FULL PAPER DOI: 10.1002/asia.201301692
The Application of CdSe Quantum Dots with Multicolor Emission as Fluorescent Probes for Cell Labeling Mei-Xia Zhao,* Yang Li, Er-Zao Zeng, and Chao-Jie Wang*[a] Abstract: Herein, highly luminescent CdSe quantum dots (QDs) with emissions from the blue to the red region of visible light were synthesized by using a simple method. The emission range of the CdSe QDs could be tuned from l = 503 to 606 nm by controlling the size of the CdSe QDs. Two amino acids, l-tryptophan (l-Trp) and l-arginine (l-Arg), were used as coating agents. The quantum yield (QY) of CdSe QDs (green color) with an optimized thickness could reach up to
52 %. The structures and compositions of QDs were examined by using X-ray diffraction (XRD) and transmission electron microscopy (TEM). Optical properties were studied by using UV/ Vis and photoluminescence (PL) spectroscopy and a comparison was made between uncoated and coated CdSe Keywords: amino acids · fluorescent probes · labeling cells · multicolor · quantum dots
Introduction
lable luminescent CdSe QDs with a simple synthetic technique under mild conditions. For fluorescent probes, the main technical problems with the surface coating is that the size of the coating on QDs with biomolecules should be maintained.[2, 6] To render the QDs water soluble, different methods have been developed to make CdSe QDs water soluble so that biomolecules can be either covalently or noncovalently attached to the surface of QDs.[7] However, it was found that, after attaching to the bio-molecules, the quantum yields (QYs) of the QDs dropped significantly and sometimes the as-prepared QDs were only slightly soluble and encountered stability problems in water due to the strong polarity of water molecules.[8] Although the mechanism of the QY decrease is still not clear, it has been attributed to surface defects generated by ligand exchange between the new molecules and the original ligand tri-n-octylphosphine oxide (TOPO) molecules.[9] Cellpenetrating peptides, such as cysteine and twin-arginine translocation (Tat), have been used to deliver QDs into living cells,[2, 10] but the delivery mechanism and behavior of intracellular QDs are still under debate. Herein, we report the synthesis and properties of CdSe QDs with emissions from the blue to the red visible-light region by using a simple synthetic method that results in high QYs. In this synthesis process, CdAc2 and Na2SeSO3 were chosen as precursors instead of precursor preparation and oleic acid was used as the capping ligand. By controlling the growth temperature of the particle, which influenced the rate at which the capping ligand attached and detached to the particles, CdSe products with emissions ranging from the green to the red visible-light region could be obtained. With increasing thickness of the CdSe QDs, the emission of the
Semiconductor quantum dots (QDs) have attracted wide interest from researchers in various disciplines in the past two decades, due to their unique size-dependent optical properties and potential applications in biological and biomedical research.[1] CdSe QDs have been the most commonly used optical material for bioconjugation.[2] The most important aspects to be considered in the surface bioconjugation of QDs are that the fundamental properties of quantum dots remain unaffected, uniform dispersion is maintained, and the conjugated molecules or functional groups become available for biological applications.[2] Although various approaches have been reported to synthesize CdSe nanocrystals,[3] only few of them could be used to synthesize CdSe nanocrystals with satisfactory size distribution and most of them require rigorous experimental conditions. For example, the preparation of CdSe QDs in organic medium often requires a high reaction temperature.[4] However, the high reaction temperature often leads to a fast reaction rate and complicated manipulation, which may limit its application in various disciplines.[5] Therefore, it is still a challenge to develop a synthetic method to prepare highly effective control[a] Dr. M.-X. Zhao, Y. Li, E.-Z. Zeng, Prof. C.-J. Wang Key Laboratory of Natural Medicine and Immune Engineering Henan University Kaifeng 475004 (China) Fax: (+ 86) 371-22864665 E-mail: [email protected] [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201301692.
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QDs. The amino acid-modified b-cyclodextrin (CD)-coated CdSe QDs presented lower cytotoxicity to cells for 48 h. Furthermore, amino acid-modified b-CD-coated green CdSe QDs in HepG2 cells were assessed by using confocal laser scanning fluorescence microscopy. The results showed that amino acid-modified b-CD-coated green CdSe QDs could enter tumor cells efficiently and indicated that biomolecule-coated QDs could be used as a potential fluorescent probe.
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obtained QDs could be tuned continuously from blue to red. The resulting QDs showed dramatically improved QYs. The QY of synthesized CdSe at 100 8C was estimated to be up to 52 % in the best case. To demonstrate the use of their high fluorescence and potential for bioimaging applications, the amino acid-conjugated CdSe QDs with green emissions were employed as a fluorescent probe for labeling tumor cells observed by using a fluorescence microscope.
