Efficient Silver Release From Ion Exchange Silver Dressings in Biologically Relevant Media

Introduction. Silver-containing wound dressings commonly are used when there is a risk of infection. These commercial antimicrobial silver dressings have various compositions that use different substrates and/or silver sources. Common silver sources are ionic silver (Ag⁺) and metallic silver (Ag⁰). A third source of silver is ion exchange silver compounds (Ag⁺ complex), in which silver ions are encapsulated in an inorganic carrier to moderate the availability of the silver ions and are released via an ion exchange mechanism. Objective. In this study, silver release of different types of silver dressings (Ag⁺, Ag⁰, Ag⁺ complex) in biologically relevant media is investigated. Materials and Methods. Simulated wound fluid (SWF) and SWF in combination with 5% bovine serum albumin were used as the extraction media. Results. The composition of the extraction media was found to strongly affect the release of silver. The silver released from most silver dressings peaked at ca 0.5 ppm of soluble silver without any protein in the extraction media. Apparent equilibria established between silver and the salts used for SWF were disrupted by the presence of the protein. This resulted in a dramatic increase in silver release over ionic solutions in the absence of the protein. Dressings differed dramatically in their silver release efficiency. The nature of the silver played a more significant role than the silver content in the dressings. Conclusions. The ion exchange (Ag⁺ complex) silver dressing was shown to be the most efficient among all the dressings tested for silver release.

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The use of silver-containing wound dressings has become standard for wounds at risk of infection.¹ Silver is a noble metal, reported to have high compatibility and low toxicity with eukaryotic cells,² and exhibits an oligodynamic effect, demonstrating bactericidal activity at very low concentrations.³ The metabolism of bacteria is adversely affected by silver ions even at minute concentration.¹ In addition, the bacteria killed by silver ions can contribute to the death of other living bacteria, which has been described as the “zombie effect.”⁴ The ionic form of silver (Ag⁺) reacts with thiols, amines, and/or other electron donor groups of proteins to prevent the growth of microorganisms.² 

While silver ions are readily available in various silver salts, including silver nitrate (AgNO₃) and silver sulfadiazine, metallic silver also has been used extensively in the form of thinly coated metal layers or as small particles (nanocrystalline silver).⁵ In all of these cases, the active components for the bactericidal activity of metallic silver are still the silver ions,^5-7 which are produced by the oxidation of silver to silver oxide (Ag₂O) in the presence of atmospheric oxygen.

Although silver/silver salt-containing dressings are widely accepted as a treatment option, there could be some associated risks, such as the dressings may generate reactive oxygen species, thus inducing DNA damage. Such degradation processes may result in allergic reactions or toxicity for humans.⁸ Allergic reactions and toxicity also may be the result of high doses or uncontrolled release of silver ions from dressings containing silver salts or metallic silver.^8,9 

To control the release of silver ions, new silver inorganic matrices have been developed, in which silver ions are encapsulated or caged in inorganic carriers. Common encapsulated/caged complexes include, but are not limited to, zeolites, phosphates, titanium dioxides, activated carbons, montmorillonite, water soluble glasses, and mesoporous silicas.^2,10,11 These materials are typically white solids, thus do not change the color of the dressings. In some cases, this approach leads to moderated and controlled release of silver ions, providing a higher clinical efficacy of these silver-containing materials.² In this paper, these caged complexes are described as ion exchange silver compounds (Ag⁺ complex) since these release silver ions (Ag⁺) by an ion-exchange mechanism.  

It is evident silver must be released as Ag⁺ to exhibit antimicrobial activity. Therefore, understanding the kinetics of release is critical to understanding the efficacy of silver dressings during use. Many previous studies of silver release kinetics from wound dressings have measured release into ultra-pure water, normal saline solution, and a human serum substitute.^12,13 Release of silver ions into an ionic solution such as normal saline may be relevant for understanding the interaction of saline-based wound cleansers with silver dressings; however, these studies are not as relevant for understanding the release of silver ions in the presence of complex mixtures of organic and nonorganic molecules and ions prevalent in actual wound exudate. It is well known complex organics such as proteins can affect the efficacy of solubilized silver ions^14,15; however, less information is available regarding the effect of biological components on the release of soluble silver from wound dressings. In addition, a comparison study of the release kinetics in biological media of ion exchange silver dressings versus metallic and ionic silver-based dressings has not been conducted. 

