Vibrational Investigations of Silver-Doped Hydroxyapatite with Antibacterial Properties

Silver-doped hydroxyapatite (Ag:HAp) was obtained by coprecipitation method. Transmission electron microscopy (TEM), infrared, andRaman analysis con�rmed the development ofAg:HApwith good crystal structure. Transmission electronmicroscopy analysis showed an uniform ellipsoidal morphology with particles from 5 nm to 15 nm. e main vibrational bands characteristic to HAp were identi�ed. e bands assigned to phosphate vibrational group were highlighted in infrared and Raman spectra. e most intense peak Raman spectrum is the narrow band observed at 960 cm. In this article Ag:HAp-NPs were also evaluated for their antimicrobial activities against gram-positive, gram-negative, and fungal strains. e speci�c antimicrobial activity revealed by the qualitative assay demonstrates that our compounds are interacting differently with the microbial targets.


Introduction
Nanotechnology is an emerging �eld which reaches out to develop a bridge between the macroscopic and the atomic or molecular levels of matter.Nowadays the research community looks for answers in the �eld of nanomaterials for the most pressing problems in medicine, electronics, optics, and environment.e most studied materials are the ones showing similarities and good compatibility with the living tissues due to the large medical applications they qualify for [1][2][3].Biomaterials, especially bioceramics belonging to calcium phosphate class, are widely investigated because of their unique properties and high similarities with human osseous tissue.In the last decade a lot of attention was directed towards hydroxyapatite (HAp) from the family of apatites, which is known to be in its natural form a major inorganic constituent of human bones and teeth [4][5][6].Having the general formula, Ca 10 (PO 4 ) 6 (OH) 2 , HAp has been widely studied for dental and orthopedic applications due to its outstanding properties of osteoconductivity and biocompatibility.In order to be able to meet the high demands that are nowadays required in the �eld of medical implants, dental implants, or bone tissue reconstruction, biomaterials such as HAp are oen improved with other signi�cant elements [7][8][9].e HAp structure allows the substitution of Ca 2+ ions with other metal ions such as Zn 2+ , Cu 2+ , and Ag + [10].As we all know, silver has been widely used since ancient times as an antimicrobial agent.Due to the outbreak of diseases and apparition of drug resistant bacterial strains, in the last years, there have been made extensive studies in order to develop new antibacterial compounds based on the effects of silver against bacterial strains.e antibacterial action of silver nanoparticles is not completely understood and in pure state it has been reported to be toxic towards human organism [11].
e result of substituting Ca 2+ ions with Ag + ions in the structure of HAp is a new compound which presents high biocompatibility, osteoconductivity, bone regeneration properties, and above all good antibacterial properties [12].
is work presents the results of infrared spectroscopic analysis and Raman investigations of silver-doped hydroxyapatite with  Ag = 0, 0.4, and 0.5.is study represents an extension of results reported in our previous work [13].e aim of this study was the evaluation of the antibacterial activity of Ag:HAp nanoparticles when the silver concentration in the samples was increased at 0.4 and 0.5.e structure and morphology of the obtained samples were characterized by transmission electron microscopy (TEM).In vitro qualitative antimicrobial activity of silver-doped hydroxyapatite samples was also investigated.

Experimental Section
e synthesis of (Ca 10− Ag  )(PO 4 ) 6 (OH) 2 , with  Ag = 0,  Ag = 0.4 and  Ag = 0.5 was carried out as reported in other papers [13].Transmission electron microscopy (TEM) studies were carried out using an FEI Tecnai 12 equipped with a low-dose digital camera from Gatan.e specimen for TEM imaging was prepared by ultramicrotomy in order to obtain thin section of about 60 nm.e powder is embedded in an epoxy resin (polaron 612) before microtomy.TEM modes used were bright �eld (BF) and selected area diffraction (SAD).e functional groups present in the prepared nanoparticles and thin �lms were identi�ed by FTIR using a spectrum BX spectrometer.To obtain the nanoparticles spectra, 1% of the nanopowder was mixed and grounded with 99% KBr.Tablets of 10 mm diameter were prepared by pressing the powder mixture at a load of 5 tons for 2 min.e 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.e microbial strains identi�cation was con�rmed by aid of �ITEK II automatic system.�ITEK cards for identi�cation and susceptibility testing were inoculated and incubated according to the manufacturer's recommendations.e results were interpreted using the soware version AMS R09.1.Microbial suspensions of 1.5 × 10 8 CFU/mL corresponding to 0.5 McFarland density obtained from 15-18 h bacterial cultures developed on solid media were used in our experiments.e tested substances were solubilised in DMSO and the starting stock solution was of 5000 g/mL concentration.e qualitative screening was performed by an adapted disk diffusion method [14][15][16][17][18].