Mei-Xia Zhao, Chao-Jie Wang et al.
have a zinc blende (sphalerite) structure. The diffraction pattern was almost the same as that of a ZnSe nanocrystal of the same size, so the main diffraction peaks could be identified as (111), (220), and (311) crystal planes. The relatively low peak height and large peak width of the (111) peak compared with the (220) peak in the diffraction pattern are due to stacking faults perpendicular to the c axis. In addition, the stronger intensity of the diffraction peaks and the appearance of the minor diffraction peak (311) indicated a better crystallization of the products. It is known that QDs are predominately used as fluorescent probes because of the size-dependent properties of QDs.[11] Here, the typical CdSe QDs sample, which was extracted from a precipitated solution of CdSe in ethanol after reacting for 10 h and dissolved in n-hexane, was first observed by using a digital camera. In the particle growth process, we obtained CdSe QDs with a full range of color emissions; photographs of the CdSe QDs samples taken under a portable UV lamp are shown in Figure 2. During the growth of the CdSe QDs, the emission color of the CdSe
Results and Discussion Figure 1 shows the X-ray diffraction (XRD) patterns of the CdSe QDs at different reaction temperatures. The patterns indicated the formation of CdSe QDs and the deep valley at 2q values of around 35 and 458 indicated that the samples
Figure 1. XRD pattern of the CdSe QDs with reaction temperatures of a) 80, b) 100, c) 120, and (d) 150 8C.
Figure 2. Photo of the fluorescent CdSe QDs with different emissions at reaction temperatures of a) 50, b) 80, c) 100, d) 120, and e) 150 8C (lex = 365 nm). Taken under UV irradiation by using a digital camera in a darkroom.
Abstract in Chinese:
gradually changed from blue to red, which reveals a moderate growth rate of CdSe QDs. These CdSe QDs remained stable under ambient conditions and remained fluorescent for at least six months, which may make them potential candidates for applications in labeling cells. Figures 3 and 4 display the typical UV/Vis absorption and photoluminescence (PL) spectra (lex = 460 nm) of the CdSe QDs with sequential increasing thickness at different temperatures. According to the UV/Vis absorption spectra in Figure 3, we found that a significant redshift i the emission band appears with increased CdSe thickness. In addition, the first excitation peak in the UV/Vis absorption spectra shifted to longer wavelengths when the QDs become thicker. The PL intensity of the QDs increased initially but then decreased as the temperature was increased. The CdSe QDs at 100 8C exhibited photoemission at l = 539 nm, with a fullwidth at half-maximum (FWHM) of 28 nm and a PL QY of
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Figure 3. UV/Vis absorbance spectra of the CdSe QDs at different growth temperatures and at the same reaction time of 10 h; a) 50, b) 80, c) 100, d) 120, and e) 150 8C.
Figure 5. TEM images of naked CdSe QDs in CHCl3 grown at a) 50 and b) 100 8C. c) l-Trp-b-CD-coated CdSe QDs in PBS and d) l-Arg-b-CDcoated CdSe QDs in PBS.
Figure 4. PL spectra of the CdSe QDs at different growth temperatures and at the same reaction time of 10 h; a) l = 503 (50 8C), b) 526 (80 8C), c) 552 (100 8C), d) 578 (120 8C), and e) 606 nm (150 8C).
52 %. The QYs of CdSe QDs at 50, 80, 120, and 150 8C were approximately 15, 28, 32, and 36 %, respectively. The UV/ Vis absorption and PL spectra indicated that, under the same reaction conditions, the particles grew faster and larger at higher temperatures. The formation of larger particles at higher growth temperatures could be explained by the kinetics of the particle formation. TEM images of the samples with different emissions are shown in Figure 5 and reveal that the CdSe QDs are nearly spherical particles with crystalline structures and possess near monodispersity. The diameters of CdSe QDs ranged from 2.6 to 4.2 nm as determined by using statistical analysis. Another objective is to encapsulate as-synthesized CdSe QDs with a water-soluble amino acid-modified b-cyclodextrin (CD) shell to obtain a water-soluble composite material. We aimed to synthesize amino acid-modified b-CD-coated CdSe QDs with green emission and research their bioactivity. Figure 6 displays the synthesis of water-soluble and biologically compatible l-Arg-b-CD and l-Trp-b-CD-coated
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Figure 6. Schematic illustration of the preparation of amino acid-modified b-CD-coated CdSe QDs (R = l-Arg, and l-Trp residues).
green CdSe QDs. The amino acid-modified b-CD was characterized by using ESI-MS, 1H NMR spectroscopy (Figures S1, S2 in the Supporting Information), and elemental analysis. The UV/Vis spectra of the CdSe QDs with green emission and the corresponding amino acid-modified b-CDcoated CdSe QDs are shown in Figure 7. There is no difference in the position or width of the absorbance bands compared with hydrophobic CdSe QDs, which suggests that the amino acid-modified b-CD-coated CdSe QDs in phosphate buffered saline (PBS) retain the optical properties of the naked CdSe QDs. The PL spectra of CdSe QDs with green emission and the corresponding amino acid-modified b-CDcoated CdSe QDs are shown in Figure 8. Compared with naked CdSe QDs, the PL spectra of the amino acid-modi-
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Figure 9. Cell viability of CdSe QDs, l-Arg-b-CD-coated CdSe QDs, and l-Trp-b-CD-coated CdSe QDs with HeLa cells, HepG2 cells, and QSG7701 cells. Cells were incubated with samples at concentrations of 20 or 40 mg mL 1 at 37 8C for 48 h.
