roxyapatite nanoparticles as multifunctional platforms for medical applications

Department of Mechanical Engineering, Es Instituto Politécnico de Setúbal, Setúbal, Po ICEMS, Instituto Superior Técnico, Technic Lisboa 1049 001, Portugal Laboratory for Bone Metabolism and Regen Universidade do Porto, Portugal MedInUP – Center for Drug Discovery and I Porto, Portugal Department of Materials and Ceramics E Materials, University of Aveiro, 3810-193 A ua.pt Max Planck Gesell, Fritz Haber Inst, Dept I Central Institute for Biomedical Technology Ulm, Germany Cite this: RSC Adv., 2015, 5, 69184


Introduction
The unprecedented developments in materials synthesis and characterization techniques at the nanoscale level are encouraging audacious approaches in matter manipulation and assembly, which are being exploited for designing nanostructures with improved or new properties and functionalities compared with their bulk counterparts.Nanomedicine is one of the numerous elds wherein such cutting-edge nanotech advancements are fostering innovative strategies similar to that of cancer theranostics. 1old nanoparticles (AuNP) are an emblematic example of biomedical nanostructures with a well-known surface plasmon resonance effect (SPR), the frequency of which is affected by nanoparticle (NP) size, shape, inter-distance and refractive index of surrounding NP media.This effect has been largely exploited for labeling and probing a wide variety of biomolecules that interfere with AuNP dispersed states and with the refractive index of the NP environment due to adsorption. 2 Such applications demand a close control of AuNP morphological attributes (size and shape) and in particular, of AuNP dispersed conditions.These prerequisites have been covered by a large number of publications and hence various proposals for AuNP production, including synthetic methods, reagents, solvents, and stabilizers, are found in recent literature.Currently, AuNPs are also being explored as photothermal contrast agents for cancer imaging and therapy. 3,4Plasmonic AuNP strategically located in tumors are used to enhance near-infrared laser absorption and heat dissipation and thus produce a local temperature increase that disturbs cellular functions, resulting in cell death.In addition, the X-ray absorbing properties of AuNPs can be also used for biodistribution data acquisition enabling non-invasive tumor imaging and 3D reconstruction. 3,5][8] Hydroxyapatite (Hap) is an appealing inorganic biomaterial.Its chemical composition, crystalline structure and particle morphology if appropriately engineered can replicate the chemical and physical attributes of the inorganic components of human bone and teeth 9,10 while offering enhanced adsorptive ability towards a considerable number of biomolecules and drugs. 11,12Addressing the combination of the exceptional properties of AuNPs and Hap and beneting from the easy functionalization of AuNPs for specic protein binding, hybrid hydroxyapatite gold nanoparticles (Hap-AuNPs) structures with a portfolio of interesting properties for various bioapplications, such as immunosensing, 13 bone-tissue regeneration 12,14,15 and improvement of hemocompatibility, 15,16 have been frequently reported.][15][16] However, gold and Hap particles are oen precipitated separately and then assembled to form the composite material. 12,13,16,17The Turkevich method 18 or a variant of it is still seen as the method of choice for AuNP synthesis, whereas no preferred method could be identied for Hap synthesis because of the successful implementation of multiple techniques. 19requently, polymers (e.g.collagen and chitosan) are required as linkers or agents for the coupling of gold and Hap. 14,15,17One simple method for producing uniform Hap nanoparticles (HapNPs) is hydrothermal synthesis in the presence of an organic chelating ion or molecule [20][21][22][23] used as a shape tailoring agent that allows the control of particle size and shape. 24,25ccording to recent reports, citrate ions could modulate prismatic HapNPs under specic hydrothermal synthetic conditions. 23he present study addresses AuNP nucleation and growth onto the surface of HapNPs for producing organized composite nanostructures.The ability of citric ions to play a dual role, i.e. as a HapNP tailoring agent and as an in situ reducing reagent of Au 3+ leading to local Au 0 nucleation and growth on HapNP facets is the engineering tool exploited herein.From an application perspective, widespread potential is anticipated, considering the benets of obtaining accurate control over the size, shape and surface chemistry of AuNPs with the high biocompatibility of HapNP.Due to bone affinity, biomimetic features and osteointegrity, HapNP is particularly suited for bone-related applications. 26,27Thus, in addition to the interest in AuNP usage as an excellent contrast element 28,29 depending on its size, the SPR tunability is envisaged with great usefulness for imparting simultaneous imaging and therapeutic abilities to Hap-AuNPs nanostructures, which makes them powerful tools for differentiated bone applications.Therefore, another point of interest is the discussion of the potential effects of HapNP assembled with AuNPs on the proliferation and differentiation of human mesenchymal stem cells due to their role in bone metabolism and regeneration, an issue that has not been covered so far.

