EPR imaging of redox-responsive hydrogels

Abstract

Hydrogels have attracted noticeable attention for various biomedical applications, including controlled drug delivery, wound dressing and healing, and tissue engineering. The significance of the stimuli-responsive degradation for sustained and "on-demand" release of the active agent, has been recently recognized. Redox-responsive disulfide-containing polymeric networks that undergo degradation when exposed to a thiol-rich environment have become particularly attractive as systems for controlled delivery. Expanding on this idea, the focus of this study was shifted from the therapeutic, to the diagnostic potential of redox-responsive hydrogels. Instead of introducing cleavable redox-active junctions into the network, in the approach used here, the bovine serum albumin (BSA) based matrix remains intact, and the stimuli-responsive indicator, 3-carbamoyl-PROXYL (3CP), is located within the protein hydrogel water pores. 3CP is an electron paramagnetic resonance (EPR) spin probe, particularly suitable as a redox environment marker, considering its sensitivity to both reduction and oxidation, observed through the probe EPR signal loss. Thermally-induced BSA hydrogel is not only a safe 3CP-indicator reservoir, in terms of biocompatibility and biodegradability, but owing to its antioxidative properties, BSA can serve as the first line of defense from reactive oxygen species (ROS), thus preserving the probe signal during the initial experiment phase. EPR imaging experiments showed that during the incubation of the 3CP-labeled BSA hydrogel with activated yeast cells, the decrease of the EPR signal intensity is dependent on the number of live cells, as well as the time of exposure. It is important to note that the observed signal loss was not attributed to 3CP diffusion out of the hydrogel, for yeast cell volumes up to 1 ml, as the spin-labeled hydrogel signal remained constant during several hours of its incubation in 1 ml of water or physiological saline. Ongoing experiments are likely to confirm the application of EPR imaging of spin-labeled hydrogels as a promising method for redox environment monitoring in human cell lines, and hopefully allow the translation of this methodology to in vivo studies.