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Effect of zinc oxide addition on antimicrobial and antibiofilm activity of hydroxyapatite: a potential nanocomposite for biomedical applications
Journal Pre-proof Effect of Zinc Oxide Addition on Antimicrobial and Antibiofilm Activity of Hydroxyapatite: A Potential Nanocomposite for Biomedical Applications Zerihun Beyene, Rupita Ghosh
PII:
S2352-4928(19)30318-6
DOI:
https://doi.org/10.1016/j.mtcomm.2019.100612
Article Number:
100612
Reference:
MTCOMM 100612
To appear in: Received Date:
9 June 2019
Revised Date:
19 August 2019
Accepted Date:
20 August 2019
Please cite this article as: Beyene Z, Ghosh R, Effect of Zinc Oxide Addition on Antimicrobial and Antibiofilm Activity of Hydroxyapatite: A Potential Nanocomposite for Biomedical Applications, Materials Today Communications (2019), doi: https://doi.org/10.1016/j.mtcomm.2019.100612
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Effect of Zinc Oxide Addition on Antimicrobial and Antibiofilm Activity of Hydroxyapatite: a Potential Nanocomposite for Biomedical Applications Zerihun Beyenea and Rupita Ghosha,b
a
Department of Biotechnology, Koneru Lakshmaiah Education Foundation, Green Fields, Vaddeswaram, Guntur District, Andhra Pradesh, India 522502 b
Corresponding Author : RUPITA GHOSH Email: [email protected] , [email protected]
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Tel No: +91 7008861886
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Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha, India 769008
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ABSTRACT
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Bacterial infections and biofilm formations are the main problems associated with implants. Hence, the aims of this study are to develop nanocomposite biomaterials having different ratios of hydroxyapatite nanoparticles (nHAP) with green and chemically synthesized zinc
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oxide (ZnO) nanoparticle (NP) and examine its antimicrobial and antibiofilm activity. The synthesized nanoparticles were characterized for phase and microstructural analysis. The nanocomposite at 90:10, 75:25 and 60:40 ratio of nHAP and ZnO NPs respectively, showed a
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different level of antimicrobial and antibiofilm activity against clinical specimen isolated gram-positive Staphylococcus aureus and gram-negative Escherichia coli. Moreover, the
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minimum inhibitory concentration (MIC) was the lowest concentration of ZnO NPs in the nanocomposite inhibiting the growth of each bacteria species and it was investigated for the given ratio of nanoparticles and found to be 0.2 mg/mL of 90:10 nanocomposite for both pathogens. The minimum bactericidal concentration (MBC) was the lowest concentration of ZnO NPs in the nanocomposite required to kill each of the bacterial species and it was found to be 0.2 mg/mL of 75:25 and 60:40 nanocomposite for S. aureus and E.coli respectively. The maximum percentage of biofilm inhibition was found at 60 (nHAP): 40 (ZnO NPs) ratio of the nanocomposites. It was 52% and 54% against S. aureus and E. coli biofilm 1
respectively, for green synthesized ZnO NPs and 51% and 52% against S. aureus and E. coli biofilm respectively, for chemically synthesized ZnO NPs. Hence, based on these results we suggest that the biomaterials containing different ratios of nHAP- ZnO NPs can be used as antimicrobial and antibiofilm materials in bone implant and bone regenerative medicine.
List of abbreviation Hydroxyapatite nanoparticles
ZnO NPs
Zinc oxide nanoparticles
CLSI
Clinical and Laboratory Standards Institute
MIC
Minimum inhibitory concentration
MBC
Minimum bactericidal concentration
TSB
Tryptone soya broth
FESEM
Field emission scanning electron microscope
JCPDS
Joint Committee on Powder Diffraction Standards
XRD
X-ray diffraction
FDA
Food and Drug Administration
GRAS
Generally Recognized As Safe
BIC
Biofilm inhibition concentration
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nHAP
Keywords: Nano-Hydroxyapatite; zinc oxide nanoparticle; antibiofilm; antimicrobial;
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minimum inhibitory concentration; minimum bactericidal concentration
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1. INTRODUCTION Hydroxyapatite (HAP, Ca10(PO4)6(OH)2) is known as a bioactive, biocompatible, and osteoconductive material chemically similar to the mineral component of bone and teeth [1]. HAP belongs to the calcium orthophosphates family, being the less soluble compound in its class in a physiological aqueous environment [1, 2]. Nano-hydroxyapatite (nHAP) has drawn great concern from researchers since they are widely applied as biomedical materials, including uses such as bone fillers, bone tissue engineering scaffolds, bioactive coatings, soft tissue repairs, drug, protein or gene loading, and delivery systems [3, 4]. 2
However, the application of HAP as biomedical materials is influenced by implant-associated infectious diseases and biofilm formation on the surface of the implant. Orthopedic implant surfaces provide suitable conditions for bacterial infection. As a result, biofilm formation is one of the most serious complications of implant surgery, and it always leads to severe physiological damage and additional costly surgical procedures [3, 5]. Pathogenic bacteria may colonize and adhere to surfaces of implanted biomaterials during the surgical process or from a hematogenous route, which is a key step in the pathogenesis of implant-associated infection [6, 7]. Based on several reports, Staphylococcus species and Escherichia coli are the most important pathogens involved in implant-associated
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infections [8]. Biofilm is a multicellular aggregation of bacteria covered with a hydrated extracellular
polymeric matrix of their own synthesis and characterized by cells that are permanently
attached to an interface with each other. Antibiotic susceptibility of bacteria embedded with a
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biofilm is less as a result, treatment requires higher doses of therapeutic agents. Therefore, the treatment of implant-associated infection using antibiotics fails to clear the pathogens.
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Revision surgery is often the only viable option [9, 10].
Different bacterial pathogens are responsible for the implant-associated infections, among
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them Staphylococcus aureus are the most common and responsible for more than 50% of infections. Other microorganisms include coagulase-negative staphylococci, streptococci, enterococci and gram-negative rods such as Escherichia coli, Pseudomonas spp and
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Enterobacter spp [11].
Zinc is an important mineral involved in biological activities such as cellular
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metabolism, DNA synthesis, and enzyme activity[12]. A zinc ion also exhibits antibacterial activity, improves biological properties, and decreases the inflammatory
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response. Moreover, the addition of Zinc in biomaterial for implant enhances osteogenesis by promoting osteoblast cell proliferation and differentiation [13]. Zinc oxide (ZnO) is listed as “Generally Recognized as Safe” (GRAS) by the FDA. Nano sized ZnO particles have antibacterial activities against a wide range of bacteria [14]. In order to prevent implant-associated infection and biofilm formation on the surface of the HAP implant, the implanted material should have an antimicrobial as well as antibiofilm properties [8]. Therefore, the aim of this study is to prepare and characterize HAP - ZnO nanocomposite at different HAP to ZnO ratio and investigate the in vitro antimicrobial and 3
antibiofilm activity of the nanocomposite against clinical specimen isolated S. aureus and E. coli bacteria. The effect of both the green and chemical synthesized ZnO nanoparticle used in the nanocomposites preparation was compared.
2. MATERIALS AND METHODS 2.1. Materials All of the chemicals used here were acquired from Merck and were of analytical grade, used without additional purification. The media purchased from Himedia Laboratories Pvt. Ltd. All experiments were performed using Double distilled water. The microorganisms, grampositive (Staphylococcus aureus) and gram-negative (Escherichia coli) were clinical
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specimen isolated bacteria, obtained from the Department of Microbiology, NRI Medical College and General Hospital, Chinakakani, Guntur, Andhra Pradesh, India. 2.2. Strains and growth condition
The microorganisms used in this work were Staphylococcus aureus and Escherichia coli,
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which are common causes of infection and biofilm formation in implanted biomaterials [15]. The bacteria were isolated from clinical specimens in the microbiology laboratory of the NRI
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Medical College and General Hospital. Inoculum of each bacterial spp was prepared by inoculating loop full bacteria from a colony into nutrient broth medium and mixed till a
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homogenous suspension was formed. The cultures were incubated at 37°C for 24h. Finally,
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bacterial numbers were adjusted at 0.5 McFarland turbidometry.