Figure 7. UV/Vis absorbance spectra of a) CdSe QDs with green emission in hexane, b) l-Arg-b-CD-coated CdSe QDs in PBS, and c) l-Trp-b-CDcoated CdSe QDs in PBS.
same condition. This suggested that the coating of amino acid-modified b-CD can decrease the cytotoxicity of naked CdSe QDs, that is, it can improve water solubility and biocompatibility of QDs. The cell viabilities of these QDs are shown in Figure 9. The IC50 values of these cells incubated with amino acid-modified b-CD-coated CdSe QDs are in the range of (42.3 4.2) to (95.6 5.4) mg mL 1. The IC50 values of the QDs found in the MTT assay at 48 h are shown in Table 1. Obviously, the cytotoxicity of the amino acid-modified b-CD-coated CdSe QDs in the three kinds of Table 1. Summary of the cytotoxicity activity of QDs. HeLa
Figure 8. PL spectra of a) CdSe QDs with green emissions in hexane, b) l-Arg-b-CD-coated CdSe QDs in PBS, and c) l-Trp-b-CD-coated CdSe QDs in PBS.
CdSe l-Arg-b-CD-coated CdSe l-Trp-b-CD-coated CdSe
QSG-7701
11.7 3.2 45.8 3.4 42.3 4.1
8.6 2.6 95.6 5.4 89.3 4.2
[a] The data are presented as mean values with standard deviations and cell viability was assessed after incubation for 48 h.
fied b-CD-coated CdSe QDs obviously shift to higher wavelengths from l = 539 to 550 and 552 nm. The PL spectra of the amino acid-modified b-CD-coated CdSe QDs are redshifted, which can be explained by their increased size due to ligand exchange. The QYs of l-Arg-b-CD-coated CdSe and l-Trp-b-CD-coated CdSe green QDs in PBS were measured and compared with the value of rhodamine B (QY = 89 %, EtOH) as a criterion at room temperature, and were found to be approximately 35 and 46 %, respectively. The TEM images (Figure 5) show that the sizes of the l-Arg-bCD-coated CdSe QDs and l-Trp-b-CD-coated CdSe QDs are virtually identical, which indicates that the modified particles are monodisperse and uniform in PBS. The cytotoxicity experiment was measured by using a standard methylthiazole tetrazolium (MTT) assay.[12] Cytotoxicity was examined by incubating the three cells (HeLa, HepG2, and QSG-7701 cells) with different concentrations of QDs for 48 h. After this incubation period, the CdSe QDs showed enhanced toxicity even at the lowest concentration of 10 mg mL 1, whereas the amino acid-modified bCD-coated CdSe QDs showed lower toxicity under the
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10.6 2.5 46.2 4.6 42.5 4.2
IC50 [mg mL 1][a] HepG2
cells was lower in our studies. Interestingly, the amino acidmodified b-CD-coated CdSe QDs showed less toxicity towards the normal QSG-7701 cells compared with other two types of cancer cells. Our data indicated that the release of Cd2 + from the QDs surface can be reduced by employing particles coated with biomolecules. Because the amino acid-modified b-CD-coated CdSe QDs showed good optical properties and minimal toxicity, their specific binding to the surface of living cells was investigated by using HepG2 cells in vitro. Figure 10 shows the flow cytometry histograms of HepG2 cells incubated with l-Arg-bCD-coated CdSe QDs. On the basis of the average fluorescence intensity of cells, it was apparent that a substantial intracellular uptake of l-Arg-b-CD-coated CdSe QDs took place in HepG2 cancer cells. The average fluorescence intensity of HepG2 cells was increased 11 times compared with the control ( 73.9 to 6.4). The results provide strong evidence for the high uptake of amino acid-modified b-CD-
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Conclusion In summary, good-quality CdSe QDs with multicolor emission were obtained by using a simple and environmentally friendly method, and the green CdSe QDs synthesized with higher QYs were coated with amino acids by using a sonochemical method in which l-Arg and l-Trp acted as surface ligands. The shape- and the size-dependent optical and magnetic properties of the CdSe QDs were studied, and these QDs presented good dispersion, uniformity, and excellent fluorescent properties. Compared with naked CdSe QDs, the PL spectra of amino acid-modified b-CD-coated green CdSe QDs with a large size distribution presented an obvious redshift. l-Arg-b-CD-coated green CdSe QDs incubated with HepG2 cells were capable of locating to the cytoplasm of cells, which indicated that the QDs could be useful for tracking cells and intracellular processes. Therefore, biomolecule-coated QDs are expected to be used in biolabeling, bioimaging, or other applications in vivo.
