Ag

Materials Science and Engineering C 51 (2015) 174–181

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

PEGylation of novel hydroxyapatite/PEG/Ag nanocomposite particles to improve its antibacterial efficacy S. Jegatheeswaran, M. Sundrarajan ⁎ Advanced Green Chemistry Lab, Department of Industrial Chemistry, School of Chemical Sciences, Alagappa University, Karaikudi-3, Tamil Nadu, India

a r t i c l e

i n f o

Article history: Received 19 September 2014 Received in revised form 6 December 2014 Accepted 9 February 2015 Available online 20 February 2015 Keywords: Hydroxyapatite Silver Nanocomposite Bioceramic Epi-fluorescence Antibacterial

a b s t r a c t Hydroxyapatite (HAp) nanocomposite particles were prepared simply in the presence of polyethylene glycol (PEG) and fabricated with silver via a sol–gel route and the physico-chemical and biological properties of these materials were investigated. The objective of this study is to inspect the crystallinity and antibacterial activity of these composite materials. PEG has been used to greatly promote biocompatibility and biodegradability of HAp. Silver nanoparticles were used for improving its bactericidal efficacy while applying composites. Nanosized HAp composite particles with PEG and nano-silver was incorporated to increase the crystalline nature of the nanocomposite. The structure of nanocomposite particles was studied by XRD, FTIR, HR-SEM, EDS and TEM analyses. Silver nanoparticles loaded on the synthesized HAp-PEG showed a synergistic antibacterial effect against Gram-negative bacterium Escherichia coli (E. coli). The controlled release of Ag+ ion from HAp-PEG-Ag nanocomposite has given good antibacterial efficacy evidenced by epi-fluorescence microscopy images during different hours. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Bioceramics are vital materials for the manufacturing of dental and bone implants based on their biocompatibility, osteoconduction, high strength, low thermal and bioinert behavior. Because the hard tissues of our body are composed of calcium phosphate ceramic-like hydroxyapatite (HAp) [1,2]. Synthetic hydroxyapatite is a bioceramic, which has been widely applied in various clinical applications such as repairing bone defects in orthopedic sites, immediate tooth replacement, augmentation of alveolar ridges, pulp capping material and maxillofacial reconstruction [3]. The human bone contains 70% weight and 50% volume of HAp. The HAp present, accounts for more than 70 wt.% and 90 wt.% of dentin and enamel respectively [4]. Pure HAp was not suitable for load bearing applications, due to its poor mechanical and biocompatibility properties. This type of materials is required to improve biocompatibility and functional behaviors. In recent days, some attempts have been made by researchers to improve and ultimately obtain similar to the bone-like mechanical properties with materials like titania, zirconia, bioglass, alumina and polymer matrixes [5]. The nanoscale materials often show a unique and consistent change in physical, chemical and biological properties compared to their macroscale counterparts [6]. HAp nanoparticles have been used as reinforcing filler in composites and have shown remarkable impacts on nanocomposite bonding agent properties compared to micro- and macro-scale fillers [7]. Integration of HAp with polymer in favor of composites, because of ⁎ Corresponding author. E-mail address: [email protected] (M. Sundrarajan).

http://dx.doi.org/10.1016/j.msec.2015.02.012 0928-4931/© 2015 Elsevier B.V. All rights reserved.

