Biocompatible CdSe−ZnS Core−Shell Quantum Dots Coated with

pubs.acs.org/langmuir © 2009 American Chemical Society

Biocompatible CdSe-ZnS Core-Shell Quantum Dots Coated with Hydrophilic Polythiols Ibrahim Yildiz,† Bridgeen McCaughan,‡ Stuart F. Cruickshank,‡ John F. Callan,*,‡ and Franc-isco M. Raymo*,† †

Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146-0431 and ‡School of Pharmacy and Life Sciences, The Robert Gordon University, Aberdeen, AB10 7FY, United Kingdom Received January 13, 2009

We designed four polymeric ligands for semiconductor quantum dots and synthesized these macromolecular constructs in four steps, starting from commercial precursors. These ligands have a poly(methacrylate) backbone with pendant thiol groups and poly(ethylene glycol) chains. The thiol groups anchor these ligands on the surface of preformed CdSe-ZnS core-shell quantum dots, and the poly(ethylene glycol) chains impose hydrophilic character on the resulting assemblies. Indeed, three of the four sets of quantum dots are soluble in aqueous environments and are stable under these conditions for days over a wide pH range (5.0-9.0). Furthermore, the polymeric coatings wrapped around the inorganic nanoparticles preserve the photophysical properties of the CdSe core and ensure relatively compact dimensions. Specifically, the luminescence quantum yield is ca. 0.4 and the hydrodynamic diameter ranges from 15 to 29 nm with the nature of the polymeric ligand. Model studies with human umbilical vein endothelial cells demonstrated that these hydrophilic quantum dots cross the cell membrane and localize either in the cytosol or in the nucleus. The length of the poly(ethylene glycol) chains appears to guide the intracellular localization of these luminescent probes. In addition, these studies indicated that these particular nanoparticles are not cytotoxic. In fact, their cellular internalization has essentially no influence on cell growth. In summary, we developed novel polymeric ligands able to impose hydrophilic character and biocompatibility on CdSe-ZnS core-shell nanoparticles. Thus, our results can lead to a new family of valuable luminescent probes for cellular imaging, based on the unique photophysical properties of semiconductor quantum dots.

Introduction Semiconductor quantum dots are inorganic particles with nanoscaled dimensions and attractive photophysical properties.1-5 They absorb radiation continuously from the deep ultraviolet to the visible region of the electromagnetic spectrum with extremely large molar extinction coefficients. In addition, they absorb pairs of near-infrared photons simultaneously with huge two-photon absorption cross sections. Upon either one- or two-photon excitation, they emit electromagnetic radiation with good quantum yields and long lifetimes. The corresponding emission bands are narrow and symmetric and can precisely be positioned across the visible and near-infrared regions with careful adjustments in the elemental composition and physical dimensions of the nanoparticles. Furthermore, quantum dots have outstanding photobleaching resistance and tolerate relatively long exposures to exciting radiation with no sign of degradation. The unique collection of photophysical properties associated with semiconductor quantum dots is encouraging * E-mail: [email protected]; [email protected]. (1) Bawendi, M. G.; Steigerwald, M. L.; Brus, L. E. Annu. Rev. Phys. Chem. 1990, 41, 477–496. (2) Alivisatos, A. P. Science 1996, 271, 933–937. (3) Efros, Al. L.; Rosen, L. Annu. Rev. Mater. Sci. 2000, 30, 475–521. (4) Yoffe, A. D. Adv. Phys. 2001, 50, 1–208. (5) Burda, C.; Chen, X. B.; Narayana, R.; El-Sayed, M. A. Chem. Rev. 2005, 105, 1025–1102. (6) Niemeyer, C. M. Angew. Chem., Int. Ed. 2003, 42, 5796–5800.

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the use of these inorganic nanoparticles in biological applications.6-16 In particular, quantum dots with an emissive CdSe core, surrounded by a ZnS shell, appear to be versatile luminescent probes for imaging and sensing protocols.17 They can conveniently be prepared by reacting inorganic precursors in the presence of organic surfactants. The resulting nanostructured constructs, however, retain the hydrophobic character of the organic components and, hence, are not soluble in water and biocompatible. Additional synthetic steps are necessary to impose hydrophilic character on the CdSeZnS core-shell assembly and ensure compatibility with biological media. Specifically, the hydrophobic organic envelope (7) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2004, 43, 6042–6108. (8) Alivisatos, A. P. Nat. Biotechnol. 2004, 22, 47–52. (9) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547–1562. (10) Gao, X.; Yang, L.; Petros, J. A.; Marshall, F. F.; Simons, J. W.; Nie, S. Curr. Opin. Biotechnol. 2005, 16, 63–72. (11) Medintz, I. G.; Uyeda, H. T.; Goldam, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435–446. (12) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538–544. (13) Pellegrino, T.; Kudera, S.; Liedl, T.; Mu~ noz Javier, A.; Manna, L.; Parak, W. J. Small 2005, 1, 48–63. (14) Somers, R. C.; Bawendi, M. G.; Nocera, D. G. Chem. Soc. Rev. 2007, 36, 579–591. (15) Callan, J. F.; de Silva, A. P.; Mulrooney, R. C.; Mc Caughan, B. J. Incl. Phenom. Macrocylc. Chem. 2007, 58, 257–262. (16) Raymo, F. M.; Yildiz, I. Phys. Chem. Chem. Phys. 2007, 9, 2036– 2043. (17) Rosenthal, S. J.; McBride, J.; Pennycook, S. J.; Feldman, L. C. Surf. Sci. Rep. 2007, 62, 111–157.

