- Email: [email protected]
Synthesis and characterization of ZrO2 nanoparticles-antimicrobial activity and their prospective role in dental care
Accepted Manuscript Synthesis and characterization of ZrO2 nanoparticles-antimicrobial activity and their prospective role in dental care John Bani Fathima, Arivalagan Pugazhendhi, Rose Venis PII:
S0882-4010(17)30631-9
DOI:
10.1016/j.micpath.2017.06.039
Reference:
YMPAT 2331
To appear in:
Microbial Pathogenesis
Received Date: 31 May 2017 Revised Date:
26 June 2017
Accepted Date: 26 June 2017
Please cite this article as: Fathima JB, Pugazhendhi A, Venis R, Synthesis and characterization of ZrO2 nanoparticles-antimicrobial activity and their prospective role in dental care, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.06.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Synthesis and characterization of ZrO2 nanoparticles-antimicrobial activity and their
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prospective role in dental care
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John Bani Fathima 1, Arivalagan Pugazhendhi 2, Rose Venis 1,*
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2 Green Processing, Bioremediation and Alternative Energies Research Group (GPBAE),
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Faculty of Environment and Labour Safety, Ton Duc Thang University (TDTU), Ho Chi Minh
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City, Vietnam. Email: [email protected]
* Corresponding Author Address Rose Venis, Ph.D
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Associate Professor
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Department of Chemistry
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St. Joseph’s College
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Tiruchirappalli
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Tamil Nadu, India.
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Tel: +91-9443115762
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Email id: [email protected]
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Department of Chemistry, St. Joseph’s College, Tiruchirappalli, Tamil Nadu, India.
Abstract
Nanomaterials are exerting a pull on deal with biological and pharmaceutical
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applications. Biomedical grade of zirconia reveals potential mechanical features of oxide
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ceramics. In this study, antimicrobial activity and anti-tooth decay applications of the
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synthesized NPs of ZrO2 were determined. The as-prepared ZrO2 NPs were characterized by UV-
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vis spectroscopy, FTIR and XRD, which determined the formation of ZrO2NPs and their
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crystalline nature. SEM analysis further revealed spherical shaped NPs and TEM analysis
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determined the size of the particles in the range of 15-21 nm, respectively. The antimicrobial
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activity of different concentrations of the synthesized ZrO2NPs was examined against gram
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positive bacteria (Bacillus subtilis and Staphylococcus aureus), gram negative bacteria
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(Escherichia coli and Pseudomonas aeruginosa), respectively. The synthesized ZrO2NPs
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displayed a better inhibitory action against Pseudomonas aeruginosa (inhibition zone size of 20
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mm) at the concentration of 100 µg/ml compared to other bacteria due to the negatively charged
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P.aeruginosa cell wall readily attracting positively charged ZrO2NPs and thereby inhibiting
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microbial actions. Moreover, the concentration of ZrO2NPs was directly proportional to their
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inhibitory actions against the tested microorganisms. Finally, the preventive role of ZrO2NPs in a
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tooth decay pathway has been elucidated. Hence, it could be concluded that the as-prepared
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ZrO2NPs possess viable biomedical applications.
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Keywords: Nanoparticles; ZrO2; Characterization; Antimicrobial Activity; Dental care.
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1. Introduction
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Nanomaterials may easily interact with humans through digestive system (digestion),
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respiratory tract (inhalation), circulatory system (blood and lymph) via injection, skin contact by
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cosmetic creams and ointments. Due to their smaller sizes, nanoparticles would easily penetrate
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into cell membrane and may reach biological system and therefore, they can easily engulf into
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living organisms and cause cellular dysfunction [1-3].
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Recent developments in the field of nanotechnology has paved way for the successful
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fabrication, characterization and alteration of the functional properties of nanoparticles for 2
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biomedical applications [4-6]. Several nanoparticles, including metal nanoparticles [7-11], own
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distinctive properties which are employed for several applications in the nanobiomedicine. It has
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been well established that nanoparticles embrace an unbelievable potential in a variety of
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biomedical applications such as successful drug delivery systems [12,13]. Though nanoparticles
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endow vital applications, researchers are facing some disadvantages due to lack of proper
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knowledge about the effects of nanoparticles on biochemical pathways and metabolic actions of
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human body. Reasonably, safety and toxicity of nanomaterials has become a questionable of
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curiosity to the public. For that reason, understanding the communications of nanomaterials with
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biological systems is mandatory for most significant scientific issues [14-17].
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Zirconium is a sturdy transition metal that bears a resemblance to titanium due to its
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strong resistance to corrosion and biomedical grade zirconia may be the one for which there is
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the largest debate among scientists, industrials or clinicians. It is widely used as an implant
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biomaterial [18], dental crown [19], femoral heads for total hip replacement [20], solid oxide fuel
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cell electrolytes [21], catalytic activity [21,22], high chemical stability and ionic conductivity
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[22,23].
