versatile nanocomposite coatings with tunable cell adhesion and bactericidity

FULL
DOI: 10.1002/adfm.200800354
PAPER

Versatile Nanocomposite Coatings with Tunable Cell Adhesionand Bactericidity**

By Martha Es-Souni,* Helge Fischer-Brandies, and Mohammed Es-Souni

TiO2-Ag nanocomposites are known for their bactericidal effect during exposure to appropriate UV radiation. While involving

hazardous radiation, and limited to accessible areas, the bactericidity of these coatings is not persistent in the absence of UV light,

which impedes their commercial application. Herein it is shown that TiO2-Ag nanocomposites can be made highly bactericidal

without the need of irradiation. Beyond this, bactericidity can even be mitigated in the presence of pre-irradiated coatings.

Biocompatibility and cell adhesion are also negligibly small for the as-processed, non-irradiated coatings, and become fairly high

when the coatings are irradiated prior to testing. This opens the possibility to pattern the coatings into areas with high and low cell

adhesion properties. Indeed by irradiating the coating through a mechanical mask it is shown that fibroblast cell adherence is

sharply confined to the irradiated area. These properties are achieved using TiO2-Ag thin films with high silver loadings of

50wt%. The films are processed on stainless steel substrates using solution deposition. Microstructural characterization by

means of X-ray diffraction, Raman, and X-ray photoelectron spectroscopy, high-resolution scanning electron microscopy, and

atomic force microscopy show a highly amorphous TiO2-AgxO nanocomposite matrix with scattered silver nanoparticles. UV

irradiation of the films results in the precipitation of a high density of silver nanoparticles at the film surface. Bactericidal

properties of the films are tested on a-haemolyzing streptococci and in-vitro biocompatibility is assessed on primary human

fibroblast cultures. The results mentioned above as to the tunable bactericidity and biocompatibility of the TiO2-Ag coatings

developed herein, are amenable to silver ion release, to catalytic effects of silver nanoparticles, and to specific wettabilities of the

surfaces.

1. Introduction

Coatings for medical devices and implants are sought to

control interfacial reactions with live tissues and body fluids.

Their functionalities may include better biocompatibility to

promote cell in-growth and better integration of implants in

the body, antibacterial, and anti-inflammatory effects.[1–4] The

need for such coatings and specific functionalities may

exemplary be illustrated in the case of stents for vascular

and arterial repair. The surface of the stents has to be

controlled in order to prevent life-threatening side effects like

[*] Dr. M. Es-Souni, Prof. H. Fischer-BrandiesClinic of OrthodonticsChristian-Albrechts UniversityArnold-Heller-Strasse 16, 24105 Kiel (Germany)E-mail: [email protected]

Prof. M. Es-SouniInstitute for Materials & Surface Technology (IMST)University of Applied Sciences, KielGrenzstrasse 3, 24149 Kiel (Germany)

[**] The authors thank Prof. F. Faupel, Technical Faculty, Kiel University, forpermission to use the XPS facility. Thanks are also due to Prof. F.Tuczek for permission to use the Raman facility and to Mrs. U.Cornelissen and Mrs. M. Schneeberg for technical assistance. Theauthors also acknowledge useful discussions with Prof. O. Jansen,Clinic of Neuroradiology, Kiel University, Dr. G. Kartopu, IMST andMr.S. Habouti, IMST. This work is sponsored by the German FederalMinistry of Education and Research (BMBF), grant no. 1761B06.

Adv. Funct. Mater. 2008, 18, 3179–3188 � 2008 WILEY-VCH Verlag

thrombosis.[5,6] Furthermore, cell in-growth in the stent

(neointimal hyperplasis) may occur but is highly undesirable

since it could lead to a new stenosis (a phenomenon designated

as in-stent restenosis in the specialized literature). The

consensus is that the in-stent restenosis rate may be as high

as 30%.[6] New stent designs and functional coatings are

thought to be helpful in alleviating these side effects.[5,7]

Although a large number of coatings on stents exist, among

them carbon, gold, and other inorganic coatings as well as drug

releasing coatings (drug eluting stents), their effect is still under

discussion as outlined in a survey study by Babapulle and

Eisenberg.[8] One way to improve the functionality of implants

is to design coatings or surface treatments that could be

tailored, also locally, to lead to the desired effect, e.g., pre-

vention or promotion of cell adhesion depending on specific

needs. Although many surface treatments particularly for

polymericmaterials, e.g., using plasma, corona treatments, etc.,

have been developed to this end, e.g., to induce hydrophilic/

hydrophobic surfaces etc., and are state of the art, work on

coatings that permit specific and local functionalization with

persistent effects is still lacking.[9–12] Onemight argue that this is

already realized in TiO2-based materials that can be func-

tionalized using UV irradiation.[13–16] Unfortunately the prop-

erties hitherto achieved, in terms of superhydrophilicity and

bactericidity, subside when the UV irradiation is shut-off.[17–20]

In the present study we propose a new strategy to develop

coatings that can be locally functionalized to achieve surfaces

GmbH & Co. KGaA, Weinheim 3179

FULLPAPER

M. Es-Souni et al. /Versatile Nanocomposite Coatings

3180

with persistent properties in terms of cell adhesion capability

and bactericidal effects. The coatings are based on TiO2-Ag

nanocomposites that are known for their bactericidal proper-

ties, although the studies published so far concentrate on

bactericidal properties during UV illumination.[21–23] How-

ever, we notice that Yuranowa et al. observed high bacteri-

cidity without exposure to light for coatings that contained

silver.[22]

The thin nanocomposite films are processed on metallic

substrates by dip-coating from a sol that contains all pre-

cursors. The main difference to previous work[17–21,23] lies in

achieving highly bactericidal effects and low cell adhesion

properties without the need of UV irradiation, while sample

areas tested after UV irradiation showmoderate effects. These

properties are obtained by a high silver loading, which leads to

highly amorphous TiO2-Ag nanocomposites. Pyrolysis and

annealing conditions have been optimized to obtain crack-free

films. Selective functionalization is conducted using photo-

irradiation through mechanical masks. The as-functionalized

surfaces exhibit intriguing properties in terms of bactericidal

activity and fibroblast adhesion capabilities that can operate

over sharp interfaces. The advantages of the proposed method

also lie in its cost-effectiveness, the possibility for large area

coating, versatility, and the high freedom in tailoring bio-

compatibility by film stoichiometry and UV irradiation.