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around the emissive inorganic core can be replaced with hydrophilic thiols18-28 or polymers29-39 or it can be overcoated with amphiphilic macromolecular constructs.40-48 In the first instance, the relatively small dimensions of the (18) Willard, D. M.; Carillo, L. L.; Jung, J.; Van Orden, A. Nano Lett. 2001, 1, 469–474. (19) (a) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016–2018. (b) Gao, X.; Chan, W. C. W.; Nie, S. J. Biomed. Opt. 2002, 7, 532–537. (20) (a) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 12142–12150. (b) Uyeda, H. T.; Medintz, I. L.; Jaiswal, J. K.; Simon, S. M.; Mattoussi, H. J. Am. Chem. Soc. 2005, 127, 3870–3878. (c) Pons, T.; Uyeda, H. T.; Medintz, I. L.; Mattoussi, H. J. Phys. Chem. B 2006, 110, 20308– 20316. (d) Susumu, K.; Uyeda, H. T.; Medintz, I. L.; Pons, T.; Delehanty, J. B.; Mattoussi, H. J. Am. Chem. Soc. 2007, 129, 13987–13996. (21) (a) Aldana, J.; Wang, Y. A.; Peng, X. J. Am. Chem. Soc. 2001, 123, 8844–8850. (b) Aldana, J.; Lavelle, N.; Wang, Y.; Peng, X. J. Am. Chem. Soc. 2005, 127, 2496–2504. (22) Wuister, S. F.; Swart, I.; van Driel, F.; Hickey, S. G.; de Mello Doneg a, C. Nano Lett. 2003, 3, 503–507. (23) Hong, R.; Fisher, N. O.; Verma, A.; Goodman, C. M.; Emrick, T.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 739–743. (24) Liu, T.; Huang, Z.; Wang, H.; Wang, J.; Li, X.; Zhao, Y.; Luo, Q. Anal. Chim. Acta 2006, 559, 120–133. :: (25) Charvet, N.; Reiss, P.; Roget, A.; Dupuis, A.; Grunwald, D.; Carayon, S.; Chandezon, F.; Livache, T. J. Mater. Chem. 2004, 14, 2638–2642. (26) Dubois, F.; Mahler, B.; Dubertret, B.; Doris, E.; Mioskowski, C. J. Am. Chem. Soc. 2007, 129, 482–483. (27) Wang, Q.; Xu, Y.; Zhao, X.; Chang, Y.; Liu, Y.; Jiang, L.; Sharma, J.; Seo, D.-K.; Yan, H. J. Am. Chem. Soc. 2007, 129, 6380–6381. (28) Liu, W.; Howarth, M.; Greytak, A. B.; Zheng, Y.; Nocera, D. G.; Ting, A. Y.; Bawendi, M. G. J. Am. Chem. Soc. 2008, 130, 1274–1284. (29) (a) Bruchez, M. P.; Morrone, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013–2016. (b) Gerion, D.; Pinaud, F.; Williams, S. C.; Parak, W. J.; Zanchet, D.; Weiss, S.; Alivisatos, A. P. J. Phys. Chem. B 2001, 105, 8861–8871. (30) Mulvaney, P.; Liz-Marzan, L. M.; Giersig, M.; Ung, T. J. Mater. Chem. 2000, 10, 1259–1270. (31) Schroedter, A.; Weller, H.; Eritja, R.; Ford, W. E.; Wessels, J. M. Nano Lett. 2002, 2, 1363–1367. (32) Potapova, I.; Mruk, R.; Prehl, S.; Zentel, R.; Basche, T.; Mews, A. J. Am. Chem. Soc. 2003, 125, 320–321. (33) (a) Kim, S.; Bawendi, M. G. J. Am. Chem. Soc. 2003, 125, 14652– 14653. (b) Kim, S.-W.; Kim, S.; Tracy, J. B.; Jasanoff, A.; Bawendi, M. G. J. Am. Chem. Soc. 2005, 127, 4556–4557. (34) Sukhanova, A.; Devy, J.; Venteo, L.; Kaplan, H.; Artemyev, M.; Oleinikov, V.; Klinov, D.; Pluot, M.; Cohen, J. H. M.; Nabiev, I. Anal. Biochem. 2004, 324, 60–67. (35) Yang, H.; Holloway, P. H.; Santra, S. J. Chem. Phys. 2004, 121, 7421– 7426. (36) (a) Tan, W. B.; Jiang, S.; Zhang, Y. Biomaterials 2007, 28, 1565–1571. (b) Tan, W. B.; Huang, N.; Zhang, Y. J. Colloid Interface Sci. 2007, 310, 464– 470. (37) Lee, J.; Yang, B.; Li, R.; Seery, T. A. P.; Papadimitrakopoulos, F. J. Phys. Chem. B 2007, 111, 81–87. (38) Yu, W. W.; Chang, E.; Falkner, J. C.; Zhang, J.; Al-Somali, A. M.; Sayes, C. M.; Johns, J.; Drezek, R.; Colvin, V. L. J. Am. Chem. Soc. 2007, 129, 2871–2879. (39) Smith, A. M.; Nie, S. J. Am. Chem. Soc. 2008, 130, 11278–11279. (40) Wu, X.; Liu, H.; Liu, J.; Hale, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 41–46. (41) Mattheakis, L. C.; Dias, J. M.; Choi, Y.-J.; Gong, J.; Bruchez, M. P.; Liu, J.; Wang, E. Anal. Biochem. 2004, 327, 200–208. (42) Petruska, M. A.; Bartko, A. P.; Klimov, V. I. J. Am. Chem. Soc. 2004, 126, 714–716. (43) (a) Pellegrino, T.; Manna, L.; Kudera, S.; Liedl, T.; Koktysh, D.; :: Rogach, A. L.; Keller, S.; Radler, J.; Natile, G.; Parak, W. J. Nano Lett. 2004, :: 4, 703–707. (b) Fern andez-Arguelles, M. T.; Yakovlev, A.; Sperling, R. A.; Luccardini, C.; Gaillard, S.; Sanz Medel, A.; Mallet, J.-M.; Brochon, J.-C.; Feltz, A.; Oheim, M.; Parak, W. J. Nano Lett. 2007, 7, 2613–2617. (44) (a) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. Nat. Biotechnol. 2004, 22, 969–976. (b) Smith, A. M.; Duan, H.; Rhyner, M. N.; Ruan, G.; Nie, S. Phys. Chem. Chem. Phys. 2006, 8, 3895–3903. (45) Nikolic, M. S.; Krack, M.; Aleksandrovic, V.; Kornowski, A.; :: Forster, S.; Weller, H. Angew. Chem., Int. Ed. 2006, 45, 6577–6580. (46) Nann, T. Chem. Commun. 2005, 1735–1736. (47) Jiang, W.; Mardyani, S.; Fischer, H.; Chan, W. C. W. Chem. Mater. 2006, 18, 872–878. (48) Yu, W. W.; Chang, E.; Falkner, J. C.; Zhang, J.; Al-Somali, A. M.; Sayes, C. M.; Johns, J.; Drezek, R.; Colvin, V. L. J. Am. Chem. Soc. 2007, 129, 2871–2879.