Hydroxyapatite synthesis
A previously reported hydrothermal precipitation method 30 was followed for HapNP synthesis.The starting precursor solution was prepared in the following manner: a 0.6 M citric acid monohydrate (C 6 H 8 O 7 $H 2 O) (Riedel-deHaën, 99.5%) solution was mixed with a 0.2 M calcium nitrate Ca(NO 3 ) 2 4H 2 O (Riedel-deHaën, 99%) solution and a 0.2 M ammonium hydrogen phosphate (NH 4 ) 2 HPO 4 (Merck, 99%) solution was then added.The pH of the solution was adjusted to 8.1 with small additions of a 25% ammonia solution (Riedel-deHaën).The nal solution was poured into a 100 ml Teon vessel, lling up 50% of the total vessel volume.The Teon vessel was then sealed in a stained-steel autoclave and subsequently transferred into an oven at 180 C wherein it was held under autogenous pressure for 24 hours.The autoclave was then quenched in water at room temperature.Finally, the precipitated particles were collected and ltered using a glass Millipore lter vessel and then dried in a desiccator.

Gold precipitation
For the precipitation of gold, 86.5 ml of a hydrogen tetrachloroaurate (HAuCl 4 ) solution was added to 500 ml of deionized water in order to obtain a 0.25 Â 10 À3 M gold solution with pH of $3.25 ml of the gold solution was then heated at $100 C for 3 min and 50 mg of HapNP was subsequently added, which was synthesized as described in Section 2.1.The resulting suspension was maintained under boiling conditions for a period of 5 min.The overall procedure was repeated for covering various reaction times, i.e. boiling times of 10 min and 20 min.The particles (Hap-AuNP) were then repeatedly washed with deionized water during ltration with a glass Millipore lter vessel and then dried in a desiccator.

Particle characterization
The morphology of all particles obtained by precipitation was evaluated through electron transmission microscopy (TEM) using a Hitachi H-9000-NA microscope operated at 200 kV and high-resolution electron transmission microscopy (HRTEM) images were obtained on a JEOL 2200 FS microscope operating at 200 kV.For TEM analysis, the particles were dispersed with 2-propanol and scattered over copper grids coated with a formvar lm, and TEM images were used to determine AuNP sizes.More than 100 particles were randomly counted to determine the particle size distribution using Image-J soware.Particle crystalline phases were identied by powder X-ray diffraction (XRD) analysis (Rigaku PMG-VH with CuKa radiation ¼ 1.5405 Å).Fourier transform infrared spectroscopy (FTIR) was performed to identify the functional groups using a Mattson Galaxy 3020 spectrophotometer.The test samples were prepared by mixing $2 mg of the precipitated particles with $300 mg of spectroscopic-grade KBr (Merck), followed by shaping the mixture into a disk.The infrared spectra were acquired in transmittance mode in the region of 4000-400 cm À1 with a resolution of 4 cm À1 .The UV-vis absorption data were collected on a Shimadzu MPC-3100 PC series spectrophotometer using an auto scan between 200 and 700 nm.The specic surface area (SSA) of the obtained particles was determined through N 2 adsorption using a Micromeritics Gemini 2370 V5 Series.The samples were thoroughly degassed for 24 h at 120 C before nitrogen adsorption.The SSA was determined based on the multipoint Brunauer-Emmett-Teller isotherm (BET). 20

