Internalization Pathways and Intracellular Fate of Poly(Lysine) Analogues

2011 
Cationic peptides and polymers are widely used as nonviral transfection agents. A fundamental understanding of the relationship between structure and translocation mechanisms, intracellular trafficking and final targeting of these molecules is still lacking to date and mostly based on empirical observations.In this contribution we systematically investigate the role of the structural parameters of the polycations on their endocytic pathway and intracellular fate in vitro. To cover all the structural parameters, a library of the polycations based on L-lysine monomer unit was constructed, covering a broad range of molecular weights and degree of branching. A new class of cationic polymers, prepared by polycondensation of L-lysine, was developed to explore the impact of very high molecular weights. The uptake pathways of the structural analogues were probed by specific inhibitors of endocytosis. The internalization kinetics was measured at a macroscopic scale by fluorescence intensity methods to identify the key structural parameters stimulating the translocation into the cytosol. The microscopic distribution and the trafficking kinetics were studied by two distinct methods: subcellular fractionation and confocal scanning laser microscopy with subsequent high throughput image analysis. The final localization in CHO cells was followed by indirect immunocytochemistry. In parallel a novel technique based on the sensitivity of the fluorescent probes to local microenvironment and assessed by triplet lifetime imaging is developed for this purpose.We found that architecture and molecular weight of polycation have profound effects on intracellular trafficking and resulting localization. Hyperbranched polylysine with extremely high molecular weight posses a unique capacity in comparison to the linear and dendritic analogues to destabilize endosomes and to escape from lysosomes, which is generally the limiting step in gene delivery.
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