Hydrophilic CdSe−ZnS Core−Shell Quantum Dots with Reactive

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Hydrophilic CdSe-ZnS Core-Shell Quantum Dots with Reactive Functional Groups on Their Surface Ibrahim Yildiz,† Erhan Deniz,† 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, Department of Pharmacy and Pharmaceutical Sciences, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, United Kingdom BT52 1SA, and §School of Pharmacy and Life Sciences, The Robert Gordon University, Aberdeen, Scotland, United Kingdom AB10 1FR

‡

Received March 15, 2010. Revised Manuscript Received April 27, 2010 We synthesized macromolecular ligands for CdSe-ZnS core-shell quantum dots incorporating multiple thiol groups, poly(ethylene glycol) chains, and either carboxylic acids or primary amines along a common poly(methacrylate) backbone. The thiol groups encourage the adsorption of these macromolecular constructs on the ZnS shell of the nanoparticles, and the poly(ethylene glycol) chains impose hydrophilic character on the resulting assemblies. Indeed, the coated quantum dots are readily soluble in water and are stable under these conditions for months over a broad pH range (4.0-12.0) and even in the presence of large salt concentrations. In addition, these nanoparticles have relatively small hydrodynamic diameters (17-30 nm) and good quantum yields (0.3-0.4). Furthermore, the pendant carboxylic acids or primary amines of the macromolecular ligands can be exploited to modify the quantum dots after the adsorption of the polymers on their surface. For example, boron dipyrromethene dyes can be connected to the hydrophilic quantum dots on the basis of amide bond formation to encourage the transfer of energy from the luminescent CdSe core to the organic dyes. Our hydrophilic nanoparticles can also cross the membrane of Chinese hamster ovarian cells and accumulate in the cytosol with limited nuclear localization. Moreover, the internalized quantum dots are not cytotoxic and have essentially no influence on cell viability. Thus, our strategy for the preparation of biocompatible quantum dots can evolve into the development of valuable luminescent probes with nanoscaled dimensions and optimal photophysical properties for a diversity of biomedical applications.

Introduction Semiconductor quantum dots are inorganic particles with nanoscaled diameters.1-5 Their elemental compositions and miniaturized dimensions translate into photophysical properties that are significantly different from those of organic chromophores. In particular, quantum dots with an emissive CdSe core of 2-6 nm in diameter,6 surrounded by a protective ZnS shell of 0.4-3.5 nm in thickness,7 tend to absorb continuously from the ultraviolet to the visible region of the electromagnetic spectrum with molar extinction coefficients in excess of 105 M-1 cm-1.6 These nanoparticles can also absorb pairs of near-infrared photons simultaneously with two-photon absorption cross sections greater than 104 GM.8 Their one- or two-photon excitation produces excitons *Corresponding authors. E-mail: [email protected] (J.F.C.); fraymo@ miami.edu (F.M.R.). (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) Yoffe, A. D. Adv. Phys. 2001, 50, 1–208. (4) Efros, Al. L.; Rosen, M. Annu. Rev. Mater. Sci. 2000, 30, 475–521. (5) Burda, C.; Chen, X. B.; Narayana, R.; El-Sayed, M. A. Chem. Rev. 2005, 105, 1025–1102. (6) Yu, W.; Qu, L.; Guo, W.; Peng, X. Chem. Mater. 2003, 15, 2854–2860. (7) Dabbousi, B. O.; Rodriguez-Viejo, J.; Mikulec, F. V.; Heine, J. R.; Mattoussi, H.; Ober, R.; Jensen, K. F.; Bawendi, M. G. J. Phys. Chem. B 1997, 101, 9463–9475. (8) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Science 2003, 300, 1434–1436. (9) Gill, R.; Willner, I.; Shweky, I.; Banin, U. J. Phys. Chem. B 2005, 109, 23715– 23719. (10) Clarke, S. J.; Hollmann, C. A.; Zhang, Z.; Suffern, D.; Bradforth, S. E.; Dimitrijevic, N. M.; Minarik, W. G.; Nadeau, J. L. Nat. Mater. 2006, 5, 409–417. (11) Tomasulo, M.; Yildiz, I.; Kaanumalle, S. L.; Raymo, F. M. Langmuir 2006, 24, 10284–10290.

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with an average lifetime of 10-20 ns,9-11 which deactivate radiatively with luminescence quantum yields of 0.3-0.5 in organic solvents.7 The corresponding emission bands are symmetric with widths at half-maximum of only 20-30 nm.7 Furthermore, these bands can precisely be positioned across the visible region with subtle adjustments in core diameter.6 In fact, mixtures of quantum dots with slightly different dimensions can be engineered to emit light of sufficiently different colors to be resolved with a conventional fluorescence microscope, offering the opportunity to implement multicolor imaging assays.12 Indeed, the unique collection of photophysical properties associated with CdSe-ZnS core-shell quantum dots is encouraging their application in biomedical research in alternative to organic dyes.13-18 Nanoparticles with a CdSe core coated with a ZnS shell can be prepared from inorganic reagents in organic solvents under the assistance of hydrophobic ligands.19 The resulting quantum dots, however, are only soluble in nonpolar environments and cannot (12) (a) Kobayashi, H.; Koyama, Y.; Barrett, T.; Hama, Y.; Regino, C. A. S.; Shin, I. S.; Jang, B. S.; Le, N.; Paik, C. H.; Choyke, P. L.; Urano, Y. ACS Nano 2007, 1, 258–264. (b) Kobayashi, H.; Hama, Y.; Koyama, Y.; Barrett, T.; Regino, C. A. S.; Urano, Y.; Choyke, P. L. Nano Lett. 2007, 7, 1711–1716. (13) Alivisatos, A. P. Nat. Biotechnol. 2004, 22, 47–52. (14) Gao, X.; Yang, L.; Petros, J. A.; Marshall, F. F.; Simons, J. W.; Nie, S. Curr. Opin. Biotechnol. 2005, 16, 63–72. (15) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547–1562. (16) Medintz, I. G.; Uyeda, H. T.; Goldam, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435–446. (17) 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. (18) Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Nat. Methods 2008, 5, 763–775. (19) Rosenthal, S. J.; McBride, J.; Pennycook, S. J.; Feldman, L. C. Surf. Sci. Rep. 2007, 62, 111–157.