Interaction of Hap-AuNPs with human mesenchymal stem cells
Human mesenchymal stem cells-bone marrow-derived (HMSCbm, Innoprot), according to the supplier, were found to stain positive for CD44 and CD90, which are characteristic markers of the population phenotype.Third subculture cells were used to evaluate the cell response to the nanoparticles.HMSC, 10 4 cells per cm 2 , was cultured in Minimum Essential Medium Eagle, alpha modication (a-MEM, Sigma-Aldrich) containing 10% fetal bovine serum (FBS, Sigma-Aldrich), 50 mg ml À1 ascorbic acid, penicillin (10 units per ml)/ streptomycin (2.5 mg ml À1 ; P/S solution, Sciencell) and 2.5 mg ml À1 of Fungizone.Aer 24 h, the medium was removed and a completely fresh culture medium containing sterilized HapNP or Hap-AuNPs-1, 10, 100 and 500 mg ml À1 -was added to the adherent cells.Cultures were further incubated for 1, 3 and 7 days without any medium change.Cultures without nanoparticles were used as controls.Incubation was carried out under a humidied atmosphere of 95% air and 5% CO 2 at 37 C. Cultures were characterized in the following manner at days 1, 3 and 7.
DNA content.DNA content was analyzed by the PicoGreen DNA quantication assay (Quant-iT™ PicoGreen® dsDNA Assay Kit, Molecular Probes Inc., Eugene), according to the manufacturer's instructions.Cultures were treated with Triton X-100 (0.1%; Sigma-Aldrich), and uorescence was measured on an Elisa reader (Synergy HT, Biotek) at wavelengths of 480 and 520 nm for excitation and emission, respectively, and corrected for reagent blank uorescence.The amount of DNA was calculated by extrapolating a standard curve obtained by running the assay with the given DNA standard.
Cellular viability.MTS 31 and LDH 32 assays were used to estimate cell viability.The MTS assay is based on the reduction of an MTS tetrazolium compound to a purple formazan product by a mitochondrial reductase enzymes system on viable cells.An MTS solution (Cell Titer 96® Aqueous One Solution Cell Proliferation Assay, Promega), 20 ml, was added to the culture medium (100 ml, 96-well plates), and cultures were incubated for 4 h (in a humidied atmosphere of 95% air and 5% CO 2 at 37 C).The absorbance was measured at 492 nm on an ELISA reader (Synergy HT, Biotek).The lactate dehydrogenase (LDH) assay is based on the reduction of NAD by the action of LDH released to the medium due to cell damage (plasma membrane integrity) or lysis.The resultant reduced NAD (NADH) is used in the stoichiometric conversion of a tetrazolium dye.Total LDH determination was performed using the lactate dehydrogenase-based in vitro toxicology assay kit (Sigma-Aldrich; St. Louis, Mo.), according to the manufacturer's instructions.The amount of LDH leakage to the medium was normalized to total LDH.Results are expressed in the percentage of cell viability compared with the control.
Gene expression by RT-PCR.Adherent 24 h HMSC were exposed to 100 mg ml À1 HapNP or Hap-AuNPs for three days and were evaluated for the expression of the housekeeping gene GAPDH (glyceraldehydes-3-phosphate dehydrogenase) and the osteoblastic genes Runt-related transcription factor 2 (RUNX-2), alkaline phosphatase (ALP), osteocalcin (OC) and osteoprotegerin (OPG).RNA was extracted using an RNeasy® Mini Kit (QIAGEN), according to manufacturer's instructions.RNA was quantied by measuring the absorbance of the samples at 260 nm.RNA, 0.5 mg, was reverse transcribed and amplied (25 cycles) with the Titan One Tube RT-PCR System (Roche) at an annealing temperature of 55 C. Table 1 shows the primers used in the RT-PCR analysis.Aer electrophoresis on 1% (w/v) agarose gel, the bands were analysed through densitometry with the ImageJ 1.41 soware.Values were normalized to the corresponding GAPDH value of each experimental condition.
ALP activity.ALP activity was evaluated in cell lysates (0.1% Triton X-100, 5 min) by the hydrolysis of p-nitrophenyl phosphate in an alkaline buffer solution (pH of $10.3; 30 min, 37 C) and colorimetric determination of the product (p-nitrophenol) at 400 nm in an ELISA plate reader (Synergy HT, Biotek).ALP activity was normalized to total protein content (quantied by Bradford's method) and expressed as nmol min À1 mg protein À1 .Statistical analysis.Three independent experiments were performed; in each experiment, six replicas were set up for the biochemical assays and two replicas for the qualitative assays.The results are presented as mean AE standard deviation (SD).Data groups were evaluated using a two-way analysis of variance (ANOVA), and no signicant differences in the pattern of the cell behavior were found.Statistical differences between the experimental groups were assessed by Bonferroni's method.Values of p # 0.05 were considered statistically signicant.

