Tunable Antibacterial Coatings That Support Mammalian Cell

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Tunable Antibacterial Coatings That Support Mammalian Cell Growth Krasimir Vasilev,*,† Vasu Sah,†,‡ Karine Anselme,§ Chi Ndi,‡ Mihaela Mateescu,§ Bjo¨rn Dollmann,‡ Petr Martinek,‡ Hardi Ys,‡ Lydie Ploux,§ and Hans J. Griesser‡ †

Mawson Institute, University of South Australia, Mawson Lakes, SA 5095, Adelaide, Australia, ‡ Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095, Adelaide, Australia, and § Institut de Science des Materiaux de Mulhouse, Universite de Haute-Alsace, France ABSTRACT Bacterial infections present an enormous problem causing human suffering and cost burdens to healthcare systems worldwide. Here we present novel tunable antibacterial coatings which completely inhibit bacterial colonization by Staphylococcus epidermidis but allow normal adhesion and spreading of osteoblastic cells. The coatings are based on amine plasma polymer films loaded with silver nanoparticles. The method of preparation allows flexible control over the amount of loaded silver nanoparticles and the rate of release of silver ions.

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ven in the 21st century infections are a tremendous problem in our society.1-5 Bacterial colonization and biofilm formation have been recognized as serious issues in areas such as medicine, food processing, marine vessels, heat exchangers, and others. Synthetic antibiotics are a common means to combat microbes; however, there are several generic problems associated with their use.1-3,6-9 A synthetic antibiotic often is specific to certain bacteria or a family of bacteria, not all bacteria can be killed by antibiotics, and most importantly bacteria can develop antibiotic resistance. All these issues have led to a renaissance of silver as an antiseptic agent in recent years. Silver has been known from the time of the ancient Greeks to have antimicrobial properties, but after the discovery of penicillin, silver was neglected, until it recently attracted once again considerable attention. Numerous studies demonstrate the capability of silver ions and nanoparticles to reduce infections and bacterial colonization on burns dressings,10,11 catheters,12,13 prostheses,14,15 arthroplasty,16 dentistry,17,18 and many other biomedical applications. Applications of antimicrobial materials involving silver extend to water treatment systems19,20 and textiles.10,21,22 Recent literature reports have explored antibacterial coatings loaded with silver nanoparticles and ions by various means, such as layer-by-layer deposition,21,23 sol-gel processes,24,25 electrochemistry,26 ion beam deposition,27 and chemical vapor deposition.28 However, the issue of the toxicity of silver to patients has been a substantial issue.29 The aim of this work is the design of novel thin polymer films loaded with silver nanoparticles which are highly efficient against bacterial colonization and at the same

time allow normal adhesion and spreading of mammalian cells, which is important when medical applications are considered. There were several criteria that needed to be fulfilled in this work. The process of preparation needed to be fast, simple, and convenient. More importantly, however, there was the need for control of the amount of silver nanoparticles loaded in the films as well as control over the rate of release of silver ions. We have used plasma polymerization to create amine-rich thin film coatings because this technique, compared to alternative approaches, allows deposition of well-adhering polymer films on practically any type of substrate without the need for surface premodification, which offers significant benefits in terms of feasibility, time, and costs. Plasma polymerization has been used in a number of applications ranging from corrosion protection30,31 to functional layers for biomaterials and medical application;32,33 however, most of the efforts have focused on the deposition of continuous and uniform films. In this work we have developed plasma polymer films containing silver nanoparticles; the approach is based on the plasma polymerization of n-heptylamine (HA) because of previous work showing that this monomer provides stable, cohesive, adherent films, for which considerable analytical data are available.34-39 Amine groups were required for the complexation of in-diffusing silver ions (see below). The appearance of nanoporous morphology upon immersion in aqueous solution in HA plasma polymer (HApp) films35 led us to conjecture that HApp coatings might provide a suitable matrix for the diffusive release of silver ions from embedded silver nanoparticles. The experimental strategy for the generation of silver nanoparticles within HA plasma polymer films employed in this work is shown in Figure 1a. First, an approximately 100 nm thick HA plasma polymer film was deposited on substrates such as a clean glass slide. Loading with silver

* To whom correspondence should be addressed. Dr Krasimir Vasilev, Mawson Institute, University of South Australia, Mawson Lakes SA 5095, Australia. Tel: +61 8 83025697. Fax: +61 8 83025689. E-mail: [email protected]. Received for review: 10/1/2009 Published on Web: 12/07/2009 © 2010 American Chemical Society