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be applied to biological preparations in their native form. In order to impose aqueous solubility, the hydrophobic ligands on the surface of the ZnS shell can be replaced with mercaptocarboxylic acids.20 The resulting nanoparticles are soluble in water, but their hydrophilic ligands tend to desorb gradually and encourage aggregation and precipitation.21 The transition from monodentate to bidentate mercaptocarboxylic acids can overcome this problem and produce water-soluble quantum dots with long-term stability.22 Nonetheless, the protic nature of carboxylic acids limits the solubility of these nanoparticles to neutral and basic environments. Under acidic conditions, these quantum dots tend to aggregate and precipitate.23,24 The introduction of a poly(ethylene glycol) chain in place of a carboxylic acid within the hydrophilic ligands can extend the stability of the nanoparticles to moderately acidic pH.23 Nonetheless, the transition from organic solvents to aqueous environments has a depressive effect on the luminescence quantum yield. The photophysical properties of the native quantum dots can be preserved by maintaining the original hydrophobic ligands in place and overcoating them with amphiphilic monomers25 or polymers.26-28 The resulting constructs have excellent quantum yields in water, and they are remarkably stable over a broad pH range. However, the amphiphilic envelope around the luminescent nanoparticle increases significantly the hydrodynamic diameter of the overall assembly. In search of strategies to prepare biocompatible CdSe-ZnS core-shell quantum dots with compact dimensions, long-term stability, and good quantum yields, we designed polymeric ligands with multiple thiol groups and poly(ethylene glycol) chains appended to a common macromolecular backbone.29 The thiol groups displace the native hydrophobic ligands and anchor these polymers to the ZnS shell of the nanoparticles. The poly(ethylene glycol) chains impose hydrophilic character on the overall assemblies. Indeed, the resulting quantum dots are soluble in water and stable for days over a broad pH range (5.0-9.0). In addition, their quantum yields approach 0.4, and their hydrodynamic diameters are close to 15 nm. Furthermore, these luminescent nanoparticles can cross cell membranes and are not cytotoxic. However, the lack of functional groups appropriate for conjugation within the polymeric ligands prevent the further modification of these biocompatible quantum dots. In order to overcome these limitations, we designed a new generation of polymeric ligands incorporating reactive functional groups suitable for conjugation. In this article, we report the synthesis of these macromolecular ligands, their adsorption on CdSe-ZnS core-shell quantum dots, the spectroscopic characterization of the resulting nanoparticles, and their conjugation to model organic chromophores. In addition, we also explored the ability of the coated nanoparticles to cross the membrane of model cells and assessed their cytotoxicity. (20) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016–2018. (21) Willard, D. M.; Carillo, L. L.; Jung, J.; Van Orden, A. Nano Lett. 2001, 1, 469–474. (22) 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. (23) Uyeda, H. T.; Medintz, I. L.; Jaiswal, J. K.; Simon, S. M.; Mattoussi, H. J. Am. Chem. Soc. 2005, 127, 3870–3878. (24) Algar, W. R.; Krull, U. J. ChemPhysChem 2007, 8, 561–568. (25) (a) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libacher, A. Science 2002, 298, 1759–1762. (b) Carion, O.; Mahler, B.; Pons, T.; Dubertret, B. Nat. Protocols 2007, 2, 2383–2390. (26) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. Nat. Biotechnol. 2004, 22, 969–976. (27) Zhou, M.; Nakatani, E.; Gronenberg, L. S.; Tokimoto, T.; Wirth, M. J.; Hruby, V. J.; Roberts, A.; Lynch, R. M.; Ghosh, I. Bioconjugate Chem. 2007, 18, 232–332. (28) 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. (29) Yildiz, I.; McCaughan, B.; Cruickshank, S. F.; Callan, J. F.; Raymo, F. M. Langmuir 2009, 25, 7090–7096.

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Figure 1. Synthesis of copolymers incorporating anchoring, hydrophilic, and connecting group along a common macromolecular backbone.

Figure 2. Monomers incorporating a poly(ethylene glycol) chain (3 and 7-9), primary amine (7), carboxylic acid (8), and dithiolane ring (10 and 12).

Results and Discussion Design and Synthesis. In order to prepare biocompatible CdSe-ZnS core-shell quantum dots with reactive functionalities on their surface, we envisaged the possibility of appending multiple anchoring, hydrophilic and connecting groups to a common macromolecular backbone (Figure 1). The anchors adsorb on the surface of preformed quantum dots, the hydrophilic tails impose aqueous solubility on the resulting assemblies, and the connectors permit the subsequent functionalization of the nanoparticles. In particular, we selected thiols, poly(ethylene glycol) chains, and either carboxylic acids or primary amines as anchoring, hydrophilic, and connecting groups, respectively, and synthesized the monomers 3, 7-10, and 12 (Figure 2) in 1-5 steps, starting from commercial precursors (Figures S1-S5). The monomers 10 and 12 incorporate a dithiolane ring, which can be reduced to the corresponding bisthiol after polymerization. The monomers 3 and 7-9 have poly(ethylene glycol) chains, which are terminated by a primary amine and carboxylic acid in 7 and 8, respectively. The reaction (Figures 1 and S6-S10) of appropriate combinations of these monomers, under the assistance of azobis(isobutyronitrile) (AIBN) in tetrahydrofuran (THF), gave the corresponding copolymers 14, 16, 18, 20, and 22 (Figure 3), after the reduction of the dithiolane rings with sodium borohydride in a mixture of methanol and water. Langmuir 2010, 26(13), 11503–11511

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Figure 3. Polymers incorporating bisthiols and poly(ethylene glycol) chains without (14 and 20) and with either primary amines (16) or carboxylic acids (18 and 22) at their termini and BODIPY fluorophores with a pendant carboxylic acid (23) or primary amine (24).

The heating of preformed CdSe-ZnS core-shell quantum dots with the hydrophilic copolymers 14, 16, 18, 20, or 22 in ethanol under argon for 3 h encouraged the adsorption of the polythiols on the nanoparticle surface in place of the native trin-octylphosphine oxide ligands. The resulting quantum dots were readily soluble in water, and according to dynamic light scattering (DLS) measurements, their hydrodynamic diameters (d in Table 1) ranged from 17 to 30 nm. These values are similar to those determined for quantum dots coated with monomeric poly(ethylene glycol) ligands terminated by a pair of thiol groups30,31 but are larger than that of similar nanoparticles coated with dihydrolipoic acid (cf. 13(1 nm).29 Nonetheless, there is no apparent correlation between the physical dimensions of the five sets of quantum dots and the structural design of their polymeric ligands. The primary amines or carboxylic acids on the surface of the nanoparticles coated with 16, 18, and 22 can be reacted with complementary functionalities to modify further the quantum dots. (30) Pons, T.; Uyeda, H. T.; Medintz, I. L.; Mattoussi, H. J. Phys. Chem. B 2006, 110, 20308–20316. (31) 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.

