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

ESPECHE TURBAY, B; REY, VALENTINA; DORADO, RITA D.; GRAMAJO FEIJOÓ, MARCELO E.; SOSA MORALES, MARCELO C; BORSARELLI, CLAUDIO D.

Carlos Paz, Córdoba

Encuentro; XIII Encuentro Latinoamericano de Fotoquímica y Fotobiología; 2017

In recent years the microbial resistance to conventional antibiotics has increased, being currently a world-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 on the cell wall of some Gram positive bacteria, and comparing with those stabilized with citrate ion (AgNP@CIT), by incubation of pathogen microorganisms under both blue-light illumination 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 growth of the surface resonance plasmon absorbance 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) and Staphylococcus aureus (St.aureus) in Muller Hinton agar and broth medium. AgNP@LZ did not show antimicrobial effect, opposite to the case of 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+ aggregates, while combined ATR-FTIR, circular dichroism, and fluorescence studies demonstrated that the covering of AgNP with LZ produces the loss α-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 of AgNP prevents or strongly reduces the delivery of Ag+ to the solution and prevents a microbicidal activity [5].Thus, the photoreduction of Ag+ in the presence LZ produces a very stable hybrid AgNP@LZ without antimicrobial activity.