Figure 1. GI XRD and Raman spectra of non- and irradiated TiO2-Agcoatings. a) GI XRD patterns of the TiO2-Ag films in the non-irradiated(nir) and irradiated (ir) states (see main text for details). The positions ofthe lines that correspond to brookite (PDF-29-1360), anatase (PDF-84-1286), rutile (PDF-21-1276), Ag (PDF-04-0783), Ag2O (PDF-12-0793), andAgO (PDF-14-0646) are also shown. b) Raman spectra of the irradiated andnon-irradiated TiO2-Ag coatings. The bands characteristic for brookite [24](&) and oxidized silver at 500 8C [26] (�) are also given.

2. Results

2.1. XRD and Raman Scattering Spectra

The TiO2-Ag films obtained on stainless steel substrates

were found to be largely amorphous. Figure 1a shows grazing

incidence X-ray diffraction (GI XRD) patterns of the films in

the non-irradiated and irradiated states. Apart from a slightly

stronger peak that can be unambiguously attributed to the 111

line of elemental Ag, only very weak lines superimposed on a

strong background are observed for the non-irradiated state.

For irradiated samples the 111 and 200 peaks of Ag become

stronger, but the main features of the patterns remain almost

unchanged. Attempts to index the different weak peaks failed,

though the presence of small amounts of anatase and/or

brookite may be hinted at. Silver oxide in its different variants,

e.g., Ag2O (PDF-12-0793) and AgO (PDF-14-0646) could not

be identified from the XRD patterns.

Further investigations of phase composition involved

Raman spectroscopy. Figure 1b displays representative Raman

spectra collected from irradiated and non-irradiated samples.

Both spectra indicate the poor crystallinity of the films, as

outlined above. Nevertheless, as many more and slightly

stronger Raman peaks can be seen in the spectrum of the

irradiated sample it can be said that the crystallinity here is

better than in the case of the non-irradiated films. The

crystalline phases present in the films were identified by

examining the spectroscopic features in the spectrum of the

irradiated sample. Main peaks occur at 155.3, 242.6, 365.2,

www.afm-journal.de � 2008 WILEY-VCH Verlag GmbH

462.5, 545.7, and 633.7 cm�1, while weaker and/or broader

features are seen at 236, �320, 414, 505, and �617 cm�1.

Although brookite (B) (orthorhombic, space group Pbca) is an

infrequently observed polymorph of TiO2, most of these

features can be attributed to its presence. In particular, the

plain peaks near 155, 243, 365, 462, and 546 cm�1 characterize

the B-phase.[24] The bands at 320, 414, 505, and 634 cm�1 can

also be assigned to brookite. It has been suggested that the

B-polymorph of TiO2 can only be realized if the material

includes a small amount of some substance built into the

structure.[25] In the present case, this is achieved by the silver

doping. The shoulder-like bands at 246 and �617 cm�1 may be

attributed to oxidized silver during the annealing treatment.[26]

& Co. KGaA, Weinheim Adv. Funct. Mater. 2008, 18, 3179–3188

FULLPAPER


No clear evidence can be found concerning the other TiO2

polymorphs, i.e., anatase (tetragonal, space group I41/amd)

and rutile (tetragonal, space group P42/mmm).

From the observations above we may conclude as to the

essentially amorphous character of the highly Ag-loaded TiO2

films processed under the conditions depicted above.

2.2. SEM and AFM Studies

The microstructures of the thin films strongly depend on

surface treatment by irradiation. Figure 2a is a low magnifica-

tion scanning electron microscopy (SEM) image of a

Figure 2. SEM and AFM micrographs of selectively irradiated samples usinmagnification SEM micrograph showing irradiated (bright) and non-irradiatedmagnification and c) FE-SEM micrograph that shows details of the microstrucirradiated surface; the bright spots are silver particles. In (c) notice the differendark-gray areas. e) and f) are AFM micrographs of the non-irradiated andtopographical changes in (f).

Adv. Funct. Mater. 2008, 18, 3179–3188 � 2008 WILEY-VCH Verl

selectively irradiated sample surface, which clearly illustrates

the changes induced by the UV irradiation. A fairly sharp

interface separates the irradiated (bright) from the non-

irradiated area. This effect can be seen well with the naked eye

owing to themetallic character of the irradiated areas. On close

examination of the non-irradiated areas (Fig. 2b) a grainy

surface is revealed with scattered white spots which, upon

energy dispersive spectrometry (EDS) analysis, are mainly

silver (see also XRD above). Further investigations of the non-

irradiated area using high-resolution field emission (FE) SEM

in the backscattered electron mode reveals the existence of

very fine silver particles (bright spots) in the size range from a

few nanometers to 20 nm (Fig. 2c). These particles appear to be

g a mechanical mask and UV irradiation as described in the text. a) Low(dark grey) surfaces. b) SEM micrograph of the non-irradiated area at lowture of the non-irradiated coating (see text for more detail). d) SEM of thet contrasts of the matrix and the confinement of silver nanoparticles to theirradiated sample surfaces, respectively. Notice the morphological and

ag GmbH & Co. KGaA, Weinheim www.afm-journal.de 3181

FULLPAPER


Figure 3. a) XPS narrow scans of silver, titanium, and oxygen of non-irradiated (�) and irradiated surfaces (�),as well as irradiated samples after argon ion etching (4min (�); 10min (�)). b) Background corrected XPSnarrow scans of silver and oxygen of cleaned, non-Ar-ion-etched surfaces of non-irradiated (�) and irradiatedsamples (�) as well as possible deconvolutions (—) of them and sum peaks (—). Deconvolution wasperformed using Gaussian functions. nir: non-irradiated surface; ir: irradiated surface.

