Fabrication , Characterization , and Antimicrobial Activity , Evaluation of Low Silver Concentrations in Silver-Doped Hydroxyapatite Nanoparticles

The aim of this study was the evaluation of (Ca 10−x Ag x )(PO 4 ) 6 (OH) 2 nanoparticles (Ag:HAp-NPs) for their antibacterial and antifungal activity. Resistance to antimicrobial agents by pathogenic bacteria has emerged in the recent years as a major public health problem worldwide. In this paper, we report a comparison of the antimicrobial activity of low concentrations silver-doped hydroxyapatite nanoparticles.The silver-doped nanocrystalline hydroxyapatite powderwas synthesized at 100C in deionisedwater. The as-prepared Ag:Hap nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), FT-IR, and FT-Raman spectroscopy. X-ray diffraction (XRD) studies demonstrate that powders obtained by coprecipitation at 100C exhibit the apatite characteristics with good crystal structure, without any new phase or impurities found. FT-IR and FTRaman spectroscopy revealed the presence of the various vibrational modes corresponding to phosphates and hydroxyl groups and the absence of any band characteristic to silver. The specific microbiological assays demonstrated that Ag:HAp-NPs exhibited antimicrobial features, but interacted differently with the Gram-positive, Gram-negative bacterial and fungal tested strains.


Introduction
Nanotechnology is at the base of recent and future developments in technological and industrial applications.In last years, the progress made in the area of engineered nanomaterials allowed us to have spectacular inside knowledge about materials at an atomic and molecular scale.Recently, biomaterials are emerging as the most studied area of materials science [1][2][3][4][5][6].Biomaterials could be defined as "implantable materials that perform their function in contact with living tissues" [7].This is a new interdisciplinary branch set to achieve new and improved materials with biological properties for use in clinical applications.The aim of this research direction is to study, facilitate, and improve the biological interactions between materials and organisms.The inorganic materials with good biological properties are intensively studied for this purpose.The most studied biomaterials are those based on calcium phosphate, which belongs to the so-called apatite's family.An important representative of the apatite's family is hydroxyapatite with the general formula Ca 10 (PO 4 ) 6 (OH) 2 .Due to its outstanding properties such as biocompatibility, osteoconductivity, and bioactivity, the hydroxyapatite (HAp) has been extensively investigated [8,9].It is found naturally in the human body as one of the major constituents of bones and teeth.HAp nanoparticles have been investigated as coatings of medical implants for use in dentistry and orthopaedics as prosthesis due to their excellent properties [10].Even though these nanoparticles have excellent biocompatibility, one of the most significant problems nowadays when using implants is the risk of infections.The development of surgical infections is common and widely spread amongst patients despite the hospitals tremendous efforts to avoid them.Due to the high costs and mortality rates, the research towards developing new compounds with high biocompatibility and antimicrobial properties is a hot topic at global scale.Silver has been used since ancient times as an efficient antimicrobial agent, being active against a wide range of microorganisms [11].In agreement with Clement and Jarrett [12], the understanding of silver toxicity to bacteria and of bacterial resistance mechanisms has a long way to go.Silver binds to many cellular components, the interaction with the membrane components probably being more important than that with the nucleic acids [12].Hydroxyapatite has the ability to achieve the substitution of Ca 2+ ions with other metal ions such as Cu 2+ , Zn 2+ , and Ag 2+ , without changing its initial structure and properties.

Experimental Section
The pH of solution was adjusted to 10 with ammonium hydroxide (NH 4 OH) in accord with [16].Nanocrystalline hydroxyapatite doped with Ag was performed by setting the  Ag from 0 to 0.1 (0 ≤  Ag ≤ 0.1) and [Ca+Ag]/P as 1.67.The AgNO 3 and Ca(NO 3 ) 2 ⋅4H 2 O were dissolved in deionised water to obtain 300 mL [Ca+Ag]-containing solution.On the other hand, the (NH 4 ) 2 HPO 4 was dissolved in deionised water to make 300 mL P-containing solution.The [Ca+Ag]containing solution was stirred at 100 ∘ C for 30 minutes.Meanwhile the pH of P-containing solution was adjusted to 10 with ammonium hydroxide (NH 4 OH) and stirred continuously for 30 minutes.The P-containing solution was added drop by drop into the [Ca+Ag]-containing solution and stirred for 2 h and the pH was constantly adjusted and kept at 10 during the reaction.When the reaction was complete, the deposited mixtures were washed several times with deionised water.The resulting material (Ag:HAp) was dried at 100 ∘ C for 72 h.
Silver doped hydroxyapatite powders (Ag:HAp) with  Ag ̸ = 0, setting the  Ag from 0.02 to 0.1 (0.02 ≤  Ag ≤ 0.1), were prepared assuming that silver ions would substitute for the calcium site in the HAp lattice.
According to Nath et al. [17], it is important to note that the acceptable reason for this phenomenon is that the radius of silver ion is bigger than that of the strontium ion (11.5%) with fewer valence electrons.It is also important to note that Ma et al. [18] and Corami et al. [19] in two separate research studies suggested some reactions for ion exchange between heavy metals and apatite particles.
The diffusion of Ag in HAp lattice can be characterized by the following equation [17]: This equation indicates that Ag can be incorporated into the lattice of HAp, which is schematically shown by [17].According to Nath et al., the complex crystal structure of HAp is described by Ca triangle around the OH column.It is worth mentioning that, in the presence of Ag, one of the Ca 2+ sites can be substituted by Ag.Nath et al. also confirmed this substitution mechanism using Raman spectroscopy by confirming the presence of Ag-O bond.Nath et al. [17] and Nazari et al. [20] showed that because of valency difference between Ca and Ag ions, a vacancy will be created in the anion site, as per the following defect reaction: As shown by Nath et al. and Nazari et al., it can be perceived that the creation of oxygen vacancy will enable the proton of OH-bond to be attracted towards Ag-defect in the HAp structure.