While it is difficult for an in vitro model to completely simulate the actual use of a wound dressing, there are several critical parameters that should be included: exposure of the dressing to wound fluids at an appropriate volume-to-surface area ratio, repeated exposures, and exposure to wound fluids with relevant levels of both ionic and organic components. In the following studies described, the ratio of dressing area to fluid volume was based on the amount of exudate that would be produced daily from a highly draining venous leg ulcer (1.2 g/cm²/24 hours).¹⁶ Serum albumin, the most abundant protein in human plasma,¹⁷ was a logical choice to simulate the organic load in wound fluid in a simple in vitro model.¹² In addition, the concentration of protein in chronic wound fluid has been reported to range from 2.6% to 5.1%, with a mean of 3.8% ± 1.3%.¹⁸ To further simplify the laboratory studies, it was reasonable to substitute the readily-available bovine serum albumin (BSA) for human serum albumin (HSA), because BSA and HSA have similar properties.¹⁹

The use of silver as an antimicrobial agent has grown significantly over the years in disparate segments of the marketplace. Silver can be found in typical consumer products and medical devices, ranging from toys to wound dressings, and has led to greater concern related to environmental toxicity.^20,21 In consideration of the environmental burden of a discarded wound dressing, the effective use of silver is just as important as its antibacterial activity. The most efficient silver dressing would release silver ions at a therapeutically consistent level throughout the duration of use, exhausting all the silver by the end of normal use. If a dressing exhausts its silver too quickly, the dressing may not be protected from contamination for the entire duration of use. Alternatively, a dressing can release silver ions too slowly and never reach exhaustion. In this case, the dressing may not achieve an effective level of free silver ions, while concomitantly, the remaining bound silver will be wasted and discarded before all the silver ions were released. Therefore, the percentage of silver remaining at the end of 7 daily exposures is an important aspect of silver release efficiency. 

The purpose of this study is to analyze the release of silver ions from wound dressings with different silver sources, including a dressing that utilizes ion exchange silver technology. The focus of this study will be on the kinetic release profile of each silver source in a medium that simulates proteinaceous wound fluid at a volume-to-surface area ratio corresponding to a heavily exuding wound over a period of several days. These analyses will enable a better understanding of the kinetics of release and the antimicrobial performance of ion exchange silver dressings during use. 

Materials

Dressings tested in this study represent several different formats and types of silver: 1 dressing containing ion exchange silver (Ag⁺ complex), 2 dressings containing ionic silver (Ag⁺), and 2 dressings containing metallic silver (Ag⁰) (Table 1). The descriptions and loadings of silver in the dressings have been reported in several publications,^22-24 including product literature and brochures. TRITEC Silver (Ag⁺ complex dressing; Milliken Healthcare Products, Spartanburg, SC) is a bilayer contact dressing incorporating a silver ion-exchange compound (silver zirconium phosphate) with Active Fluid Management (Milliken Healthcare Products) technology. ACTICOAT 7 with SILCRYST Nanocrystals (Ag⁰ dressing A; Smith + Nephew, Fort Worth, TX) is a multilayer contact dressing in which the silver treatment is a physical vapor deposition process. Silverlon Surgical Dressing (Ag⁰ dressing B; Argentum Medical, Geneva, IL) contains a nylon electroplated with Ag⁰. 3M Tegaderm Ag Mesh Dressing with Silver (Ag⁺ dressing A; 3M, St Paul, MN) consists of silver sulfate deposited onto a nonwoven cotton fabric. AQUACEL Ag Dressing (Ag⁺ dressing B; ConvaTec Inc, Bridgewater, NJ) consists of silver chloride (sodium carboxymethylcellulose). 

Methods

The amount of silver content in the Ag⁺ complex dressing was determined by ashing of the sample followed by an acid digestion of the ash with nitric acid (HNO₃) and hydrofluoric acid (HF), a dangerous substance for which proper handling protocols were followed. Measurement of zirconium in the digested solution was carried out by inductively coupled plasma optical emission spectrometry (ICP-OES). Silver content of Ag⁺ dressing A was determined by soaking a known amount of dressings in 50 mL of 70% HNO₃ over 48 hours. The solution then was diluted and measured for silver concentration using ICP-OES. The concentration of silver in the other commercial samples has been reported in the literature (Table 2).²⁴ 

