(681a) Design of Biodegradable Polycations for Localized Gene Delivery from Layer-By-Layer Coatings

The layer-by-layer (LbL) method of assembling polyelectrolyte multilayers has become one of the most promising coating methods capable of mimicking cellular microenvironments and releasing nucleic acids from the surfaces of biomedical devices. Despite extensive knowledge on the LbL film assembly, important questions remain regarding the film disassembly, for example, what are the timing and structure of the released species and how can these factors be designed to facilitate programmable nucleic acid delivery? Successful translation of the LbL technology to clinical use requires that the film degrades in physiological conditions. We therefore utilize extracellular reducing microenvironment of redox-active membrane proteins to degrade bioreducible disulfide-containing poly(amido amine)s (PAAs) in order to release the bioactive components from the film. LbL films containing PAAs and DNA were assembled and their reductive disassembly was studied by in situ AFM, fluorescence spectroscopy, and dynamic light scattering. The PAA/DNA LbL films underwent fast bulk degradation and released microparticles. We discovered that by placing the poly(ethylenimine) (PEI) layer strategically throughout the film we were able to achieve highly controlled slow release of DNA nanoparticles. We further improved transfection by terminating the LbL film with a fibronectin top layer, incorporating hyaluronic acid inside the film, and crosslinking the film. In order to improve the inherent transfection efficiency of PAAs, we incorporated a highly transfecting monomer, 5-amino-1-pentanol (APOL), into the PAA molecular structure. The buffering capacity of the new APOL-containing PAA in the relevant intracellular pH range was higher than PEI and PAA without APOL.  The transfection efficiency of the APOL PAA was confirmed by in vitro experiments using HEK 293, NIH 3T3, and MC 3T3 cells. The highly transfecting polyplexes can be incorporated directly into the LbL film. Our study contributes new molecular designs for efficient nucleic acid delivery.