Synthesis of functionalized hydroxyapatite nanosized particles (HapNP)
Literature reports on hydroxyapatite synthetic methods are plentiful.The choice of a particular synthetic methodology is normally dictated by the particle morphology and/or crystallinity criteria.In the present case, a previously reported hydrothermal precipitation method 23 was selected to obtain crystalline HapNP with nanometric dimensions and well-dened facets easily imaged using electron microscopy.In addition, other issues, including the surface chemistry of the precipitated particles, were taken into account.
Fig. 1(a) shows an image of HapNP as synthesized by the hydrothermal procedure.As observed, the particles display a light-yellowish colour.The crystalline phase composition of these particles is revealed by the XRD pattern presented in Fig. 2 (curve (a)), the diffraction peaks of which can be assigned to hydroxyapatite (Hap), according to the JCPDS no.09-0432.4][35] Furthermore, several features attributed to the organic groups are also identied in a range from 1680 cm À1 to 1350 cm À1 (Fig. 3(b)).The band at 1384 cm À1 may be ascribed to CH 2 scissoring, and carboxylate groups differently coordinated to the Hap surface under monodentate or bidentate congurations 6,30 may account for the bands at 1402, 1458, 1577 and 1669 cm À1 .[38][39] The particle morphology assessed by TEM and shown in Fig. 4(a) and (b) can be well described by a hexagonal prismatic shape with a width (w) of between 20 and 30 nm and a length (l) ranging from 50 nm to 100 nm.These nanoscale dimensions of the prismatic hydroxyapatite nanoparticles (HapNP) account for a large specic surface area of 55 m 2 g À1 and an aspect ratio (l/w) larger than 1.7.Fig. 4(b) shows the HRTEM image of a HapNPs wherein well-dened lattice fringe patterns are observed.The distance between adjacent lattice fringes corresponds to an interplanar distance of 0.65 nm that can be indexed to the d-spacing values of the hydroxyapatite crystal (010) planes, which are parallel to the c axis.The applied citratemediated hydrothermal synthesis thus favored preferential HapNP growth along the c-axis, which resulted in elongated particle morphology.
The interaction of citric acid with the HapNP surface and its effects on HapNP growth have been frequently addressed, from both experimental and theoretical perspectives.Calcium phosphate precursor solutions containing citric acid have been reported to favor the precipitation of HapNP with elongated shapes under varied conditions of temperature and pressure. 23ccording to computer simulation studies, 33,40 a strong binding of citric acid or of citrate ion to Hap lateral prismatic facets may slow their growth rates and as a result, enable their dominant expressions on the nal particle morphology.Such theoretical predictions might explain the current results assuming that citrate ions at an early stage of the hydrothermal processing and citrate-derived moieties at a later stage of the precipitation perform as lateral growth inhibitors, hence contributing to particle shapes.An accurate illustration of citrate's role under the temperature and pressure regime of this study requires a complete follow-up of the interplay among Hap nucleation, growth and citrate degradation progress, which is now an ongoing study.