Figure 10. Flow cytometry data of l-Arg-b-CD-coated CdSe QDs in HepG2 cells.
coated CdSe QDs in cells. The possible mechanism of intracellular uptake of amino acid-modified QDs is that both clathrin-mediated endocytosis and the electrostatic interaction between the positive charge of the amino acids and the negatively charged plasma membrane played important roles in the intracellular delivery of QD.[13] To further confirm the cell labeling of amino acid-modified b-CD-coated CdSe QDs, HepG2 cells were treated with l-Arg-b-CD-coated CdSe QDs. Confocal microscopy was used to investigate cell labeling by the green QDs and observe their fluorescent emission. The green QD samples were excited by using a green laser (l = 480 nm), and the nuclei, stained with Hoechst 33342 solution, were excited by using a blue laser (l = 405 nm). In Figure 11, many green
Experimental Section Materials and Reagents Selenium powder (99.99 %), Na2SO3 (98.0 %), oleic acid (OA, 90 %), and [Cd(Ac)2]·2 H2O were purchased from Alfa Aesar (Karlsruhe, Germany). The selenium precursor solution of Na2SeSO3 was prepared by heating selenium powder (5 mmol) and Na2SO3 (6 mmol) in of distilled water (50 mL) at reflux for 3 h. b-Cyclodextrin (b-CD), triethanolamine, p-toluenesulfonyl chloride, l-tryptophan (l-Trp), l-arginine (l-Arg), ethanol, NaOH, and n-hexane were obtained from Shanghai Chemical Factory, China. b-CD was recrystallized three times from distilled water and dried under vacuum for 12 h at 95 8C before use. Fetal bovine serum (FBS) was purchased from Gibco (Life Technologies AG, Switzerland). Dulbecco minimum essential medium (DMEM) was from Invitrogen Corporation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst 33342 were from Sigma. Other organic solvents were purchased from EM Sciences. Characterization Powder X-ray diffraction (XRD) of the products were carried out by using a Shimadzu XRD-6000 X-ray diffractometer equipped with CuKa radiation (l = 0.154060 nm) and recorded at a scanning rate of 0.02 s 1; 2q ranged from 15–708. The transmission electron micrograph (TEM) measurements were performed by using a JEOL JEM-200CX microscope. UV/Vis absorption spectra were acquired by using a PerkinElmer Lambda-850 UV/Vis spectrometer. Fluorescence spectra were acquired by using a PerkinElmer Ls55 fluorescence spectrometer with excitation at l = 480 nm. QYs for TOPO-coated CdSe in hexane and amino acidmodified b-CD-coated CdSe in PBS were evaluated against rhodamine B (RhB). Confocal microscopy images were obtained by using a confocal laser scanning fluorescence microscopy equipped with a laser at l = 480 nm.
Figure 11. Confocal microscopy imaging of l-Arg-b-CD-coated CdSe QDs in HepG2 cells. a) Fluorescent images of HepG2 cells with l-Arg-bCD-coated CdSe QDs, b) fluorescent images of Hoechst 33342-stained HepG2 cells, c) an overlay of fluorescent images of the l-Arg-b-CDcoated CdSe QDs labeled HepG2 cells. (HepG2 cells were treated with 40 mg mL 1 of l-Arg-b-CD-coated CdSe QDs).
fluorescent dots are present in the cytoplasm of the cells, which shows that the green l-Arg-b-CD-coated CdSe QDs can permeate the cell membrane and enter the cells. Notably, no obvious heavy aggregates settled in the cytoplasm. Therefore, the high brightness of the QDs made it possible to record fluorescent images for cells or in vivo and leads to the observation of QD localization. These results indicate the potential of using biomaterial-coated QDs systems for cell fluorescent labeling.
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Synthesis of l-Amino Acid-Modified b-Cyclodextrin l-Amino acid-modified b-cyclodextrin was synthesized based on a previous method with slight modification.[14] Briefly, monoACHTUNGRE[6-O-(p-toluene-sulfonyl)]-b-cyclodextrin (0.77 mmol) and amino acid (l-Trp and l-Arg; 2.3 mmol) were dissolved in water (30 mL) that contained triethanolamine (20 mL) at 85 8C with stirring for 24 h under N2. After evaporation of most of the solvent under reduced pressure, the resulting solution was poured into vigorously stirred anhydrous ethanol (500 mL), and the resultant mixture was stored in a refrigerator 18 h to produce a pale yellow precipitate. The solid product was collected by centrifugation and then
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purified by chromatography on a Sephadex G-25 column with deionizated water as the eluent, followed by repeated recrystallization from water and ethanol to give pale yellow product. Elemental analysis calcd (%) for l-Arg-b-CD complex (C48H82O36N4·10 H2O): C 39.18, H 6.99, N 3.81; found: C 39.32, H 6.65, N 3.81; elemental analysis calcd (%) for l-Trp-bCD complex (C53H80O36N2·6 H2O): C 44.54, H 6.49, N 1.96; found: C 44.28, H 6.28, N 1.99.
laser scanning fluorescence microscopy was used to detect the fluorescence in the cells by using l = 488 nm laser to excite the QDs.