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In particular, the reaction of 16 with 23 (Figure 3) and of either 18 or 22 with 24, under the assistance of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) in dimethyl sulfoxide (DMSO), resulted in the attachment of the boron dipyrromethene (BODIPY) fluorophores to the hydrophilic nanoparticles. Absorption and Emission Spectroscopy. The absorption and emission spectra (e.g., a and b in Figure 4) of aqueous dispersions of CdSe-ZnS core-shell quantum dots coated with 14, 16, 18, 20, or 22 show the characteristic band gap absorption and emission of the inorganic core at ca. 530 and 540 nm, respectively, with luminescence quantum yields (φ in Table 1) ranging from 0.3 to 0.4. These values are slightly greater than that determined for similar quantum dots coated with dihydrolipoic acid (cf. 0.27 ( 0.01),29 suggesting that the polymer ligands shield the inorganic nanoparticles from the aqueous environment more effectively than their monomeric counterpart. The absorption and emission spectra of the quantum dots remain essentially unaffected for all ligands, even after maintaining the nanoparticles in water for months under ambient conditions. Furthermore, the nature of the linkages connecting the macromolecular DOI: 10.1021/la1010488

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Table 1. Hydrodynamic Diameter (d) and Luminescence Quantum Yield (O) of CdSe-ZnS Core-Shell Quantum Dots Emitting at 540 nm in H2O at 20 °C

14 16 18 20 22

d (nm)

φ

28 ( 4 22 ( 6 17 ( 3 30 ( 4 26 ( 2

0.39 ( 0.03 0.38 ( 0.04 0.40 ( 0.04 0.35 ( 0.03 0.30 ( 0.02

Figure 4. Absorption (a, 1.4 μM) and emission (b, 0.3 μM, λEx = 350 nm) spectra of CdSe-ZnS core-shell quantum dots coated with 16 in H2O at 20 °C. Figure 6. Absorption spectra of CdSe-ZnS core-shell quantum

dots coated with 16 before (a, 0.9 μM) and after (b-d, 0.21 μM) conjugation of increasing amounts of 23 and of 23 (e, 0.7 μM) in PBS (pH = 7.2) at 20 °C. Emission spectra (λEx = 443 nm) of CdSe-ZnS core-shell quantum dots coated with 16 before (f, 0.7 μM) and after (g-i, 0.21 μM) conjugation of increasing amounts of 23 and of 23 (j, 2.7 μM) in PBS (pH = 7.2) at 20 °C.

Figure 5. Dispersions (0.5 μM) of CdSe-ZnS core-shell quantum dots coated with 14 in H2O without (a) and with (b) NaCl (1 M) under ultraviolet illumination. Dispersions (0.5 μM) of CdSe-ZnS core-shell quantum dots coated with 14 in PBS at a pH of 2.0 (c), 4.0 (d), 6.0 (e), 8.0 (f), 10.0 (g), and 12.0 (h) under ultraviolet illumination.

backbone to the hydrophilic and anchoring side chains has no influence on the temporal spectral evolution. The three polyamides 14, 16, and 18 and the two polyesters 20 and 22 have all excellent long-term stability. Similarly, the photophysical properties of these quantum dots do not change in the presence of large 11506 DOI: 10.1021/la1010488

amounts of sodium chloride (a and b in Figure 5) and do not vary with pH in the 4.0-12.0 range (d-h in Figure 5). However, the nanoparticles precipitate at a pH of 2.0 (c in Figure 5). The pH range accessible with these nanoparticles is significantly wider than that tolerated by their commercial counterparts and its comparable to that associated with quantum dots coated with monomeric poly(ethylene glycol) ligands with terminal methoxy groups.32 The absorption spectra (a-d in Figure 6) of quantum dots coated with 16, recorded before and after conjugation with increasing amounts of 23, show the appearance of the characteristic BODIPY absorption at 529 nm. Indeed, this band resembles that of 23 (e in Figure 6), and its absorbance indicates the average number of BODIPY dyes per quantum dot to range from 1.3 to 2.2. Furthermore, this band is positioned in the same range of wavelengths where the nanoparticles emit (f in Figure 6). Moreover, the hydrodynamic diameter of the coated quantum dots (Table 1) suggests the distance of the conjugated dyes from the emissive CdSe core to be less than 10 nm. This relatively short separation together with the significant overlap between the emission of the quantum dots and the absorption of the fluorophores ensure the transfer of energy from the inorganic core to the organic dyes upon excitation. Consistently, the emission spectra (g-i in Figure 6), recorded after the attachment of increasing amounts of 23 to the nanoparticles, show a pronounced decrease in the emission intensity associated with the quantum dots with the concomitant appearance of another band at longer wavelengths. (32) Mei, B. C.; Susumu, K.; Medintz, I. L.; Delehanty, J. B.; Mountziaris, T. J.; Mattoussi, H. J. Mater. Chem. 2008, 18, 4949–4958.

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Figure 8. Emission intensity in the background, cytosol, and nucleus of CHO cells in image b of Figure 7.

Figure 7. Confocal luminescence images of CHO cells recorded with two-photon excitation (λEx=740 nm, λEm=494-560 nm) before (a) and after (b) incubation in the presence of CdSe-ZnS coreshell quantum dots (500 nM) coated with 16 for 48 h.

This emission resembles that of 23 (j in Figure 6) and is associated with the sensitized fluorescence of the conjugated dyes. The quantum dots coated with 18 and 22 show a similar behavior to those coated with 16 after the attachment of BODIPY dyes to their pendant carboxylic acids. Once again, the absorption spectra (Figures S12 and S13), recorded before and after the conjugation of 24 to these nanoparticles, reveal the appearance of the characteristic BODIPY absorption. The corresponding emission spectra (Figures S12 and S13) show a decrease in the luminescence of the inorganic core with the concomitant appearance of an additional emission band for the sensitized emission of the organic dyes. Cell Imaging and Cytotoxicity Assays. The ability of CdSeZnS core-shell quantum dots coated with 16, 18, 20, or 22 to cross the membrane of Chinese hamster ovarian (CHO) cells was assessed by confocal microscopy with two-photon excitation (740 nm). Specifically, CHO cells were imaged before (a in Figure 7) and after (b in Figure 7 and a-c in Figure S14) incubation with the nanoparticles (500 nM) for 48 h. The resulting images reveal luminescence within the cells only after their incubation with the quantum dots. In particular, all four sets of luminescent nanoparticles cross the cell membrane and accumulate preferentially in the cytosol (Figure 8) with limited localization in the nucleus. The exact mechanism of accumulation is not clear at this stage, but quantum dot endocytosis is known to result in endosomal localization.33 Consistently, the punctuate intracellular distribution of 16, 18, 20, and 22 suggests nanoparticle accumulation in restricted cytosolic compartments, such as endosomes/vesicles. (33) (a) Zhang, L. W.; Yu, W. W.; Colvin, V. L.; Monteiro-Riviere, N. A. Toxicol. Appl. Pharmacol. 2008, 228, 200–211. (b) Zhang, L. W.; Monteiro-Riviere, N. A. Toxicol. Sci. 2009, 110, 138–155-211.

