Abstract
Introduction
Human-hair derived keratin (KOS) protein has been selected in this investigation for its ability to bind antibiotic compounds and provide sustained release while withstanding harsh proteolytic environments such as inflamed, damaged tissue. The need to control local flora has been recognized as an imperative for wound healing, as recovery is significantly hampered by infection. This study investigates the synthesis of KOS-based particulate matter, developed using acid-precipitation, to load and release the water-soluble antibiotic ciprofloxacin (CIP). We hypothesize that ionically bound CIP release is tied to the degradation of KOS, therefore, bacterial metabolism, which produces proteolytic enzymes, will trigger CIP release thereby creating a novel self-extinguishing delivery system for contaminated skin wounds.
Methods
Ciprofloxacin hydrochloride was solubilized in deionized water (pH 5.3) under constant stirring. Freeze-dried KOS powder was added for an ultimately 5% w/v and 0.8% w/v solution of KOS and CIP, respectively. To improve the stability of KOS a water-soluble diglycidyl ether crosslinker was added to solutions and stirred for 24 hours. CIP-loaded protein was precipitated out by a hydrochloric acid induced pH reduction. Samples were collected and frozen at -20 °C prior to lyophilization, thus forming the stable product. Degradation of KOS and commensurate release of CIP were measured using a bicinchoninic acid (BCA) assay and fluorescent measurements of hydrated material supernatant. The reduction of bacterial colonies was validated by a broth inhibition assay whereby CIP-loaded KOS or unloaded KOS controls where hydrated in bacterial-laden broth cultures of Pseudomonas aeruginosa or Methicillin-resistant Staphylococcus aureus. Cultures were sampled at 24, 48, or 72 hours and plated to quantify colony-forming units.
Results
The presence of CIP in the KOS protein was confirmed and release rates follow similar patterns to that of KOS degradation. CIP-loaded proteins significantly reduce bacterial colony presence in concentrated inoculant solutions up to 72 hours.
Conclusions
CIP release does appear to coincide with KOS degradation, which is bolstered in the presence of infectious levels of bacteria. Ongoing studies aim to observe more robust models of infection and more controlled antibiotic release.
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