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nanoparticles are retained, after the displacement of the native hydrophobic ligands. However, the hydrophilic thiols do not effectively insulate the emissive CdSe core from the aqueous environment, and hence, their attachment to the nanoparticles causes a decrease in luminescence quantum yield. Furthermore, thiols tend to desorb gradually from the ZnS shell of the quantum dots, leading to the aggregation and precipitation of the nanoparticles. In the second and third instances, the polymer coating preserves the photophysical properties of the CdSe-ZnS core-shell assembly and protects it from the aqueous environment. Nonetheless, it also leads to a significant enhancement in the physical dimensions of the nanostructured assembly in most cases, complicating intracellular delivery and tissue penetration. Thus, viable strategies to overcome the limitations associated with the protocols developed so far to passivate the surface of semiconductor quantum dots with hydrophilic components must be identified to facilitate the application of these promising luminescent probes in biology. In search of methods to prepare biocompatible quantum dots with compact dimensions and excellent photophysical properties, we envisaged the possibility of combining the ability of thiol groups to anchor ligands on the surface of CdSe-ZnS core-shell assemblies with that of hydrophilic polymers to protect the inorganic core while ensuring solubility in water. Specifically, we designed a series of macromolecular constructs incorporating multiple thiol groups and poly (ethylene glycol) tails along a polymer backbone. In this article, we report the synthesis and properties of CdSe-ZnS core-shell quantum dots coated with these polymeric ligands as well as their use as luminescent probes for intracellular imaging and an assessment of their cytotoxicity.

Results and Discussion Design and Synthesis of the Ligands. Dihydrolipoic acid (1 in Table 1) is a convenient ligand for the preparation of water-soluble CdSe-ZnS core-shell quantum dots.20 Its two thiol groups adsorb on the surface of the ZnS shell, and its pendant carboxylic acid imposes hydrophilic character on the resulting nanoparticles. These nanostructured constructs, however, are soluble in water only at neutral or moderately basic pH. Under these conditions, the carboxylic acids on the surface of the nanoparticles are converted into carboxylates, facilitating the solvation of the quantum dots. Furthermore, these nanoparticles tend to form intracellular aggregates, complicating their applications in cell imaging.49 The esterification of 1 with poly(ethylene glycol), prior to adsorption on the quantum dots, extends the solubility of the nanoparticles to moderately acidic pH and prevents aggregation.20 Nonetheless, the luminescence quantum yield remains fairly modest (0.25-0.30), and the limited number of anchoring thiol groups per poly(ethylene glycol) chain can eventually result in the desorption of the hydrophilic ligands from the surface of the nanoparticles. On the basis of these observations, we envisaged the possibility of assembling macromolecular ligands, bearing multiple thiol groups and poly(ethylene glycol) chains along a polymer backbone. Specifically, we designed the three polymers 14-16 (Figure 1), differing in the length (x) of their poly(ethylene

(49) (a) Voura, E. B.; Jaiswal, J. K.; Mattoussi, H.; Simon, S. M. Nat. Med. 2004, 10, 993–998. (b) Jaiswal, J. K.; Goldman, E. R.; Mattoussi, H.; Simon, S. M. Nat. Methods 2004, 1, 73–78.

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Article Table 1. Luminescence Quantum Yield (O) and Hydrodynamic Diameter (d ) of the CdSe-ZnS Core-Shell Quantum Dotsa Ligand φ d (nm) 1 0.27 ( 0.01 13 ( 1 15 0.35 ( 0.02 21 ( 2 16 0.41 ( 0.02 29 ( 5 22 0.38 ( 0.02 15 ( 3 a φ was measured in H2O at 20 °C, using fluorescein as standard. d was measured under the same conditions by dynamic light scattering. The structure of 1 is shown below:

glycol) chains, and the copolymer 22 (Figure 2), incorporating two poly(ethylene glycol) segments. We synthesized the polymers 14-16 in four steps, starting from the corresponding poly(ethylene glycol)s 2-4 (Figure 1). Specifically, we coupled one end of 2-4 with (()-R-lipoic acid, under the assistance of N,N0 -dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP), to give the esters 5-7. We reacted the other end with methacryloyl chloride, in the presence of triethylamine, to generate diesters 8-10. Then, we polymerized these compounds, under the influence of 2,20 -azobis(2-methylpropionitrile) (AIBN), to produce 11, 12, and 13 with number