2.3. Nanoparticle synthesis 2.3.1. Hydroxyapatite
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HAP was prepared by chemical co-precipitation method using Ca(NO3)2·4H2O and (NH4)2HPO4 as starting materials and ammonia solution as agents for pH adjustment as
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described elsewhere [16]. In brief, an aqueous suspension of 0.24 M Ca(NO3)2·4H2O was prepared. Then a solution of (NH4)2HPO4 was slowly added dropwise to the Ca(NO3)2·4H2O solution maintaining Ca/P ratio of 1.67. The pH of the solution was maintained at 11 by dropwise addition of ammonia solution. This can be explained by the following reaction: 10𝐶𝑎(𝑁𝑂3 )2 . 4𝐻2 𝑂 + 6(𝑁𝐻4 )2 𝐻𝑃𝑂4 + 8𝑁𝐻4 𝑂𝐻 → 𝐶𝑎10 (𝑃𝑂4 )6 (𝑂𝐻)2 + 20𝑁𝐻4 𝑁𝑂3 + 46𝐻2 0
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The solution was continuously stirred for 2 hrs. Then the precipitated HAP was removed from the solution by centrifugation method at the rotation speed of 3000 rpm. The resulting powder was dried at 80°C in a hot air oven and then calcinated at 700°C for 2h in an electric furnace. 2.3.2. Zinc oxide nanoparticle preparation Green synthesis The synthesis of ZnO nanoparticle was carried out by mixing the plant extract (Coriandrum sativum) with aqueous zinc acetate dihydrate solution [Zn(CH3COO)2·2H2O]. The input parameters of the synthesis process were identified and optimized. Then the synthesis and
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characterization studies were carried out at optimum input conditions. Preparation of Plant Extract
The Coriandrum sativum plant leaf was used as a reducing and capping agent to synthesize
ZnO NPs as indicated elsewhere [17]. The fresh plants were purchased from the local market
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in Vijayawada, Andhra Pradesh, India. The surface of the plant leaves was cleaned using
distilled water to remove the minor organic contents. To get the plant extract, 10 grams of the
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leaf was added to 200 mL of distilled water and heated at 70°C for 30 minutes. The liquid extract was separated through filtration and then the extract was cooled at atmospheric temperature.
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Green synthesis of ZnO Nanoparticles
0.2M of Zn (CH3COO) 2·2H2O was prepared and the plant extract was added to the solution
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in the 1:1 ratio (v/v). The mixture was continuously stirred and heated at 70oC. Sodium hydroxide was added drop by drop to it till light yellow precipitate was formed. Then the precipitate was washed with distilled water and finally ethanol followed by drying in a hot air
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oven and finally calcinated at 500oC for 2 hours in an electric furnace.
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Synthesis of ZnO Nanoparticles by Chemical Precipitation Method ZnO NPs were synthesized by direct precipitation method using Zn(CH3COO)2·2H2O solution and potassium hydroxide (KOH) as precursors and a precipitating agent respectively as described elsewhere [18]. An aqueous solution of 0.2 M Zn(CH3COO)2·2H2O and 0.4 M KOH were prepared with deionized water. The KOH solution was slowly added into Zn(CH3COO)2·2H2O solution at room temperature under vigorous stirring. This resulted in the formation of a white suspension. The white product was centrifuged at 5000 rpm for 20
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min and washed three times with distilled water followed by absolute alcohol. The obtained product was calcinated at 500 °C for 3 h after drying in an oven. 2.4. HAP- ZnO nano-composite preparation HAP - ZnO nanocomposite was prepared by the liquid mixing method. Nano HAP powder suspension was mixed with ZnO nanoparticles powder suspension at a different ratio. The nHAP: ZnO NPs ratio was 90:10, 75:25 and 60:40 respectively. Ethanol was used as the organic solvent medium. The suspension was sonicated to dissolve the mixture completely. The formed nanocomposite was finally dried at 100°C in a hot air oven. The batches used for the process are given in Table 1. For each batch of the nanocomposite, 0.2 mg/ mL
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concentration was used for all testes in this research.
Table 1. The batch of nanocomposites at different HAP: ZnO ratio HAP content
ZnO content
Sample designation
1
90%
10% Green method
G.1
2
90%
10% Chemical method
3
75%
25% Green method
4
75%
25% Chemical method
5
60%
6
60%
7
100%
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40% Green method
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40% Chemical method
G.2 C.2 G.3 C.3 H
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0%
C.1
2.5. Phase Analysis
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The synthesized calcined HAP powders and both chemically and green synthesized ZnO nanoparticles phase analysis was done individually in an X-ray powder diffractometer (XRD) (Rigaku, Japan). The composite powders with varying ZnO content were also analyzed for phase confirmation and to observe the composite nature even after mixing. The diffractometer was fitted with a Ni filter of 0.154 nm and the diffraction patterns were measured using CuKα (λ=1.5418 Ǻ) radiation generated at a voltage of 40 kV and current of 40 mA. The samples were scanned in the interval 20˚<θ<50˚ at a scanning rate 20˚/min with a step size of 0.05˚ in a continuous mode. XRD analysis of the samples was performed using 6
X’pert High Score software in comparison with the reference powder diffraction data given by the Joint Committee on Powder Diffraction Standards (JCPDS). 2.6.
Microstructural study
The microstructural study to observe the morphology of the individual nanoparticles and composites were done in field emission scanning electron microscope (FESEM) (FEI, Nano Nova, Netherland). The samples were gold coated for 240 s in a sputter coater in an Ar atmosphere before loading in FESEM. The electrons at 15 kV sources were used to develop relevant information about the samples. Antimicrobial activity
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2.7.
The HAP-ZnO nanocomposite prepared at different nHAP to the ZnO NPs ratio was
examined for its antimicrobial activity. This was determined according to the Clinical and Laboratory Standards Institute (CLSI) procedure, using the agar diffusion test which was
performed using Müller-Hinton agar [19, 20]. The diffusion technique was carried out by
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pouring agar into Petri dishes to form 4 mm thick layers. After the agar was solidified the
medium were inoculated and spread with a bacterial suspension of approximately 1-2 X 108
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CFU/mL using spreader. The wells were prepared by punching with a 6 mm diameter standard sterile cork borer. These wells were filled up with 50 μL of the HAP-ZnO
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nanocomposite solution to be tested. Each concentration of the composite was tested for each culture in triplicates and the assay was performed twice. Antimicrobial activity against each bacterial spp (S. aureus and E. coli bacteria) was observed, and the diameters of the inhibition
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zones (mm) around the wells were measured after 24 and 48 h of incubation at 37°C. The lowest concentration that inhibited the growth was determined by a comparison among the
microbe.
