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Department of Applied Chemistry, Royal Melbourne Institute of Technology, GPO Box 2476V, Melbourne, Victoria 3001, Australia.
a Author for correspondence. Fax Int +61-3-9639-1321; e-mail C.RIX{at}rmit.edu.au
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Abstract |
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Key Words: indexing terms: silver chloride • solubility • biological fluids • membranes • burns
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The process of wound healing follows three phases: the inflammatory response, the migratory response, and the proliferation response. The inflammatory response that occurs has two functions: (a) the prevention of hemorrhage at the wound site by attracting coagulation components and subsequent thrombosis of small vessels, and (b) the provision of essential cells to the wound site to begin the process of healing and repairing of the damaged tissue (8).
Blood vessels in an acute burn wound are vasodilated and have an increased permeability, so when silver sulfadiazine is applied to a wound during this inflammatory stage, which may last for several days, silver is likely to be absorbed. Once the inflammatory response has subsided, the migratory response begins to form granulation tissue (consisting of collagenous material and mucopolysaccharides) and a barrier begins to form between the wound surface and the previously exposed vascular space. Epithelial cells also migrate to the wound site during this phase, forming an additional barrier, and damaged blood vessels within the wound begin to regrow. The migratory response may last for several days before the proliferation phase, which results in scar formation. The time required for the proliferation phase to be completed is dependent on the severity and size of the wound. For large burn wounds (>40% body surface area), the proliferation phase may last for weeks or months, and skin grafts may be required if the body's own healing factors have been destroyed by either the burn injury or infection. When this phase is completed, the epidermis will have been restored to a normal thickness and the collagenous material, forming the scar tissue, will have become more organized. The fibroblasts responsible for synthesizing the scar tissue will have disappeared from the wound site and most of the blood vessels will have been restored. Although this is a superficial description of burn wound healing, it has allowed the problem of silver absorption to be considered during the various stages of healing.
Silver absorption does not occur when silver sulfadiazine is placed on intact skin; however, when the compound is placed on an acute burn wound, silver absorption is at a maximum for the first 14 days during the inflammatory and migratory phases of wound healing (6)(9). After this time, silver absorption decreases.
We have recently studied the interaction of silver sulfadiazine and silver chloride in direct contact with various biological fluids (10), and the results clearly showed that silver absorption was greatly enhanced in the presence of normal serum components such as chloride, peptides, and proteins. However, the study had limited applicability since it only modeled the chemical interactions of silver sulfadiazine placed on an acute burn wound, where the compound was in direct contact with circulating blood.
The current study describes the chemical interactions of silver salts
when separated from various biological fluids by membrane-mimetic
materials. This provides a more realistic model for the chemical
interactions of silver sulfadiazine placed on a burn wound that has
progressed in its healing and contains granulation tissue. The
membranes used in the present study were quite similar (~40 µm
thick with a molecular-mass cutoff of 12–14 kDa), and they were chosen
to include a hydrophobic membrane (polyethylene) and three hydrophilic
membranes (cellulose, chitosan, and collagen). The hydrophobic membrane
was selected to model intact skin, whereas the hydrophilic membranes
were selected to model granulation tissue, which consists of a
collagen–polysaccharide matrix. Cellulose (poly
-D-glucose) was chosen as a simple polysaccharide
that does not possess silver-binding abilities, i.e., no amino
(-NH2) or thiol (-SH) functional groups, whereas
chitosan (poly -D-aminoglucose), which contains amino
groups, was chosen as a simple polysaccharide with silver-binding
capacity, and collagen (a polypeptide, essentially polyproline), which
contains amino, peptide, and thiol groups, was selected as a protein
with silver-binding abilities.
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Materials and Methods |
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Sulfadiazine analysis was performed on a Model U-3200 spectrophotometer (Hitachi, Tokyo, Japan).
All glassware was washed in nitric acid (1.5 mol/L) for 48 h, then thoroughly rinsed with deionized water.
reagents
To prepare all solutions, we used water purified in a high-grade
MilliQ deionizer system (Millipore, Melbourne, Australia) with a
resistance >14 M
. All chemicals used to synthesize the required
silver salts and any solutions needed for this study were of analytical
grade or better.
