Effect of light on physicochemical and biological properties of nanocrystalline silver dressings

Abstract

The purpose of this study was to characterize the interactive effects of light and aging on physicochemical properties and antimicrobial activity of nanocrystalline silver wound dressings Acticoat that might find their way into the environment. Nanocrystalline silver films deposited on high-density polyethylene were exposed to a light source that mimics natural daylight for various time lengths and characterized using atomic force microscopy (AFM), X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and secondary ion mass spectroscopy (SIMS). Bactericidal efficacy of the light exposed nanocrystalline silver was measured using a log reduction assay. A change in color from dark blue to purple and then brown was observed after exposure to light. The change in color was attributed to an increase in surface roughness as emphasized by the AFM imaging. A decrease in silver oxide thickness followed by an increase in silver sulfide and silver carbonate content was observed during SIMS analysis. Interestingly, the XPS analysis confirmed this result by the breakage of the Ag–O bonds and formation of silver sulfide in the nanocrystalline matrix. Even though nanocrystalline silver exposed to light for four weeks maintained its bactericidal capacity, the observed physicochemical changes (grain growth, chemical composition changes) suggest that given sufficient time, nanocrystalline silver dressings should deactivate in time, in the environment.

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Introduction

Nanotechnology is a rapidly expanding field with nanomaterials revolutionizing the areas of aerospace engineering, nanoelectronics, environmental remediation, agriculture, and medical healthcare.^1,2 The large selection of products containing nanomaterials increases the certainty that nanoparticles and nanostructures will eventually reach the environment presenting unknown consequences.³ Despite the fast growing market for nanomaterials, large knowledge gaps exist regarding the possible risks associated with human and environmental exposure to these products.^4,5 Identification of the potential toxic effects of nanomaterials and preventing harm to humans and the environment is of great importance for the widespread implementation and consent of nanotechnology.^6,7 The first step in assessing the potential hazards associated with exposure to nanomaterials, is to understand how environmental conditions, such as light or heat exposure may change their physical and chemical characteristics.⁸

The most prominent metal employed in nanoformulation is silver.⁹ Silver has been recognized for its potential application in medical healthcare since 1884, when Crede first used 1% silver nitrate to treat and prevent ocular infections.^10,11 Later, during World War I, silver compounds became a major weapon against wound infections.¹² In 1965, Moyer et al. first used 0.5% silver nitrate solution in the burn arena and this was followed by the development in 1968 of a topical antibacterial cream containing 1% silver sulfadiazine for the treatment of burns.¹³

Silver nanoproducts are indicated as one of the fastest growing materials in the area of nanotechnology industry.¹⁴ The prevalence of silver nanoparticles and nanostructures in consumer products makes it essential to elucidate how variables, such as light exposure, could change their physical and chemical properties.^15,16 In recent years, metallic silver has been subjected to modern engineering technologies leading to nanocrystalline materials that exhibit advanced morphologies and properties. The exceptionally strong antimicrobial activity of silver remains the major driver for the development of silver nanoparticles and nanocrystalline materials.^17,18

Nanocrystalline silver represents one of the most successful modern nanomaterials that differs in both physical and chemical properties from micro- or macro-crystalline silver and from silver salts.¹⁹ The distinguishing characteristics of nanocrystalline silver are mainly attributed to its high surface area to volume ratio, which allows a larger number of atoms to interact with their surroundings and potentially results in higher reactivity.^20,21 As reported by Fan and Bard (2002), nanocrystalline silver presents a remarkable property namely, it dissolves to release Ag⁰ clusters, Ag⁺, and a complex hydroxyl form, whereas other silver sources release only Ag⁺.²² It is this unique dissolution process that gives nanocrystalline silver its unusual biological bifunctionality including antimicrobial and anti-inflammatory properties.²³

Numerous studies have recently been conducted to increase the understanding of the impact of environmental conditions such as pH, ionic strength, and electrolyte composition on the surface charge, aggregation and dissolution behavior of capped and uncoated silver nanoparticles.^24,25 The results show that all the studied variables influence the fate of nanoparticles in the environment. In contrast, only a few research papers discuss the instability of nanocrystalline materials when exposed to environmental factors such as heat or light.

Polycrystalline materials such as nanocrystalline silver absorb light and this energy can initiate chemical reactions on their surface which would otherwise not occur.²⁶ Moreover, sunlight irradiation is believed to influence the toxicity of silver nanomaterials in the environment, particularly since silver nanomaterials are UV light sensitive.²⁷ Taylor et al. (2005) showed that heat treatment of nanocrystalline silver dressings has a significant impact on their chemical and biological properties.²⁸ Whereas at temperature between 23 and 37 °C the initial decomposition of the silver–oxygen bonds occurred, temperatures above 75 °C for 24 h inactivated the dressings, greatly decreasing their effectiveness both as short-term bactericidal agents, demonstrated using the log reduction test, and as long-term inhibitors of bacterial growth, determined from the corrected zone of inhibition (CZOI) assay. In another study, high quality nanocrystalline diamond samples have been investigated prior and after UV exposure at room temperatures.²⁹ The UV treatment induced an oxidation of the surface which was originally hydrogen-terminated. This phenomenon was verified macroscopically by the change of the surface wettability and by the increase of the oxygen content as evaluated by XPS.²⁹ Both these studies confirm the instability of nanocrystalline materials under the influence of external factors when compared to their bulk counterparts.