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For more than two decades, biomedical grade of Zirconia was used to solve the problem
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of alumina brittleness and the consequent potential failure of implants. Nowadays, more than
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600,000 of zirconia femoral heads have been implanted worldwide, especially in the US and in
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Europe. Biomedical grade of zirconia reveals the potential mechanical features of oxide
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ceramics. It is involved in phase transformation toughening, which increases its crack breeding
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resistance [24]. Furthermore, ZrO2 have wide applications due to outstanding biocompatibility,
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increasing strength, and low wear cost, but in biomedical industries there is no clear information
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about metabolic interactions and toxicity of zirconium.
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Bacterial infections are the main reasons for chronic infections and mortality. Antibiotics
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are recommended for bacterial infectivity as they are economic and effective as well [25].
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Antibiotics directly impair the cell wall synthesis, translational machinery and DNA replication
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processes of bacteria. Bacterial resistance to antibiotics inhibits the aforementioned mechanisms
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via expression of relevant enzymes [26-28]. Previously it has been reported that zirconia based
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ceramics are chemically inert materials. They allow excellent cell adhesion, and no adverse
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systemic reactions have been related [29]. Hence, novel substances which could attach to the
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bacterial cells without penetrating into the cell are needed to overcome the antibiotic resistance.
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It is recognized that nanoparticles could perform the required action clearly [30].
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Hence, the present study has focused on biogenic synthesis of ZrO2NPs and promoting
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their biological applications with special emphasis on antimicrobial activities. The prepared
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ZrO2NPs were characterized by UV-vis, FTIR, XRD, TEM and analysis.
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2. Materials and methods
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2.1 Equipments
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UV-vis spectrophotometer (Varian, Carry 5000) spectrophotometer was used to measure
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absorption with 1-cm path length quartz cuvettes, FTIR spectrophotometer (Thermo Nicolet,
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Avatar 370) was employed for embedded function group identification and Bruker AXS
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Advance powder X-ray diffractometer was used for characterization of crystalline nature of
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sample. Morphological analyses were carried out using Scanning Electron Microscopy (SEM)
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with a JEOL Model JSM - 6390LV instrument and Jeol/JEM 2100 High Resolution
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Transmission Electron Microscope.
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2.2 Chemicals and microbial cultures Zirconium nitrate hexahydrate (Zr(NO3)4.5H2O, and urea were purchased from HiMedia.
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All the chemicals used in this work were of analytical grades. Milli-Q distilled water was used
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for nanoparticle synthesis. Gram positive (Bacillus subtilis MTCC 1305, Staphylococcus aureus
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MTCC 3160) and gram negative bacteria (E. coli, P. aeruginosa MTCC 2453) were procured
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from Microbial Type Culture Collection (MTCC) and used as test organisms. All the cultures
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were stored and maintained at 4◦C.
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2.3 Synthesis of ZrO2 nanoparticles
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Synthesis of ZrO2NPs was carried out as follows: 0.5 mmol of Zr(NO3)4.5H2O and a
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trivial quantity of urea (CO(NH2)2 were dissolved in 70 ml of deionized water and the mixture
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was continuously stirred. Then, the homogenous solution was transferred into a 100 ml conical
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flask, teflon lined and autoclaved for 12 h at 180º C. Once the process was completed, the
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solution was shifted to room temperature and allowed to cool. Finally, a white precipitate was
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formed. The precipitate was washed numerous times with plenty of distilled water and absolute
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ethanol and subsequently dried overnight at 353 K. Eventually, the white precipitate was claimed
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in air at 450ºC for 15 h.
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2.4 Characterization of ZrO2 nanoparticles The morphology and the size of the ZrO2NPs were characterized by Scanning Electron
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Microscopy (SEM). ZrO2 NPs size, morphology and distribution were examined by the High
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Resolution Transmission Electron Microscope. UV-vis spectrophotometer was used to measure
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the absorption spectra of the synthesized nanoparticles in the range of 175-3300 nm. The
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readings were taken at intervals of 1nm with the scan rate 600 nm/min. The as-prepared
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nanoparticles were subjected to functional group analysis using FTIR spectrophotometer.
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The samples were prepared using KBr pellet method in 1:99 ratios at room temperature.
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The samples were scanned in the spectral range of 4000-400 cm-1 with the resolution of 2 cm-1.
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X-ray diffraction (XRD) patterns were collected using a powder X-ray diffractometer. The XRD
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pattern of synthesized sample was obtained inside the special XRD cell designed to avoid the
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reaction of air sensitive samples with atmospheric oxygen [31].
