Selective Intracellular Delivery of Dexamethasone into Activated Endothelial Cells Using an E-Selectin-Directed Immunoconjugate

Abstract

In chronic inflammatory diseases, the endothelium is an attractive target for pharmacological intervention because it plays an important role in leukocyte recruitment. Hence, inhibition of endothelial cell activation and consequent leukocyte infiltration may improve therapeutic outcome in these diseases. We report on a drug targeting strategy for the selective delivery of the anti-inflammatory drug dexamethasone to activated endothelial cells, using an E-selectin-directed drug-Ab conjugate. Dexamethasone was covalently attached to an anti-E-selectin Ab, resulting in the so-called dexamethasone-anti-E-selectin conjugate. Binding of the conjugate to E-selectin was studied using surface plasmon resonance and immunohistochemistry. Furthermore, internalization of the conjugate was studied using confocal laser scanning microscopy and immuno-transmission electron microscopy. It was demonstrated that the dexamethasone-anti-E-selectin conjugate, like the unmodified anti-E-selectin Ab, selectively bound to TNF-α-stimulated endothelial cells and not to resting endothelial cells. After binding, the conjugate was internalized and routed to multivesicular bodies, which is a lysosome-related cellular compartment. After intracellular degradation, pharmacologically active dexamethasone was released, as shown in endothelial cells that were transfected with a glucocorticoid-responsive reporter gene. Furthermore, intracellularly delivered dexamethasone was able to down-regulate the proinflammatory gene IL-8. In conclusion, this study demonstrates the possibility to selectively deliver the anti-inflammatory drug dexamethasone into activated endothelial cells, using an anti-E-selectin Ab as a carrier molecule.

Factors modulating the delivery and effect of enzymatic cargo conjugated with antibodies targeted to the pulmonary endothelium

“…Targeted drug delivery to the endothelium may help to improve treatment of diseases affecting the vasculature [1][2][3][4][5][6][7]. Targeted drug delivery systems (DDSs) designed to achieve this goal consist of affinity vectors (e.g., antibodies directed to endothelial surface determinants) coupled either directly to pharmacological cargoes (e.g., therapeutic enzymes) or to drugloaded vehicles or carriers (e.g., liposomes or polymer nanocarriers) [8][9][10][11][12][13][14].…”

Targeting of angiogenic endothelial cells at sites of inflammation by dexamethasone phosphate–containing RGD peptide liposomes inhibits experimental arthritis

“…Arg-Gly-Asp (RGD) sequence-containing peptides with a cyclic conformation have been developed as specific ligands for ␣v␤3 integrin (12). Most reports on VEC-specific modulation as antiinflammatory therapy are limited to in vitro studies (9,13,14). To date only one report has described in vivo efficacy, in a study of a systemically applied dicyclic RGD peptide targeting a proapoptotic peptide directed to synovial neovasculature in murine collagen-induced arthritis (4).…”

“…Cellular adhesion molecules or growth factor receptors that are preferentially expressed on VECs at inflamed sites may function as receptors for such recognition (1,3,8,9). The integrin ␣v␤3 is strongly up-regulated on angiogenic endothelium at sites of inflammation (1,10,11).…”

Targeted siRNA delivery to diseased microvascular endothelial cells—Cellular and molecular concepts

“…Initially employed drugs include the cell death inducing molecules doxorubicin (38) and the proapoptotic heptapeptide dimer D(KLAKLAK)2 (39). Moreover, anti-inflammatory enzymes that provide anti-oxidant protection (40) and corticosteroids that inhibit intracellular signaling and concomitant proinflammatory gene expression (16,22) have been formulated and were shown to improve disease status. Also, targeted delivery of antisense oligonucleotides has been investigated in detail in the last 15 years (41).…”

“…cells, that they are not avidly shed, and that they become internalized upon ligand binding and intracellularly processed when intracellular drug release is a prerequisite. E-selectin is an internalizing receptor that routes its ligands including antibodies and antibody modified-liposomes to the lysosomal compartment (21)(22)(23). This feature substantially contributes to its outstanding quality as a target to be exploited for intracellular delivery of small RNAs.…”