High-purity argon (CIG, Melbourne, Australia) was used as the purge gas for silver analysis with graphite furnace AAS and instrument-grade air (CIG), and industrial-grade acetylene (CIG) was used for the flame when flame AAS was required.
Calibrators.
Silver calibrators for graphite furnace AAS
were prepared from a 50 µg/L silver nitrate stock solution
(equivalent to 31.8 µg/L silver) that was freshly prepared from
analytical grade silver nitrate (Deak International, Melbourne,
Australia) dissolved in 0.2% Aristar nitric acid. A working curve was
regularly prepared by using the AAS autosampler, which diluted the 50
µg/L silver nitrate solution with 0.2% Aristar nitric acid to give
concentrations of 5, 10, 15, 20, 25, 30, 40, and 50 µg/L silver
nitrate.
Silver calibrators for flame AAS were derived from a 100 mg/L silver nitrate stock solution (equivalent to 63.5 mg/L silver) that was freshly prepared as above, and that was serially diluted to give concentrations of 1, 2, 3, 4, 5, and 10 mg/L silver nitrate.
Sulfadiazine calibrators were prepared from a stock solution that was freshly prepared from Sigma-grade sulfadiazine (Sigma Chemical Co., St. Louis, MO), by using 3 mL of 2 mol/L sodium hydroxide per 100 mg of sulfadiazine to aid dissolution of the solid. This was diluted to give concentrations of 1, 2, 3, 4, 5, and 10 mg/L sulfadiazine.
Silver salt preparation.
Silver sulfadiazine and silver
chloride were prepared by reacting equimolar concentrations (0.1 mol/L)
of ammoniacal solutions of silver nitrate and the appropriate anion in
the dark (11). White pastes were formed, which were
filtered off and washed several times with MilliQ water before being
dried at 60 °C for 10 h under reduced pressure. Silver
sulfadiazine was a white "fluffy" solid; silver chloride was white
and "clumpy."
silver sulfadiazine creams
SilvazineTM (Smith and Nephew, Melbourne, Australia)
was used as purchased, and a 10 g/kg silver sulfadiazine cream was
manufactured in our laboratory according to a formulation provided by
Steve Blackbourn of the Royal Perth Hospital (Pharmacy Manufacturing
Services), Perth, Australia.
sample preparation
The experiments were carried out in 500-mL polycarbonate
containers fitted with lids through which a polycarbonate tube (i.d. 30
mm, length 60 mm), supporting a membrane, could be inserted. The
membranes were attached to the lower end of the tube by Teflon tape and
held in place by a rubber O-ring seal (see Fig. 1
).
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The containers were filled with 400 mL of the appropriate medium and the pH adjusted with 0.2 mol/L sodium hydroxide or 0.2 mol/L nitric acid to within the range 7–7.5. The lid, with the tube holding the membrane inserted, was fitted onto the container and 1 g of paste containing the appropriate silver salt or 5 g of silver sulfadiazine cream was placed into the tube and onto the membrane. The pastes were prepared in MilliQ water containing either 200 g/L silver sulfadiazine or 80 g/L silver chloride, giving the same amount of silver for a given mass of paste. The tube was lowered into the medium until the membrane was immersed ~3 mm below the surface, and the medium was continuously stirred with a Teflon-coated magnetic stirring bar for the duration of the experiment. The polycarbonate containers were placed on thermal insulation mats made from cardboard to prevent stray heat from the stirring apparatus heating the medium. This was not a trivial precaution, since other studies in our laboratory have shown the solubility of silver sulfadiazine, in particular, to be significantly affected by temperature. The experiments were carried out in the dark, at laboratory temperature (22 °C), for periods of up to 5 days (120 h). Samples were collected at appropriate times and passed through a 0.45-µm Teflon filter and acidified, then stored in the dark in acid-washed glassware that was covered with Parafilm until the end of the experiment. The samples were then analyzed for soluble silver and sulfadiazine.
