pubs.acs.org/Langmuir © 2010 American Chemical Society
Polyvalent Lactose-Quantum Dot Conjugate for Fluorescent Labeling of Live Leukocytes Min Yu,†,z Yang Yang,‡,z Rongcheng Han,† Qiang Zheng,† Lijun Wang,† Yuankai Hong,† Zhongjun Li,*,‡ and Yinlin Sha*,† † Single-Molecule and Nanobiology Laboratory, Department of Biophysics, School of Basic Medical Sciences and Biomed-X Center and ‡State Key Laboratory of Natural and Biomimetic Drugs, Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. z These authors contributed equally to this work.
Received December 1, 2009. Revised Manuscript Received January 12, 2010 Oligosaccharides play crucial roles in many biorecognition processes by the so-called “cluster glycosidic effect”. We here report a facile synthesis of lactose-CdSeS/ZnS quantum dot conjugate (Lac-QDs) by use of 1-thiol-β-D-lactose via ligand exchange, which exhibits significantly high affinity and specificity to leukocytes in contrast to the monovalent lactose. Structural analyses indicate that there are about 132 lactosyl molecules assembled on single QDs and the hydrodynamic diameter is small, close to 8.2 nm. Further, Lac-QDs display good fluorescence and physicochemical stability in physiological conditions, as well as extremely low cytotoxicity. These properties facilitate the use of Lac-QDs in fluorescent labeling of live leukocytes.
1. Introduction Nanosized semiconductor crystals (namely, quantum dots, QDs) have recently attracted considerable interest in the biological area for their unique optical properties, such as precisely tunable and sharp photoluminescence (PL), broad absorption cross section, high quantum yield (QY), and good photostability.1,2 Since Nie’s group3 first reported the delivery of CdSe/ZnS QDs into live cells by conjugating with transferrin protein, diverse bioconjugated QDs have been developed and used in biological imaging, labeling, and sensing, both in vivo and in vitro.4-8 In this research, biomolecules with unusual affinities (e.g., antibody, protein/peptide, and DNA) are grafted onto QD surfaces to endow QDs with expected functions, such as biorecognition and targeting. It is acknowledged that carbohydrates exhibit excellent water solubility and structural diversity, and play crucial roles in many biological and pathological processes. For example, oligosaccharides in a cellular glycocalyx can mediate cell adhesion through specific carbohydrate-carbohydrate or carbohydrateprotein interactions. In comparison with polyvalent and complex saccharides showing highly specific affinity in natural processes, many monomeric saccharides, however, generally display extremely low affinity. This property hampers their use in studying *Corresponding author. Fax þ86 10 8280 1278; e-mail
[email protected]. cn (Y. Sha); e-mail
[email protected] (Z. Li).
(1) Alivisatos, P Nat. Biotechnol. 2004, 22(1), 47–52. (2) Klostranec, J. M.; Chan, W. C. W. Adv. Mater. 2006, 18(15), 1953–1964. (3) Chan, W. C. W.; Nie, S. M. Science 1998, 281(5385), 2016–2018. (4) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4(6), 435–446. (5) 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(5709), 538–544. (6) Nie, S. M.; Xing, Y.; Kim, G. J.; Simons, J. W. Annu. Rev. Biomed. Eng. 2007, 9, 257–288. (7) Tholouli, E.; Sweeney, F.; Barrow, E.; Clay, V.; Hoyland, J. A.; Byers, R. J. Am. J. Pathol. 2008, 216(3), 275–285. (8) Weissleder, R.; Kelly, K.; Sun, E. Y.; Shtatland, T.; Josephson, L. Nat. Biotechnol. 2005, 23(11), 1418–1423. (9) Branderhorst, H. M.; Liskamp, R. M. J.; Visser, G. M.; Pieters, R. J. Chem. Commun. 2007, 47, 5043–5045.