The silver release was divided into 3 parts. The goal of the first part of the study was to determine the kinetics of silver release from wound dressings into a simulated wound fluid (SWF; NaCl: 142 mM, CaCl₂: 2.5 mM) that is isotonic to actual wound fluid. Release kinetics were determined in the SWF over several days with daily exchange of SWF. Samples of each dressing (surface areas of the wound contact side of the dressing were 19.6 cm²) were placed in 130-mL polypropylene bottles.^25,26 The bottles were filled with 100 mL of SWF solution using a volumetric flask. This solution was shaken at room temperature in an orbital shaker at 150 rpm for 24 hours. After 24 hours, 9.5 mL of the solution was removed and transferred to a labeled 15-mL tube. Then, 0.5 mL of 70% HNO₃ was added to each tube. The concentration of silver ion was determined against 2 ppm Ag⁺ standard using ICP-OES. The dressing from the remaining solution was removed, allowed to drip for 1 minute to reduce transfer of excess solution, and placed into a new 130-mL polypropylene bottle filled with 100 mL of SWF. This process was repeated every 24 hours for 7 consecutive days, with the dressings removed each day and placed into fresh SWF solution. Each day, the solution was collected and measured for silver concentration using ICP-OES.

In the second part of the study, release of silver ions into SWF supplemented with BSA was determined for different dressings. Investigators prepared 1%, 2.5%, and 5% BSA solutions by adding the required amount of BSA in the SWF. Samples of each dressing (surface area of 19.6 cm²) were placed into 100 mL of SWF supplemented with 1%, 2.5%, or 5% BSA (Fraction V; Sigma-Aldrich, St Louis, MO). The samples were allowed to sit at room temperature for the desired time period with no replacement of the fluid. The samples were diluted 10-fold before measuring in the atomic absorption spectrophotometer (AAS); this instrument was more appropriate to measure silver in a solution containing proteins than ICP-OES. The AAS was calibrated to read silver using silver standards from 0.5 ppm to 2.5 ppm. Concentration of Ag⁺ was calculated based on the reading from AAS and dilution factor. 

In the third part of the study, the dressings were exposed to clinically relevant conditions without modification to account for absorption capacities of the dressings. To achieve this, samples (surface area of 19.6 cm²) of each dressing were placed in 24 g of SWF supplemented with 5% BSA. The dressings were removed daily and placed into fresh solution for 7 consecutive days to mimic the continuous exposure of exudate to the wound dressing over time. For each study, a sample of the solution was collected daily and measured for silver concentration using AAS; this instrument was more appropriate to measure silver in a solution containing proteins than ICP-OES.

Silver release in SWF

As previously described, the nature or form of active silver components vary among different commercial dressings. The common silver sources are metallic or ionic silver. This study investigated ion exchange Ag⁺ complex dressings and compared the results against traditional dressings with metallic or ionic silver. Silver content varies from 0.5% to more than 20% in these dressings and from less than 0.1 mg/cm² to more than 5 mg/cm². Silver content is summarized in Table 2.²⁴ It may be important to recognize silver content per unit area is more relevant than weight percent of silver, since density of the dressings can be very different and the size of the dressings used is related to the area of the wound being treated. It is interesting to note that silver content is typically lower for dressings with Ag⁺ when compared with Ag⁺ complex, while Ag⁰ dressings typically have the highest silver content. The high loadings of silver found in these dressings may indicate Ag⁰ is not the most efficient silver source.

To understand the kinetics of silver release from different silver sources, the amount of bioavailable silver in SWF was determined through 7 daily exposures. Regardless of dressing type or type of silver treatment, silver ion release into the solution reaches an apparent solution equilibrium at around 0.5 ppm (Figure 1). Because of this equilibrium, the concentration of silver ions released does not depend on the amount of silver in the dressing, provided the dressing is capable of releasing enough silver ions to reach the equilibrium concentration. It is known that chloride ions at the levels present in SWF affect the solubility of Ag⁺. However, based solely on Le Châtelier’s principle²⁷ of the common ion effect, the concentration of silver ions should be about 0.14 ppb. This is calculated from the molar solubility of silver chloride (1.34 x 10^-10 M of silver) and the solubility product constant (K_sp(AgCl) = 1.8 x 10^-10). The excess silver release is explained by the formation of lyophobic colloids due to the adsorption of ions of the same charge (eg, silver chloride [AgCl] in excess Ag⁺ or Cl^-). The adsorbed ions repel each other, preventing collision and precipitation of the colloidal particles. The colloidal particles of AgCl are very small particles of AgCl crystals. Solvation of the adsorbed ions keeps the colloidal particles in suspension. Overall, silver release is controlled by several factors related to the solubility of AgCl, including the solubility product constant (k_sp), Le Châtelier’s principle, and the lyophobic colloidal nature of the solution.²⁸ 