the action of polymer aggregation of nanoparticles, enhances subsequently to improve the expected compatibility [8]. Novel biodegradable polymers with specific properties are in great demand in tissue engineering applications. Biodegradable polymers can be classified as natural or synthetic polymers according to the source. Synthetic biodegradable polymers have found more versatile and diverse biomedical applications owing to their tolerable designs or modifications [9]. The composites of HAp and synthetic biodegradable polymers that can compensate for the weak mechanical properties of HAp have become of great interest [10]. Polyethylene glycol (PEG) is a well-known synthetic biodegradable polymer and it has been applied to numerous biomedical applications. The reported results stated on the fabrication of photo-cross-linked and degradable PEG hydrogels with tunable mechanical properties. The composition of HAp-PEG shows a significant effect on the size of HAp with increases of PEG concentration in composites. PEG is an important factor for controlling particle size, crystal phase and degree of aggregation in these HAp nanoparticles [11,12]. Inorganic materials like metal and metal oxides have attracted lots of attention over the past decades due to its ability to withstand harsh process conditions. Metal nanoparticles are one of the interesting research fields in material science, due to the development of nanotechnology and it has been greatly applied in biomedical applications [13]. Synthesis of noble metal nanoparticles are applied in many applications such as catalysis, electronics, optics, environmental, and antimicrobial, and biotechnology, an area of constant interest. Among, various metal nanoparticles, silver nanoparticles have received scientific attention because their physical and chemical properties are more effective from the bulk materials. The antibacterial properties of silver nanoparticles are

S. Jegatheeswaran, M. Sundrarajan / Materials Science and Engineering C 51 (2015) 174–181

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Fig. 1. Schematic diagram and probable mechanism of HAp-PEG-Ag nanocomposite formation.

well known from ancient days with condition that the nanoparticles have to be stable and easily dispersed in water [14–19]. Silverhydroxyapatite (Ag-HAp) nanocomposite has been synthesized without a high degree of agglomeration and gives a great antimicrobial activity. It can be a promising antimicrobial biomaterial for implant and reconstruction surgery applications [20]. Silver doped with hydroxyapatite nanoparticles gives great crystallinity and is expected to display a greater biological efficacy in terms of osteointegration [21]. The researchers have developed HAp nanocomposite synthesis through many techniques such as hydrothermal process [22], mechanochemical methods [23], electrochemical deposition [24], co-precipitation [25] and sol–gel methods [26]. Among these methods, the sol–gel method has been used in the fabrication of high quality nanopowders. This method achieves ultra-homogeneity of the several components on a molecular scale with lower preparation temperature, which saves energy, cost and the ability to form unique composition [27]. In this work reports the novel HAp-PEG-Ag nanocomposite was synthesized using an easy and low cost sol–gel method to study on their antibacterial activity. This nanocomposite has well crystallized and it has a similar particle size due to the action of PEG and Ag materials. Its chemical composition, crystal phase, particle size, and purity were characterized using FT-IR, XRD, HR-SEM, EDS and TEM analyses. The antibacterial activity of the nanocomposite was studied using the Gram negative bacterium E. coli.

Fig. 2. X-ray diffraction (XRD) patterns of HAp, HAp-PEG and HAp-PEG-Ag nanocomposites.

2. Materials and methods 2.1. Materials Polyethylene glycol (PEG) with a molecular weight of 6000 g/mol was purchased from Sigma-Aldrich. Silver nitrate (AgNO3), calcium nitrate tetrahydrate (Ca (NO3)2·4H2O), ammonium dihydrogen phosphate (NH4H2PO4), sodium borohydrate (NaBH4) and sodium hydroxide pellets (NaOH) were purchased from Merck in analytical grades. All the solutions were prepared with ultrapure water (Millipore, 18.2 MΩ cm). Himedia supplied nutrient broth, nutrient agar and agar– agar for carrying out the antibacterial testing. 2.2. Preparation of HAp nanoparticles A sol–gel method was used for synthesis of HAp nanoparticles, HApPEG and HAp-PEG-Ag nanocomposites. Ca(NO3)2 and NH4H2PO4 served as the sources of calcium (Ca) and phosphorous (P) for HAp. The Ca/P molar ratio was maintained at 1.67 [28] and the solution of phosphorus precursor (0.77 g in the 50 mL ultra-pure water) was added drop-wise to the stirred calcium nitrate solution (2.36 g in the 50 mL of ultrapure water). For the pH adjusting 0.1 M NaOH solution was added during the reaction and pH was maintained at 10–11. After mixing, the sol– gels were stirred for 6 h at room temperature. Finally, the precipitate

Fig. 3. FT-IR spectrum of HAp, HAp-PEG and HAp-PEG-Ag nanocomposites.

