I. INTRODUCTION
Silver has long been used for its antimicrobial properties. However, the delivery systems available, often in the form of a salt, have been the limiting factor to successful biological use of this noble metal. Nanotechnology and the ability to deliver silver from a nanocrystalline structure has and will markedly improve the biologic value of silver. These advances in crystal chemistry will likely have a dramatic impact on the microbiology, as well as biology of wound healing and control of inflammation. This review will attempt to describe the past, present and future uses of silver in biologic systems focusing on its biological properties on “wounds”.

II. HISTORICAL OVERVIEW OF MEDICINAL SILVER AND WOUNDS
Silver has been used for centuries to prevent and treat a variety of diseases, most notably infections. It has been well documented that silver coins were used in ancient Greece and Rome as a disinfectant for the storage of water and other liquids. (1,2) More recently, NASA still uses silver to maintain water purity on the space shuttle. Silver has extremely potent antimicrobial properties, as only one part per 100 million of elemental silver is an effective antimicrobial in a solution. Free silver ions, or radicals, are known to be the active antimicrobial agent. In order to achieve a bactericidal effect, silver ions must be available in solution at the bacterial surface. Efficacy depends on the aqueous concentration of these ions. Silver ions appear to kill micro-organisms instantly by blocking the respiratory enzyme system (energy production), as well as altering microbe DNA and the cell wall, while having no toxic effect on human cells in vivo.

 

Silver in solution has been used as an antimicrobial for wound management for nearly a century. However, crystalline silver is quite insoluble in water and in dilute acids making the available silver cation concentration, inadequate for use as an antimicrobial on a wound surface. Beginning in the 1920’s, a small electrical charge was passed thru water and silver crystals in order to obtain an effective silver (electro-colloidal) ion solution to be used topically on wounds. The charged silver solutions (electro-colloidal) were approved in the 1920’s by the FDA for use as an antibacterial agent.(3) Some wound centers still use these solutions although silver ions in solution are quite unstable. In addition, to its recognized antibacterial properties, beginning with the electro-colloidal elemental form, silver solutions have been reported to improve the healing of “indolent wounds” and to “regenerate damaged tissue”. The description of decreased rubor in wounds also reflects an anti-inflammatory property of silver.

More recent information has provided, at least a hypotheses as to the mechanism of silver’s pro-healing and anti-inflammatory effects. Initial literature reports on the use of pure silver, mainly in the electro-colloidal form, occurred prior to the 1940’s when pure silver was still being used. After 1940 a host of systemic antibiotics became prevalent, decreasing the use of silver except as a topical agent. During this transition, silver was complexed as a salt (e.g. silver nitrate and silver sulfadiazine) or other compound (e.g. silver protein) to increase the available silver ion concentration. These silver complexes remain a popular topical antimicrobial agent for the care of wounds. Silver itself is considered to be non-toxic to human cells in vivo.(4) The only reported complication is the cosmetic abnormality argyria caused by precipitation of silver salts in the skin and leading to a blue-gray color.(2)

Although not yet defined, there remains some concerns about the potential of high tissue levels of silver altering enzyme function. Dr. Carl Moyer(4) in 1965 introduced the use of a 0.5% silver nitrate solution for burn wound management. The silver nitrate was a more stable compound and replaced colloidal silver. During the same time period, Dr. Charles Fox(5) developed another silver compound for burns, silver sulfadiazine. The sulfadiazine is composed of propylene glycol, stearyl alcohol and isopropanolol. This compound was formulated as a water soluble cream to be applied twice a day to a wound surface instead of a continuous soak required of silver nitrate for continued silver delivery. Over the past 40 years silver sulfadiazine has become the most popular anti-microbial silver delivery system.(4-7) However, both nitrate and sulfadiazine impair fibroblast and epithelial proliferation, impairing healing.(8) Some of the biological properties of topically applied silver which have been identified include.(1-10) Although electro-colloidal silver is still used, advances in the field of nanotechnology were required before a new form of silver was available for use in biological systems.(11) The mechanism of the decrease in MMP activity is yet unknown as is the reduction in wound surface zinc, a necessary co-factor for MMP activity(9). Advances in the field of nanotechnology have provided a new form of silver available for use in biological systems.(6) With the current availability of nanocrystalline silver, it is likely that a large number of biological effects of silver will be identified.