Author Contributions M. X. Zhao, Y. Li and E.-Z. Zeng performed the experiments. M. X. Zhao, Y. Li, E.-Z. Zeng, and C.-J. Wang reviewed, analyzed, and interpreted the data. M. X. Zhao and C.-J. Wang wrote the paper. All authors discussed the results and commented on the manuscript.
Preparation of CdSe QDs QDs with different emission wavelengths were prepared according to a modified literature approach.[15] Typically, [Cd(Ac)2]·2 H2O (0.133 g) dissolved in deionized water (5 mL) was added with stirring to a solution of OA (4 mL) and NaOH (0.35 g) in C2H5OH/deionized water (20 mL, 15:5 v/v). Na2SeSO3 solution (5 mL, 0.1 m) was added to the mixture. After reacting for about 5 min, the mixture was transferred to several autoclaves and sealed. These autoclaves were held at the designed temperature (50, 80, 100, 120, and 150 8C) for 10 h. When the reaction was complete, the products deposited in the autoclaves were dissolved in nhexane, then the raw products were separated by ethanol precipitation followed by centrifugation. Afterwards, the precipitation was repeatedly purified from n-hexane and ethanol. Finally, the final CdSe quantum dot products were redissolved in n-hexane and used in the following measurements.
Acknowledgements This work was supported by the National Natural Science Foundation of China (U1204201), the Postdoctoral Science Surface Foundation of China (no. 2012M511570), and the Department of Education Science and Technology Research Key Projects of Henan (13A150063).
[1] a) D. Zhang, G. Li, H. Li, Y. Lu, Chem. Asian J. 2013, 8, 26 – 40; b) P. Wu, X.-P. Yan, Chem. Soc. Rev. 2013, 42, 5489 – 5521; c) F. Zhao, Y. Zhao, Y. Liu, X. Chang, C. Chen, Y. Zhao, Small 2011, 7, 1322 – 1337; d) V. Biju, T. Itoh, A. Anas, A. Sujith, M. Ishikawa, Anal. Bioanal. Chem. 2008, 391, 2469 – 2495. [2] a) V. Biju, Chem. Soc. Rev. 2014, 43, 744 – 764; b) V. Biju, T. Itoh, M. Ishikawa, Chem. Soc. Rev. 2010, 39, 3031 – 3056. [3] a) W. R. Siah, A. P. LaGrow, M. J. Banholzer, R. D. Tilley, Cryst. Growth Des. 2013, 13, 2486 – 2492; b) M. M. Moghaddam, M. Baghbanzadeh, A. Sadeghpour, O. Glatter, C. O. Kappe, Chem. Eur. J. 2013, 19, 11 629 – 11 636; c) M. M. Moghaddam, M. Baghbanzadeh, A. Keilbach, C. O. Kappe, Nanoscale 2012, 4, 7435 – 7442; d) S. Taniguchi, M. Green, S. B. Rizvi, A. Seifalian, J. Mater. Chem. 2011, 21, 2877 – 2882; e) X. Jiang, M. Ahmed, Z. Deng, R. Narain, Bioconjugate Chem. 2009, 20, 994 – 1001; f) L. Qu, X. Peng, J. Am. Chem. Soc. 2002, 124, 2049 – 2055. [4] a) A. J. Morris-Cohen, M. T. Frederick, G. D. Lilly, E. A. McArthur, E. A. Weiss, J. Phys. Chem. Lett. 2010, 1, 1078 – 1081; b) Z. A. Peng, X. G. Peng, J. Am. Chem. Soc. 2001, 123, 183 – 184; c) C. B. Murray, D. J. Norris, M. G. Bawendi, J. Am. Chem. Soc. 1993, 115, 8706 – 8715. [5] a) Y. A. Yang, H. Wu, K. R. Williams, Y. C. Cao, Angew. Chem. Int. Ed. 2005, 44, 6712 – 6715; Angew. Chem. 2005, 117, 6870 – 6873; b) L. H. Qu, W. W. Yu, X. G. Peng, Nano Lett. 2004, 4, 465 – 469. [6] a) W. Xiong, D. D. Hickstein, K. J. Schnitzenbaumer, J. L. Ellis, B. B. Palm, K. E. Keister, C. Ding, L. Miaja-Avila, G. Dukovic, J. L. Jimenez, M. M. Murnane, H. C. Kapteyn, Nano Lett. 2013, 13, 2924 – 2930; b) J. Chen, H. Jiao, W. Li, D. Liao, H. Zhou, C. Yu, Chem. Asian J. 2013, 8, 276 – 281; c) Y. Li, Y. Zhou, H.-Y. Wang, S. Perrett, Y. Zhao, Z. Tang, G. Nie, Angew. Chem. Int. Ed. 2011, 50, 5860 – 5864; Angew. Chem. 2011, 123, 5982 – 5986; d) R. Viswanatha, D. D. Sarma, Chem. Asian J. 2009, 4, 904 – 909; e) X. Gao, L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, S. Nie, Curr. Opin. Biotechnol. 2005, 16, 63 – 72. [7] a) C.-P. Huang, C.-F. Chao, M.-Y. Shen, T.-M. Chen, Y.-K. Li, Chem. Asian J. 2012, 7, 2848 – 2853; b) N. Varghese, K. Biswas, C. N. Rao, Chem. Asian J. 2008, 3, 1435 – 1442; c) X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, S. Weiss, Science 2005, 307, 538 – 544; d) K. Narayanan, S. K. Yen, Q. Dou, P. Padmanabhan, T. Sudhaharan, S. Ahmed, J. Y. Ying, S. T. Selvan, Sci. Rep. 2013, 3, 2184, DOI: 10.1038/srep02184; e) S. T. Selvan, Biointerphases 2010, 5, FA110 – FA115; f) F. Pinaud, D. King, H.-P. Moore, S. Weiss, J. Am. Chem. Soc. 2004, 126, 6115 – 6123; g) T. Pellegrino, L. Manna, S. Kudera, T. Liedl, D. Koktysh, A. L. Rogach, S. Keller, J. Rdler, G. Natile, W. J. Parak, Nano Lett. 2004, 4, 703 – 707. [8] a) F. A. Esteve-Turrillas, A. Abad-Fuentes, Biosens. Bioelectron. 2013, 41, 12 – 29; b) D. Painuly, A. Bhatt, V. K. Krishnan, J. Biomed.