Templating gold precipitation with HapNP
HapNP synthesized by a hydrothermal technique were used as substrates for AuNP growth.The effects of gold precipitation time were examined to assess its impact on the gold particle size and size distribution.
Gold particle crystal structure, morphology and SPR.HapNP aer submitting to the gold precipitation experimentation during various reaction times.The original yellowish colour of HapNP gives way to a purple colour as precipitation occurs, hence anticipating the presence of nanometric-sized gold particles in the Hap-AuNPs powders. 19Further evidence of gold precipitation is provided by the XRD patterns shown in Fig. 2(b)-(d), wherein the peak detected at 2q $ 38 was iden-tied as the more intense metallic gold peak, 41 according to the JCPDS no.071-4614.4(a) are now uniformly spotted with nanosized dark dots, the dimensions of which vary approximately from 1.5 to 2.5 nm as the synthesis time increases from 5 min (Fig. 5(a)) to 20 min (Fig. 5(b)).These are nanosized dots of metallic gold that were precipitated on HapNP facets, accounting for the HapNP colour variation previously described.It was also found that Hap-AuNPs aer 20 min of reaction time apparently have a lower density of larger AuNPs dots on their surfaces as documented by the particle-size distribution curves (Fig. 5(c)).This may be explained by an Ostwald ripening mechanism favouring the growth of larger AuNPs at the expense of the smaller. 19Although the dots become larger, they apparently preserve the spherical shape, which is known to be the lowest-energy shape, oen observed among metal nanoparticles obtained from the reaction of metal salts with reducing agents. 42The present strategy proved to be highly reproducible, consistently coupling uniform spherical AuNPs to HapNPs.
Fig. 6 shows the UV-vis spectra of as-prepared HapNP (a) and of Hap-AuNPs aer 5 min (b) and 20 min (c) of synthesis.No UV-vis band is detected in the range of 300-800 nm for asprepared HapNP, which conrms previous reports. 41In contrast, a characteristic SPR band of AuNPs is clearly observed around 550 nm in the Hap-AuNPs spectra.The observed SPR accounts for the strong colour changes observed on the particles 29 and conrms the formation of spherical AuNP by this synthetic method, which is in good agreement with the TEM results.Moreover, it was observed that the SPR band is similar for the two selected precipitation times, which may be related to the small variation in particle size, which is insignicant for detection by using UV-vis in powders.The SPR band shi from the well-known value of 520 nm reported for isotropic spherical gold particles (<20 nm) dispersed in water 43,44 to the observed value herein of $550 nm may reect the inuence of the substrate (Hap) refractive index (1.64),which is larger than that of water (1.33), to which the surrounding medium is generally referred. 45A similar shiing effect was also reported for gold nanoparticles of $5.5 nm deposited over SiO 2 /Si substrates. 46echanism of gold precipitation.The process of AuNPs growth on the HapNP surface was also followed by FTIR spectral analysis for the purpose of assessing the modications on     precipitation proceeds (Fig. 3(b)).In this spectral slot wherein citrate-derived species containing carboxylate groups coordinated differently to HapNP surfaces are expected to be detected, 47 several features are observed to have disappeared or undergone a strong intensity decrease as observed for the bands at 1384, 1567 and 1670 cm À1 , although the bands at 1402 cm À1 and 1458 cm À1 remain unaffected by the Au synthetic process.These results indicate that some adsorbed species containing COO À groups may be appropriately congured to participate in Au nucleation, 44 whereas other groups appear to be less strategic in the metallic precipitation.
As referred by Kumar et al., 48 gold precipitation in a bulk solution by the Turkevich method can be systematized according to the following steps.An initial step comprises the oxidation of citrate (CH 2 COO À ) 2 C(OH)COO À to an acetone dicarboxylic acid (CH 2 COO À ) 2 CO accompanied by the reduction of auric salt (AuCl 3 ) to aurous salt (AuCl).The subsequent step requires the disproportionation of AuCl into gold atoms (Au 0 ) and AuCl 3 , as schematized by the equation 3AuCl / 2Au 0 + AuCl 3 .This last step requires various aurous chloride molecules to be in a nearby condition, which is facilitated by an acetone dicarboxylic species that tethers molecular aurous chloride.
In this study, gold precipitation took place on the HapNP surface, most likely on the lateral prismatic faces as conrmed by TEM images.When HapNPs are added to the reagent HAuCl 4 aqueous solution (pH of $3), HapNPs acquire a neutral or weakly positive zeta potential 23 that favours electrostatic attachment of negatively charged auric complexes, i.e. [AuCl 3 -OH] À and/or [AuCl 2 OH 2 ] À , which are abundant species in an HAuCl 4 solution at pH 3-4. 49,50It is herein proposed that [R(COO À ) x ] ads groups undergo oxidation under the thermal conditions of the precipitation process (T ¼ 100 C), thus enabling the reduction of the auric complexes on the HapNP surface to aurous species and mimicking the role of acetone dicarboxylic acid reported by Kumar et al. 48Furthermore, the vicinity of the resulting aurous species will present an advantageous condition for the disproportionation reaction to proceed towards Au 0 nucleation.
Although previous reports have shown that gold precipitation may take place on pristine oxide surfaces wherein O À or OH À are the protagonist surface species interacting with solution gold complexes, [49][50][51] long precipitation times are thus required ($1 h) compared with the few minutes reported in this study.The applied HapNPs thus behaved as a substrate that prompted the heterogeneous nucleation of AuNPs without the addition of any reducing agent such as NaBH 4 , one of the most commonly used reagents for gold ion reduction. 44Another method for the deposition of gold on the surface of Hap nanoparticles was reported by J.-D.Wang et al. 52 By using urea as a precipitating agent, a gold precursor (Au(OH) 3 ) was precipitated, which was later converted to metallic gold upon heat treatment at 350 C. Therefore, compared with that synthesis route, the present method offers a straightforward way to produce metallic gold, exempting any precipitating agent or intermediate calcination step.The carboxylic species attached to the HapNP surface are thus a strategic tool for in situ gold reduction and nucleation hence leading to AuNPs uniformly distributed on HapNP facets, without the need of any additional surfactant for stabilization. 19Being attached to HapNP surfaces, AuNPs are not prone to aggregation, thereby eliminating potential drawbacks associated with aggregates.
Recent reports examined the multiple functionalities attributed to AuNP, several of which in a concerted manner may convey performance synergy in the eld of therapeutics. 53,54In the present case, the coupling of AuNPs to HapNPs imparts an SPR effect to the resulting composite nanostructure, which could be further exploited for thermotherapy: AuNPs are envisaged to empower HapNPs with hyperthermic capability for treating near-surface tumours or skin-type cancers wherein laser requirements are less strict as compared to tumours located deep within bodily tissue for which a laser light in the near-infrared (NIR) region of the biological water window (650-900 nm) would be recommended. 29Moreover, HapNPs can also behave as drug delivery systems (DDS) 55 due to their ability in the adsorption and release of therapeutic drugs. 55The endowment of a thermal response afforded by AuNPs to such DDS could open the possibility of customizing a HapNP-based DDS to a target of interest.It was reported that the addition of a thermo-responsive coating to a DDS would allow for the use of induced thermal stimulus to trigger drug delivery only at the local area of interest, 56 thus conning the drug effects to the unhealthy tissue while minimizing drug losses before the diseased cells are reached.In addition to thermotherapeutic benets, AuNPs may also provide imaging capabilities as demonstrated in a recent study wherein the ability of gold nanoparticles to perform as a CT imaging contrast agent for tumour imaging was reported. 54Therefore, the hydroxyapatite-based nanostructures (Hap-AuNPs) engineered in the present study offer promising characteristics for thermotherapeutic and imaging functionalities.The exploration of such attributes will warrant signicant interest because it may lead to the expansion of hydroxyapatite applications beyond its wellknown medical use in hard-tissue regeneration.