3182

confined to the matrix areas with

dark-gray contrast. They prob-

ably precipitated from these areas

leading to silver depletion, which

would explain their darker con-

trast. The bright areas of the

matrix most probably contain a

higher silver loading in solution

(high Z silver backscatters more

electrons) in comparison to the

dark-gray ones. Based on these

observations it is thought that

the TiO2-AgxO matrix undergoes

a demixing into two solid solu-

tions, one supersaturated solid

solution where metallic silver

particle precipitation takes place

and a less saturated, metastable

TiO2-AgxO solid solution.

UV irradiation leads to the

precipitation of a high density of

silver particles, as illustrated in

Figure 2d. Stereographic quanti-

tative image analysis of the silver

particles’ distribution on the irra-

diated surface, using commercial

software, yields a surface area

fraction of 12% of silver particles,

which exhibits a mean chord

length of 80 nm. It should be

pointed out that the microstruc-

tures examined here were all

irradiated for a constant time of

20min. Shorter or longer irradiation times lead to smaller or

coarser particles, respectively. The atomic force microscopy

(AFM) images in Figures 2e,f also demonstrate the morpho-

logical and topographical changes that occur at the surface with

particle coarsening and surface roughening (compare the z-

range of the micrographs).

2.3. XPS Investigations

Elemental chemical environments have been investigated

using XPS analysis on irradiated and non-irradiated sample

surfaces. Additional analysis was performed on both samples

after argon ion etching for 4 and 10min. Figure 3 and Table 1

summarize the results obtained.

It is noticed that the Ag peak maxima are located at very

similar binding energies for both sample surfaces. Deconvolu-

tion of the Ag 3d5/2 peak shown in Figure 3b yields two peak

maxima at 368.3 and 367.7 eV. The first peak can be attributed

to metallic silver and the second to silver oxide, probably

AgxO.[27] On the non-irradiated surface, the relative intensities

of both peaks are quite similar, which indicates that the surface

has roughly equal amounts of metallic and oxidized silver,


whereas the surface of the irradiated sample reveals a higher

amount of metallic silver. Upon argon ion etching the apparent

Ag peak maxima of both surfaces are shifted to lower binding

energies, which can be correlated with the predominant

presence of silver oxide. The Ti peak is located for all samples

between 458.3 and 459 eV, where the irradiated surface is

characterized by the highest binding energy. Significant shifts

upon argon ion etching were not observed. These peaks can

solely be attributed to Ti4þ.[27] Since neither peak-broadening

nor shoulders were observed at lower binding energies, the

presence of Ti3þ species (with peak maxima between 456 and

456.9 eV) can be ruled out.[28,29] However, it is the O 1s peak

that shows a shape most specific to the irradiated sample

surface. As shown in Figure 3, the double peak can be

deconvoluted into three separate peaks, one lying at a very

similar energy to the non-irradiated sample surface or in the

bulk, while the other peaks are located at higher binding

energies of 532.2 and 533.2 eV. According to the literature,

these higher binding energies may be attributed to H–O

bonds.[28,30] We may conclude that UV irradiation induces

hydroxide bonding at the sample surface, wherein the OH

groups are thought to be mainly chemisorbed to the silver

particles present at the surface.


FULLPAPER


Table 1. Apparent binding energies of the elements Ag, Ti, and O. ir: irradiated surface; nir: non-irradiated surface. The peak energies are given after argonion cleaning (surface) and argon ion etching for 4 and 10min, which correspond to a depth of 4 and 8 nm, respectively. The peak energies are also shownafter deconvolution and attributed to different chemical environments.

Species BE apparent BE after deconvolution Attributed to

Surface After Ar ion etching Surface

4 min 10 min

nir ir nir ir nir ir nir ir

Ag 3d5/2 368.0 368.1 367.9 367.7 367.9 367.8 368.3 368.3 Ag0 [27]

367.7 367.7 Ag in AgxO [27]

Ti 2p3/2 458.6 459.0 458.5 458.3 458.6 458.5 Ti in TiO2 [27]

O 1s 529.8 530.2 530.0 529.8 530.0 530.0 530.3 Ti/Ag oxide [27]

532.2 532.2

533.2 adsorbed HO on Ag/Ti [28,30]]

2.4. In-vitro Bactericidal and Cytotoxicity Effects

In the following we present the effects of the TiO2-Ag

coatings on bacteria cultures and fibroblast adhesion and

proliferation properties. Although TiO2-Ag has been reported

to be a bactericidal coating, the present work shows that the

effect can be tailored by UV-light irradiation of the thin

films.[21–23] Curiously, the irradiated thin films are far less

bactericidal than the non-irradiated ones. This is evident from

Figure 4 which shows typical proliferation curves of the

bacteria tested with and without coated samples. As can be

seen, sample-free bacterial suspensions (NC) exhibit a typical

sigmoidal proliferation behavior. In the presence of the

coated samples, specific responses are observed depending

on the surface functionalization. Non-irradiated samples fully

inhibit bacterial proliferation while the irradiated ones

provoke only a net decrease in the proliferation rate. These

findings are confirmed by the results of the colony forming

units (CFU) tests. Compared with sample free cultures upon

Figure 4. Bacteria proliferation (Bacteria) in the absence (NC –&–) andpresence of irradiated (Bacteriaþ ir –�–) and non-irradiated (Bacter-iaþnir – –) TiO2-Ag coatings. The proliferation is monitored photometri-cally in terms of turbidity measurements at 860 nm. Absorption effects, i.e.,in the absence of bacteria, from medium (Med &) or samples (Medþ ir(�) and Medþ nir ( )) are also shown for comparison.


which 76 colonies formed, the irradiated samples had nearly

half this number (30), whereas non-irradiated surfaces induced

a 15-fold reduction (5).

The biotoxicity of the coated samples on primary human

fibroblast cultures was assessed by a BrdU proliferation test for

an incubation period of 24 h. Similarly to the bacteria cultures,

the response of the fibroblast cultures to the presence of the

coated samples strongly depends on the surface functionaliza-

tion. The results obtained are presented in Figure 5a. The

proliferation of fibroblasts grown in the presence of irradiated

sample surfaces attain approximately 80% of the cells’

proliferation grown in sample free medium (NC), whereas

non-irradiated surfaces reduce the proliferation down to 12%

of the NC.

