LL37 and collagen-binding domain-mediated LL37 binding with type I collagen: Quantification via QCM-D

Abstract

Antimicrobial peptide (AMP)-loaded biomaterials may represent a viable alternative for stimulating wound healing while protecting against infections. Previously, to develop an efficient delivery system for the cathelicidin antimicrobial peptide, LL37, our lab modified LL37 with a collagen-binding domain derived from collagenase (cCBD) as an anchoring unit to collagen-based wound dressings. However, a direct quantification of unmodified LL37 and cCBD-LL37 binding with collagen has not been performed. In this study, we used quartz crystal microbalance with dissipation monitoring (QCM-D), immunohistochemistry (IHC), and atomic force microscopy (AFM) to establish and characterize an adsorbed layer of type I collagen on the QCM-D sensor and quantify peptide-collagen binding. A collagen deposition protocol was successfully established by measuring concentration-dependent deposition of collagen in QCM-D, and collagen self-assembly was observed by IHC and AFM. Hydrophobicity is known to affect the behavior of collagen adsorption. Therefore, we compared the deposition of collagen on hydrophilic SiO2-coated sensors vs. hydrophobic polystyrene (PS)-coated sensors via QCM-D, and found that the hydrophobic surface yielded more collagen adsorption, which suggested that hydrophobic surfaces are preferable for collagen layer establishment. There was no significant difference between LL37 and cCBD-LL37 binding with collagen, but the cCBD-LL37 showed better retention on the collagen after washing with PBS, indicating that there is an advantage to using cCBD as an anchoring unit to collagen. Collectively, these results provide important information on cCBD-mediated AMP-binding mechanisms and establish an effective method for quantifying peptide-collagen binding.