Interaction of Hap-AuNPs with human mesenchymal stem cells
As previously mentioned, because of the similarity of Hap to the mineral phase of bone tissue and thus its natural bone affinity and biocompatibility, a range of bone-tissue-related applications are envisaged for HapNPs. 26,27In a previous study, HapNPs similar to those reported herein proved to be non-toxic to MG63 osteoblastic cells in terms of cell viability/proliferation, F-actin cytoskeleton organization and apoptosis rate.In addition, they increased the expression of ALP and BMP-2. 23In order to assess the potential of the synthesized Hap-AuNPs for bone-related applications, the particles obtained aer 5 min of reaction time were selected to be cultured with human bone marrowderived mesenchymal stem cellsdue to their potential in bone regenerative approaches.HMSC were cultured under standard culture conditions, with the medium supplemented by ascorbic acid, because of its role in the synthesis of the bone collagenous extracellular matrix 57 but in the absence of any osteogenic inducer.HMSC cell response to the NPs was assessed for cell viability/proliferation and osteoblastic-related markers.DNA content.The DNA content of control cultures (in the absence of NPs) increased throughout the culture time.Comparatively, exposure to HapNP or Hap-AuNPs (1-500 mg ml À1 ) did not result in any obvious effects.As the DNA content reects the number of cells present in the cultures, this observation suggests that the two nanoparticles did not interfere in HMSC proliferation.Results are shown in Fig. 7(a).
Cellular viability.The viability of HMSC grown with the NPs was assessed by MTS reduction and LDH release assays.Results for the MTS assay, mostly based on the reduction ability of cell mitochondrial dehydrogenases by viable cells, showed similar values for the control and NPs-exposed cultures, as shown in Fig. 7(b).However, cultures exposed to 10 and 100 mm ml À1 NPs for 7 days displayed a slight increase in metabolic activity, which attained statistical signicance for Hap-AuNPs nanoparticles.On the other hand, cultures treated with 500 mg ml À1 HapNP or Hap-AuNPs, for a period of seven days, displayed a slight decrease, suggesting lower reduction ability.On the LDH assay shown in Fig. 7(c), the LDH released in the medium throughout the culture time revealed low values on the control cultures and those exposed to 1-100 mg ml À1 NPs, suggesting minimal deleterious effects on cell membrane integrity; however, an increase was observed in the presence of the higher nanoparticle levels (500 mg ml À1 ), following three and seven days of exposure, suggesting a somewhat compromised cell viability.Nevertheless, the increase on the percentage of LDH leakage was only around 9% aer seven days of exposure, compared with $6.5% in the control cultures.These observations are in line with that observed in the MTS assay.
F-actin cytoskeleton.The observation of cultures under CLSM, following staining for F-actin cytoskeleton and nucleus counterstaining, corroborated the low toxicity prole of HapNP and Hap-AuNP particles.The cell adhesion to the culture substrate and the subsequent cytoplasmic expansion were similar in both the absence and presence of the nanoparticles.On day 1, cells displayed an elongated morphology, establishing elementary cell-to-cell contacts.A high cell growth rate was then noticed.On day 3, areas of high cell density were already visualized and on day 7, cultures showed conuent zones with continuous cellular multilayers.Cells exhibited a well-organized F-actin cytoskeleton with intense staining at the cell boundaries, prominent nucleus and ongoing cell division, which are signs of mechanical integrity and a healthy behaviour.Representative images are shown in Fig. 8 for the cultures exposed to 100 mg ml À1 NPs.The F-actin cytoskeleton, which is highly concentrated just beneath the plasma membrane, provides structural stability and elasticity to the cell undergoing substrate adaptation, but it is also a key player in the cellular mechano-transduction mechanisms modulating complex signalling pathways, such as Rho family GTPases, which affects overall cellular behaviour. 58Thus, these results further support the cytocompatibility of HapNP and Hap-AuNPs towards mesenchymal stem cells.
