(16g) A Modified Polyethylenimine for Light-Triggered DNA Release

Viruses protect their genome and after endosome escape can disassemble to release their genetic contents. These two functions of protection and triggered-release are competing functionalities that have been difficult to engineer into nonviral transfection reagents. In order to mimic viral condensation of DNA, packaging and protection, and triggered release, we have designed a novel photolabile monomer, P25M, for plasmid DNA (pDNA) delivery. P25M was synthesized via a 6-step reaction scheme and consists of three functional domains: 1) a cationic domain of 25 kDa polyethylenimine (PEI) to electrostatically condense the pDNA; 2) a polymerizable domain (a methacrylamide moiety) for crosslinking the pDNA within the polyplex; and 3) a photolabile nitrobenzyl domain for triggered release by 365 nm light (Fig. 1). The formation of P25M was verified through ¹H NMR. In addition to establishing the chemical structure, the functionality of each domain was verified. HPLC studies showed that upon exposure to 365 nm light (time = 4 min, distance = 20 cm), P25M (retention time = 0.99 min) was cleaved into two products (retention time = 0.34 and 1.85 min). Similar results were also found after P25M had been polymerized with VA-044, an azo-initiator. Agarose gels confirmed that P25M, essentially a modified PEI, condensed pDNA when the N/P ratio was ³ 4. Dynamic light scattering confirmed that these polyplexes were suitable for in vitro transfection (polyplex diameter < 500 nm) both before and after polymerization with VA-044 and overnight incubation in 150 mM NaCl. Finally, heparin challenge with agarose gels confirmed that once polymerized, the polyplexes retained the pDNA. This was true for three different polymerizing agents, VA-044, ammonium persulfate, and Grubb's 2nd generation catalyst. Unpolymerized polyplexes were unable to resist pDNA release in the heparin challenge. Preliminary transfection experiments showed that the polyplexes were able to transfect two different cell lines (BAEC and CHO-K1). The goal of this work is to create vehicles for spatial, temporal, and metered delivery of DNA.