The proliferation andmorphology of the NC and cells grown

on locally irradiated coatings using a mechanical mask were

studied using SEM on critical point dried samples (Fig. 5b) and

light microscopy on Giemsa stains (Fig. 5c). Figure 5b shows

that cells actually adhere on the irradiated part of the sample

(which can easily be distinguished owing to its brighter

contrast); the cells exhibit normal fibroblast morphology, as

depicted in Figure 5c. In contrast there is barely any cell

adhesion on the non-irradiated areas of the sample. We notice

that cell adhesion can actually be tailored at a sharp interface.

That some fibroblast can be seen beyond the interface is a

result of the irradiation technique, which involves mechanical

masks that cannot be fit without a gap to the substrate, thus

leading to a diffuse interface between the irradiated and non-

irradiated areas.

3. Discussion

3.1. Film Formation and Microstructure

When pure TiO2 films are prepared from a precursor

solution that contains acetylacetone-stabilized Ti-isopropoxide

in methoxyethanol, only nanocrystalline anatase is formed at

500 8Cwith a crystallite size of approximately 13 nm.[31] Sol–gel

TiO2-Ag thin films prepared from sols with a silver/Ti molar

ratio of 0.1 were recently investigated by Page et al. after


FULLPAPER


Figure 5. Results of biotoxicity testing on fibroblast cultures (a) andmorphological studies (b,c). a) Assessment of the biotoxicity of the coat-ings in the presence of fibroblast cell cultures (F-Culture). Blanks consist ofthe medium with and without coated samples. The NC is a sample-free cellculture. Proliferation testing was assessed by immunological detection ofBrdU incorporation into cellular DNA using horseradish peroxidase-labeledanti-BrdU conjugates. ABTS (2,2-azino-bis(3-ethylbenzthiazoline-6-sulfo-nic acid)) is used as a substrate for the peroxidase. ir: irradiated; nir: non-irradiated samples. b) Low magnification SEM micrograph of critical pointdried fibroblasts grown on a selectively irradiated sample. Notice theexclusive cell adhesion on the irradiated side and the sharp interface thatseparates the irradiated (brighter surface on the left) from the maskedcoating. Also notice the elongated fibroblast cell morphology, characteristicfor healthy cells, which indicates a good biocompatibility of the surface. c)Light micrograph of Giemsa-stained fibroblasts on the irradiated samplesurface showing morphological details of healthy fibroblast cells.

3184 www.afm-journal.de � 2008 WILEY-VCH Verlag GmbH

annealing at 500 8C in air (Ag concentrations up to 20% were

used for X-ray absorption near edge structure (XANES)

spectra).[23] Their results show nometallic silver and a decrease

in crystallinity with increasing film thickness. They also claim

the observation of anatase and Ag2O, at least for thinner films,

from their XRD and XANES patterns. Epifani et al. used sols

of the same molar ratio with silver ions being stabilized by an

excess amount of pyridine in SiO2 and TiO2 base sols.[32] Their

XRD patterns of coatings also annealed at 500 8C in air were

attributed to anatase and fine particles of elemental silver

rather than to silver oxide. These authors point out that the

amount of complexing agent is at least seven fold higher in the

case of doped titania nanocomposites than for silica, because of

a lower complex stability and the higher electronegativity of Ti.

Furthermore, it has been previously reported that Cu, Pt, and

Ag can stabilize brookite in sol–gel-processed TiO2 materials

depending on the concentration and precursor type.[33–35] In

particular, Ag was shown to stabilize nanocrystalline brookite

even at annealing temperatures of 500 8C, when it was used at a

high concentration, e.g., 30%, from AgNO3 precursors.[35]

Therefore, it appears that specific microstructures can be

obtained for doped TiO2 films depending on precursor

solutions, concentration of doping elements, and processing

conditions. In this work the high silver loading is responsible

for the formation of the microstructures described above and

discussed in the following.

The thin films were processed from sols that contained a

molar ratio of 0.74AgNO3/TiIV-isopropoxide, where silver

ions were stabilized by an excess amount of pyridine. It is

conceivable that in the present study, because of the elevated

silver concentration and the mentioned stability characteristics

of the Ag–pyridine complex, a certain amount of the total

silver could be built into the TiO2 network. Since tetravalent Ti

ions in TiO2 would be substituted by monovalent Ag ions, a

high density of defects, e.g., oxygen vacancies and/or inter-

stitial Ti ions, should be generated. According to Kroger–Vink

notation, the Ag incorporation reactions may be written as:

Ag2Oþ TiTi þ OO ! 2Ag000TiþTiO2 þ 3V��O (1)

Ag2Oþ TiTi þ OO þ VI ! 2Ag000Ti þ TiO2 þ Ti��I þ V��O (2)

where Ag000Ti is a monovalent Ag ion on a tetravalent Ti ion

site, which leads to a negatively charged defect (charge �3),

V��O is an oxygen vacancy (positively charged defect, charge

þ2), VI denotes an interstitial vacancy and Ti��I is an

interstitial, positively charged Ti ion defect (charge þ4).[36]

First principle calculations show that interstitial Ti defects

could form spontaneously in anatase because of their low

formation energy whereas a slightly higher formation energy

was calculated for oxygen vacancies.[37] In the present work

the only crystalline phase observed is brookite. Since

brookite has a more open structure than anatase (brookite

(PDF-29-1360) has the highest cell volume among the TiO2


FULLPAPER


polymorphs), we expect it to accommodate similar defects

with probably higher concentrations.The deconvoluted XPS curves, including those for the ion

etching experiments, reveal that elemental silver is the pre-

dominant species on the surface, whereas oxidized silver is

found mainly in the underlying layers. The binding energies

reported in the literature for Ag–O in AgO and Ag2O are very

close, making a specific assignment difficult.[27] However, the

Raman features at 246 and �617 cm�1 may be attributed to

oxidized silver during heat treatment. In fact, it is known that

during thermal decomposition of silver oxide, e.g., by a heat

treatment at 500 8C like in the present case, metallic silver may

incorporate oxygen as surface and bulk species. The Raman

spectra obtained in this case differ from those of bulk AgO/

Ag2O, which exhibits main peaks at 241, 460, 617, 800, and

968 cm�1 and coincide well with the results found in this

work.[26]