Nanocrystalline silver dressings, commercially available as Acticoat (Smith and Nephew, Inc.), are the world's number one selling nanosilver dressings used in burn units around the globe. Due to its widespread application, Acticoat eventually ends up in the environment presenting unknown consequences. The present study explores the interactive effects of light and aging on the physicochemical properties of nanocrystalline silver wound dressings and relates them to the antimicrobial activity of this nanomaterial. The microstructural evolution of nanocrystalline silver dressings exposed to light up to four weeks was explored in detail using atomic force microscopy (AFM), X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Then, the bactericidal effect of nanocrystalline silver was evaluated using a log-reduction assay.

Materials and methods

Acticoat is composed of three layers with the two outer layers, each of 1000 nm thickness, consisting of nanocrystalline silver films sputtered onto high-density polyethylene (HPDE).³⁰ The inner layer represents absorbent rayon/polyester non-woven gauze and the three layers are spot welded together. The nanocrystalline silver coated layers are produced by magnetron sputtering (physical vapor deposition) onto HPDE in an argon atmosphere with trace oxygen levels (4%). During this process, the silver atoms (99.99% purity) are layered down atom by atom onto the coating, forming polynanocrystalline columnar structures.³¹ The ammonium soluble part of the total silver in Acticoat is approximately 45%.³¹ Sterile nanocrystalline silver dressings (Acticoat, Smith & Nephew Inc., Largo, FL, expiry date December, 2014) were obtained from a commercial supplier. Prior to testing, three silver dressings were cut into 150 mm × 150 mm squares that were then exposed to a light source, in ambient air, at room temperature for 24 h, 1 week, 2 weeks, and 4 weeks. Three pieces of Acticoat (25.4 mm × 25.4 mm) were cut out of each exposed nanosilver dressing and further used for the log reduction assay and silver dissolution tests. Three pieces of Acticoat (10 mm × 10 mm) were also cut out of each exposed nanosilver dressing and used for AFM, SEM, XPS, and SIMS analyses. All nanosilver dressings were prepared in the same manner to ensure the starting conditions for all of the experiments were the same.

For the irradiation of the samples, a cool-white fluorescent wide spectrum lamp (GRO-LUX, Osram Sylvania) was used. This lamp mimics the natural daylight and has a spectral range from 380–750 nm. Nanocrystalline silver was exposed to an illuminance of 2000 lux which was measured with a digital illuminance meter (Nicety LX 802). This illuminance was close to that of indirect sunlight measured at the time of the experiment, approximately 5000 lux and did not increase the temperature on the sample surface. The conditions for this experiment are less extreme than what might be expected under bright sunlight which provides illuminance of approximately 100 000 lux.

The ambient atmosphere composition in Edmonton, Alberta, Canada is known to be rich in H₂S, O₃, CO, CO₂, and NO₂ as a result of the oil sands exploration in the nearby regions. Table S1 in ESI† Data provides daily average concentrations of some ambient air pollutants in Edmonton, Alberta, Canada.

Surface physicochemical characterization

Nanocrystalline silver was characterized by AFM, SEM, ToF-SIMS, XRD, and XPS. SEM, ToF-SIMS, and XPS were performed under high-vacuum conditions (<10⁻⁸ Torr).

Atomic force microscopy (AFM). Small pieces of nanocrystalline silver dressing (10 × 10 mm) attached to glass slides were analyzed by AFM to determine if structural changes occurred after exposure to light. The AFM characterization was performed using a Molecular Force Probe 3D (MFP 3D) from Asylum Research (Santa Barbara, CA) controlled with IGOR PRO software (Wavemetrics, Portland, OR). The MFP 3D instrument gives improved control over the z position of the cantilever relative to the sample by using an absolute position sensor. All the topographic images were acquired in tapping mode of operation. The scan rate ranged from 0.5 to 1 Hz and the tip velocity was maintained between 25 and 50 μm s⁻¹. Silicone probes (AC240TS) with nominal spring constants of 0.5–4.4 N m⁻¹ were purchased from Asylum Research (Santa Barbara, CA) and used for AFM imaging.

The IGOR PRO software allows the calculation of the R_rms (root mean square)-roughness and comparison of the nanocrystalline silver surfaces before and after exposure to light. R_rms is calculated by eqn (1):

where z_i is the height value of each single data point in the image, z⁻ the mean value of all height values in the image and n the number of data points within the image.

Scanning electron microscopy (SEM). Small pieces of nanocrystalline silver dressing (10 × 10 mm) were analyzed by SEM to detect any changes in structure after exposure to light. The SEM was performed on a JAMP-9500F Auger microprobe (JEOL) at the Alberta Centre for Surface Engineering and Science (ACSES, University of Alberta). This instrument offers the highest spatial resolution available in a microprobe (3 nm). All samples were attached directly to a specimen mount and were not cleaned prior to imaging as such a process could alter the nanostructures. Images were taken at magnifications from 20 to 500 000 times, using an accelerating voltage of 15 kV, and a working distance of approximately 24 mm. The SEM experiments were performed in triplicate for each experimental condition.

X-Ray photoelectron spectroscopy (XPS). XPS measurements were performed to identify the chemical species present on the surface of the light exposed nanocrystalline silver. The XPS analysis was conducted on an AXIS Ultra XPS Spectrometer (Kratos Analytical, Shimadzu Corp., Kyoto, Japan) at the Alberta Center for Surface Engineering and Science (ACSES, University of Alberta). The base pressure in the analytical chamber was lower than 2 × 10⁻⁸ Pa. The X-rays were generated using an Al Mono (Kα) (hm = 1486.6 eV) source operated at 210 W. The spectra were collected at normal 90° take-off angle, with analyzer pass energy of 160 eV for survey spectra, and 20 eV for high-resolution spectra. The instrument was calibrated on the basis of the C 1s peak. Casa XPS software (Version 2.3.9, © 1999–2005 Neal Fairley) was applied for data processing. The spectra fitting and component analysis was performed using the high-resolution spectra. No charge corrections to the binding energy values were introduced. The XPS experiments were performed in triplicate for each experimental condition.