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concentrations as well as between the green and chemical methods of synthesis for each
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2.8. MIC and MBC determination The MIC of the HAP-ZnO nanocomposite against clinical specimens isolate bacteria, that were S. aureus and E. coli were determined using a standard broth dilution method according to CLSI procedures [19, 20]. Tryptone soya broth (TSB) was used to determine MIC and MBC, as antibacterial assay media. For each of the nanocomposite, 0.2 mg/mL was used with adjusted bacterial concentration (0.10 at 625 nm (1×108 CFU/mL, 0.5 McFarland’s standard). The positive control used in this study was TSB containing only test bacterial concentrations and negative control contained only the sterile broth and incubated at 37ºC for 24 h. The 7
MIC was defined as the lowest concentration of ZnO NPs in the HAP-ZnO nanocomposite inhibiting the growth of each bacteria spp. The MIC was noted by the visual turbidity of the tubes both before and after incubation and it was done in triplicate to confirm its value for the tested bacteria. The minimum bactericidal concentration (MBC) is the lowest concentration of ZnO NPs in the nanocomposites required to kill a particular bacterium (MBC, mg/mL). It was determined by taking 100 µL of each sample from test tubes to remain clear (with no turbidity) in MIC and sub-cultured in test tubes containing 1 mL of fresh medium and incubated for 24 h at 37ºC. 2.9. Antibiofilm Activity
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The biofilm development inhibition ability of the HAP-ZnO nanocomposite at different
nHAP to ZnO NPs ratio was determined using polystyrene microtiter plate for both clinical
isolate S. aureus and E. coli as described previously [21, 22]. Pure colonies of S. aureus and E. coli isolates were aseptically picked up and used to inoculate sterile TSB, then incubated
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for 24 h at 37ºC. Overnight grown cultures were diluted in sterile TSB to match 0.5
McFarland turbidity standard which is equivalent to 1.5 × 108 CFU/mL. These bacterial
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suspensions were further diluted in the ratio 1:100 in TSB, which was supplemented with 2% (w/v) glucose and 2% (w/v) sodium chloride, and were mixed with nanocomposite (0.2 mg/
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mL) to be tested in different test tubes, for each of bacterial species. A total of 200 μL of these cell suspensions for each nanocomposite was transferred to each of three parallel wells of a 96-well plate. Wells having TSB (supplemented with 2% (w/v)
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glucose and 2% (w/v) sodium chloride) containing bacterial suspension without nanocomposite as positive control and without bacteria and nanocomposite as negative control were used. After incubation of the plate at 37oc for 24h, the content of each well was
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discarded and washed three times using 200 μL of phosphate buffer saline in order to remove non-adherent cells, subsequently, were dried in an inverted position. The adherent biofilm
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was fixed using 95% ethanol and stained with 100 μL/well of 1% crystal violet at room temperature for 5 minutes. After biofilm staining, the crystal violet solution was removed and the biofilms were washed three times with 200 μL distilled water. Subsequently the water was removed and the wells were dried for 2h. Finally, the adherent dye was dissolved using 30% acetic acid (S. aureus) and 20% acetone (E. coli). The optical density values of triplicate samples were determined using Microplate Reader. The percentage of biofilm inhibition was calculated using the following formula [23].
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% of Inhibition = (𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷570 𝑛𝑚 − 𝑡𝑒𝑠𝑡 𝑂𝐷𝑛𝑚 ⁄𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷570 𝑛𝑚 ) ∗ 100
3. RESULT 3.1. Phase Analysis The XRD patterns for individually synthesized powders of ZnO and HAP were shown in Fig 1 and also the HAP-ZnO nanocomposite with varying ZnO content. The characteristic diffraction peaks for both green synthesized (Fig 1a) and chemically synthesized (Fig 1b) ZnO were observed at position 2ɵ = 32.18°, 34.02°, 36.67°, 47.95° corresponds to (100), (002), (101) and (102) crystal planes respectively. It matches with the JCPDS card no. 750576. It had the hexagonal crystal system with a space group of P63mc. The three highest characteristic peaks for HAP (Fig 1c) were observed at position 25.97°, 31.87°, 33.02°
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corresponding to (002), (211) and (300) crystal planes respectively. It matches with the
JCPDS card no. 09-0432. It had the hexagonal crystal system with a space group of P63/m. The XRD pattern of HAP-ZnO nanocomposite, G1, G2, G3 where green synthesized ZnO
was used and C1, C2, C3 where chemically synthesized ZnO was used, are shown in Fig 1d-i
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respectively. Sharp characteristic peaks at 36.67°, corresponding to (101) crystal plane was observed for ZnO NPs. However, other diffraction peaks for ZnO NPs appeared at 32.18°,
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34.02° were overlapped with the characteristic diffraction peaks of nano-HAP at 31.87° and 34.17°. In addition, sharp peaks were observed for nano-HAP in the XRD patterns of -HAP-
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ZnO nanocomposite. The intensity of the ZnO peaks were found to increase with the increase
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in content for both the green and chemically synthesized ZnO in the HAP-ZnO composites.
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Fig 1. XRD pattern for (a) calcined green synthesized ZnO, (b) calcined chemically synthesized ZnO, (c) calcined HAP, (d) HAP-ZnO nanocomposite (90:10), (e) HAP-ZnO
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nanocomposite (75:25), (f) HAP-ZnO nanocomposite (60:40) using green synthesized ZnO and (g) HAP-ZnO nanocomposite (90:10), (g) HAP-ZnO nanocomposite (75:25), (g) HAP-
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ZnO nanocomposite (60:40) using chemically synthesized ZnO. 3.2. Microstructural study
The micrograph of the chemically precipitated calcined HAP, chemically and green synthesized calcined ZnO powders, are shown in Fig 2. The microstructure as observed under the microscope showed the shape and particle size of the powders. The ZnO nanosized particles were found to be agglomerated. It was observed that the shape of both the green (Fig 2a) and chemically synthesized (Fig 2b) ZnO nanoparticles was more or less spherical. Though very few elongated grains were noticed for the chemically synthesized 10
ZnO NPs, the particle size of the chemically synthesized powder was found to be much larger than that of the green synthesized powder. The 700 °C calcined HAP particles (Fig 2c) are merely spherical and non-uniform in shape. The range for the particle size of the
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synthesized powder was found to be within the nano range.
Fig 2. Micrograph showing (a) chemically synthesized ZnO, (b) green synthesized ZnO, (c)
3.3.
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chemically coprecipitated calcined HAP nanoparticles Antimicrobial activities
The test result of the HAP - ZnO nanocomposite showed that at the given ratio, the
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nanocomposite has an antimicrobial effect against both gram-positive (S. aureus) and gramnegative (E. coli). Pure HAP (100%) did not show any antibacterial activity against both
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bacteria, as shown in Table 2 and Fig 3. All the tested concentrations of nanocomposite showed antimicrobial activities against both bacteria (Fig 3), S. aureus is being more sensitive to the minimum concentration of ZnO NPs than E. coli as presented in Table 2.
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The zone of inhibition was different between pathogens and the zone size increased when the ratio of ZnO NPs was increased in the nanocomposite for each bacteria species. The
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comparison between green and chemical methods of ZnO NP showed almost the same level of antimicrobial activity with no significant difference. The intensity of antibacterial activity
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of the nanocomposite was dependent on the concentration of ZnO NP (Table 2).
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Table 2. Diameter of zone of inhibition by different ratio of HAP-ZnO nanocomposite against S. aureus and E.coli. No
Sample
Inhibition zone diameter in mm S. aureus E.coli
1 2 3 4 5 6 7
G.1 C.1 G.2 C.2 G.3 C.3 H
3.0 ± 0.3 3.0 ± 0.6 10.0 ±0.6 10.0 ± 1 14.0 ± 0.3 16.0 ± 0.3 -
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1.0± 0.3 2.0 ± 0.3 6.0 ± 0.6 8.0 ± 0.3 9.0± 0.3 11.0 ± 0.3 -
Fig 3. Zone of inhibition of HAP - ZnO nanocomposite at different ratio of HAP to ZnO for
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both green and chemical synthesis method of ZnO NPs, against clinical sample isolated a) S. aureus b) E.coli and c) pure HAP on both bacteria.
