Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides

Abstract

Cell-penetrating peptides (CPPs) are able to traverse cellular membranes and deliver macromolecular cargo. Uptake occurs through both endocytotic and nonendocytotic pathways, but the molecular requirements for efficient internalization are not fully understood. Here we investigate how the presence of tryptophans and their position within an oligoarginine influence uptake mechanism and efficiency. Flow cytometry and confocal fluorescence imaging are used to estimate uptake efficiency, intracellular distribution and toxicity in Chinese hamster ovarian cells. Further, membrane leakage and lipid membrane affinity are investigated. The peptides contain eight arginine residues and one to four tryptophans, the tryptophans positioned either at the N-terminus, in the middle, or evenly distributed along the amino acid sequence. Our data show that the intracellular distribution varies among peptides with different tryptophan content and backbone spacing. Uptake efficiency is higher for the peptides with four tryptophans in the middle, or evenly distributed along the peptide sequence, than for the peptide with four tryptophans at the N-terminus. All peptides display low cytotoxicity except for the one with four tryptophans at the N-terminus, which was moderately toxic. This finding is consistent with their inability to induce efficient leakage of dye from lipid vesicles. All peptides have comparable affinities for lipid vesicles, showing that lipid binding is not a decisive parameter for uptake. Our results indicate that tryptophan content and backbone spacing can affect both the CPP uptake efficiency and the CPP uptake mechanism. The low cytotoxicity of these peptides and the possibilities of tuning their uptake mechanism are interesting from a therapeutic point of view.

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties

“…Consistent with our proposed endocytic neuroprotective mechanism, we have demonstrated that poly-lysine (K10) is only weakly neuroprotective in a cortical neuronal glutamic acid excitotoxicity model (Meloni et al, 2015). It is also likely that other amino acids can influence the endocytic properties of cationic peptides in both a positive and negative manner as has been demonstrated for tryptophan (W; Rydberg et al, 2012;Bechara et al, 2013) and alanine (A; Yang et al, 2014), respectively. Indeed, we have now confirmed that tryptophan and alanine amino acids within arginine-rich peptides respectively increase and decrease neuroprotective efficacy in a glutamic acid excitotoxicity model (Fig.…”

“…Additionally, there is evidence that tryptophan residues within basic peptides can also promote proteoglycan binding and endocytosis (Bechara et al, 2013;Rydberg et al, 2012), while alanine resides have been shown to impede peptide-proteoglycan binding . Interestingly, the replacement of 1 to 3 arginine residues in poly-arginine-6 with the equivalent number of tryptophan residues increases the ability of the peptide to block NMDA receptor activity in amphibian oocytes (Ferrer-Montiel et al, 1988).…”

Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9

“…It has been shown that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts electrostatically with sugars [41]. It is noteworthy that the free uptake studies of CPPs indicate that tryptophan content and backbone spacing within basic peptide also affect uptake efficiency [42], [43].…”

Noncovalently Associated cell-penetrating Peptides for Gene Delivery Applications

“…Cationic CPPs interact with the negatively charged phosphate backbone of genetic material (e.g., pDNA and siRNA) through electrostatic interaction [62,66]. This process can result in genetic material condensation and protection from nuclease enzyme digestion [67].…”

“…Polyarginine peptides have a strong tendency to electrostatically interact with both siRNA [85,86] and with DNA [66]. One study indicated that as with PLL and other polycations, the length of polyarginine may affect transfection efficiency and cytotoxicity.…”