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carbohydrate-involved interactions.9-11 Interestingly, there is evidence that some artificial assemblies with multivalent display of oligosaccharide ligands can efficiently promote biorecognitions.12 Recent advances in glyconanobiology research further reveal that, by conjugating with nanoparticles (NPs; e.g., gold NPs), some oligosaccharides with low affinity exhibit significant enhancement of specificity and binding affinity.10,13 This is ascribed to the fact that the oligosaccharide assemblies on the NP surface can efficiently promote the “cluster glycoside effect”14,15 by multivalent display of sugar ligands. Taking advantage of oligosaccharides in order to functionalize QDs has been implemented by a few groups in recent years.16-22 However, to use QD-oligosaccharide conjugates as biological labels, the main challenge is the complex and difficult synthesis of carbohydrate derivatives. Leukocytes (also called “white blood cells”) are cells belonging to the immune system, which defend the body against infectious diseases and foreign substances. When exposed to inflammatory stimuli, the number of leukocytes often increases to a certain amount. During such a period, some epithelia are activated and (10) De la Fuente, J. M.; Penades, S. Biochim. Biophys. Acta 2006, 1760(4), 636– 651. (11) Pukin, A. V.; Branderhorst, H. M.; Sisu, C.; Weijers, C. A. G. M.; Gilbert, M.; Liskamp, R. M. J.; Visser, G. M.; Zuilhof, H.; Pieters, R. J. ChemBioChem 2007, 8(13), 1500–1503. (12) Dwek, R. A. Chem. Rev. 1996, 96(2), 683–720. (13) De la Fuente, J. M.; Penades, S. Glycoconjugate J. 2004, 21(3-4), 149–163. (14) De la Fuente, J. M.; Barrientos, A. G.; Rojas, T. C.; Rojo, J.; Canada, J.; Fernandez, A.; Penades, S. Angew. Chem., Int. Ed. 2001, 113(12), 2217–2321. (15) Lundquist, J. J.; Toone, E. J. Chem. Rev. 2002, 102, (2), 555-578. (16) Babu, P.; Sinha, S.; Surolia, A. Bioconjugate Chem. 2007, 18(1), 146–151. (17) Chen, Y. F.; Ji, T. H.; Rosenzweig, Z. Nano Lett. 2003, 3(5), 581–584. (18) De la Fuente, J. D. M.; Penades, S. Tetrahedron Asymm. 2005, 16(2), 387– 391. (19) Earhart, C.; Jana, N. R.; Erathodiyil, N.; Ying, J. Y. Langmuir 2008, 24(12), 6215–6219. (20) Kikkeri, R.; Lepenies, B.; Adibekian, A.; Laurino, P.; Seeberger, P. H. J. Am. Chem. Soc. 2009, 131(6), 2110–2112. (21) Niikura, K.; Nishio, T.; Akita, H.; Matsuo, Y.; Kamitani, R.; Kogure, K.; Harashima, H.; Ijiro, K. ChemBioChem 2007, 8(4), 379–384. (22) Robinson, A.; Fang, J. M.; Chou, P. T.; Liao, K. W.; Chu, R. M.; Lee, S. J. ChemBioChem 2005, 6(10), 1899–1905.
Published on Web 02/01/2010
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express adhesion molecules to recruit leukocytes, thus developing inflammatory indications. It has been revealed that sugar moieties, such as lactosylceramide (Galβ4GlcβCer), and Gg3 (GalNAcβ4Galβ4GlcβCer) of epithelial cells play an important role in leukocyte-epithelium interaction, which recognizes β2 integrin (CD11b/CD18) on the leukocyte surface.23,24 A recent report further demonstrates that synthetic di- and tetravalent lactosyl derivatives can specifically bind to CD11b on leucocytes.25 By mimicking the CD11b-binding oligosaccharide cluster of epithelia, we designed the polyvalent Lac-QDs to achieve specifically labeling of leukocytes. Here, we report facile synthesis of CdSeS/ZnS QD-lactose conjugate (Lac-QDs) by use of 1-thiol-substituent lactosyl derivative, 1-thiol-β-D-lactose, as the coordinating ligand. The preparation of 1-thiol-β-D-lactose can be easily performed by using lactose as the starting material. The prepared Lac-QDs exhibit significant affinity and specificity to leukocytes, while the monovalent lactose had no such efficacy. Moreover, the luminescent Lac-QDs have a small hydrodynamic diameter up to 8.2 nm, and display good colloidal stability and almost no cytotoxicity. These properties facilitate the use of Lac-QDs in fluorescent labeling of live leukocytes.