ToF-SIMS depth profiling. SIMS depth profiling analysis was carried out using a ToF-SIMS IV instrument (ION-ToF, GmbH, Munster Germany), located at the Alberta Center for Surface Engineering and Science (ACSES, University of Alberta). For the depth profiling, two ion beams operated in the dual beam mode were used. The primary analysis beam (Bi⁺) generated the secondary ions which were analyzed in a time of flight mass analyzer; the Bi⁺ gun was operated at 25 keV. During the flight time, a second beam delivered by a Cs⁺ thermal ionization source was used as the sputtering source; the Cs⁺ gun was operated at 1 keV. By alternating the two ion beams with a flood gun pulse in between cycles, a depth profile was acquired. Metallic silver, carbon, oxygen, silver oxide, silver sulfide, silver carbonate and silver cluster species weights were analyzed. The ToF-SIMS experiments were performed in triplicate for each experimental condition.

X-Ray diffraction (XRD). XRD analysis of the light exposed nanocrystalline silver dressings was performed on a Rigaku Geigerflex 2173 X-ray diffractometer equipped with a cobalt tube (the average wavelength 1–78 899 A), a graphite monochromator and a scintillation detector. The samples were mounted using two sided tape onto glass slides and placed into the Rigaku Geigerflex XRD machine. Diffraction data were collected between 20 and 100° 2θ at a speed of 2 degrees two-theta per minute and a step size of 0.02. The peaks in the X-ray diffraction pattern were identified in terms of the Bragg angle, 2θ. The XRD experiments were performed in triplicate for each experimental condition.

Silver dissolution tests. The release of silver from light exposed nanocrystalline silver in distilled water was measured using Atomic Absorption Spectroscopy (AAS). The distilled water was purified on a Milli-Q_plus apparatus (Millipore) to a resistivity higher than 18.2 MΩ cm⁻¹. These experiments were performed in triplicate and the results were the average of three independent experiments.
(a) Soluble silver. Pieces of 25.4 × 25.4 mm nanocrystalline silver coated HDPE were individually submersed in glass vials containing 5 mL of distilled water. The vials were wrapped in aluminum foil to prevent silver photoreduction and precipitation and incubated without agitation for 24 hours at 37 °C. After incubation, the silver dressings were removed aseptically, allowed to drip into the vials for approximately 10 s, and then discarded. The resulting extracts were then acidified in 9% nitric acid/0.9% tartaric acid to ensure that all silver released was in solution and submitted for total silver analysis by AAS.
(b) Total silver. Pieces of 25.4 × 25.4 mm nanocrystalline silver coated HDPE were individually submersed in glass vials and 15 mL of 1 : 2 nitric acid solution (65% w/w, Fisher Scientific, Canada) were added into the vials containing the sample to be digested. The samples were then heated at 105 °C ± 10 °C for twenty minutes. At the end of the time period, the vials were cooled to room temperature and the solutions submitted for silver analysis by AAS.

Biological characterization

Log reduction assay. In this study, the log reduction assay was performed to determine the ability of light exposed nanocrystalline silver to kill bacteria in 30 min and to compute the rate of bacterial killing. Pseudomonas aeruginosa (ATCC 27317), a Gram-negative bacterium found throughout the environment and the cause of numerous opportunistic infections in humans, was suspended in trypticase soy broth (TSB, Difco, Sparks, MD) medium and used to test the antimicrobial activity of nanocrystalline silver. Initially, P. aeruginosa was grown overnight (16 hours) in 100 mL TSB with incubation at 37 °C under gyratory shaking (RPM × 120). The method used was similar to that employed by Wright et al.²³ (1998), with the main difference that the inoculum volume was increased from 200 to 300 μL. One milliliter of bacterial culture obtained from overnight cultures was added to 100 mL of fresh TSB and incubated at 37 °C for 3–6 h to obtain approximately 1.1 × 10⁹ CFU of P. aeruginosa per mL. Then, P. aeruginosa suspensions were used to inoculate 25.4 × 25.4 mm pieces of nanocrystalline silver. The inoculated dressing was incubated for 30 min at 37 °C. After removal of the dressings from the incubator, each one was placed in 2.7 mL of a salt, polysorbate, sodium thioglycollate (SPS) bacterial recovery solution containing 0.85% [w/v] NaCl, 1% [v/v] polysorbate 20, and 0.1% [w/v] sodium thioglycollate. The SPS solution inactivates the nanosilver dressing.

Each dressing suspended in SPS was vortexed vigorously and the resulting bacterial recovery solution was serially diluted (i.e. 10⁻² to 10⁻⁷ dilutions) in phosphate-buffered saline (PBS, pH 7.0). Three 20 μL drops of each dilution were plated on tryptic soy agar (TSA). The plates were then incubated at 37 °C for 24 h and the numbers of bacterial colonies were counted. The logarithms of the starting numbers and surviving numbers of bacteria were determined and the log reductions were calculated as the difference between the log of the initial number of bacteria and the log of the final surviving number of bacteria. These experiments were performed in triplicate, and the results were the average of three independent experiments.

Results

Nanocrystalline silver morphology and physicochemical characterization

After exposing the nanocrystalline silver to a light source that mimics natural daylight for 1 day, 1 week, 2 weeks, and 4 weeks, we noticed a change in color from dark blue, Fig. 1a (for the reference sample) to purple, Fig. 1c, and then, brown (Fig. 1d) after 2 and 4 weeks of exposure, respectively.

