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

Purpose: A novel system, based on biosensor DNA tethered to a nanoparticle, was developed for the treatment of retinopathy of prematurity. Methods: The construction of a five-layered nanoparticle was visualized with gel electrophoresis. Transcriptionally active PCR products (TAP) containing the biosensor sequence, were bioconjugated to the surface of magnetic nanoparticles yielding biosensor tethered magnetic nanoparticles (MNP). The biosensor was based on an enhanced green fluorescent protein (EGFP) reporter gene driven by an enhanced antioxidant response element (ARE). Image analysis and flow cytometry were used to characterize MNP delivery and biosensor activity. Results: The MNP penetrated dividing and migrating cells more often than quiescent endothelial cells in a wound-healing iin vitro assay. Prussian blue staining demonstrated that more cells have nanoparticle cores than are transfected. When compared to naked TAP alone, MNP transfected more cells in a dose dependent manner. Both the biosensor alone and MNP induce gene expression in the presence of hyperoxia, greater than 1.5 fold over normoxic controls. These data also show that the MNP had a signal to noise ratio of 0.5 greater than the plasmid form of the biosensor as demonstrated by flow cytometry. Conclusions: This approach has the potential to allow the endothelial cells of the retinal vasculature to prevent or treat themselves after hyperoxic insult, rather than systemic treatment to protect or treat only the retina.