Figure 1. Synthesis of the ligands 14-16. 7092 DOI: 10.1021/la900148m

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average molecular weights (Mn) of 16.5  103, 36.6  103, and 50.3  103, respectively. Finally, we reduced the dithiolane rings of these polymers with sodium borohydride to afford the target ligands 14-16. We prepared the copolymer 22 in four steps starting from the two poly(ethylene glycol) precursors 17 and 19 (Figure 2). In particular, we coupled these compounds with (()-R-lipoic acid and methacrylic acid, under the influence of DCC and DMAP, to generate the esters 18 and 20, respectively. Then, we copolymerized these methacrylates, in the presence of AIBN, to produce 21 with a Mn of 63.5  103 and a ratio of 4:3 (y:z) between the two segments. Finally, we reduced the dithiolane ligands of this copolymer with sodium borohydride to afford the target ligand 22. Synthesis and Properties of the Quantum Dots. We prepared CdSe-ZnS core-shell quantum dots coated with trin-octylphosphine oxide, following a literature procedure.50 Then, we reacted the resulting nanoparticles with 1, 14, 15, 16, or 22 in ethanol and isolated the modified quantum dots after precipitation, centrifugation, and filtration steps. After this treatment, the nanoparticles became readily soluble in water with the exception of those coated with 14. Furthermore, their 1H nuclear magnetic resonance (NMR) spectra do not show the characteristic resonances of tri-n-octylphosphine oxide, indicating that the thiol groups of the hydrophilic ligands can effectively displace the native hydrophobic species on the surface of the quantum dots. The absorption spectrum of the native CdSe-ZnS coreshell quantum dots shows a band gap absorption at 551 nm in chloroform. The corresponding emission spectrum reveals an emission band at 563 nm with a quantum yield of 0.55. The replacement of the hydrophobic tri-n-octylphosphine oxide surfactants with the hydrophilic ligands 1, 15, 16, or 22 and the transition from chloroform to water results in minor wavelength shifts but a noticeable decrease in quantum yield. In particular, the band gap absorption moves to 546 nm (a in Figure 3) and the emission shifts to 564 nm (b in Figure 3). Interestingly, the quantum yield drops to 0.27 for 1 (Table 1), but only to 0.35, 0.38, and 0.41 for 15, 16, and 22, respectively. Thus, the polymeric ligands 15, 16, and 22 protect the emissive CdSe core from the aqueous environment more effectively than the monomeric ligand 1. Furthermore, the quantum dots coated with 15, 16, and 22 remain soluble in water even under acidic conditions, and in fact, their emissive behavior does not change with pH in the 5.0-9.0 range (Supporting Information Figure S1). In addition, the nanoparticles coated with these polymers are remarkably stable and can be stored in aqueous solution for several days with virtually no change in their emission spectra (Supporting Information Figure S2). Presumably, the multiple thiol anchoring groups associated with each polymer prevent the desorption of these ligands from the ZnS shell of the quantum dots and ensure long-term stability. Dynamic light scattering measurements revealed the hydrodynamic diameter of the quantum dots coated with 1 to be 13 nm (Table 1). This parameter increases to 21 and 29 nm with the transition from the monomer 1 to the polymers 15 and 16, respectively, but is only 15 nm for the quantum dots coated with the copolymer 22. Indeed, the poly(ethylene glycol) chain, separating the anchoring thiol groups from the main polymer backbone, of 22 is (50) Tomasulo, M.; Yildiz, I.; Kaanumalle, S. L.; Raymo, F. M. Langmuir 2006, 22, 10284–10290.

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Figure 2. Synthesis of the ligand 22.

Figure 3. Absorption (a, 0.9 μM) and emission (b, 0.05 μM, λEx = 350 nm) spectra of CdSe-ZnS core-shell quantum dots coated with 15 in H2O at 20 °C.

significantly shorter than that of 15 and 16 and ensures the formation of compact nanostructured assemblies. Thus, this particular macromolecular ligand offers the opportunity to retain the relatively small dimensions of the inorganic CdSe-ZnS core-shell assembly, while offering optimal protection to the emissive core. Cellular Imaging. In order to assess the biocompatibility of the hydrophilic nanoparticles, we incubated human umbilical vein endothelial cells (HUVECs) with CdSe-ZnS core-shell quantum dots (500 nM), coated with 1, 15, 16, or 22, for 48 h. In control experiments, HUVECs were also maintained under identical conditions in the absence of the luminescent nanoparticles. The resulting images (a in Figure 4 and Supporting Information Figures S1-S4) reveal luminescent features only in cells incubated with the quantum dots and are qualitatively representative of three Langmuir 2009, 25(12), 7090–7096

identical experiments. All four sets of quantum dots appear to localize intracellularly, according to the corresponding cross-sectional profiles (b in Figure 4 and Supporting Information Figures S2-S4). These luminescence profiles were recorded using a standard 50 μm line, drawn axially through the images, and the resulting intensities are normalized to measurements taken from cells in the absence of any quantum dots (b in Supporting Information Figure S3) and plotted as a percentage increase. Specifically, the line profiles for the quantum dots coated with 1, 15, and 22 (b in Figure 4 and Supporting Information Figures S2 and S3) suggest that the luminescent nanoparticles are predominantly in the cytosol, while those coated with ligand 16 (b in Supporting Information Figure S6) indicate nuclear localization. The distinct behavior of the quantum dots coated with 16 must be a result of the structural differences between this particular polymer and the other three ligands. In fact, the long poly(ethylene glycol) chains of 16 translate into a hydrodynamic diameter for the corresponding quantum dots that is greater than those of the other three nanoparticles (Table 1). Luminescence measurements (Supporting Information Figure S7), taken from a standard 5 μm2 area in ten cells from each experiment, revealed an intensity enhancement relative to the control experiment of (1004 ( 152)% for 1, (999 ( 99)% for 15, (1350 ( 89)% for 16, and (3198 ( 553)% for 22. Interestingly, these data indicate an approximately 3fold increase in luminescence intensity per μm2 for the quantum dots coated with 22 relative to those coated with 1, 15, or 16. This trend is presumably a result of the ability of the nanoparticles coated with 22 to enter HUVECs more effectively than those coated with 1, 15, or 16 and, possibly, is a consequence of their relatively small hydrodynamic diameter (Table 1). DOI: 10.1021/la900148m

