Development of novel cell penetrating peptides for therapeutic protein delivery

Abstract

Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehi-cles for various therapeutics. However, there are some concerns about non-specific penetra-tion and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides on the cancer cell surface, BR2 enters cells via lipid-mediated macropinocytosis in concentration-dependent manner. Moreover, BR2 showed higher membrane translocation efficiency than a well-known CPP Tat (49-57). The protein delivery efficiency of BR2 was proved by fusion with the 28-kDa enhanced green fluorescent protein (EGFP). BR2-fused EGFP was successfully delivered into HeLa cells and distributed mainly within the cytoplasm. Furthermore, the capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced significant growth inhibition and triggered a higher degree of apoptotic cell death of K-Ras-mutated HCT116 cells than Tat-fused scFv. Furthermore, BR2-fused scFv inactivated GTP-Ras proteins insi de tumor cells mediated via neutralization by specific binding between mutated Ras and BR2-scFv fusion proteins. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.