Fig. 1 Photographic images of the same piece of nanocrystalline silver dressing (25.4 mm × 25.4 mm) showing the evolution in the properties for the same spot, namely the change in color with light exposure to 2000 lux: (a) unexposed; (b) 1 week exposure; (c) 2 weeks exposure; (d) 4 weeks exposure.

Atomic force microscopy (AFM). Fig. 2 presents representative AFM amplitude (a, c, e and g) and 3D (b, d, f and h) images of the reference nanocrystalline silver dressing (a and b) together with nanocrystalline silver samples exposed to light for 1 week (c and d), 2 weeks (e and f), and 4 weeks (g and h). The AFM images were taken on different areas of the three pieces of nanocrystalline silver exposed to light for various time lengths. All the AFM images presented the same characteristics.

Fig. 2 Representative AFM amplitude images of nanocrystalline silver (a, c, e and g) and 3D surface plots of AFM height images (b, d, f and h). (a and b) Unexposed to light; (c and d) 1 week; (e and f) 2 weeks; and (g and h) 4 weeks exposure to light.

AFM images for the reference nanocrystalline silver (Fig. 2a and b) show a porous structure with many distinct agglomerates of grains and/or particles. After light exposure, we notice that the columnar structures are getting thinner (Fig. 2d and f) and smaller (Fig. 2h). These visual observations were accompanied by calculation of surface roughness. As shown in Table S2,† the root mean square roughness R_rms of the film increases from 26 ± 0.2 nm for the reference sample to 36.5 ± 0.05 nm for the nanocrystalline silver exposed to light for 4 weeks (5 μm scan).

Scanning electron microscopy (SEM). Fig. 3 shows representative high resolution SEM images (×50 000) of the reference sample (a) and the nanocrystalline silver exposed to light for 1 week (b), 2 weeks (c), and 44 weeks (d). The SEM experiments were performed at different locations on the three pieces of nanocrystalline silver dressings exposed to light.

Fig. 3 Representative SEM images of nanocrystalline silver samples exposed to light: (a) reference sample; (b) 1 week; (c) 2 weeks; and (d) 4 weeks exposure to light.

The morphology of the reference sample is representative of columnar structures with secondary branching of equiaxed grains (Fig. 3a). The large depth of field of the high resolution SEM reveals the general openness of the structure and the porosity between the columns. The points of interest for the reference sample (Fig. 3a) are the nanocrystalline fine structures about 20–30 nm in diameter on the surface of the columnar grains. In the case of the nanocrystalline silver film exposed to 2000 lux for 1 week (Fig. 3b), the nanometer size crystals on top of the columnar grains are no longer visible. This trend continues for the 2 weeks (Fig. 3c) and 4 weeks (Fig. 3d) exposed nanocrystalline silver samples and is accompanied by a visible increase in surface roughness.

X-Ray diffraction (XRD). XRD was performed to study the structural changes of the nanocrystalline silver exposed to light. Fig. 4 presents the XRD patterns for the reference sample and for nanocrystalline silver exposed to light for 1 week, 2 weeks, and 4 weeks. With light exposure, it is noticeable a decrease in height of the silver oxide peak followed by an increase in the Ag/Ag₂O peak.

Fig. 4 XRD patterns of light exposed nanocrystalline silver.

The crystal grain diameter d for silver and silver oxide, was estimated from the full width at half maximum of the Ag (200) and Ag₂O (111) peaks using Scherrer eqn (2).

where k, the Scherrer constant, is the shape factor taken equal to 0.9, λ is the X-ray wavelength, β is the line broadening at half the maximum intensity (FWHM) in radians, and θ is the Bragg angle.

Table S3 in ESI† Data summarizes the results of the XRD analysis. We notice a decrease in size for the silver oxide grains from 17.66 ± 0.02 nm for the reference sample to about 15 ± 0.20 nm for the nanocrystalline silver exposed to light for 4 weeks. There is an inverse trend for the silver grains for which we see an increase in size with exposure to light from 15.18 ± 0.01 nm for the reference sample to 22.7 ± 0.50 nm for the samples exposed to light for 4 weeks.

X-Ray photoelectron spectroscopy (XPS). The change in chemical composition of the few uppermost layers of nanocrystalline silver with exposure to light was investigated by XPS. Generally, identification of chemical species based on peak position within the XPS spectra involves peak deconvolution.³² In order to investigate the presence of any silver compounds, the Ag 3d_5/2 spectrum for the reference sample was deconvoluted into three components as shown in Fig. 5a. It is evident that the width and position of the Ag 3d_5/2 peak for the reference sample is due to overlapping of the peaks related to Ag–Ag, Ag–O, and Ag–C bonds, with Ag 3d_5/2 peaks positioned at about 368.2, 367.8, and 367.7 eV. In the case of nanocrystalline silver exposed to light for 2 weeks (Fig. 5b), the width and position of the Ag 3d_5/2 peak is due to overlapping of four peaks related to Ag–Ag, Ag–O, Ag–C, and Ag–S bonds with Ag 3d_5/2 peaks positioned at about 368.2, 367.8, 367.7, and 368.1 eV. The width and position of the Ag 3d_5/2 peak for nanocrystalline silver exposed to light for 4 weeks (Fig. 5c) is due to overlapping of four peaks related to Ag–Ag, Ag–O, Ag–C, and Ag–S bonds with Ag 3d_5/2 peaks positioned at about 368.2, 367.8, 367.7, and 368.1 eV.

Fig. 5 Examples of deconvolution of Ag 3d_5/2 XPS spectra for nanocrystalline silver: (a) not exposed to light; (b) exposed to light for 2 weeks; and (c) exposed to light for 4 weeks. The solid line represents the experimental data and the colored peaks are the corresponding deconvoluted peaks.