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3.4. MIC and MBC
After 24 hours of incubation under the aerobic condition at 37ºC, there was no turbidity noticed in all the test tubes having each of the bacteria, except for the test tube that contains
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pure HAP (100%). MIC and MBC results are summarized in Table 3 and shown in Fig 4. The nanocomposite has antimicrobial activity at the lowest concentration of ZnO NP that is 90:10
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(HAP to ZnO ratio) for both bacteria (S. aureus and E. coli). This indicates the MIC value of nanocomposite for both bacteria is 0.2 mg/mL of 90:10 (HAP to ZnO ratio) and the result has no significant difference between green and chemical methods of synthesis of ZnO NP. The MBC value was found to be 0.2 mg/mL of 75:25 (HAP to ZnO ratio) and 60:40 (HAP to ZnO ratio) for both S. aureus and E. coli respectively. In other words, 0.2 mg/mL of nanocomposite having 25% of ZnO NP and 75% HAP can kill S. aureus as well as 0.2 mg/mL of 40% of ZnO NP and 60 % of HAP can kill E. coli. The result showed that E. coli
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needs more concentration of ZnO NP to be eradicated from implant material. The MIC and MIB values between green and chemical synthesis of ZnO NP shows a similar outcome. Table 3. MIC and MBC values of the tested nanocomposites at 0.2 mg/mL concentration Sample names
S. aureus
E.coli
MIC
MBC
MIC
MBC
HAP to ZnO ratio
G1
90:10
×
×
C1
90:10
×
×
G2
75:25
×
C2
75:25
×
G3
60:40
C3
60:40
H
100
×
×
×
×
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= Represent the ratio is effective (yes) × = Represent the ratio that has no effect (no)
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Abbreviation
Fig 4. Figure showing test tubes containing different ratios of HAP-ZnO nanocomposite
for determining MIC and MBC value 3.5. Antibiofilm activity The clinical specimen isolated S. aureus and E. coli biofilm development inhibition ability of the HAP - ZnO nanocomposite was investigated using crystal violet staining, showed in Fig 13
5. The graphs showed (Fig 6) that all the examined ratio of HAP - ZnO concentrations inhibited the activity of biofilm formation at different levels of inhibition percentage. The nanocomposites, G1, and C1 which inhibited S. aureus biofilm development at 13% and 15%, respectively, E.coli biofilm development at 7.8% and 6.5%, respectively, were found to be a biofilm inhibition concentration (BIC) for both strains. The percentages of inhibition for G2 and C2 were 32% and 33% for S. aureus and 17.4% and 19.5% for E.coli. For the G3 composite, biofilm inhibitions were 52% and 54% for S. aureus and E.coli respectively, and for C3 inhibition of biofilm were 51% and 52% for S. aureus and E.coli biofilm respectively. As the concentration of ZnO nanoparticle increased the percentage (%) of inhibition also
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increased. The pure HAP (H) has no effect on biofilm development. The level of biofilm inhibition between the green and chemical methods of synthesis of ZnO was almost the same, there was no significant difference.
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Fig 5. Biofilm formation in microtiter plate. The effect of the nanocomposite on biofilm of (a) E.coli and (b) S. aureus
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PERCENT OF INHIBITION
G1 C1 G2 C2 G3 C3 H
60 50 40 30 20 10
A
B
HAP- ZnO CONCENTRATION
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Fig 6. Biofilm inhibition assay using different ratios of HAP to ZnO nanocomposite against clinical specimen isolated S. aureus (A) and E.coli (B)
4. DISCUSSION
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Nano-hydroxyapatite has been applied in orthopedic and maxillofacial surgery as replacements of bone graft, fillers and spacers [24, 25]. In recent times, there is great
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concern about complications caused by infection and biofilm formation on the surface of implanted biomaterial by bacteria and fungi [26, 27]. Nanoparticles made of metal oxide exhibit antimicrobial and antibiofilm property. Composite biomaterials should have an
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antimicrobial and antibiofilm activity to avoid implant failure because of bacterial infection [28, 29]. There are several suggested antibacterial mechanisms of ZnO nanoparticles mostly related to the large active surface area, bacterial cells are killed
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because of electrostatic interactions between ZnO NPs and cell walls [30].
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In this paper, nHAP was synthesized using the chemical co-precipitation method. ZnO NPs were prepared using chemical methods and green methods using the Coriandrum sativum plant. HAP - ZnO nanocomposite, having different ratios of nHAP to ZnO NPs was prepared by the liquid mixing method. Each of the nano particles and nanocomposite prepared was characterized by XRD and FESEM. The antimicrobial activity, MIC, MBC, and antibiofilm activity at a given ratio, for each nanocomposite was investigated against both clinical specimen isolated gram-positive (S. aureus) and gram-negative (E. coli) bacteria. Chemically and green synthesized ZnO and HAP were successfully prepared and were 15
mixed together to form the composite. The phase analysis was done to identify the phases present in the samples. The XRD patterns showed diffraction lines characteristic of ZnO, both present in standards and in other literature [31]. Single and pure phase ZnO powders were observed with the absence of any other impurity phase even in minor quantity. Therefore the calcination temperature was optimized at 500 °C. Similarly, all XRD peaks show diffraction lines characteristic of HAP, both present in standards and in literature [16]. It is observed from the phase analysis study of the composite that only the reactant phases, namely HAP and ZnO, and no other new reaction product phases for all the compositions. This indicates that both the reactant phases are chemically stable, and
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there is no reaction amongst them. Hence, all the compositions behave as a true composite.
It was observed from the antimicrobial and antibiofilm study, that all ratios of the nHAP: ZnO NPs in the nanocomposite applied in this study exhibited different levels of
antimicrobial and antibiofilm activity, shown in Table 2, Fig 3 and Fig 4. Related antibiotic
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activity of HAP: ZnO composite were reported by Saha et al. [32]. It was found that the
composite antimicrobial and antibiofilm activity might be because of the minimum amount of
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ZnO NPs used for the nanocomposite preparation and was effective at a given ratio. These results supported the previous report by Bhowmick et al. that, antimicrobial activity and
oxide was added [28].
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protein adsorption ability of nanocomposite were increased as nano-hydroxyapatite-zinc
As the ratio of ZnO NPs increases, a zone of inhibition and a percent of biofilm inhibition of
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nanocomposite increased for both bacteria. This is because of the antimicrobial and antibiofilm activity of ZnO NPs. This was in agreement with Vijayakumar & Vaseeharan [30] who stated that the zone size increased when the concentration of Cl-ZnO NPs was
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increased and it was also in agreement with Sangeetha et al. [33], who reported that, increasing the concentration of ZnO nanoparticles increased the bacterial inhibition
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consistently in wells of agar medium. The minimum concentration of ZnO NPs in the nanocomposite (G1 and C1) was more effective against S. aureus than E. coli. These results followed a similar trend where the better inhibition effect of organically modified montmorillonite clay supported chitosan/ hydroxyapatite-zinc oxide nanocomposites was observed against gram-positive bacteria compared to gram-negative bacteria [34]. Pure nano HAP did not show any effect on bacteria and its biofilm development as it has no antibiotic and antibiofilm activity, which is supporting the result by Sahithi, K., et al. [35]. It was found
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that nHAP acted like inert material thereby not supporting or inhibiting bacterial cell growth. Bhowmick et al. reported that the addition of ZnO NPs to the HAP significantly increased the human osteoblastic MG-63 cell proliferation on bone extracellular matrix. This shows that HAP: ZnO composite has no negative effect on human osteoblastic MG63 cells and has good cytocompatibility, signifying a positive prospect for successful bone regeneration [36].
5. CONCLUSION In conclusion, nanocomposite biomaterial having different ratios of nHAP to ZnO NPs were synthesized and characterized by powder XRD and FESEM. Antimicrobial and antibiofilm activity of each nanocomposite was investigated against clinical specimen isolated gram-
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positive S. aureus and gram-negative E. coli. As the ratio of ZnO NPs increases in the nanocomposite, good antimicrobial and antibiofilm properties were observed to prevent bacterial infection in orthopedic implants. The MIC against both bacteria was found by
adding 10% of ZnO NPs to 90% of nHAP at 0.2 mg/mL of nanocomposite and MBC the
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value was found to be 0.2 mg/mL of 75:25 (HAP to ZnO ratio) and 60:40 (HAP to ZnO ratio) for both S. aureus and E. coli respectively. The maximum biofilm inhibition was found by the
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addition of 40% ZnO NPs to 60% nHAP for both pathogens. This study is limited to the effect of the nanocomposite on bacteria and its biofilm; further studies are required to prepare nano composite scaffold for implant application and to know the effect of different ratio of
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Acknowledgements
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ZnO NPs in nHAP as well as its mechanical properties.
The authors thankfully acknowledge Dr. B.Jana Kram from the Department of Microbiology
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NRI Medical College and General Hospital, for providing the clinical specimen isolated S. aureus and E.coli cultures. We also thankfully acknowledge the extended support of Dr.