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Fig. 4. HR-SEM images of pure HAp, HAp-PEG and HAp-PEG-Ag in different magnifications.

was centrifuged for 5 min at 10,000 rpm to remove any air bubbles, then dried in hot air oven at 70 °C for 2 h. 2.3. PEGylation of HAp and HAp-Ag nanocomposite particles About 4 g of polyethylene glycol was dissolved in 40 mL of ultrapure water under vigorous stirring by a magnetic stirrer and then the polymer solution was kept for 12 h overnight at room temperature to remove the air bubbles trapped in the viscous solution. Ca (NO3)2 solution was added into the above solution with continuous stirring for 6 h, followed by NH4H2PO4 which has added the NaOH solution for pH adjusting agent. The composite gel was centrifuged to remove any air bubbles. For the preparation of silver incorporated HAp/PEG nanoparticles were obtained by adding a solution of 1 mM of silver nitrate (AgNO3) to the gel-like HAp/PEG composite suspension during synthesis and stirred for another 6 h. The nanocomposite particles were separated by centrifugation at 9000 rpm for 45 min and then the supernatants

were discarded. To prepare silver nanoparticles using the reducing agent 0.5 M of NaBH4. The van der Waals attraction between the Ag and HAp-PEG became nanocomposite particles. Hence, these particles were formed with HAp-PEG-Ag nanofillers. Successfully prepared HAp, HAp-PEG and HAp-PEG-Ag nanomaterials were dried at 70 °C in an air oven. Dried samples were ground to form powder for 20 min using a vibrating agate ball mill and to ensure similar particle size. 2.4. Antibacterial property 2.4.1. Agar well diffusion method The antibacterial activity of the synthesized HAp-PEG-Ag nanocomposite was screened against Escherichia coli (G–) using agar well diffusion method with some modifications [29]. In brief, the bacterial cell suspension of 1 × 106 colony forming unit (cfu) mL−1 was inoculated using sterile cotton swabs on the Mueller Hinton Agar (MHA) plates. Experimental wells of 6 mm in diameter on the plates were loaded with 20 μg mL−1 concentrations of test materials and incubated for 24 h at


177

Fig. 5. EDS image of HAp-PEG-Ag nanocomposite for elemental composition and weight percentage diagram.

37 °C. The experiment included a positive control (Ampicillin alone) and a negative control (Milli Q water without nanocomposite). All the experiments were carried out in triplicate. The zone of inhibition was measured using Antibiotic Zonescale (HiMedia, India). Reduction in bacterial viability was measuring total viable counts at different time intervals (0, 6 and 12 h) and bacterial cell death visualized by epi-Fluorescence microscopy. Fluorescein isothiocyanide (FITC) and propidium iodide (PI) dual stains were used for living and dead cells on bacterium. Propidium iodide penetrates only to damaged cells and binds to the DNA emitting a red color, whereas FITC remains exterior to undamaged cell walls giving rise to green emission [30].

2.4.2. Bacteriostatic (MIC) and bactericidal concentration (MBC) The MIC and MBC of the nanocomposite were determined according to the method of Ruparelia et al. Fourty microlitres (∼ 1 × 106 cells mL−1) of E. coli (G–) bacterial strain was added to 4 mL of Luria Bertani (LB) broth. The different concentrations (10 to 50 μg mL − 1) of the HAp-PEG-Ag nanocomposite gel was added to the test tubes containing the test bacterial strain. After 24 h of incubation, the MIC results were noted by checking the turbidity of the bacterial growth and the MBC were determined by streaking a loop full of the bacterial culture on the MHA plates, incubated at 37 °C for 24 h. The MBC is the concentration at which the bacteria are completely killed.

