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

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Cell-penetrating peptides (CPPs) facilitate molecular cargo transport across cellular membranes, making them valuable for in vivo applications.
• Existing CPPs face challenges including rapid protease degradation and cytotoxicity at high concentrations.
• Strategies to enhance CPP stability and reduce toxicity involve restricting peptide flexibility and limiting length.

Purpose of the Study:

• To design and characterize novel 30-mer peptides combining short CPP sequences with collagen-like folding domains.
• To evaluate the cellular internalization, stability, and toxicity of these engineered peptides.
• To assess the role of collagen-like folding in CPP efficiency and functionality.

Main Methods:

• Peptides were designed with specific CPP domains (hexa-arginine or arginine/glycine) and collagen-like folding domains (proline-hydroxyproline-glycine repeats).
• Cellular uptake was assessed using techniques to measure membrane penetration.
• Stability against enzymatic degradation was tested in human serum.
• Cytotoxicity was evaluated at various peptide concentrations.

Main Results:

• The designed triple helical peptides efficiently penetrated cellular membranes.
• Folded peptides demonstrated significant stability against enzymatic degradation in human serum.
• These peptides exhibited minimal toxicity compared to conventional CPPs.
• Non-folded peptides or those lacking CPP domains failed to cross cellular membranes.

Conclusions:

• Triple helical cell-penetrating peptides offer enhanced stability and reduced toxicity.
• The collagen-like triple helical structure is crucial for efficient cellular internalization and functionality.
• These engineered CPPs represent a promising advancement for molecular cargo delivery in biological systems.