Enhanced Delivery of Biomolecules into Caco2 Cells Based on the Cell-Penetrating Ability of Keratin Peptides.

Abstract

Keratin, as a promising bioresource, possesses significant potential for diverse biological applications due to its favorable biocompatibility, low toxicity, biodegradability, and cell adhesion ability. However, there are few studies on the cell-penetrating ability of keratin peptides (KEPs) for biomolecule delivery. Therefore, this study explored the cell-penetrating ability of KEPs with different molecular weights (Mw) on Caco2 cells using fluorescein-labeled insulin (FITC-INS) as the target intracellular biomolecule. The potential cell-penetrating mechanism was elaborated by combining cellular investigation with the physicochemical characterization of KEPs. The result shows that the KEPs <3 kDa (KEP1) exhibited the highest cell-penetrating ability at 2 mg/mL, allowing efficient delivery of FITC-INS into Caco2 cells without covalent bonding. The cellular uptake mechanism was energy-dependent, mainly involving macropinocytosis. The further fractionation of KEP1 reveals that the most effective components consisted of 8-19 amino acids, including specific hydrophobic peptides (e.g., RVVIEPSPVVV and IIIQPSPVVV), PPII amphipathic peptides (e.g., PPPVVVTFP and FIQPPPVVV), and Cys-rich peptides (e.g., LCAPTPCGPTPL and CLPCRPCGPTPL). Additionally, analysis of the secondary and tertiary structure and amino acid composition illustrated that KEP1 exhibited rich hydrophobic residues and disulfide bonds, which probably contributed to its cell-penetrating ability, as opposed to its small particle size and electrostatic interactions. This study reveals the cell-penetrating ability of KEPs, thus highlighting their potential as biomaterials for noncovalently delivering biomolecules.