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2.5 Antibacterial assay of ZrO2 nanoparticles
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The antimicrobial activity of the ZrO2NPs was evaluated with well disc diffusion method
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[32]. The experiment was conducted against reference gram positive (Bacillus subtilis and
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Staphylococcus aureus) and gram negative bacteria (E. coli and P. aeruginosa) procured from
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Microbial Type Culture Collection (MTCC), Chandigarh, India. An inoculum size of 105 cells/ml
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was used to spread on the Mueller-Hinton Agar (MHA, HiMedia, India). In Brief, 20 ml of MHA
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was poured into petri dishes and allowed to solidify. Then, 6mm thick sterile discs were placed
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appropriately on petridishes. Finally, different concentrations of ZrO2Nps (20, 40, 80 and 100
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µg/ml) were loaded on each disc and 50% ethanol was used as a negative control. All the plates
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were incubated at 37 °C for 24 h and the respective inhibition zones were measured. Each test
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was performed in triplicates under the same set of conditions for reproducibility [33].
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3. Results and discussion
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3.1 UV–Vis and FTIR absorption spectra
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Fig. 1 represented the UV-vis absorption spectrum for the synthesis of ZrO2NPs in the
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scan range of 175 – 3300 nm. In this study the formation of ZrO2NPs was confirmed through the
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appearance of milky white precipitate. The milky colloidal solution indicated the conversion of
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Zr(NO3)4.5H2O into nanosized ZrO2 colloidal particles [34]. Further, their physical properties
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were examined using UV-Visible spectroscopy and FTIR techniques. The synthesis of nanosized
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ZrO2 colloidal particles was thus confirmed with the absorption spectra at 300 nm and 824 nm
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(Fig. 1). The UV-vis peaks denoted the transition of an inner shell electron to conduction.
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Various forms of Zr including ions, atoms or clusters strongly interacted with water molecules to
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form ZrO2, which was quickly quenched in liquid solution to form nanoparticles [34].
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The FTIR spectrum was used to establish the functional groups of nanoparticles. Fig. 2
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represents the FTIR absorption spectrum of the as-prepared nanoparticles. Spectrum of ZrO2NPs
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had shown peaks at 3428.96 cm-1 due to OH stretching, at 1634.05 cm-1 because of OH bending
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and at 492.73 cm-1 denoting Zr-O band, respectively. Based on the fingerprint characters of the
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peak positions, shapes and intensities along with the essential components in the materials were
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observed.
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exposed cluster appearance with crystal nature was indicated. However, spherical structures of
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size less than 20 nm with irregular surface morphologies denoted an increased grain size due to
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the increase in temperature leading to crystalline as well as grain growth. The stony appearance
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might have been due to the agglomeration of ZrO2NPs of size 20–50 nm [35].
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Fig. 4 shows the TEM reflection and size allocation of ZrO2NPs, which were a mixture of
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diverse sizes and shapes. The particles were almost spherical or irregular spherical with particle
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size ranging between 15 nm to 21 nm. For instance, the diameters of ZrO2NPs were observed to
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be 15.77 nm, 20.76 nm and 21.54 nm, respectively.
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XRD is of great importance in the microstructure characterization of complex, multiphase
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and single phase materials. The application of XRD enables not only qualitative and quantitative
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phase analysis but also microstructure characterization (crystallite size, lattice distortions and
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dislocation densities, stacking faults and twins probability). In the XRD pattern of ZrO2Nps, the
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diffraction peaks were observed at 2?? values of 28.9º, 60.13º and 62.7º corresponding to the
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characteristic diffraction planes (1 1 1), (3 0 2) and (3 1 1) of ZrO2, respectively. The well-
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known peaks have been exploited to estimate the grain size of sample. Grain size estimated using
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the relative intensity peak for ZrO2Nps was found to be above 20 nm and enhancement in the
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sharpness of XRD peaks denoted that the particles were crystalline in nature. All diverse peaks in
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Fig. 5 were associated to ZrO2Nps and harmonized to Joint Committee for Powder Diffraction
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Studies (JCPDS) [36].
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3.3 Antibacterial activity
Nanomaterials reveal strong inhibiting effects towards a broadened spectrum of bacterial
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strains. As per various research understandings, the metal oxides transmit the positive charge
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while the microorganisms hold negative charge. Hence, electromagnetic attractions between
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microorganisms and metal oxides lead to oxidization and finally result in the death of
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microorganisms [37].
