Novel Artificial Cell Surface Receptors and Related Molecular Probes

Open Access
Cai, Sutang
Graduate Program:
Master of Science
Document Type:
Master Thesis
Date of Defense:
September 29, 2008
Committee Members:
  • Blake R Peterson, Thesis Advisor
  • Endosome Disruption
  • Drug Delivery
  • Molecular Probes
  • Cell Surface Receptors
Many receptors on the surface of mammalian cells actively promote the uptake of specific cell impermeable ligands via receptor-mediated endocytosis. To mimic this process, we synthesized artificial cell surface receptors that comprise N-alkyl derivatives of 3beta-cholesterylamine linked to ligand binding motifs. These synthetic receptors enable the uptake of proteins and other cellimpermeable molecules, a strategy termed “synthetic receptor targeting”. To improve the efficiency of synthetic receptors and extend their utility as molecular probes, tools for drug delivery, and agents for cellular targeting, we report here studies of structure-activity relationships (SAR). Receptors with different linkers between 3beta-cholesterylamine and a ligand binding motif were designed, synthesized, and evaluated. It was found that installation of glutamic acid subunits in the linker region of synthetic receptors greatly enhances their cellular activities. Receptors with glutamic acids associate with plasma membrane of living mammalian cells much faster and to a greater extent and can deliver more ligands compared to receptors lacking these subunits. Although synthetic receptors can promote the internalization of many cell impermeable bioactive molecules, trapping of this cargo in endosomes is a major limitation for many applications. This limitation is also commonly observed with many other endocytic delivery systems. To circumvent this issue, we synthesized two different 3beta-cholesterylamine derivatives: one was linked to a pH-dependent membrane-lytic dodecapeptide, and the second comprised a disulfide-linked fluorophore coupled through glutamic acid subunits. When added to living cells, membranes of early / recycling endosomes were selectively disrupted, resulting in cleavage of the disulfide and release of the fluorophore into the cytosol and nucleus. The ability of this system to deliver cargo into and release disulfidelinked cargo from relatively nonhydrolytic early / recycling endosomes may be useful for the delivery of a variety of sensitive molecules to diverse intracellular targets. We also studied the feasibility of using small artificial cell surface receptors as a generic drug delivery system. The studies showed that a synthetic receptor comprising 3beta-cholesterylamine linked to the antibiotic vancomycin and bearing two glutamic acid residues in the linker region significantly enhanced the uptake of a fluorophore linked to a L-Lys-D-Phe-D-Ala tripeptide. The high efficiency of this delivery system may have potential application in the delivery of drugs and other cell impermeable molecules.