| We have developed a novel gene delivery scaffold based on DNA plasmid condensation with colloidal gold/polyethylenimine conjugates. This scaffold system was designed to enable systematic study of the relationships between DNA complex physical properties and transfection efficiency. Using an enhanced green fluorescent protein (EGFP)-coding reporter plasmid and Chinese hamster ovary (CHO) and human hepatoma (HepG2) cells, we have measured the transfection efficiencies of our complexes using flow cytometry and their cytotoxicities using the trypan blue assay. We have also assayed complex particle morphologies using atomic force microscopy, dynamic light scattering, and a novel plasmon absorbance peak position analysis. In our initial proof-of-principle assay, we found that we achieved high rates of transfection relative to the commonly used polycationic condensation agents calcium phosphate and LipofectAMINE™, with comparably low cytotoxicities. In particular, we had the best success with complexes with a charge ratio of ∼8 and diameters of a few hundred nanometers.; We then studied the effects of PEI properties and plasma membrane targeting ligand incorporation on controlling particle morphologies and thereby transfection efficiencies. PEI molecular weight and branching ratio were found to dramatically affect both the morphologies and the transfection efficiencies of complexes formed; targeting ligand incorporation was not found to significantly affect morphology or to enhance performance. Finally, we investigated plasma membrane transport and vector-DNA unpackaging as potential intracellular gene delivery process bottlenecks. We used fluorophore-conjugated colloidal gold to study overall uptake across the plasma membrane as a function of charge ratio and particle size, and found that the plasma membrane excludes complexes larger than ∼450 nm. With a computational gene delivery model, we found that the timing of DNA release is crucial, and in particular, that stimulating nuclear release is a key to improving expression efficiency. |