Based on these results and taking into consideration

Figure 2c we may conclude that our as-processed films are

composed of a main amorphous TiO2 matrix that contains

silver in its network, nanocrystalline brookite, as well as ele-

mental and oxidized silver. The incorporation of Ag ions into

the oxide network is thought to result in a high concentration of

defects that lead to the above microstructure after annealing at

500 8C.In TiO2, electron–hole pairs are induced by photo-irradiation

with photon energies higher than the band gap of TiO2 (3.3 eV)

according to Reaction (3).[13] In the case of silver-doped

titania, it is conceived that reduction of silver ions occurs by

trapping an electron fromReaction (3) to form elemental silver

following Reaction (4):

TiO2 �!hn

TiO2 þ e0 þ h�

(3)

Agþ þ 1e0 ! Ag0 ð4Þ

It should be pointed out that irradiation with light in the vis/

IR region is sufficient to induce silver reduction in the samples

presented here.[39] This can be explained by the broad

absorption edge of brookite which has been reported by

Zallen et al. to extend throughout the visible region, in contrast

to anatase and rutile, which show steep optical absorption

bands with bandgap energies of 3.3 and 3.06 eV, respec-

tively.[40]

The fact that silver nanoparticles grow to an approximate

mean size of 80 nm (Fig. 2d) on irradiated surfaces is certainly

amenable to the fast solid-state diffusion of silver and subse-

quent silver particle ripening. The temperature rise of approxi-

mately 130 8C during irradiation appears to be sufficient to

drive the process. It is likely that the nanocrystalline nature of

the film, the high density of vacancies, and surface diffusion are

responsible for the fairly high kinetics of the process, despite

the relatively low heating temperature. This leads to the high

area fraction of the silver particles at the surface and their fairly


coarse size seen as in Figure 2d. However, as inferred from

XRD, Raman spectroscopy, and XPS, the nature of the matrix

and the number of phases present are not altered by the

irradiation process.

3.2. Microstructure Effects on Bactericidity and Cell

Biotoxicity

As shown above, the surface microstructure of the TiO2-Ag

nanocomposite coatings can be changed using a simple and

versatile irradiation process. The response of bacteria and

fibroblast cultures to these coatings is highly dependent on the

film microstructure, i.e., on whether it has been irradiated or

not. We have clearly shown that the coatings are highly

bactericidal and biotoxic (at least for the bacteria strain and

fibroblast culture tested here) when they are in the as-

processed, i.e., non-irradiated state, whereas the irradiated

samples are characterized by a moderate bactericidity and

satisfactory biocompatibility.

The catalytic effects of pure TiO2 in its different polymorphs

on pollutant degradation and its bactericidal properties are

usually related to the generation of electron–hole pairs upon

photo-irradiation, as described above.[13] The literature

concerning these effects and their mechanisms is abundant

and need not be further discussed here. Still there have been

attempts undertaken in order to increase the photocatalytic

effect. In this respect, the addition of some noble metals to

TiO2 has been shown to be favorable.[35,38,41,42] However, for

applications that involve antibacterial effects, more work was

devoted to Ag-doping by photodeposition of silver on TiO2

from AgNO3 solutions or by sol–gel processes.[21–23] The

higher photocatalytic and bactericidal effects of these coatings

with regard to pure TiO2 have been discussed in terms of a

more efficient separation of TiO2 charge carriers (because of

the reduction of silver ions).[21–23,35,41,42] Furthermore, metallic

silver is a well known catalyst of oxygen reduction, which leads

to structural damage to bacteria.[43] Common to these studies is

the fact that a measurable bactericidal (photocatalytic) effect

of Ag-doped TiO2 samples is achieved only during UV

irradiation on previously activated, i.e., irradiated, coatings,

and this constitutes the main difference to the results reported

in the present work.

The stoichiometry chosen, i.e., a high silver loading of 50wt%,

and the particular microstructure achieved in our as-

processed TiO2-Ag nanocomposites confer to them high

bactericidal and biotoxic properties, making irradiation during

use superfluous. As demonstrated by our results, these prop-

erties can be mitigated by irradiating the surface prior to use.

The mechanisms that govern these effects could partly be

amenable to silver ion release and the effects of silver

nanoparticles on catalyzing the formation of highly active

oxygen/organic radicals. The release rate of silver ions from the

non-irradiated sample in water, determined photometrically

over the first 24 h, amounts to 4.4mg cm�2 day�1, which is

markedly higher than that measured for the irradiated sample


FULLPAPER


Figure 6. Schematic representation of TiO2-Ag nanocomposite microstructures and their properties in termsof bactericidal effects and cell adhesion. The as processed, non-irradiated nanocomposites exhibit highbactericidal effects and high biotoxicity. After irradiation the nanocomposites show moderate bactericidityand higher biocompatibility. When the nanocomposites are selectively irradiated, e.g., usingmechanical masks,fibroblast cell cultures adhere to the irradiated areas with a sharp confinement.

3186

(0.06mg cm�2 day�1). The higher

silver ion release rate of the non-

irradiated samples can be ratio-

nalized in terms of the co-exis-

tence of metastable TiO2-AgxO

solid solutions and nanometer-

scale silver particles at the sur-

face (see above) that lead to

greater silver ion leaching. The

catalytic effect of the silver

nanoparticles for the reduction

of oxygen, which leads to the

formation of highly reactive oxy-

gen species, also contributes to

bactericidity and cell toxicity.

TiO2 and metallic silver are

known to form a Schottky con-

tact. Since the electron work

function of silver (4.7 eV) is

higher than the electron affinity

of TiO2 (3.9 eV), adsorbed oxy-

gen will extract electrons from

Ag, which in its turns will extract electrons from the

semiconducting oxide TiO2.[44,45] This results in highly

oxidizing O2� species and a depleted adjacent oxide layer.

