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 the 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 [proline-hydroxyproline-glycine])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.

Keywords: cell-penetrating peptides; collagen peptides; enzymatic degradation; internalization; intracellular delivery; triple helix.

Figure 1

Circular Dichroism spectra of peptides listed in table 1 at 37°C. Arrows indicate peak characteristic for triple helical conformation.

Figure 2

Circular Dichroism for triple helical peptides: thermal unfolding shows that the peak (224 nm) is eliminated in a cooperative transition which indicates the presence of a triple helix. The first derivative of molar elipticity is used to indicate the temperature of helix to coil transition (T_m).

Figure 3

Cellular uptake of peptides listed in table 1 by 3T3 Swiss mice fibroblasts. Cells were incubated with 15 μM peptide in PBS for 30 min. The images were taken by confocal microscope in bright field, with FITC filter, DAPI filter (for coloclization studies), and all images were digitally combined. Panel A: peptides containg folding and CPP domains with DAPI: V1 and V2, panel B: control peptides FL8, FL4 and V2R. Panel C: peptides containg folding and CPP domains without DAPI: V1 and V2

Figure 4

Cellurar uptake of peptides by E6 Jurkat human leukemia cells measured with flow cytomerty. Incubation time: 10 min at 37°C.

Figure 5

Peptide degradation performed in 25% human serum/DMEM at 37ºC. Supernatant of peptide after digestion was analyzed by HPLC (integraded peak area) and ploted with respect to digestion time. Error bars represent standard deviation calculated from 3 repeats.

Figure 6

Histogram of the percentage of viable cells (3T3 fibroblasts) after 30 min. measured with the Trypan Blue assay after the addition of 15 μM peptides normalized with respect to the control (no incubation). The error bars reflect standard deviation calculated from three repeats on two different plates.

Figure 7

Schematic representation of V1 and V2 peptides. The peptide is folded into triple helix within the POGn sequence (folding domain). The CPP domain R₆ or (RRG)₂ is located at C-terminus. The N-terminus is modified with FITC.

Figure 8

Cellurar uptake of peptides by E6 Jurkat human leukemia cells measured with flow cytomerty : histogram of mean fluorescence measured after Jurkat cells were incubated for 10 min. in 15 μM peptide solution.

Figure 9

Peptide degradation performed in 25% human serum/DMEM at 37ºC. Plot of pseudo first order enzymatic digestion analyzed by HPLC (integraded peak area) for peptides that undergo decomposition (T5, V2R). Error bars represent standard deviation calculated from 3 repeats.