2. Materials and Methods 2.1. Chemicals. All chemicals were used directly without further purification: cadmium oxide (CdO, 99.0%, Shuanghuan Weiye Reagent Co.), sulfur powder (S, 99.5%, Sinopharm Chemical Reagent Co.), selenium powder (Se, 99.95%, Meixing Chemical Reagent Co.), oleic acid (OA, 97%, Kermel Chemical Reagent Co.), tri-n-octylphosphine (TOP, 99%, Fluka), tri-noctylamine (TOA, 97%, Yuanyue Chemical Co.), dimethyl sulfoxide (DMSO, 99%, Sigma-Aldrich), poly(maleic anhydride-alt1-tetradecene) (PMA, Sigma-Aldrich). Zinc oxide (ZnO, 99.0%), chloroform, and ethanol were purchased from Beijing Chemical Co. Ultrapure water (18.2 MΩ cm-1) was used in the experiments. 2.2. Instruments. Absorption spectra were acquired with a TU-1901 UV-vis spectrophotometer (Beijing Puxi Tongyong Co.). PL spectra were recorded on an F-4500 fluorescence spectrophotometer (Hitachi). Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a JEOL 300 MHz spectrometer (LA 300), and chemical shifts of the 1H NMR spectra were measured in reference to internal Me4Si (δ = 0 ppm). Fourier transform infrared (FTIR) spectra were acquired on a NEXUS470 FTIR spectrometer (Nicolet) using KBr pellets in the range from 4000 to 400 cm-1. Samples were dried overnight using a freeze-drier (Alpha 1-4, Christ). Data were collected at 8 cm-1 resolution with 256 scans. Transmission electron microscopy (TEM) and energy-dispersive X-ray (EDX) microanalysis were performed on an FEI Tecnai F30 high-resolution transmission electron microscope, operating at 300 kV with a point-to-point resolution of 0.205 nm. The samples were prepared by pipetting one drop of the nanoparticle suspension onto the carbon-coated copper grid and allowing to settle for 20 s. The residual solution was wicked away using an absorbent tissue. The nanoparticle size analysis was conducted by using Image J 1.34s (available at http:// rsb.info.nih.gov/ij/). Atomic force microscopy (AFM) imaging was conducted on a MultiMode atomic force microscope (23) Arnaout, M. A. Blood 1990, 75(5), 1037–1050. (24) Zen, K.; Parkos, C. A. Curr. Opin. Cell Biol. 2003, 15(5), 557–564. (25) Zhao, Z. H.; Li, Q.; Hu, J. L.; Li, Z. J.; Liu, J. H.; Liu, A. H.; Deng, P.; Zhang, L.; Gong, X. W.; Zhao, K. S.; Zhang, S. Q.; Jiang, Y. Glycoconjugate J. 2009, 26(2), 173–188.