Silver release in biologically relevant media

Although release of soluble silver ions from wound dressings into a wound fluid based on salts or SWF alone is interesting and important for understanding the release of silver ions and relevant to wound cleansers and saline used to irrigate wounds, this situation may not be clinically relevant to a silver dressing placed onto an exuding wound. In order to investigate the silver ion release in biologically relevant media, biological components (as would be found in wound fluid) should be introduced into the test media.

The wound environment is difficult to analyze due to the complex nature of the wound exudate. Plasma proteins such as albumin are present in ample concentration in wound exudate and have multiple functions, such as maintaining osmotic pressure, immunity, and transport of macromolecules.²⁹ The average dialysate protein concentration in exuding chronic ulcers is reported to be 0.5 mg/L, while the concentration of protein in plasma from chronic wound fluid is much higher, reported to be 2.6 g/100 mL to 5.1 g/100 mL with a mean of 3.8 g/100 mL.¹⁸ Serum albumin is the most abundant protein in plasma at a typical concentration of 5 g/100 mL (~5%).³⁰ Bovine serum albumin is very similar to HSA both in chemical and physical properties.²¹ Therefore, the present authors investigated the nature of silver ion release in the SWF supplemented with various concentrations of BSA.  

The addition of BSA to SWF resulted in a substantial increase in the silver ions released from 3 of the 5 silver dressings tested (Table 3). The efficacy of silver dressings at different levels of BSA (1%, 2.5%, and 5% in SWF) also was examined, and the rate of silver ion release increased dramatically with higher levels of BSA (Figure 2). The amount of silver ion release in the presence of BSA in SWF is 10 to 100 times the amount released in SWF alone. The result indicates silver ion release from silver dressings is enhanced by the presence of proteins, such as those commonly found in blood and wound fluids. This experiment suggests proteins like BSA can bind to released silver ions and accelerate the release of silver ions.

This enhanced silver ion release phenomenon was observed for other commercial dressings with different types of silver sources, but the magnitude of the effect varied substantially (Figure 3). The 1-day and 7-day silver ion release data of the dressings are summarized in Table 3. The silver ion release data indicate the amount of bioavailable silver in a dressing is not solely dependent on the amount of silver found in the dressing.³⁰  

Although the oxidation states of silver in Ag⁰ dressing A and Ag⁰ dressing B are identical (Ag⁰), these silver dressings behave very differently in the presence of BSA. Ag⁰ dressing A uses nanocrystalline silver and Ag⁰ dressing B uses a homogeneous uniform silver coating. The authors presume the higher surface area of the nanocrystalline silver leads to stronger interactions with BSA in SWF. 

Another important consideration is the duration of silver ion release under clinically relevant conditions. As previously described, this study attempted to simulate several critical parameters involved with the use of silver wound dressings, including the exposure of dressings to SWF with proteins over a 7-day period and mimicking the composition and hydrodynamics (volume-to-surface area; fresh fluid daily) of wound fluid in a heavily exuding wound. Although the typical exudate production of a wound is reported to be about 0.5 g/cm² per 24 hours, the exudate levels in the heaviest draining wounds are reported to be 1.2 g/cm² per 24 hours.¹⁶ Considering these clinically relevant factors, the authors measured the release of silver ions from each silver dressing over 7 days immersed in 24 g of fresh SWF with 5% BSA every day. Silver ion release appears to be at its peak within the first 24 hours and remains relatively steady for the next 7 days for all silver types tested, except for the Ag⁺ dressings. Ag⁺ dressing A and Ag⁺ dressing B release silver ions so rapidly in the SWF with 5% BSA that after 2 days all silver is exhausted. Notably, this condition occurred only with a high concentration of protein loading (ie, 5%); typical wounds have lower protein content.¹⁸ The levels of daily silver ion release from silver dressings are shown in Figure 4. All dressings exhibited the same bolus effect with the highest silver ion release occurring on the first day of exposure (Figure 4). After the high initial release rate, the rate of release decreases substantially for all dressings, resulting in a lower sustained release.  

