Acta Biomaterialia 9 (2013) 5100–5110

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Acta Biomaterialia

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Electron storage mediated dark antibacterial action of bound silver nanoparticles:Smaller is not always better

Huiliang Cao a, Yuqin Qiao a, Xuanyong Liu a,⇑, Tao Lu a, Ting Cui a, Fanhao Meng a, Paul K. Chu b

a State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences,Shanghai 200050, People’s Republic of Chinab Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 July 2012Received in revised form 26 September2012Accepted 11 October 2012Available online 17 October 2012

Keywords:NanoparticleIon implantationSilverAntibacterialTitanium oxide

1742-7061/$ - see front matter � 2012 Acta Materialhttp://dx.doi.org/10.1016/j.actbio.2012.10.017

⇑ Corresponding author. Tel./fax: +86 21 5241 2409E-mail address: xyliu@mail.sic.ac.cn (X. Liu).

Size tunable silver nanoparticles (Ag NPs) are synthesized and incorporated into titanium oxide coatings(TOCs) by manipulating the atomic-scale heating effect of silver plasma immersion ion implantation (AgPIII). The resulting Ag NPs/TOC composite coatings possess electron storage capability that gives rise toboth controlled antibacterial activity and excellent compatibility with mammalian cells. The precipitationbehavior of these Ag NPs is qualitatively constrained by the classical nucleation theory. Both photolumi-nescence (PL) spectra and fluorescence microscopy results demonstrate that larger Ag NPs (5–25 nm) arebetter at reserving electrons than smaller ones (�4 nm). The antibacterial activities of the as-sprayed andAg PIII treated TOCs show that Ag NPs with a different size act distinctively to bacteria: large particlesinduce serious cytosolic content leakage and lysis of both Staphylococcus aureus and Escherichia coli cellswhile small ones do not. The excellent activity of larger Ag NPs against bacteria is highly related to theirstronger electron storage capability, which can induce accumulation of adequate valence-band holes (h+)at the titanium oxide side, arousing oxidation reactions to bacterial cells in the dark. Moreover, the in vitrocell culture assay (using both MG63 and MC3T3 cells) reveals no significant cytotoxicity and even goodcytocompatibility on the Ag PIII treated samples. Our results show that, by taking advantage of the bound-ary property between Ag NP and titanium oxide, the antibacterial activity of Ag NPs can be accurately con-trolled. This study provides a distinct criterion for the design of nanostructured surfaces such that theirosteoblast functions and antibacterial activity are perfectly balanced.

� 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Bacterial contamination and the associated risk of infectionconstitute one of the most serious complications related to medicaldevices [1,2]. Because of its excellent photocatalytic activity,titanium oxide can prevent adhesion of microbes [3]. It is generallyrecognized that the biocide process involves excitation of titaniumoxide with photons of energy at least equal to the band gap energyproducing electron/hole (e�/h+) pairs, which further induce strongredox reactions destroying bacterial cells [4]. Although dopingwith oxygen vacancies [5], nitrogen [6] and noble metals [7] mayfacilitate charge separation in titanium oxide [8] and dramaticallyimprove its light-activable bactericidal activity, the effect will fadeaway when the light is turned off [9,10], limiting their applicationsunder dark conditions.

Moreover, antibacterial strategies targeting bacterial membranefunctions are promising in treating persistent infections [11,12].Silver nanoparticles (Ag NPs) are known to interact with the mem-

ia Inc. Published by Elsevier Ltd. A

.

brane constituents, causing structural changes, degradation andeventual cell death [13], but the exact mechanism is still contro-versial due to the difficulty in discerning the relative effects of allthe possible antimicrobial actions rendered by mobile Ag NPs. Inrecent years, controlling the antibacterial actions of Ag NPs isbecoming the major research field. Gunawan et al. reported thatAg NPs possesses a reversible photo-switching antibacterial prop-erty when they are deposited on a semiconductor support [14].Prucek et al. demonstrated that targeted transport of Ag NPs canbe realized by combining magnetic iron oxide and antimicrobialAg NPs together [15]. These studies indicate that the antibacterialperformance of Ag NPs is highly related to the properties of theirservice environments. Our previous study shows that the localcathodic reactions of Ag NPs due to the micro-galvanic effect facil-itate the formation of proton depleted regions, resulting in thedeath of bacterial cells [16]. However, the biocide activity of thoseAg NPs is basically determined by the galvanic current, that is, thecontinuous electron transfer efficiency between Ag NPs and thetitanium matrix. Thus, when Ag NPs embedded in a material thatis not so good at conducting as titanium, the micro-galvanic effectcontrolled antibacterial activity of Ag NPs may be weakened.

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Table 2Details of the Ag PIII parameters for the samples involved.

Samples Ag PIII parameters

0.5 h-30Ag PIII Bias voltage 30 kV, duration 0.5 h1.0 h-30Ag PIII Bias voltage 30 kV, duration 1.0 h1.5 h-30Ag PIII Bias voltage 30 kV, duration 1.5 h1.0 h-14Ag PIII Bias voltage 14 kV, duration 1.0 h

H. Cao et al. / Acta Biomaterialia 9 (2013) 5100–5110 5101

Actually, when Ag NPs contact with a semiconductor such as tita-nium oxide, a so-called Schottky barrier will be established due tothe Fermi level alignment at the metal (Ag NP)/semiconductor(titanium oxide) interface [17], endowing the Ag NPs with electrontrapping capability. The stored electrons on the Ag NPs can arouseaccumulation of valence-band holes (h+) on the semiconductorside, and lead to notable oxidation reactions and biocide actionthat are independent of light illuminations.

