SiRNA versus pharmacological inhibition of endocytic pathways for studying cellular uptake of cell penetrating peptides and other drug delivery vectors

Abstract

Cell-penetrating peptides (CPPs) have the potential to deliver numerous therapeutic macromolecules into cells including peptides, proteins, and nucleic acids. Under defined conditions endocytosis is thought to be of significant importance for CPP entry but identifying the exact uptake mechanism and pathway(s) involved has been difficult. Multiple pathways have been reported to contribute to uptake, including macropinocytosis and those regulated by clathrin and cavaeolin-1. This project aims enhance the use of cell penetrating peptides as drug delivery vectors by developing new technologies to study their mechanisms of uptake. Traditionally studies investigating the uptake of these molecules, and other drug delivery vectors, have been performed using chemical inhibitors but these are often toxic and lack specificity [1]. We have developed siRNA-based assays to silence endocytic proteins that have previously been shown to regulate distinct endocytic pathways. The effect of depleting these proteins was then assessed to investigate their roles in mediating the uptake of well characterised endocytic probes and CPPs. Two cell lines were predominantly used, HeLa (cervical cancer epithelial) and A431 (human epithelial carcinoma). Endocytic proteins clathrin heavy chain, flotillin-1, dynamin II, caveolin-1 and P21-activated kinase (PAK-1) were depleted using single siRNA sequences; siRNA against GFP was used as a control. In siRNA treated cells the uptake of fluorescent endocytic markers including; Alexa488-transferrin (clathrin mediated endocytosis), 40 kDa FITC Dextran (fluid phase uptake and macropinocytosis), FITC conjugated anti-CD59 antibody (flotillin-1 dependent uptake) and the uptake of Alexa488 CPPs (RRRRRRRRGC-Alexa488-R8, and GRKKRRQRRRPPQ-Alexa488-HIV-TAT) were measured by flow cytometry. Protein depletion was assessed from protein lysates using SDS PAGE and Western blotting. Overall, the data shows that siRNA transfection method could effectively reduce expression of clathrin heavy chain, caveolin-1 and flotillin-1 from HeLa cells and this then allowed for us to study effects on endocytosis of various probes. PAK-1 has been shown to regulate macropinocytosis and we show that the induction of macropinocytosis and PAK-1 expression are highly cell line dependent. Paralleled with this was our findings that cationic CPPs induce an increase in fluid phase uptake of dextran and the extent of this was cell line dependent. Comparative analysis of these experiments with those performed using pharmacological inhibitors, allowed us to determine the usefulness of this approach for drug delivery research.