Fig. 6 presents a comparison of the Ag 3d_5/2 peaks for the reference sample and nanocrystalline silver exposed to light for 4 weeks, after normalizing the data. We notice that increasing the light exposure of nanocrystalline silver leads to a shift of Ag 3d_5/2 peak from a binding energy of 367.8 eV (corresponding to Ag–O bond) for the reference sample to 368.1 eV (corresponding to Ag–S bond) for nanocrystalline silver exposed to light for 4 weeks.

Fig. 6 Comparison of Ag 3d_5/2 peaks for the reference sample and nanocrystalline silver exposed to light for 4 weeks after data normalization.

Fig. 7 contains representative XPS deconvolution peaks for the O 1s spectra of nanocrystalline silver not exposed to light (Fig. 7a), exposed to light for 2 weeks (Fig. 7b), and exposed to light for 4 weeks (Fig. 7c). Three major components related to O–O, O–Ag, and O–C bonds are clearly observed in all the samples (Fig. 7) and they are positioned at binding energies of 530.8, 529.3, and 531.6 eV.

Fig. 7 Summary of XPS intensities and deconvolution of O 1s peaks for nanocrystalline silver dressings: (a) not exposed to light; (b) exposed to light for 2 weeks; (c) exposed to light for 4 weeks. The solid line represents the experimental data and the colored peaks are the corresponding deconvoluted peaks.

Fig. 8 presents a comparison of the O 1s peaks for the reference sample and nanocrystalline silver exposed to light for 2 and 4 weeks after normalizing the data. With exposure to light, the O–Ag bonds break (right peak) so that, after 4 weeks of light exposure the amount of silver oxide in the dressing diminishes.

Fig. 8 Comparison of O 1s peaks for the reference sample and nanocrystalline silver exposed to light for 2 and 4 weeks after data normalization.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS). The depth profiling SIMS was performed to analyze the changes in nanocrystalline silver film chemistry after exposure to light for various time lengths. SIMS depth profiles were obtained to detect the presence, depth, and concentration of various silver elements and species.³³ Fig. 9 presents a summary of the depth profiles for elements and species that comprise the nanocrystalline silver films: (a) not exposed to light; (b) exposed to light for 2 weeks; (c) exposed to light for 4 weeks. The distribution of various ions and compounds is plotted versus the sputtering time.

Fig. 9 ToF-SIMS depth profiling as a function of sputtering time. Summary of the depth profiles for elements and species that comprise the nanocrystalline silver dressing: (a) not exposed to light; (b) exposed to light for 2 weeks; (c) exposed to light for 4 weeks.

In Fig. 9, three regions can be identified: (i) the nanocrystalline silver film (pink), (ii) the film/substrate interface (blue), and (iii) the HDPE substrate (gray). The first region of the reference sample (not exposed to light) (Fig. 9a) is characterized by a plateau of the Ag, and AgO profiles. At about 420 s, the intensity of C starts to increase marking the beginning of the silver film/substrate interfacial region in which the intensity of Ag and AgO drops. The intensity of C becomes stable after approximately 600 s of sputtering, indicating the end of the interfacial region and entry in the bulk substrate. In the first region of the reference sample, we notice approximately the same quantities of AgO and Ag⁰ and trace amounts of AgS and Ag₂CO₃. Whereas the C content remains constant in the third region of the reference sample, Ag is continuously decreasing and AgO is completely removed from the system.

The first layer of the sample exposed to light for 2 weeks, (Fig. 9b) shows a plateau in Ag and AgO profiles with almost equal quantities of metallic Ag and AgO and an increase in AgS and Ag₂CO₃ content. The same trend follows the elements and compounds in the silver film region of the sample exposed to light for 4 weeks (Fig. 9c). In Fig. 9b and c, the beginning of the interfacial region is emphasized by a sharp decrease in Ag, AgO, AgS, and Ag₂CO₃, with AgO, AgS, Ag₂CO₃ completely disappearing from the system at the onset of the third region.

Since the nanocrystalline silver layer is porous, the depth of the crater etched during SIMS profiling, respectively the etch rate could not be measured directly. However, the etch time in the graph can be scaled to nanocrystalline silver depth (reference sample, Fig. 9a) assuming that the end of the profile at etch time of 800 seconds corresponds to a thickness of 1000 nm which was reported by Burrell and Morris (1998).³¹

Consequently, assuming a sputtering rate of 75 nm min⁻¹, we can estimate the approximate depth of the various Ag products formed. The ToF-SIMS depth profile results in Fig. 9c indicate the individual thickness of AgS and Ag₂CO₃ layers to be about 750 nm for the sample exposed to light for 4 weeks. The AgO layer starts to decrease sharply at about 500 s in the reference sample which corresponds to a thickness of 625 nm. After light exposure, the thickness of the AgO decreases to about 500 nm. This decrease in silver oxide is emphasized in the AFM 3D images (Fig. 2) by the thinning of the columnar structures and also in the XRD results, where the peak for silver oxide becomes shorter (Fig. 4).

Atomic absorption spectroscopy (AAS). Fig. S1† shows that the total silver content present in the reference sample (∼220 mg L⁻¹ or 3 mg in⁻²) it is also found in the nanocrystalline silver samples exposed to light for 1 week, 2 weeks and 4 weeks.

Fig. S2† shows a slight increase in soluble silver content with exposure to light for four weeks.