2.4.3. Analysis of growth curve Bacterial colony counting is a convincing technique for the analysis of antibacterial activity of nanomaterials. Antibacterial activity of the synthesized HAp-PEG-Ag nanocomposite was tested by a standard microdilution method leading to the inhibition of the bacterial growth. E. coli bacterial strain was allowed to grow in 10 mL of LB broth. This media was supplemented with nanocomposite ranging from 10 to 50 μg mL− 1 and the bacterial cultures were incubated at 37 °C. The growth of test bacterial strains in broth was indexed by measuring the optical density (OD) at 595 nm at regular time intervals using UV–vis spectrophotometer (UV-1800, Shimadzu). The growth curve was plotted between OD595 nm and time [31]. 2.4.4. Statistical analysis The data were expressed as mean ± SD. The analysis of variance (one-way ANOVA; P b 0.05) was performed for the mean value of antibacterial properties of nanocomposite against E. coli bacterial strain. All the analyses were carried out using SPSS v16.0 (SPSS Inc., USA). 2.5. Characterizations X-ray powder diffraction (XRD) profiles were obtained from computer controlled XRD system JEOL IDX 8030 with Ni filtered Cu KR

Fig. 6. TEM image of PEGylated HAp-PEG-Ag nanocomposite in different magnifications.

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nanocomposite. The nanocomposites have been slowly releasing the silver nanoparticles by weak hydrogen bonding breakage during the antibacterial activity.

3.2. X-ray diffraction studies

Fig. 7. Nanoparticles analyzed for HAp-PEG-Ag nanocomposite by particle size analyzer.

radiation with an automatic Philips diffractometer to use record the crystallinity. The intensity data for the pure HAp and HAp nanocomposite particles were collected over a 2θ range of 35–70° at a scan rate of 0.3° 2θ/min. The surface properties of the samples were studied using scanning and transmission electron microscopes. SEM is widely employed for determination of the morphology and size distribution of prepared composite materials. Morphology of nanocomposites was carried out (by using a Qunata 250 FEG scanning electron microscope operating at 10 kV). The size and linkage of the nanocomposite were obtained with Hitachi H 7500 (Tokyo, Japan) transmission electron microscopes (TEM), operating at 80 kV and 200 kV, respectively. The FT-IR analysis of the prepared nanomaterials was performed by Perkin Elmer make Model Spectrum RX1 (range 4000 cm−1–400 cm−1). The antibacterial activity of HAp-PEG-Ag nanocomposite particles depends on the distribution of Silver nanoparticles on the bacterial cell membrane with respect to incubation time was visualized by Epi-fluorescence microscopy (E200 Coolpix; Nikon, Tokyo, Japan). 3. Results and discussion 3.1. Probable mechanism A schematic description of the conceivable mechanism of HAp/PEG/ Ag nanocomposite formation has been shown in Fig. 1. PEG is an easy water soluble polymer and it has been treated with Ca(NO3)2 and NH4H2PO4 for Ca2+ activation by active oxygen species that could provide an electron bond network to adsorb Ca2+ of HAp and then PO3− 4 and OH− easily combine with divalent Ca2+ by electrostatic interaction to form apatite nucleation sites [32]. The reaction Ca2+ has anchored on PEG by ion polar interaction to form ionic bonds. This bond formation should proceed by nucleophile attack of divalent Ca2 + cations onto the two pairs of lone pair electrons of oxygen from PEG [33]. The positive charge of the Ag nanoparticles is interacting with the hydroxyl group of PEG to form [Ag (PEG)] core due to the presence of van der Waals force between the negatively charged oxygen species present in the molecular structure of PEG [34]. Therefore, HAp and Ag nanoparticles uniformly distributed in PEG by PEGylation to form HAp/PEG/Ag

Table 1 Antibacterial assay of HAp-PEG-Ag nanocomposite and pure AgNPs against E. coli. Bacterial pathogens

E. coli

Concentration of nanocomposite (μg mL−1)

Zone of inhibition (mm)