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equipped with a Type IV controller (Veeco). About 10 μL of Lac-QD solution in water was pipetted onto a fresh cleaved mica surface and incubated for about 10 min. The specimen was gently rinsed with water and dried under a flow of nitrogen. AFM images were recorded in “tapping mode” in air and analyzed by using Nanoscope III v. 5.12 r2 (Veeco). The hydrodynamic diameters of the nanoparticles were measured on a Particle Sizing NICOMPTM 380 ZLS system. Lac-QDs were dispersed in PBS at a concentration of 0.05 mg mL-1. The particle size distribution analysis was conducted by the NICOMP number-weighted distribution analysis.26 The thermal properties of CdSeS/ZnS QDs and Lac-QDs were studied by using a thermoanalyzer, model Q 600 SDT (TA Co.), at a constant heating rate of 10 °C/min from 25 to 950 °C under a nitrogen atmosphere. Differential interference contrast (DIC) and fluorescent imaging were performed on an IX 71 inverted microscope with a 60 oil immersion objective (NA 1.45; Olympus), equipped with an EM-CCD (iXon DV-887; Andor). The digital images were collected and analyzed by using IP Lab software (BD Biosciences Bioimaging). 2.3. Preparation of Lac-QDs. CdSeS/ZnS QDs were prepared by the method described in the literature with minor modifications.27 Typically, CdO (0.05 g), oleic acid (OA, 0.46 g), and tri-n-octylamine (TOA, 15 mL) were mixed in a three-necked flask and heated to 300 °C under an argon atmosphere to get a clear solution. A stock solution of Se (2.1 mg) and S (12.4 mg) in 1.0 mL TOP was swiftly injected into the hot solution, and the reaction was allowed to proceed at 280 °C for 1 min. ZnS stock solution (a mixture of 0.2 mM ZnO powder in 1.0 mL oleic acid and 0.2 mM S powder in 1.0 mL TOP) was then added dropwise (ca. 1 drop per second) at 220-240 °C. CdSeS/ZnS QDs were obtained with ethanol sedimentation and repeatedly washed with ethanol. The prepared CdSeS/ZnS QDs displayed a PL emission (λem, max 551 nm) with the full width at the halfmaximum (fwhm) less than 30 nm. Supporting Information Figure S1 shows the HRTEM and EDX analyses of CdSeS/ ZnS QDs. The synthesis of 1-thiol-β-D-lactose was described in the Supporting Information (SI). The as-prepared CdSeS/ZnS QDs (13 mg) was first dispersed in chloroform (10 mL). A solution of 1-thiol-β-D-lactose (65 mg) in 50 mM phosphate buffered saline (PBS, 30 mL; pH 8.0) was added, and the mixture was vigorously stirred for 2 h at room temperature. After standing for a while, two layers separated naturally. The upper aqueous layer was collected and washed with chloroform. The Lac-QDs were collected by centrifugation (30 000 rpm for 5 min), and the nanoparticles were redispersed in PBS or ultrapure water. Three cycles were conducted to obtain the Lac-QD powder and stored at 4 °C before use. 2.4. Leukocyte and Hela Cells Preparation, Labeling, and Fluorescent Imaging. Leukocyte preparation was performed in accordance with the guidelines of the National Institute of Health of USA. Whole blood was withdrawn from SpragueDawley rats, and the leukocytes isolation was carried out according to the literature.25 Briefly, the heparinized whole blood was mixed with red blood cell lysis buffer (KHCO3 1.0 g, NH4Cl 8.3 g, EDTA-Na2 37 mg, add sterile water to 1000 mL, adjust pH to 7.2 with HCl) at a ratio of 1:10 (v/v). The mixture was incubated at room temperature for 5 min with intermittent shaking. The reaction was stopped by dilution of the mixture with 20 vol of isotonic PBS (150 mM; pH 7.4). The cells were centrifuged at 400 g for 5 min, the supernatant was removed, and the cell pellet (26) Choi, Y. S.; Mecke, A.; Orr, B. G.; Holl, M. M. B.; Baker, J. R. Nano Lett. 2004, 4(3), 391–397. (27) Jang, E.; Jun, S.; Pu, L. Chem. Commun. 2003, 24, 2964–2965.
