(210g) Study of Collagen Tethered Antimicrobial Peptide Binding and Antimicrobial Activity

Authors: 
Wei, Z., Worcester Polytechnic Institute
Lozeau, L. D., Worcester Polytechnic Institute
Rolle, M. W., Worcester Polytechnic Institute
Camesano, T. A., Worcester Polytechnic Institute
Recent work from our lab has demonstrated that collagen tethering of antimicrobial peptides (AMPs) using collagen binding domains (CBDs) has potential in delivering novel, non-cytotoxic, antimicrobial, and pro-healing therapies to chronic wounds and will not promote bacterial resistance. Our lab designed two chimeric versions of human-derived 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, optimizing the formation of the deposited collagen films, in terms of the appropriate number of binding sites and optimal loading CBD-LL37 concentration for treating different types of bacteria needs to be further studied. The goal of this project is to study collagen film deposition, to quantify CBD-LL37 binding concentration, retention over time and adsorbed surface density onto collagen type I, and to observe antimicrobial activity on E. coli and Methicillin-resistant S. aureus in quartz crystal microbalance with dissipation monitoring (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 film, respectively. To realize our goal, we first studied the concentration-dependent viscoelastic deposition of collagen type I films onto SiO2 or Au substrates by measuring frequency and dissipation in QCM-D. We then studied time- and concentration-dependent interactions of CBD-LL37 with these films and quantified the theoretical number of binding sites between CBD-LL37 and collagen. To study the antimicrobial activity, we introduced E. coli or Methicillin-resistant S. aureus into QCM-D over AMP functionalized collagen and monitored real-time changes in frequency and dissipation. We also performed ex situ antimicrobial assays which consisted of sample preparation, plate culture, and colony counting steps to determine the antimicrobial activity of collagen tethered CBD-LL37. Collagen films were then visualized ex situ using SEM. The results of these studies will help to optimize concentration-dependent tethering of CBD-LL37 onto collagen-based substrates and provide insight on the best approach to using collagen-tethered AMPs to efficiently prevent and treat bacterial wound infections.
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