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Antimicrobial activity of nanoparticles was determined by disc diffusion method against
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E. coli, P. aeruginosa, S. aureus and B. subtilis as shown in Fig. 6. The results show that
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ZrO2NPs had potential inhibitory action against P. aeruginosa and S. aureus and a good
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inhibition against E. coli while there was no action against B. subtilis (Fig. 7). It was also
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observed that the zone of inhibition values increased linearly with the increase in concentration
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of the nanoparticles, which was in accordance with the previous research reports [13, 38]. In
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another study, MIC values of silver nanoparticles against the chosen dental caries and
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periodontal disease microorganisms were obvious in the range of 25–75 µg/mL [12]. A better
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antibacterial against gram negative bacteria may due to the negatively charged cell wall of
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bacteria being ruptured by positively charged zirconium ions from zirconium nanoparticles and
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finally ensuing in cell necrosis. The similar understanding was witnessed for silver nanoparticles
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[12]. It has also been proposed that decrease in intracellular ATP levels could lead to
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destabilization in the outer and plasma membranes. Moreover, membrane proteins may also have
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been affected leading to cellular damage and eventually, cell death [13, 39]. In a previous study,
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reported by Banerjee et al. [40], ZrO2 nanostructures showed antibacterial activity against only E.
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coli bacteria and no activity for S. aureus and fungus, while the complexes showed antibacterial
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activity against both gram positive and negative bacteria, i.e., S. aureus and E. coli. Thus,
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ZrO2NPs proved to be a potential antibacterial agent against both gram positive and gram
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negative microorganisms though revealed to be most effective against gram negative bacteria.
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3.4 Role of ZrO2NPs in dental care
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The accumulation of bacteria on teeth produces acid, which stimulates corrosion on the
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teeth surfaces. This process is known as tooth decay or dental caries. Various bacteria
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accumulate on teeth, gums and tongue, and produce plague on teeth ie supragingival plaque.
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Once bacteria accumulate on teeth and interact with food containing sugars, acid is produced,
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which is known as demineralization. If acid penetrates on teeth, enamel is dissolved ending up
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in cavity formation.
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It has been previously established that silver nanoparticles could easily approach to the
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target site and deliver the drug molecule to the hydrophobic surface of the bacterial cell and
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could interfere with the bacterial growth signaling pathway. In this context, coupling of AgNPs
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with drugs (Azithromycin and Clarithromycin) divulged potential activity against dental carries
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[22].
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Zirconia material is used as ceramic surface of metal implants and it aids to improve
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biocompatibility in the oral cavity but exposure of fibro integration of implants has been
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observed by histological examination [41]. With this understanding, the as-prepared ZrO2NPs
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may probably overcome the above noted issues and provide first-class protection to the teeth.
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ZrO2NPs would bind on the surface of bacteria and stop the metabolic action with food, thereby
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preventing the synthesis of acid and enamel corrosion. Thus, the outer coating of ZrO2NPs to
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teeth provides external strength to the teeth and increases their lifespan as shown in Fig. 8.
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Conclusion
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The present study has illustrated the synthesis of ZrO2NPs and their subsequent
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characterization using UV-vis spectroscopy, FTIR, XRD and SEM.
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indicated the transformation of ZrO2 into ZrO2 NPs which was also confirmed through functional
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group and bonding of nanosized colloidal particles by FTIR. ZrO2NPs were of crystalline nature
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and of size 15 nm – 21 nm as identified by XRD, SEM and TEM analysis. The antimicrobial
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The UV-visible peaks
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activity of ZrO2NPs observed against gram positive and gram negative bacteria revealed that
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ZrO2NPs had potential inhibitory action against
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aeruginosa at higher concentrations due to their negatively charged cell surfaces. In this
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context, the feasibility of exploiting ZrO2NPs in averting tooth decay has been elucidated via
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comprehending the tooth decay pathway. As the conclusion of this research, ZrO2NPs could be
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recommended for applications in dental care and other relevant biomedical applications upon
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conducting further in vitro and in vivo studies, respectively.
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Figure legends
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Fig. 1. UV-Vis spectrum of ZrO2NPs
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Fig. 2. FTIR spectrum of ZrO2NPs
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Fig. 3. SEM micrograph of ZrO2NPs
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Fig. 4. TEM and size distribution ZrO2NPs
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Fig. 5. XRD patterns of ZrO2NPs
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Fig. 6. Antibacterial effects of ZrO2NPs
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Fig. 7. Antibacterial activity of ZrO2NPs determined by disc diffusion method S- Standard (100
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µg/ml), C- Control (100 µg/ml), 1- ZrO2 NPs (100 µg/ml), 2- ZrO2NPs (80 µg/ml), 3- ZrO2NPs (40 µg/ml) and 4- ZrO2NPs (20 µg/ml).
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Fig. 8. Role of ZrO2NPs in dental care
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Highlights
ZrO2NPs - antimicrobial activity and anti-tooth decay applications were determined
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ZrO2NPs was examined against various gram positive bacteria and gram negative
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bacteria.