Temperature-dependent silver release

In addition to protein content, temperature may play a crucial role in silver ion release kinetics. Human body temperature is about 37°C. The silver release was studied for 2 dressings (Ag⁺ complex dressing and Ag⁰ dressing A) at room temperature (22°C) and at typical body temperature (37°C); the results are summarized in Figure 5. Overall, temperature does not play a significant role in silver ion release. Within the scope of this study, silver release at room temperature appears to be directionally consistent with silver release at body temperature. It is interesting to note that the 2 silver sources (Ag⁺ complex dressing and Ag⁰ dressing A) appear to be impacted somewhat differently by changes in temperature, which warrants further study.  

Residual silver after exposure

Silver ion release data over 7 days is shown in Figure 4. Further analysis of silver ion release efficiency is shown in Figure 6. The authors determined the percentage of silver remaining in the dressings based on the amount of silver released and the amount of silver originally contained in the dressing samples based on reported values or as analyzed by ICP-OES (Table 2²⁴). In the worst case, 1 type of dressing (Ag⁺ dressing A) exhausted most of its silver reservoir after only 1 day of exposure (Figure 6). Results from this study indicate there is no perfect dressing in terms of effective and complete release of silver ion for 7 days of use. Ag⁺ dressings A and B readily released 50% to 100% of the silver reservoir in the first 2 days of exposure. Ag⁰ dressing B sustained a high release of silver but still contained more than 90% of the silver reservoir at the end of the target duration of use. The most efficient dressing in terms of these 2 parameters was the Ag⁺ complex dressing, which sustained silver release for 7 consecutive daily exposures and released more than 60% of its silver content.

In the current study, only a few representative dressings were investigated from 3 categories. Although the study clearly demonstrates the higher efficiency of this particular ion exchange Ag⁺ complex dressing, other Ag⁺ dressings may not behave similarly. Some silver ion sources with advanced technology or silver ion with higher oxidation state may behave very differently. In addition, the antimicrobial efficacy of released silver ions from different dressings may behave differently, irrespective of the concentration of Ag⁺ in the media.

In typical SWF, the silver ions released from most silver dressings peaked at Ca 0.5 ppm of soluble silver. Apparent equilibria established between silver ions and salts used for SWF (NaCl and CaCl₂) were disrupted by the presence of protein (eg, BSA), resulting in a dramatic increase in silver ion release over ionic solutions with no protein. All tested dressings responded similarly to increasing concentrations of protein, regardless of the dressing format, form, nature of silver source, or initial silver content. It is clear from this study that organics, such as proteins, act to shift the equilibrium of soluble silver. Temperature does not seem to play a significant role in controlling the release of silver ions. 

Dressings differed dramatically in the efficiency of silver release. It is evident the nature of silver is more relevant to its release than the silver content of the dressings. The percentage of silver depleted from the dressings after exposure to clinically relevant amounts of exudate ranged from 100% (silver reservoir was depleted after only 1 day of exposure) to greater than or equal to 60% after 7 days. The Ag⁺ complex dressing was the most effective among all the dressings tested for silver ion release efficiency. Further studies will focus on the antimicrobial efficacy of released silver ions from different dressings and whether microbes themselves can act as a reservoir for silver ions. 

Authors: Rajib Mondal, PhD¹; Matthew Foote, MS¹; Andrew Canada, PhD¹; Mark Wiencek, PhD²; Martin E. Cowan, PhD¹; and Cristina Acevedo, PhD¹

Note: The authors would like to acknowledge Dr. Geoffrey Haas, Dr. Warren Gerhardt, and Brian Lindsay for technical discussions and George Gorrell for ICP-OES and AAS analysis.

Affiliations: ¹Milliken Healthcare Products, Spartanburg, SC; and ²Contec Inc, Spartanburg, SC

Correspondence: Rajib Mondal, PhD, Research Scientist, Milliken Healthcare Products, Medical, 920 Milliken Road, Spartanburg, SC 29303; Rajib.Mondal@milliken.com 

Disclosure: Drs. Mondal, Canada, Cowan, and Acevedo and Mr. Foote are employees of Milliken Healthcare Products. Dr. Wiencek discloses no financial or other conflict of interest.