In this respect the specific behavior of the non-irradiated

samples can be attributed to the presence of nanometer-scale

silver at the surface (see above), since the high surface to

volume ratio of these nanoparticles would impart them higher

catalytic activity. It should, however, be mentioned that in the

present work we tested bactericidity only on gram positive

bacteria, which are known to be more prone to attack from

oxygen radicals and to the toxic effects of released silver ions

because of their cell envelope characteristics.[23,46]

The mechanisms above are certainly responsible for the

bactericidal effect. The fact that the irradiated samples exhibit

a lower bactericidal effect can be explained by the lower

concentration of released silver ions and the larger silver

particle size. However, silver ion release alone cannot explain

the fairly sharp interface that marks the fibroblast proliferation

area, e.g., Figure 5b, since fast ionic transport in aqueous

solution would alleviate the silver deficit in the irradiated areas.

We hinted that the specific wettabilities of the surfaces

involved might also contribute to the confinement of cell

adhesion to the irradiated areas. Indeed, using the sessile drop

method, contact angles of 99 8� 2 8 and 30 8� 5 8 were found

for the non-irradiated and irradiated samples, respectively.

UV-irradiation induced hydrophilicity of TiO2 surfaces is fairly

well known and relies on the radiation-induced electron–hole

pair formation described above.[15,16] In the present films the

generation of electrons serve to reduce the silver ions while the

trapped holes are supposed to transfer to H2O to form OH

radicals and induce hydrophilicity. Furthermore, while

the hydrophilicity of TiO2 is known to degrade upon storing

in the dark, in the present TiO2-Ag nanocomposites both the

hydrophilicity and hydrophobicity are persistent even after


sonification and aging of the films.[17] We surmize that hydro-

philicity is largely a result of the presence of Ag particles on the

surface of the irradiated samples and the adsorbed OH groups,

evident from the XPS results above.

Thus, we may conclude that the biotoxicity and the ability to

locally tailor it in the present films relies on concurrent

mechanisms that involve contributions from interactions of the

silver nanoparticles and silver ions as well as the surface charge

(which governs wetting) with the live medium.

The effects of the TiO2-Ag nanocomposite microstructure

on bactericidity and biotoxicity are schematically illustrated in

Figure 6.

4. Conclusions

In conclusion, the salient result of the present work concerns

the tailoring of the biotoxicity and bactericidal effect using sol–

gel TiO2-Ag nanocomposite coatings. We have shown that

high silver loading and silver ion stabilization in the precursor

solution leads to specific microstructures, which involve meta-

stable TiO2-AgxO solid solutions, and show high bactericidal

and biotoxic properties in the as-processed state. When the

microstructure is irradiated with UV a high density of silver

particles is formed at the surface, and this confers rather

moderate bactericidal and biotoxic properties to the TiO2-Ag

nanocomposites. We have also shown that when the samples

were selectively irradiated usingmasks, fibroblast cell adhesion

was confined to the irradiated areas, making a sharp interface

with the non-irradiated area where no cell adhesion occurred.

These findings may open new possibilities for functionalizing

biomedical devices and programming their responses to micro-

organisms and cell adhesion.


FULLPAPER


The intriguing bactericidal effect of the non-irradiated

coatings offers additional application possibilities; the coatings

can be applied to tiles in hospitals, operation rooms, and to

medical devices for the persistent prevention of contamination,

without the need of UV irradiation. Furthermore, these

coatings certainly have anti-fouling properties that should

constitute an additional field of application for them.

5. Experimental

Substrate, Precursor Solutions, and Coatings: In the present paper acommercially available annealed stainless steel (Good Fellow, AISI316L, sheet of 30� 30 cm2 and 0.1mm thickness) was used as asubstrate. For dip coating, rectangular samples (4� 2 cm2) were cutwith scissors, cleaned ultrasonically in 99.9% ethanol, and dried. Inorder to avoid diffusion between substrate and film, an SiO2 bufferlayer was deposited, pyrolyzed at 300 8C, and finally annealed at 500 8Cfor 20min. The SiO2-terminated substrates were subsequently coatedwith a 300 nm TiO2-Ag thin film using dip coating, pyrolysis, andannealing as above. Details of the sol preparation and coatingprocedures are given below.

Silica deposition was performed with a 0.8 M tetraethoxysilane(TEOS, ABCR, Germany) sol having a molar composition ofTEOS/2-methoxyethanol (MEO) (Fluka)/HCl (30%, Roth)/distilledH2O of 1:12.5:2.5� 10�4:2.8. For a final volume of 100mL, 2 g ofpoly(ethylene glycol) 400 (PEG400, Alfa Aesar, Germany) was addedto promote film formation and prevent film cracking.

For the TiO2-Ag precursor solution preparation a modifiedprocedure of that reported by Epifani et al. was adopted [32]. Asilver solution was first prepared by mixing a 1: 15: 5 molar ratio ofAgNO3 (MaTeck GmbH), pyridine (Fluka), and MEO. The silvernitrate solution was then mixed at room temperature with a stabilizedtitanium oxide solution that contained TiIV-isopropoxide (Fluka),acetylacetone (Fluka), distilled H2O, and MEO with molar ratios of1: 0.5: 4: 3.8, and stirred for 30min. The clear yellow solution obtainedwas subsequently diluted with MEO to a final concentration of 0.7 M

and filtered through 0.2mm nylon filters (Roth, Germany). For thesame reasons as those given above, PEGwas added to the final solutionat the same concentration. Using this precursor solution, a nominal Agcomposition of 50wt.-% should be obtained; this was confirmed in ananalytical scanning electron microscope (Philips XLC 30CP) usingEDS analysis.

UV Irradiation: Irradiation of TiO2 base coatings was performedfor 20min in a closed chamber using a UV lamp (Supratec UV-HighPressure lamp, Radium; 250 nm< l< 400 nm). For direct qualitativecomparison of fibroblast adhesion and morphology studies, UV-selective irradiation of the sample surface was carried out through ashadowmask that had an area of 1 cm2, the opposite side being entirelyirradiated.

Prior to bacteria/cell proliferation testing, samples were cut withscissors into rectangles (1� 2 cm2 for bacteria, 0.7� 0.3 cm2 forfibroblast culture), cleaned ultrasonically in ethanol, and heat sterilizedat 134 8C for 5min (Stat IM 5000S, Autoclave, SciCan).

