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.

The effective delivery of therapeutics and imaging agents to a selected group of cells has been at the forefront of biomedical research. Unfortunately, the identification of the unique cell surface targets for cell selection remains a major challenge, particularly if cells within the selected group are not identical. Here we demonstrate a novel approach to cell section relying on a thermo-responsive peptide-based nanocarrier. The hybrid peptide containing cell-penetrating peptide (CPP) and collagen (COLL) domains is designed to undergo coil-to-helix transition (folding) below physiological temperature. Since only helical form undergoes effective internalization by the cells, this approach allows effective temperature-discriminate cellular uptake. The cells selected for uptake are locally cooled down thus enabling the carrier to fold and subsequently internalize. Our approach demonstrates a generic method as selected cells could differ from the adjacent cells or could belong to the same cell population. The method is fast (< 15 min) and selective; over 99.6% of cells in vitro internalized the peptide carrier at low temperatures (15°C), while less than 0.2% internalized at 37°C. In vivo results confirm the high selectivity of the method. The potential clinical applications in mixed cell differentiation carcinoma, most frequently encountered in breast and ovarian cancer, are envisioned.