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Figure 9. Viability of CHO cells incubated with of CdSe-ZnS core-shell quantum dots coated with 16, 18, 20, or 22 for 48 h at increasing nanoparticle concentrations (logarithmic scale) relative to that of cells incubated without quantum dots.

The toxicity of CdSe-ZnS core-shell quantum dots coated with 16, 18, 20, or 22 to CHO cells was assessed at increasing nanoparticle concentrations by the Trypan Blue assay.34 This particular organic dye stains exclusively dead/dying cells and offers the opportunity to determine the fraction of living cells (viability). The resulting plots (Figure 9) show that the four sets of quantum dots have negligible influence on cell viability up to a concentration of 250 nM, and hence, they are not cytotoxic under these experimental conditions.

Conclusions The radical copolymerization of methacrylate monomers with pendant dithiolane rings, poly(ethylene glycol) chains, and either carboxylic acids or primary amines generates multicomponent macromolecular ligands for semiconductor quantum dots. Indeed, these polymers adsorb on the surface of preformed CdSe-ZnS core-shell quantum dots, after the chemical reduction of their dithiolane rings to bisthiols. The resulting nanostructured assemblies have compact hydrodynamic diameters, are soluble in water, are stable over broad pH ranges and in the presence of salts, and have good quantum yields. In addition, the carboxylic acids or primary amines of the macromolecular ligands permit the subsequent conjugation of the hydrophilic nanoparticles to complementary reactants. The attachment of BODIPY fluorophores, for example, can be exploited to assemble nanoparticle-dye constructs undergoing energy transfer from the inorganic to the organic components upon excitation. The hydrophilic quantum (34) Freshney, R. I. Culture of Animal Cells ; A Manual of Basic Techniques; Wiley: New York, 2005.

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dots are able to cross the membrane of CHO cells, accumulate preferentially in the cytosol, and have essentially no influence on cell viability. Hence, our multicomponent macromolecular ligands can lead to the development of valuable luminescent probes for biomedical applications based on the outstanding photophysical properties of semiconductor quantum dots.

Experimental Procedures Materials and Methods. Chemicals were purchased from commercial sources and used as received with the exception of CH2Cl2 and THF, which were distilled over CaH2 and Na/benzophenone, respectively; methacrylic acid, which was vacuum distilled; AIBN, which was crystallized twice from methanol. Compounds 5 and 11 were prepared adapting literature procedures.35,36 Compound 23 and CdSe-ZnS core-shell quantum dots were prepared according to reported protocols.11,37 All reactions were monitored by thin-layer chromatography, using aluminum sheets coated with silica (60, F254). 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 and PDI of the polymers 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. DLS experiments were performed in quartz cells (3  3 mm) with a Coulter N4 Plus apparatus, operating at a wavelength of 632.8 nm (10 mW) with an orthogonal geometry. The samples were dissolved in H2O (3 mL), filtered through Pall Corp. syringe filters (0.1 μm) five times, and stored. The concentration was adjusted to ensure scattering intensities in the range from 5  104-1  106 counts/s. Nanoparticle size was calculated by averaging the values of five runs of 300 s in unimodal size mode. 1. A solution of Et3N (2.27 mL, 16 mmol) in THF (20 mL) was added dropwise over the course of 20 min to a solution of poly(ethylene glycol) methyl ether (Mn = 2000, 10 g, 5 mmol) and MeSO2Cl (1.16 mL, 15 mmol) in THF (80 mL) maintained at 0 °C under Ar. The mixture was allowed to warm up to ambient temperature and stirred for 24 h under these conditions. Then, a solution of NaHCO3 (420 mg, 5 mmol) and NaN3 (1.1 g, 17 mmol) in H2O (100 mL) was added, and the resulting mixture was concentrated under heating to half of its original volume. After heating under reflux for 24 h and cooling down to ambient temperature, the mixture was diluted with H2O (50 mL) and extracted with CHCl3 (3  50 mL). The organic phase was dried over NaSO4, and the solvent was distilled off under reduced pressure to afford 1 (8 g, 80%) as a white solid. 1H NMR (300 MHz, CDCl3): δ=3.38 (3H, s), 3.39 (2H, t, 13 Hz), 3.53-3.87 (180H, m). 2. PPh3 (3.0 g, 11 mmol) was added to a solution of 1 (7.7 g, 4 mmol) in THF (50 mL) maintained at ambient temperature under Ar for 7 h. After the addition of H2O (3.0 mL, 167 mmol), the mixture was stirred for a further 24 h. Then, the solvent was distilled off under reduced pressure, and the residue was dissolved in aqueous HCl (0.5 M, 100 mL) and washed with EtOAc (3  50 mL). The aqueous layer was diluted with aqueous solutions of NaOH (1 M, 100 mL) and NaCl (1 M, 50 mL) and extracted with CHCl3 (3  70 mL). The organic phase was dried over Na2SO4, and the solvent was distilled off under reduced pressure to afford 2 (5 g, 65%) as a white solid. 1H NMR (400 MHz, CDCl3): δ = 2.87 (2H, t, 13 Hz), 3.38 (3H, s), 3.49-3.88 (180H, m). (35) Susumu, K.; Mei, B. C.; Mattoussi, H. Nat. Protocols 2009, 4, 424–436. (36) Bhang, S. H.; Won, N.; Lee, T.-J.; Jin, H.; Nam, J.; Park, J.; Chung, H.; Park, H.-S.; Sung, Y.-E.; Hahn, S. K.; Kim, B.-S.; Kim, S. ACS Nano 2009, 3, 1389–1398. (37) Tomasulo, M.; Deniz, E.; Alvarado, J.; Raymo, F. M. J. Phys. Chem. C 2008, 112, 8038–8045.