Composite

AgNPs

Composite

AgNPs

MIC

MBC

MIC

MBC

10

20

10

25

14.6 ± 0.54

13.5 ± 0.5

The typical X- ray diffraction patterns of pure HAp, HAp-PEG and HAp-PEG-Ag were recorded in the 2θ range of 10–70 θ are shown in Fig. 2. The characteristic peaks of HAp at 26.5 θ, 31.9 θ, 32.8 θ, 39.8 θ, 47.7 θ, 49.5 θ and 61.6 θ are consistent with the lattice planes (210), (112), (300), (310), (312), (321) and (214) that obeyed the same ICDD-PDF no. 09-432 with the hexagonal crystal structure. The reflections at 35.8°, 38.1°, 44.3° and 55.0° are consistent with (100), (111), (200) and (006) lattice planes corresponding to silver present in the HAp-PEG-Ag nanocomposite particles and only four characteristic peaks of HAp appear in the composite spectra to confirm the maximum incorporation of Ag in the nanocomposite. The peaks of HAp and HAp-PEG were very broad and more noise observed in the X-ray diffractogram due to amorphous nature of pure hydroxyapatite particles. However, the characteristic peaks of HAp-PEG are broader and less noisy compared with pure HAp particles. Hence, hydroxyapatite nanoparticles are comprised of a dense network structure of interpenetrating PEG polymer chains covalently cross-linked to increase the crystallinity of HAp-PEG nanocomposite particles [35]. Therefore, PEG played a very important role in activating and progressing the particle nature of nanocomposites. The characteristic peaks arising from Ag crystals increase the crystallinity of composites, which means that amorphous HAp nanopowder has strong crystallinity with Ag and it has been specified that Ag nanoparticles were mostly incorporated in the nanocomposite.

3.3. Functional group modifications by FT-IR analysis In order to illustrate the intermolecular interaction between components in the system, FT-IR spectrum was taken and shown in Fig. 3. These spectra exhibited the functional group presence of HAp, HApPEG and HAp-PEG-Ag nanocomposite particles have performed to characterize the chemical structure for nanocomposite particles. The materialization of apatite nanocomposites on the substrates is in the form of broad FT-IR bands centered at 569 cm− 1, 603 cm− 1, 963 cm−1, 1045 cm−1 and 1091 cm−1. These peaks correspond to the first symmetric P–O band and the fourth symmetric P–O band has identified the stretching vibration of the PO34 −. The well-defined and sharp peaks, occurring at 569 cm−1 and 603 cm−1 have related to the fourth phosphate bond. In addition, the third phosphate band has well defined peaks at 963 cm−1 and 1045 cm−1. While the sharp peaks at 633 cm−1 and 3470 cm−1 have appeared due to the structural –OH of the apatite. The peak around 1084 cm− 1 and 1748 cm− 1 attributed to the stretching of carbonyl groups (C_O and C\O) bond from the PEG. These peaks seemed due to the polymer that was effectively combined with a large amount of Ca2+ through the strong electrostatic attraction presence of negatively charged C–O–C group of PEG to form a covalent bond [36]. Adsorption bands available at 874 cm− 1, 1420 cm− 1 and 1465 cm−1 correspond to carbonate adsorption. It reveals that carbonate ions are produced in the HAp nanocomposite particles. A possible explanation for this phenomenon is that atmospheric CO2 might have been adsorbed by apatite at ambient temperature. The spectrum of HAp-PEG-Ag shows greatly dominated bonds compared to pure HAp and HAp-PEG due to its ion polar interaction with Ag. The hydroxyl network around 3470 cm−1 has a broad covered area that causes positive silver ion interacting with more hydroxyl groups of HAp [37]. The intensity peaks of phosphate groups have been decreased in the HAp-PEG-Ag due to apatite formation was controlled by a large number of Ag nanoparticles present in the composite.


179

Fig. 8. Epi-fluorescent microscopic images of bacterial species E. coli a) initial, b) 6 h and c) 12 h during interfacial contact studies in the presence of HAp-PEG-Ag nanocomposite.