In order to explore how to control the antibacterial activity ofAg NPs by adjusting their electron storage capability, in this work,size tunable Ag NPs are incorporated into the plasma-sprayed tita-nium oxide coatings (TOCs) by manipulating the atomic-scaleheating (ASH) effect of silver plasma immersion ion implantation(Ag PIII). The antibacterial activities of these bound Ag NPs in thedark indicate that the Ag NPs/titanium oxide system possesses tun-able electron trapping capability targeting the bacterial membrane,and the ability highly depends on the size of the incorporated AgNPs.

2. Materials and methods

2.1. Sample fabrication and characterization

2.1.1. Plasma spraying of titanium oxide coating (TOC)Plasma spraying was carried out on an atmospheric plasma

spray system (APS-2000, Sulzer Metco, Switzerland). The sprayingparameters are listed in Table 1. Commercial TiO2 powders wereused to deposit titanium oxide layers onto the 10 mm square CpTi (Grade 2) plates (with a thickness of 1 mm).

2.1.2. Ag plasma immersion ion implantation (Ag PIII)The as-sprayed TOC samples were transferred into the chamber

of a plasma immersion ion implantation system where silver wasimplanted into the TOC side using a filtered cathodic arc plasmasource. The Ag PIII process was detailed in our previous report[16]. The involved TOCs treated with various implantation param-eters are detailed in Table 2.

2.1.3. Surface chemistry and structure characterizationThe surface morphologies of the Ag PIII treated TOCs were

examined by scanning electron microscopy (SEM; JEOLJSM-6700F, Japan). X-ray diffraction (XRD) spectra were acquiredon a PHILIPS X’Pert MPO Pro X-ray diffractometer with Cu Ka radi-ation at 40 kV and 40 mA under the conventional Bragg diffraction(CBD) modes. The Ag depth profiles and chemical states weredetermined by X-ray photoelectron spectroscopy (XPS) (Physicalelectronics PHI 5802). Both the planar and cross-sectional viewsof the Ag PIII treated TOCs were investigated by transmission elec-tron microscopy (TEM; JEM2100F). The TEM specimens werethinned using a Gatan 691 ion-thinning system. Photolumines-cence spectra were obtained using a SHIMADZU RT-5301PL Spec-tro Fluorophotometer.

2.1.4. Silver releaseThe Ag PIII treated TOCs were incubated for various periods in

10 ml of pure water at 37 �C without stirring. The amounts of re-leased silver were determined by analyzing the resulted solutions

Table 1Parameters for plasma spraying of titanium oxide coating (TOC).

Plasma gas Ar 40 slpm Powder feed rate 20 g/minPlasma gas H2 12 slpm Current 600 ASpray distance 100 mm Voltage 60 VPowder carrier gas Ar 3.0 slpm Angle of feed powder 90

via inductively coupled plasma optical emission spectrometry(ICP-OES).

2.2. Antibacterial activity and cytocompatibility evaluation

2.2.1. Antibacterial testsThe antibacterial activities on the as-sprayed and Ag PIII treated

TOCs were evaluated by the bacterial counting method usingStaphylococcus aureus and Escherichia coli. Staphylococcus aureuswas cultured in tryptic soy broth (TSB) or TSB agar plates whileE. coli was cultured in Luria–Bertani (LB) broth or LB agar plates.The samples were sterilized in an autoclave at 121 �C for 40 min.A bacterial suspension with a concentration of 1.0 � 108 cfu ml�1

was introduced onto the samples to a density of 0.06 ml cm�2.The samples with the bacterial suspension were incubated at37 �C for 24 h in the dark. For quantitative analysis of the reductionrate, the samples with the bacterial suspension (after the incuba-tion) were put into each test tube with 5 ml physiological saline.The test tube was vigorously vortexed for over 60 s using a vortexmixer to detach the bacteria from the coating surface. Subse-quently, the detached bacterial suspension was serially diluted inten-fold steps with sterile physiological saline. Then 200 ll of thediluted bacterial suspension was inoculated onto TSA (for S. aureus)or LB (for E. coli) agar plates. After incubation at 37 �C for 24 h, theactive bacteria were counted in accordance with the National Stan-dard of China GB/T 4789.2 protocol. For SEM observations, the bac-teria were put on the sample, incubated at 37 �C for 24 h in thedark, fixed and dehydrated according to the same procedures (de-tailed in Section 2.2.3) used on the osteoblast-like cells.

2.2.2. Cell proliferation and viabilityThe osteoblast-like cell line MG63 and MC3T3 cell line from

mouse (Cells Resource Center, Shanghai Institutes of BiologicalScience, Shanghai, P.R. China) were seeded on the as-sprayed andAg PIII treated TOC to evaluate the cytocompatibility accordingto the AlamarBlue™ assay (AbD Serotec Ltd, UK). The cell cultureprocess was detailed in our previous report [16]. Briefly, five sam-ples were tested for each incubation period (up to 9 days). Aftereach incubation period, the culture medium was removed and1.0 ml of the fresh medium with 5% AlamarBlue™ was added toeach well. After incubation for 5 h, 100 ll of the culture mediumwas transferred to a 96-well plate for measurement. Accumulationof reduced AlamarBlue™ in the culture medium was determinedby an enzyme labeling instrument (BIO-TEK, ELX 800) at extinctionwavelengths of 570 nm and 600 nm. The operation procedures andcalculation of cell proliferation or viability of cells followed theinstruction of the AlamarBlue™ assay.