Bactericidal efficacy of light exposed nanocrystalline silver

Log reduction assay. In Fig. S3,† the log reduction of P. aeruginosa suspended in TSB medium is plotted versus time for the reference sample and nanocrystalline silver exposed to light for 2 and 4 weeks, respectively. The reference sample yielded approximately a 5.0 log₁₀ reduction (this is the maximum reduction possible with a 10⁸ CFU inoculum and a 10³ CFU detection limit). In this range, no statistically countable numbers of colonies (between 20 and 200 colonies) were recovered from the lowest dilutions. No significant difference in log reduction was detected for the nanocrystalline silver samples exposed to light for various time intervals. Notably, nanocrystalline silver exposed to light and inoculated with P. aeruginosa maintains its bactericidal capacity, where the definition of bactericidal is a dressing capable of producing a log₁₀ reduction greater than three.

Discussion

This study emphasizes the microstructural evolution and physicochemical changes of nanocrystalline silver dressings exposed to a light source that mimics natural daylight for up to four weeks. The results presented herein reveal important implications for human and environmental health.

As shown in Fig. 1, nanocrystalline silver exposed to light undergoes color changes from blue (reference sample) to purple and brown after 2 and 4 weeks of exposure, respectively. It is well known that optical properties, an important characteristic of metal nanoparticles and nanofilms, depend strongly on the particle size, shape, and proximity to each other.³⁴ For example, according to Liz-Marzán³⁴ (2004) bulk Au looks yellowish in reflected light whereas thin Au films look blue in transmission and the characteristic blue color changes to orange, through several tones of purple and red, as the particle size is reduced to approximately 3 nm. These color effects belong exclusively to metals which are known to possess free electrons (essentially Au, Ag, Cu, and the alkali metals) and emerge as a result of changes in metals surface plasmon resonance when exposed to visible spectrum.³⁵ Therefore, we expect that the variation in color that we observed for nanocrystalline silver samples exposed to light may occur as a result of the modification in surface roughness accompanied by the change in chemical composition.

As presented in the AFM images in Fig. 2, the nanosized columnar structures become thinner with light exposure. In an interesting study, Taylor et al. (2005) showed that silver oxide pins the columnar silver structures on nanocrystalline silver surface and prevents them from growing.²⁸ However, when exposed to temperatures as low as 30 °C, the Ag–O bond starts to break and metallic silver is formed. Their observation was reinforced by the XPS analysis which showed that Ag–O bonds started to break at temperatures between 23 and 37 °C. In another study, Gorham et al. (2012) showed that silver nanoparticles underwent a decrease in diameter upon UV-exposure.¹⁵

Consequently, we suspect that some of the silver oxide on the nanocrystalline silver matrix is reduced to metallic silver as this process requires the presence of an electron that would probably be generated through a photo process. As a result, the layer of silver oxide becomes narrower and the columnar structures look thinner (Fig. 2). The same conversion route for silver oxide reduction on nanoparticles surface was reported by Shi et al.²⁷

The visual observations in Fig. 2 were supported by the calculation of R_rms as shown in Table S2.† The R_rms of the film increases from 26 ± 0.2 nm for the reference sample to 36.5 ± 0.05 nm for the nanocrystalline silver exposed to light for 4 weeks (5 μm scan). The SEM images in Fig. 3 confirm the increase in surface roughness and show a loss of fine features for nanocrystalline silver with exposure to light. Moreover, the XRD results emphasize a decrease in height of the silver oxide peak followed by an increase in the Ag/Ag₂O peak (Fig. 4). This observation is in agreement with the AFM 3D images (Fig. 2) that emphasize a thinning of the columnar structures that are pinned by silver oxide.

The crystal grain diameter d calculated for silver and silver oxide (Table S3†) using Scherrer eqn (2) presents a decrease in size for the silver oxide grains exposed to light followed by an inverse trend for the silver grains that increase in size with light exposure. These results are in agreement with the previous observations that show a decrease in silver oxide content and an increase in metallic silver.

The XPS analysis in this study shows that Ag–O bonds break with light exposure and Ag₂S and Ag₂CO₃ are formed in the system (Fig. 5). As Ag–O bonds break, more metallic silver is produced in the nanocrystalline matrix. Furthermore, nanosilver reacts with H₂S in the ambient atmosphere leading to Ag₂S. Moreover, some of the silver oxide reacts with CO₂ in the environment, in the presence of humidity, producing Ag₂CO₃. These products account for the change in color observed in Fig. 1. Raju and Kumar (2011) showed that metallic silver nanostructures, the result of photodissociation of silver oxide, are highly reactive chemical species and very sensitive to O₃ and H₂S in the atmosphere.³⁶ Moreover, oxidizing species such as O₃ can enhance the formation rate of Ag₂S.¹²

In gaseous environments, it is known that thermal decomposition of Ag₂O to produce O₂ and metallic silver takes place at temperatures as low as 30 °C.³⁷ In aqueous environments, decomposition of Ag₂O colloids to produce silver nanoparticles takes place spontaneously at room temperature.³⁷ Peyser et al. (2002) showed that illumination of nanocrystalline silver oxide samples with blue and UV mercury lamp excitation produces Ag nanoclusters that range in size from 2 to 8 atoms.³⁸

The ToF-SIMS results in Fig. 9 display an increase in AgS and Ag₂CO₃ with light exposure accompanied by a sharp decrease in AgO content starting at about 400 s (∼500 nm) followed by the complete removal of this compound from the system at the onset of the third region. In the reference sample, the AgO layer starts to decrease at about 500 s which corresponds to a thickness of 625 nm. The difference in thickness explains the thinning of the columnar structures in Fig. 2 and the XRD results, where the peak for silver oxide becomes shorter (Fig. 4) after exposure to light.

According to the work of Blanton et al. (2011) and Krylova and Dukstiene (2013) silver sulphide should present XRD peaks in the 2θ range between 20 to 30°.^39,40 The XRD peaks for high definition polyethylene (HDPE), which is used as substrate for the nanocrystalline dressing, are in the same range as silver sulphide peaks and their intensity exceeds the intensity of the silver sulphide peaks. As a result, the silver sulphide peaks are not visible in the XRD pattern in Fig. 4.