Microstructure Analysis: For phase analysis both XRD and Ramanscattering experiments were performed. XRD patterns of the filmswere obtained in GI mode on a Seifert 3000 PTS 4-circle-diffracto-meter, with Cu Ka radiation (l¼ 1.5418 A). The GI angle was fixed at5 8. Raman spectra of TiO2-Ag films were collected using a Dilor X-Yspectrometer and the 514.5 nm line of an Arþ laser at 20K. The poweron the sample was 30 mW.

The microstructure was characterized by means of conventionalSEM (Philips XL 30) equipped with EDS (EDAX CDU Leapdetector), field emission SEMwith a Schottky emitter (FE-SEM, Zeiss


Ultraplus, Germany), and AFM (SIS Nano-Station, Germany). Ele-mental chemical environments were investigated by XPS (Omicron,Full Lab, Germany) using a monochromatic Al Ka X-ray source at10�9mbar. For depth analysis, argon ion etching was conducted at anacceleration voltage of 160V, a pressure of 9.7� 10�9mbar, and acurrent of 8mA. These gentle sputtering conditions were chosen inorder to avoid sputtering artifacts, i.e., reduction of ionic species, whichcan arise from high energy beams.

Silver Ion Release: The leaching of silver ions from irradiated/non-irradiated samples incubated in doubly distilled water was assessedphotometrically (mQuant, Bio-Tek Instr. Inc) using the method pro-posed by El Ghamry and Frei [47]. Leaching was performed in triplicateon 8 cm2 sample surfaces for 24 h at 37 8C in a humid atmospheresupplied with 5% CO2. The method mentioned above allows themonitoring of the concentration of released silver ions by measuringthe absorption at 550 nm of a ternary [Ag(phenanthroline)2]2

þTBF2�

complex that is formed by the addition of phenanthroline and Eosin Y(2,4,5,7-Tetra bromo fluorescein) to a silver-containing aqueoussolution. A calibration was run with molar silver ion concentrationsof 2.91� 10�5, 4.36� 10�5, 5.82� 10�5, and 7.27� 10�5. Distilledwater was taken as the blank. The molar extinction coefficient of31 311 M

�1 cm�1, measured for the complex above, was in good agree-ment with the values reported in the literature [47,48]. Photometricmeasurements were performed on eight aliquots of each sample.

Bacteria Proliferation Tests: Bacteria testing was carried out withgram-positive, non-pathogenic a-haemolyzing streptococci from throatsmear cultures of healthy persons. Bacteria were cultured for 24 h onagar plates (Columbia III Agar supplemented with 5% sheep blood,BD Diagnostic Systems). After 24 h the colonies were selected,suspended in 30mL brain heart broth (Fluka), and incubated at 37 8C.After 90min incubation the turbidity was determined photometricallyat 890 nm (mQuant, Bio-Tek Instr. Inc) and, where necessary, adjustedwithmedium.On reaching 0.09ODper 200mL the test was started withthe addition of metal substrates in the non-irradiated and irradiatedstates, in 4mL of bacterial suspension. Bacteria without substrateswere taken as negative controls (NC). Pure medium samples with andwithout substrates were taken as blank. Turbidity measurements wereperformed for increasing time intervals (0, 90, 150, 210, 270, 330,390min) on 200mL aliquots in triplicate to enable the monitoring ofbacterial growth. Aliquots of each culture type were taken after180min (log-phase), diluted to 1: 1000, and plated on agar plates inorder to determine the colony forming units (CFU) capacity of each.

Cell Culture and Proliferation Testing: Primary fibroblasts fromhuman explants cultured in a-MEM (Sigma) supplemented with 10%FCS (HyClone), 2.5mg mL�1 of AmphotericinB, 100U mL�1 ofpenicillin, and 100mg mL�1 of Streptomycin (Biochrom) were used.Cell culture and proliferation testing by BrdU incorporation intocellular DNA (BrdU Labelling and Detection Kit III, Roche) werecarried out as described elsewhere [49]. An amount of 8� 103 cells permicro-well were brought in contact with the samples to be tested. After24 h, proliferation was quantified photometrically (OD405 nm/OD490nm)by peroxidase activity measurement (peroxidase being the label of theanti BrdU POD, Fab fragments). Blanks consisted of cell free mediumwith and without coated samples, whereas sample free fibroblastcultures were taken as NC.

Morphological Studies and Wettability Testing: Fibroblasts(3.5� 104 per cm2) were cultured on coated and partly irradiatedsamples for 24 h. Light microscopy investigations were conducted onfixed cells (2.5% glutaraldehyde, Merck) stained with Giemsa (PAA).SEM was performed on critical point dried fibroblast cultures.

Wettabilitiy tests were performed using the sessile drop method anda commercial video-based contact-angle measurement system (Cruss-G10, Hamburg, Germany).

Received: March 12, 2008Revised: June 12, 2008

Published online: September 29, 2008


FULLPAPER


3188

[1] D. J. Balazs, H. M. Mokbul, E. Brombacher, G. Fortunato, E. Korner,

D. Hegemann, Plasma Process. Polym. 2007, 4, 380.

[2] A. Duran, A. Conde, A. Gomez Coedo, T. Dorado, C. Garcıa, S. Cere,

J. Mater. Chem. 2004, 14, 2282.

[3] A. B. Sanghvi, K. P. H. Miller, A. M. Belcher, C. E. Schmidt, Nat.

Mater. 2005, 4, 496.

[4] R. G. Nuzzo, Nat. Mater. 2003, 2, 207.

[5] L. K. Keffer, Nat. Mater. 2003, 2, 357.

[6] E. R. Edelmann, C. Rogers, Am. J. Cardiol. 1998, 81, 4E.

[7] J.M.Garasic, E. R. Edelman, J. C. Squire, P. Seifert, M. S.Williams, C.

Rogers, Circulation 2000, 101, 812.

[8] M. N. Babapulle, M. J. Eisenberg, Circulation 2002, 106, 2859.

[9] M. R. Wertheimer, L. Martinu, E. M. Liston, J. Adhes. Sci. Technol.