ZrO2NPs in averting tooth decay has been elucidated via comprehending the tooth decay
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pathway.
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S0882-4010(17)30631-9
DOI:
10.1016/j.micpath.2017.06.039
Reference:
YMPAT 2331
To appear in:
Microbial Pathogenesis
Received Date: 31 May 2017 Revised Date:
26 June 2017
Accepted Date: 26 June 2017
Please cite this article as: Fathima JB, Pugazhendhi A, Venis R, Synthesis and characterization of ZrO2 nanoparticles-antimicrobial activity and their prospective role in dental care, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.06.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Synthesis and characterization of ZrO2 nanoparticles-antimicrobial activity and their
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prospective role in dental care
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John Bani Fathima 1, Arivalagan Pugazhendhi 2, Rose Venis 1,*
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2 Green Processing, Bioremediation and Alternative Energies Research Group (GPBAE),
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Faculty of Environment and Labour Safety, Ton Duc Thang University (TDTU), Ho Chi Minh
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City, Vietnam. Email: [email protected]
* Corresponding Author Address Rose Venis, Ph.D
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Associate Professor
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Department of Chemistry
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St. Joseph’s College
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Tiruchirappalli
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Tamil Nadu, India.
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Tel: +91-9443115762
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Email id: [email protected]
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Department of Chemistry, St. Joseph’s College, Tiruchirappalli, Tamil Nadu, India.
Abstract
Nanomaterials are exerting a pull on deal with biological and pharmaceutical
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applications. Biomedical grade of zirconia reveals potential mechanical features of oxide
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ceramics. In this study, antimicrobial activity and anti-tooth decay applications of the
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synthesized NPs of ZrO2 were determined. The as-prepared ZrO2 NPs were characterized by UV-
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vis spectroscopy, FTIR and XRD, which determined the formation of ZrO2NPs and their
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crystalline nature. SEM analysis further revealed spherical shaped NPs and TEM analysis
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determined the size of the particles in the range of 15-21 nm, respectively. The antimicrobial
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activity of different concentrations of the synthesized ZrO2NPs was examined against gram
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positive bacteria (Bacillus subtilis and Staphylococcus aureus), gram negative bacteria
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(Escherichia coli and Pseudomonas aeruginosa), respectively. The synthesized ZrO2NPs
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displayed a better inhibitory action against Pseudomonas aeruginosa (inhibition zone size of 20
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mm) at the concentration of 100 µg/ml compared to other bacteria due to the negatively charged
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P.aeruginosa cell wall readily attracting positively charged ZrO2NPs and thereby inhibiting
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microbial actions. Moreover, the concentration of ZrO2NPs was directly proportional to their
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inhibitory actions against the tested microorganisms. Finally, the preventive role of ZrO2NPs in a
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tooth decay pathway has been elucidated. Hence, it could be concluded that the as-prepared
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ZrO2NPs possess viable biomedical applications.
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Keywords: Nanoparticles; ZrO2; Characterization; Antimicrobial Activity; Dental care.
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1. Introduction
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Nanomaterials may easily interact with humans through digestive system (digestion),
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respiratory tract (inhalation), circulatory system (blood and lymph) via injection, skin contact by
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cosmetic creams and ointments. Due to their smaller sizes, nanoparticles would easily penetrate
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into cell membrane and may reach biological system and therefore, they can easily engulf into
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living organisms and cause cellular dysfunction [1-3].
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Recent developments in the field of nanotechnology has paved way for the successful
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fabrication, characterization and alteration of the functional properties of nanoparticles for 2
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biomedical applications [4-6]. Several nanoparticles, including metal nanoparticles [7-11], own
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distinctive properties which are employed for several applications in the nanobiomedicine. It has
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been well established that nanoparticles embrace an unbelievable potential in a variety of
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biomedical applications such as successful drug delivery systems [12,13]. Though nanoparticles
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endow vital applications, researchers are facing some disadvantages due to lack of proper
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knowledge about the effects of nanoparticles on biochemical pathways and metabolic actions of
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human body. Reasonably, safety and toxicity of nanomaterials has become a questionable of
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curiosity to the public. For that reason, understanding the communications of nanomaterials with
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biological systems is mandatory for most significant scientific issues [14-17].
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Zirconium is a sturdy transition metal that bears a resemblance to titanium due to its
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strong resistance to corrosion and biomedical grade zirconia may be the one for which there is
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the largest debate among scientists, industrials or clinicians. It is widely used as an implant
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biomaterial [18], dental crown [19], femoral heads for total hip replacement [20], solid oxide fuel
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cell electrolytes [21], catalytic activity [21,22], high chemical stability and ionic conductivity
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[22,23].
