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Endocytosis of monomeric ICAM-1 targeting moieties provides intracellular transport of active enzyme conjugates



Wyatt N. Vreeland, Abraham H. Abouzeid, Silvia Muro, Rasa Ghaffarian


Purpose: Multimeric targeting of drugs to intercellular adhesion molecule 1 (ICAM-1), a receptor expressed on various cell types and disease conditions, facilitates intracellular drug uptake via cell adhesion molecule (CAM) endocytosis. While uptake by multimeric targeting was achieved using drug conjugates or nanocarriers bearing multiple copies of ICAM-1-targeted ligands (antibodies or peptides), ICAM-1 targeting using a single (monomeric) ligand was not thought to induce endocytosis. However, although reduced relative to multimeric strategies, our recent study revealed significant uptake of monomeric anti-ICAM in endothelial cells. Herein, we explore whether this phenomenon stands for other ICAM-1-expressing cell types, e.g. a gastrointestinal epithelial cell model, and examine the potential of monomeric ICAM-1 targeting in providing specific endocytic uptake of an active model drug cargo. Control or TNFα-activated human colorectal adenocarcinoma Caco-2 cells expressing ICAM-1 were used to model healthy or disease-like conditions, respectively. Cells were incubated with anti-ICAM vs. non-specific IgG antibodies, both alone or conjugated to horseradish peroxidase (HRP), a well-established model enzyme cargo. SDS-PAGE, Western blot, and asymmetrical flow field-flow fractionation (AF4) were used to characterize conjugates. Cell binding and uptake of antibodies or conjugates were assessed by fluorescence microscopy and spectrophotometric enzyme activity assays. Results: Monomeric anti-ICAM showed specific binding (8-fold over IgG) and significant uptake (~50% by 1 h) via endocytosis, in both control and TNFα-activated cells. Amiloride inhibited uptake by ~50%, characteristic of the CAM pathway. Similarly, monomeric anti-ICAM-HRP conjugates showed specific binding (90-fold over IgG-HRP conjugates) as well as significant uptake (~50% by 1 h) and intracellular enzyme activity (6.4 pM; 9-fold over unconjugated HRP), which was reduced by 44% in the presence of amiloride
Journal of Controlled Release


drug targeting, monomeric vs. multimeric targeting, receptor-mediated endocytosis, antibody-enzyme conjugate, ICAM-1, gastrointestinal epithelial cells
Created July 27, 2016, Updated November 10, 2018