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by immersion in NaBH4, the color of the glass substrate changed to yellowish-brownish due to the presence of silver nanoparticles. In Figure 1c UV-vis spectra of a sample loaded with Ag+ (black) and the same sample after reduction in NaBH4 (red) are shown. The new peak at a wavelength of about 420 nm that appears after reduction is due to the plasmon resonance of silver nanoparticles embedded in the film. Using Mie theory to model optical properties arising from silver nanoparticles in the plasma polymer matrix shows that the position of the plasmon resonance band corresponds to nanoparticles of average diameter of 25 nm. In situ QCM analysis showed that the loaded amount of silver nanoparticles is in the range of 500 ng/cm2 (Figure S1 in Supporting Information). Next we aimed to control the amount of loaded silver nanoparticles. We conducted a series of studies to document the influence of the Ag+ loading time, time of reduction, and thickness of the HApp film on the amount of embedded silver nanoparticles. The intensity of the plasmon resonance peak serves as a quantitative measure for the amount and size of silver nanoparticles within the film. All studies were conducted with HApp films deposited at an rf power of 40 W. The choice of power for plasma polymerization was based on previous studies of the effect of plasma power on the chemistry of HA pp films.35,37 The influence of the time of immersion in AgNO3 on the final amount of silver nanoparticles within the film was evaluated based on the intensity of the absorption maxima and is shown in Figure 2a. All samples were prepared using identical plasma polymerization conditions and were exposed to NaBH4 for the same time. In the time frame of 120 min, the absorption intensity of the plasmon resonance of silver nanoparticles increased with the time of immersion in AgNO3. The increase was steeper in the first 40 min. After 1 h of immersion the absorption maximum still increases, but at a slower rate. These data show that by increasing the time of immersion in AgNO3 solution up to 2 h, more silver ions can be complexed within the HApp film and as a consequence more silver nanoparticles are formed after reduction in NaBH4. Notably, increasing the Ag+ loading time did not lead to significant changes of the wavelength of the plasmon resonance, which was 420 ((2) nm, which indicates that particles of the same size were produced irrespective of the loading. In addition, we examined the effect of the reduction time on the amount of embedded silver nanoparticles (Figure 2b). Identical conditions of plasma polymerization and immersion in AgNO3 were used for all samples. The reduction proceeded with a high rate in the first 10 min. The amount of silver nanoparticles almost doubled after 30 min. Between 30 and 60 min the rate of reduction decreased. The wavelength of the adsorption maxima did not change in the first 30 min, remaining at about 420 nm, but longer times of immersion in NaBH4 led to a slight shift to a longer wavelength, of ∼424 nm.

FIGURE 1. Loading silver nanoparticles in HApp films. (a) Schematic representation of the experimental strategy for the fabrication of silver nanoparticles within amine plasma polymer films: (1) plasma deposition of n-heptylamine; (2) loading with silver ions by immersion in AgNO3 solution; (3) reduction of silver ions by immersion in NaBH4 solution. (b) Glass samples after deposition of a 100 nm of HApp layer; after loading with Ag+ ions, and after reduction with NaBH4. (c) UV-vis absorption spectra of samples after loading with silver ions (black) and after reduction by NaBH4 (red).

ions was implemented by placing samples in a solution of AgNO3 for a predetermined time. In this loading step we have taken advantage of the well-known fact that amine groups can complex silver ions, according to

Ag+ + 2RNH2 ) [Ag[RNH2)2]+ Reduction of silver ions (Ag+) to metallic silver was subsequently achieved by immersion of samples in a solution of NaBH4, which acts as a reducing agent for the synthesis of silver nanoparticles from silver ions. An example of the outcome of our experimental strategy is shown in Figure 1b. After the deposition of a 100 nm thick HApp layer the glass substrate is still transparent, maintaining its optical properties. After immersion in AgNO3, no visible change of the optical properties of the substrate is observed. After reduction of the complexed Ag+ ions to Ag0 © 2010 American Chemical Society