2.2.3. Cell morphology observationAfter each time point, the samples were taken out and rinsed

with a phosphate buffered saline (PBS) solution (pH = 7.2) twiceto remove the unattached cells and fixed with 3% glutaraldehydesolution in a sodium cacodylate buffer (pH = 7.4, Gibco, Invitrogen)for 30 min after removal from the culture plate. Prior to SEM, thespecimens were dehydrated in a series of ethanol solutions (30,50, 75, 90, 95 and 100 vol.%) for 10 min each sequentially, withthe final dehydration conducted in absolute ethanol (twice)

5102 H. Cao et al. / Acta Biomaterialia 9 (2013) 5100–5110

followed by drying in the hexamethyldisilizane (HMDS) ethanolsolution series.

2.2.4. Fluorescence microscopyFor chromosomal DNA staining, the as-sprayed and Ag PIII trea-

ted TOCs are sterilized in an autoclave at 121 �C for 40 min and putinto a 24-well culture plate (Costar, USA). A solution containing S.aureus cells at a concentration of � 108 cfu ml�1 is introduced ontothe sample with a density of 0.06 ml cm�2. The samples togetherwith the bacterial solution are incubated in the dark at 37 �C for12 h, and then the samples are rinsed with a PBS solution(pH = 7.2) twice to remove the unattached cells and the residualculture medium. Afterwards, 500 ll solution of Hoechst 33342(suspended in PBS, 10 lg ml�1) is added to each well, incubatedat 37 �C for 12 min, rinsed with PBS twice, fixed with a 4% parafor-maldehyde (PFA) solution at ambient temperature for 10 min andrinsed with PBS twice again. Finally, the samples are observed un-der an optical microscope (Olympus GX71 equipped with a UVlight source for excitation of Hoechst 33342) by immersing thefront of the sample in a glycerol solution containing 10% water(Fig. S1, Supplementary materials). For nucleus DNA staining(mammalian cells), rat bMSCs were cultured on the Ag NPs/TOCsurfaces for 24 h according to the literature [18], and then their nu-clei are stained with Hoechst 33342. The staining process andobservation procedure are similar (without glycerol solution) tothat for bacteria.

2.3. Statistical analysis

Statistically significant differences (p) between the variousgroups were measured using one-way analysis of variance and Tu-key’s multiple comparison tests. All statistical analysis was carriedout with a GraphPad Prism 5 statistical software package.

3. Results

3.1. Fabrication and characterization of bound silver nanoparticles

Fig. 1 shows the microstructure evolution on the plasma-sprayed TOC surface after undergoing Ag PIII at 30 kV for 0.5 h,1.0 h and 1.5 h (designated as 0.5 h–30Ag PIII, 1.0 h–30Ag PIIIand 1.5 h–30Ag PIII, respectively). The as-sprayed TOC, rough onthe micro-scale (Fig. 1a) but smooth on the nano-scale (the insertin the upper-right corner of Fig. 1a), contains rutile and anatasephases of TiO2 and titanium sub-oxides (mainly Ti3O5) as the majorphases according to the powder X-ray diffraction spectrum (the in-sert in the lower left corner of Fig. 1a). After Ag PIII, nanoparticlescan be detected from all kinds of samples (Fig. 1b, c and d), but theparticle size and distribution are different among them. The parti-cles comprise a mixture of two distinctive size groups on 1.0 h–30Ag PIII and 1.5 h–30Ag PIII, which is contrast to the 0.5 h–30AgPIII. This result is depicted more apparently by the lower magnifi-cation SEM images (Fig. S2, Supplementary materials). Althoughthe particle size distributions in 0.5 h–30Ag PIII, 1.0 h–30Ag PIIIand 1.5 h–30Ag PIII are entirely different, the chemical states of sil-ver in these surfaces are similar. As shown in Fig. 1e, the X-ray pho-toelectron spectroscopy (XPS) Ag 3d spectra show no noticeablebinding energy shift between the three Ag PIII samples, implyingthat the silver chemical states in these samples are about the same.The Ag 3d doublet at �374.1 eV (Ag 3d3/2) and 368.1 eV (Ag 3d5/2) correspond to metallic silver [19]. As shown by the XPS depthprofiles in Fig. 1f, the amount of Ag increases with implantationtime but the total penetration depth of Ag ceases to increase after1.0 h, suggesting that the previous implanted silver atoms act asbarriers which reduce penetration depth of the coming silver ions

and just rest them at the outermost surface as the Ag PIII proceduresustained for over 1.0 h. This is consistent with rise style in Ag con-centrations at the outermost surface. The Ag concentration peak is�4.0 at.%, 5.5 at.% and 8.5 at.% for the 0.5 h-, 1.0 h- and 1.5 h–30AgPIII, respectively.

The 1.5 h–30 Ag PIII sample was explored by TEM techniques.Fig. 2a displays the typical bright-field (BF) TEM image of the pla-nar view of the particles on 1.5 h–30Ag PIII together with the cor-responding energy-dispersive X-ray spectroscopy (EDS) spectrum.High-resolution transmission electron microscopy (HR-TEM) indi-cates that the particles have multiple twined structures and arewell bound to the substrate coating. As shown in the HR-TEM lat-tice pattern (Fig. 2b) of the encircled area in Fig. 2a, multiple direc-tions of the {111} planes can be discerned from a single particle.The corresponding fast Fourier transform (FFT) pattern shows theparticle is a single-crystal silver (Fig. 2c). Cross-sectional TEMexamination reveals that the Ag NPs in 1.5 h–30Ag PIII are disper-sive underneath the surface (Fig. S3, Supplementary materials).The structure of these Ag NPs suggests that their precipitationbehavior is dictated by the principle of minimum energy.

