Silver nanoparticles obtained by the 'green' synthesis combined or not with calcium glycerophosphate and tyrosol: antimicrobial, anti-inflammatory effect and in the transcriptomic response in oral pathogens

Author(s)

José Antonio Santos Souza

• Document type: Doctoral Thesis
• Press: Araçatuba. 2018-11-27.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araçatuba
• Defense date: 2018-11-01
• Advisor: Alberto Carlos Botazzo Delbem

Abstract

The aim of this study was to evaluate the antimicrobial/antibiofilm activities and the cell viability of silver nanoparticles (AgNPs) obtained by a ‘green’ synthesis associated or not to β-calcium glycerophosphate (CaGP) against Streptococcus mutans and Candida species (reference strains and oral clinical isolates including azole-resistant strains). The effect of these nanocompounds in combination with tyrosol (TYR), fluconazole (FLC), nystatin (NYT) and amphotericin B (AmB) also was evaluated. Furthermore, we evaluated, comparatively, the transcriptome alterations of cells of C. glabrata CBS138 after exposition to AgNPs and to silver ion (Ag+). Initially, AgNPs were synthesized by reducing silver nitrate with extracts of different parts (peel, leaves and seeds) of a pomegranate (Punica granatum L.) associated or not to CaGP. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal/Bactericidal Concentrations (MFC and MBC) of the nanocomposites (AgNPs and AgNPs-CaGP) associated or not to TYR against S. mutans and C. albicans were determined by the microdilution method. And, the action of the AgNPs combined with FLC, NYT and AmB also was evaluated in planktonic condition against oral clinical isolates of Candida species. The effect of the nanocomposites associated or not to TYR on cell viability of fibroblasts (L929) and cytokines production was evaluated through MTT and ELISA assays, respectively. The number of cells undergoing filamentation was quantified in C. albicans biofilms formed in the presence of AgNPs with or without fetal bovine serum (FBS). The effect of AgNPs in inhibit the formation of biofilm (in C. albicans and C. glabrata) also was investigated through PrestoBlue assay and scanning electron microscope. Biofilms of S. mutans and Candida species (12 and 24 h) were treated with nanocomposites (AgNPs and AgNPs-CaGP) associated or not to TYR for 24 h and, then, the viability of these biofilms was determined through PrestoBlue assay and colony forming units (CFUs). Moreover, a transcriptome analysis was performed using microarrays to determine which genes are up- or down-regulated as response to AgNPs (in the planktonic state and forming biofilms) and to Ag+ ion (forming biofilms). Antimicrobial solutions synthesized in this study (AgNPs and AgNPs-CaGP) presented antimicrobial activity against tested microorganisms. Furthermore, lower values of MIC were obtained when these nanocompounds were associated to TYR, FLC, NYT and AmB showing a synergistic effect. AgNPs and AgNPs-CaGP were not toxic to L929 cells, increased the stem cell factor production and did not promote significant alterations in the Interleukine-6 release. The nanocomposites associated to TYR did not present cytotoxic effect on L929 cultures, except for the higher concentration (AgNPs – 39.05 µg/mL + TYR – 1.25 mM). The incubation in the presence of the AgNPs drastically reduced the number of cells exhibiting hyphae, this effect being observed either in the presence or absence of FBS. The amount of biofilm formed by Candida species in the presence of the AgNPs was reduced by more than 50% the one formed in the absence of the nanoparticles, this reduction being further increased to more than 90% when the concentration of the AgNPs was increased. After 24 h of treatment with the nanocompounds (AgNPs and AgNPs-CaGP) in S. mutans biofilms, there was a significant reduction in the number of CFUs being similar to chlorhexidine. A reduction in the viability of C. glabrata biofilms also was observed after exposition to AgNPs (24 h). However, the nanocomposites (AgNPs and AgNPs-CaGP) associated or not to TYR were not effective against C. albicans biofilms. After exposition to AgNPs and to Ag+ ion, alterations in the transcriptome of cells of C. glabrata CBS138 were observed: in the planktonic state, genes responsible by the methionine and lysine biosynthesis are up-regulated, and genes related with the transmembrane transport are down-regulated. And, finally, the cells exposed to AgNPs responded differently in relation to the Ag+ ion, indicating that the AgNPs might present a different mechanism of action. All these results may help guide therapeutic decisions with formulation containing AgNPs or AgNPs-CaGP associated or not with different compounds in patients with dental caries and oral candidiasis. (AU)

• FAPESP's process: 15/00825-5 - Effect of the calcium glycerophosphate associated with nanoparticles of colloidal silver obtained by the 'green' synthesis about biofilms of Candida albicans and Streptococcus mutans
• Grantee: José Antonio Santos Souza
• Support Opportunities: Scholarships in Brazil - Doctorate