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Ritwik Sarkar, Professor of Department of Ceramic Engineering, National Institute of Technology, Rourkela during material characterization.
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PII:
S2352-4928(19)30318-6
DOI:
https://doi.org/10.1016/j.mtcomm.2019.100612
Article Number:
100612
Reference:
MTCOMM 100612
To appear in: Received Date:
9 June 2019
Revised Date:
19 August 2019
Accepted Date:
20 August 2019
Please cite this article as: Beyene Z, Ghosh R, Effect of Zinc Oxide Addition on Antimicrobial and Antibiofilm Activity of Hydroxyapatite: A Potential Nanocomposite for Biomedical Applications, Materials Today Communications (2019), doi: https://doi.org/10.1016/j.mtcomm.2019.100612
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Effect of Zinc Oxide Addition on Antimicrobial and Antibiofilm Activity of Hydroxyapatite: a Potential Nanocomposite for Biomedical Applications Zerihun Beyenea and Rupita Ghosha,b
a
Department of Biotechnology, Koneru Lakshmaiah Education Foundation, Green Fields, Vaddeswaram, Guntur District, Andhra Pradesh, India 522502 b
Corresponding Author : RUPITA GHOSH Email: [email protected] , [email protected]
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Tel No: +91 7008861886
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Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha, India 769008
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ABSTRACT
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Bacterial infections and biofilm formations are the main problems associated with implants. Hence, the aims of this study are to develop nanocomposite biomaterials having different ratios of hydroxyapatite nanoparticles (nHAP) with green and chemically synthesized zinc
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oxide (ZnO) nanoparticle (NP) and examine its antimicrobial and antibiofilm activity. The synthesized nanoparticles were characterized for phase and microstructural analysis. The nanocomposite at 90:10, 75:25 and 60:40 ratio of nHAP and ZnO NPs respectively, showed a
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different level of antimicrobial and antibiofilm activity against clinical specimen isolated gram-positive Staphylococcus aureus and gram-negative Escherichia coli. Moreover, the
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minimum inhibitory concentration (MIC) was the lowest concentration of ZnO NPs in the nanocomposite inhibiting the growth of each bacteria species and it was investigated for the given ratio of nanoparticles and found to be 0.2 mg/mL of 90:10 nanocomposite for both pathogens. The minimum bactericidal concentration (MBC) was the lowest concentration of ZnO NPs in the nanocomposite required to kill each of the bacterial species and it was found to be 0.2 mg/mL of 75:25 and 60:40 nanocomposite for S. aureus and E.coli respectively. The maximum percentage of biofilm inhibition was found at 60 (nHAP): 40 (ZnO NPs) ratio of the nanocomposites. It was 52% and 54% against S. aureus and E. coli biofilm 1
respectively, for green synthesized ZnO NPs and 51% and 52% against S. aureus and E. coli biofilm respectively, for chemically synthesized ZnO NPs. Hence, based on these results we suggest that the biomaterials containing different ratios of nHAP- ZnO NPs can be used as antimicrobial and antibiofilm materials in bone implant and bone regenerative medicine.
List of abbreviation Hydroxyapatite nanoparticles
ZnO NPs
Zinc oxide nanoparticles
CLSI
Clinical and Laboratory Standards Institute
MIC
Minimum inhibitory concentration
MBC
Minimum bactericidal concentration
TSB
Tryptone soya broth
FESEM
Field emission scanning electron microscope
JCPDS
Joint Committee on Powder Diffraction Standards
XRD
X-ray diffraction
FDA
Food and Drug Administration
GRAS
Generally Recognized As Safe
BIC
Biofilm inhibition concentration
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nHAP
Keywords: Nano-Hydroxyapatite; zinc oxide nanoparticle; antibiofilm; antimicrobial;
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minimum inhibitory concentration; minimum bactericidal concentration
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1. INTRODUCTION Hydroxyapatite (HAP, Ca10(PO4)6(OH)2) is known as a bioactive, biocompatible, and osteoconductive material chemically similar to the mineral component of bone and teeth [1]. HAP belongs to the calcium orthophosphates family, being the less soluble compound in its class in a physiological aqueous environment [1, 2]. Nano-hydroxyapatite (nHAP) has drawn great concern from researchers since they are widely applied as biomedical materials, including uses such as bone fillers, bone tissue engineering scaffolds, bioactive coatings, soft tissue repairs, drug, protein or gene loading, and delivery systems [3, 4]. 2
However, the application of HAP as biomedical materials is influenced by implant-associated infectious diseases and biofilm formation on the surface of the implant. Orthopedic implant surfaces provide suitable conditions for bacterial infection. As a result, biofilm formation is one of the most serious complications of implant surgery, and it always leads to severe physiological damage and additional costly surgical procedures [3, 5]. Pathogenic bacteria may colonize and adhere to surfaces of implanted biomaterials during the surgical process or from a hematogenous route, which is a key step in the pathogenesis of implant-associated infection [6, 7]. Based on several reports, Staphylococcus species and Escherichia coli are the most important pathogens involved in implant-associated
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infections [8]. Biofilm is a multicellular aggregation of bacteria covered with a hydrated extracellular
polymeric matrix of their own synthesis and characterized by cells that are permanently
attached to an interface with each other. Antibiotic susceptibility of bacteria embedded with a
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biofilm is less as a result, treatment requires higher doses of therapeutic agents. Therefore, the treatment of implant-associated infection using antibiotics fails to clear the pathogens.
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Revision surgery is often the only viable option [9, 10].
Different bacterial pathogens are responsible for the implant-associated infections, among
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them Staphylococcus aureus are the most common and responsible for more than 50% of infections. Other microorganisms include coagulase-negative staphylococci, streptococci, enterococci and gram-negative rods such as Escherichia coli, Pseudomonas spp and
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Enterobacter spp [11].
Zinc is an important mineral involved in biological activities such as cellular
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metabolism, DNA synthesis, and enzyme activity[12]. A zinc ion also exhibits antibacterial activity, improves biological properties, and decreases the inflammatory
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response. Moreover, the addition of Zinc in biomaterial for implant enhances osteogenesis by promoting osteoblast cell proliferation and differentiation [13]. Zinc oxide (ZnO) is listed as “Generally Recognized as Safe” (GRAS) by the FDA. Nano sized ZnO particles have antibacterial activities against a wide range of bacteria [14]. In order to prevent implant-associated infection and biofilm formation on the surface of the HAP implant, the implanted material should have an antimicrobial as well as antibiofilm properties [8]. Therefore, the aim of this study is to prepare and characterize HAP - ZnO nanocomposite at different HAP to ZnO ratio and investigate the in vitro antimicrobial and 3
antibiofilm activity of the nanocomposite against clinical specimen isolated S. aureus and E. coli bacteria. The effect of both the green and chemical synthesized ZnO nanoparticle used in the nanocomposites preparation was compared.
2. MATERIALS AND METHODS 2.1. Materials All of the chemicals used here were acquired from Merck and were of analytical grade, used without additional purification. The media purchased from Himedia Laboratories Pvt. Ltd. All experiments were performed using Double distilled water. The microorganisms, grampositive (Staphylococcus aureus) and gram-negative (Escherichia coli) were clinical
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specimen isolated bacteria, obtained from the Department of Microbiology, NRI Medical College and General Hospital, Chinakakani, Guntur, Andhra Pradesh, India. 2.2. Strains and growth condition
The microorganisms used in this work were Staphylococcus aureus and Escherichia coli,
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which are common causes of infection and biofilm formation in implanted biomaterials [15]. The bacteria were isolated from clinical specimens in the microbiology laboratory of the NRI
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Medical College and General Hospital. Inoculum of each bacterial spp was prepared by inoculating loop full bacteria from a colony into nutrient broth medium and mixed till a
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homogenous suspension was formed. The cultures were incubated at 37°C for 24h. Finally,
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bacterial numbers were adjusted at 0.5 McFarland turbidometry.