Ag NPs can be synthesized and bound by implanting silver. Dur-ing silver ion implantation, energetic silver ions are stopped by thesubstrate, and some of their kinetic energy is converted into ther-mal energy in a confined region within a very short time. The pro-cess is termed atomic-scale heating (ASH) [20]. What is more, onaccount of the distinctive non-line-of-light nature of Ag PIII, silverions are penetrated in an immersion configuration and heating viarandom ASHs which may evoke silver concentration fluctuations inthe TOC surface, i.e., the thermal energy converted from thoseincoming ions may dislodge the stationary silver atoms to diffuse,enabling the formation of critical clusters and growth of stablenanoparticles, or dismissing the Ag atoms composed of the clus-ters. Accordingly, the variation of silver concentration in the outersurface can alter the precipitating behavior of silver particles onTOC. In the case of Ag PIII at 30 kV (Fig. 1f), the silver depth profilesees a continuous silver concentration increase in the depth of 5 to55 nm as the implantation time is increased from 0.5 to 1.0 h, but itceases to increase when the duration exceeds 1.0 h. As a result, anabrupt silver concentration increase in the outermost surface(depth of 0–5 nm) can be detected in 1.5 h–30 Ag PIII: within thesame interval of 0.5 h, the peak silver concentration increased to3.0 at.% when the Ag PIII duration was prolonged from 1.0 h to1.5 h, which is twice of that (1.5 at.%) prolonged from 0.5 h to1.0 h. Such an abrupt increase in silver concentration likely ex-plains the size distribution variation of the bound Ag NPs(Fig. S2, Supplementary materials). The effects of silver concentra-tion on the precipitating behavior of Ag NPs could be qualitativelyunderstood by the classical nucleation theory. The relationship be-tween the number (Ns) of the precipitated spherical clusters andsaturation degree (S) of silver concentration in the outer surfacecan be expressed as Eq. (1) [21]:

Ns ¼ NACeqS� exp �DGs

RT

� �ð1Þ

where NA is Avogadro’s constant, S can be further defined as the ra-tio between the solute concentrations at saturation (Cs) and equilib-rium (Ceq) conditions, RT is the product of ideal gas constant (R) andthe absolute temperature (T) and the excess free energy (DGs) canbe described by Eq. (2) [21]:

DGs ¼ 4pr2c� 43pr3 RT ln S

Vmð2Þ

where c is the surface tension of the cluster and Vm is the molar vol-ume of the bulk crystal. Under the supersaturated conditions, i.e.S > 1, DGs decreases with increasing cluster radius (r) and the clus-

Fig. 1. SEM images of (a) the as-sprayed TOC with XRD spectrum and higher magnification image inserted, (b) 0.5 h–30 Ag PIII, (c) 1.0 h–30 Ag PIII and (d) 1.5 h–30 Ag PIII,accompanied by the corresponding Ag 3d XPS spectra (e) and the depth profiles of silver (f).

H. Cao et al. / Acta Biomaterialia 9 (2013) 5100–5110 5103

ters become stable. Substituting Eq. (2) into Eq. (1) gives the criticalclusters number Ns as Eq. (3):

Ns ¼ NACeq � exp �4pr2cRT

� �� Sð

4pr33Vmþ1Þ ð3Þ

Therefore, under supersaturated conditions (S > 1), Ns is di-rectly proportional to S to the power of ð4pr3

3Vmþ 1Þ which is larger

than one, suggesting that a slight increase of saturation degree(S) may result a notable increase in cluster distribution density.Furthermore, the minimum radius of a nucleus (rm) that can growspontaneously under the supersaturated conditions is defined as

the value of r at which DGs is maximum. Consequently, based onEq. (2), setting dDGs=dr ¼ 0 allows the determination of rm as Eq.(4) [21]:

rm ¼2cVm

RT ln Sð4Þ

According to Eq. (4), rm is inversely proportional to ln S, indicat-ing that an increase in S results in a reduced minimum nucleus ra-dius (rm). The predictions of Eqs. (3) and (4) are consistent with themicrostructure evolution in Fig. 1, which demonstrates that low-density, large-size group A Ag NPs are formed in the low saturationdegree region (Fig. 1b) whereas the high-density, small-size group

Fig. 2. Planar TEM view of the 1.5 h–30Ag PIII surface: (a) BF image acquired at low magnification with its corresponding EDS spectrum being inserted, (b) HR-TEM latticepattern of the encircled area in (a) and (c) the corresponding fast Fourier transform (FFT) of (b).

Table 3Energy gain (in eV) of a silver ion with a mean charge state of Q � 2.

Eo QeVs Ei Ec E_{o}^{sum}

69.00a 6.00E4b 2.10b 2.95a 29.10a

a From reference [20].b Calculated with Vs � 30 kV which is approximate to the bias voltage in the

present discussion.

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B Ag NPs are formed in the high saturation degree region (Fig. 1cand d). The change in the saturation degree is evidenced by theabrupt increase of silver contraction in the outermost surface asthe Ag PIII duration is prolonged (Fig. 1f).

