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J. A. MacKay, A. Jashnani, P.-Y. Hsueh, S. Janib, S. Hamm-Alvarez; Controlled Ocular Drug Delivery Using Peptide-Mediated Phase Separation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4150. doi: https://doi.org/.
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Affecting over 3.2 million Americans, dry eye syndrome is a common disorder characterized by decreased tear production that leads to significant complications. Symptoms of dry eye can be mollified using drugs in eye drops. After ocular administration, tears wash drugs away from the eye within minutes. Due to rapid clearance, ocular formulations must be given frequently, every 2 to 8 hours. To improve the treatment of eye disorders, better approaches are required to sustain ocular retention of drugs. To achieve this, we are exploring biodegradable elastin-like polypeptides (ELPs), which are thermally responsive polymers composed of a pentapeptide repeat (VPGXG). ELPs reversibly phase separate in response to multiple variables, which makes them attractive candidates for local retention. For example, ELPs have characteristic inverse phase transition temperatures (Tt), below which they are soluble and above which they self-associate. By adjusting Tt between room temperature and body temperature, we are developing ELPs that may be administered as drops in order to increase ocular retention of proteins and small molecules.
We developed three libraries of synthetic ELP genes using recursive directional ligation with a range of transition temperatures. This technique produces high molecular weight polypeptides and enables precise control over the polypeptide length, sequence, and Tt. ELP phase behavior was used to efficiently purify these biopolymers out of bacterial lysates.
For several ELPs (15-40 kD), we characterized the Tt over a range of concentrations (5-100 uM) using UV-Vis spectrophotometry. This approach was used to compare the transition properties of ELPs in saline versus rabbit tears. We found that lacrimal gland secretions, which contain proteases, lipids, sugars, and other enzymes, had little influence on ELP behavior. Using SDS PAGE, we characterized the rate of degradation of ELPs both in tears and in the presence of defined proteases. ELPs were not degraded in tears over a period of two hours.
ELPs were developed with Tt between 25-35°C, which was unchanged in tear fluid, and there was limited degradation of aggregated ELPs in tears. These results suggest that formation of viscous ELP aggregates may be a useful platform for controlled ocular drug delivery. We are currently evaluating the influence of ELP phase behavior on ocular residence time and drug activity.
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