Synthesis of Amino Acid-Modified b-CD-Coated CdSe Amino acid-modified b-CD-coated CdSe QDs were formed by surface modification of the QDs. The synthesis was based on our previous method with slight modification.[16] A mixture of purified CdSe QDs (0.016 m) in chloroform (2.0 mL) and amino acid-modified b-CD (10 mg mL 1) in water (2.0 mL) was sonicated for 4 h. Triethanolamine (25 %, w/v) was added to raise the pH of the mixture above 10.0 to initiate surfactant exchange. Then ethyl acetate was added to precipitate the nanocrystal complexes and purify the nanocrystals from side products and unreacted precursors. Cell Culture HepG2 (human hepatocellular liver carcinoma cell), HeLa, and QSG7701 cells were maintained in DMEM medium supplemented with 10 % (v/v) heat-inactivated FBS and 1 % (v/v) penicillin–streptomycin (100 U mL 1 penicillin and 100 mg mL 1 streptomycin), and cultured in a 5 % CO2 atmosphere at 37 8C. Thereafter, the medium was replaced every third day. The adherent cells were allowed to reach 80 % confluence (7–10 days for the first passage). Cells were passaged in culture, and passage 3 cells (P3) were used for experiments. Cell Viability Assay The cytotoxicity of the samples was assayed by using a modified 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, according to a previous method.[17] Briefly, cells were seeded into 96-well plates at 5 104 cells per well. After 24 h, various concentrations (5, 10, 20, 50, 100, and 200 mg mL 1) of QDs were subsequently added and incubated for 48 h. MTT (20 mL, 2.5 mg mL 1) was added to each well and the plates were incubated for 4 h at 37 8C under 5 % CO2. After the medium was removed, DMSO (100 mL) was added to each well. The OD570 of QDs was measured at l = 690 nm by using an Infinite M200 monochromator-based multifunction microplate reader. Cell Labeling Fluorescent images were obtained from l-Arg-b-CD-conjugated CdSe QDs in HepG2 cells and were recorded by using confocal laser scanning fluorescence microscopy. HepG2 cells were seeded in culture medium that contained 10 % FBS in a utensil (35 mm diameter) that contained glass cover-slips (15 mm diameter) and incubated for 24 h at 37 8C and 5 % CO2. Pristine l-Arg-b-CD-conjugated CdSe QD probes containing medium solution were then added to the cells at a concentration of 40 mg mL 1. The cells were incubated at 37 8C in 5 % CO2 for 6 h, and then washed with PBS. After washing, the cell nuclei were stained by using Hoechst 33342 solution and mounted in glycerol buffer. Confocal
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[9] [10]
[11] [12]
[13] A. Anas, T. Okuda, N. Kawashima, K. Nakayama, T. Itoh, M. Ishikawa, V. Biju, ACS Nano 2009, 3, 2419 – 2429. [14] a) M.-X. Zhao, Q. Xia, X.-D. Feng, X.-H. Zhu, Z.-W. Mao, L.-N. Ji, K. Wang, Biomaterials 2010, 31, 4401 – 4408; b) Y. Liu, B. H. Han, S. X. Sun, T. Wada, Y. Inoue, J. Org. Chem. 1999, 64, 1487 – 1493. [15] P. Yang, M. Ando, N. Murase, Colloids Surf. A 2011, 390, 207 – 211. [16] M.-X. Zhao, H.-F. Huang, Q. Xia, L.-N. Ji, Z.-W. Mao, J. Mater. Chem. 2011, 21, 10290 – 10297. [17] H. Z. Wang, C. H. Chang, C. P. Lin, M. C. Tsai, J. Ocul. Pharmacol. Ther. 1996, 12, 35 – 43. Received: December 20, 2013 Published online: February 24, 2014
Nanotechnol. 2013, 9, 257 – 266; c) P. Babu, S. Sinha, A. Surolia, Bioconjugate Chem. 2007, 18, 146 – 151. F. Dubois, B. Mahler, B. Dubertret, E. Doris, C. Mioskowski, J. Am. Chem. Soc. 2007, 129, 482 – 483. a) S. B. Lowe, J. A. G. Dick, B. E. Cohen, M. M. Stevens, ACS Nano 2012, 6, 851 – 857; b) V. Biju, S. Mundayoor, R. V. Omkumar, A. Anas, M. Ishikawa, Biotechnol. Adv. 2010, 28, 199 – 213. J. Rodriguez-Viejo, K. F. Jensen, H. Mattoussi, J. Michel, B. O. Dabbousi, M. G. Bawendi, Appl. Phys. Lett. 1997, 70, 2132 – 2134. H. S. Baek, J. Y. Yoo, D. K. Rah, D.-W. Han, D. H. Lee, O.-H. Kwon, J.-C. Park, Yonsei Med. J. 2005, 46, 579 – 583.