The said abrupt silver concentration increase in the outermost sur-face also indicates that controlling the penetration depths of theincoming energetic silver ions is important in adjusting the size distri-bution of bound Ag NPs. As previously mentioned, the resting of a sil-ver ion is mainly an energy transformation process, and the trajectorydepth of an arriving atom can be reduced by down-regulating its pre-vious energy gain. In the present case, the silver ions produced by acathodic arc are subjected to three zones of acceleration. The first isnear the cathode spot that leads to the initial ion kinetic energy(Eo). The second zone is in the space-charge sheath between the sil-ver plasma and TOC surface. By applying a negatively biased volt-age (�Vs) to the TOC coating, additional kinetic energy (QeVs,where e is the elementary charge) is given to the ion of charge stateQ as it passes through this zone. The third is in the nano-scalevicinity of the TOC surface where an image charge acceleration in-duced kinetic energy (Ei) is added. In addition, the silver ion alsopossesses a significant potential energy which includes cohesiveenergy Ec , excitation energy of bound electrons Ee, and ionizationenergy EQ . The ionization energy EQ is defined as the energyneeded to remove a bound electron from an ion of charge stateQ , forming an ion with a charge state of Q þ 1 [20]. To calculatethe ionization energy of a multiply charged silver ion, one needsto add the ionization energies of all the ionization steps (Esum

Q ),i.e. Esum

Q ¼PQ�1

i¼0 EQ ðiÞ. The general expression for the total energygain of an incident silver ion can be described by Eq. (5) [20]:

Et ¼ Eo þ QeVs þ Ei þ Ec þ Ee þXQ�1

i¼0

EQ ðiÞ ð5Þ

The reported and calculated components of the energy gain fora silver ion (with the mean charge state of Q � 2) impacting theTOC surface are listed in Table 3. The charge state value of Q � 2

is about the mean charge reported in the literature [22]. The con-tribution of the electronic excitation energies (Ee) is relativelysmall [23] and neglected in Table 3. The kinetic energy gainedvia image charge acceleration Ei is calculated according to Eq. (6)initially proposed for metallic substrate [24]:

Ei ¼w2

XQ�1

j¼0

2ðQ � jÞ � 1ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi8ðQ � jÞ þ 2

p ð6Þ

Given the work function of the as-sprayed TOC surface,w � 4:13eV [25], we may determine the image charge induced ki-netic energy, Ei � 2:10 eV .

Based on the data in Table 3, the entire energy gain of a singlesilver ion is far more than the minimum displacement energy (inthe range of 10–40 eV [20]), and hence, it is sufficient to dislodgeand move stationary silver atoms in the TOC matrix. A silver ionmay gain 99.8% of its integral energy after passing through thehigh-voltage sheath, implying that the total energy gain by a silverion is predominated by the sheath voltage, i.e. the bias voltage(�Vs) applied on the TOCs. Therefore, the penetration depth of asilver ion and consequently the saturation degree of silver in theoutermost surface can be manipulated by simply adjusting the biasvoltage. Fig. 3a shows the typical microstructure of the as-sprayedTOC after undergoing Ag PIII at 14 kV for 1.0 h (designated as 1.0 h-14 Ag PIII). Although the size distribution style (Fig. 3b) is different

Fig. 3. SEM microstructure of the TOC Ag PIII-treated at 14 kV for 1.0 h (a), and the corresponding particle size distribution (b), Ag 3d XPS spectrum (c), and depth profile ofsilver (d).

Fig. 4. Photoluminescence (PL) spectra of the as-sprayed and Ag PIII treated TOCs,kEx = 300 nm.

H. Cao et al. / Acta Biomaterialia 9 (2013) 5100–5110 5105

to that at 30 kV (Fig. 1b, c and d), the chemical state of silver(indicated by the Ag 3d spectrum in Fig. 3c) is similar to that at30 kV (Fig. 1e). Because the total energy gain by a silver ion (Et,14

� 2.80 � 104 eV) at 14 kV is about one-half of that at 30 kV (Et,30

� 6.00 � 104 eV), and accordingly, for the same duration, the satu-ration degree of silver in the outermost surface at 14 kV can reach ahigh level faster than at 30 kV (the peak silver concentration for1.0 h-14 Ag PIII is 6.6 at.%, which is 1.1 at.% higher than that of1.0 h–30 Ag PIII, Figs. 3d and 1f). Consequently, the particles fabri-cated at 14 kV (1.0 h-14Ag PIII) (Fig. 3b) are generally smaller thanthat produced at 30 kV (Fig. S2b, Supplementary materials), but thecorresponding particle distribution density exhibits the reversetrend, which are consistent with Eqs. (3) and (4).

3.2. Electron storage behaviors

The intrinsic electron storage capability of the bound Ag NPscan be evaluated using photoluminescence (PL) spectra [26]. De-creased PL signals were detected on all the Ag PIII treated TOCsas compared with that of the as-sprayed TOC (Fig. 4), indicatingthat Ag NPs indeed serve as efficient electron traps preventingthe recombination of electrons and holes. Moreover, the PL signaldecreases for 1.0 h-14 Ag PIII and 0.5 h–30 Ag PIII are comparable,though the particle distribution density of the former one is �23times larger than that of the latter one, revealing that large boundAg NPs (0.5 h–30 Ag PIII) are firmer than small ones (1.0 h-14 AgPIII) in electron trapping. Even so, it may be argued that the PL sig-nals presented in Fig. 4 are detected by putting the samples inatmosphere air, and it may be different in a solution due to dis-charge of the electrons. Nonetheless, we notice that extracellularelectron transport is an important behavior of bacteria by whichthey produce energy for cell growth and maintenance [27–29],and the bacterial chromosome is specifically attached to the cellmembrane to maintain its proper localization [30]. Thus, we

hypothesize that the extracellular electron transfer process of bac-teria can be mimicked by exciting the dye-stained chromosomaldeoxyribonucleic acid (DNA), producing electrons that are readyto transfer from chromosome to bacterial membrane and finallyto the surface of a sample. In order to prove the claim, S. aureuscells are cultured on the Ag/TOC surface for 12 h in the dark (in or-der to hold a proper bacterial population, the culture duration isshorter than that in antibacterial activity test), so that some ofthem can differentiate and adhere to the coatings. Then the chro-mosomal deoxyribonucleic acid (DNA) is stained with Hoechst33342 (a DNA specific blue dye with excitation/emission light of�350/461 nm), which only interacts physically with DNA [31].Afterwards, the electron discharging events at surfaces of the Ag