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We have also explored the effect of the film thickness on the amount of silver nanoparticles. Initially we expected to increase the loading of silver in the films with increasing thickness. Figure 2c shows, however, that increasing the thickness in the range 0-100 nm leads to a rapid increase in the amount of silver nanoparticles followed above 100 nm by the amount of silver nanoparticles remaining constant to a film thickness of ∼200 nm and then gradually decreasing. Our approach of the synthesis of silver nanoparticles within HApp films was based on earlier findings35 that showed that immersion in aqueous solution leads to extraction of low-molecular-weight water-soluble fractions of the films, thus creating a nanoporous structure, which allows the loading of Ag+ ions and the subsequent formation of silver nanoparticles within the films. The data presented in Figure 2c suggest that increasing the plasma polymerization time leads to a gradual change of the chemistry and properties of the HApp films, probably an increase in the cross-link density, and thus smaller numbers of pores that are accessible for loading with silver ions. Control over the rate of release of silver ions from the films is an important issue since excessive doses of silver ions may have undesired consequences in medical applications, such as tissue staining (argyria). In an aqueous medium the silver nanoparticles loaded into HApp films undergo oxidation and subsequently silver ions are eluted from the films (Figure 3). In order to control the release of silver ions from the films, we adopted a convenient approach which consists of depositing another thin layer of HApp on top of the film loaded with silver nanoparticles. This approach amounts to the addition of a diffusion barrier that enables control of the rate of release of silver ions by adjusting the thickness of the HApp overlayer. Silver release studies were carried out by immersion in phosphatebuffered saline (PBS) solution for 20 days and measuring the reduction in the content of silver nanoparticles within the films by the change in the intensity of the plasmon resonance peak. Figure 3 shows that deposition of an additional HA plasma polymer film of thickness of 6 nm (circles) achieves a reduction of the rate of release of silver ions compared to films without such an additional pp film (squares). Increasing the thickness of the overlayer to 12 nm (triangles) and 18 nm (blue triangles) led to further reductions of the rate of release of silver ions, and within 20 days only 35% and 20%, respectively, of the silver content were released. Thus, a HApp overlayer of predetermined thickness can serve to adjust the release rate to a value intended for a specific application, over a time frame of several weeks. The antibacterial properties of silver nanoparticle loaded films were assessed with cultures of Staphylococcus epidermidis, a bacterium causing serious problems related to implant infections.40 For all samples the HApp film thickness was 100 nm and the rf power of polymerization was 40 W. Silver nanoparticles were loaded using a time of immersion in AgNO3 of 1 h and a time of reduction in NaBH4 of 30 min.

FIGURE 2. Control over the amount of loaded silver nanoparticles. (a) Influence of the time of immersion in AgNO3 on the amount of Ag nanoparticles in the film, for HApp films 100 nm thick and a time of immersion in NaBH4 of 1 h. (b) Influence of the time of immersion in NaBH4 on the amount of Ag nanoparticles in the film, with HA films 100 nm thick and a time of immersion in AgNO3 of 1 h. (c) Influence of the thickness of the HA pp film on the amount of Ag nanoparticles in the film, with a time of immersion in AgNO3 of 1 h and a reduction time of 30 min. The solid lines are guides for the eye, not fits.

Our interpretation of this trend is in terms of the mechanism of nucleation and growth of silver nanoparticles. In the first 30 min nucleation occurs and particles grow to a size similar for all particles. The shift in the plasmon resonance peak to longer wavelengths upon longer reduction times can be explained in terms of consuming remaining silver ions within the HApp film for additional growth of existing nanoparticles and perhaps some Ostwald ripening. © 2010 American Chemical Society

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FIGURE 3. Control over the release of silver ions: Achieved by depositing an additional layer of HApp on top of the silver nanoparticles loaded film, as shown on the right. The graph on the left shows the kinetics of release of silver ions over 20 days of immersion in PBS: black squares, silver loaded films as prepared; red circles, after deposition of a 6 nm thick HApp overlayer; green triangles, after deposition of a 12 nm thick HApp overlayer; blue triangles, after deposition of a 18 nm thick HApp overlayer.

FIGURE 4. Antibaterial efficiency. Staphylococcus epidermidis adhesion and colonization after 4 h on (a) HApp only (no silver), (b) HA film loaded with silver nanoparticles, (c) HA film loaded with silver nanoparticles and covered with a additional layer 6 nm thick of HApp. (d, e, and f) Biofilm formation after 24 h on samples loaded with silver nanoparticles covered with an additional layer of HApp of thickness (d) 6 nm, (e) 12 nm, and (f) 18 nm. In (d), (e), and (f), “pp” denotes plasma polymer only and SN denotes films loaded with silver nanoparticles.