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The Application of CdSe Quantum Dots with Multicolor Emission as Fluorescent Probes for Cell Labeling Mei-Xia Zhao,* Yang Li, Er-Zao Zeng, and Chao-Jie Wang*[a] Abstract: Herein, highly luminescent CdSe quantum dots (QDs) with emissions from the blue to the red region of visible light were synthesized by using a simple method. The emission range of the CdSe QDs could be tuned from l = 503 to 606 nm by controlling the size of the CdSe QDs. Two amino acids, l-tryptophan (l-Trp) and l-arginine (l-Arg), were used as coating agents. The quantum yield (QY) of CdSe QDs (green color) with an optimized thickness could reach up to
52 %. The structures and compositions of QDs were examined by using X-ray diffraction (XRD) and transmission electron microscopy (TEM). Optical properties were studied by using UV/ Vis and photoluminescence (PL) spectroscopy and a comparison was made between uncoated and coated CdSe Keywords: amino acids · fluorescent probes · labeling cells · multicolor · quantum dots
Introduction
lable luminescent CdSe QDs with a simple synthetic technique under mild conditions. For fluorescent probes, the main technical problems with the surface coating is that the size of the coating on QDs with biomolecules should be maintained.[2, 6] To render the QDs water soluble, different methods have been developed to make CdSe QDs water soluble so that biomolecules can be either covalently or noncovalently attached to the surface of QDs.[7] However, it was found that, after attaching to the bio-molecules, the quantum yields (QYs) of the QDs dropped significantly and sometimes the as-prepared QDs were only slightly soluble and encountered stability problems in water due to the strong polarity of water molecules.[8] Although the mechanism of the QY decrease is still not clear, it has been attributed to surface defects generated by ligand exchange between the new molecules and the original ligand tri-n-octylphosphine oxide (TOPO) molecules.[9] Cellpenetrating peptides, such as cysteine and twin-arginine translocation (Tat), have been used to deliver QDs into living cells,[2, 10] but the delivery mechanism and behavior of intracellular QDs are still under debate. Herein, we report the synthesis and properties of CdSe QDs with emissions from the blue to the red visible-light region by using a simple synthetic method that results in high QYs. In this synthesis process, CdAc2 and Na2SeSO3 were chosen as precursors instead of precursor preparation and oleic acid was used as the capping ligand. By controlling the growth temperature of the particle, which influenced the rate at which the capping ligand attached and detached to the particles, CdSe products with emissions ranging from the green to the red visible-light region could be obtained. With increasing thickness of the CdSe QDs, the emission of the
Semiconductor quantum dots (QDs) have attracted wide interest from researchers in various disciplines in the past two decades, due to their unique size-dependent optical properties and potential applications in biological and biomedical research.[1] CdSe QDs have been the most commonly used optical material for bioconjugation.[2] The most important aspects to be considered in the surface bioconjugation of QDs are that the fundamental properties of quantum dots remain unaffected, uniform dispersion is maintained, and the conjugated molecules or functional groups become available for biological applications.[2] Although various approaches have been reported to synthesize CdSe nanocrystals,[3] only few of them could be used to synthesize CdSe nanocrystals with satisfactory size distribution and most of them require rigorous experimental conditions. For example, the preparation of CdSe QDs in organic medium often requires a high reaction temperature.[4] However, the high reaction temperature often leads to a fast reaction rate and complicated manipulation, which may limit its application in various disciplines.[5] Therefore, it is still a challenge to develop a synthetic method to prepare highly effective control[a] Dr. M.-X. Zhao, Y. Li, E.-Z. Zeng, Prof. C.-J. Wang Key Laboratory of Natural Medicine and Immune Engineering Henan University Kaifeng 475004 (China) Fax: (+ 86) 371-22864665 E-mail: [email protected] [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201301692.
