Intracellular Trafficking Mechanism and Cytosolic Protein Interactions of a Non Viral Gene Delivery Vector: Studies Based on Transferrin Conjugated Pullulan-PEI
Chandra P. Sharma.
Compared to viral vectors the intracellular barriers faced by non-viral vectors are high, which lead to lesser transfection efficiency. The various limiting steps include endocytosis, endosomal escape, intracellular stability and mobility, unpacking and nuclear localization. The understanding of these mechanisms for overcoming these barriers is essential for developing gene delivery vectors which can really be used for appropriate clinical/medical applications. Transferrin conjugated pullulan-PEI (PPETf) was developed for neuronal cell targeted gene delivery which was evaluated for its transfection efficiency. The stability of the polymer-DNA nanoplexes in the presence of plasma and cytosolic proteins was evaluated by agarose gel electrophoresis. Time dependent uptake of the nanoplexes by using labeled polymer and YOYO tagged plasmid was employed to understand the uptake mechanism and the sequence of unpacking of DNA from the vector. The uptake pathway, endosomal escape, trafficking etc. are evaluated in the presence of endocytic and microtubilin inhibitors to understand the mechanism of nanoplexes trafficking into and within the cell.
Keywords: Gene delivery, hemocompatibility, intracellular trafficking, polyethyleneimine, pullulan, Cytosolic Protein Interactions, Transferrin
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