Introduction: Prebiotic chemistry is associated with the chemical origin of life; how simple non-living molecules (such as HCN oligomers) formed amino acids, nucleic acids and proteins. Building on the knowledge collected in the field of prebiotic chemistry over more than 50 years, we have been able to show that polymeric coatings can be deposited using the p-toluene sulphonate form of aminomalonitrile (AMN, the trimer of HCN). The robust, adhesive polymeric surface coatings are formed via neutralisation and spontaneous polymerisation in slightly basic, aqueous conditions[1]. The resulting coatings are chemically complex, peptidic in nature with a high content of nitrogenous heterocycles. The AMN surfaces are non-cytotoxic and by incorporation of silver, we have additionally introduced antibacterial properties as measured against the biofilm forming bacterial strains S. epidermidis (gram-positive) and P. aeruginosa (gram-negative).
Materials and Methods: Aminomalonitrile p-toluenesulphonate (AMN, Sigma-Aldrich) solutions were prepared in 100 mM phosphate buffer (pH 8.5, 10 mg/mL). Samples were incubated upside for 24 hours at 25 °C. Incorporation of silver was achieved by incubation in AgNO3 for 24 hours (1 nM to 100 mM), rinsed and incubated in water for a further 24 hours. XPS was used to determine the incorporation of Ag and atomic composition of the coatings. Quantification of biofilm formation on the AMN and Ag impregnated AMN coatings was performed via a crystal violet assay, (using S. epidermidis (ATCC 35984) and P. aeruginosa (ATCC 27853)) and cell toxicity and attachment of mouse fibroblasts (ATCC NCTC clone 929 adipose mouse) were observed via photomicroscopy and quantified using an MTS assay, after 24 hours.
Results and Discussion: The AMN coatings have been shown to be highly nitrogenous, non-cytotoxic and facilitate attachment of L929 mouse fibroblast cells equivalent to tissue culture polystyrene control surfaces. As silver containing coatings are of particular interest in antimicrobial surface applications, we investigated the potential for silver impregnation of the AMN coating, with the nitrogenous nature of the polymeric surfaces expected to provide a suitable environment for the complexation of metal ions. XPS analysis of the AMN coated surfaces after incubation in AgNO3 indicated that silver was detected in the films when the silver nitrate concentration was >10-5 M (Figure 1A). Analysis of the Ag MVV Auger region of the XPS spectrum has indicated that some of the incorporated Ag exists in its reduced form, Ag(0), in coatings treated with higher concentrations of AgNO3 (0.01 and 0.1 M).
The silver treated AMN surfaces were evaluated for their ability to prevent biofilm growth of two well-known biofilm forming bacterial strains S. epidermidis (gram-positive) and P. aeruginosa (gram-negative). The results show a concentration dependent anti-biofilm activity with biofilm growth sharply dropping off on surfaces that have been incubated with AgNO3 at a concentration greater than 10-4 M (Figure 1B).
Conclusion: Prebiotic chemistry inspired polymeric coatings based on aminomalonitrile have shown to be highly biocompatible and to facilitate the growth and attachment of mammalian cells. Incubation in silver nitrate solutions impregnated the AMN coatings with silver, when a concentration of 10-4 M and above were used. This same concentration was found to be optimal in terms of exhibiting antibacterial effects against two biofilm forming bacterial strains, S.epidermidis and P.aeruginosa.
References:
[1] Thissen, H., Evans, R.A. & Koegler, A. Hydrogen cyanide-based polymer surface coatings and hydrogels, WO2013170308-A1.