siRNA depletion of endocytic proteins and pathways for analysing the cellular uptake of cell penetrating peptides as vectors for drug delivery

• Main Author: Alsoraj, Monerah
• Published: Cardiff University 2011
• Subjects: 615.1
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.585146

Cell-penetrating peptides (CPPs) have the potential to deliver a host of macromolecular therapeutics into cells including peptides, proteins, and nucleotides. The mechanism by which they are internalised has been hotly-disputed but is important if improvements are to be made in their delivering capacities. Endocytosis is thought to be of significant importance but identifying the exact uptake mechanism has been difficult due predominantly to a lack of specific tools. Multiple pathways have been reported to contribute to uptake, including macropinocytosis and those regulated by clathrin and cavaeolin-1. The aim of this thesis was to utilise siRNA-depletion to develop cell models with defects in specific endocytic proteins and pathways that could then be utilised to study the uptake of drug delivery vectors such as CPPs. Targeted pathways were those regulated by clathrin heavy chain, dynamin-2, caveolin-1, flotillin-1 and P21-activated kinase (PAK-1). Significant variation between cell lines emerged in the expression of these proteins and the ease with which they could be depleted. Single siRNA sequences were, however, discovered that effectively depleted these proteins and using a variety of endocytic probes the effects of depletion could be determined. Eventually, model cell lines were generated that were measurably defective in at least one of the five different endocytic pathways and these were tested to determine routes utilised by two well characterised CPPs, HIV-Tat and octaarginine. Only cells depleted of pak-1 protein and thus macropinocytosis were defective in CPP uptake. Further analysis revealed defective actin organisation in these cells that could have caused the effects and support data presented here and elsewhere on actin disruption with cytochalasin D. With comparative studies using pharmacological inhibitors of endocytic pathways these methods provide new tools to study drug delivery systems as shown here for CPPs and also for polyplexes through a collaboration with the University of Ghent, Belgium.