In vitro testing of genotoxic and apoptotic potential of two synthesized nanomaterials by DNA fragmentation assay

Abstract

Nanomaterials represent a relatively new and developing class of materials that are widely used because of their unique mechanical and physiochemical properties. Due to the particles’ size and the ability to easily enter the cells and interact with different cellular components, nanoparticles can exhibit toxic effects in biological systems, so it is necessary to conduct a thorough in vitro biocompatibility testing. Our aim was to examine the in vitro genotoxic and apoptotic potential of two different synthesized nanomaterials intended for bone tissue engineering and regenerative applications: biphasic calcium phosphate coated with poly- D,L-lactide-co-glycolide (CP/PLGA) and cobalt-substituted hydroxyapatite (CoHAp), by DNA fragmentation assay. NB4 cells (acute promyelocytic leukemia cell line) were incubated with different concentrations of CP/PLGA and CoHAp nanomaterials for 24 hours. Cells cultured in standard medium, without nanomaterials, were used as negative control and cells treated with hydrogen peroxide were used as positive control. After incubation with materials the genomic DNA was extracted from the cells and applied on horizontal agarose gel electrophoresis. The results of DNA fragmentation assay were observed on gel documentation system. Higher examined concentrations of CP/PLGA caused DNA fragmentation at some extent with DNA laddering at approximately 180 bp which is characteristic for later stages of apoptosis, while DNA laddering was not seen in the case of CoHAp at the same concentrations. Although both materials are in the form of nanoparticles, a different effect in apoptotic DNA fragmentation was observed which suggests that the chemical composition of nanomaterials notably affects the genotoxicity and apoptosis potential in addition to the particle size.