III. NANOTECHNOLOGY
The property of matter depends on size and many of the chemical and physical characteristics change significantly when matter is reduced in size.11,12 Nanotechnology is a general term that refers to a relatively new frontier of scientific endeavor. The prefix “nano” signifies one-billionth. Therefore, a nanometer is one-billionth of a meter, a nanogram is one-billionth of a gram. Ten hydrogen atoms placed side by side measures one nanometer in length. Silver crystals sputtered under normal vapor deposit conditions result in tightly adherent crystals of 100-900nm in diameter (Figure 1). Decreasing crystal volume by nanotechnology markedly increases the exposed surface area of the crystal (Figure 2) which increases the available surface for chemical reactions to take place over a shorter time period. Decreasing the particle size will also, in general, change the physical/chemical properties of the material. Examples of changed properties resulting from nano-sized metals, include increased superconductivity and increased optical and electrical properties. Nanosizing can also lead to a more economical utilization of expensive materials-meaning that can use less material because the reactions are more efficient. Although not yet specifically defined, it is clear that some of the properties of silver in a nanocrystal are quite different than the typical crystal.(11-15) A large portion of the silver is available as grain or interphase boundaries, considered by some to be a new form of matter.

Orientation relationships are very different for the silver. Also some of the silver appears to be in an oxidized form. Increasing the variety of oxidized silver species would be expected to increase solubility and lead to a much higher overall reactivity. The antimicrobial nanocrystalline silver film to be discussed is produced, using a magnetron sputtering process whereby the silver atoms are layered down atom by atom in the presence of trace levels of oxygen. The process results in a much higher energized state in that there is a large number of defects in the crystal lattice. These defects include both point defects (i.e. missing, misplaced or foreign atoms) and line defects (i.e. grain boundaries, sub grain boundaries, twins, etc.). The thickness of the antimicrobial silver layer is approximately one micrometer. The layer is activated to release silver clusters of Ag0, silver cations and silver radicals that may have overall positive or negative charges with the addition of water including that from the wound surface. The currently used nanocrystalline silver delivery system has the following composition. There are two silver coated high density polyethylene meshes between which is a rayon polyester layer mainly used as an absorptive layer for wound exudates. This structure allows for the release of silver well above antimicrobial levels for a period of days compared to minutes or hours seen with silver salts and complexes.(13,14)

IV. BIOLOGIC PROPERTIES OF NANOCRYSTALLINE SILVER COMPARED TO OTHER SILVER PRODUCTS

a) Antimicrobial Properties(3,4,16,17)
As can be seen in Table 3 the aqueous concentration of silver ions released from the nanocrystalline film is approximately 3% of that released from a 0.5% silver nitrate or a 1% silver sulfadiazine cream. However, the biological properties of the silver released from nanocrystals are much greater. Silver resistance has been reported in the literature and is mediated through one of two pathways. Either the silver is tied up in the cell wall and membranes, or it is actively transported out of the cell. Bacterial organisms that have either one of these resistance mechanisms, which are effective up to 1000 µg/mL Ag+, have been tested against the nanocrystalline silver coated dressing. These tests showed that these organisms were susceptible to the silver produced by the nanocrystals, but not to Ag+ from silver nitrate. These findings, as will be described, strongly suggest that other species of silver besides Ag+ are released from the nanocrystals.

The lower amount Ag+ released should also decrease the potential of silver toxicity to cells, if it exists, by a substantial margin when compared to the other silver agents. The nanocrystal coating contains 0.84-1.34 mg silver/cm2 of dressing, is resistant to abrasion, does not adhere to the wound and is flexible. In another study nanocrystalline silver was extracted from the silver delivery system Acticoat by incubating the dressing in nanopure water at 37°C in a shaking incubator, and silver concentrations were measured using atomic absorption spectrophotometry. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using five bacterial isolates of clinical interest, and results were compared for nanocrystal silver, silver nitrate and silver sulfadiazine, based upon total silver as shown in Table 4. Nanocrystalline silver had similar MIC and MBC values when compared to three silver containing agents. Kill kinetics were also studied, using 2.0 cm x 2.0 cm pieces of silver dressing, and the same sized pieced of dressing impregnated with either silver nitrate (100µl of 1% solution. This results in a final concentration of 0.5% silver nitrate) or silver sulfadiazine (370mg of a 1% cream). Bacterial survival was measured using a plate counting technique. Nanocrystal silver demonstrated the fastest kill times for the five bacteria used. In most instances with Nanocrystal silver, bacterial survival was undetectable 30 minutes after inoculation, whereas at least 2-4 hours elapsed before no viable cells were detected with silver nitrate or silver sulfadiazine. These findings strongly suggest that silver species in addition to Ag+, are released from the nanocrystalline film which are responsible for the more potent antimicrobial properties. To date the nanocrystalline silver system kills all microbes found in a wound including fungi and all current antibiotic resistant strains such as vancomycin resistant enterococcus (VRE) and methicillin resistant Staphylococcus aureus (MRSA).18)

