Abstract
The increased need for macromolecular therapeutics, such as peptides, proteins and nucleotides, to reach intracellular targets necessitates more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell penetrating peptides (CPPs) can transport a range of macromolecules into cells, either through direct plasma membrane translocation or endocytosis. All known endocytic pathways involve cell-cortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Here using flow cytometry, confocal microscopy and a variety of actin inhibitors we identify how actin disorganisation in different cell types differentially influences the cellular entry of three probes: the CPP octaarginine – Alexa488 conjugate (R8-Alexa488), octaarginine conjugated Enhanced Green Fluorescent Protein (EGFP-R8), and the fluid phase probe dextran. Disrupting actin organisation in A431 skin epithelial cells dramatically increases the uptake of EGFP-R8 and dextran, and contrasts strongly to inhibitory effects observed with transferrin and R8 attached to the fluorophore Alexa488. This demonstrates that uptake of the same CPP can occur via different endocytic processes depending on the conjugated fluorescent entity. Overall this study highlights how cargo influences cell uptake of this peptide and that the actin cytoskeleton may act as a gateway or barrier to endocytosis of drug delivery vectors.
Highlights
Cell penetrating peptides (CPPs) are a group of short sequences typically containing 5–30 amino acids that have been extensively investigated as carriers for intracellular delivery of various cargos including genetic material, peptides, proteins and nanoparticles[1,2,3,4] Numerous efforts have been made to unveil the mechanisms of CPP translocation to the cytoplasm and cytosol of cells, and it is well accepted that two modes of cell entry exist: direct membrane translocation, which may be energy and temperature independent, and uptake via one or more energy dependent endocytic pathways[5,6]
Of interest are observations that some CPPs under defined experimental conditions may induce plasma membrane effects similar to that seen upon growth factor activation[19,20,21] and in line with this that they promote the concomitant uptake of dextran, a well characterised marker of fluid phase endocytosis[22,23,24]
In our previous studies using flow cytometry we showed that cytochalasin D (Cyt D) inhibited the uptake of Alexa488-CPPs R8 and Tat in two different cell models, HeLa and A43133
Summary
Cell penetrating peptides (CPPs) are a group of short sequences typically containing 5–30 amino acids that have been extensively investigated as carriers for intracellular delivery of various cargos including genetic material, peptides, proteins and nanoparticles[1,2,3,4] Numerous efforts have been made to unveil the mechanisms of CPP translocation to the cytoplasm and cytosol of cells, and it is well accepted that two modes of cell entry exist: direct membrane translocation, which may be energy and temperature independent, and uptake via one or more energy dependent endocytic pathways[5,6]. Macropincytosis is induced in response to growth factor activation such as epidermal growth factor (EGF) binding to the EGF receptor, initially leading to extensive actin-dependent ruffling on the plasma membrane. This induces a “gulping effect” manifest as an increased uptake of extracellular fluid[13,14,16]. Tools used routinely to examine the roles of the actin cytoskeleton in various cellular processes, including endocytosis and CPP entry are pharmacological/chemical inhibitors. The most notable such agent is the fungal www.nature.com/scientificreports/. Together the data indicate that actin organisation has very different influences on uptake of these octaarginine and fluid phase conjugates and that actin could be targeted to enhance cellular uptake of drug delivery vectors
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