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growth promotes the oxidized state. The absorbance and/or emission of the dye can continually be monitored, and thus, cell proliferation can be probed. In this study, we monitored changes in absorbance at 595 nm over a period of 72 h. The resulting temporal absorbance profiles (Figure 5) demonstrate that there is no significant difference in cell growth, at the concentrations and time frames investigated, in the presence of the nanoparticles, as determined using the Mann-Whitney nonparametric analysis.52

Conclusions

Figure 4. Fluorescence image (a) of HUVECs incubated with CdSe-ZnS core-shell quantum dots coated with 22. The asterisk indicates the cell (inset) used for the determination of the crosssectional profile (b) of the relative luminescence intensity.

Thiol groups and poly(ethylene glycol) chains can be appended to a common poly(methacrylate) backbone to generate hydrophilic ligands for semiconductor nanoparticles. The resulting macromolecular constructs adsorb on the surface of preformed CdSe-ZnS core-shell quantum dots and impose solubility in water on the resulting assemblies. The coated nanoparticles are stable in aqueous environments for days over a broad pH range (5.0-9.0) and have good luminescence quantum yields (0.35-0.41) and relatively small hydrodynamic diameters (15-29 nm). Furthermore, they cross the membrane of HUVECs, localize either in the cytosol or in the nucleus, depending on the nature of the ligand, and are not cytotoxic. In principle, the termini of the pendant poly (ethylene glycol) chains of these polymeric ligands can easily be modified to permit the conjugation of the hydrophilic and biocompatible quantum dots to biomolecules. Thus, these novel macromolecular ligands can offer the opportunity to develop valuable luminescent probes, based on the unique photophysical properties of quantum dots, for cellular imaging applications.

Experimental Procedures

Figure 5. Temporal evolution of the mean net absorbance at 595 nm for HUVECs incubated with alamarBlue (10% v/v) in the absence (a) and in the presence of CdSe-ZnS core-shell quantum dots (500 nM) coated with 1 (b), 15 (c), 16 (d) or 22 (e). Cytotoxicity Assays. Having established the ability of the quantum dots coated with 1, 15, 16, and 22 to label HUVECs, we tested whether these nanoparticles displayed any potentially cytotoxic effects using the alamarBlue proliferation assay.51 Specifically, we incubated HUVECs with alamarBlue (10% v/v) and the quantum dots (500 nM) coated with 1, 15, 16, or 22. This assay gives a measure of cellular respiration by detecting the redox state of the growth medium. Cell growth results in a reduced environment and so changes the alamarBlue from its oxidized (blue) state to its reduced (red) state. Continued proliferation of the cells maintains the reduced alamarBlue, whereas inhibition of (51) Nociari, M. M.; Shalev, A.; Benias, P.; Russo, C. J. Immunol. Meth. 1998, 213, 157–167.

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Materials and Methods. Chemicals were purchased from commercial sources and used as received with the exception of CH2Cl2 and tetrahydrofuran (THF), which were distilled over CaH2 and Na/benzophenone, respectively. (()-R-Dihydrolipoic acid (1) and CdSe-ZnS core-shell quantum dots, coated with tri-n-octylphosphine oxide, were prepared according to literature procedures.50,53 All reactions were monitored by thinlayer chromatography, using aluminum sheets coated with silica (60, F254). Gel permeation chromatography (GPC) was performed with a Phenomenex Phenogel 5-μm MXM column (7.8  300 mm) operated with a Varian ProStar system, coupled to a ProStar 330 photodiode array detector, in THF at a flow rate of 1.0 mL min-1. Monodisperse polystyrene standards (2700-200 000) were employed to determine the Mn of the polymers and their polydispersity index (PDI) from the GPC traces. NMR spectra were recorded with Bruker Avance 300 and 400 spectrometers. Absorption spectra were recorded with a Varian Cary 100 Bio spectrometer, using quartz cells with a path length of 0.5 cm. Emission spectra were recorded with a Varian Cary Eclipse spectrometer in aerated solutions. Dynamic light scattering (DSL) experiments were performed in quartz cells (3  3 mm2) with a Coulter N4 Plus apparatus, operating at a wavelength of 632.8 nm (10 mW) and with an orthogonal geometry. The samples were dissolved in H2O (3 mL), filtered through Pall Corporation syringe filters (100 μm) five times, and stored at 20 °C for 20 min before measurement. The concentration was adjusted to ensure scattering intensities in the range from 5  104 to 1  106 counts per s. The data were analyzed (52) Conover, W. J. Practical Nonparametric Statistics; Wiley: New York, 1998. (53) Gunsalus, I. C.; Barton, L. S.; Gruber, W. J. Am. Chem. Soc. 1956, 78, 1763–1768.

