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

Authors: A. Costescu; C. S. Ciobanu; S. L. Iconaru; R. V. Ghita; C. M. Chifiriuc; L. G. Marutescu; D. Predoi
Journal: Journal of Nanomaterials
 (2013)

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## Abstract

The aim of this study was the evaluation of(Ca10-xAgx)(PO4)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 powder was synthesized at 100°C in deionised water. 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 100°C exhibit the apatite characteristics with good crystal structure, without any new phase or impurities found. FT-IR and FT-Raman 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.


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## Body

## 1. 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 [1–6]. 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 Ca10(PO4)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 Ca2+ ions with other metal ions such as Cu2+, Zn2+, and Ag2+, without changing its initial structure and properties.In this paper, we report a novel compound based on low silver concentrations in silver doped hydroxyapatite nanoparticles with high biocompatibility, synthesized at low temperature using the coprecipitation method. Characterization of the Ag:Hap(0≤xAg≤0.1) using X-ray diffraction (XRD) and transmission electron microscopy (TEM) confirmed that the product consists of pure HAp ellipsometric nanoparticles. The antimicrobial properties of (Ca10-xAgx)(PO4)6(OH)2, with 0≤xAg≤0.1 nanoparticles were evaluated on Gram-negative (Escherichia coli ATCC 25922, E. coli 714, K. pneumoniae 2968) and Gram-positive (Bacillus subtilis) bacterial strains as well as Candida krusei 963 yeast.


## 2. Experimental Section

### 2.1. Sample Preparation

The reagents used for the synthesis of silver doped hydroxyapatite were ammonium dihydrogen phosphate [(NH4)2HPO4] (Alpha Aesare, Germany, 99.99% purity), calcium nitrate [Ca(NO3)2·4H2O] (Alpha Aesare, Germany, 99.99% purity), silver nitrate AgNO3 (Alpha Aesare, Germany, 99.99% purity), and ammonium hydroxide NH4OH (Alpha Aesare, Germany, 99.99% purity).The synthesis of(Ca10-xAgx)(PO4)6(OH)2, with xAg=0, xAg=0.02, xAg=0.05, xAg=0.07, and xAg=0.1 was carried out as reported [13, 14]. The synthesis of Ag:HAp with xAg=0 was carried out as reported [13, 14] by the following reaction [15]:
(1)10Ca(NO3)2·4H2O+6(NH4)2HPO4+2NH4OH⟶Ca10(PO4)6(OH)2+20NH4NO3+46H2OThe pH of solution was adjusted to 10 with ammonium hydroxide (NH4OH) in accord with [16]. Nanocrystalline hydroxyapatite doped with Ag was performed by setting the xAg from 0 to 0.1 (0≤xAg≤0.1) and [Ca+Ag]/P as 1.67. The AgNO3 and Ca(NO3)2·4H2O were dissolved in deionised water to obtain 300 mL [Ca+Ag]-containing solution. On the other hand, the (NH4)2HPO4 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 (NH4OH) 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) withxAg≠0, setting the xAg from 0.02 to 0.1 (0.02≤xAg≤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]:
(2)Ca10(PO4)6(OH)2+2xAg→O2Ca10-xAg2x(PO4)6(OH)2+xCaOThis 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 Ca2+ 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:
(3)2Ag→Ca2+2AgCa′+VO∞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.


### 2.2. Sample Characterization

The X-ray diffraction measurements for the Ag:HAp samples were recorded using a Bruker D8 Advance diffractometer, with nickel filtered CuKα(λ=1.5418Å) radiation with a high efficiency one-dimensional detector (Lynx Eye type) operated in integration mode. The diffraction patterns were collected in the 2θ 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 of1.5×108 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 μg/mL concentration. The quantitative assay was performed by liquid microdilution method [21–27].


