Engineered Sumoylation-Deficient Prdx6 Mutant Protein-Loaded Nanoparticles Provide Increased Cellular Defense and Prevent Lens Opacity

Abstract

Aberrant Sumoylation-mediated protein dysfunction is involved in a variety of oxidative and aging pathologies. We previously reported that Sumoylation-deficient Prdx6K(lysine)122/142R(Arginine) linked to the TAT-transduction domain gained stability and protective efficacy. In the present study, we formulated wild-type TAT-HA-Prdx6WT and Sumoylation-deficient Prdx6-loaded poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) to further enhance stability, protective activities, and sustained delivery. We found that in vitro and subconjuctival delivery of Sumoylation-deficient Prdx6-NPs provided a greater protection of lens epithelial cells (LECs) derived from human and Prdx6−/−-deficient mouse lenses against oxidative stress, and it also delayed the lens opacity in Shumiya cataract rats (SCRs) than TAT-HA-Prdx6WT-NPs. The encapsulation efficiencies of TAT-HA-Prdx6-NPs were ≈56%–62%. Dynamic light scattering (DLS) and atomic force microscopy (AFM) analyses showed that the NPs were spherical, with a size of 50–250 nm and a negative zeta potential (≈23 mV). TAT-HA-Prdx6 analog-NPs released bioactive TAT-HA-Prdx6 (6%–7%) within 24 h. Sumoylation-deficient TAT-HA-Prdx6-NPs provided 35% more protection by reducing the oxidative load of LECs exposed to H2O2 compared to TAT-HA-Prdx6WT-NPs. A subconjuctival delivery of TAT-HA-Prdx6 analog-NPs demonstrated that released TAT-HA-Prdx6K122/142R could reduce lens opacity by ≈60% in SCRs. Collectively, our results demonstrate for the first time that the subconjuctival delivery of Sumoylation-deficient Prdx6-NPs is efficiently cytoprotective and provide a proof of concept for potential use to delay cataract and oxidative-related pathobiology in general.

Hydralazine Revives Cellular and Ocular Lens Health-Span by Ameliorating the Aging and Oxidative-Dependent Loss of the Nrf2-Activated Cellular Stress Response

“…The relative values of target genes were normalized to the control using corresponding β-actin as an internal control. Phospholipase A 2 (PLA 2 ) activity was carried out in accordance with the manufacturer's protocol (EnzChek Phospholipase A2 kit; E10217, Invitrogen, Waltham, MA, USA) and our published protocol [3,81,83]. Briefly, SRA-hLECs and mLECs were treated with different concentrations of Hyd for 24 h. Thereafter, total protein was isolated and quantified by BCA protein assay (ThermoFisher Scientific, Waltham, MA, USA).…”

“…Glutathione (GSH) peroxidase activity was quantified according to company's protocol (Glutathione Peroxidase activity kit, Cat No. ADI-900-158, Enzo Life Sciences, Farmingdale, NY, USA) and our published protocol [3,81,83]. Total cell lysate was prepared from Hydtreated or untreated SRA-hLECs or mLECs.…”

“…Primers used were as follows: forward primer 5 -GACAGAGTTGAGCTCCACACAG-3 and reverse primer 5 -CACGTCCTCGAGAAGCAGAC-3 . Expression and purification of recombinant protein TAT-HA-Prdx6 was prepared as described in our published report [83,86]. Hyd and N-acetyl-L-cysteine (NAC) were purchased from Sigma (Catalog No.…”

“…The lenses were observed routinely. After 48 h, oxidative stress lenses were photographed using Nikon SMZ 745T microscope fitted with a Nikon camera and a computer loaded with an analysis software program [51,54,83,87]. To examine the effects of Hyd, the lenses were processed to measure levels of ROS, lipid peroxidation, protein carbonyls and oxidative DNA damage, and Nrf2 and antioxidant genes, as described above and in the Results and Legends Sections.…”

Lipid Nanostructures for Antioxidant Delivery

“…The second article describes a formulative study, aimed at producing nanoparticles constituted of poly lactic-co-glycolic acid, for SUMOylation-deficient Prdx6 delivery to the eye [ 7 ]. This recombinant protein can be employed in the treatment of diseases related to oxidative stress, such as cataractogenesis [ 8 ].…”