RESUMEN
Purpose: To determine the impact of hypothermia on the barrier function of donor corneal endothelium, thereby enhancing the success of corneal transplantation. Methods: Primary cultures of porcine endothelial cells were subjected to hypothermia (15 h; 4°C). The impact on microtubule assembly, peri-junctional actomyosin ring (PAMR), and ZO-1 was assessed by immunocytochemistry with and without pretreatment with a microtubule-stabilizing agent (Epothilone B; EpoB; 100 nM) and a p38 MAP kinase inhibitor (SB-203580; 20 µM). In addition, EpoB-loaded PLGA nanoparticles (ENPs) prepared by nanoprecipitation technique and coated with poly-L-lysine (PLL-ENPs) were administered one-time for sustained intracellular delivery of EpoB. Results: Exposure to hypothermia led to microtubule disassembly concomitant with the destruction of PAMR and the displacement of ZO-1 at the cellular periphery, suggesting a loss in barrier integrity. These adverse effects were attenuated by pretreatment with EpoB or SB-203580. PLL-ENPs possessed a zeta potential of â¼26 mV and a size of â¼110 nm. Drug loading and entrapment efficiency were 5% (w/w) and â¼87%, respectively, and PLL-ENPs showed a biphasic release in vitro: burst phase (1 day), followed by a sustained phase (â¼4 weeks). Pretreatment with PLL-ENPs (0.4 mg/mL) for 24 h stabilized the microtubules and opposed the hypothermia-induced damage to PAMR and the redistribution of ZO-1. Conclusions: Hypothermia induces microtubule disassembly via activation of p38 MAP kinase and subsequently breaks down the barrier function of the endothelium. Sustained intracellular delivery of EpoB using nanoparticles has the potential to overcome endothelial barrier failure during prolonged cold storage of donor cornea.