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For more than two decades, biomedical grade of Zirconia was used to solve the problem
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of alumina brittleness and the consequent potential failure of implants. Nowadays, more than
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600,000 of zirconia femoral heads have been implanted worldwide, especially in the US and in
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Europe. Biomedical grade of zirconia reveals the potential mechanical features of oxide
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ceramics. It is involved in phase transformation toughening, which increases its crack breeding
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resistance [24]. Furthermore, ZrO2 have wide applications due to outstanding biocompatibility,
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increasing strength, and low wear cost, but in biomedical industries there is no clear information
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about metabolic interactions and toxicity of zirconium.
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Bacterial infections are the main reasons for chronic infections and mortality. Antibiotics
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are recommended for bacterial infectivity as they are economic and effective as well [25].
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Antibiotics directly impair the cell wall synthesis, translational machinery and DNA replication
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processes of bacteria. Bacterial resistance to antibiotics inhibits the aforementioned mechanisms
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via expression of relevant enzymes [26-28]. Previously it has been reported that zirconia based
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ceramics are chemically inert materials. They allow excellent cell adhesion, and no adverse
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systemic reactions have been related [29]. Hence, novel substances which could attach to the
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bacterial cells without penetrating into the cell are needed to overcome the antibiotic resistance.
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It is recognized that nanoparticles could perform the required action clearly [30].
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Hence, the present study has focused on biogenic synthesis of ZrO2NPs and promoting
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their biological applications with special emphasis on antimicrobial activities. The prepared
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ZrO2NPs were characterized by UV-vis, FTIR, XRD, TEM and analysis.
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2. Materials and methods
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2.1 Equipments
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UV-vis spectrophotometer (Varian, Carry 5000) spectrophotometer was used to measure
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absorption with 1-cm path length quartz cuvettes, FTIR spectrophotometer (Thermo Nicolet,
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Avatar 370) was employed for embedded function group identification and Bruker AXS
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Advance powder X-ray diffractometer was used for characterization of crystalline nature of
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sample. Morphological analyses were carried out using Scanning Electron Microscopy (SEM)
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with a JEOL Model JSM - 6390LV instrument and Jeol/JEM 2100 High Resolution
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Transmission Electron Microscope.
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2.2 Chemicals and microbial cultures Zirconium nitrate hexahydrate (Zr(NO3)4.5H2O, and urea were purchased from HiMedia.
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All the chemicals used in this work were of analytical grades. Milli-Q distilled water was used
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for nanoparticle synthesis. Gram positive (Bacillus subtilis MTCC 1305, Staphylococcus aureus
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MTCC 3160) and gram negative bacteria (E. coli, P. aeruginosa MTCC 2453) were procured
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from Microbial Type Culture Collection (MTCC) and used as test organisms. All the cultures
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were stored and maintained at 4◦C.
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2.3 Synthesis of ZrO2 nanoparticles
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Synthesis of ZrO2NPs was carried out as follows: 0.5 mmol of Zr(NO3)4.5H2O and a
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trivial quantity of urea (CO(NH2)2 were dissolved in 70 ml of deionized water and the mixture
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was continuously stirred. Then, the homogenous solution was transferred into a 100 ml conical
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flask, teflon lined and autoclaved for 12 h at 180º C. Once the process was completed, the
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solution was shifted to room temperature and allowed to cool. Finally, a white precipitate was
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formed. The precipitate was washed numerous times with plenty of distilled water and absolute
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ethanol and subsequently dried overnight at 353 K. Eventually, the white precipitate was claimed
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in air at 450ºC for 15 h.
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2.4 Characterization of ZrO2 nanoparticles The morphology and the size of the ZrO2NPs were characterized by Scanning Electron
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Microscopy (SEM). ZrO2 NPs size, morphology and distribution were examined by the High
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Resolution Transmission Electron Microscope. UV-vis spectrophotometer was used to measure
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the absorption spectra of the synthesized nanoparticles in the range of 175-3300 nm. The
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readings were taken at intervals of 1nm with the scan rate 600 nm/min. The as-prepared
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nanoparticles were subjected to functional group analysis using FTIR spectrophotometer.
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The samples were prepared using KBr pellet method in 1:99 ratios at room temperature.
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The samples were scanned in the spectral range of 4000-400 cm-1 with the resolution of 2 cm-1.
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X-ray diffraction (XRD) patterns were collected using a powder X-ray diffractometer. The XRD
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pattern of synthesized sample was obtained inside the special XRD cell designed to avoid the
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reaction of air sensitive samples with atmospheric oxygen [31].