## 2.1. Sample Preparation

The reagents used for the synthesis of silver doped hydroxyapatite were ammonium dihydrogen phosphate [(NH4)2HPO4] (Alpha Aesare, Germany, 99.99% purity), calcium nitrate [Ca(NO3)2·4H2O] (Alpha Aesare, Germany, 99.99% purity), silver nitrate AgNO3 (Alpha Aesare, Germany, 99.99% purity), and ammonium hydroxide NH4OH (Alpha Aesare, Germany, 99.99% purity).The synthesis of(Ca10-xAgx)(PO4)6(OH)2, with xAg=0, xAg=0.02, xAg=0.05, xAg=0.07, and xAg=0.1 was carried out as reported [13, 14]. The synthesis of Ag:HAp with xAg=0 was carried out as reported [13, 14] by the following reaction [15]:
(1)10Ca(NO3)2·4H2O+6(NH4)2HPO4+2NH4OH⟶Ca10(PO4)6(OH)2+20NH4NO3+46H2OThe pH of solution was adjusted to 10 with ammonium hydroxide (NH4OH) in accord with [16]. Nanocrystalline hydroxyapatite doped with Ag was performed by setting the xAg from 0 to 0.1 (0≤xAg≤0.1) and [Ca+Ag]/P as 1.67. The AgNO3 and Ca(NO3)2·4H2O were dissolved in deionised water to obtain 300 mL [Ca+Ag]-containing solution. On the other hand, the (NH4)2HPO4 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 (NH4OH) 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) withxAg≠0, setting the xAg from 0.02 to 0.1 (0.02≤xAg≤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]:
(2)Ca10(PO4)6(OH)2+2xAg→O2Ca10-xAg2x(PO4)6(OH)2+xCaOThis 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 Ca2+ 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:
(3)2Ag→Ca2+2AgCa′+VO∞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.


## 2.2. Sample Characterization

The X-ray diffraction measurements for the Ag:HAp samples were recorded using a Bruker D8 Advance diffractometer, with nickel filtered CuKα(λ=1.5418Å) radiation with a high efficiency one-dimensional detector (Lynx Eye type) operated in integration mode. The diffraction patterns were collected in the 2θ 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 of1.5×108 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 μg/mL concentration. The quantitative assay was performed by liquid microdilution method [21–27].