Figure 4a shows representative images. The HApp film without silver allows bacteria to readily adsorb and start colonizing the surface within 4 h. The situation is very different when bacteria are exposed to silver nanoparticle loaded films for the same time (Figure 4b). Much of the staining occurs by dye uptake into the plasma polymer film, very few bacterial cells are evident, and they appear to have a rounded shape indicative of poor attachment. Figure 4c shows the same effectiveness in preventing bacterial attachment when the silver nanoparticle loaded HApp layer is covered with an additional thin pp polymer film to reduce the rate of release of silver ions. Few bacteria are able to attach to the surface, and the silver ions prevent them from dividing and colonizing the surfaces. © 2010 American Chemical Society

Results of tests performed over 24 h with films loaded with silver nanoparticles and subsequently coated with an HApp overlayer of thicknesses 6, 12, and 18 nm are shown in panels d, e, and f of Figure 4. In each test, six wells were coated only with HA pp film and another six wells with HApp loaded with silver nanoparticles. Safranin staining was used to detect bacterial biofilms. The photographs show rapid colonization of the samples without silver nanoparticles. In contrast, all films containing silver nanoparticles, regardless of the rate of silver release, show complete (100%) inhibition of bacterial adsorption. For medical devices, the potential toxicity of antibacterial coatings to mammalian cells is an important criterion. Hence identical coatings as used for bacterial studies were tested 205

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FIGURE 5. Osteoblastic cell adhesion and spreading. after 24 h on (a) glass control; (b) HApp film (sample 1); (c) HApp film loaded with silver nanoparticles (sample 2); (d-f) same as (c) but coated with an overlayer of HApp of thickness 6 nm (d) (sample 3), 12 nm (e) (sample 4), and 18 nm (f) (sample 5); (g) cell numbers and (h) individual cell areas after 72 h on HApp and HApp films loaded with silver nanoparticles without and with additional HApp of different thickness.

with human SaOs-2 osteoblastic cells. Panels a-f of Figure 5 show fluorescence microscopy images of cells after 24 h in culture. On a glass surface (Figure 5a) cells are rounded and clustered. When the surface is coated with HApp film (Figure 5b) cells are spread, presenting a morphology characteristic of healthy cells of this type, in agreement with an earlier study reporting good anchorage-dependent mammalian cell attachment to HApp surfaces.41 When coatings are loaded with silver nanoparticles (Figure 5c), the number of attached cells is significantly reduced due to the toxicity of the nanoparticles. However, applying an HApp overlayer of increasing thickness enables cells to attach in greater numbers (Figure 5, panels d, e, and f). The cell numbers on samples without and with silver nanoparticles after 72 h are shown in Figure 5g. Cell numbers increase with increasing thickness of the additional HApp film controlling the rate of release of silver ions from the HApp films. Averaged cell © 2010 American Chemical Society

areas estimated from the images in panels b-e of Figure 5 are plotted in Figure 5h and show comparable cell spreading area to the sample without silver nanopatricles. Thus, by appropriate selection of the silver ion outdiffusion kinetics via an HApp overlayer of specific thickness, it is feasible to maintain efficient prevention of bacterial attachment while allowing osteoblasts to attach and spread on the surface. This balance, achievable by the control afforded by our multilayer coating design, holds great promise for minimizing deleterious effects on human tissue from implants coated with our coatings. In summary, we report a novel method for the generation of antibacterial coatings involving plasma polymerization and in situ synthesis of silver nanoparticles within the films. The advantage of using plasma polymerization is that these films can be deposited onto practically any type of material and device. Our method of fabrication of antibacterial 206

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coatings is fast, consisting of three straightforward steps with the total sample preparation time in the range of 2 h. We have also demonstrated that we can control the amount of loaded silver nanoparticles by the time of loading with silver ions, by the time of reduction to silver nanoparticles, and by the thickness of the HA plasma polymer layer. We have also demonstrated control over the release of silver ions by depositing a thin plasma polymer overlayer. Bacterial studies with Staphylococcus epidermidis show complete inhibition of bacterial colonization. Moreover, the coatings can be tuned to be acceptable to osteoblast cells, which is important in medical device applications. This work demonstrates that via careful material design it is possible to generate surfaces inhibiting bacterial colonization but still allowing attachment of mammalian cells. The coating presented in this work may open new horizons for the design of a next generation of antibacterial coatings. Supporting Information Available. Description of all experimental procedures and QCM measurement to determine the amount of loaded silver nanoparticles. This material is available free of charge via the Internet at http:// pubs.acs.org. REFERENCES AND NOTES (1)

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