11508 DOI: 10.1021/la1010488

3. A solution of DCC (0.74 g, 3.6 mmol) in CH2Cl2 (20 mL) was added dropwise over the course of 20 min to a solution of 2 (4.00 g, 2 mmol), DMAP (44 mg, 0.36 mmol), and methacrylic acid (258 mg, 3.6 mmol) in CH2Cl2 (80 mL) maintained at 0 °C under Ar. The mixture was allowed to warm up to ambient temperature and was stirred for 24 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 (19:1, v/v)] to afford 3 (2.75 g, 68%) as a white solid. 1H NMR (400 MHz, CDCl3): δ=1.96 (3H, s), 3.37 (3H, s), 3.44-3.81 (180H, m), 5.31 (1H, s), 5.70 (1H, s), 6.44 (1H, bs). 4. A solution of Et3N (7.67 mL, 55 mmol) in THF (30 mL) was added dropwise over the course of 30 min to a solution of poly(ethylene glycol) (Mn =600, 10 g, 17 mmol) and MeSO2Cl (3.86 mL, 50 mmol) in THF (80 mL) maintained at 0 °C under Ar. The mixture was allowed to warm up to ambient temperature and stirred for 24 h under these conditions. Then, a solution of NaHCO3 (1.43 g, 15 mmol) and NaN3 (3.9 g, 60 mmol) in H2O (100 mL) was added, and the resulting mixture was concentrated under heating to half of its original volume. After heating under reflux for 24 h and cooling down to ambient temperature, the mixture was diluted with H2O (50 mL) and extracted with CHCl3 (3 50 mL). The organic phase was dried over Na2SO4, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography [SiO2:CHCl3/MeOH (19:1, v/v)] to afford 4 (8 g, 80%) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ = 3.39 (4H, t, 10 Hz), 3.53-3.81 (48H, m). 5. A solution of PPh3 (3.57 g, 13.6 mmol) in EtOAc (125 mL) was added dropwise over the course of 20 min to a solution of 4 (8 g, 12 mmol) in HCl (1 M, 25 mL) maintained at 0 °C under Ar. The mixture was allowed to warm up to ambient temperature and stirred for 15 h under these conditions. The aqueous phase was separated, extracted with EtOAc (3  25 mL), diluted with aqueous NaOH (1 M, 100 mL) and NaCl (1 M, 50 mL), and extracted with CHCl3 (3  60 mL). The organic phase was dried over Na2SO4, and the solvent was distilled off under reduced pressure to afford 5 (5.59 g, 70%) as colorless oil. 1H NMR (400 MHz, CDCl3): δ = 2.85 (2H, t, 10 Hz), 3.38 (2H, t, 10 Hz), 3.50 (2H, t, 10 Hz), 3.59-3.75 (48H, m). 6. A solution of DCC (1.17 g, 3.6 mmol) in CH2Cl2 (20 mL) was added dropwise over the course of 20 min to a solution of 5 (2.0 g, 3.33 mmol) and methacrylic acid (431 mg, 5 mmol) in CH2Cl2 (80 mL) maintained at 0 °C under Ar. The mixture was allowed to warm up to ambient temperature and stirred for 24 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 (19:1, v/v)] to afford 6 (1.9 g, 95%) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ=1.96 (3H, s), 3.39 (2H, t, 10 Hz), 3.48-3.81 (48H, m), 5.32 (1H, s), 5.71 (1H, s), 6.48 (1H, bs). 7. PPh3 (1.5 g, 5.7 mmol) was added to a solution of 6 (2 g, 2.9 mmol) in THF (40 mL) maintained at ambient temperature under Ar for 2 h. After the addition of H2O (3.0 mL, 167 mmol) and stirred 24 h, the solvent was distilled off under reduced pressure. The residue was dissolved in aqueous HCl (1.0 M, 30 mL) and washed with EtOAc (3  25 mL). The aqueous layer was diluted with aqueous NaOH (1 M, 50 mL) and NaCl (1 M, 50 mL) and extracted with CHCl3 (3  50 mL). The organic phase was dried over Na2SO4, and the solvent was distilled off under reduced pressure to afford 7 (1.6 g, 80%) as colorless oil. 1H NMR (400 MHz, CDCl3): δ = 2.06 (3H, s), 2.87 (2H, t, 10 Hz), 3.493.69 (48H, m), 5.31 (1H, s), 5.75 (1H, s), 6.44 (1H, bs). 8. A solution of succinic anhydride (285 mg, 2.9 mmol) in CH2Cl2 (10 mL) was combined with a solution of 7 (1.0 g, 1.4 mmol) and Et3N (398 μL, 2.9 mmol) in CH2Cl2 (10 mL) maintained at ambient temperature under Ar for 15 h. The mixture was diluted with aqueous HCl (1 M, 60 mL) and extracted with CHCl3 (360 mL). The organic phase was dried over Na2SO4, and the solvent was distilled off under reduced pressure to afford 8 (0.7 g, 61%) as Langmuir 2010, 26(13), 11503–11511

