Comparing the effect of gold and silver nanoparticles on human dermal fibroblasts using Cellomics high-content screening system

摘要

Introduction: Gold nanoparticles (GNPs) and silver nanoparticles (SNPs) are widely used in the field of biomedicine, and their biosafety must not be overlooked. Cellomics high-content screening (HCS) system is a combination of cell biology with automated high resolution microscopy and robotic handling, providing detailed analyses of cytotoxicity of biomaterials based on parallel analyses of multiple markers. The aim of this study is to compare the influence of GNPs and SNPs on cell membrane permeability, cell adhesion, mitochondrial membrane potentials, and apoptosis of human dermal fibroblasts (HDFs) with the HCS system. Materials and methods: GNPs and SNPs were synthesized by water-phase reaction and their size and morphology were observed by transmission electron microscope (TEM). HDFs were exposed to 200μM GNPs and SNPs for 1,4,8,24,48 and 72h, respectively, and their effects were analyzed by the HCS system. Cells were dyed with Sytox and membrane permeability was assayed by calculating relative fluorescent intensity of Sytox. For focal adhesion observation, HDFs were stained using a FAK100 kit. Mitochondrial membrane potentials were monitored by determining relative amounts of dual emissions from mitochondrial JC-1 monomers or aggregates by JC-1 probing. Cell apoptosis detection were carried out using PI/Hoechst dyeing method. Results and discussion: TEM images showed that GNPs and SNPs were generally spherical with size of ～20 nm. HCS analyses showed that relative fluorescent intensity of Sytox in GNP-treated HDFs had no obvious difference compared to untreated cells; while significant increase was detected after cells were treated with SNPs for 48 and 72h, indicating that SNPs destroyed cell membranes after 48h. Fig. 1 displays the focal adhesion of untreated, GNP- or SNP-treated HDFs for 72h. No distinct alteration in number, area of focal adhesion and nucleus were observed in GNP-treated cells; while in SNP-treated groups, the amount and area of focal adhesion was significantly reduced and was accompanied with a decrease of number and area of nucleus, indicating that SNPs inhibited cell adhesion and proliferation. JC-1 staining results revealed that mitochondrial membrane potentials in HDFs did not changed after GNPs treatment, while the potentials increased after treatment of SNPs for 8h, implying that SNPs could induce early apoptosis. PI/Hoechst dyeing results suggested that SNPs induced HDFs later apoptosis after 4h, while GNPs did not. In summary, GNPs did not induce toxicity at the cellular level, while SNPs destroyed membrane, restrained cell adhesion, increased mitochondrial membrane potentials, induced apoptosis, inhibited cell proliferation, and finally led to cytotoxicity. Fig. 1. Focal adhesion of HDFs observed on HCS platform (a) Untreated HDFs, (b) HDFs exposed to GNPs for 72h, (c) HDFs exposed to SNPs for 72h. Green: Focal adhesion, Blue: nucleus. Conclusion: With the Cellomics HCS system, the influence of GNPs and SNPs on HDFs was compared, and the toxicity effect of SNPs was illustrated. The obtained results had a good consistency with our previous conclusions from multi-biomics data analysis.