Peptide internalization enabled by folding: triple helical cell-penetrating peptides

Abstract

Cell-Penetrating Peptides (CPPs) are known as efficient transporters of molecular cargo across cellular membranes. Their properties make them ideal candidates for in vivo applications. However, challenges in development of effective CPPs still exist: CPPs are often fast degraded by proteases and large concentration of CPPs required for cargo transporting can cause cytotoxicity. It was previously shown that restricting peptide flexibility can improve peptide stability against enzymatic degradation and limiting length of CPP peptide can lower cytotoxic effects. Here we present peptides (30-mers) that efficiently penetrate cellular membranes by combining very short CPP sequences and collagen-like folding domains. The CPP domains are hexa-arginine (R6) or arginine/glycine (RRGRRG). Folding is achieved through multiple proline-hydroxyproline-glycine (POG)n repeats that form a collagen-like triple helical conformation. The folded peptides with CPP domains are efficiently internalized, show stability against enzymatic degradation in human serum, and have minimal toxicity. Peptides lacking correct folding (random coil) or CPP domains are unable to cross cellular membranes. These features make triple helical cell penetrating peptides promising candidates for efficient transporters of molecular cargo across cellular membranes.

ECM turnover-stimulated gene delivery through collagen-mimetic peptide-plasmid integration in collagen

“…CMP-modification has been demonstrated to not only enhance vector availability/stability in the delivery site, but also to provide a ligand promoting cellular uptake through endocytic pathways linked to high efficiency expression. Additional studies have demonstrated that the CMPs’ unique triple helical structure is integral in achieving enhanced cellular binding and cell penetration [78]. For instance, CMPs were successfully used to enhance liposome targeting of melanoma cells through specific ligand/receptor interactions, whereas no binding to non- targeted cells was recorded [79].…”

Thermoresponsive Collagen/Cell Penetrating Hybrid Peptide as Nanocarrier in Targeting-Free Cell Selection and Uptake

“…29 Lowering the temperature from physiological value affects not only peptide folding, but also the effectiveness of nanocarrier cellular uptake due to changes induced in the cell membrane.…”

“…For all conditions, the fluorescence signal is observed within cell cytoplasm, as previously shown for tagged peptides that carry an RRGRRG sequence in the CPP domain. 29 If the desired location of peptide carrier is nucleus, the CPP domain can be altered to R 6 , what causes accumulation of peptide in the nuclear bodies that contain high concentration of nucleic acids. 29 …”

“…29 If the desired location of peptide carrier is nucleus, the CPP domain can be altered to R 6 , what causes accumulation of peptide in the nuclear bodies that contain high concentration of nucleic acids. 29 …”

Cellular Uptake of IgG Using Collagen-Like Cell-Penetrating Peptides

“…10,11) Herein, we report the strategy for conjugating the CPP to IgG, properties of the complexes, and its delivery and intracellular localization.…”