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Abstract
When an inflammatory stimulus is given, vascular endothelial cells express various cell adhesion molecules including the vascular cell adhesion molecule (VCAM)-1. In this study, the possibility of specifically delivering anti-inflammatory drugs to activated endothelial cells by utilizing VCAM-1 as a target receptor was explored by loading celecoxib, a selective cyclooxygenase-2 inhibitor, into liposomes coupled to the Fab′ fragment against VCAM-1. Anti-VCAM-1-Fab′-conjugated liposomes were prepared by forming an amide linkage between amino groups of Fab′ and the carboxylic group of glutaryl-N-phosphatidylethanolamine in liposomes using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a cross-linker in the presence of sulpho-N-hydroxysuccinimide. The coupling of Fab′ to phospholipids constituting liposomes was confirmed by SDS-PAGE analysis. Under our optimized conjugation conditions, 130.0 µg Fab′ was coupled to 1 µmol liposomes. Immunoblotting analysis showed that VCAM-1 protein expression could be induced by incubating human umbilical vein endothelial cells (HUVEC) with TNF-α. Confocal laser microsopy analysis revealed that Fab′ conjugation to liposomes selectively increased liposomal uptake in TNF-α-pre-stimulated (VCAM-1-expressed) HUVECs, but not in cells without VCAM-1 expression. The concentration of celecoxib loaded in Fab′-conjugated liposomes was 281.1 ± 29 µg/mL, suggesting that liposomal loading also helped to overcome the limitations in celecoxib administration caused by its poor water solubility. Celecoxib loaded in Fab′-conjugated liposomes inhibited prostaglandin E2 (PGE2) production induced by TNF-α-pre-stimulation more efficiently than when loaded in conventional liposomes. Therefore, Fab′-conjugated liposomes served as a drug delivery system with dual functions: targeted delivery and solubilizing capacity.