The membranes used included polyethylene (freezer bag, commercially available); cellulose (commercially available); chitosan membranes cast from 10 g/L acetic acid solution and washed in 0.25% ammonia solution before use, with chitosan (75–80% amino content) prepared in our laboratories from prawn shells according to the method of Mima et al. (12); and collagen, which had been washed in acetone for 30 min (Naturin Collagen Casting; Devro Pty, Sydney, Australia). Comparative diffusion studies involving a cellulose dialysis material with a known molecular-mass cutoff of 12–14 kDa and involving sulfadiazine as a test solute indicated that the chitosan, cellulose, and collagen membranes had a molecular-mass cutoff in the same range (12–14 kDa), whereas polyethylene did not allow any passage of the sulfadiazine solute through it.
Each membrane was studied with the following media: (a) 0.1 mol/L sodium nitrate containing 0.028 mol/L sodium hydrogen carbonate; (b) synthetic serum electrolyte solution (SSES) (0.103 mol/L NaCl, 0.028 mol/L NaHCO3, 0.002 mol/L Na2HPO4, and 0.002 mol/L Na2SO4); (c) solution b containing 0.43 g/L glutathione (Sigma grade); this gave a 1:1 ratio of silver to glutathione if all the silver in 1 g of paste reacted; and (d) human serum. The serum in this study was used in accordance with the rules and regulations set out by the Royal Melbourne Institute of Technology for dealing with human products, and was provided by the Institute's Department of Hematology, which had obtained it from the Blood Bank of Victoria.
analytical procedure
For silver analysis by graphite furnace AAS we used a wavelength
of 328.1 nm, a slitwidth of 0.7 nm, and a 20-µL sample volume. The
method was similar to that described by Wan et al. (9) for
the determination of silver in blood and urine. Samples with a silver
concentration >50 µg/L (as silver nitrate) were diluted before
analysis. The optimum conditions found were 140 °C for the drying
temperature, 600 °C for the ashing temperature, and 1900 °C for
the atomization temperature. The purge gas was high-purity argon for
all steps except the ashing step, where instrument-grade air was used
to remove any volatile materials such as organic matter. The gas flow
rate was 300 mL/min for all steps except the read step, when the flow
was stopped for 5 s while the analytical signal was read.
Automatically integrated peak areas were used for calculation of
results. The tube clean-up step had a temperature of 2650 °C. This
procedure was used for both calibrators and samples.
For silver analysis by flame AAS we used a wavelength of 328.1 nm with a slitwidth of 0.7 nm. Samples with silver concentrations >10 mg/L (as silver nitrate) were diluted before analysis. The signal peak areas were automatically integrated over 5 s to calculate the results.
Sulfadiazine analysis was by a modified Bratton and Marshall method (13), with measurements at three wavelengths: Readings at 420 and 650 nm were used to set the baseline, and the peak height was measured at 542 nm.
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Results |
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Figure 2
shows the concentrations of soluble silver and sulfadiazine in
0.1 mol/L sodium nitrate over a 5-day period, when an aqueous silver
sulfadiazine paste was placed above each of the various membranes.
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For a polyethylene membrane, no soluble silver or sulfadiazine could be detected in the medium, indicating that neither silver nor sulfadiazine passed through the hydrophobic membrane.
For a cellulose membrane, the soluble silver and sulfadiazine concentrations in the medium were similar to each other and increased with time towards the expected equilibrium concentration of 2.61 µmol/L. This value had been determined previously when silver sulfadiazine was placed in direct contact with the medium (10), and required ~10 h to reach equilibrium. In contrast, the time taken to equilibrate silver sulfadiazine when separated by a cellulose membrane was ~8 times longer, and is clearly an indication of the reduced mobility of silver and sulfadiazine as they pass through the cellulose. The data obtained for the cellulose experiment was used as a comparison for the other two hydrophilic membranes (chitosan and collagen) to observe how the amino and thiol functional groups influenced the dissociation of the silver sulfadiazine and the movement of silver and sulfadiazine across these membranes.
