Microscopy Research and Technique 2010-09-01

Structural and functional consequences of poly(ethylene glycol) inclusion on DNA condensation for gene delivery.

Peter G Millili, Joshua A Selekman, Kory M Blocker, David A Johnson, Ulhas P Naik, Millicent O Sullivan

Index: Microsc. Res. Tech. 73 , 866-877, (2010)

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Abstract

Polycationic polymers have been used to condense therapeutic DNA into submicron particles, offering protection from shear-induced or enzymatic degradation. However, the spontaneous nature of this self-assembly process gives rise to the formation of multimolecular aggregates, resulting in significant polyplex heterogeneity. Additionally, cytotoxicity issues and serum instability have limited the in vivo efficacy of such systems. One way these issues can be addressed is through the inclusion of poly(ethylene glycol) (PEG). PEG has known steric effects that inhibit polyplex self-aggregation. A variety of PEGylated gene delivery formulations have been previously pursued in an effort to take advantage of this material's benefits. Because of such interest, our aim was to further explore the consequences of PEG inclusion on the structure and activity of gene delivery vehicle formulations. We explored the complexation of plasmid DNA with varying ratios of a PEGylated trilysine peptide (PEG-K(3)) and 25-kDa polyethylenimine (PEI). Atomic force and scanning electron microscopy were utilized to assess the polyplex size and shape and revealed that a critical threshold of PEG was necessary to promote the formation of homogeneous polyplexes. Flow cytometry and fluorescence microscopy analyses suggested that the presence of PEG inhibited transfection efficiency as a consequence of changes in intracellular trafficking and promoted an increased reliance on energy-independent mechanisms of cellular uptake. These studies provide new information on the role of PEG in delivery vehicle design and lay the foundation for future work aimed at elucidating the details of the intracellular transport of PEGylated polyplexes.