2.3. Nanoparticle synthesis 2.3.1. Hydroxyapatite
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HAP was prepared by chemical co-precipitation method using Ca(NO3)2·4H2O and (NH4)2HPO4 as starting materials and ammonia solution as agents for pH adjustment as
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described elsewhere [16]. In brief, an aqueous suspension of 0.24 M Ca(NO3)2·4H2O was prepared. Then a solution of (NH4)2HPO4 was slowly added dropwise to the Ca(NO3)2·4H2O solution maintaining Ca/P ratio of 1.67. The pH of the solution was maintained at 11 by dropwise addition of ammonia solution. This can be explained by the following reaction: 10𝐶𝑎(𝑁𝑂3 )2 . 4𝐻2 𝑂 + 6(𝑁𝐻4 )2 𝐻𝑃𝑂4 + 8𝑁𝐻4 𝑂𝐻 → 𝐶𝑎10 (𝑃𝑂4 )6 (𝑂𝐻)2 + 20𝑁𝐻4 𝑁𝑂3 + 46𝐻2 0
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The solution was continuously stirred for 2 hrs. Then the precipitated HAP was removed from the solution by centrifugation method at the rotation speed of 3000 rpm. The resulting powder was dried at 80°C in a hot air oven and then calcinated at 700°C for 2h in an electric furnace. 2.3.2. Zinc oxide nanoparticle preparation Green synthesis The synthesis of ZnO nanoparticle was carried out by mixing the plant extract (Coriandrum sativum) with aqueous zinc acetate dihydrate solution [Zn(CH3COO)2·2H2O]. The input parameters of the synthesis process were identified and optimized. Then the synthesis and
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characterization studies were carried out at optimum input conditions. Preparation of Plant Extract
The Coriandrum sativum plant leaf was used as a reducing and capping agent to synthesize
ZnO NPs as indicated elsewhere [17]. The fresh plants were purchased from the local market
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in Vijayawada, Andhra Pradesh, India. The surface of the plant leaves was cleaned using
distilled water to remove the minor organic contents. To get the plant extract, 10 grams of the
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leaf was added to 200 mL of distilled water and heated at 70°C for 30 minutes. The liquid extract was separated through filtration and then the extract was cooled at atmospheric temperature.
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Green synthesis of ZnO Nanoparticles
0.2M of Zn (CH3COO) 2·2H2O was prepared and the plant extract was added to the solution
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in the 1:1 ratio (v/v). The mixture was continuously stirred and heated at 70oC. Sodium hydroxide was added drop by drop to it till light yellow precipitate was formed. Then the precipitate was washed with distilled water and finally ethanol followed by drying in a hot air
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oven and finally calcinated at 500oC for 2 hours in an electric furnace.
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Synthesis of ZnO Nanoparticles by Chemical Precipitation Method ZnO NPs were synthesized by direct precipitation method using Zn(CH3COO)2·2H2O solution and potassium hydroxide (KOH) as precursors and a precipitating agent respectively as described elsewhere [18]. An aqueous solution of 0.2 M Zn(CH3COO)2·2H2O and 0.4 M KOH were prepared with deionized water. The KOH solution was slowly added into Zn(CH3COO)2·2H2O solution at room temperature under vigorous stirring. This resulted in the formation of a white suspension. The white product was centrifuged at 5000 rpm for 20
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min and washed three times with distilled water followed by absolute alcohol. The obtained product was calcinated at 500 °C for 3 h after drying in an oven. 2.4. HAP- ZnO nano-composite preparation HAP - ZnO nanocomposite was prepared by the liquid mixing method. Nano HAP powder suspension was mixed with ZnO nanoparticles powder suspension at a different ratio. The nHAP: ZnO NPs ratio was 90:10, 75:25 and 60:40 respectively. Ethanol was used as the organic solvent medium. The suspension was sonicated to dissolve the mixture completely. The formed nanocomposite was finally dried at 100°C in a hot air oven. The batches used for the process are given in Table 1. For each batch of the nanocomposite, 0.2 mg/ mL
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concentration was used for all testes in this research.
Table 1. The batch of nanocomposites at different HAP: ZnO ratio HAP content
ZnO content
Sample designation
1
90%
10% Green method
G.1
2
90%
10% Chemical method
3
75%
25% Green method
4
75%
25% Chemical method
5
60%
6
60%
7
100%
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40% Green method
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40% Chemical method
G.2 C.2 G.3 C.3 H
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0%
C.1
2.5. Phase Analysis
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The synthesized calcined HAP powders and both chemically and green synthesized ZnO nanoparticles phase analysis was done individually in an X-ray powder diffractometer (XRD) (Rigaku, Japan). The composite powders with varying ZnO content were also analyzed for phase confirmation and to observe the composite nature even after mixing. The diffractometer was fitted with a Ni filter of 0.154 nm and the diffraction patterns were measured using CuKα (λ=1.5418 Ǻ) radiation generated at a voltage of 40 kV and current of 40 mA. The samples were scanned in the interval 20˚<θ<50˚ at a scanning rate 20˚/min with a step size of 0.05˚ in a continuous mode. XRD analysis of the samples was performed using 6
X’pert High Score software in comparison with the reference powder diffraction data given by the Joint Committee on Powder Diffraction Standards (JCPDS). 2.6.
Microstructural study
The microstructural study to observe the morphology of the individual nanoparticles and composites were done in field emission scanning electron microscope (FESEM) (FEI, Nano Nova, Netherland). The samples were gold coated for 240 s in a sputter coater in an Ar atmosphere before loading in FESEM. The electrons at 15 kV sources were used to develop relevant information about the samples. Antimicrobial activity
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2.7.
The HAP-ZnO nanocomposite prepared at different nHAP to the ZnO NPs ratio was
examined for its antimicrobial activity. This was determined according to the Clinical and Laboratory Standards Institute (CLSI) procedure, using the agar diffusion test which was
performed using Müller-Hinton agar [19, 20]. The diffusion technique was carried out by
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pouring agar into Petri dishes to form 4 mm thick layers. After the agar was solidified the
medium were inoculated and spread with a bacterial suspension of approximately 1-2 X 108
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CFU/mL using spreader. The wells were prepared by punching with a 6 mm diameter standard sterile cork borer. These wells were filled up with 50 μL of the HAP-ZnO
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nanocomposite solution to be tested. Each concentration of the composite was tested for each culture in triplicates and the assay was performed twice. Antimicrobial activity against each bacterial spp (S. aureus and E. coli bacteria) was observed, and the diameters of the inhibition
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zones (mm) around the wells were measured after 24 and 48 h of incubation at 37°C. The lowest concentration that inhibited the growth was determined by a comparison among the
microbe.