For chitosan, which is an amino cellulose, the soluble silver concentration in the medium never exceeded about one-tenth the maximum expected concentration. The soluble sulfadiazine concentration was much greater than this value, indicating that although the silver salt had indeed dissociated, the released silver did not readily pass into solution but was bound by the membrane, while the sulfadiazine was free to diffuse through the membrane into the medium. The rate at which sulfadiazine appeared in the medium was much greater than for the cellulose experiment because of the enhanced dissociation of the salt.
For collagen, which is essentially a polyproline protein containing both peptide and some amino and thiol groups, the soluble silver concentration was slightly greater than with chitosan, although it remained lower than the maximum expected value for the entire experiment, indicating that the collagen membrane also bound the silver. Initially, the soluble sulfadiazine concentration remained as low as the silver concentrations in the medium, but it gradually increased with time up to the expected equilibrium value by the end of the experiment. The increased sulfadiazine concentration together with the depressed silver concentration in solution indicates that the silver salt had dissociated, but that a portion of the silver had become immobilized by being bound to the collagen membrane, similar to the behavior with chitosan.
When the same experiments were performed with silver chloride instead
of silver sulfadiazine, the concentration of soluble silver was as
shown in Fig. 3
. The solubility of silver chloride in 0.1 mol/L sodium nitrate
(11.61 µmol/L) is greater than that of silver sulfadiazine (2.61
µmol/L), so the soluble silver concentration will be greater for
silver chloride than silver sulfadiazine (10).
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In the case of silver chloride supported on the hydrophilic membranes (cellulose, chitosan, and collagen), the soluble silver concentration in the medium remained below the maximum expected concentration of 11.61 µmol/L for the duration of the experiment.
For cellulose, the soluble silver concentration steadily increased, reaching about one-quarter of the maximum expected value by the end of the experiment (120 h).
For chitosan, the soluble silver concentration was very low for the entire duration of the experiment (less than one-tenth of the maximum value expected), and these concentrations were similar to those obtained in the silver sulfadiazine experiment. This result was not entirely unexpected since chitosan is able to bind silver, so this interaction would keep silver concentrations in the medium low.
For collagen, the soluble silver concentration increased at a rate greater than the cellulose experiment, but then leveled off to about one-third the maximum expected concentration halfway through the experiment and remained at that concentration until the end of the investigation.
When the two types of 10 g/kg silver sulfadiazine creams (commercial
and laboratory manufactured) were placed above the cellulose membrane,
the movement of silver and sulfadiazine across the membrane was much
slower than that obtained when an aqueous silver sulfadiazine paste was
used. Nevertheless, the sulfadiazine concentrations finally reached
were similar to those when silver sulfadiazine paste was used above the
cellulose membrane, whereas the silver concentrations were less than
one-tenth the value obtained for the paste (data not shown in Fig. 2
).
These studies in 0.1 mol/L sodium nitrate clearly indicate the influence of each membrane on the dissociation of the silver salts and the movement of silver and sulfadiazine across the membranes.
influence of medium
Figure 4
illustrates the change in concentration of soluble silver and
sulfadiazine in the chloride-containing medium SSES when a silver
sulfadiazine paste was placed above the various membranes.
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For polyethylene, the soluble silver and sulfadiazine concentrations in the medium (SSES) were below the limit of detection, indicating that neither silver nor sulfadiazine passed through the membrane, even if dissociation had occurred in the paste above the membrane. For the three hydrophilic membranes (cellulose, chitosan, and collagen), the soluble silver concentrations in SSES eventually reached a similar value, close to the maximum expected equilibrium concentration of 1.48 µmol/L (10). The soluble sulfadiazine concentrations steadily increased during the experiments, reaching close to the maximum obtainable concentration (1400 µmol/L) by the end of the study period. In each case, a white precipitate had formed above the membrane that turned a purple-blue color when exposed to light, indicating the formation of silver chloride. For all the silver salts studied in this work, the behavior of silver chloride when exposed to light was unique, as it was the only salt to turn purple-blue. This demonstrated that silver sulfadiazine had dissociated, allowing the sulfadiazine to move through the membrane into the medium below, while the chloride from the medium had diffused through the membrane and reacted with the dissolved silver in the paste reservoir to form solid silver chloride and some soluble dichloroargentate(I) anion (AgCl2-). The sulfadiazine concentration was much greater than the silver concentration from the beginning of the experiment, and in this instance the dissociation of silver sulfadiazine had been enhanced by its reaction with chloride. Since these samples all reached the maximum expected silver concentration within 24 h, it is clear that the membranes did not impede the diffusion of silver when it was bound in the dichloroargentate(I) anion, the major silver species in solution.