3.4. Surface morphology using scanning electron spectroscopy

3.5. Energy dispersive spectroscopic analysis

The external morphology of nanocomposite particles was studied by scanning electron microscopy (SEM). Fig. 4 displayed SEM photographs in different magnifications of amorphous, poor crystalline and crystalline natured particles of pure HAp; HAp-PEG and HAp-PEG-Ag nanocomposite particles were formed. The Fig. 4 (a, a1) exhibits SEM observations that revealed the amorphous powder of pure HAp and Fig. 4 (b, b1) proved the presence of HAp particles into the polymer matrix and typical images of HAp-PEG show improved particle behavior compared to HAp (pure). Because inorganic crystals have a high affinity with PEG polymer matrix, the interface between inorganic and organic phases was indistinguishable. Usually HAp particles have been dispersed in respectable solvents for less aggregation and convenient use of biomedical applications. Akhilesh et al., has reported that the PEG polymer can inhibit the agglomeration of inorganic nanomaterials. Because the oxygen group in the PEG was responsible for the lower aggregation level in HAp-PEG [38], PEG molecules surrounded metal ion Ca2+ in this case to form small and well-dispersed particles. Fig. 4 (c, c1) of HAp-PEG-Ag nanocomposites shows that , the doping component silver highly influence on the surface morphology. The morphology identifications designated that the nanoparticles with good crystal structure could be made using the sol gel method at room temperature. The HAp-PEG-Ag nanocomposite shows non-agglomerated nanocomposite particles. These nanocomposite particles have been suitable for cell adhesion and attachment as well as nutrient delivery to the site of tissue regeneration.

Energy dispersive spectroscopy (EDS) was employed to investigate the elemental composition presenting the nanocomposite particles. The results in Fig. 5 established that only Ca, P, Ag, C and O appear in HAp-PEG-Ag nanocomposite particles. EDS analysis showed that the HAp nanocomposite had a Ca/P ratio equal to 1.67 same as the Ca/P ratio of natural bone and C, O peaks responsible for the PEG present in the nanocomposite. Finally, the presence of an Ag peak is predicted and observed in the spectrum specifying the incorporated silver nanoparticles. Also, note that no other impurity including carbonaceous species was present in the composites. 3.6. Transmission electron microscopy and particle size analysis Transmission electron microscopy (TEM) was performed to observe the fabrication of Ag nanoparticles on HAp-PEG nanocomposites. As compared with SEM, more complicated procedures for specimen preparation is needed. However, it has even a higher resolving power. Fig. 6 shows the morphology image of HAp-PEG-Ag nanocomposite particles that revealed a hexagonal structure. It can be seen that HAp crystals in three component nanocomposite particles have a good dispersive property and display a meticulous hexagonal lattice structure. The particle size of HAp in the nanocomposite was measured by PSA as shown in Fig. 7. The cumulative size distribution of nanoparticles approximately starts from the range of 1 nm to 150 nm. The PSA study demonstrates that the higher size distribution of nanoparticles exhibit sizes of

Fig. 9. Effects of the different concentrations of HAp-PEG-Ag nanocomposite and pure AgNPs on E. coli growth kinetics.

