Nanoparticle tethered antioxidant response element as a biosensorfor oxygen induced toxicity in retinal endothelial cells

[摘要] Purpose: A novel system, based on biosensor DNA tethered to ananoparticle, was developed for the treatment of retinopathy ofprematurity.Methods: The construction of a five-layered nanoparticle wasvisualized with gel electrophoresis. Transcriptionally active PCRproducts (TAP) containing the biosensor sequence, were bioconjugated tothe surface of magnetic nanoparticles yielding biosensor tetheredmagnetic nanoparticles (MNP). The biosensor was based on an enhancedgreen fluorescent protein (EGFP) reporter gene driven by an enhancedantioxidant response element (ARE). Image analysis and flow cytometrywere used to characterize MNP delivery and biosensor activity.Results: The MNP penetrated dividing and migrating cells more oftenthan quiescent endothelial cells in a wound-healing in vitro assay.Prussian blue staining demonstrated that more cells have nanoparticlecores than are transfected. When compared to naked TAP alone, MNPtransfected more cells in a dose dependent manner. Both the biosensoralone and MNP induce gene expression in the presence of hyperoxia,greater than 1.5 fold over normoxic controls. These data also show thatthe MNP had a signal to noise ratio of 0.5 greater than the plasmid formof the biosensor as demonstrated by flow cytometry.Conclusions: This approach has the potential to allow theendothelial cells of the retinal vasculature to prevent or treatthemselves after hyperoxic insult, rather than systemic treatment toprotect or treat only the retina.

[学科分类] 生物化学/生物物理