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2.5 Antibacterial assay of ZrO2 nanoparticles
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The antimicrobial activity of the ZrO2NPs was evaluated with well disc diffusion method
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[32]. The experiment was conducted against reference gram positive (Bacillus subtilis and
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Staphylococcus aureus) and gram negative bacteria (E. coli and P. aeruginosa) procured from
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Microbial Type Culture Collection (MTCC), Chandigarh, India. An inoculum size of 105 cells/ml
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was used to spread on the Mueller-Hinton Agar (MHA, HiMedia, India). In Brief, 20 ml of MHA
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was poured into petri dishes and allowed to solidify. Then, 6mm thick sterile discs were placed
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appropriately on petridishes. Finally, different concentrations of ZrO2Nps (20, 40, 80 and 100
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µg/ml) were loaded on each disc and 50% ethanol was used as a negative control. All the plates
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were incubated at 37 °C for 24 h and the respective inhibition zones were measured. Each test
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was performed in triplicates under the same set of conditions for reproducibility [33].
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3. Results and discussion
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3.1 UV–Vis and FTIR absorption spectra
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Fig. 1 represented the UV-vis absorption spectrum for the synthesis of ZrO2NPs in the
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scan range of 175 – 3300 nm. In this study the formation of ZrO2NPs was confirmed through the
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appearance of milky white precipitate. The milky colloidal solution indicated the conversion of
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Zr(NO3)4.5H2O into nanosized ZrO2 colloidal particles [34]. Further, their physical properties
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were examined using UV-Visible spectroscopy and FTIR techniques. The synthesis of nanosized
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ZrO2 colloidal particles was thus confirmed with the absorption spectra at 300 nm and 824 nm
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(Fig. 1). The UV-vis peaks denoted the transition of an inner shell electron to conduction.
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Various forms of Zr including ions, atoms or clusters strongly interacted with water molecules to
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form ZrO2, which was quickly quenched in liquid solution to form nanoparticles [34].
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The FTIR spectrum was used to establish the functional groups of nanoparticles. Fig. 2
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represents the FTIR absorption spectrum of the as-prepared nanoparticles. Spectrum of ZrO2NPs
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had shown peaks at 3428.96 cm-1 due to OH stretching, at 1634.05 cm-1 because of OH bending
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and at 492.73 cm-1 denoting Zr-O band, respectively. Based on the fingerprint characters of the
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peak positions, shapes and intensities along with the essential components in the materials were
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observed.
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exposed cluster appearance with crystal nature was indicated. However, spherical structures of
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size less than 20 nm with irregular surface morphologies denoted an increased grain size due to
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the increase in temperature leading to crystalline as well as grain growth. The stony appearance
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might have been due to the agglomeration of ZrO2NPs of size 20–50 nm [35].
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Fig. 4 shows the TEM reflection and size allocation of ZrO2NPs, which were a mixture of
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diverse sizes and shapes. The particles were almost spherical or irregular spherical with particle
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size ranging between 15 nm to 21 nm. For instance, the diameters of ZrO2NPs were observed to
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be 15.77 nm, 20.76 nm and 21.54 nm, respectively.
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XRD is of great importance in the microstructure characterization of complex, multiphase
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and single phase materials. The application of XRD enables not only qualitative and quantitative
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phase analysis but also microstructure characterization (crystallite size, lattice distortions and
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dislocation densities, stacking faults and twins probability). In the XRD pattern of ZrO2Nps, the
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diffraction peaks were observed at 2?? values of 28.9º, 60.13º and 62.7º corresponding to the
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characteristic diffraction planes (1 1 1), (3 0 2) and (3 1 1) of ZrO2, respectively. The well-
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known peaks have been exploited to estimate the grain size of sample. Grain size estimated using
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the relative intensity peak for ZrO2Nps was found to be above 20 nm and enhancement in the
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sharpness of XRD peaks denoted that the particles were crystalline in nature. All diverse peaks in
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Fig. 5 were associated to ZrO2Nps and harmonized to Joint Committee for Powder Diffraction
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Studies (JCPDS) [36].
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3.3 Antibacterial activity
Nanomaterials reveal strong inhibiting effects towards a broadened spectrum of bacterial
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strains. As per various research understandings, the metal oxides transmit the positive charge
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while the microorganisms hold negative charge. Hence, electromagnetic attractions between
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microorganisms and metal oxides lead to oxidization and finally result in the death of
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microorganisms [37].
