Hydrophobicity is a key determinant in the activity of arginine-rich cell penetrating peptides

Abstract

To deliver useful biological payloads into the cytosolic space of cells, cell-penetrating peptides have to cross biological membranes. The molecular features that control or enhance this activity remain unclear. Herein, a dimeric template of the arginine-rich HIV TAT CPP was used to establish the effect of incorporating groups and residues of various chemical structures and properties. A positive correlation is established between the relative hydrophobicity of these additional moieties and the ability of the CPP conjugates to deliver a peptidic probe into live cells. CPP conjugates with low hydrophobicity lead to no detectable delivery activity, while CPPs containing groups of increasing hydrophobicity achieve intracellular delivery at low micromolar concentrations. Notably, the chemical structures of the hydrophobic groups do not appear to play a role in overall cell penetration activity. The cell penetration activity detected is consistent with endosomal escape. Leakage assays with lipid bilayer of endosomal membrane composition also establish a positive correlation between hydrophobicity and membrane permeation. Overall, these results indicate that the presence of a relatively hydrophobic moiety, regardless of structure, is required in a CPP structure to enhance its cell penetration. It also indicates that simple modifications, including fluorophores used for cell imaging or small payloads, modulate the activity of CPPs and that a given CPP-conjugate may be unique in its membrane permeation properties.

Structural Dissection of Epsin‐1 N‐Terminal Helical Peptide: The Role of Hydrophobic Residues in Modulating Membrane Curvature

“…In addition, the relative hydrophobicities of the peptides were assessed by measuring their retention times in analytical reverse‐phase high‐performance liquid chromatography (RP‐HPLC). The order of elution has been suggested to correlate well with the hydrophobicity of the folded peptides, as molecules with a higher hydrophobicity should have a higher affinity to the HPLC stationary phase [28] . Here, the retention time of EpN18‐L was the largest, and we observed an order of EpN18‐L>EpN18‐R≥EpN18≥EpN18‐L6W>EpN18‐K>EpN18‐Q>EpN18‐N>EpN18‐V (Table 1, R t ).…”

“…The order of elution has been suggested to correlate well with the hydrophobicity of the folded peptides, as molecules with a higher hydrophobicity should have a higher affinity to the HPLC stationary phase. [28] Here, the retention time of EpN18-L was the largest, and we observed an order of EpN18…”

Elucidating the Impact of Payload Conjugation on the Cell-Penetrating Efficiency of the Endosomal Escape Peptide dfTAT: Implications for Future Designs for CPP-Based Delivery Systems

“…The fluorescence that remains trapped in the endosomes is also partially masked. Recently, to facilitate quantification, we have used AlexaFluor 488-labeled Histone H1 (AF488-H1) as a probe of endosomal escape. , When co-incubated with dfTAT-like CPPs, AF488-H1 enters cells with minimal degradation and intrinsically accumulates in nuclei. The number of cells that display nuclear staining can then be counted (above a detection threshold) and a ratio of cytoplasmic/endosomal versus nuclear signal established .…”

“…This suggests a direct or indirect contribution of ST or SC in fusion. In this context, it is worth noting that the introduction of hydrophobic residues to the dfTAT structure can amplify its membrane activity . In light of this, ST and SC might impart additional hydrophobicity to 1 , thereby augmenting interactions with lipids and promoting bilayer fusion.…”

Cell-Penetrating Peptides (CPPs) as Therapeutic and Diagnostic Agents for Cancer

“…Since then, numerous comparisons have been made differentiating the uptake potential of positively charged, short peptide chains against their long or net-neutral counterparts. Specifically, it was shown that short homopolymers of arginine (R) had higher uptake compared to other amino acids including polylysine chains [ 20 , 21 , 22 , 23 ]. Attempts to utilize and mimic this ability have been undertaken, achieving excellent results.…”