|
Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver.
We are developing a self-assembling non-viral in vivo gene delivery vehicle based on poly-l-lysine and plasmid DNA. We have characterized poly-l-lysines of different chain lengths for DNA condensation and strength of DNA binding. Poly-l-lysine chains >20 residues bound DNA efficiently in physiological saline, while shorter chains did not. Attachment of asialoorosomucoid to PLL increased the PLL chain length required for efficient DNA binding in saline and for efficient DNA condensation. By electron microscopy, poly-l-lysine/DNA polyplexes appeared as toroids 25-50 nm in diameter or rods 40-80 nm long; conjugation of asialoorosomucoid to the polylysine component increased the size of resulting polyplexes to 50-90 nm. In water, poly-l-lysine and asialoorosomucoid-PLL polyplexes have effective diameters of 46 and 87.6 nm, respectively. Polyplexes containing only poly-l-lysine and DNA aggregated in physiological saline at all charge ratios and aggregated at neutral charge ratios in water. Attachment of asialoorosomucoid lessened, but did not eliminate, the aggregation of PLL polyplexes, and did not result in efficient delivery of polyplexes to hepatocytes. Conjugation of polyethylene glycol to poly-l-lysine sterically stabilized resulting polyplexes at neutral charge ratios by shielding the surfaces. For efficient in vivo gene delivery, polyplexes will need to be sterically stabilized to prevent aggregation and interaction with serum components.
|
