Application of Antimicrobial Peptides on the development of antibiotic-free wound care dressings

Chronic wounds infections (CWI) are a rising global problem, being responsible for considerable morbidity and decreased life quality. Resulting from microorganism colonization and biofilm establishment, CWI occur in patients with underlying pathologies that hinder wound healing. Current therapies have proven to be insufficient, with the rise and dissemination of antibiotic-resistant microorganisms. Herein, a new antibiotic-free chitosan-based dressing material with antimicrobial peptides (AMPs) was developed to mitigate CWI. AMPs are small peptides with broad spectrum of activity and low propensity for inducing resistance. Unfortunately, AMPs are easily degraded in vivo and can self-aggregate. Hence, covalent immobilization may be an efficient strategy to improve bioavailability. In this context, Dhvar5 (LLLFLLKKRKKRKY) was covalently immobilized onto chitosan through the highly efficient photoinitiated thiol-norbornene “click” chemistry. Dhvar5 is a synthetic AMP derived from histatin-5 with promising antimicrobial activity. Chitosan is a biopolymer amenable to functionalization with antimicrobial, hemostatic and prohealing properties. To allow the chemistry, chitosan was functionalized with a norbornene group (Nor- Chit). Dhvar5 modified with a spacer and a cysteine either at N- or C-terminal, was used to chemoselectively immobilize the AMP. The immobilization was confirmed by Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy. Proteolytic stability of the immobilized AMP was investigated with trypsin by a competitive assay with N- -benzoylarginine ethylester. However, the results were inconclusive due to a pH mismatch. NorChit-Dhvar5 coatings were produced by spin-coating, to assess its potential in attributing antimicrobial properties to wound dressings. NorChit-Dhvar5 coatings were characterized by ellipsometry and water contact angle, and its antimicrobial efficacy was evaluated against Staphylococcus epidermidis using a LIVE/DEAD Bacterial Viability Kit. Both NorChit-Dhvar5 (Nt and Ct) coatings revealed improved antimicrobial activity in comparison to chitosan, with lower adhered live bacteria percentages ( 75%). NorChit- Dhvar5 (Nt) also presented lower number of total adhered bacteria (less 35%). Therefore, NorChit-Dhvar5 represent promising biomaterial for future antimicrobial wound dressing development