO2 incubator at 37 8C. Cells were washed with warm PBS and fresh medium containing TiO2
Fig. 1. (a) Propionibacterium jensenii and (b) observation of pleomorphic rods by Gram staining procedure at a magnification of 100.
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S. Babitha, P.S. Korrapati / Materials Research Bulletin 48 (2013) 4738–4742
Fig. 2. (a) UV–visible, (b) X-RD and (c) FTIR spectrum of TiO2 nanoparticle.
Fig. 3. (a) TEM and (b) FE-SEM image of TiO2 nanoparticle with (c) EDAX.
NPs at various concentrations (5–100 mg/ml) was added in triplicates. after 24 h incubation with TiO2 NPs at 37 8C, the cells were treated with MTT (5 mg/ml) for 3 h. The formazan complex formed was solubilized with DMSO (dimethyl sulphoxide) and the absorption was measured at 570 nm with a reference wavelength of 630 nm, in a micro plate reader (Biorad). The percentage of viability was calculated using the optical density of the control and treated cells.
photographed and the wound margins were traced to determine the area of contraction throughout the healing period. Granulation tissue was removed every third day and the dressings were replaced till the healing was complete. The experiment was carried out after obtaining approval from the institutional animal ethics committee.
2.5. In-vivo biocompatibility study
The organism on the basis of 16S rDNA technique and the biochemical parameters was identified to be Propionibacterium jensenii [KC545833], a probiotic, high G + C rich, pleomorphic rod shaped gram-positive bacteria (Fig. 1). The G + C content of the sequence were estimated to be 57.49% using Seqool software [21].
2.5.1. Preparation of collagen – TiO2 wound dressing Nanosized titanium dioxide particles possess the property of enhancing mechanical strength. Various concentrations of TiO2 NPs (5–100 mg/ml) were dispersed in the reconstituted collagen (5 mg/ml Collagen, 0.2 M phosphate buffer, 2 M NaCl, and 1.25 N NaOH) film and assessed for their mechanical strength using Instron tensile tester at an extension rate of 5 mm/min. Dumb-bell shaped pieces of 20 mm length, 0.02 mm thickness and width of 12 mm were cut from the reconstituted collagen – TiO2 films for measuring the stress strain characteristics. The ideal concentration of TiO2 dispersed collagen film was observed to be 25 mg/ml giving a good mechanical strength of 33.37 MPa. The films were used for further studies on animal experiment. 2.5.2. Open excision wound model Female wistar rats (12 Nos) weighing 120 g were selected and their dorsal surface below the cervical region was shaved off the pelage. Uniform sized open excision type wounds of 5 cm2 were created under mild anaesthesia (intraperitoneal injection of pentobarbitone sodium (50 mg/kg). The control group (6 Nos) was treated with reconstituted collagen film while the experimental group (6 Nos) was dressed with collagen film containing TiO2 NPs and were covered with sterile cotton gauze. The wounds were
3. Results and discussion
Fig. 4. Cytotoxicity of synthesized TiO2 NPs.
S. Babitha, P.S. Korrapati / Materials Research Bulletin 48 (2013) 4738–4742
4741
Fig. 5. (a) In vivo wound healing efficiency of TiO2 NPs. (b) Percentage of wound contraction rate in control and TiO2 treated rats.
The UV–visible spectrum showed a well defined surface plasmon resonance peak (Fig. 2a). The cut off wavelength of TiO2 nanoparticles was observed at 382 nm which was subsequently used for calculating the band gap (Ebg) using the formula. The band gap value of TiO2 was found to be 3.247 eV which confirms the excitation absorption peak correspond to anatase form of nano TiO2. The crystal phase of the TiO2 was confirmed using XRD (Fig. 2b). The line broadening of the diffraction peaks indicated that the synthesized materials were in nanometer range. The diffraction patterns showed that the anatase phase (a = 3.782 A˚, JCPDS no: 84-1286) was formed, and the crystallite size was calculated using Scherrer’s formula. The average particle size was found to be 65 nm. The identified 2u peaks at 25.378, 37.818, 47.988, 62.548, 74.838 matching the planes 101, 004, 200, 204 and 215 confirmed the characteristics of biologically synthesized TiO2 NPs crystallites. FTIR spectrum of the TiO2 NPs (Fig. 2c) revealed the purity owing to the observation of bands at 1235, 1643 and 1068 cm1 which were assigned to the bending vibrations of primary and secondary amines and carboxylic groups respectively. The band observed at 2924 cm1 confirms the bound carboxylic groups with reference to Ti–O–Ti in accordance to Hardy et al. [22]. The presence of this carboxylic group probably attributes to the nucleation site for nanoparticle formation owing to the ability of co-ordination with metal ions. A broad peak at 3411 cm1, the other peaks at 1549 cm1, 1408 cm1 correspond to the O–H stretching of alcoholic groups, C5 5C ring stretching and bending vibration of CH2 in the lipids and proteins respectively. The amide linkage between the bacterial proteins and the TiO2 during the reaction was confirmed by the presence of a peak at 1235 cm1. The uniform shape and size of the nanoparticle and their polydispersed nature is depicted in the (Fig. 3a) ranging between 10 and 80 nm. This data is in line with XRD studies. The difference in size observed amongst the synthesized nanoparticles could be attributed to variation in time scales during the process of bacterial synthesis. The FE-SEM studies clearly showed that the nanoparticles were smooth and spherical in shape and ranged in size from 15 to 80 nm (Fig. 3b). The EDAX spectrum recorded in the spot profile mode from one of the densely populated TiO2 nanoparticle area with the binding energy region of 0–20 keV is shown in (Fig. 3c). The peak
from the spectrum revealed the presence of Ti and O at 4.5 and 0.5 keV. The atomic percentage of Ti and O was 54.73 and 45.27 respectively. The most prominent signals were corresponding to that of titanium while the lesser owing chlorine, oxygen and ka atoms were probably due to X-ray emission from proteins and enzymes present in the cell wall of the biomass. No haemolysis or cell death with reference to NIH/3T3 cell lines was observed with TiO2 NPs even at a concentration of 100 mg/ml (Fig. 4). This clearly demonstrates the non toxic nature of the nanoparticles. Further, the in vivo wound healing experiment in wistar rat model showed that the synthesized TiO2 nanoparticles have an ability to enhance wound healing. The rats treated with collagen film containing TiO2 NPs nanoparticles (25 mg/ml) showed a complete healing in 14 days, while the control group containing only collagen film healed in 18 days (Fig. 5). The enhanced wound closure rate observed in the experimental group might be attributed to the sustained release of Ti ions from the TiO2 NPs incorporated wound dressing. 4. Conclusion Eco friendly synthesis of TiO2 NPs using a highly efficient Propionibacterium species isolated from the coal fly ash effluent is illustrated. This process of synthesis resulted in a well dispersed uniform sized anatase form of nanoparticles that are highly stable, biocompatible and cost effective thereby offering several advantages over conventional methods. The present study provides scope for the synthesis of non toxic nanoparticles with enhanced wound healing activity. Acknowledgements The authors thank the Director, CSIR – CLRI for his constant support. The first author gratefully acknowledges the DST – INSPIRE, New Delhi for the fellowship (IF110583). References [1] B. Nair, T. Pradeep, Cryst. Growth Des. 2 (2002) 293–298. [2] G. Rajakumar, A. Abdul Rahuman, S. Mohana Roopan, V. Gopiesh Khanna, G. Elango, C. Kamaraj, Spectrochim. Acta Part A 91 (2012) 23–29.
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