Extracellular domains of internalizing cell surface receptors are often targeted to enable drug delivery through the mechanism of receptor-mediated endocytosis. To circumvent natural receptors required for endocytic drug delivery, we constructed a small artificial cell surface receptor comprising the membrane anchor N-alkyl-3β-cholesterylamine linked to a D-Phe-D-Ala motif that binds the glycopeptide antibiotic vancomycin. By mimicking membrane association and trafficking properties of cholesterol, this cholesterol-derived synthetic receptor functions as a prosthetic molecule, inserting into plasma membranes of mammalian cells, and rapidly cycling between the cell surface and intracellular endosomes. Human HeLa cells treated with this receptor gained the ability to internalize vancomycin by endocytosis, enabling this cell-impermeable antibiotic to eradicate the lethal intracellular pathogen Listeria monocytogenes, and preserving the viability of the host cells in vitro. Moreover, when combined with the synthetic receptor, vancomycin penetrated the blood brain barrier, accumulating in the brain and other tissues of mice in vivo. Because of their ability to define new pathways across biological membrane barriers, small synthetic cell surface receptors represent promising tools for drug delivery.Receptor-mediated drug delivery is a Trojan horse approach for the release of therapeutics into cells, tissues, or organs. By coupling drugs to ligands of cell surface receptors, ligand-drug conjugates can accumulate in specific tissues and can be internalized by cells through the mechanism of receptor-mediated endocytosis. 1 Receptors targeted in this way include the low density lipoprotein (LDL) receptor, 2 transferrin receptors, 3 and folate receptors. 4 Receptors involved in the passage of nutrients across the blood-brain barrier through the related mechanism of receptor-mediated transcytosis have been similarly targeted to deliver drugs and imaging agents into the brain. 5 This latter strategy is of significant interest because less than 2% of all drugs are capable of penetrating the blood-brain barrier. 5We report here synthetic receptor-mediated drug delivery. This approach was applied to the glycopeptide antibiotic vancomycin (1, Figure 1, Panel A), a drug of last resort against bacterial pathogens such as methicillin-resistant Staphylococcus aureus. 6 By binding to peptidoglycan precursors that terminate in the peptide sequence D-Ala-D-Ala, vancomycin (1) blocks crosslinking of the bacterial cell wall, providing an effective mode of action against gram-positive bacteria. However, the high polarity and consequent low cell permeability of glycopeptide E-mail: brpeters@chem.psu.edu. antibiotics render these drugs ineffective against bacteria capable of replicating either in the cytoplasm of mammalian host cells or in organs such as the brain that are protected by membrane barriers. NIH Public AccessWe hypothesized that a synthetic receptor capable of promoting the endocytosis of vancomycin (1) might allow this...