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was resuspended in PBS at a concentration of about 1 106 mL-1. The cell suspension (90 μL) was transform to Eppendorf tubes (0.5 mL) and incubated with Lac-QDs (10 μL, 1.0 mg mL-1) or Alexa-labeled lactose (10 μL, 0.6 mg mL-1; the synthesis was described in SI). The labeled cells were centrifuged and washed with PBS to remove unbonded Lac-QDs or Alexa-labeled lactose. The cell suspension (100 μL) was transferred to a 35 mm Petri dish for DIC and fluorescent imaging. Hela cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing fetal bovine serum (FBS, 10%; Beijing Xinjingke Biotech. Co.) and penicillin/streptomycin (1%, Beijing Xinjingke Biotech. Co.). The cultured cells were trypsinized and resuspended in DMEM with FBS (10%) and 1% penicillin/streptomycin at a concentration of about 1 106 mL-1. The cell suspension (90 μL) was transferred to 35 mm Petri dishes. After 24 h of incubation, the cells were carefully rinsed with PBS (pH 7.4). For each Petri dish, DMEM (90 μL) and Lac-QDs (10 μL, 1.0 mg mL-1) added and incubated for 12 h. The cells were carefully rinsed with PBS to remove unbonded LacQDs, and the serum-free medium (100 μL) was added to the Petri dishes for DIC and fluorescent imaging. 2.5. Phototabilities against pH and Reductive Glutathione (GSH). The experiments were performed on an F-4500 fluorescence spectrophotometer (Hitachi) equipped with a temperature controller. The excitation and emission wavelengths are set at 400 and 551 nm, respectively. The split widths of excitation and emission were set at 2.5 nm. A quartz cuvette with a path length of 1 cm was used. The concentration of LacQDs was about 0.1 mg mL-1 for all experiments. To test the pHrelevant photostability, the PL spectra of Lac-QDs were recorded at different pH values, 37 °C. HCl and NaOH were used to adjust the pH from 4 to 9. The PL measurement was repeated three times at each pH point. To reveal the photostability of Lac-QDs against GSH, we prepared the Lac-QD solution in PBS containing 2.0 μM GSH (Beijing Biodee Biotech. Co.). The PL spectra were recorded at different times (1, 15, 30, 60, 120, 180, and 300 min). The time-course curve was obtained by plotting the PL intensity (551 nm) with incubation times. During the experiments, the quartz cuvette was sealed and no extra protection against O2 was conducted. 2.6. Cytotoxicity Measurement. The cytotoxicity measurements were performed according to a previously reported method.28 Briefly, Hela cells were trypsinized and resuspended in DMEM containing 10% FBS and 1% penicillin/streptomycin. The cells were seeded at a density of 5000 cells per well in a 96-well plate. After 24 h of incubation at 37 °C with 5% CO2 (the following steps were carried out under the same conditions), the cells were washed with PBS (100 mM Na2HPO4, 20 mM KH2PO4, 137 mM NaCl, 27 mM KCl, pH 7.4). The Lac-QDs and poly(maleic anhydride-alt-1-tetradecene) coated QDs (PMAQDs; the synthesis was described in SI) solutions with different concentrations (10 μL, 0.1-5.0 mg mL-1) and DMEM (90 μL) were added to the wells. After 24 h of incubation, the supernatant was removed, and the cells were washed with PBS three times. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Amresco) solution (10 μL, 0.5 mg mL-1) was then added to each well. After 4 h of incubation, the medium was discarded and the intracellular water-insoluble formazan blue was collected by DMSO (100 μL). The optical absorbance was measured at 570 nm on a BioRad model 550 microplate reader. Each data point was collected by averaging that of six wells, and the untreated cells were used as controls. The data analyses were (28) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2004, 4(1), 11–18.
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Yu et al. Scheme 1.
a
a (a) Schematic illustration of ligand exchange of TOPO (tri-n-octylphosphine oxide) and OA (oleic acid)-capped CdSeS/ZnS QDs with 1-thiol-β-D-lactose. (b) Lac-QDs hold a multivalent lactosyl ligand displaying; between the adjacent ligands, the spacing is about 1.31.6 nm, and the angle is about 19°.
conducted by Student’s t-test. Differences with P values of