Results and Discussions
Figure 1 exhibits the TEM images of pure HAp ( Ag = 0) and Ag:HAp ( Ag = 0.4, and  Ag = 0.5) with low resolution.As shown in Figure 1, pure HAp and Ag:HAp exhibit an ellipsometric morphology, which is consistent with the SEM results [13].
It can be seen from the HRTEM image of Ag:HAp (Figure 2(a)) that the crystalline phase of hydroxyapatite with well-resolved lattice fringes can be observed.e distances between the adjacent lattice fringes (2.72 Å) agree well with the d 300 spacing of the literature values (2.872 Å) (ICDD-PDF no.9-432).All 3 samples exhibit a uniform ellipsoidal morphology with particles from 5 nm to 15 nm.e Figure 2(b) shows a selected area electron diffraction (SAED) pattern recorded from an area containing a large number of ellipsometric nanoparticles.e rings in the SAED pattern can be indexed as (002), ( 210), ( 211), (310), ( 222), (213), (004) and (304) re�ections of the hexagonal HAp, in agreement with the XRD results.No extra re�ections are observed and we can therefore conclude that the product consists of pure HAp ellipsometric nanoparticles.
ese results are well consistent with the XRD results [13], revealing that the doping components have little in�uence on the surface morphology of the samples.e morphology identi�cations 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, 0.4, and 0.5) synthesized at 100 ∘ C by coprecipitation method.Figure 3 shows the FT-IR results obtained from Ag:HAp-NPs when the  Ag increases from 0 to 0.5.e 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 synthesis products [19][20][21].
ese data clearly revealed 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.e peak observed at 634 cm −1 is attributed to the characteristic stretching and vibrational modes of structural OH groups [22,23].e band at 1630 cm −1 corresponds to the adsorbed H 2 O [23].
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 4. e OH − vibrational bands expected in the region of 630 cm −1 are not clearly detected.is behavior is in good accord with the previous studies [31].
We assigned the bands present at 1026 cm −1 ( 3 ), 1047 cm −1 ( 3 ), and 1073 cm −1 ( 3 ) to the asymmetric  3 (P-O) stretching.e internal modes of the PO 4 3− tetrahedral  1 frequency (960 cm −1 ) corresponds to the symmetric stretching of P-O bonds.e 616 cm −1 , 590 cm −1 , and 576 cm −1 bands arise from  4 PO 4 [28].e vibrational bands at 446 cm −1 ( 2 ) and 431 cm −1 ( 2 ) are attributed to the O-P-O bending modes.e band at 1070 cm −1 ( 1 ) attributed to CO 3 2− impurity is by the strong intensity PO 4 F 4: Raman spectra of the Ag:HAp samples synthesized with  Ag = 0, 0.4, and 0.5.weak intensities and were not detected [32].Water vibrational modes give rise to weak intensity stretching and bending T 1: e results of the qualitative screening of the antimicrobial activity of Ag:HAp compounds against different gram-positive and gramnegative bacteria, as well as fungal strains.

Microbial strains
Ag = 0  Ag = 0.4  Ag = 0. bands in Raman spectra.ese water bands, expected at about the same wave number in FTIR spectra, were not observed in Raman spectra.e qualitative antimicrobial activity of the tested compounds performed using stock solutions of 5 mg/mL obtained in dimethylsulfoxide (DMSO) allowed the selection of the active compounds, by the occurrence of high inhibition zones around the spotted compound.e results of the qualitative screening of the antimicrobial activity of Ag:HAp compounds against different gram-positive and gram-negative bacteria, as well as fungal strains are presented in Table 1.e tested compounds proved to be very active on C. krusei strain, irrespective of the Ag concentration.On the other hand, our tests proved them to be also active on P. aeruginosa 1397, S. aureus 0364, K. pneumoniae 2968, and E. cloacae 61R.e tested compounds, at certain concentrations, were also active against E. coli 714, E. coli ATCC 25922, and B. subtilis.
For the samples with  Ag = 0 the antibacterial activity was not present in the case of E. faecalis ATCC 29212, B. subtilis, E. coli ATCC 25922, K. Pneumoniae 2968, E. cloacae 61R, and S. aureus 0364 bacterial strains.On the other hand it showed an inhibitory effect on the fungal strain C. krusei 963 and the gram-negative strain P. aeruginosa 1397.
For  Ag = 0.4, the tested samples presented a good antibacterial activity both on gram-positive, gram-negative, and fungal strains, excepting E. coli 714 and E. faecalis ATCC 29212.
e samples with  Ag = 0.5 had a good antibacterial effect for all the tested microbial strains, except for E. faecalis ATCC29212.

Conclusions
Ag:HAp nanoparticles with  Ag = 0, 0.4, and 0.5 were prepared by coprecipitation method at low temperature and characterized by TEM, FT-IR, and FT-Raman.TEM investigations con�rmed the nanocrystalline nature of the synthesized powder and the ellipsometric morphology of the Ag:HAp particles.All 3 samples exhibit a uniform ellipsoidal morphology with particles from 5 nm to 15 nm.Bands characteristics of PO 4 3− tetrahedral apatite's structure are clearly observed by infrared and Raman analysis.e results of the qualitative screening of the antimicrobial activity of Ag:HAp compounds against different gram-positive and gram-negative bacterial strains proved to be very good irrespective of the Ag concentration.e speci�c antimicrobial activity revealed by the qualitative assay demonstrates that our compounds interacted differently with the microbial targets.

F 1 :F 2 :F 3 :
e TEM images of pure HAp ( Ag = 0) and Ag:HAp ( Ag = 0.4 and  Ag = 0.5) with low resolution.HRTEM image and SAED analysis of Ag:HAp with  Ag = 0.5.Transmittance infrared spectra of the Ag:HAp samples synthesized with  Ag = 0, 0.4, and 0.5.band at 1073 cm −1 .e other CO 3 modes  3 ,  4 , and  2 have band positions not obscured by the PO 4 bands, but they have