Abstract
A recent theory suggests that endocytosis is involved in uptake and intracellular transport of electrotransfected plasmid DNA (pDNA). The goal of the current study was to understand if approaches used previously to improve endocytosis of gene delivery vectors could be applied to enhancing electrotransfection efficiency (eTE). Results from the study showed that photochemically induced endosomal escape, which could increase poly-L-lysine (PLL)-mediated gene delivery, decreased eTE. The decrease could not be blocked by treatment of cells with endonuclease inhibitors (aurintricarboxylic acid and zinc ion) or antioxidants (L-glutamine and ascorbic acid). Chemical treatment of cells with an endosomal trafficking inhibitor that blocks endosome progression, bafilomycin A1, resulted in a significant decrease in eTE. However, treatment of cells with lysosomotropic agents (chloroquine and ammonium chloride) had little effects on eTE. These data suggested that endosomes played important roles in protecting and intracellular trafficking of electrotransfected pDNA.
Highlights
Electrotransfection is a non-viral method for gene delivery
Since it is known that both dextran uptake and PLL-mediated gene delivery occur via endocytosis [38,39,40,41,42], the data described above indicated that the photochemical internalization (PCI) technique could significantly enhance gene delivery through facilitation of plasmid DNA (pDNA) escape from endosomes
The study showed that electrotransfection efficiency (eTE) could be significantly decreased by PCI-induced endosomal escape at different stages during intracellular transport, or inhibition of endosomal progression by a chemical inhibitor, bafilomycin A1
Summary
Electrotransfection is a non-viral method for gene delivery It facilitates delivery of naked plasmid DNA (pDNA) into cells through application of a pulsed electric field [1]. Results from numerical simulations have predicted that the lifetime of pores, which are larger than the size of pDNA, is on the order of 10 msec [4,5]. This time scale is several orders of magnitude shorter than the time frame of pDNA uptake, which has been observed to be on the order of 10 min [6,7]. A few studies investigated endocytic pathways involved in electric pulse-mediated internalization of pDNA, based on treatments of cells
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