Photoreduction of Ag+ in the presence of lysozyme produces stable hybrid nanoparticles (AgNP@LZ) without bactericidal effect

M. BEATRIZ ESPECHE TURBAY; VALENTINA REY; RITA D. DORADO; MARCELO E. GRAMAJO FEIJOÓ; MARCELO C. SOSA; CLAUDIO D. BORSARELLI

Villa Carlos Paz

Congreso; XIII Encuentro Latinoamericano de Fotoquímica y Fotobiología (XIII ELAFOT 2017); 2017

In recent years the microbial resistance to conventional antibiotics has increased, being currently a word-wide concern. Hence, the development of new therapies in order to eliminate strains of multiresistant microorganisms is mandatory. In this context the use of nanomaterials with antibiotic activity has become recognized such as silver nanoparticles (AgNP), which are able to inactivate different strains of microorganisms, mainly due to the delivery of Ag+ as microbicide [1].Additionally, hybrid nanomaterials based on the conjugation of AgNP with biomolecules are also used for antibiotic purposes [2]. Therefore, in this report we evaluated a possible synergistic antibacterial effect of hybrid AgNP stabilized with lysozyme (AgNP@LZ), a protein with lytic activity of on the cell wall of some Gram (+) bacteria, and comparing with those stabilized with citrate ion (AgNP@CIT), by incubation of pathogen microorganism under both blue-ligth illuminated and dark conditions. The in situ preparation of both AgNP in the presence of LZ or CIT was performed by UVB photo-cleavage of Igarcure-2959 and subsequent ketil radical reduction of Ag+ [3], and the formation of the AgNP was monitored following the absorbance growth of surface resonance plasmon with maximum around 400 nm. The determination of the Minimum Inhibitory Concentration (MIC) of both nanomaterials was evaluated on 4 strains of clinical interest Escherichia coli (E.coli), Klebsiella pneumoniae (K. pneumoniae), Serratia marcescens (S. marcenscens) y Staphylococcus aereus (St.aereus) in Muller Hinton agar and broth medium, and the results showed that AgNP@LZ did not show antimicrobial effect, opposite as to happen with AgNP@CIT and control tests of LZ and Ag+. This striking result led us to explore the reasons of the loss of antimicrobial effect of AgNP@LZ. Binding studies by stationary absorbance and fluorescence spectroscopies indicated the formation of a LZ/Ag+ adduct (Kb = 4500 M-1), while combined ATR-FTIR, circular dichroism , and fluorescence studies demonstrated that the covering of LZ onto AgNP produces the loss -helix conformation of the protein [4] together with oxidation of Trp residues, e.g. W62, and consequent loss of its lytic activity. Additionally, the tightly protein shell formed on the surface AgNP prevents or strongly reduced the delivery of Ag+ to the solution to observe a microbicide activity [5]. Thus, the present work shows that the photoreduction of Ag+ in presence LZ produces very stable hybrid AgNP@LZ without antimicrobial activity.