5106 H. Cao et al. / Acta Biomaterialia 9 (2013) 5100–5110

NPs/TOC composites are monitored on a fluorescence microscopeby immersing the front (the surface to where bacteria adhere) ofthe samples in a glycerol solution containing 10% water. Fig. 5 pre-sents a series of movie frames captured on the fluorescence micro-scope following the previous mentioned procedure. Undercontinuous UV irradiation, quenching of fluorescence dye (Hoechst33342, blue light) is prevalent on all the samples, but the bubblingdynamics is different. Bubble formation can be observed on the as-sprayed TOC and 1.0 h-14 Ag PIII at the very beginning of UV illu-mination (marked in Fig. 5a–0 and b-0), though the bubbling speedon the 1.0 h-14 Ag PIII (Movie 2, Supplementary materials) isslower than that on the as-sprayed TOC (Movie 1, Supplementarymaterials). The bubbling velocity on 0.5 h–30 Ag PIII is the smallestamong the three samples studied. No bubble forms within the illu-mination of the first 14.3 s (Fig. 5c) and there only two bubblesstart to burst on the surface after continuous irradiation for�16.9 s (Movie 3, Supplementary materials). The quenching ofthe fluorescence dye is probably a sign for the occurrence of extra-celluar electron transfer. And the forming of bubbles is likely theresult of electron discharging at the materials/solution interface[32]. This size-dependent electron storage behavior of the Ag NPsis highly correlated to their antibacterial actions which will be dis-cussed later.

3.3. Antibacterial activity

As demonstrated in the previous sections, Ag NPs with regu-lated size distribution can be fabricated and bound to plasma

Fig. 5. A series of movie frames captured under a fluorescence microscope depicting theand Ag-PIII TOCs illuminated with UV for about 0 s (i-0), 7.2 s (i-1), and 14.3 s (i-2), withrespectively. S. aureus cells, before the illumination, are cultured on the samples and theiMovies 1, 2, and 3 in Supplementary materials for details.

sprayed TOCs by manipulating the unique atomic-scale heating(ASH) effect of the silver plasma immersion ion implantation (AgPIII). These bound Ag NPs provide us with good platforms to ex-plore their antibacterial mechanisms. In order to eliminate thephotocatalysic biocide effects of TOC and evaluate the biocide effi-ciency bound Ag NPs, S. aureus and E. coli cells are introduced ontoboth the as-sprayed (control) and Ag PIII TOCs (the 1.0 h-14 Ag PIII,0.5 h–30 Ag PIII, and 1.5 h–30 Ag PIII), and incubated in the dark at37 �C for 24 h. The morphologies of the S. aureus cells seeded onthese samples were observed by SEM. As shown in Fig. 6, bacterialcolonies are predominant on the as-sprayed and 1.0 h-14 Ag PIIITOC surfaces, but not on the 0.5 h–30 Ag PIII and 1.5 h–30 Ag PIIIsurfaces, indicating that S. aureus can grow relatively well and rap-idly on the former two surfaces but can hardly survive on the lattertwo surfaces. Further observations demonstrate that the cell mem-brane of the bacteria on the as-sprayed is normal without apparentdamage (Fig. 6a) while some of that on the 1.0 h-14Ag PIII arerough with ‘‘blebs’’ (the insert in Fig. 6b) and cytosolic contentleakage happens occasionally (arrowed in Fig. 6b). Cytosolic con-tent leakage and cell lysis is more serious on the 0.5 h–30 Ag PIIIand 1.5 h–30 Ag PIII (Fig. 6c and d, and Fig. S4, Supplementarymaterials). In order to determine the bactericidal efficacy of theAg PIII surfaces, the attached bacteria are detached and re-culti-vated on agar according to the bacteria counting method. The re-sults indicate that all the Ag PIII surfaces can reduce proliferationof S. aureus. The reduction rate is �98% and 99% for 0.5 h–30 Ag PIIIand 1.5 h–30 Ag PIII, respectively, and that for the 1.0 h-14 Ag PIIIis less than 70% (Fig. S5, Supplementary materials). These results

kinetics of fluorescence dye quenching and gas bubble bursting from the as-sprayedi = a, b and c representing the as-sprayed TOC, 1.0 h-14 Ag PIII and 0.5 h–30 Ag PIII,

r chromosomal DNA is stained with Hoechst 33342. Please watch the corresponding

Fig. 6. SEM morphologies of the S. aureus cells cultivated in the dark for 24 h on (a) as-sprayed TOC, (b) 1.0 h-14 Ag PIII, (c) 0.5 h–30 Ag PIII and (d) 1.5 h–30 Ag PIII, with thecorresponding higher magnification images of the circled regions inserted.

Fig. 7. SEM morphologies of the E. coli cells cultivated in the dark for 24 h on (a) 1.0 h-14 Ag PIII, (c) 0.5 h–30 Ag PIII; (b) and (d) are the corresponding higher magnificationimages of the circled regions in (a) and (c), respectively.