When the same experiments were performed with solid silver chloride
instead of silver sulfadiazine, the concentration of soluble silver in
the medium (SSES), shown in Fig. 5
, approached the expected equilibrium value (1.48 µmol/L)
similar to that for silver sulfadiazine.
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When the two types of 10 g/kg silver sulfadiazine creams were placed
above the cellulose membrane, the movement of silver varied for both
creams, with the laboratory-manufactured cream showing similar results
to that obtained with silver sulfadiazine paste, whereas the
proprietary cream gave concentrations that were one-tenth of this
value. The movement of sulfadiazine was slower than that obtained when
silver sulfadiazine aqueous paste was used, with the concentrations
reaching only one-fiftieth of the concentrations obtained with the
paste (data not shown in Fig. 4
).
These experiments indicate that the chloride-containing medium SSES causes significant dissociation of the silver salts, and silver (and sulfadiazine) become free to diffuse across the membranes.
influence of ligand
Figure 6
illustrates the change in concentration of soluble silver and
sulfadiazine in SSES in the presence of the silver-binding ligand,
glutathione (a tripeptide), when a silver sulfadiazine paste was placed
above each of the various membranes. The mole ratio of glutathione
dissolved in the medium to silver in the paste was 1:1.
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For polyethylene, neither silver nor sulfadiazine was detected in the initial stages of the experiment, although halfway through the experiment (after about 60 h), trace amounts of silver and sulfadiazine were detected, indicating that they had migrated across the membrane.
For the experiments involving the hydrophilic membranes (cellulose, chitosan, and collagen), the soluble silver and sulfadiazine concentrations never reached the maximum obtainable equilibrium concentration (1400 µmol/L). For cellulose, the soluble silver and sulfadiazine concentrations increased steadily, with the silver concentration reaching about one-third of the expected concentration and the sulfadiazine concentration reaching about half that expected by the end of the experiment. During the experiment, the colorless medium gradually changed to a yellow color, as the glutathione from the medium reacted with the silver to form a soluble colored complex that had been observed in previous studies. In addition, a white precipitate had formed above the membrane that turned a purple-blue color when exposed to light, indicating the formation of silver chloride. For chitosan and collagen, a similar trend in the sulfadiazine concentrations was found to that for the cellulose experiment; however, the soluble silver concentration in the medium was significantly less than the concentrations observed in the cellulose experiment because of the silver-binding abilities of both the chitosan and collagen membranes.
When the same experiments were performed with silver chloride instead
of silver sulfadiazine, the concentration of soluble silver was as
illustrated in Fig. 7
.
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The results show that the soluble silver concentration increased steadily with time, reaching about one-seventh of the expected concentration by the end of the experiment. During that time, the colorless medium gradually changed to a yellow color, indicating formation of the glutathione complex. These experiments demonstrate the influence of a potential biological ligand, glutathione, on the dissociation of the silver salts and the movement of silver and sulfadiazine across the membranes, and clearly indicate that the formation of soluble glutathione complexes enhances silver transport through the hydrophilic membranes.
When each of the two types of 10 g/kg silver sulfadiazine creams was
placed above the cellulose membrane, the movement of silver and
sulfadiazine across the membrane into SSES plus glutathione was much
slower than that obtained with a silver sulfadiazine aqueous paste. The
sulfadiazine concentrations were one-tenth (commercial cream) to
two-thirds (laboratory-manufactured cream) the concentration obtained
when silver sulfadiazine paste was used above the cellulose membrane,
whereas in each case the silver concentrations were less than
one-twentieth the concentration obtained from the paste (data not shown
in Fig. 6
).
