Mechanistic Study of DNA Delivery from Self-Assembled Nanolayered Films

Abstract

The delivery of nucleic acids to modulate gene expression levels can enable highly specific and durable therapeutic effects. Formulation to protect nucleic acid cargoes and direct them to target tissues is critical to the success of these promising therapies. Delivery of nucleic acids, such as plasmid DNA, must overcome both systemic and local cellular barriers. Surfacemediated gene delivery bypasses systemic trafficking obstacles by localizing DNA release within the cellular microenvironment. Local delivery has several advantages including increased efficacy at the target site and reduced off-target effects. Layer-by-layer (LbL) self-assembly is a promising method to incorporate DNA in nanolayered thin film surface coatings for controlled, localized release. Although the potential for DNA delivery via layer-by-layer films has been reported, details in the mechanism or factors influencing the success of delivery have not been explored.

In this thesis, we designed LbL-assembled DNA multilayer films for localized gene delivery. We present a mechanistic investigation of factors impacting in vitro DNA transfection efficacy. Using pre-formed DNA-polymer complexes as a model system, we identified relative polymer to DNA content in polyplexes as a key driver of effective transfection. We then explored the impact of LbL assembly parameters on DNA multilayer film composition and release kinetics, and how these subsequently influence transfection efficacy in vitro. Finally, we characterized the film releasate to elucidate how cells interact with DNA multilayer films. Rapid release of DNA complexed with polymer was found to enable the greatest transfection efficiency. The findings described here will contribute to rational design of more effective LbL films for DNA delivery.