(186o) AFM Study of DNA Release Dynamics

Synthetic polymers are promising alternatives to virus-based gene delivery vectors due to safety concerns. Disulfide-containing synthetic polycations are gaining attention because of their high transfection efficiency and low cytotoxicity. In this study, bioreducible poly (amido amine)s (PAA) with different disulfide content, molecular weight, and polymer architecture were used to condense DNA into polyplex nanoparticles. The DNA release process was studied by AFM in simulated physiologic conditions with 1,4-dithiothreitol (DTT) as the reducing agent. The high transfection efficiency and low cytotoxicity are due to redox sensitivity of the disulfide bonds that break down via thiol-disulfide exchange reaction in cell nucleus. In-situ AFM shows that polyplexes share a common release pathway induced by DTT that begins with morphological change from metastable nanostructures into the more favorable toroid structure. Then toroids interact with each other by aggregation and fusion. Finally, DNA wormlike chains gradually unravel from the polyplex resulting in loose loops/tails that are held by a central compact core. On the other hand, polyplex disassembly due to polyelectrolyte exchange with albumin or heparin lowers transfection efficiency. DNA molecules released outside the nucleus are subjected to DNAse degradation. AFM was used to investigate DNA release pathway through polyelectrolyte exchange with heparin. The AFM results at the single polyplex level reveal likely connections between nanostructures and delivery and transfection efficiency of polyplexes.