Mechanistic and Kinetic Insights into Cellular Uptake of Biomimetic Dinitrosyl Iron Complexes and Intracellular Delivery of NO for Activation of Cytoprotective HO-1

Abstract

Dinitrosyl iron unit (DNIU), [Fe(NO) 2 ], is a natural metallocofactor for biological storage, delivery, and metabolism of nitric oxide (NO). In the attempt to gain a biomimetic insight into the natural DNIU under biological system, in this study, synthetic dinitrosyl iron complexes (DNICs) [(NO) 2 Fe(μ-SCH 2 CH 2 COOH) 2 Fe(NO) 2 ] (DNIC−COOH) and [(NO) 2 Fe(μ-SCH 2 CH 2 COOCH 3 ) 2 Fe(NO) 2 ] (DNIC−COOMe) were employed to investigate the structure−reactivity relationship of mechanism and kinetics for cellular uptake of DNICs, intracellular delivery of NO, and activation of cytoprotective heme oxygenase (HO)-1. After rapid cellular uptake of dinuclear DNIC−COOMe through a thiol-mediated pathway (t max = 0.5 h), intracellular assembly of mononuclear DNIC [(NO) 2 Fe(SR)-(S Cys )] n− /[(NO) 2 Fe(SR)(S Cys-protein )] n− occurred, followed by O 2 -induced release of free NO (t max = 1−2 h) or direct transfer of NO to soluble guanylate cyclase, which triggered the downstream HO-1. In contrast, steady kinetics for cellular uptake of DNIC− COOH via endocytosis (t max = 2−8 h) and for intracellular release of NO (t max = 4−6 h) reflected on the elevated activation of cytoprotective HO-1 (∼50−150-fold change at t = 3−10 h) and on the improved survival of DNIC−COOH-primed mesenchymal stem cell (MSC)/human corneal endothelial cell (HCEC) under stressed conditions. Consequently, this study unravels the bridging thiolate ligands in dinuclear DNIC−COOH/DNIC−COOMe as a switch to control the mechanism, kinetics, and efficacy for cellular uptake of DNICs, intracellular delivery of NO, and activation of cytoprotective HO-1, which poses an implication on enhanced survival of postengrafted MSC for advancing the MSC-based regenerative medicine.