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concentrations as well as between the green and chemical methods of synthesis for each
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2.8. MIC and MBC determination The MIC of the HAP-ZnO nanocomposite against clinical specimens isolate bacteria, that were S. aureus and E. coli were determined using a standard broth dilution method according to CLSI procedures [19, 20]. Tryptone soya broth (TSB) was used to determine MIC and MBC, as antibacterial assay media. For each of the nanocomposite, 0.2 mg/mL was used with adjusted bacterial concentration (0.10 at 625 nm (1×108 CFU/mL, 0.5 McFarland’s standard). The positive control used in this study was TSB containing only test bacterial concentrations and negative control contained only the sterile broth and incubated at 37ºC for 24 h. The 7
MIC was defined as the lowest concentration of ZnO NPs in the HAP-ZnO nanocomposite inhibiting the growth of each bacteria spp. The MIC was noted by the visual turbidity of the tubes both before and after incubation and it was done in triplicate to confirm its value for the tested bacteria. The minimum bactericidal concentration (MBC) is the lowest concentration of ZnO NPs in the nanocomposites required to kill a particular bacterium (MBC, mg/mL). It was determined by taking 100 µL of each sample from test tubes to remain clear (with no turbidity) in MIC and sub-cultured in test tubes containing 1 mL of fresh medium and incubated for 24 h at 37ºC. 2.9. Antibiofilm Activity
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The biofilm development inhibition ability of the HAP-ZnO nanocomposite at different
nHAP to ZnO NPs ratio was determined using polystyrene microtiter plate for both clinical
isolate S. aureus and E. coli as described previously [21, 22]. Pure colonies of S. aureus and E. coli isolates were aseptically picked up and used to inoculate sterile TSB, then incubated
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for 24 h at 37ºC. Overnight grown cultures were diluted in sterile TSB to match 0.5
McFarland turbidity standard which is equivalent to 1.5 × 108 CFU/mL. These bacterial
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suspensions were further diluted in the ratio 1:100 in TSB, which was supplemented with 2% (w/v) glucose and 2% (w/v) sodium chloride, and were mixed with nanocomposite (0.2 mg/
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mL) to be tested in different test tubes, for each of bacterial species. A total of 200 μL of these cell suspensions for each nanocomposite was transferred to each of three parallel wells of a 96-well plate. Wells having TSB (supplemented with 2% (w/v)
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glucose and 2% (w/v) sodium chloride) containing bacterial suspension without nanocomposite as positive control and without bacteria and nanocomposite as negative control were used. After incubation of the plate at 37oc for 24h, the content of each well was
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discarded and washed three times using 200 μL of phosphate buffer saline in order to remove non-adherent cells, subsequently, were dried in an inverted position. The adherent biofilm
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was fixed using 95% ethanol and stained with 100 μL/well of 1% crystal violet at room temperature for 5 minutes. After biofilm staining, the crystal violet solution was removed and the biofilms were washed three times with 200 μL distilled water. Subsequently the water was removed and the wells were dried for 2h. Finally, the adherent dye was dissolved using 30% acetic acid (S. aureus) and 20% acetone (E. coli). The optical density values of triplicate samples were determined using Microplate Reader. The percentage of biofilm inhibition was calculated using the following formula [23].
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% of Inhibition = (𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷570 𝑛𝑚 − 𝑡𝑒𝑠𝑡 𝑂𝐷𝑛𝑚 ⁄𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷570 𝑛𝑚 ) ∗ 100
3. RESULT 3.1. Phase Analysis The XRD patterns for individually synthesized powders of ZnO and HAP were shown in Fig 1 and also the HAP-ZnO nanocomposite with varying ZnO content. The characteristic diffraction peaks for both green synthesized (Fig 1a) and chemically synthesized (Fig 1b) ZnO were observed at position 2ɵ = 32.18°, 34.02°, 36.67°, 47.95° corresponds to (100), (002), (101) and (102) crystal planes respectively. It matches with the JCPDS card no. 750576. It had the hexagonal crystal system with a space group of P63mc. The three highest characteristic peaks for HAP (Fig 1c) were observed at position 25.97°, 31.87°, 33.02°
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corresponding to (002), (211) and (300) crystal planes respectively. It matches with the
JCPDS card no. 09-0432. It had the hexagonal crystal system with a space group of P63/m. The XRD pattern of HAP-ZnO nanocomposite, G1, G2, G3 where green synthesized ZnO
was used and C1, C2, C3 where chemically synthesized ZnO was used, are shown in Fig 1d-i
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respectively. Sharp characteristic peaks at 36.67°, corresponding to (101) crystal plane was observed for ZnO NPs. However, other diffraction peaks for ZnO NPs appeared at 32.18°,
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34.02° were overlapped with the characteristic diffraction peaks of nano-HAP at 31.87° and 34.17°. In addition, sharp peaks were observed for nano-HAP in the XRD patterns of -HAP-
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ZnO nanocomposite. The intensity of the ZnO peaks were found to increase with the increase
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in content for both the green and chemically synthesized ZnO in the HAP-ZnO composites.
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Fig 1. XRD pattern for (a) calcined green synthesized ZnO, (b) calcined chemically synthesized ZnO, (c) calcined HAP, (d) HAP-ZnO nanocomposite (90:10), (e) HAP-ZnO
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nanocomposite (75:25), (f) HAP-ZnO nanocomposite (60:40) using green synthesized ZnO and (g) HAP-ZnO nanocomposite (90:10), (g) HAP-ZnO nanocomposite (75:25), (g) HAP-
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ZnO nanocomposite (60:40) using chemically synthesized ZnO. 3.2. Microstructural study
The micrograph of the chemically precipitated calcined HAP, chemically and green synthesized calcined ZnO powders, are shown in Fig 2. The microstructure as observed under the microscope showed the shape and particle size of the powders. The ZnO nanosized particles were found to be agglomerated. It was observed that the shape of both the green (Fig 2a) and chemically synthesized (Fig 2b) ZnO nanoparticles was more or less spherical. Though very few elongated grains were noticed for the chemically synthesized 10
ZnO NPs, the particle size of the chemically synthesized powder was found to be much larger than that of the green synthesized powder. The 700 °C calcined HAP particles (Fig 2c) are merely spherical and non-uniform in shape. The range for the particle size of the
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synthesized powder was found to be within the nano range.
Fig 2. Micrograph showing (a) chemically synthesized ZnO, (b) green synthesized ZnO, (c)
3.3.
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chemically coprecipitated calcined HAP nanoparticles Antimicrobial activities
The test result of the HAP - ZnO nanocomposite showed that at the given ratio, the
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nanocomposite has an antimicrobial effect against both gram-positive (S. aureus) and gramnegative (E. coli). Pure HAP (100%) did not show any antibacterial activity against both
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bacteria, as shown in Table 2 and Fig 3. All the tested concentrations of nanocomposite showed antimicrobial activities against both bacteria (Fig 3), S. aureus is being more sensitive to the minimum concentration of ZnO NPs than E. coli as presented in Table 2.
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The zone of inhibition was different between pathogens and the zone size increased when the ratio of ZnO NPs was increased in the nanocomposite for each bacteria species. The
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comparison between green and chemical methods of ZnO NP showed almost the same level of antimicrobial activity with no significant difference. The intensity of antibacterial activity
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of the nanocomposite was dependent on the concentration of ZnO NP (Table 2).
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Table 2. Diameter of zone of inhibition by different ratio of HAP-ZnO nanocomposite against S. aureus and E.coli. No
Sample
Inhibition zone diameter in mm S. aureus E.coli
1 2 3 4 5 6 7
G.1 C.1 G.2 C.2 G.3 C.3 H
3.0 ± 0.3 3.0 ± 0.6 10.0 ±0.6 10.0 ± 1 14.0 ± 0.3 16.0 ± 0.3 -
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1.0± 0.3 2.0 ± 0.3 6.0 ± 0.6 8.0 ± 0.3 9.0± 0.3 11.0 ± 0.3 -
Fig 3. Zone of inhibition of HAP - ZnO nanocomposite at different ratio of HAP to ZnO for
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both green and chemical synthesis method of ZnO NPs, against clinical sample isolated a) S. aureus b) E.coli and c) pure HAP on both bacteria.
