Biosynthesis of silver nanoparticles using Sambucus nigra L. fruit extract for targeting cell death in oral dysplastic cells

The study aims to evaluate the impact of silver nanoparticles, phytosynthesized with polyphenols from Sambucus nigra L. (SN) fruit extract (AgSN), on dysplastic oral keratinocytes (DOK) and human gingival fibroblasts (HGF) in terms of cell viability and apoptosis. The morphology and ultrastructure of treated cells as well as the mechanisms involved in cell death induction were investigated in DOK cultures. The structure of AgSN was studied by using the appropriate analysis tools such as UV-Vis, transmission electron microscopy, Raman spectroscopy, dynamic light scattering (DLS) and zeta potential assessment. DOK and HGF were treated either with silver nanoparticles capped with Sambucus nigra L. extract or with SN extract. Untreated cells were used as controls. Viability was determined by MTS assay. Transmission electronic microscopy (TEM) was used to evaluate the intracellular localization of the nanoparticles at 4 and 24 h. Annexin V-FITC/propidium iodide staining and the expressions of p53, BAX, BCL2, NFkB, phosphorylated NFkB (pNFkB), pan AKT, pan phosphoAKT, LC3B and gamma H(2)AX were evaluated to quantify the cell death. ELISA measurements of TNF-alpha and TRAIL was used for the study of the inflammatory response. Oxidative stress damage induced by nanoparticles was assessed by the malondialdehyde (MDA) level. Silver nanoparticles stimulated HGF proliferation and significantly diminished DOK viability at doses higher than 20 mu g/ml. TEM analysis demonstrated the internalization of silver nanoparticles and showed ultrastructural changes of cells such as the appearance of vacuoles, autophagosomes, endosomes. AgSN inhibited the pro-survival molecules and regulators of apoptosis, diminished oxidative stress and inflammation and induced cell death through various mechanisms: necrosis, autophagy and DNA lesions. SN extract had antioxidant and anti-inflammatory effect and increased the DNA lesions and autophagy in DOK cells. Silver nanoparticles protected the normal cells and induced cell death in dysplastic cells by different mechanisms thus offering beneficial effects in the treatment of oral dysplasia.