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Yildiz et al. with the CONTIN algorithm built into the size-distribution process (SDP) of the Coulter N4 Plus software. Intensity distributions were converted into weight distributions by using the Mie scattering theory approximation incorporated within the software. Particle sizes were estimated by SDP weight and intensity analyses. Assembly size was calculated by averaging the values of five runs of 300 s. Synthesis of 5-7. A solution of DCC (0.88 g, 4.3 mmol) in CH2Cl2 (20 mL) was added dropwise over the course of 30 min to a solution of 2 (Mn = 600; 39 mmol), 3 (Mn = 1000; 39 mmol), or 4 (Mn = 2000; 39 mmol), thioctic acid (0.8 g, 3.9 mmol), and DMAP (57 mg, 0.39 mmol) in CH2Cl2 (300 mL) maintained at 0 °C under Ar. The reaction mixture was allowed to warm up to ambient temperature and stirred for 15 h under these conditions. The resulting precipitate was filtered off, and the filtrate was concentrated under reduced pressure. After the addition of saturated aqueous NaCl (200 mL), the mixture was extracted with MeCO2Et (200 mL). Then, the solvent of the organic phase was distilled off under reduced pressure, and the residue was purified by column chromatography [SiO2:CHCl3/ MeOH (14:1, v/v)] to afford the product as a yellow oil. 5 (62%): 1 H NMR (300 MHz, CDCl3) δ = 1.30-1.60 (2H, m), 1.65-1.72 (4H, m), 1.88-1.95 (1H, m), 2.36 (2H, t, 15 Hz), 2.44-2.48 (1H, m), 2.70-2.80 (1H, bs), 3.11-3.19 (2H, m), 3.55-3.70 (50H, m), 4.24 (2H, t, 10 Hz). 6 (50%): 1H NMR (300 MHz, CDCl3) δ = 1.30-1.55 (2H, m), 1.64-1.74 (4H, m), 1.88-1.95 (1H, m), 2.35 (2H, t, 15 Hz), 2.44-2.48 (1H, m), 2.65-2.76 (1H, bs), 3.113.17 (2H, m), 3.55-3.70 (85H, m), 4.23 (2H, t, 10 Hz). 7 (60%): 1 H NMR (400 MHz, CDCl3) δ = 1.32-1.56 (2H, m), 1.63-1.68 (4H, m), 1.85-1.90 (1H, m), 2.33 (2H, t, 15 Hz), 2.44-2.48 (1H, m), 2.60-2.70 (1H, bs), 3.08-3.14 (2H, m), 3.55-3.70 (175H, m), 4.23 (2H, t, 10 Hz). Synthesis of 8-10. A solution of methacryloyl chloride (2.0 g, 19 mmol) in CH2Cl2 (20 mL) was added dropwise over the course of 10 min to a solution of 5, 6, or 7 (3.8 mmol) and triethylamine (1.9 g, 18.8 mmol) in CH2Cl2 (100 mL) maintained at 0 °C under Ar. The reaction mixture was allowed to warm up to ambient temperature and stirred for 15 h under these conditions. The resulting solution was washed with aqueous NaHCO3 (0.1 M, 3  50 mL), aqueous HCl (0.1 M, 2  50 mL), and saturated aqueous NaCl (2  50 mL). The organic phase was dried over MgSO4, and the solvent was distilled off under reduced pressure to afford the product as a yellow liquid. 8 (78%): 1H NMR (400 MHz, CDCl3) δ = 1.37-1.53 (2H, m), 1.64-1.68 (4H, m), 1.85-1.90 (1H, m), 1.93 (3H, s), 2.34 (2H, t, 15 Hz), 2.42-2.50 (1H, m), 2.66-2.74 (1H, bs), 3.08-3.14 (2H, m), 3.55-3.70 (50H, m), 4.22 (2H, t, 10 Hz), 4.29 (2H, t, 10 Hz), 5.56 (1H, s), 6.12 (1H, s). 9 (84%): 1H NMR (400 MHz, CDCl3) δ = 1.32-1.49 (2H, m), 1.57-1.64 (4H, m), 1.81-1.87 (1H, m), 1.86 (3H, s), 2.27 (2H, t, 15 Hz), 2.42-2.50 (1H, m), 2.60-2.70 (1H, bs), 3.02-3.12 (2H, m), 3.55-3.70 (85H, m), 4.14 (2H, t, 10 Hz), 4.22 (2H, t, 10 Hz), 5.49 (1H, s), 6.10 (1H, s). 10 (83%): 1H NMR (300 MHz, CDCl3) δ = 1.32-1.50 (2H, m), 1.56-1.65 (4H, m), 1.80-1.87 (1H, m), 1.91 (3H, s), 2.32 (2H, t, 15 Hz), 2.42-2.50 (1H, m), 2.70-2.80 (1H, bs), 3.01-3.13 (2H, m), 3.55-3.73 (175H, m), 4.19 (2H, t, 10 Hz), 4.26 (2H, t, 10 Hz), 5.54 (1H, s), 6.10 (1H, s). Synthesis of 11-13. A solution of 8, 9, or 10 (2.3 mmol) and AIBN (19 mg, 0.1 mmol) in degassed THF (10 mL) was heated under reflux and Ar for 24 h. After cooling down to ambient temperature, the solvent was distilled off under reduced pressure. The residue was dissolved in CH2Cl2 (2 mL) and treated with Et2O (50 mL) to cause the precipitation of a solid, which was filtered off after centrifugation. This procedure was repeated three times to afford the product as a yellow solid. 11 (1.2 g): GPC: Mn = 16 500, PDI = 1.6; 1H NMR (300 MHz, CDCl3) δ = 0.81-1.27 (3H, m), 1.32-1.55 (2H, m), 1.52-1.70 (4H, m), 1.80-1.87 (1H, m), 1.89-1.94 (3H, m), 2.32 (2H, t, 15 Hz), 2.42-2.50 (1H, m), 2.65-2.77 (1H, bs), 3.01-3.13 (2H, m), 3.55-3.73 (50H, m), 4.05-4.30 (4H, m). 12 (2.1 g): GPC: Langmuir 2009, 25(12), 7090–7096

