Grafting protein ligand monolayers onto the surface of microparticles for probing the accessibility of cell surface receptors.
Coupling of a specific ligand to vaccines or drugs can be a powerful aid to route these compounds to a certain target cell population. However, if the targeted receptor is buried in a glycocalyx, binding of the ligand may be sterically hindered or even abolished, especially when the ligand is attached to bulky payloads. The antigen-transporting M cells that cover the gut-associated lymphoid tissue have a less pronounced glycocalyx than neighboring enterocytes. Such architectural differences might provide a possibility for targeting micro- or nanoparticulate vaccines to the mucosal immune system. To investigate the influence of the glycocalyx on the accessibility of cell surface receptors, we developed a system where a monolayer of ligand molecules is coupled in spatially aligned manner onto the surface of microparticles. On the basis of fluorescent carboxylate-modified particles of 1 micron diameter, different synthetic strategies were tested. Particles were first modified to display aldehyde functions on their surface, then protein ligands were coupled via Schiff base formation. The performance of the particles was tested on cultured mouse fibroblasts using the B subunit of cholera toxin as ligand and the plasma membrane glycolipid ganglioside G(M1) as receptor. Cholera toxin B subunit-coated microparticles generated by one of our synthetic pathways exhibited specific binding to fibroblasts which could be blocked with soluble cholera toxin B subunit. As particles as small as 50 nm and any proteinaceous ligand may be used, this system provides a versatile means for monitoring receptor accessibilities in vitro and in vivo.
Institut für Infektiologie, Zentrum für Molekularbiologie der Entzündung (ZMBE), Westfälische Wilhelms-Universität Münster, von-Esmarch-Strasse 56, D-48129 Münster, Germany. frey@uni-muenster.de
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Frey
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Meckelein
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Schmidt
D016475:3T3 Cells; D000447:Aldehydes / Q000737:chemistry; D000818:Animals; D001360:Avidin / Q000378:metabolism; D001665:Binding Sites; D002264:Carboxylic Acids / Q000737:chemistry; D002460:Cell Line; D005347:Fibroblasts; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D008863:Microspheres; D011506:Proteins / Q000737:chemistry*; D011956:Receptors, Cell Surface / Q000187:drug effects*
D000447:Aldehydes; D002264:Carboxylic Acids; D005456:Fluorescent Dyes; D008024:Ligands; D011506:Proteins; D011956:Receptors, Cell Surface; D001360:Avidin