title: Intra- and trans-cellular delivery of enzymes by direct conjugation with non-multivalent anti-ICAM molecules.
abstract: Intercellular adhesion molecule 1 (ICAM-1) is a cell-surface protein overexpressed in many diseases and explored for endocytosis and transcytosis of drug delivery systems. All previous evidence demonstrating ICAM-1-mediated transport of therapeutics into or across cells was obtained using nanocarriers or conjugates coupled to multiple copies of anti-ICAM antibodies or peptides. Yet, transport of therapeutics linked to non-multivalent anti-ICAM ligands has never been shown, since multivalency was believed to be necessary to induce transport. Our goal was to explore whether non-multivalent binding to ICAM-1 could drive endocytosis and/or transcytosis of model cargo in different cell types. We found that anti-ICAM was specifically internalized by all tested ICAM-1-expressing cells, including epithelial, fibroblast and neuroblastoma cells, primary or established cell lines. Uptake was inhibited at 4°C and in the presence of an inhibitor of the ICAM-1-associated pathway, rather than inhibitors of the clathrin or caveolar routes. We observed minimal transport of anti-ICAM to lysosomes, yet prominent and specific transcytosis across epithelial monolayers. Finally, we coupled a model cargo (the enzyme horseradish peroxidase (HRP)) to anti-ICAM and separated a 1:2 antibody:enzyme conjugate for non-multivalent ICAM-1 targeting. Similar to anti-ICAM, anti-ICAM-HRP was specifically internalized and transported across cells, which rendered intra- and trans-cellular enzyme activity. Therefore, non-multivalent ICAM-1 targeting also provides transport of cargoes into and across cells, representing a new alternative for future therapeutic applications via this route.
authors.affiliation: Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA.
authors.forename: Rasa
authors.initials: R
authors.lastname: Ghaffarian
authors.affiliation: Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA.
authors.forename: Niksa
authors.initials: N
authors.lastname: Roki
authors.affiliation: National Institute of Standards and Technology, Gaithersburg, MD, USA.
authors.forename: Abraham
authors.initials: A
authors.lastname: Abouzeid
authors.affiliation: National Institute of Standards and Technology, Gaithersburg, MD, USA.
authors.forename: Wyatt
authors.initials: W
authors.lastname: Vreeland
authors.affiliation: Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA; Institute of Bioscience & Biotechnology Research, University of Maryland, College Park, MD, USA. Electronic address: muro@umd.edu.
authors.forename: Silvia
authors.initials: S
authors.lastname: Muro
mesh_terms: D000818:Animals; D018938:Caco-2 Cells; D002966:Clathrin / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods*; D004705:Endocytosis; D042783:Endothelial Cells / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000008:administration & dosage* / Q000378:metabolism / Q000493:pharmacokinetics*; D006801:Humans; D018796:Immunoconjugates / Q000008:administration & dosage / Q000378:metabolism* / Q000493:pharmacokinetics; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D051379:Mice
chemical_list: D002966:Clathrin; D018796:Immunoconjugates; D018799:Intercellular Adhesion Molecule-1; D006735:Horseradish Peroxidase
keywords: Antibody-enzyme conjugate; Drug targeting, monomeric vs. multivalent targeting; ICAM-1; Receptor-mediated endocytosis; Transcytosis