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40–60 nm. The average particle size of the hexagonal HAp is 50–70 nm in the range was confirmed by the particle size analysis (PSA) (Fig. 7). These hexagonal HAp crystalline particles present in the composite bonded with Ag nanoparticles, which were identified by maximum covered dark area to specify the Ag fabricated on the nanocomposites. The white arrow marking indicated the dark silver particles and the dark arrow (double side) identified the PEG linkage between HAp. From TEM results, the silver greatly interacted with PEGylated HAp to the construction of nanocomposites. The diffraction patterns shown in Fig. 2(c) suggest that the particles are pure crystalline in nature. The XRD patterns are almost the same depending on the crystallinity and phase composition according to the TEM results. 3.7. Antibacterial activity of HAp-PEG-Ag nanocomposites The consumption of bioceramic–polymeric nanocomposite has opened new treatment avenues in the advanced nanobiotechnological research. In the present investigation, prepared HAp-PEG-Ag nanocomposite exhibited a broad spectrum of antibacterial activity against the test bacterial pathogen. The zone of inhibition against E. coli was registered at 14.6 ± 0.54 mm and the pure Ag nanoparticles were recorded at 13.5 ± 0.5 mm (Table 1). The HAp-PEG-Ag nanocomposite is thus proved to reduce the bacterial densities significantly even at the least concentration. The results of the composite have given excellent antibacterial activity showed by epi-fluorescence microscopic images during different time intervals to increase cell death evidenced by mostly the yellow and red staining and very little green was present in it (Fig. 8). However, the broth dilution method inferred the MIC (10 μg mL−1) and MBC (20 μg mL−1) of nanocomposite against E. coli (Table 1), whereas the bacteriostatic and bactericidal concentrations of antibiotic drug were recorded at 5 and 15 μg mL−1 respectively. Studies by some of the researchers have established the nanomaterial impregnated nanocomposite as an effective bactericidal material against the various microorganisms [39]. However, there is little information available for the nanocomposite alone in the biomedical applications. In our study we established that the nanocomposite alone has been proved effective antibacterial agents against the test bacterial pathogen. Optical densities were measured and plotted as a function of time at regular intervals between 0 and 24 h with various concentrations (0–50 μg mL− 1) of nanocomposite (Fig. 9). Dose-dependent growth dynamics of test bacterial pathogenic strain can be used to assess the relative rate and extent of bactericidal activity of nanomaterials [40]. The growth visibilities of the bacterial strain are treated with various concentrations of the HAp-PEG-Ag nanocomposite. The results showed that the introduction of Ag impregnated nanocomposite affected the growth dynamics effectively as compared to the control. Growth of the bacteria was effectively reduced with an increased concentration of nanocomposite, in particular 30 and 40 μg mL−1 concentrations of the HAp-PEG-Ag nanocomposite act as effective bactericides and there was an abrupt reduction of bacterial growth [41]. The nanocomposite depicted efficient bacteriostatic and bactericidal properties due to their better efficiency with microorganisms. Earlier researchers have already proved that the different combinations of nanocomposite show an effective antibacterial property against E. coli [42]. The exact inhibitory mechanism of Ag nanomaterial and nanocomposite on microorganisms is still unknown, the possible mechanism involves the disruption of cell wall of the bacteria which causes structural damages and destabilizes the respiratory chain dehydrogenase and finally causes cell death [43]. The present study supports that the nanocomposite was found to be the potent source of antibacterial biocides against pathogenic bacteria. In the future, the necessary efforts will elucidate the antibacterial effect of nanocomposite impregnated AgNPs and the bacterial contact may improve the antibacterial mechanism by damaging the bacterial cell wall and entering of nanocomposite or nanoparticles to the cells to kill the bacteria and further it can be developed as an antibacterial agent in biomedical research. These results confirmed that the HAp-

