(162z) Characterization of LL37 Binding to Collagen through Collagen-Binding Domains (CBDs)

Increasing bacterial resistance to antibiotics has become a fast-emerging global crisis and developing new therapeutic strategies to prevent infection and promote chronic wound healing is paramount. Natural antimicrobials, known as antimicrobial peptides (AMPs), have a broad spectrum of targeted organisms and modest direct antimicrobial function. Specifically, LL37, the only antimicrobial peptide found in humans, demonstrates these properties and promotes wound healing. However, the major challenges in the application of AMP-based therapies are the poor understanding of their mechanism, narrow therapeutic ratio, and toxicity to mammalian cells. Recent work from our lab has demonstrated that binding AMPs to wound dressing materials using collagen binding domains (CBDs) has potential in delivering novel, non-cytotoxic, antimicrobial, and pro-healing therapies to chronic wounds. Our lab designed two chimeric versions of LL37 with C-terminal CBDs derived from collagenase (cCBD-LL37) and fibronectin (fCBD-LL37), and we found that the peptides were antimicrobial but not cytotoxic when adsorbed onto collagen. However, the CBD-mediated LL37 binding collagen mechanism needs to be further studied. The goal of this project is to study collagen deposition, to quantify CBD-LL37 binding, and to understand the mechanism of CBD-mediated LL37 binding to collagen. To realize our goal, we first studied the concentration-dependent viscoelastic deposition of collagen type I onto SiO₂ substrates by measuring frequency and dissipation in quartz crystal microbalance with dissipation (QCM-D). The frequency and dissipation are two main measurements of QCM-D which can reflect the mass adsorption and the viscoelastic properties of the deposited material, respectively. To observe the morphology of deposited collagen, we applied immunohistochemistry (IHC) to collagen coated sensor crystals. Alternatively, we developed an enzyme-linked immunosorbent assay (ELISA) to quantify the binding between cCBD-LL37 and collagen adsorbed to the plate. In the future, we will also perform atomic force microscopy (AFM) to further analyze the physical properties of deposited collagen. The impacts of these studies will help to develop a novel antimicrobial peptide delivery system and elucidate a general mechanism of CBD-mediated binding of AMPs.