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1 Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Pharmacology and Targeted Therapeutics Program of the Institute of Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, PA, USA
2 Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA
3 Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA, USA
4 Department of Pharmacology and Targeted Therapeutics Program of the Institute of Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, PA, USA
5 Division of Pulmonary, Allergy, and Critical Care Medicine, Emory University, Atlanta, GA, USA
* To whom correspondence should be addressed. E-mail: muzykant{at}mail.med.upenn.edu.
Targeting nanocarriers (NCs) loaded by antioxidant enzymes (e.g., catalase) to endothelial cell adhesion molecules (CAMs) alleviates oxidative stress in the pulmonary vasculature. However, antioxidant protection is transient, since CAM-targeted catalase is internalized, delivered to lysosomes and degraded. To design means to modulate the metabolism and longevity of endothelial cell (EC) targeted drugs we identified and manipulated cellular elements controlling the uptake and intracellular trafficking of nanocarriers targeted to InterCellular Adhesion Molecule 1 (anti-ICAM/NCs). BAPTA, thapsigargin, amiloride and EIPA inhibited anti-ICAM/NC uptake by ECs and actin rearrangements induced by anti-ICAM/NCs (required for uptake), suggesting that member(s) of Na+/H+ exchanger family proteins (NHEs) regulate these processes. Consistent with this hypothesis, a siRNA specific for the plasmalemma NHE1, but not the endosome-associated NHE6, inhibited actin remodeling induced by anti-ICAM/NCs and internalization. Anti-ICAM/NC binding to ECs stimulated formation of a transient ICAM-1/NHE1 complex. One hour after uptake, ICAM-1 dissociated from NHE1 and anti-ICAM/NCs were transported to NHE6-positive vesicles en route to lysosomes. Inhibition of PKC (an activator of intracellular NHEs) accelerated nanocarrier lysosomal trafficking. In contrast, monensin, which enhances the endosomal sodium influx and proton efflux maintained by NHE6, inhibited delivery of anti-ICAM/NCs to lysosomes by switching their trafficking to a plasma membrane recycling pathway. This markedly prolonged the protective effect of catalase-coated anti-ICAM/NCs. Therefore: i) NHE1 and NHE6 regulate distinct phases of anti-ICAM/NC uptake and trafficking; ii) pharmacological agents affecting these regulatory elements alter the itinerary of anti-ICAM/NC intracellular trafficking; and, iii) these agents modulate duration of the therapeutic effects of targeted drugs.
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