Enhanced\nDelivery of Biomolecules into Caco2 Cells\nBased on the Cell-Penetrating Ability of Keratin Peptides

Keratin, as a promising bioresource,\npossesses significant potential\nfor diverse biological applications due to its favorable biocompatibility,\nlow toxicity, biodegradability, and cell adhesion ability. However,\nthere are few studies on the cell-penetrating ability of keratin peptides\n(KEPs) for biomolecule delivery. Therefore, this study explored the\ncell-penetrating ability of KEPs with different molecular weights\n(Mw) on Caco2 cells using fluorescein-labeled insulin (FITC-INS) as\nthe target intracellular biomolecule. The potential cell-penetrating\nmechanism was elaborated by combining cellular investigation with\nthe physicochemical characterization of KEPs. The result shows that\nthe KEPs <3 kDa (KEP1) exhibited the highest cell-penetrating ability\nat 2 mg/mL, allowing efficient delivery of FITC-INS into Caco2 cells\nwithout covalent bonding. The cellular uptake mechanism was energy-dependent,\nmainly involving macropinocytosis. The further fractionation of KEP1\nreveals that the most effective components consisted of 8–19\namino acids, including specific hydrophobic peptides (e.g., RVVIEPSPVVV\nand IIIQPSPVVV), PPII amphipathic peptides (e.g., PPPVVVTFP and FIQPPPVVV),\nand Cys-rich peptides (e.g., LCAPTPCGPTPL and CLPCRPCGPTPL). Additionally,\nanalysis of the secondary and tertiary structure and amino acid composition\nillustrated that KEP1 exhibited rich hydrophobic residues and disulfide\nbonds, which probably contributed to its cell-penetrating ability,\nas opposed to its small particle size and electrostatic interactions.\nThis study reveals the cell-penetrating ability of KEPs, thus highlighting\ntheir potential as biomaterials for noncovalently delivering biomolecules.