On the nature of silver ions in complex media

Antimicrobial biocides are commonly used to present the growth of bacteria on surfaces and within materials. They are typically added in small quantities to many applications to prevent bacterial growth on the treated object. Silver is increasingly used in many applications due to the aim to replace organic chemical agents by inorganic additives. Examples of applications are bacteriostatic water filters for household use or swimming pool algaecides and numerous devices, ranging from consumer commodities like mobile phones, refrigerators, and clothes to medical devices like catheters, implant surfaces, and plasters. To meet the diversity of application types, many different forms of silver compounds have been developed to serve this market. In particular, there is little information on the types of transformations that silver nanoparticles will undoubtedly undergo in real, complex environments during long-term aging, and the impact of these transformations on their distribution in the environment, bioavailability, and toxicity potential. The biocidal action results from the interaction of silver ions with bacteria. The most potent compounds for a high silver release are soluble silver salts like silver nitrate or silver acetate. These are fully water soluble with a high silver ion release rate. Therefore they are often used as control in cell experiments to elucidate the biological effect of silver nanoparticles. However, in the case of free silver nanoparticles the interactions can be more complex and catalytic reactions on the particle surface which depend on the size and shape of the nanoparticles can render the system very complex. If AgNO3 is used as control, it is tacitly assumed, that the free silver ion concentration is the same as that in the added AgNO3. This obviously cannot be true because of the presence of a whole set of proteins, biomolecules and inorganic ions like Cl- and H2PO4- in the biological medium. These will react with the silver ions in one or the other way. We report on experiments on the behaviour of silver ions in biologically relevant concentrations in different media, from physiological salt solution over phosphate-buffered saline solution to cell culture media. For dissolution and immersion experiments PVP-coated silver nanoparticles were synthesized by reduction with glucose in the presence of PVP. The final silver concentration in all dispersions was determined by atomic absorption spectroscopy. The dissolution of silver nanoparticles was followed in long-term experiments out of a dialysis tube which was permeable only for silver ions. In case of immersion experiments, the nanoparticles and all precipitates were isolated by ultracentrifugation, redispersed in pure water and again subjected to ultracentrifugation. The particles were analyzed by scanning electron microscopy, energy-dispersive X-ray spectroscopy and X-ray powder diffraction. The dissolution requires the presence of dissolved oxygen. If no oxygen is present, only a very small fraction of silver is dissolved, possibly by traces of oxygen in the experimental setup. An oxidizing agent like H2O2 clearly enhances the dissolution. The presence of NaCl, either in pure form or as PBS, strongly slows down the dissolution, probably due to silver chloride formation. Cysteine has a clearly inhibiting effect with almost no dissolution of the silver nanoparticles whereas glucose has a decelerating effect but leads to a similar final dissolved fraction. This suggests that cysteine adsorbs onto the silver nanoparticle surface with its thiol group and prevents the oxidation. In contrast, glucose slows down the dissolution, but clearly did not prevent the oxidation on a longer time scale. We have extended the studies by mixing silver nanoparticle dispersions with different media of increasingly biological nature. The solutions/dispersions were stirred for equilibration and then subjected to ultracentrifugation. All precipitates and nanoparticles were isolated by this way and then analyzed. The results show that both initially present silver ions and released silver ions are mainly precipitated as AgCl if chloride is present. Only in the absence of chloride, glucose is able to reduce Ag+ to Ag0. The initially present silver nanoparticles were recovered in all cases. Silver phosphate was not observed in any case, probably due to the moderate pH (around 7) at which phosphate is mostly protonated to hydrogen phosphate and dihydrogen phosphate. We can conclude that released silver ions precipitate mostly as AgCl in biological media, and that most cell culture studies where silver ions are used as control are in fact studying the effect of colloidal silver chloride on the cells. To prove this assumption, human mesenchymal stem cells (hMSC) were cultured in the presence of silver chloride nanoparticles (diameter 120 nm), and the viability of the cells was analyzed by fluorescence microscopy. In general, we clearly observed that pure silver nanoparticles have lower toxicity to hMSC compared to silver chloride nanoparticles with a comparable total silver dose. Silver acetate in the biological medium had a comparable toxicity to hMSC compared to silver chloride nanoparticles with the same total silver dose.