Pyridylhydrazone-based PEGylation for pH-reversible lipopolyplex shielding

PEGylation, which is reversed after the therapeutic agent reaches the target cell, presents an attractive feature for drug, protein or nucleic acid delivery. For bioreversible surface shielding of DNA lipopolyplexes, the bifunctional, endosomal pH-cleavable reagent OPSS-PEG-HZN-Chol [ω-2-pyridyldithio poly (ethylene) glycol α-(butyraldehyde) (N(1)-cholesteryloxycarbonyl-1, 2-diaminoethane amidocarboxy) pyridyl hydrazone] was synthesized. Both OPSS-PEG-HZN-Chol and its corresponding pH stable analog OPSS-PEG-Chol form micelles which remain stable and shielded at pH 7.4 and 37 °C as demonstrated by size exclusion chromatography. At endosomal pH 5.4, OPSS-PEG-HZN-Chol micelles are destroyed within 30 min at 37 °C, while OPSS-PEG-Chol micelles remain stable. Lipopolyplexes composed of DNA condensed with polyethylenimine (PEI), phospholipids including dioleoyl phosphatidylethanolamine (DOPE) and pH-labile OPSS-PEG-HZN-Chol were prepared by the ethanol injection technique. The optimal formulation yielded particles of 160 nm size and a zeta potential of +7 mV. At physiological neutral conditions pyridylhydrazone-based PEGylated lipopolyplexes were as stable as standard PEGylated lipopolyplexes. At pH 5.4, an increase in size was observed only for pH-reversible HZN lipopolyplexes. Transfection with plasmids encoding for luciferase or EGFP resulted in an upto 40-fold enhancement in gene expression with the reversibly PEGylated lipopolyplexes compared to the stably PEGylated ones. Incorporation of a synthetic peptidic ligand for transferrin receptor targeting further improved transfection efficiency in transferrin overexpressing tumor cells. Cellular association and uptake studies by flow cytometry and confocal laser scanning microscopy with an Alexa-labeled PEG shield revealed intracellular deshielding in case of the HZN lipopolyplexes.