Quantification and Characterization of Collagen-Binding Domain-Mediated LL37 Binding with Type I Collagen for Chronic Wound Healing

Wei, Ziqi

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Quantification and Characterization of Collagen-Binding Domain-Mediated LL37 Binding with Type I Collagen for Chronic Wound Healing

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The escalating challenge posed by antibiotic-resistant bacteria in chronic wound infections necessitates the development of alternative therapeutic strategies. Antimicrobial peptides (AMPs), such as the human-derived LL37, have emerged as promising broad-spectrum candidates. LL37 not only exhibits antimicrobial effects but also possesses wound healing properties. However, the clinical application of LL37 has been hampered by its systemic cytotoxicity and limited in vivo stability. The strategy of anchoring AMPs to surfaces has been suggested as an effective way to deliver bioactive AMPs, potentially reducing cytotoxicity and enhancing stability. As the fundamental structural protein in human tissues, collagen provides an ideal scaffold for wound repair. In our previous work, we modified the antimicrobial peptide LL37 by incorporating a collagen-binding domain (cCBD) to serve as an anchoring unit for collagen-based wound dressings. Our previous results indicated that the incorporation of cCBD maintained the antimicrobial efficacy of LL37 against wound-associated pathogens, ensured stability over a 14-day release period, and did not exhibit cytotoxicity to human fibroblasts. However, there remains a lack of direct quantification for the binding of unmodified LL37 and cCBD-LL37 to collagen, and the binding mechanism of cCBD-LL37 to collagen remains to be elucidated. To quantify the binding of LL37 and cCBD-LL37 to collagen, our study utilized a suite of analytical techniques. Quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to quantitatively assess the adsorption kinetics of both LL37 and cCBD-LL37 on type I collagen. This approach was complemented by immunohistochemistry (IHC) and atomic force microscopy (AFM), which facilitated the characterization and confirmation of type I collagen layer formation on QCM-D sensors. Our protocol enabled concentration-dependent measurements of collagen deposition via QCM-D. Acknowledging hydrophobicity’s influence on collagen adsorption, we compared the deposition of collagen on hydrophilic silicon dioxide (SiO2) coated sensors with that on hydrophobic polystyrene (PS)-coated sensors. The results indicated a preferential adsorption to hydrophobic surfaces, implying their suitability for forming collagen layers. Upon establishing the collagen layer, we proceeded to quantify peptide-collagen interactions using QCM-D. While cCBD-LL37 and LL37 exhibited comparable initial binding affinities to collagen, the cCBD-LL37 displayed greater retention after phosphate-buffered saline (PBS) washes, indicating the cCBD’s efficacy as an anchoring unit for durable collagen attachment. According to the information provided by QCM-D and Voigt-Kelvin viscoelastic modeling, we proposed hypothesized models of LL37 and cCBD-LL37 binding to collagen. Our model suggested that the LL37 peptides bound to collagen non-specifically and may have also exhibited LL37 peptide-peptide interactions on the surface. On the other hand, the cCBD-LL37 bound to collagen non-specifically or had peptide-peptide interactions, in addition to cCBD-LL37 peptide binding through the collagenase binding domain end. In progressing with our quantification of LL37 and cCBD-LL37 binding to collagen, we sought to further understand the mechanism of their interaction with collagen. We hypothesized that the positively-charged LL37 may bind non-specifically to the negatively-charged groups on the collagen surface. As cCBD-LL37 retains the same positive charge with LL37, non-specific interactions are likely one of the components during binding, but the cCBD may also aid in the formation of high affinity binding. To confirm the role of the electrostatic interactions in LL37 and cCBD-LL37 binding to collagen, the binding of LL37 and cCBD-LL37 under varying ionic strength and pH was measured. Analysis of electrostatic interactions indicated that both LL37 and cCBD-LL37 interacted with collagen primarily through initial long-rang electrostatic forces, which then potentially transitioned into more specific, close-range interactions, possibly of a hydrophobic nature. Circular dichroism (CD) spectroscopy was employed to investigate the structural stability of the peptides, demonstrating that the cCBD-LL37 maintained a more defined conformation under various ionic strengths and pH levels, compared to unmodified LL37. This enhanced structural integrity of cCBD-LL37 suggests a potential for greater antimicrobial activity and resilience in physiological environments. Our studies also explored into the release dynamics of both peptides from the collagen matrices. We observed that the release was influenced by the ionic strength of the surrounding medium. Indicating an electrostatically driven mechanism. Notably, the presence of cCBD augmented the retention of LL37 on the collagen scaffold, an attribute that could prove advantageous in creating more effective and lasting antimicrobial surfaces for clinical use. This study provides an effective strategy for the quantification of AMP and cCBD-AMP binding to collagen. Furthermore, our findings contribute to the broader understanding of cCBD-AMP interactions with collagen-based scaffolds, providing crucial insights that will significantly inform the development and refinement of cCBD-AMP delivery systems for future biomaterial implementation.

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