Timed-release polymers as novel transfection reagents

Abstract

Development of novel agents that mediate nucleic acid delivery into cells has widespread application from basic cell biology to gene therapy. Enabling subsequent gene expression relies on the efficient delivery of DNA into the nucleus. In this work, we have developed a series of polymers designed to release DNA, via a self-catalysed hydrolysis mechanism, in a time-dependent manner to test if release of DNA near the time of cell division (which typically occurs every 24 h in mammalian cells) would result in an increase in levels of gene expression. We utilize a transient gene expression system to test our delivery potential. Our results show that the polymers are able to bind to DNA for up to 24 h and in some cases 48 h before release, thus providing sufficient time for endosomal escape and transport to the nucleus. Polymer A-C3, which bound DNA for up to 48 h, was able to achieve the highest levels of transfection efficiency. Using a GFP reporter gene, up to 95% of cells were positive for gene expression, which was much greater than the commercially available Freestyle Max. This work demonstrates a link between protection of DNA against degradation and high levels of transfection, indicating that protection of DNA is also a limiting factor in successful transfection. We postulate that due to the strong binding of the polymers to the DNA and the large size of the polyplexes, which are significantly larger than the nuclear pores, entry into the nucleus occurs through passive transport during cell division and nuclear envelope breakdown.