## 3. Results and Discussions

Figure1 shows the XRD patterns of pure HAp (xAg=0), Ag:HAp (xAg=0.02, xAg=0.05, xAg=0.07, and xAg=0.1) and the standard data for the hexagonal hydroxyapatite. For pure HAp (xAg=0) and Ag:HAp (xAg=0.02,  xAg=0.05, xAg=0.07, and xAg=0.1), the diffraction peaks can be well indexed to the hexagonal Ca10(PO4)6(OH)2 in P63mspace 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].Figure 1
Comparative representation of the experimental XRD patterns of the Ag:HAp samples synthesizedxAg=0, xAg=0.02, xAg=0.05, xAg=0.07, and xAg=0.1, and the characteristic lines of hydroxyapatite according to the ICDD-PDF number 9-432.The absence of other phases in XRD pattern of Ag:HAp samples demonstrates that the Ag+ ions have successfully substituted Ca2+ 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. Figure2 shows TEM images of Ag:HAp (xAg=0). As shown in Figure 1, Ag:HAp nanoparticles exhibited an ellipsoidal morphology which is consistent with the SEM results [13].Figure 2
The TEM images of Ag:HAp(xAg=0) at low resolution.Figure3 exhibits the TEM images of Ag:HAp (xAg=0.02, xAg=0.05, xAg=0.07, and xAg=0.1) at low resolution. No obvious morphological changes are observed between the initial HAp and the corresponding Ag:HAp (xAg=0.02, xAg=0.05, xAg=0.07, and xAg=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.Figure 3
The TEM images of Ag:HAp (xAg=0.02, xAg=0.05, xAg=0.07, and xAg=0.1) at low resolution.It can be seen from the HRTEM image of Ag:HAp (Figure4(a)) the crystalline phase of hydroxyapatite with well-resolved lattice fringes. The distances between the adjacent lattice fringes (2.81 Å and 1.94 Å) agree well with the d211 and d222 spacing values from the literature (ICDD-PDF no. 9-432). All the samples exhibit a uniform ellipsoidal morphology with particles from 5 nm to 15 nm. Figure 4(b) shows a selected area electron diffraction (SAED) pattern recorded from an area containing a large number of ellipsometric nanoparticles. The rings in the SAED pattern can be indexed as (002), (210), (211), (310), (222), (213), (004), and (304) reflections of the hexagonal HAp, in agreement with the XRD results. No extra reflections are observed and we can therefore conclude that the product consists of pure HAp ellipsometric nanoparticles.HRTEM image and SAED analysis of Ag:HAp withxAg=0.05.
(a)
(b)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,Ca10-xAgx(PO4)6(OH)2, (xAg=0, xAg=0.02, xAg=0.05, xAg=0.07, and xAg=0.1) synthesized at 100°C by coprecipitation method. Figure 5 shows the FT-IR results obtained from Ag:HAp-NPs when the xAg 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].Figure 5
Transmittance infrared spectra of the Ag:HAp samples synthesized withxAg=0, xAg=0.02, xAg=0.05, xAg=0.07, and xAg=0.1.The data clearly reveals the presence of various vibrational modes corresponding to phosphate and hydroxyl groups. For all the samples, the presence of strong OH vibration peak could be noticed. The peak observed at 634 cm−1 is attributed to the characteristic stretching and vibrational modes of structural OH groups [33]. The band at 1630 cm−1 corresponds to the adsorbed H2O [33]. The broad bands in the regions 1600–1700 cm−1 and 3200–3600 cm−1 correspond to H–O–H bands of water lattice [34].Bands characteristics ofPO43- tetrahedral apatite’s structure are clearly observed at 473 cm−1, 563 cm−1, 603 cm−1, 960 cm−1, and 1095–1033 cm−1 [35, 36]. The peak at 473 cm−1 is attributed to PO4 ν2. The peaks at 563 cm−1 and 603 cm−1 belong to PO4 ν4. The peak at 960 cm−1 is attributed to PO4 ν1 and the peaks at 1095–1033 cm−1, to PO4 ν3. A CO32- band occurred in the spectra at 1452 and 1400 cm−1. The bands at 875 cm−1 are attributed to HPO42- ions [37, 38].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].Figure 6
Raman spectra of the Ag:HAp samples synthesized withxAg=0, xAg=0.02, xAg=0.05, xAg=0.07, and xAg=0.1.We assigned the bands present at 1026 cm−1 (ν3), 1047 cm−1 (ν3), and 1074 cm−1 (ν3) to the asymmetric ν3 (P–O) stretching. The internal modes of the PO43- tetrahedral ν1 frequency (960 cm−1) correspond to the symmetric stretching of P–O bonds. The 616 cm−1, 590 cm−1, and 576 cm−1 bands arise from ν4 PO4 [37]. The vibrational bands at 429 cm−1 (ν2) and 449 cm−1 (ν2) are attributed to the O–P–O bending modes. The band at 1070 cm−1 (ν1) attributed to CO32- impurity was obscured by the intensity of PO4 band at 1074 cm−1. The other CO3 modes ν3, ν4, and ν2 have band positions not obscured by the PO4 bands, but they have weak intensities and were not detected [40]. 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 Table1. The synthesis allowed a good control over the chemical composition of Ag:HAp powders (xAg=0, xAg=0.02, xAg=0.05, xAg=0.07, and xAg=0.1), with the Ca/P and (Ca+Ag)/P atomic ratios being close to the stoichiometric value of 1.67.Table 1
Chemical analyses of samples.
Samples
Ca/P (molar ratio)
Ag (%)
(Ca+Ag)/P (molar ratio)
x
Ag = 0
1.668
—
—
x
Ag = 0.02
1.673
1.89
1.675
x
Ag = 0.05
1.668
4.91
1.673
x
Ag = 0.07
1.666
6.97
1.669
x
Ag = 0.1
1.665
9.98
1.667The antimicrobial activity of Ag:HAp(0≤xAg≤0.1) nanoparticles was tested using strains belonging to the most commonly encountered pathogens: E. coli ATCC 25922, E. coli 714, K. pneumoniae 2968, B. subtilis, and C. krusei 963. The results of microbiological assays of Ag:HAp (0≤xAg≤0.1) nanoparticles are shown in Figures 7(a)–7(e). The Ag:HAp (xAg=0) nanoparticles exhibited no inhibitory effect on the microbial growth as compared to the positive growth control (C+) (Figure 7(a)). The Ag:HAp nanoparticles with xAg=0.02 exhibited an inhibitory effect on the growth of K. pneumoniae at the first two tested concentrations and of C. krusei 963 at the first three tested concentrations (Figure 7(b)). The Ag:HAp nanoparticles with higher concentrations of Ag (xAg=0.05; 0.07 and 0.1) showed an improved antimicrobial activity against the same two pathogens, that is, K. pneumoniaeand C. krusei,but maintained for the entire range of the tested concentrations, from 5 to 0.01 mg/mL (Figures 7(c), 7(d), and 7(e)). Starting with the xAg=0.05, the tested nanoparticles started to exhibit also an inhibitory effect against one of the two E. colitested strains, that is, E. coliATCC 25922, which is totally susceptible to antibiotics, being recommended as a reference strain for performing susceptibility testing. In exchange, the second E. coli714 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 xAg=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. subtilisstrain, 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.Antibacterial activity of Ag:HAp-NPs (xAg=0,0.02,0.05,0.07, and 0.1) on E. coli ATCC 25922, E. coli 714, K. pneumoniae 2968, B. subtilis,  and C. krusei strains.
(a)
(b)
(c)
(d)
(e)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 μg is needed while the rod shaped particles need a total of 50 to 100 μg 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.


## 4. 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.



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*Source: 194854-2013-05-26.xml*