Nanocrystalline silver preserves its antimicrobial activity even after exposure to light for four weeks (Fig. S3†). Taylor et al. (2005) showed that a silver grain size larger than 32 nm on nanocrystalline silver surface reduces considerably its bactericidal activity.²⁸ Other studies in the literature report that the toxicity of silver nanoparticles and nanostructured materials can be affected by the particle size. In this study, the silver grain size increased from approximately 15 nm for the reference sample to approximately 22 nm for nanocrystalline silver exposed to light for four weeks (Table S2†) indicating that a longer exposure to light might render its surface as ineffective antimicrobial agent.

Moreover, not only the increase in grain size but also the presence of Ag₂S on nanocrystalline silver surface exposed to light can modify its antimicrobial capabilities. For example, Choi et al. (2009) reported that the toxicity of silver nanoparticles on nitrifying bacteria is considerably reduced once they form Ag_xS_y complexes or precipitates with sulfide added to the system.⁴¹

Whereas the total silver content in the nanocrystalline silver dressing exposed to light remains constant over time, the soluble silver content presents a slight increase. This increase in soluble silver might be due to the transformation of metallic silver in silver sulfide and silver carbonate with light exposure.

Conclusions

In summary, surface reduction of silver oxide is one of the consequences of light exposure and provides a concise explanation for the observations in this study. The chemical and structural changes nanocrystalline silver underwent herein did not affect its antimicrobial activity. Nanocrystalline silver is capable of releasing silver even after exposure to light for four weeks and this aspect could impact the environment. However, the physicochemical changes observed in this study (grain growth, chemical composition changes) accompanied by exposure to a more intense source of illuminance (bright sunlight) suggest that given sufficient time, nanocrystalline silver dressings should inactivate in time, in the environment.

Acknowledgements

We gratefully acknowledge the financial support from the NRC-NSERC-EC-BDC Nanotechnology Initiative Grant.