H. Cao et al. / Acta Biomaterialia 9 (2013) 5100–5110 5107

clearly demonstrate that the antibacterial activity of 1.0 h-14 AgPIII is inferior to that of the 0.5 h–30 Ag PIII, although the boundAg NPs on 1.0 h-14 Ag PIII are smaller (with a mean size at�4 nm, Fig. 3b) than that on the latter one (Fig. S2b). This is alsotrue for E. coli cells. As shown in Fig. 7, most of the E. coli cellscan tightly adhere to 1.0 h-14 Ag PIII by expressing many pili(Fig. 7a and b) while lysis and cytosolic content leakage are preva-lent on 0.5 h–30 Ag PIII (Fig. 7c and d). This is also consistent with

the quantitative analysis results (Fig. S6, Supplementarymaterials).

3.4. Response of mammalian cells

A successful surface modification procedure should balance theantibacterial activity and the cytocompatibility of the resulted sur-faces. Therefore, the response of mammalian cells was studied by

Fig. 8. Reduction of AlamarBlue for MG63 cells cultured for periods of time onvarious surfaces: (a) cell density in the suspension is �1 � 105 cell ml�1, and SEMmorphology of the MG63cells cultured for 1 day on 1.5 h–30Ag PIII (b), with thehigher magnification image of the circled area in (b) inserted. �� p < 0.05.

5108 H. Cao et al. / Acta Biomaterialia 9 (2013) 5100–5110

using the alamarBlue™ assay. As Fig. 8a shows, the proliferationand vitality of MG63 cells on 0.5 h–30 Ag PIII and 1.5 h–30 Ag PIIIare comparable to that on the as-sprayed TOC. The individual andclustered MG63 cells cover the surfaces and complete cell spread-ing is also observed. The MG63 cells, after being cultured for 1 day,can adhere to the 1.5 h–30Ag PIII surface through extending manyfilopodia (the insert in Fig. 8b). The response of MC3T3 cells is alsopositive (Fig. S7, Supplementary materials). The in vitro cell cultureassay reveals no significant cytotoxicity and even excellent cyto-compatibility on the Ag PIII samples. It suggests that the biocidalaction of bound Ag NPs is specific to bacteria. The mechanism willbe discussed in the next section.

4. Discussions

By manipulating the unique immersion atomic-scale heating(ASH) effect of Ag PIII, size tunable Ag NPs were bound to plasmasprayed titanium oxide coatings, including 1.0 h-14 Ag PIII,0.5 h–30 Ag PIII and 1.5 h–30 Ag PIII. It was demonstrated thatthe precipitation behavior of these Ag NPs is qualitativelyconstrained by the classical nucleation theory. Bound particles ofa relative larger population can be obtained by enhancing thesaturation degree of silver in the surface, but particle sizes are re-duced. And bias voltage is the major factor which controls the sat-uration degree of silver in the outermost surface. Thus, we can

conveniently obtain bound silver nanoparticles with various sizesand distributions by properly carrying out Ag PIII at selected biasvoltages. The electron storage behavior of these Ag NPs is sizedependent. Both photoluminescence spectra (Fig. 4) and fluores-cence microscopy (Fig. 5) results demonstrate that larger Ag NPs(5–25 nm) are better at reserving electrons than smaller ones(�4 nm). Electrons can reside on the Ag particles because of boththe Schottky barrier and the Helmholtz capacitance establishedseparately at the Ag NP/TOC interface [17] and the particle/solutioninterface [33,34]. The electron storage on the metal nanoparticlesshifts the apparent Fermi level of the metal/semiconductor to morenegative potentials, and the shift in the Fermi level is greater withdecreasing particle size [35]. Consequently, for a given populationof Ag NPs, only the fraction of particles smaller than a critical sizepossesses a potential that is negative enough to discharge at theparticle/solution interface. That is why large bound Ag NPs (5–25 nm, 0.5 h–30 Ag PIII, Fig. 5c) can more effectively prevent elec-tron discharge than small bound Ag NPs (�4 nm, 1.0 h-14 Ag PIII,Fig. 5b).

The antibacterial activity was studied by separately introducingS. aureus and E. coli onto these samples and incubating them in thedark. The experimental results clearly demonstrate that the activ-ity of 1.0 h-14 Ag PIII against bacteria (both S. aureus and E. coli) isinferior to that of 0.5 h–30 Ag PIII, although its particle size is smal-ler and distribution density is �23 times greater than the latter one(the distribution density for the former is �1.34 � 103 parti-cles lm�2 while that for the latter is �0.058 � 103), and its peaksilver concentration is �1.65 times larger than that in the latterone, indicating that the difference in antibacterial activity is notsimply a matter of silver concentration. It may be argued thatthe antibacterial activity of Ag NPs may arise from silver ion(Ag+) release [36]. However, in the present study, less than a10 ppb Ag cm2 can be found even after incubating the bound AgNPs in pure water for over 70 days (at 37 �C), implying that silverrelease is minimal and it cannot be the answer to the distinct per-formance of 1.0 h-14 Ag PIII and 0.5 h–30 Ag PIII.

It is generally recognized that the biocide ability of titaniumoxide (TO) stems from the photo-generated electron/hole pairs(e�/h+, Reaction (7)), which facilitate the simultaneous occurrenceof both oxidation and reduction reactions [37]. The resulted holescould react directly with the contacted bacterial membrane lipids,or indirectly by reacting with the adsorbed water (H2O) or hydrox-ide ions (OH�) and producing hydroxyl radicals OH� according toReactions (8), (9) to perform chemical transformations on the bio-molecules [38,39]. The generated electrons (e�) can be scavengedby acceptor species in the aqueous media, such as protons (H+)by Reaction (10) [40].