PEG nanocomposite has given good delivery of silver ions on the bacterial surface to reduce the survival of bacteria. This composite released silver ions to give effective antibacterial property, low toxicity and biocompatibility with human cells. 4. Conclusion This work describes the successful preparation of novel HAp-PEG-Ag nanocomposite particles and its application in improving the crystallinity and antibacterial behavior for biomedical applications. The nanoparticle was made by an ionic gelation method using the PEG covalent bond cross-linking with HAp and silver ions confirmed by FTIR. Amorphous HAp particles improved its crystallinity by PEG and silver, revealed by X-ray diffraction pattern. SEM and TEM images exhibited the agglomeration of nanocomposite particles reduced by Ag+ ions to develop the exact hexagonal structure with good interfacial bonding and mechanical interlocking by the polymer matrix. HAp-PEG-Ag nanocomposite can be a promising antibacterial agent as it has been interacting and inactivating the vital enzymes of bacteria to lose its replication ability leading to cell death and giving a significant bactericidal activity against E. coli (G–) bacteria. This composite is therefore a good candidate for bone fixation devices. Acknowledgment This research was carried out through the financial support of the University Grands Commission (UGC — BSR fellowship for sciences) India. We have gratefully acknowledged the Department of Physics and Department of Industrial Chemistry, Alagappa University for the providing XRD and SEM facilities. The authors would also like to gratefully acknowledge Dr. V. Sri Ramkumar, Post-doctoral Scientist, UGCDSK PDF, Dept. of Environmental Biotechnology, School of Environmental Sciences, Bharathidasan University, Tiruchirappalli, Tamilnadu, India and Dr. S. Selvam, Post doctoral scientist, Laser and sensor application laboratary, Pusan National Univarsity, Busan, South Korea for the help with antimicrobial application studies. References [1] Roya Zandpersa, Latest biomaterials and technology in dentistry, Dent. Clin. N. Am. 58 (2014) 135–158. [2] Zahra Babaei, Mohsen Jahanshahi, Sayed Mohmed Rabiee, The fabrication of nanocomposites via calcium phosphate formation on gelatin–chitosan network and the gelatin influence on the properties of biphasic composites, Mater. Sci. Eng. C 33 (2013) 370–375. [3] Ying Zhang, Yong Wang, The effect of hydroxyapatite presence on the degree of conversion and polymerization rate in a model self-etching adhesive, Dent. Mater. 28 (2012) 237–244. [4] Laleh Solhi, Mohammad Atai, Azizollah Nodehi, Mohammad Imani, Azadeh Ghaemi, Kazem Khosravi, Poly(acrylic acid) grafted montmorillonite as novel fillers for dental adhesives: synthesis, characterization and properties of the adhesive, Dent. Mater. 28 (2012) 369–377. [5] Mahnaz Enayati -Jazi, Mehran Solati -Hashjin, Ali Nemati, Farhad Bakhshi, Synthesis and characterization of hydroxyapatite/titania nanocomposites using in situ precipitation technique, Superlattice. Microst. 51 (6) (2012) 877–885. [6] Yong X. Gan, Structural assessment of nanocomposites, Micron 43 (7) (2012) 782–817. [7] M.R. Nikpour, S.M. Rabiee, M. Jahanshahi, Synthesis and characterization of hydroxyapatite/chitosan nanocomposite materials for medical engineering applications, Compos. Part B 43 (4) (2012) 1881–1886. [8] C. Albano, R. Perera, Catenoh, A. Karam, G. Gonzalez, Prediction of mechanical properties of composites of HDPE/HA/EAA, J. Mech. Behav. Biomed. Mater. 4 (2011) 467–475. [9] Huayu Tian, Zhaohui Tang, Xiuli Zhuang, Xuesi Chen, Xiabin Jing, Biodegradable synthetic polymers: preparation, functionalization and biomedical application, Prog. Polym. Sci. 37 (2012) 237–280. [10] M. Jayabalan, K.T. Shalumon, M.K. Mitha, K. Ganesan, M. Epple, Effect of hydroxyapatite on the biodegradation and biomechanical stability of polyester nanocomposites for orthopaedic applications, Acta Biomater. 6 (2010) 763–775. [11] Yao-Hsuan Tseng, Chien-Sheng Kuo, Yuan Yao Li, Chin-Pao Haang, Polymer-assisted synthesis of hydroxyapatite nanoparticle, Mater. Sci. Eng. C 29 (2009) 819–822. [12] N. Rameshbabu, T.S. Sampath Kumar, T.G. Prabhakar, V.S. Sastry, K.V. Murty, K. Prasad Rao, Antibacterial nanosized silver substituted hydroxyapatite: synthesis and characterization, J. Biomed. Mater. Res. A 80 (2007) 581–591.

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