Track: Formulation and Delivery - Biomolecular - Drug Delivery - Other Routes of Administration - Transdermal and Topical
Category: Poster Abstract
Localized Delivery of Glucose Oxidase and Catalase Enzymes in a Self-Assembling Peptide Hydrogel: Potential for Treatment of Diabetic Chronic Wounds
Purpose: Hypoxia and hyperglycemia in chronic diabetic wounds impedes several healing pathways resulting in delayed and improper healing. Here we present work to use glucose oxidase (GOx) and catalase (CAT) enzymes for the controlled delivery of dissolved oxygen to the wound site from a self-assembling peptide (SAP) hydrogel. Z15_EAK is an SAP formed through the fusion of staphylococcal protein A with EAK, providing an Fc-binding domain. Previous works have established the use of Z15_EAK for extended release of IgG in vivo from 8-28 days. GOx and CAT enzymes will be attached to the Z15_EAK through biotin linkage with polyclonal anti-biotin antibody (anti-BA). The attached enzymes will be then linked the Z15_EAK SAP through the Fc binding domain. Dissolved oxygen will be produced in the presence of glucose as GOx catalyzes H2O2 to provide the substrate for CAT. The advantage of the Z15_EAK system will be localization of the reactions to wound sites and sustained action mediated by release kinetics from the Z15_EAK hydrogel. Methods: Biotinylation was performed using NHS ester at a 20:1 molar ratio of biotin to enzyme. The reaction was confirmed using Agilent microfluidic SDS-PAGE of the non-biotinylated enzyme fraction isolated via streptavidin-agarose beads. Binding of biotinylated enzymes to anti-BA will be confirmed by incubation of biotinylated enzymes with anti-BA then using SDS-PAGE to observe loss of anti-BA band. Association of the anti-BA/ enzyme complex to Z15_EAK will be confirmed relative to neat anti-BA through isothermal titration calorimetry. Activity of GOx and CAT in the complex will be confirmed using a quantitative peroxide kit. GOx activity will be quantified through incubation with glucose and the increase in H2O2 measured at various time points. Conversely, CAT will be incubated with H2O2 and the decrease over time quantified. GOx/ CAT Z15_EAK system will be confirmed initially in vitro by incubation of the combined system in a solution with glucose and the variation in H2O2 measured at various ratios of GOx: CAT. Results: CAT biotinylation was confirmed through SDS-PAGE with the ratio of biotinylated: non-biotinylated CAT of approximately 2: 1. CAT activity post-biotinylation and after attachment to streptavidin-agarose beads was confirmed qualitatively by incubation in 0.1% H2O2 and observation of bubbles. Conclusion: Biotinulated CAT was generated for use in an Fc-binding hydrogel with the ultimate purpose of controlled delivery of dissolved oxygen directly to a wound site. The synchronous use of GOx and CAT in a hydrogel system will allow for oxygen production at specific concentrations while localizing the enzymes to the site of action.
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