b) Pro-healing Properties
Although silver in an electro colloidal form, had been reported to improve the healing of indolent wounds in the early 20th century, that finding disappeared with the use of silver salts and complexes. Recently there have been several reported studies of improved re-epithelialization rates across partial thickness wounds with silver in the nanocrystalline form. The mechanism, although unknown at present, does not appear to be due to silver’s antimicrobial action.(5,19,21)

c) Anti-inflammatory Properties
Increased wound inflammation not only accentuates pain but markedly impairs healing. Several heavy metals have been reported to decrease surface inflammation, the most recognized being gold. Wound surface inflammation has been reported to be decreased with the use of nanocrystalline silver. Excess metalloproteinases (MMP) are known to increase inflammation by both increasing inflammatory cell exudates and also leading to a non-healing chronic wound. A characteristic of this type of wound is excess surface MMP activity, decreased inhibitory MMP activity and degradation of growth factors by the MMP’s.(9,2,23) Nanocrystalline silver has been shown both in vitro and in vivo to decrease but not prevent MMP activity as some activity is needed to remove devitalized tissue. The mechanism for this action also remains unknown. Decreasing the necessary zinc activity required for MMP’s, is one possibility. The other is an effect on the expression or release of pro-inflammatory cytokines.

V.THEORETICAL CONSIDERATIONS
The biological evidence suggests that active silver moieties other than Ag+ must be released from nanostructured silver coatings. The evidence includes; much higher kill rates of bacteria and fungi with up to thirty times less silver than conventional silver salts like AgNO3 and silver sulfadiazine; the modulation of MMP’s; the modulation of TNF-a; improved dermal regeneration; the reduction of exudate; and the induction of apoptosis. The mechanisms of action for these reactions are not understood but there is some evidence as to what the mechanisms might be. It has been established that the dissolution process results in the production of at least two chemical entities. Ag+ has been observed both by specific ion electrode and electrochemical analysis such as scanning electrochemical microscopy (SECM). Ag0 has been observed using SECM techniques15. A higher oxidation state in the sputtered coatings has also been observed using cyclic voltametry (R. Burrell Pers. Comm). It is also known that silver oxide complexes will react with water to produce silver hydroxide species. It is postulated that the highly active surface of nanocrystalline silver with silver present in various oxidation states may produce unusual metastable silver hydroxide compounds. It is believed that a series of complex metastable silver hydroxyl anions could be produced at the surface of the crystals. The complex hydroxyls would be metastable and thus have an ability to migrate into the surrounding environment and interact with it. The rapid kill of bacteria and fungi suggests a rapid route of uptake of silver from nanocrystalline silver. Since orthophosphate uptake is known to be very rapid, we speculate that other metal oxides, with a similar configuration, may be mistakenly taken up.

Thus, if none of the chemical species produced include silver hydroxide complexes with the general formula Agx(OH)y (charge = x-y), then it is conceivable that uptake could occur through the orthophosphate pathway. Such a scenario would explain the rapid uptake and kill of microorganisms as well as the susceptibility of silver resistant organisms. The presence of Ag0 suggests that there must be clusters present as it is unlikely that a bare atom such as Ag0 could exist on its own. These clusters may exist as uncharged or charged entities, but it is unknown what the biological activity might be. It is known that other heavy metals such as Au and Pt have unique biological properties including anti-inflammatory and apoptosis induction (anti-tumour?) activity. Since these activities have not been observed with Ag+ in the past, but they have been observed with dissolution products of nanocrytalline silver, it is postulated that it is the other species released, including Ag0, that may be partly or wholly responsible for the unusual biological properties.

VI. SUMMARY
The application of nano-technology to develop nanocrystalline silver has led to a highly effective topical antimicrobial agent due not only to a more rapid release of silver cation onto a wound surface, compared to other available silver antimicrobial agents, but likely the release of other silver species as well. In addition, nanocrystalline silver appears to have overall positive biological effects on wound inflammation, healing and likely on a host of as yet undetermined reactions. Current available data only scratches the surface of the potential theoretical benefits of nanocrystalline silver.

Robert H. Demling,
Dottore in Medicina; Professore di Chirurgia, Harvard Medical School; Direttore Centro Ustioni, Brigham & Women’s Hospital Boston, MA 02115

Robert E. Burrell
Ph.D. Westaim Corporation Fort Saskatchewan Alberta, Canada T8L 3W4