Targeting Cell Surface Receptors with Ligand-Conjugated Nanocrystals Sandra J. Rosenthal,* ,² Ian Tomlinson, ² Erika M. Adkins, ‡ Sally Schroeter, ‡ Scott Adams, ‡ Laura Swafford, ² James McBride, ² Yongqiang Wang, ² Louis J. DeFelice, ‡ and Randy D. Blakely ‡ Contribution from the Department of Chemistry, and the Department of Pharmacology, Vanderbilt UniVersity School of Medicine, Vanderbilt UniVersity, NashVille, Tennessee 37235 Received September 25, 2000. Revised Manuscript Received September 10, 2001 Abstract: To explore the potential for use of ligand-conjugated nanocrystals to target cell surface receptors, ion channels, and transporters, we explored the ability of serotonin-labeled CdSe nanocrystals (SNACs) to interact with antidepressant-sensitive, human and Drosophila serotonin transporters (hSERT, dSERT) expressed in HeLa and HEK-293 cells. Unlike unconjugated nanocrystals, SNACs were found to dose- dependently inhibit transport of radiolabeled serotonin by hSERT and dSERT, with an estimated half- maximal activity (EC 50) of 33 (dSERT) and 99 µM (hSERT). When serotonin was conjugated to the nanocrystal through a linker arm (LSNACs), the EC50 for hSERT was determined to be 115 µM. Electrophysiology measurements indicated that LSNACs did not elicit currents from the serotonin-3 (5HT3) receptor but did produce currents when exposed to the transporter, which are similar to those elicited by antagonists. Moreover, fluorescent LSNACs were found to label SERT-transfected cells but did not label either nontransfected cells or transfected cells coincubated with the high-affinity SERT antagonist paroxetine. These findings support further consideration of ligand-conjugated nanocrystals as versatile probes of membrane proteins in living cells. Introduction Semiconductor nanocrystals such as those consisting of CdSe (Figure 1) have received substantial attention due to their size- tunable optical properties. 1 Nanocrystals, or “quantum dots”, have the same crystal structure as the bulk material but consist of only a few hundred to a few thousand atoms. Due to incomplete surface passivation resulting in dangling bonds at the nanocrystal surface, nanocrystals have a low fluorescent quantum yield. However, when a “core” nanocrystal is passi- vated in a “shell” of a wider bandgap semiconductor material, the resulting nanocrystal becomes highly luminescent. 2-4 Core/ shell nanocrystals have several advantages over organic mol- ecules as fluorescent labels for biological applications, including resistance to photodegradation, improved brightness, and size- dependent, narrow-emission spectra that enable the monitoring of several processes simultaneously. 5,6 Additionally, their absorption spectrum is continuous above the band gap so that any standard, inexpensive, excitation source can be used to excite the nanocrystal. To target fluorescent nanocrystals to biological molecules, core/shell nanocrystals must be deriva- tized. Recently, two reports demonstrated the use of ligand- conjugated, fluorescent nanocrystals as biological labels. 5,6 In a two-color experiment using mouse fibroblasts, Bruchez et al. 5 used small, green fluorescent core/shells coated with methoxy- silylpropylurea and acetate groups to stain the cell nucleus and larger, red fluorescent core/shells coated with biotin to label F-actin filaments preincubated with phalloidin-biotin and streptavidin. In a second experiment, Chan and Nie 6 linked the protein transferrin to the surface of core/shells and found the nanocrystal-transferrin conjugates were internalized by HeLa cells through receptor-mediated endocytosis. In a slightly different application, Chunyang et al. 7 conjuagated the protein trichosanthin to the surface of core/shell nanocyrstals and found that the enzymatic activity of the protein was not inhibited when conjugated to the quantum dot. Furthermore, these nanoconju- gates were found to aggregate in the cytoplasm of human choriocarinoma cells. 7 One specific use of ligand-conjugated nanocrystals that has not been explored is the targeting of these probes to cell surface receptors, ion channels, and transporters via specific organic ligands or drugs. Chemical signaling in the nervous system involves the coordinated action of a large number of these cell surface proteins. Among these proteins are the neurotransmitter ² Department of Chemistry. ‡ Department of Pharmacology, Vanderbilt University School of Medi- cine. (1) For a review of the size-dependent properties of semiconductor nanocrystals, see: Alivisatos, A. P. J. Phys. Chem. 1996, 100, 13226-13239. (2) Hines, M. A.; Guyot-Sionnest, P. J. Phys. Chem. 1996, 100, 468-471. (3) Dabbousi, B. O.; Rodriguez-Viejo, Mikulec, F. V.; Heine, J. R.; Mattoussi, H.; Ober, R.; Jensen K. F.; Bawendi M. G. J. Phys. Chem. B 1997, 101, 9463-9475. (4) Peng, X.; Schlamp, M. C.; Kadananich, A. V.; Alivisatos, A. P. J. Am. Chem. Soc. 1997, 119, 7019-7029. (5) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (6) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016-2018 (7) Chunyang, Z.; Hui, M.; Nie, S.; Yao, D.; Lei, J.; Dieyan, C. Analyst 2000, 125, 1029-1031. Published on Web 04/05/2002 4586 9 J. AM. CHEM. SOC. 2002, 124, 4586-4594 10.1021/ja003486s CCC: $22.00 © 2002 American Chemical Society