Yildiz et al. a colorless oil. 1H NMR (300 MHz, CDCl3): δ=1.95 (3H, s), 2.54 (2H, t, 8 Hz), 2.64 (2H, t, 7 Hz), 3.47-3.69 (48H, m) 5.32 (1H, s), 5.71 (1H, s), 6.57 (1H, bs), 6.95 (1H, bs). 9. A solution of DCC (1.17 g, 3.6 mmol) in CH2Cl2 (20 mL) was added dropwise over the course of 20 min to a solution of poly(ethylene glycol) methyl ether (Mn =2000, 10 g, 5 mmol), DMAP (244 mg, 2 mmol), and methacrylic acid (860 mg, 10 mmol) in CH2Cl2 (80 mL) maintained at 0 °C under Ar. The mixture was allowed to warm up to ambient temperature and stirred for 24 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 (19:1, v/v)] to afford 9 (6 g, 60%) as a white solid. 1H NMR (300 MHz, CDCl3): δ = 1.95 (3H, s), 3.38 (3H, s), 3.543.88 (180H, m), 4.30 (2H, t, 5.32, 10 Hz), 5.57 (1H, s), 6.13 (1H, s). 10. A solution of DCC (1.72 g, 8.3 mmol) in CH2Cl2 (20 mL) was added dropwise over the course of 20 min to a solution of 2-hydroxyethyl methacrylate (1.0 g, 7.7 mmol), thioctic acid (1.32 g, 6.4 mmol), and DMAP (157 mg, 1.3 mmol) in CH2Cl2 (70 mL) maintained at 0 °C under Ar. The reaction mixture was allowed to warm up to ambient temperature and stirred for 24 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] to afford 10 (1.69 g, 69%) as a yellow oil. FABMS: m/z = 318 [M þ H]þ. 1H NMR (400 MHz, CDCl3): δ = 1.35-1.50 (2H, m), 1.63-1.70 (4H, m), 1.87-1.92 (1H, m), 1.95 (3H, s), 2.35 (2H, t, 15 Hz), 2.35-2.55 (1H, m), 3.10-3.18 (2H, m), 3.48-3.58 (1H, m), 4.33 (4H, s), 5.59 (1H, s), 6.17 (1H, s). 13C NMR (400 MHz, CDCl3): δ = 18.7, 25.0, 29.1, 34.3, 35.0, 38.9, 40.6, 56.7, 62.4, 62.8, 126.5, 136.3, 167.5, 173.6. 11. A solution of DCC (1.2 g, 5.8 mmol) in CH2Cl2 (10 mL) was added dropwise over the course of 20 min to a solution of thioctic acid (1.0 g, 4.9 mmol) and NHS (669 mg, 5.8 mmol) in CH2Cl2 (75 mL) maintained at 0 °C under Ar. The 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 added dropwise over the course of 20 min to a solution of 1,2-ethylenediamine (1.63 mL, 24.3 mmol) in CH2Cl2 (75 mL) maintained at 0 °C under Ar. The mixture was allowed to warm up to ambient temperature and stirred for 3 h under these conditions. The resulting precipitate was filtered off, and the filtrate was washed with aqueous NaCl (0.1 M, 3  50 mL) and HCl (0.5 M, 50 mL). The aqueous layer was diluted with aqueous NaOH (1 M, 50 mL) and NaCl (1 M, 50 mL) and extracted with CH2Cl2 (50 mL). The organic phase was dried over Na2SO4, and the solvent was distilled off under reduced pressure to afford 11 (0.80 g, 66%) as a yellow gel. FABMS: m/z = 249 [M þ H]þ. 1H NMR (400 MHz, CDCl3): δ = 1.46-1.50 (2H, m), 1.65-1.73 (4H, m), 1.90-1.93 (1H, m), 2.20 (2H, t, 15 Hz), 2.45-2.47 (1H, m), 2.83 (2H, t, 12 Hz), 3.12-3.17 (2H, m), 3.30 (2H, t, 15 Hz), 3.55-3.59 (1H, m), 5.98 (1H, bs). 13C NMR (400 MHz, CDCl3): δ = 25.8, 29.3, 34.9, 36.8, 40.6, 41.8, 42.4, 56.8, 173.5. 12. A solution of DCC (2.40 g, 12 mmol) in CH2Cl2 (15 mL) was added dropwise over the course of 20 min to a solution of methacrylic acid (0.84 g, 10 mmol) and NHS (1.34 g, 12 mmol) in CH2Cl2 (60 mL) maintained at 0 °C under Ar. The 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 added to a solution of 11 (0.8 g, 3.2 mmol) in CH2Cl2 (40 mL) maintained at ambient temperature under Ar. The mixture 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 [CHCl3/MeOH (19:1, v/v)] to afford 12 (0.62 g, 62%) as a yellow solid. FABMS: m/z = 317 [M þ H]þ. 1H NMR (400 MHz, CDCl3): δ = 1.43-1.46 (2H, m), 1.60-1.70 (4H, m), 1.86-1.90 (1H, m), 1.95 (3H, s), 2.20 (2H, t, 15 Hz), 2.43-2.45 (1H, m), 3.11-3.17 (2H, m), 3.43 (4H, s), 3.52-3.55 (1H, m), 5.34 Langmuir 2010, 26(13), 11503–11511

Article (1H, s), 5.75 (1H, s), 6.56 (1H, bs), 6.88 (1H, bs). 13C NMR (400 MHz, CDCl3): δ=18.9, 25.8, 29.2, 35, 36.7, 38.9, 40.2, 40.6, 41.3, 56.8, 120.64, 139.8, 169.7, 174.8. 13. A solution of 3 (1.2 g, 0.6 mmol), 12 (121 mg, 0.4 mmol), and AIBN (4 mg, 0.03 mmol) in degassed THF (8 mL) was heated at 75 °C for 24 h in a sealed vial under Ar. After cooling down to ambient temperature, the mixture was transferred to a centrifuge tube and diluted with THF (2 mL). Consecutive aliquots (1 mL) of hexane were added with vigorous shaking until the formation of a yellow precipitate was observed. After centrifugation, the precipitate was separated from the supernatant and dissolved in THF (10 mL). Once again, consecutive aliquots (1 mL) of hexane were added with vigorous shaking until the formation of a yellow precipitate was observed. After centrifugation, the solid residue was separated from the supernatant and dried under reduced pressure to afford 13 (0.6 g) as a yellow solid. GPC: Mn =56 896, PDI= 1.88. 1H NMR (400 MHz, CDCl3): δ=0.83-1.08 (10H, m), 1.141.21 (5H, m), 1.35-1.46 (10H, bs), 1.47-1.81 (20H, bs), 1.841.91 (15H, bs), 2.40-2.90 (25H, m), 3.02-3.17 (15H, m), 3.17 (6H, s), 3.46-3.81 (360H, m). 14. A mixture of 13 (0.5 g) and NaBH4 (50 mg, 1.3 mmol) in MeOH/H2O (2:1, v/v, 15 mL) was stirred at ambient temperature for 2 h. After dilution with aqueous NaCl (1 M, 85 mL) and extraction with CHCl3 (3  50 mL), the organic phase was dried over Na2SO4, and the solvent was distilled off under reduced pressure to afford 14 (0.45 g) as a white solid. 1H NMR (400 MHz, CDCl3): δ = 0.85-1.10 (12H, m), 1.14-1.21 (5H, m), 1.35-1.46 (10H, bs), 1.47-1.81 (20H, bs), 1.84-1.91 (15H, bs), 2.38-2.97 (25H, m), 3.02-3.17 (13H, m), 3.17 (6H, s), 3.46-3.81 (360H, m). 15. A solution of 3 (1.2 g, 0.6 mmol), 12 (91 mg, 0.3 mmol), 7 (65 mg, 0.1 mmol), and AIBN (4 mg, 0.03 mmol) in degassed THF (8 mL) was heated at 75 °C for 24 h in a sealed vial under Ar. After cooling down to ambient temperature, the mixture was transferred to a centrifuge tube and diluted with THF (2 mL). Consecutive aliquots (1 mL) of hexane were added with vigorous shaking until the formation of a yellow precipitate was observed. After centrifugation, the precipitate was separated from the supernatant and dissolved in THF (10 mL). Once again, consecutive aliquots (1 mL) of hexane were added with vigorous shaking until the formation of a yellow precipitate was observed. After centrifugation, the solid residue was separated from the supernatant and dried under reduced pressure to afford 15 (0.6 g) as a yellow solid. GPC: Mn = 50 167, PDI = 1.76. 1H NMR (400 MHz, CDCl3): δ = 0.96-1.81 (7H, bs), 1.81-1.96 (3H, m), 2.12-2.30 (2H, m), 2.32-2.81 (2H, m), 3.12-3.17 (1H. m) 3.37 (2H, s), 3.51-3.80 (108H, m). 16. A mixture of 15 (0.5 g) and NaBH4 (50 mg, 1.3 mmol) in MeOH/H2O (2:1, v/v, 15 mL) was stirred at ambient temperature for 2 h, diluted with aqueous NaCl (1 M, 85 mL), and extracted with CHCl3 (3  50 mL). The organic phase was dried over Na2SO4, and the solvent was distilled off under reduced pressure to afford the product 16 (0.47 g) as a white solid. 1H NMR (400 MHz, CDCl3): δ = 0.87-1.80 (8H, m), 1.81-1.96 (3H, m), 2.12-2.30 (2H, m), 2.32-2.95 (4H, m), 3.12-3.17 (1H, m) 3.37 (2H, s), 3.51-3.80 (115H, m). 17. A solution of 3 (1.2 g, 0.6 mmol), 12 (91 mg, 0.3 mmol), 8 (77 mg, 0.1 mmol), and AIBN (4 mg, 0.03 mmol) in degassed THF (8 mL) was heated at 75 °C for 24 h in a sealed vial under Ar. After cooling down to ambient temperature, the mixture was transferred to a centrifuge tube and diluted with THF (2 mL). Consecutive aliquots (1 mL) of hexane were added with vigorous shaking until the formation of a yellow precipitate was observed. After centrifugation, the precipitate was separated from the supernatant and dissolved in THF (10 mL). Once again, consecutive aliquots (1 mL) of hexane were added with vigorous shaking until the formation of a yellow precipitate was observed. After centrifugation, the solid residue was separated from the supernatant and dried under reduced pressure to afford 17 (0.7 g) as a yellow solid. GPC: Mn = 46 232, PDI = 1.73. 1H NMR (400 MHz, DOI: 10.1021/la1010488