Sample Characterization.
The X-ray diffraction measurements for the Ag:HAp samples were recorded using a Bruker D8 Advance diffractometer, with nickel filtered Cu K  ( = 1.5418Å) radiation with a high efficiency onedimensional detector (Lynx Eye type) operated in integration mode.The diffraction patterns were collected in the 2 range 20 ∘ -70 ∘ , with a step of 0.02 ∘ and 34 s measuring time per step.Transmission electron microscopy (TEM) studies were carried out using a FEI Tecnai 12 equipped with a low dose Gatan digital camera.The specimen for TEM imaging was prepared by ultra microtomy in order to obtain thin section of about 60 nm.The powder was embedded in an epoxy resin (polaron 612) before microtomy.TEM modes used were bright field (BF) and selected area diffraction (SAD).The functional groups present in the prepared nanoparticles and thin films were identified by FTIR using a Spectrum BX spectrometer.In order to obtain the nanoparticles spectra, 1% of the nanopowder was mixed and ground with 99% KBr.Tablets of 10 mm diameter were prepared by pressing the powder mixture at a load of 5 tons for 2 min.The spectrum was recorded in the range of 500 to 4000 cm −1 with 4 cm −1 resolution.Micro-Raman spectra on powders were performed in a backscattering geometry at room temperature and in ambient air, under a laser excitation wavelength of 514 nm, using a Jobin Yvon T64000 Raman spectrophotometer under a microscope.Chemical analyses of samples were performed by HITACHI Z-8100 Polarized Zeeman Atomic Spectrophotometer performed at the Fonctionnement et Evolution des Biogéosystèmes Continentaux (Institut des Sciences de la Terre d' Orléans, France).
For the antimicrobial assays, microbial suspensions of 1.5 × 10 8 CFU/mL corresponding to 0.5 McFarland density obtained from 15 to 18 h bacterial cultures developed on solid media were used.The tested substances were solubilised in DMSO and the starting stock solution was of 5000 g/mL concentration.The quantitative assay was performed by liquid microdilution method [21][22][23][24][25][26][27].