Figure 8
shows the change in concentration of soluble silver and
sulfadiazine in human serum over a 5-day period, when silver
sulfadiazine was separated from the medium by the various hydrophilic
membranes.
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The results show that the soluble silver and sulfadiazine concentrations increased for the duration of the experiments, although the rate of increase was slower for silver than for sulfadiazine. However, neither silver nor sulfadiazine reached their maximum expected concentration of 145 µmol/L and 1400 µmol/L, respectively (10), with both reaching only about one-fifth of their expected value. The lower concentrations of silver compared with that of sulfadiazine indicate that the serum proteins from the medium impede the movement of silver across the membrane (possibly by adsorption to the membrane surface) while allowing the sulfadiazine to pass through into the serum, albeit at a reduced rate when compared with the other media.
When these same experiments were performed with silver chloride instead
of silver sulfadiazine, the concen- tration of soluble silver in
the medium (shown in Fig. 9
) increased slightly with time but reached only about
one-sixtieth of the maximum expected value by the end of the
experiment.
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When each of the two types of 10 g/kg silver sulfadiazine creams was
placed above the cellulose membrane, the movement of silver and
sulfadiazine across the membrane into human serum was much slower than
that obtained when silver sulfadiazine aqueous paste was used. The
silver and sulfadiazine concentrations reached were only about one-half
the concentration obtained when silver sulfadiazine paste was used
above the cellulose membrane (data not shown in Fig. 8
).
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Discussion |
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1) Non-interactive medium of 0.1 mol/L sodium nitrate:
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2) Chloride-containing medium of SSES:
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3) Ligand-or protein-containing medium of SSES:
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We have discussed these interactions in detail in a previous paper (10). However, to study a more appropriate model for solute absorption at a healing wound, we have interposed several different membranes between the silver salt and the medium. We recognize that the mem-branes chosen do not perfectly mimic the phospholipidmembrane of living cells, but we believe they provide an adequate model to explore passive solute diffusion and some of the chemical interactions likely between the solute and the membrane components. Indeed, the cellulose and chitosan membranes used in this study are known to be permeable to gases, and have also been used as dialysis membranes; thus, we believe they are best regarded as semipermeable, with interconnected channels or voids extending through the entire membrane so allowing passive diffusion of water and low-molecular-mass solutes. Indeed, comparison of the membranes used in this study with a standard cellulose dialysis membrane (molecular-mass cutoff 12–14 kDa) indicated similar diffusion characteristics, and so we do not expect the molecular size to be a critical factor in controlling the solute diffusion through the membranes, except in the case of serum proteins. Thus, restrictions on the movement of soluble materials will primarily depend on the chemical compatibility of the membrane and solute, e.g., a nonpolar membrane such as polyethylene is expected to show little affinity for water, and thus no passage of soluble salts in aqueous solution, whereas the presence of polar functional groups will allow free movement of water, but particular functional groups present on the membrane may impose additional restrictions on solute movement, and this is likely with chitosan and collagen, which contain polar functional groups capable of binding silver. In this manner an interactive membrane will act as a heterogeneous complexing agent towards the solute, and we can define an equilibrium constant, Kim, for such an interaction. Thus, when either silver salt is separated from the medium by a hydrophilic membrane, the previous equilibria may also include an additional interaction with the membrane. The influence of the membrane on the dissociation of the silver salt and the movement of the silver ion and its accompanying anion can be observed when the medium is noninteractive, i.e., 0.1 mol/L sodium nitrate solution. The following general equilibria are then established :
where Kim = equilibrium constant for the interaction of the membrane with the solute.