Article Mn = 36 600, PDI = 1.9; 1H NMR (300 MHz, CDCl3) δ = 0.70-1.21 (3H, m), 1.33-1.58 (2H, m), 1.59-1.70 (4H, m), 1.80-1.87 (1H, m), 1.89-1.94 (3H, m), 2.32 (2H, t, 15 Hz), 2.42-2.50 (1H, m), 2.63-2.74 (1H, bs), 3.01-3.13 (2H, m), 3.55-3.73 (88H, m), 4.05-4.30 (4H, m). 13 (2.2 g): GPC: Mn = 50 300, PDI = 1.9; 1H NMR (300 MHz, CDCl3) δ = 0.72-1.58 (5H, bs), 1.59-1.70 (4H, m), 1.80-1.87 (1H, m), 1.89-1.94 (3H, m), 2.34 (2H, t, 15 Hz), 2.42-2.50 (1H, m), 2.60-2.73 (1H, bs), 2.95-3.15 (2H, m), 3.55-3.73 (177H, m), 4.05-4.30 (4H, m). Synthesis of 14-16. NaBH4 (100 mg, 2.6 mmol) was added over the course of 30 min to a solution of 11, 12, or 13 (1.0 g) in MeOH/H2O (2:1, v/v, 15 mL) stirred at ambient temperature. The reaction mixture was stirred for a further 2 h, diluted with aqueous NaCl (1 M, 85 mL). and extracted with CHCl3 (3  50 mL). The organic phase was dried over MgSO4, and the solvent was distilled off under reduced pressure to afford the product as a colorless oil. 14 (0.9 g): 1H NMR (300 MHz, CDCl3) δ = 0.82-1.23 (3H, bs), 1.27-1.75 (9H, bs), 1.80-1.89 (1H, m), 1.90-1.97 (3H, m), 2.32 (2H, t, 15 Hz), 2.50-2.82 (3H, bs), 2.85-3.01 (1H, bs), 3.50-3.80 (50H, m), 4.10-4.30 (4H, m). 15 (0.9 g): 1H NMR (400 MHz, CDCl3) δ = 0.76-1.24 (3H, bs), 1.25-1.72 (9H, bs), 1.80-1.90 (1H, m), 1.93-2.04 (3H, m), 2.34 (2H, t, 15 Hz), 2.50-2.82 (3H, bs), 2.88-3.01 (1H, bs), 3.553.83 (90H, m), 4.12-4.23 (4H, m). 16 (0.9 g): 1H NMR (400 MHz, CDCl3) δ = 0.72-1.23 (3H, bs), 1.25-1.72 (9H, bs), 1.75-1.86 (1H, m), 2.02-2.12 (3H, m), 2.32 (2H, t, 15 Hz), 2.55-2.82 (3H, bs), 2.90-3.04 (1H, bs), 3.55-3.83 (180H, m), 4.15-4.28 (4H, m). Synthesis of 18. A solution of DCC (2.0 g, 10 mmol) in CH2Cl2 (20 mL) was added dropwise over the course of 30 min to a solution of 17 (Mn = 526; 3.0 g, 6 mmol), thioctic acid (1.7 g, 8 mmol), and DMAP (118 mg, 0.8 mmol) in CH2Cl2 (100 mL) maintained at 0 °C under Ar. The reaction mixture was allowed to warm up to ambient temperature and was stirred for 15 h under these conditions. The resulting precipitate was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by column chromatography [SiO2:CHCl3/ MeOH (14:1, v/v)] to afford 18 (3.9 g, 95%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ = 1.31-1.49 (2H, m), 1.63-1.69 (4H, m), 1.82-1.89 (1H, m), 1.94 (3H, s), 2.34 (2H, t, 15 Hz), 2.42-2.50 (1H, m), 2.62-2.73 (1H, bs), 3.06-3.17 (2H, m), 3.52-3.75 (40H, m), 4.22 (2H, t, 10 Hz), 4.29 (2H, t, 10 Hz), 5.57 (1H, s), 6.11 (1H, s). Synthesis of 20. A solution of DCC (1.5 g, 7 mmol) in CH2Cl2 (10 mL) was added dropwise over the course of 30 min to a solution of 19 (Mn = 2000; 10 g, 5 mmol), methacrylic acid (0.5 g, 6 mmol), and DMAP (79 mg, 1.2 mmol) in CH2Cl2 (100 mL) maintained at 0 °C under Ar. The reaction mixture was allowed to warm up to ambient temperature and stirred for 15 h under these conditions. The resulting precipitate was filtered off and distilled under reduced pressure. The residue was purified by column chromatography [SiO2:CHCl3/MeOH (14:1, v/v)] to afford 20 (3.9 g, 95%) as a white solid. 1H NMR (400 MHz, CDCl3) δ = 1.91 (3H, s), 3.34 (3H, s), 3.50-3.70 (180H, m), 4.25 (2H, t, 10 Hz), 5.54 (1H, s), 6.17 (1H, s). Synthesis of 21. A solution of 18 (1.0 g, 1.2 mmol), 20 (1.5 g, 0.7 mmol), and AIBN (17 mg, 0.1 mmol) in degassed THF (10 mL) was heated under reflux and Ar for 24 h. After cooling down to ambient temperature, the solvent was distilled off under reduced pressure. The residue was dissolved in CH2Cl2 (2 mL) and treated with Et2O (50 mL) to cause the precipitation of a solid, which was filtered off after centrifugation. This procedure was repeated three times to afford 21 (1.8 g) as a yellow solid. GPC: Mn = 63 500, PDI = 1.8. 1H NMR (300 MHz, CDCl3) δ = 0.60-1.15 (8H, m), 1.15-1.50 (12H, m), 1.63 (6H, bs), 1.841.91 (4H, m), 2.32 (3H, t, 15 Hz), 2.50 (6H, bs), 3.10 (4H, bs), 3.34 (4H, s), 3.55-3.73 (220H, m), 4.06-4.19 (7H, bs). Synthesis of 22. NaBH4 (100 mg, 2.7 mmol) was added over the course of 30 min to a solution of 21 (1.0 g) in MeOH/H2O DOI: 10.1021/la900148m