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3.4. MIC and MBC
After 24 hours of incubation under the aerobic condition at 37ºC, there was no turbidity noticed in all the test tubes having each of the bacteria, except for the test tube that contains
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pure HAP (100%). MIC and MBC results are summarized in Table 3 and shown in Fig 4. The nanocomposite has antimicrobial activity at the lowest concentration of ZnO NP that is 90:10
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(HAP to ZnO ratio) for both bacteria (S. aureus and E. coli). This indicates the MIC value of nanocomposite for both bacteria is 0.2 mg/mL of 90:10 (HAP to ZnO ratio) and the result has no significant difference between green and chemical methods of synthesis of ZnO NP. The MBC value was found to be 0.2 mg/mL of 75:25 (HAP to ZnO ratio) and 60:40 (HAP to ZnO ratio) for both S. aureus and E. coli respectively. In other words, 0.2 mg/mL of nanocomposite having 25% of ZnO NP and 75% HAP can kill S. aureus as well as 0.2 mg/mL of 40% of ZnO NP and 60 % of HAP can kill E. coli. The result showed that E. coli
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needs more concentration of ZnO NP to be eradicated from implant material. The MIC and MIB values between green and chemical synthesis of ZnO NP shows a similar outcome. Table 3. MIC and MBC values of the tested nanocomposites at 0.2 mg/mL concentration Sample names
S. aureus
E.coli
MIC
MBC
MIC
MBC
HAP to ZnO ratio
G1
90:10
×
×
C1
90:10
×
×
G2
75:25
×
C2
75:25
×
G3
60:40
C3
60:40
H
100
×
×
×
×
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lP
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= Represent the ratio is effective (yes) × = Represent the ratio that has no effect (no)
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Abbreviation
Fig 4. Figure showing test tubes containing different ratios of HAP-ZnO nanocomposite
for determining MIC and MBC value 3.5. Antibiofilm activity The clinical specimen isolated S. aureus and E. coli biofilm development inhibition ability of the HAP - ZnO nanocomposite was investigated using crystal violet staining, showed in Fig 13
5. The graphs showed (Fig 6) that all the examined ratio of HAP - ZnO concentrations inhibited the activity of biofilm formation at different levels of inhibition percentage. The nanocomposites, G1, and C1 which inhibited S. aureus biofilm development at 13% and 15%, respectively, E.coli biofilm development at 7.8% and 6.5%, respectively, were found to be a biofilm inhibition concentration (BIC) for both strains. The percentages of inhibition for G2 and C2 were 32% and 33% for S. aureus and 17.4% and 19.5% for E.coli. For the G3 composite, biofilm inhibitions were 52% and 54% for S. aureus and E.coli respectively, and for C3 inhibition of biofilm were 51% and 52% for S. aureus and E.coli biofilm respectively. As the concentration of ZnO nanoparticle increased the percentage (%) of inhibition also
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increased. The pure HAP (H) has no effect on biofilm development. The level of biofilm inhibition between the green and chemical methods of synthesis of ZnO was almost the same, there was no significant difference.
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Fig 5. Biofilm formation in microtiter plate. The effect of the nanocomposite on biofilm of (a) E.coli and (b) S. aureus
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PERCENT OF INHIBITION
G1 C1 G2 C2 G3 C3 H
60 50 40 30 20 10
A
B
HAP- ZnO CONCENTRATION
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0
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Fig 6. Biofilm inhibition assay using different ratios of HAP to ZnO nanocomposite against clinical specimen isolated S. aureus (A) and E.coli (B)
4. DISCUSSION
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Nano-hydroxyapatite has been applied in orthopedic and maxillofacial surgery as replacements of bone graft, fillers and spacers [24, 25]. In recent times, there is great
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concern about complications caused by infection and biofilm formation on the surface of implanted biomaterial by bacteria and fungi [26, 27]. Nanoparticles made of metal oxide exhibit antimicrobial and antibiofilm property. Composite biomaterials should have an
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antimicrobial and antibiofilm activity to avoid implant failure because of bacterial infection [28, 29]. There are several suggested antibacterial mechanisms of ZnO nanoparticles mostly related to the large active surface area, bacterial cells are killed
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because of electrostatic interactions between ZnO NPs and cell walls [30].
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In this paper, nHAP was synthesized using the chemical co-precipitation method. ZnO NPs were prepared using chemical methods and green methods using the Coriandrum sativum plant. HAP - ZnO nanocomposite, having different ratios of nHAP to ZnO NPs was prepared by the liquid mixing method. Each of the nano particles and nanocomposite prepared was characterized by XRD and FESEM. The antimicrobial activity, MIC, MBC, and antibiofilm activity at a given ratio, for each nanocomposite was investigated against both clinical specimen isolated gram-positive (S. aureus) and gram-negative (E. coli) bacteria. Chemically and green synthesized ZnO and HAP were successfully prepared and were 15
mixed together to form the composite. The phase analysis was done to identify the phases present in the samples. The XRD patterns showed diffraction lines characteristic of ZnO, both present in standards and in other literature [31]. Single and pure phase ZnO powders were observed with the absence of any other impurity phase even in minor quantity. Therefore the calcination temperature was optimized at 500 °C. Similarly, all XRD peaks show diffraction lines characteristic of HAP, both present in standards and in literature [16]. It is observed from the phase analysis study of the composite that only the reactant phases, namely HAP and ZnO, and no other new reaction product phases for all the compositions. This indicates that both the reactant phases are chemically stable, and
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there is no reaction amongst them. Hence, all the compositions behave as a true composite.
It was observed from the antimicrobial and antibiofilm study, that all ratios of the nHAP: ZnO NPs in the nanocomposite applied in this study exhibited different levels of
antimicrobial and antibiofilm activity, shown in Table 2, Fig 3 and Fig 4. Related antibiotic
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activity of HAP: ZnO composite were reported by Saha et al. [32]. It was found that the
composite antimicrobial and antibiofilm activity might be because of the minimum amount of
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ZnO NPs used for the nanocomposite preparation and was effective at a given ratio. These results supported the previous report by Bhowmick et al. that, antimicrobial activity and
oxide was added [28].
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protein adsorption ability of nanocomposite were increased as nano-hydroxyapatite-zinc
As the ratio of ZnO NPs increases, a zone of inhibition and a percent of biofilm inhibition of
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nanocomposite increased for both bacteria. This is because of the antimicrobial and antibiofilm activity of ZnO NPs. This was in agreement with Vijayakumar & Vaseeharan [30] who stated that the zone size increased when the concentration of Cl-ZnO NPs was
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increased and it was also in agreement with Sangeetha et al. [33], who reported that, increasing the concentration of ZnO nanoparticles increased the bacterial inhibition
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consistently in wells of agar medium. The minimum concentration of ZnO NPs in the nanocomposite (G1 and C1) was more effective against S. aureus than E. coli. These results followed a similar trend where the better inhibition effect of organically modified montmorillonite clay supported chitosan/ hydroxyapatite-zinc oxide nanocomposites was observed against gram-positive bacteria compared to gram-negative bacteria [34]. Pure nano HAP did not show any effect on bacteria and its biofilm development as it has no antibiotic and antibiofilm activity, which is supporting the result by Sahithi, K., et al. [35]. It was found
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that nHAP acted like inert material thereby not supporting or inhibiting bacterial cell growth. Bhowmick et al. reported that the addition of ZnO NPs to the HAP significantly increased the human osteoblastic MG-63 cell proliferation on bone extracellular matrix. This shows that HAP: ZnO composite has no negative effect on human osteoblastic MG63 cells and has good cytocompatibility, signifying a positive prospect for successful bone regeneration [36].
5. CONCLUSION In conclusion, nanocomposite biomaterial having different ratios of nHAP to ZnO NPs were synthesized and characterized by powder XRD and FESEM. Antimicrobial and antibiofilm activity of each nanocomposite was investigated against clinical specimen isolated gram-
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positive S. aureus and gram-negative E. coli. As the ratio of ZnO NPs increases in the nanocomposite, good antimicrobial and antibiofilm properties were observed to prevent bacterial infection in orthopedic implants. The MIC against both bacteria was found by
adding 10% of ZnO NPs to 90% of nHAP at 0.2 mg/mL of nanocomposite and MBC the
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value was found to be 0.2 mg/mL of 75:25 (HAP to ZnO ratio) and 60:40 (HAP to ZnO ratio) for both S. aureus and E. coli respectively. The maximum biofilm inhibition was found by the
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addition of 40% ZnO NPs to 60% nHAP for both pathogens. This study is limited to the effect of the nanocomposite on bacteria and its biofilm; further studies are required to prepare nano composite scaffold for implant application and to know the effect of different ratio of
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Acknowledgements
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ZnO NPs in nHAP as well as its mechanical properties.
The authors thankfully acknowledge Dr. B.Jana Kram from the Department of Microbiology
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NRI Medical College and General Hospital, for providing the clinical specimen isolated S. aureus and E.coli cultures. We also thankfully acknowledge the extended support of Dr.
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Ritwik Sarkar, Professor of Department of Ceramic Engineering, National Institute of Technology, Rourkela during material characterization.
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