Results and Discussions
Figure 1 shows the XRD patterns of pure HAp ( Ag = 0), Ag:HAp ( Ag = 0.02,  Ag = 0.05,  Ag = 0.07, and  Ag = 0.1) and the standard data for the hexagonal hydroxyapatite.For pure HAp ( Ag = 0) and Ag:HAp ( Ag = 0.02,  Ag = 0.05,  Ag = 0.07, and  Ag = 0.1), the diffraction peaks can be well indexed to the hexagonal Ca 10 (PO 4 ) 6 (OH) 2 in P6 3 m space group (ICDD-PDF No. 9-432).The XRD analysis of Ag:HAp nanoparticles did not give the characteristic peak for silver or other phases.The XRD patterns of HAp and Ag:HAp also demonstrate that powders made by coprecipitation at 100 ∘ C with low silver concentrations in silver doped hydroxyapatite nanoparticles exhibit the apatite characteristics with good crystal structure and no new phase or impurity is found in good accord with our previous studies [13,14,28,29].
The absence of other phases in XRD pattern of Ag:HAp samples demonstrates that the Ag + ions have successfully substituted Ca 2+ ions without affecting the crystal structure of the original HAp.This result is in agreement with previous studies conducted by Ravindran et al., in 2010 [29], and Shirkhanzadeh et al., in 1995 [30].
The morphology and particle size of nanopowders were determined by TEM observation.Figure 2 shows TEM images of Ag:HAp ( Ag = 0).As shown in Figure 1, Ag:HAp nanoparticles exhibited an ellipsoidal morphology which is consistent with the SEM results [13].
Figure 3 exhibits the TEM images of Ag:HAp ( Ag = 0.02,  Ag = 0.05,  Ag = 0.07, and  Ag = 0.1) at low resolution.No obvious morphological changes are observed between the initial HAp and the corresponding Ag:HAp ( Ag = 0.02,  Ag = 0.05,  Ag = 0.07, and  Ag = 0.1), and new fine particles were formed.The Ag:HAp nanoparticles preserve the morphology of pure HAp prepared by coprecipitation method [14,28].The long axis corresponded to the c-axis of the hexagonal HAp structure.These results are well consistent with the XRD results revealing that the doping components have little influence on the surface morphology of the samples.The morphology identifications indicated that the nanoparticles with good crystal structure could be made by coprecipitation method at low temperature.
FT-IR spectroscopy was performed in order to investigate the functional groups present in nanohydroxyapatite, Ca 10− Ag  (PO 4 ) 6 (OH) 2 , ( Ag = 0,  Ag = 0.02,  Ag = 0.05,  Ag = 0.07, and  Ag = 0.1) synthesized at 100 ∘ C by coprecipitation method.Figure 5 shows the FT-IR results obtained from Ag:HAp-NPs when the  Ag increases from 0.05 to 0.3.The absorption peak in the region of 1600-1700 cm −1 ascribed to O-H bending mode is evidence of the presence of absorbed water in the synthesized products [31,32].
Complementary information can be obtained from Raman spectroscopy.Raman spectra of Ag:HAp from 1200 cm −1 to 400 cm −1 is shown in Figure 6.The OH − vibrational bands expected in the region of 630 cm −1 are not clearly detected.This behavior is in good accord with the previous studies [39].
Water vibrational modes give rise to weak intensity stretching and bending bands in Raman spectra.These water bands, expected at about the same wave number in FTIR spectra, were not observed in Raman spectra.The results on chemical analyses of samples are reported in Table 1.The synthesis allowed a good control over the chemical composition of Ag:HAp powders ( Ag = 0,  Ag = 0.02,  Ag = 0.05,  Ag = 0.07, and  Ag = 0.1), with the Ca/P and (Ca+Ag)/P atomic ratios being close to the stoichiometric value of 1.67.
The antimicrobial activity of Ag:HAp (0 ≤  Ag ≤ 0.1) nanoparticles was tested using strains belonging to the most commonly encountered pathogens: E. coli   Starting with the  Ag = 0.05, the tested nanoparticles started to exhibit also an inhibitory effect against one of the two E. coli tested strains, that is, E. coli ATCC 25922, which is totally susceptible to antibiotics, being recommended as a reference strain for performing susceptibility testing.In exchange, the second E. coli 714 strain, which is a clinical strain, resistant to beta-lactam antibiotics, proved to be resistant to almost all tested nanoparticles, irrespective to the silver ions concentration, except the Ag:HAp nanoparticles doped  Ag = 0.1, demonstrating that a potential cross-resistance mechanism could be responsible for the concomitant resistance to antibiotics and silver ions in this strain.As for B. subtilis strain, only the nanoparticles doped with the highest concentration of silver exhibited an inhibitory effect.These results are clearly demonstrating that the antimicrobial effect of the silver doped nanoparticles is dependent on the structure of the microbial cell wall.Kreibig and Vollmer [40], Morones et al. [41], and Pal et al. [42] demonstrated that the nanosize of the nanoparticles assures a large surface contact area with the microbial cells and, hence, a better interaction with the microbial target will occur.Raimondi et al. [43] and Bai et al. [36], studying the inhibition of bacterial growth by differentially shaped nanoparticles, showed that the antibacterial efficacy of the nanoparticles depends on the shape of nanoparticles.In their studies, Pal et al. [42] show that, in the case of spherical nanoparticles, total silver content of 12.5 g is needed while the rod shaped particles need a total of 50 to 100 g of silver content.Our study demonstrates that the antibacterial activity of Ag:HAp nanoparticles is dependent on the silver concentration, with the intensity of the inhibitory effect increasing proportionally from 0.01 mg/mL to 5 mg/mL.In order to obtain a large and effective antimicrobial spectrum, including the Gram-negative, Gram-positive bacterial and fungal strains, a higher Ag:Hap concentration of 5 mg/mL is required for this type of nanoparticles.However, further studies are needed to evaluate the cytotoxicity of the nanoparticles doped with this silver concentration, before recommending them for in vivo clinical applications.

Conclusions
The Ag:HAp nanoparticles obtained by coprecipitation method at low temperature have good crystal structure and doping components have little influence on the surface morphology of the samples.The DRX analysis of Ag:Hap did not exhibit the characteristic peak for silver.Low silver concentrations are required to be effective against Gram-negative bacterial and fungal strains, but, however, the intensity of the antimicrobial effect against these strains is increasing with the silver ions concentration.Only high Ag:HAp concentration of 5 mg/mL was effective against the Gram-positive bacterial strains.The antimicrobial properties of Ag:HAp nanoparticles are supporting their potential use for various medical applications, for example, textile dressings, orthopedic and dental prostheses, or implants with improved resistance to microbial colonization.

Table 1 :
Chemical of samples.