TOþ hv ! hþ þ e� ð7Þ

hþ þH2O! OH� þHþ ð8Þ

hþ þ OH ! OH� ð9Þ

2e� þ 2Hþ ! H2 ð10Þ

Nevertheless, the point is that the results presented in Figs. 6and 7 are obtained by incubating the samples in the dark, and thereis no light irradiation during the experimental process (Reaction(7) did not occur). In fact, although photo-generated electrons donot appear, the physical Schottky contact structure of those bound-aries at Ag NPs/TOC, i.e. the electron trapping capabilities of thesebound Ag NPs, remain, and the extracelluar electron transfer pathsof bacterial cells remain. That is, Reactions (8)–(10) still may bearoused when ‘‘electron supply’’ in the dark is kept up with the de-mand. As mentioned before, the bound Ag NPs possess electron

Fig. 9. Illustration for extracelluar electron transfer stimulated biocide action of Ag/TOC composites in the dark. That is, electrons are transferred from the bacterialmembranes to the TOC surface, stored on the Ag NPs (‘‘bacterial charging’’), and induce valence-band hole (h+) accumulation at the TOC side that explains cytosolic contentleakage (oxidation).

H. Cao et al. / Acta Biomaterialia 9 (2013) 5100–5110 5109

storage behavior and the bacterial cells are negatively charged.Accordingly, as shown in Fig. 9, in the dark, the ‘‘bacterial charging’’process occurs, that is, electrons generated by bacteria are readilytransferred from the bacterial membranes to the TOC surface andfinally stored on the Ag NPs due to the Schottky barrier effect atthe Ag NP/TOC interface which blocks electron–hole recombina-tion [17] as well as the Helmholtz capacitance effect at the AgNP/solution interface which limits, to some extent, the release ofaccumulated electrons to the adjacent solution [33,34]. Conse-quently, valence-band holes (hþVB) accumulate at the TOC side adja-cent the boundaries at Ag NPs/TOC, leading to notable oxidationreactions and biocide action. And these valence-band holes (h+)may directly react with the membrane lipids based on the electro-static effects or stimulate catalytic oxidation according to Eq. (9)[38,39], inducing pore formation on the outer membrane and even-tually cell lysis. The effect of hole accumulation on bacterial mem-brane, indicated by cytosolic content leakage, is prevalent on0.5 h–30 Ag PIII (Figs. 6c and S4c) and 1.5 h–30 Ag PIII (Figs. 6dand S4d), whereas it is undetectable on 1.0 h-14Ag PIII (Fig. 6band S4b) where sufficient valence-band hole accumulations arenot likely to exist because of the difference in electron storage. It

must be pointed out, though, that electron supply by bacteria isnot as abundant and uninterrupted as that from continuousphoto-excitation of titanium oxide. Accordingly, in order to arouseReactions (8)–(10), in the dark, the bound Ag NPs should ‘‘use’’ theelectrons economically; this explains why large bound Ag NPs (5–25 nm) induce serious cytosolic content leakage and lysis of both S.aureus and E. coli cells while small ones (�4 nm) do not. Moreimportantly, although fluorescence quenching for chromosomalDNA is violent (Fig. 5b), the quenching for nucleus DNA of bMSCcells is not apparent (Fig. S8, Supplementary materials). The resultindicates that mammalian and bacterial cells interact differently tothe same materials, explaining why the in vitro cell culture assaydoes not reveal any significant cytotoxicity (Figs. 8 and S7).

5. Conclusions

In summary, size tunable Ag NPs can be fabricated and bound toplasma-sprayed TOCs by manipulating the atomic-scale heating ef-fect of silver plasma immersion ion implantation. The precipitationbehavior of these bound Ag NPs is qualitatively constrained by the

5110 H. Cao et al. / Acta Biomaterialia 9 (2013) 5100–5110

classical nucleation theory. Antibacterial performance of the as-sprayed and Ag PIII treated TOCs in the dark show that Ag NPs withdifferent size behavior distinctively in promotion of the biocidereactions on TOCs: large particles (5–25 nm) stimulate tougheroxidation reactions than small ones (�4 nm). These oxidationreactions explain the cytosolic content leakage and lysis of bothS. aureus and E. coli. The dark biocide actions of these bound AgNPs are likely aroused by extracellular electron transport behaviorof bacteria. This study presents a novel and controlled methodol-ogy to design nanostructure coatings specifically targeting thebacterial membrane functions.

Acknowledgements

Joint financial support from the National Basic Research Programof China (973 Program, 2012CB933600), National Natural ScienceFoundation of China (31100675, 51071168 and 81271704), Shang-hai Science and Technology R&D Fund under grant 11JC1413700,Science Foundation for Youth Scholar of State Key Laboratory of HighPerformance Ceramics and Superfine Microstructures (SKL201103)and Innovation Fund of SICCAS (Y26ZC3130G), Hong Kong ResearchGrants Council (RGC) General Research Funds (GRF) No. CityU112510, and City University of Hong Kong Applied Research Grant(ARG) No. 9667038 are acknowledged.

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figs. 1–9, are difficult tointerpret in black and white. The full colour images can be found inthe on-line version, at http://dx.doi.org/10.1016/j.actbio.2012.10.017.

Appendix B. Supplementary data

Supplementary data associated with this article can be found, in theonline version, at http://dx.doi.org/10.1016/j.actbio.2012.10.017.

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