A membrane that has no significant silver binding ability, such as
cellulose, has Kim 
0 and so is expected to
produce similar equilibrium concentrations for the silver ion and
its accompanying anion in the medium as determined by the solubility
product (Ksp) of the silver salt used, as though
it was in direct contact with the medium. However, the time taken to
reach equilibrium will be dependent on the rate of diffusion of these
ions through the membrane (see cellulose data in Fig. 2
); thus, the
kinetics of membrane transport will be an important factor in
determining how much silver moves through the membrane. When a membrane
has silver-binding abilities, such as chitosan and collagen, then
Kim >0 and the soluble silver concentration in the
medium will be low compared with its accompanying anion as the membrane
binds the silver and prevents it from migrating into the medium. Thus,
the silver concentration in the medium will be dependent on the ability
of the membrane to bind silver. When silver sulfadiazine was used
experimentally, this trend was clearly observed; however, the trend was
not as clear with silver chloride, as chloride was not analyzed (see
Figs. 2
and 3
). In addition, when a silver salt is placed in a
partially hydrophobic cream above the membrane, the mobilization of
silver from the cream and into the aqueous medium via the membrane will
be slower compared with the silver salt being placed above the membrane
as an aqueous paste, because of the hydrophobic nature of the cream and
the reduced rate of diffusion of polar components from it.
Experimentally, both silver sulfadiazine creams studied showed this
behavior, with the mobility of sulfadiazine being faster than silver,
although it was still slower compared with the aqueous silver
sulfadiazine paste. For a membrane that is hydrophobic in nature and
has no silver-binding abilities, such as polyethylene, ionic species
will be unable to pass through the membrane into the medium, unless an
ion-pairing agent is available to assist in transport or an uncharged
complex is formed between the ion and an appropriate ligand. In the
present study with polyethylene, neither of these processes appears to
occur.
When the medium is changed from the noninteractive 0.1 mol/L sodium nitrate solution to the chloride-containing SSES, the following general equilibria are established:
As shown in the above equilibria
, the chloride from the
medium can diffuse above the membrane to react with the silver salt to
form both insoluble solid silver chloride and the soluble
dichloroargentate(I) anion, which can then diffuse back into the
medium. The formation of the dichloroargentate(I) anion above the
membrane prevents other species, such as the membrane, from complexing
the silver unless the affinity to bind silver is greater than that for
chloride. Therefore, a membrane that has no silver-binding abilities
(cellulose), where Kim 0, is expected to
produce a silver concentration that is determined by the
dichloroargentate(I) anion equilibrium. Experimentally, the value
obtained ranged between 1.27 to 1.75 µmol/L, which was in good
agreement with the previously determined value of 1.48 µmol/L
(10). Because there is a vast excess of chloride in the
SSES medium, all the silver sulfadiazine can ultimately be converted to
silver chloride according to the reaction:
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). The time
taken to reach this latter concentration is dependent on the rate of
diffusion of chloride through the membrane and the rate of diffusionof
sulfadiazine back again. In addition, a significant amount of
silver chloride should form as a result of the above reaction, and this
was indeed noted in every case in which the medium contained chloride.
When a membrane has silver-binding abilities (chitosan and collagen),
where Kim >0, the concentration of soluble silver will
be determined by both the formation of the dichloroargentate(I) anion
and the ability of the membrane to bind the silver ion
[Ag+ (aq)] or the dichloroargentate(I) complex. For
the chitosan and collagen membranes, the interaction of the membrane
was not as significant compared with the formation of the
dichloroargentate(I) anion because of the large excess of chloride in
the SSES medium (see Fig. 4
), and so again the concentration of silver
reached the value of 1.27–1.75 µmol/L. The accompanying sulfadiazine
anion should thus reach the maximum obtainable concentration of 1400
µmol/L, a value in good agreement with the experimental results, with
the time to reach this concentration being dependent on the rate
of diffusion through the membrane. A membrane that is hydrophobic in
nature and has no silver-binding abilities, such as polyethylene, will
not allow ionic species through the membrane and into the medium,
regardless of whether dissociation occurs above the membrane or not,
and this is borne out by the results in Fig. 4
.
