DNA delivery in vitro via surface release from multilayer assemblies with poly(glycoamidoamine)s

Abstract

Localized controlled release of nucleic acid therapeutics could be an effective way to reduce the extracellular barriers associated with systemic delivery. Herein, we have used the layer-by-layer film deposition approach to construct ultrathin multilayer assemblies for in vitro controlled release of plasmid DNA (pDNA). Layer-by-layer assemblies containing alternate layers of cationic poly(l-tartaramidopentaethylenetetramine) (T4), and anionic pDNA were fabricated. The film thickness and the absorbance at 260 nm for different T4/pDNA multilayer assemblies were characterized by ellipsometry and UV-vis spectrophotometry, respectively. The results indicated an increased loading capacity of pDNA with respect to an increase in the number of T4/pDNA bilayers deposited. For the controlled-release studies we incubated the bilayers coated on quartz slides in phosphate-buffered saline (PBS) at 37 degrees C and collected the media at different incubation time points. The collected PBS samples were characterized for pDNA release by complexing solutions containing the released pDNA with Lipofectamine 2000 and following cellular pDNA uptake via flow cytometry and GFP gene expression assays with HeLa cells. The study showed that the multilayer films started to release pDNA after 1 day of incubation and increased after 7 days of incubation. Assays monitoring green fluorescent protein (GFP) expression in HeLa cells indicated that about 20% of the cells were positive for GFP expression at all sample time points up to 11 days. Although an increase in cells positive for Cy5-pDNA was found as the incubation time increased, the number of cells positive for GFP expression remained constant over the same time frame.