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Article (2:1, v/v, 15 mL) stirred at ambient temperature. The reaction mixture was stirred for a further 2 h, diluted with aqueous NaCl (1 M, 85 mL), and extracted with CHCl3 (3  50 mL). The organic phase was dried over MgSO4, and the solvent was distilled off under reduced pressure to afford 22 (0.78 g) as a colorless solid. 1H NMR (300 MHz, CDCl3) δ = 0.60-1.50 (20H, m), 1.63-1.90 (10H, bs), 2.32-2.60 (9H, bs), 3.10 (4H, bs), 3.34 (4H, s), 3.55-3.73 (210H, m), 4.06-4.19 (8H, bs). Synthesis of the Quantum Dots. A suspension of CdSe-ZnS core-shell quantum dots (5 mg) and 1, 14, 15, 16, or 22 (800 mg) in EtOH (2 mL) was stirred for 15 h at 65 °C under Ar. After cooling down to ambient temperature, the solvent was distilled off under reduced pressure. The residue was suspended in hexane/CHCl3/EtOH (8:1:1, v/v/v, 10 mL), and the supernatant was removed after centrifugation. The resulting viscous liquid was dissolved in H2O (3 mL) and filtered through Pall Corporation syringe filters (0.1 μm) once and Millipore centrifuge filters (>100 kDa) three times to produce the modified quantum dots as a reddish gel. Cell Imaging. HUVECs were cultured in glucose-free Glasgow’s minimal essential medium, supplemented with glucose (5 mM) and incubated at 37 °C with O2/CO2/air (20:5:75, v/v/v). Upon reaching 60-80% confluence, the cells were incubated for a further 48 h in the presence of CdSe-ZnS core-shell quantum dots (500 nM) coated with 1, 15, 16, or 22. After washing with phosphate buffered saline (PBS), the cells were imaged with a Leica DMI4000B inverted epifluorescent microscope (Leica Microsystems CMS GmbH, Germany). Excitation wavelengths were in the 340-380 nm range, and the emitted light passed through a 425 nm long-pass filter. Images were analyzed using ImageJ (v 1.38) software (U.S. National Institute of Health, Bethesda, Maryland, USA) with all fluorescent intensities being normalized (I I0-1) to measurements taken from cells in the absence of any quantum dots. Cytotoxicity Assays. HUVECs were cultured in glucose-free Glasgow’s minimum essential medium and supplemented with glucose (5 mM), foetal bovine serum (10% v/v), penicillin (200 U mL-1), streptomycin (200 μg mL-1), and glutamine (2 mM). After reaching confluency, they were harvested by trypsinization and diluted to a density of 5  104 cells mL-1. An aliquot (100 μL) of this suspension was added to the inner 60

7096 DOI: 10.1021/la900148m

Yildiz et al. wells of two 96-well plates with the outermost cells containing phosphate buffered saline (200 μL) at 37 °C. The plates were incubated at 37 °C with O2/CO2/air (20:5:75 v/v/v) for 24 h to allow the cells to adhere and proliferate before adding fresh media (80 μL), containing CdSe-ZnS core-shell quantum dots (500 nM) coated with 1, 15, 16, or 22; each of these inner wells was then supplemented with alamarBlue (10% v/v, AbD Serotec, UK). alamarBlue is a redox-active dye that can quantitatively measure cell proliferation and relative cytotoxicity as it exhibits both fluorescence and absorbance change in the appropriate oxidation-reduction range for cellular metabolic reduction.51 As the cells grow, innate metabolic activity renders the surrounding environment reducing. This causes the alamarBlue to change from its oxidized (blue) state to its reduced (red) form. Continued proliferation of the cells maintains this reduced state, whereas the inhibition of growth promotes the oxidized form. The absorbance and/or emission of the dye can continually be monitored to probe cell proliferation. Experiments were repeated six times and directly compared with a blank (media without cells) and a negative control (medium with cells). The plates were allowed to incubate for 30 min, after spiking with alamarBlue, before recording the initial absorbance at time zero. The absorbance was recorded at regular intervals over a period of 72 h at a wavelength of 595 nm in a Bio-Tex FL600 Microplate Fluorescence Reader.

Acknowledgment. I.Y. and F.M.R. thank the National Science Foundation (CAREER Award CHE-0237578 and CHE-0749840) and the University of Miami for financial support. B.M.C., S.F.C., and J.F.C. thank the Engineering and Physical Sciences Research Council (UK) and the Robert Gordon University for financial support. Supporting Information Available: Emission intensity of quantum dots coated with 15, 16, and 22 at various pH values. Temporal evolution of the emission intensity of quantum dots coated with 15, 16, and 22. Fluorescence images of HUVECs incubated without and with quantum dots coated with 1, 15, or 16. Average luminescence intensity in images of HUVECs incubated without and with quantum dots coated with 1, 15, 16, or 22. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2009, 25(12), 7090–7096