When glutathione was included in the SSES, the following general equilibria were established:
Although not shown in the above equilibria , the chloride from the medium can diffuse through the membrane to react with the silver salt to form silver chloride and the dichloroargentate(I) anion, which can then diffuse back into the medium. In addition, the glutathione from the medium can also diffuse through the membrane; however, it was observed to occur at a slower rate than chloride, no doubt as a result of their differing concentration gradients. Indeed, as the chloride concentration was much greater than the glutathione concentration in the medium, chloride is expected to preferentially diffuse through the membrane to form solid silver chloride and the soluble dichloroargentate(I) anion above the membrane, and then the glutathione can react to form a silver glutathione complex. The rate at which silver appears in the medium will thus be dependent on the diffusion of both the silver glutathione and the dichloroargentate(I) complexes that may form above the membrane.
When a membrane has no silver binding abilities (cellulose), where
Kim 0, the soluble silver concentration will
ultimately be determined by the formation of the silver glutathione
complex. Since the silver-to-glutathione mole ratio was 1:1, the
equilibrium concentration expected for soluble silver in the medium is
the maximum obtainable concentration of 1400 µmol/L; however, this
was not achieved over the duration of this study, and the silver
concentrations reached only one-seventh to one-third of this value (see
Figs. 6
and 7
). The accompanying sulfadiazine anion was also expected
to reach the maximum obtainable concentration; this was not achieved
over the duration of this study since the sulfadiazine concentrations
only reached about three-quarters of this value (see Fig. 6
). When a
membrane has silver-binding-abilities (chitosan and collagen), with
Kim >0, the silver-concentration will be
determined by the formation of the silver glutathione complex and the
ability of the membrane to bind both the silver ion and the complexed
forms of silver. In this experiment, the membranes that bound the
silver ultimately determined the soluble silver concentration in the
medium. The accompanying sulfadiazine anion was expected to reach the
maximum obtainable concentration of 1400 µmol/L; however, this was
not achieved over the duration of this study and the silver
concentrations reached only about one-fourteenth to one-seventh of this
value (see Figs. 6
and 7
). A membrane that has no silver-binding
abilities and is hydrophobic in nature (polyethylene) will not allow
ionic species to pass through into the medium, regardless of whether
dissociation occurs above the membrane or not, although if a neutral
silver complex was formed, as is possible with glutathione, then some
silver movement through the porous membrane and into the medium would
be possible. The data presented in Fig. 6
indicate that this does not
occur to any significant extent.
When the medium is changed to human serum, the same type of equilibria
are established as those for SSES containing glutathione, except serum
proteins and amino acids replace the glutathione. The serum proteins
are not expected to diffuse through the membranes because of their
large molecular size (cf dialysis materials); however, the equilibrium
concentration of silver in the medium was expected to be similar to
that when the silver salts were in direct contact with human serum. The
rate at which this occurs will be dependent on the ability of silver to
diffuse across the membrane, principally as the dichloroargentate(I)
anion, and then, at the membrane–medium interface, the proteins can
react with the dichloroargentate(I) anion. The results in Fig. 8
show a
similar trend to those for glutathione, although both silver and
sulfadiazine concentrations are lower, indicating the poorer complexing
ability of the serum proteins compared with the glutathione.
Our results clearly demonstrate that each membrane retards the movement of soluble silver into the medium as it acts as a diffusion-limiting barrier, but, in addition, the rate of movement is dependent on the membrane properties. Hydrophilic membranes with no silver-binding abilities allow silver through at a faster rate than a membrane that can bind silver. Moreover, silver salts in aqueous pastes release silver more readily and equilibrate much faster than those in partially hydrophobic creams.
Thus, our experimental results indicate that a significant amount of silver can be absorbed during the early phases of wound treatment, especially if the burn injury is large, as has been observed in some studies (5)(6)(7). However, as wound healing progresses, and granulation tissue forms in the burn wound, the rate of silver absorption from topically applied silver sulfadiazine will not only decrease as a result of a barrier forming, but also because the tissue is likely to bind silver to a significant degree. Therefore, silver is expected to be retained by the superficial layers of the eschar as the burn wound heals, a result that has been found in various animal studies (3)(4) and a feature that is no doubt important in limiting silver absorption and hence minimizing its toxic potential.
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), chitosan membrane used;
(
), collagen membrane used; (•) polyethylene membrane used.
Broken horizontal line, expected equilibrium value.
