Prdx6-Loaded PLGA Nanoparticles Protect Lens Epithelial Cells From Oxidative Stress And Prevent Lens Opacity

Abstract

Purpose : Oxidative stress-evoked aberrant Sumoylation of proteins is linked to aging etiopathologies. We previously reported that TAT transduction domain fused Prdx6 mutated at Sumoylation sites, Prdx6^{K(lysine)122/142R(Arginine)}, enhanced protection of human lens epithelial cells (hLECs) by escaping Sumoylation. Herein, we formulated TAT-His-Prdx6 and TAT-His-Prdx6^K122/142R-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to further enhance protective efficiency in vitro and in vivo.

Methods : PLGA-based TAT-Prdx6(TAT-His-Prdx6-PLGA) and TAT-Prdx6^K122/142R(TAT-His-Prdx6^K122/142R-PLGA) NPs were prepared by nano-precipitation method. The properties of TAT-His-Prdx6 and TAT-His-Prdx6^K122/142R NPs were characterized by dynamic light scattering(DLS) and atomic force microscopy (AFM). Release assay was done using Prdx6-ELISA. The Prdx6-NPs were delivered to rat lenses cultured under H₂O₂ stress or delivered subconjuctivally to Shumiya Cataract Rat (SCR), to assess the formulation’s protective ability. MTS and Dye assays examined cell viability and reactive oxygen species (ROS) levels. Western blot and ELISA analyzed Prdx6 levels using His/Prdx6 antibodies. Two-tailed Student’s t-test and one–way ANOVA were used for statistical analysis.

Results : Prdx6 analog loaded PLGA NPs (~56-62% efficiency) were cytocompatible. DLS and AFM analyses showed that the NPs were spherical and of submicron size (220-250nm), with a negative zeta potential of ~23 mV. Release studies revealed that encapsulated Prdx6 analogs were biologically active with ~6-7.5% cumulative release within 24h, followed by slower release thereafter and retained the Prdx6 integrity (~35kDa). NPs with TAT-Prdx6^K122/142R (4µg/ml) provided 35% more protection (p<0.05), with ROS reduction in hLECs exposed to H₂O₂ (50µM/72h) compared to TAT-Prdx6-NPs. Lenses cultured with TAT-Prdx6^K122/142R-NPs were clear compared to control lenses facing H₂O₂ stress (100µM/72h). Subconjuctival delivery of Prdx6 formulations revealed that TAT-Prdx6^K122/142R-NPs released the Prdx6 in vivo and could reduce lens opacity by 58% and delayed cataractogenesis in SCR.

Conclusions : A single application of TAT-Prdx6^K122/142R-NPs provided increased cytoprotection and prevented the cataractogenesis compared to TAT-Prdx6-NPs in vitro and in vivo, providing a proof of concept for potential delivery of TAT-Prdx6 ^K122/142R via NPs to delay cataract formation.

This is a 2021 ARVO Annual Meeting abstract.