Osteoblastic gene expression.In addition, gene expression by a reverse-transcription polymerase chain reaction (RT-PCR) was performed to investigate osteogenic signal expression in the presence of HapNP and Hap-AuNPs (100 mg ml À1 ), shown in Fig. 9(a).Hap-AuNPs greatly induced the expression of Runx2 ($100%) compared with the slight increase observed in HapNPs.This is an interesting nding, as Runx2 is the earliest transcription factor for the osteogenic differentiation pathway, and in addition, it is a master regulator for the expression of multiple late-stage genes as a determinant for osteoblastic differentiation Fig. 8 Representative confocal laser scanning microscopy images of HMSC cultured for 3 and 7 days in the presence of HapNPs and Hap-AuNPs, 100 mg ml À1 .Cultures were stained for the cytoskeleton (green) and nucleus (red).Cellular morphology and organization of the cell layer were similar in control and NP-exposed cultures.Bar: 50 mm.and bone formation. 57Both the particles showed a trend for an increase in the expression of Col 1, the main component of the bone extracellular matrix, synthesized during the proliferative stage of the osteoblastic differentiation pathway. 57It can be noted that Hap and Hap-AuNPs had a signicant impact in ALP expression with more than a fourfold increase in the expression level of this early osteoblastic differentiation marker.Osteocalcin expression, a late osteoblastic differentiation marker, with a role in the regulation of crystal growth, 57 was not particularly affected by HapNP or Hap-AuNPs.This suggests that these particles appear to not interfere in the regulatory mechanisms of the matrix mineralization.The expression of OPG, a key molecule in the interplay between osteoblasts and osteoclasts during bone remodelling, was not affected by Hap but was slightly reduced in the presence of Hap-AuNPs.
ALP activity.Cultures exposed to HapNP and Hap-AuNPs (100 mg ml À1 ) were also assessed for ALP activity.In line with the increased gene expression for this enzyme, ALP activity was greatly induced by the two nanoparticles.The inductive effect was observed already aer one day of exposure but greatly increased at day 3 and 7 ($150% and $80%, respectively; Fig. 9(b)).ALP, a hallmark of osteoblastic differentiation, is synthesized during the early matrix formation and maturation periods.This enzyme has an essential role in the onset of matrix mineralization, by providing phosphate ions which, together with calcium ions, ensure the chemical conditions for the osteoblastic-mediated mineral deposition. 570][61][62][63] However, regarding the present HapNP, the observed cell response is in line with previous reports showing low cytotoxicity of Hap nanoparticles towards osteoblastic cells, in addition to their ability to modulate cell proliferation and differentiation events.Upon exposure to HapNP, increased proliferation was found in bone marrowderived mesenchymal stem cells, 63 and enhanced osteoblastic differentiation features were also observed for these cells, 59,63 in addition to the osteoblastic cell lines hFOB 1. 19 (ref.62) and MG63. 23On the other hand, cytotoxicity/biocompatibility studies involving AuNPs coupled with hydroxyapatite are rare or even non-existent.In regard to AuNPs, the osteoblastic cell response also showed variable results.In the murine preosteoblastic cell line MC3T3-E1, AuNPs did not affect cellular viability; 64 in another study, they promoted their viability and osteogenic differentiation, depending on the particle size. 65In mesenchymal stem cells, AuNPs promoted viability and osteogenic differentiation, 66 but they were also reported to be toxic to these stem cells, depending on the particle size. 67AuNp aggregation states also inuence cellular uptake and cytotoxicity as reported for various cell types, 68,69 although not following a simple rule of thumb as aggregated AuNP may result in cytotoxicity or favour cell growth depending on the aggregate size, which in turn conditions its internalization. 68The conicting results could also arise from the variability of the applied toxicity assays, cellular systems, and chemical/physical properties of the nanoparticles; moreover, the dosing parameters and exposure times to AuNPs vary, which make comparisons difficult.In the present study, the synthesized Hap-AuNPs showed low cytotoxicity, and this may reect the use of a system in which small-sized AuNPs are xed to a HapNP template and thus inhibited from undergoing agglomeration.Hap-AuNPs also exhibited the ability to enhance early phenotype markers for osteogenic differentiation in HMSC, i.e.ALP expression and activity.Furthermore, the considerable increase in the expression of Runx2 is signicant, pointing to a higher commitment of mesenchymal stem cells toward the osteogenic lineage in the presence of these particles.In this regard, it has been reported that AuNPs are able to activate the p38 MAPK signalling pathway in the MSCs, which leads to the up-regulation of the osteogenic master transcription factor Runx2, driving MSCs to differentiate toward osteoblast cells. 66he potential of AuNPs for use in numerous different biological applications has led to strong interest in their cellular and tissue compatibility.In this regard, the response of the synthesized Hap-AuNPs towards mesenchymal stem cells seems to substantiate interesting features in bone-related applications from imaging and drug delivery to regenerative approaches.However, their ability to affect the fate of mesenchymal stem cells, i.e. by the enhancement of osteogenic differentiation pathways, should also be carefully considered in bone-related and other biomedical applications.