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Article CDCl3): δ = 0.9-1.33 (4H, m), 1.37-1.47 (1H, bs), 1.60-1.67 (2H, m), 1.76-1.97 (4H, m), 2.17-2.70 (3H, m), 3.12-3.18 (1H, bs), 3.38 (2H, s), 3.51-3.80 (112H, m). 18. A mixture of 17 (0.5 g) and NaBH4 (50 mg, 1.3 mmol) in MeOH/H2O (2:1, v/v, 15 mL) was stirred at ambient temperature for 2 h, diluted with aqueous NaCl (1 M, 85 mL), titrated with aqueous HCl (1 M) until the pH was 2-3, and extracted with CHCl3 (3  50 mL). The organic phase was dried over Na2SO4, and the solvent was distilled off under reduced pressure to afford 18 (0.44 g) as a white solid. 1H NMR (400 MHz, CDCl3): δ = 0.85-1.31 (4H, m), 1.37-1.47 (1H, bs), 1.60-1.67 (1H, m), 1.761.97 (3H, m), 2.17-2.95 (4H, m), 3.12-3.18 (1H, bs), 3.38 (2H, s), 3.51-3.80 (116H, m). 19. A solution of 3 (123 mg, 0.4 mmol), 10 (1.2 g, 0.6 mmol), and AIBN (4 mg, 0.02 mmol) in degassed THF (8 mL) was heated at 75 °C for 24 h in a sealed vial under Ar. After cooling down to ambient temperature, the mixture was transferred to a centrifuge tube and diluted with THF (2 mL). Consecutive aliquots (1 mL) of hexane were added with vigorous shaking until the formation of a yellow precipitate was observed. After centrifugation, the precipitate was separated from the supernatant and dissolved in THF (10 mL). Once again, consecutive aliquots (1 mL) of hexane were added with vigorous shaking until the formation of a yellow precipitate was observed. After centrifugation, the solid residue was separated from the supernatant and dried under reduced pressure to afford 19 (0.57 g) as a yellow solid. GPC: Mn =62 777, PDI=1.9. 1H NMR (400 MHz, CDCl3): δ=0.86-1.17 (2H, m), 1.20-1.33 (1H, m), 1.32-1.46 (2H, bs), 1.58-1.82 (5H, m), 1.85-2 (3H, m), 2.10-2.53 (9H, m), 3.04-3.22 (3H, m), 3.36 (5H, s), 3.45-3.80 (290H, m), 4.08-4.25 (10H, m). 20. A mixture of 19 (0.5 g) and NaBH4 (50 mg, 1.3 mmol) in MeOH/H2O (2:1, v/v, 15 mL) was stirred at ambient temperature for 2 h, diluted with aqueous NaCl (1 M, 85 mL), and extracted with CHCl3 (350 mL). The organic phase was dried over Na2SO4, and the solvent was distilled off under reduced pressure to afford 20 (0.47 g) as a white solid. 1H NMR (400 MHz, CDCl3): δ = 0.83-1.20 (3H, m), 1.20-1.33 (1H, m), 1.33-1.48 (2H, bs), 1.601.84 (5H, m), 1.85-2 (3H, m), 2.10-2.53 (9H, m), 3.04-3.22 (2H, m), 3.36 (5H, s), 3.45-3.80 (285H, m), 4.10-4.28 (9H, m). 21. A solution of 3 (92 mg, 0.29 mmol), 10 (1.2 g, 0.58 mmol), methacrylic acid (8 mg, 0.10 mmol), and AIBN (4 mg, 0.02 mmol) in degassed THF (8 mL) was heated at 75 °C for 24 h in a sealed vial under Ar. After cooling down to ambient temperature, the mixture was transferred to a centrifuge tube and diluted with THF (2 mL). Consecutive aliquots (1 mL) of hexane were added with vigorous shaking until the formation of a yellow precipitate was observed. After centrifugation, the precipitate was separated from the supernatant and dissolved in THF (10 mL). Once again, consecutive aliquots (1 mL) of hexane were added with vigorous shaking until the formation of a yellow precipitate was observed. After centrifugation, the solid residue was separated from the supernatant and dried under reduced pressure to afford 21 (0.55 g) as a yellow solid. GPC: Mn = 77 599, PDI = 1.76. 1H NMR (400 MHz, CDCl3): δ=0.84-1.22 (3H, m), 1.25-1.40 (1H, m), 1.421.52 (1H, bs), 1.54-1.72 (2H, bs), 1.85-2.0 (2H, m), 2.30-2.53 (2H, m), 3.02-3.22 (1H, m), 3.37 (2H, s), 3.44-3.80 (100H, m), 4.08-4.25 (5H, m). 22. A mixture of 21 (0.5 g) and NaBH4 (50 mg, 1.3 mmol) in MeOH/H2O (2:1, v/v, 15 mL) was stirred at ambient temperature for 2 h, diluted with aqueous NaCl (1 M, 85 mL), titrated with aqueous HCl (1 M) until the pH was 2-3, and extracted with CHCl3 (3  50 mL). The organic phase was dried over Na2SO4, and the solvent was distilled off under reduced pressure to afford 22 (0.5 g) as a white solid. 1H NMR (400 MHz, CDCl3): δ=0.881.40 (5H, m), 1.40-1.55 (1H, bs), 1.58-1.75 (2H, bs), 1.85-2.0 (2H, m), 2.30-2.53 (2H, m), 3.02-3.22 (2H, m), 3.37 (2H, s), 3.44-3.80 (110H, m), 4.06-4.27 (5H, m). 24. A solution of N,N0 -dicyclohexylcarbodiimide (29 mg, 0.41 mmol) in CH2Cl2 (10 mL) was added dropwise over the course of 11510 DOI: 10.1021/la1010488