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Antimicrobial activity of nanoparticles was determined by disc diffusion method against
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E. coli, P. aeruginosa, S. aureus and B. subtilis as shown in Fig. 6. The results show that
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ZrO2NPs had potential inhibitory action against P. aeruginosa and S. aureus and a good
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inhibition against E. coli while there was no action against B. subtilis (Fig. 7). It was also
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observed that the zone of inhibition values increased linearly with the increase in concentration
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of the nanoparticles, which was in accordance with the previous research reports [13, 38]. In
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another study, MIC values of silver nanoparticles against the chosen dental caries and
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periodontal disease microorganisms were obvious in the range of 25–75 µg/mL [12]. A better
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antibacterial against gram negative bacteria may due to the negatively charged cell wall of
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bacteria being ruptured by positively charged zirconium ions from zirconium nanoparticles and
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finally ensuing in cell necrosis. The similar understanding was witnessed for silver nanoparticles
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[12]. It has also been proposed that decrease in intracellular ATP levels could lead to
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destabilization in the outer and plasma membranes. Moreover, membrane proteins may also have
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been affected leading to cellular damage and eventually, cell death [13, 39]. In a previous study,
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reported by Banerjee et al. [40], ZrO2 nanostructures showed antibacterial activity against only E.
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coli bacteria and no activity for S. aureus and fungus, while the complexes showed antibacterial
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activity against both gram positive and negative bacteria, i.e., S. aureus and E. coli. Thus,
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ZrO2NPs proved to be a potential antibacterial agent against both gram positive and gram
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negative microorganisms though revealed to be most effective against gram negative bacteria.
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3.4 Role of ZrO2NPs in dental care
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The accumulation of bacteria on teeth produces acid, which stimulates corrosion on the
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teeth surfaces. This process is known as tooth decay or dental caries. Various bacteria
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accumulate on teeth, gums and tongue, and produce plague on teeth ie supragingival plaque.
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Once bacteria accumulate on teeth and interact with food containing sugars, acid is produced,
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which is known as demineralization. If acid penetrates on teeth, enamel is dissolved ending up
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in cavity formation.
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It has been previously established that silver nanoparticles could easily approach to the
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target site and deliver the drug molecule to the hydrophobic surface of the bacterial cell and
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could interfere with the bacterial growth signaling pathway. In this context, coupling of AgNPs
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with drugs (Azithromycin and Clarithromycin) divulged potential activity against dental carries
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[22].
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Zirconia material is used as ceramic surface of metal implants and it aids to improve
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biocompatibility in the oral cavity but exposure of fibro integration of implants has been
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observed by histological examination [41]. With this understanding, the as-prepared ZrO2NPs
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may probably overcome the above noted issues and provide first-class protection to the teeth.
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ZrO2NPs would bind on the surface of bacteria and stop the metabolic action with food, thereby
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preventing the synthesis of acid and enamel corrosion. Thus, the outer coating of ZrO2NPs to
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teeth provides external strength to the teeth and increases their lifespan as shown in Fig. 8.
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Conclusion
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The present study has illustrated the synthesis of ZrO2NPs and their subsequent
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characterization using UV-vis spectroscopy, FTIR, XRD and SEM.
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indicated the transformation of ZrO2 into ZrO2 NPs which was also confirmed through functional
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group and bonding of nanosized colloidal particles by FTIR. ZrO2NPs were of crystalline nature
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and of size 15 nm – 21 nm as identified by XRD, SEM and TEM analysis. The antimicrobial
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The UV-visible peaks
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activity of ZrO2NPs observed against gram positive and gram negative bacteria revealed that
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ZrO2NPs had potential inhibitory action against
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aeruginosa at higher concentrations due to their negatively charged cell surfaces. In this
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context, the feasibility of exploiting ZrO2NPs in averting tooth decay has been elucidated via
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comprehending the tooth decay pathway. As the conclusion of this research, ZrO2NPs could be
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recommended for applications in dental care and other relevant biomedical applications upon
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conducting further in vitro and in vivo studies, respectively.
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Figure legends
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Fig. 1. UV-Vis spectrum of ZrO2NPs
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Fig. 2. FTIR spectrum of ZrO2NPs
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Fig. 3. SEM micrograph of ZrO2NPs
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Fig. 4. TEM and size distribution ZrO2NPs
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Fig. 5. XRD patterns of ZrO2NPs
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Fig. 6. Antibacterial effects of ZrO2NPs
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Fig. 7. Antibacterial activity of ZrO2NPs determined by disc diffusion method S- Standard (100
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µg/ml), C- Control (100 µg/ml), 1- ZrO2 NPs (100 µg/ml), 2- ZrO2NPs (80 µg/ml), 3- ZrO2NPs (40 µg/ml) and 4- ZrO2NPs (20 µg/ml).
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Fig. 8. Role of ZrO2NPs in dental care
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Highlights
ZrO2NPs - antimicrobial activity and anti-tooth decay applications were determined
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ZrO2NPs was examined against various gram positive bacteria and gram negative
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bacteria.
ZrO2NPs in averting tooth decay has been elucidated via comprehending the tooth decay
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pathway.
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