Conclusions
Gold nanoparticles (AuNPs) were successfully synthesized on the surface of hydroxyapatite nanoparticles (HapNP).The presence of organic species containing carboxylate groups on HapNP surfaces obtained by the synthetic processes of the HapNPs were a key condition for enabling in situ ionic gold reduction to metal gold, which resulted in spherical AuNPs with a diameter of few nanometers.The presence of nanometric gold on the resulting composite particle (Hap-AuNPs) imparts an SPR effect to the nal particles, which may be further explored for imaging and therapeutic purposes.Hap-AuNP interaction with mesenchymal stem cells converged to conrm the nanoparticles cytocompatibility and also their enhancing osteoblastic differentiation properties.In addition, this prole substantiates Hap-AuNPs as promising materials for bone-related applications.

Fig. 1 (
Fig. 1 Optical images of hydroxyapatite nanoparticles: (a) as synthesized at 180 C (HapNPs) and after submission to gold precipitation (Hap-AuNPs) during (b) 5 min, (c) 10 min and (d) 20 min.The particle colour evolution denotes the presence of metallic gold.

Fig. 5 (
Fig. 5(a) and (b) shows the HRTEM images of the Hap-AuNPs particles collected aer various precipitation times.It is clearly observed that the prismatic HapNP previously imaged in Fig.4(a) are now uniformly spotted with nanosized dark dots, the dimensions of which vary approximately from 1.5 to 2.5 nm as the synthesis time increases from 5 min (Fig.5(a)) to 20 min (Fig.5(b)).These are nanosized dots of metallic gold that were precipitated on HapNP facets, accounting for the HapNP colour variation previously described.It was also found that Hap-AuNPs aer 20 min of reaction time apparently have a lower density of larger AuNPs dots on their surfaces as documented by the particle-size distribution curves (Fig.5(c)).This may be explained by an Ostwald ripening mechanism favouring the growth of larger AuNPs at the expense of the smaller.19Although the dots become larger, they apparently preserve the spherical shape, which is known to be the lowest-energy shape, oen observed among metal nanoparticles obtained from the reaction of metal salts with reducing agents.42The present strategy proved to be highly reproducible, consistently coupling uniform spherical AuNPs to HapNPs.Fig.6shows the UV-vis spectra of as-prepared HapNP (a) and of Hap-AuNPs aer 5 min (b) and 20 min (c) of synthesis.No UV-vis band is detected in the range of 300-800 nm for asprepared HapNP, which conrms previous reports.41In contrast, a characteristic SPR band of AuNPs is clearly observed around 550 nm in the Hap-AuNPs spectra.The observed SPR accounts for the strong colour changes observed on the particles29 and conrms the formation of spherical AuNP by this synthetic method, which is in good agreement with the TEM results.Moreover, it was observed that the SPR band is similar for the two selected precipitation times, which may be related to the small variation in particle size, which is insignicant for detection by using UV-vis in powders.The SPR band shi from the well-known value of 520 nm reported for isotropic spherical gold particles (<20 nm) dispersed in water43,44 to the observed value herein of $550 nm may reect the inuence of the substrate (Hap) refractive index (1.64),which is larger than that of water(1.33), to which the surrounding medium is generally referred.45A similar shiing effect was also reported for gold nanoparticles of $5.5 nm deposited over SiO 2 /Si substrates.46Mechanism of gold precipitation.The process of AuNPs growth on the HapNP surface was also followed by FTIR spectral analysis for the purpose of assessing the modications on

Fig. 2 X
Fig. 2 X-ray diffraction patterns of hydroxyapatite nanoparticles: (a) as produced at 180 C (HapNPs) and after submission to gold precipitation (Hap-AuNPs) during (b) 5 min, (c) 10 min and (d) 20 min.The peak detected at 2q $ 38 is assigned to metallic gold.

Fig. 4
Fig. 4 (a) TEM images showing the elongated prismatic shape of HapNPs.(b) The hexagonal base of prismatic particles is confirmed on a particle top view.

Fig. 5
Fig. 5 HRTEM images of Hap-AuNPs collected after two different precipitation times: (a) 5 min and (b) 20 min.The particles are spotted with nanosized dark dots of metallic gold (AuNP), which were precipitated on HapNP facets.(c) AuNP size distribution precipitated after 5 min and 20 min.

Fig. 6
Fig. 6 UV-vis spectra of (a) HapNPs synthesized at 180 C and after reacting with the gold solution (Hap-AuNPs) during (b) 5 min and (c) 20 min.The characteristic surface plasmon resonance band of gold nanoparticles is clearly observed at $552 nm for the Hap-AuNPs.

Fig. 7
Fig. 7 (a) DNA content, (b) MTS reduction and (c) LDH leakage of HMSC cultured for 1, 3 and 7 days in the presence of HapNPs and Hap-AuNPs, 1-500 mg ml À1 .NPs did not interfere with the cellular proliferation and metabolic activity in the range 1-100 mg ml À1 , but exposure to 500 mg ml À1 caused a slight decrease in cellular viability.*Significantly different from control (absence of NPs).

Fig. 9
Fig. 9 (a) Gene expression and (b) ALP activity of HMSC cultured in the presence of HapNPs and Hap-AuNPs, 100 mg ml À1 ; (a) cultures with 3 days; (b) cultures with 1, 3 and 7 days.*Significantly different from control (absence of NPs).Hap-AuNPs caused a significant increase in the expression of Runx2, and both NPs increased ALP expression and activity.

Table 1
Primers used on RT-PCR analysis