Yildiz et al. 20 min to a solution of 23 (50 mg, 0.12 mmol), 1,6-diaminohexane (0.14 g, 1.2 mmol), and 4-(dimethylamino)pyridine (4 mg, 0.03 mmol) in CH2Cl2 (40 mL) maintained at 0 °C under Ar. The reaction mixture was allowed to warm up to ambient temperature and stirred for 24 h under these conditions. The resulting precipitate was filtered off, and the filtrate was washed with H2O (350 mL). The organic phase was dried over Na2SO4, and the solvent was distilled off under reduced pressure. The residue was purified by column chromatography [SiO2:CHCl3/MeOH/Et3N (19:1:0.1, v/v/v)] to afford 24 (30 mg, 48%) as a red powder. FABMS: m/z=524 [M þ H]þ. 1H NMR (400 MHz, CDCl3): δ=0.98 (6H, t, 8 Hz), 1.27 (6H, s), 1.37-1.82 (8H, m), 2.30 (4H, q, 8 Hz), 2.54 (6H, s), 2.87 (2H, t, 8 Hz) 3.49 (2H, t, 8 Hz), 7.39 (2H, d, 8 Hz), 8.03 (2H, d, 8 Hz). Ligand Exchange. A dispersion of CdSe-ZnS core-shell quantum dots in hexane (0.1 mM, 1 mL) was diluted with EtOH (20 mL) and subjected to centrifugation. The supernatant was discarded, and the solid residue was dispersed in CHCl3 (20 mL) and diluted with a solution of 14, 16, 18, 20, or 22 (450 mg) in CHCl3 (40 mL). The resulting mixture was concentrated under heating to an oily slurry. The residue was dispersed in EtOH (3 mL) and heated at 70 °C for 3 h in a sealed vial under Ar. After cooling down to ambient temperature, the mixture was diluted with EtOH (5 mL) and transferred to a centrifuge tube. Consecutive aliquots (1 mL) of hexane were added with vigorous shaking until the formation of a precipitate was observed. After centrifugation, the precipitate was separated from the supernatant, dispersed in H2O (10 mL), and filtered through syringe filters (0.2 and 0.1 μm, Pall Corp.) and four times through centrifuge filters (100 kDa, Millipore) to produce the modified quantum dots, which were dispersed in H2O (3 mL) and stored under these conditions. Conjugation A. A dispersion of quantum dots coated with either 18 or 22 (3.4 μM, 400 μL) in PBS (0.1 M, pH = 7.2) was combined with solutions of 24 (0.66 mM) in DMSO (10.3 μL), EDC (10.4 mM, 25.9 μL) in PBS (0.1 M, pH = 7.2), and sulfoNHS (9.2 mM,146.8 μL) in PBS (0.1 M, pH = 7.2). The mixture was stirred at ambient temperature for 4 h and purified by sizeexclusion chromatography [GE Healthcare PD-10, PBS (0.1 M, pH = 7.2)] to afford the modified quantum dots. Conjugation B. A dispersion of quantum dots coated with 16 (3.4 μM, 400 μL) in PBS (0.1 M, pH = 7.2) was combined with solutions of 23 (0.66, 1.32, or 2.64 μM) in DMSO (10.3 μL), EDC (10.4 mM, 25.9 μL) in PBS (0.1 M, pH = 7.2), and sulfo-NHS (9.2 mM, 146.8 μL) in PBS (0.1 M, pH=7.2). The mixture was stirred at ambient temperature for 4 h and purified by size-exclusion chromatography [GE Healthcare PD-10, PBS (0.1 M, pH=7.2)] to afford the modified quantum dots. Cell Imaging. CHO cells were cultured in F-12 nutrient mixture and supplemented with Foetal Bovine Serum (10%), penicillin (200 U mL-1), streptomycin (200 μg mL-1), and glutamine (2 mM). After reaching confluence, the cells were harvested by trypsinization and seeded at a density of 1104 cells mL-1 in a sixwell plate containing one sterile cover slide (22 mm  22 mm) in each well. The cells were incubated at 37 °C with O2/CO2/air (20:5:75, v/v/v) overnight and then in the presence of CdSe-ZnS core-shell quantum dots (500 nM) coated with 16, 18, 20, and 22 for a further 48 h. The coverslips were removed, washed with PBS (pH = 7.2), and fixed onto a glass slide for imaging. The images were recorded on an inverted Leica SP5 confocal/multiphoton microscope, using a two-photon excitation wavelength of 740 nm and collecting the emission between 494 and 560 nm. A control slide was prepared and imaged under the same conditions by removing the coverslips before incubation of the cells with the quantum dots. Cytotoxicity Assays. CHO cells were cultured in F-12 nutrient mixture and supplemented with Foetal Bovine Serum (10%), penicillin (200 U mL-1), streptomycin (200 μg mL-1), and glutamine (2 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 Langmuir 2010, 26(13), 11503–11511

Yildiz et al. for a further 48 h in the presence of various concentrations (0-250 nM) of CdSe-ZnS core-shell quantum dots coated with 16, 18, 20, or 22. The cells were then harvested by trypsinization and resuspended in Hams-F12 media and Trpyan Blue (1:1, v/v). After incubation at ambient temperature for 5 min, the cells were counted using a hemocytometer and their viability was determined.

Acknowledgment. I.Y., E.D., and F.M.R. thank the National Science Foundation (CAREER Award CHE-0237578 and

Langmuir 2010, 26(13), 11503–11511

Article CHE-0749840) and the University of Miami for financial support. J.F.C. and B.M.C. thank the Engineering and Physical Sciences Research Council (UK) and the University of Ulster for financial support.

Supporting Information Available: Synthetic schemes; absorption and emission spectra of quantum dots coated with 18 or 22; images of cells incubated in the presence of quantum dots coated with 18, 20, or 22. This material is available free of charge via the Internet at http://pubs.acs.org.

DOI: 10.1021/la1010488

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