1993, 7, 1091.

[10] D. Klee, R. V. Villari, H. Hocker, B. Dekker, C. Mittermayer, J.

Mater. Sci.: Mater. Med. 1994, 5, 592.

[11] Y. I. Yoon, H. S. Moon, W. S. Lyoo, T. S. Lee, W. H. Park, J. Colloid

Interface Sci. 2008, 320, 91.

[12] W. Xu, X. Liu, Eur. Polym. J. 2003, 39, 199.

[13] E. Pelizetti, C. Minero, Electrochem. Acta. 1993, 38, 47.

[14] A. Mills, S. J. Le Hunte, J. Photochem. Photobiol. A 1997, 108, 1.

[15] R. Wang, K. Hashimoto, A. Fujishima, M. Chikuni, E. Kujima, A.

Kitamura, M. Shimohigashi, T. Watanabe, Nature 1997, 388, 431.

[16] K. Funakoshi, T. Nonami, J. Am. Chem. Soc. 2006, 89, 2782.

[17] T. Watanabe, A. Nakajima, R. Wang, M. Minabe, S. Koizumi, A.

Fujishima, K. Hashimoto, Thin Solid Films 1999, 351, 260.

[18] S. S. Block,V. P. Seng,D.W.Goswami, J. Sol. EnergyEng. 1997, 119, 85.

[19] P. Maness, S. Smolinski, D. M. Blake, Z. Huang, E. J. Wolfrum, W. A.

Jacoby, Appl. Environ. Microbiol. 1999, 65, 4049.

[20] J. A. Ibanez, M. Litter, R. A. Pizzaro, J. Photochem. Photobiol. A

2003, 157, 81.

[21] M. Machida, K. Norimoto, T. J. Kimura,Am. Ceram. Soc. 2005, 88, 95.

[22] T. Yuranova, A. G. Rincon, C. Pulgarin, D. Laub, N. Xantopoulos, H.

J. Mathieu, J. Kiwi, J. Photochem. Photobiol. A 2006, 181, 363.

[23] K. Page, R. G. Palgrave, I. P. Parkin, M. Wilson, S. L. P. Savin, A. V.

Chadwick, J. Mater. Chem. 2007, 17, 95.

[24] G. A. Tompsett, G. A. Bowmaker, R. P. Cooney, J. B. Metson, K. A.

Rodgers, J. M. Seakins, J. Raman Spectrosc. 1995, 26, 57.

[25] Y. S. Bobovich, O. S. Dymshits, A. A. Zhilin, M. Y. Tsenter, T. I.

Chuvaea, J. Appl. Spectrosc. 1989, 50, 937.


[26] I. E. Wachs, in:Handbook of Raman Spectroscopy: From the Research

Laboratory to the Process Line, (Eds: I. R. Lewis, H. G. M. Edwards),

Marcel Dekker, New York 2001, pp. 821–822.

[27] NIST X-ray Photoelectron Spectroscopy Database, Version 3.4, 2000,

http//:srdata.nist.gov/xps/.

[28] O. Oji, J. V. Wood, J. Downes, J. Mater. Sci.: Mater. Med. 1999, 10,

869.

[29] W. Gopel, J. A. Anderson, D. Frankel, M. Jaehnig, K. Phillips, J. A.

Schafer, G. Rocker, Surf. Sci. 1984, 139, 333.

[30] D. B. Haddow, P. F. James, R. J. vanNoort, J. Mater. Sci. : Mater. Med.

1996, 7, 255.

[31] M. Es-Souni, G. Kartopu, A. Piorra, M. Es-Souni, C. H. Solterbeck, H.

Fischer-Brandies, Phys. Status Solidi A 2008, 2, 305.

[32] M. Epifani, C. Giannini, L. Tapfer, L. J. Vasanelli, Am. Ceram. Soc.

2000, 83, 2385.

[33] X. Bokhimi, A. Morales, O. Novaro, Chem. Mater. 1997, 9, 2616.

[34] E. Sanchez, T. Lopez, R. Gomez, X. Bokhimi, A. Morales, O. Novaro,

J. Solid State Chem. 1996, 122, 309.

[35] J. Yu, J. Xiong, B. Cheng, S. Liu, Appl. Catal. B: Environ. 2005, 60,

211.

[36] F. A. Kroger, H. Vink, J. Solid State Phys. 1956, 3, 307.

[37] S. Na-Phattalung, M. F. Smith, K. Kim, M. H. Du, S. H. Wei, S. B.

Zhang, S. Limpijumnong, Phys. Rev. B 2006, 73, 125 205.

[38] D. P. Cozzoli, E. Fanizza, R. Comparelli, M. L. Curri, A. Agostiano,

Chem. B 2004, 108, 9623.

[39] M. Es-Souni, German Patent Application DE 10 2007 019 166.0,

2007.

[40] R. Zallen, M. P. Moret, Solid State Commun. 2006, 137, 154.

[41] S. Rengaraj, X. Z. Li, J. Mol. Catal. A: Chem. 2006, 243, 60.

[42] V. Iliev, D. Tomova, L. Bilyarska, A. Eliyas, L. Petrov,Appl. Catal. B:

Environ. 2006, 63, 266.

[43] K. Yashida, M. Tanagawa, S. Matsumoto, T. Yamada, M. Atsita, Eur.

J. Oral Sci. 1999, 107, 290.

[44] R. Konenkamp, Phys. Rev. B 2000, 61, 11 057.

[45] Y. Shimizu, M. Egashira, MRS Bull. 1999, 24, 18.

[46] A. Ewald, S. Gluckermann, R. Thull, U. Gbureck, Biomed. Eng.

OnLine 2006, 5, 22.

[47] M. T. El-Ghamry, R. W. Frei, Anal. Chem. 1968, 40, 1986.

[48] K. Basavahia, P. Nagegowda, J. Iran. Chem. Soc. 2004, 1, 106.

[49] M. Es-Souni, H. Fischer-Brandies, M. Es-Souni, J. Biomed. Mater.

Res. A. 2007, 80, 159.


versatile nanocomposite coatings with tunable cell adhesion and bactericidity

Documents