Synthesis and analysis of artificial cell surface receptors and molecular probes

The efficient delivery of macromolecules to living cells presents a formidable challenge to the development of effective macromolecular therapeutics and cellular probes. We describe here novel synthetic compounds that function as artificial cellular receptors and enable efficient cellular uptake of diverse proteins by promoting non-covalent interactions with cholesterol and sphingolipid-rich lipid raft subdomains of cellular plasma membranes. These compounds comprise N-alkyl derivatives of 3beta-cholesterylamine linked to protein ligands. Molecular recognition between antibodies or streptavidin and cognate artificial receptors in cellular plasma membranes promotes receptor-mediated endocytosis, which renders target proteins completely internalized within 4 h of protein addition. Diverse mammalian cell lines are susceptible to this protein delivery strategy including Jurkat lymphocytes, THP-1 macrophage-monocytes, MCF-7 breast cancer cells, and Chinese Hamster Ovary cells. Structurally related receptors substituting the protonated steroidal amines with isosteric amides are less effective molecular transporters due to diminished affinity of the synthetic compounds for cellular plasma membranes. Confocal fluorescence microscopy revealed that this novel delivery system functionally mimics the initial cellular penetration steps of Cholera toxin, which undergoes clathrin- and non-clathrin mediated endocytosis upon binding to the lipid raft-associated natural product ganglioside GM1. The synthetic compounds described here represent designed cell surface receptors capable of targeting specific proteins into diverse mammalian cells by hijacking the molecular machinery used to organize cellular membranes. This technology has potential applications in drug delivery and provides a novel probe of biological processes at cellular plasma membranes.