References

1. E. J. E. Stuart, K. Tschulik, D. Omanovic, J. T. Cullen, K. Jurkschat, A. Crossley and R. G. Compton, Electrochemical detection of commercial silver nanoparticles: identification, sizing and detection in environmental media, Nanotechnology, 2013, 24, 444002.
2. N. Arshi, F. Ahmed, M. S. Anwar, S. Kumar, B. H. Koo, J. Lu and C. G. Lee, Novel and cost-effective synthesis of silver nanocrystals: a green synthesis, ACS Nano, 2011, 6, 295–300.
3. N. Feliu and B. Fadeel, Nanotoxicology: no small matter, Nanoscale, 2010, 2, 2514–2520.
4. A. Kumar, A. Dhawan and R. Shanker, The need for novel approaches in ecotoxicity of engineered nanomaterials, J. Biomed. Nanotechnol., 2011, 7, 79–80.
5. P. D. Dwivedi, A. Tripathi, K. M. Ansari, R. Shanker and M. Das, Impact of nanoparticles on the immune system, J. Biomed. Nanotechnol., 2011, 7, 193–194.
6. W. Liu, Y. Wu, C. Wang, H. C. Li, T. Wang, C. Y. Liao, L. Cui, Q. F. Zhou, B. Yan and G. B. Jiang, Impact of silver nanoparticles on human cells: effect of particle size, Nanotoxicology, 2010, 4(3), 319–330.
7. F. Giralt and R. Rallo, Modelling the Environmental and Human Effects of Nanomaterials, Compendium of Projects in the European NanoSafety Cluster, 2013, pp. 197–202.
8. V. Colvin, The potential environmental impact of engineered nanomaterials, Nat. Biotechnol., 2003, 21, 166–170.
9. S. W. P. Wijnhoven, J. G. M. Willie, W. J. G. M. Peijnenburg, A. Carla, C. A. Herberts, W. I. Hagens, A. G. Oomen, E. H. W. Heugens, B. Roszek, J. Bisschops, I. Gosens, D. Van De Meent, S. Dekkers, W. H. De Jong, M. van Zijverden, A. J. A. M. Sips and R. E. Geertsma, Nano-silver – a review of available data and knowledge gaps in human and environmental risk assessment, Nanotoxicology, 2009, 3, 109–138.
10. N. Grier, Silver and its compounds, in Disinfection, sterilization and preservation, ed. S. S. Block, Lea & Febiger, Philadelphia, 1983, p. 375.
11. A. Schmidt, Gonorrheal ophthalmia neonatorum: Historic impact of Credé's eye prophylaxis, in Pediatric infectious diseases revisited, ed. H. Schroten and S. Wirth, Birkhäuser Verlag Basel, Switzerland, 2007, p. 95.
12. X. Chen and H. J. Schluesener, Nanosilver: a nanoproduct in medical application, Toxicol. Lett., 2008, 176(1), 1–12.
13. C. A. Moyer, L. Brentano, D. L. Grevens, H. W. Margraf and W. W. Monafo, Treatment of large human burns with 0.5% silver nitrate solution, Arch. Surg., 1965, 90, 812–867.
14. M. Ahamed, M. S. Alsalhi and M. K. Siddiqui, Silver nanoparticle applications and human health, Clin. Chim. Acta, 2010, 411, 1841–1848.
15. J. M. Gorham, R. I. MacCuspie, K. L. Klein, D. H. Fairbrother and R. D. Holbrook, UV-induced photochemical transformations of citrate-capped silver nanoparticle suspensions, J. Nanopart. Res., 2012, 14, 1–16.
16. S. D. Sarma, Life cycle of a nanosilver based water candle filter: Examining issues of toxicity, risks, challenges and policy implications, J. Biomed. Nanotechnol., 2011, 7, 83–84.
17. D. Dellasega, A. Facibeni, F. Di Fonzo, M. Bogana, A. Polissi, C. Conti, C. Ducati, C. S. Casari, A. L. Bassi and C. E. Bottani, Nanostructured Ag(4)O(4) films with enhanced antibacterial activity, Nanotechnology, 2008, 19, 475602.
18. R. Birringer, Nanocrystalline materials, Mater. Sci. Eng., A, 1989, 117, 33–43.
19. P. Taylor, A. Ussher and R. Burrell, Impact of heat on nanocrystalline silver dressings part I: Chemical and biological properties, Biomaterials, 2005, 26, 7221–7229.
20. A. Nel, T. Xia, L. Madler and N. Li, Toxic potential of materials at the nanolevel, Science, 2006, 31, 622–627.
21. R. Warriner and R. E. Burrell, Infection and the chronic wound: A focus on silver, Adv. Skin Wound Care, 2005, 18, 2–12.
22. F. Fan and A. Bard, Chemical, electrochemical, gravimetric, and microscopic studies on antimicrobial silver films, J. Phys. Chem. B, 2002, 106, 279–287.
23. J. Wright, K. Lam, A. Buret, M. Olson and R. Burrell, Early healing events in a porcine model of contaminated wounds: effects of nanocrystalline silver on matrix metalloproteinases, cell apoptosis, and healing, Wound Repair Regen., 2002, 10, 141–151.
24. B. El, A. M. Luxton, T. P. Silva, R. G. Scheckel, K. G. Suidan and T. M. Tolaymat, Impact of environmental conditions (pH, ionic strength, and electrolyte type) on the surface charge and aggregation of silver nanoparticles suspensions, Environ. Sci. Technol., 2010, 44, 1260–1266.
25. J. Fabrega, S. R. Fawcett, J. C. Renshaw and J. R. Lead, Silver nanoparticle impact on bacterial growth: effect of pH, concentration, and organic matter, Environ. Sci. Technol., 2009, 43, 7285–7290.
26. R. Wiesinger, M. Schreiner and C. Kleber, Investigations of the interactions of CO(2),O(3) and UV light with silver surfaces by in situ IRRAS/QCM and ex situ TOF-SIMS, Appl. Surf. Sci., 2010, 256, 2735–2741.
27. J. P. Shi, C. Y. Ma, B. Xu, H. W. Zhang and C. P. Yu, Effect of light on toxicity of nanosilver to Tetrahymena pyriformis, Environ. Toxicol. Chem., 2012, 31, 1630–1638.
28. P. Taylor, O. Omotoso, J. Wiskel, D. Mitlin and R. Burrell, Impact of heat on nanocrystalline silver dressings part II: Physical properties, Biomaterials, 2005, 26, 7230–7240.
29. G. Speranza, S. Torrengo, L. Minati, M. Filippi, A. Castellino, C. Manfredotti, C. Manfredotti, M. Dipalo, A. Pasquarelli, E. Kohn, H. El-Hajj and E. Vittone, Characterization of UV irradiated nanocrystalline diamond, Diamond Relat. Mater., 2008, 17, 1194–1198.
30. S. Sant, K. Gill and R. Burrell, Novel duplex antimicrobial silver films deposited by magnetic sputtering, Philos. Mag. Lett., 2000, 80, 249–256.
31. R. E. Burrell and L. R. Morris. Anti-microbial coating for medical device, US Pat. No. 5753251, 1998.
32. K. M. Mc Evoy, M. J. Genet and C. Dupont-Gillain, Principal component analysis: a versatile method for processing and investigation of XPS spectra, Anal. Chem., 2008, 80, 7226–7238.
33. E. Niehuis and T. Grehl, Dual beam depth profiling, in ToF-SIMS – surface analysis by mass spectrometry, ed. J. C. Vickerman and D. Briggs, IM Publications and Surface Spectra Limited, UK, 2001, p. 753.
34. L. M. Liz-Marzan, Nanometals: Formation and color, Mater. Today, 2004, 7, 26–31.
35. U. Kreibig and M. Vollmer, Optical Properties of Metal Clusters, Springer-Verlag, Berlin, 1995.
36. N. R. C. Raju and K. J. Kumar, Photodissociation effects on pulsed laser deposited silver oxide thin films: Surface-enhanced resonance Raman scattering, J. Raman Spectrosc., 2011, 42, 1505–1509.
37. O. A. D. Gallardo, R. Moiraghi, M. A. MacChione, J. A. Godoy, M. A. Pérez and E. A. Coronado, Silver oxide particles/silver nanoparticles interconversion: Susceptibility of forward/backward reactions to the chemical environment at room temperature, RSC Adv., 2012, 2, 2923–2929.
38. L. A. Peyser, T. H. Lee and R. M. Dickson, Mechanism of Ag_n nanocluster photoproduction from silver oxide films, J. Phys. Chem. B, 2002, 106, 7725–7728.
39. T. Blanton, S. Misture, N. Dontula and S. Zdzieszynski, In situ high-temperature X-ray diffraction characterization of silver sulfide, Ag₂S, Powder Diffr., 2011, 36, 114–118.
40. V. Krylova and N. Dukstiene, Synthesis and Characterization of Ag₂S Layers Formed on Polypropylene, J. Chem., 2013, 987879.
41. O. Choi, T. E. Clevenger, B. Deng, R. Y. Surampalli, L. Ross Jr and Z. Hu, Role of sulfide and ligand strength in controlling nanosilver toxicity, Water Res., 2009, 43, 1879–1886.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra15745a

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