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QDs. The amino acid-modified b-cyclodextrin (CD)-coated CdSe QDs presented lower cytotoxicity to cells for 48 h. Furthermore, amino acid-modified b-CD-coated green CdSe QDs in HepG2 cells were assessed by using confocal laser scanning fluorescence microscopy. The results showed that amino acid-modified b-CD-coated green CdSe QDs could enter tumor cells efficiently and indicated that biomolecule-coated QDs could be used as a potential fluorescent probe.
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obtained QDs could be tuned continuously from blue to red. The resulting QDs showed dramatically improved QYs. The QY of synthesized CdSe at 100 8C was estimated to be up to 52 % in the best case. To demonstrate the use of their high fluorescence and potential for bioimaging applications, the amino acid-conjugated CdSe QDs with green emissions were employed as a fluorescent probe for labeling tumor cells observed by using a fluorescence microscope.
Mei-Xia Zhao, Chao-Jie Wang et al.
have a zinc blende (sphalerite) structure. The diffraction pattern was almost the same as that of a ZnSe nanocrystal of the same size, so the main diffraction peaks could be identified as (111), (220), and (311) crystal planes. The relatively low peak height and large peak width of the (111) peak compared with the (220) peak in the diffraction pattern are due to stacking faults perpendicular to the c axis. In addition, the stronger intensity of the diffraction peaks and the appearance of the minor diffraction peak (311) indicated a better crystallization of the products. It is known that QDs are predominately used as fluorescent probes because of the size-dependent properties of QDs.[11] Here, the typical CdSe QDs sample, which was extracted from a precipitated solution of CdSe in ethanol after reacting for 10 h and dissolved in n-hexane, was first observed by using a digital camera. In the particle growth process, we obtained CdSe QDs with a full range of color emissions; photographs of the CdSe QDs samples taken under a portable UV lamp are shown in Figure 2. During the growth of the CdSe QDs, the emission color of the CdSe
Results and Discussion Figure 1 shows the X-ray diffraction (XRD) patterns of the CdSe QDs at different reaction temperatures. The patterns indicated the formation of CdSe QDs and the deep valley at 2q values of around 35 and 458 indicated that the samples
Figure 1. XRD pattern of the CdSe QDs with reaction temperatures of a) 80, b) 100, c) 120, and (d) 150 8C.
Figure 2. Photo of the fluorescent CdSe QDs with different emissions at reaction temperatures of a) 50, b) 80, c) 100, d) 120, and e) 150 8C (lex = 365 nm). Taken under UV irradiation by using a digital camera in a darkroom.
Abstract in Chinese:
gradually changed from blue to red, which reveals a moderate growth rate of CdSe QDs. These CdSe QDs remained stable under ambient conditions and remained fluorescent for at least six months, which may make them potential candidates for applications in labeling cells. Figures 3 and 4 display the typical UV/Vis absorption and photoluminescence (PL) spectra (lex = 460 nm) of the CdSe QDs with sequential increasing thickness at different temperatures. According to the UV/Vis absorption spectra in Figure 3, we found that a significant redshift i the emission band appears with increased CdSe thickness. In addition, the first excitation peak in the UV/Vis absorption spectra shifted to longer wavelengths when the QDs become thicker. The PL intensity of the QDs increased initially but then decreased as the temperature was increased. The CdSe QDs at 100 8C exhibited photoemission at l = 539 nm, with a fullwidth at half-maximum (FWHM) of 28 nm and a PL QY of
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Figure 3. UV/Vis absorbance spectra of the CdSe QDs at different growth temperatures and at the same reaction time of 10 h; a) 50, b) 80, c) 100, d) 120, and e) 150 8C.
Figure 5. TEM images of naked CdSe QDs in CHCl3 grown at a) 50 and b) 100 8C. c) l-Trp-b-CD-coated CdSe QDs in PBS and d) l-Arg-b-CDcoated CdSe QDs in PBS.
Figure 4. PL spectra of the CdSe QDs at different growth temperatures and at the same reaction time of 10 h; a) l = 503 (50 8C), b) 526 (80 8C), c) 552 (100 8C), d) 578 (120 8C), and e) 606 nm (150 8C).
52 %. The QYs of CdSe QDs at 50, 80, 120, and 150 8C were approximately 15, 28, 32, and 36 %, respectively. The UV/ Vis absorption and PL spectra indicated that, under the same reaction conditions, the particles grew faster and larger at higher temperatures. The formation of larger particles at higher growth temperatures could be explained by the kinetics of the particle formation. TEM images of the samples with different emissions are shown in Figure 5 and reveal that the CdSe QDs are nearly spherical particles with crystalline structures and possess near monodispersity. The diameters of CdSe QDs ranged from 2.6 to 4.2 nm as determined by using statistical analysis. Another objective is to encapsulate as-synthesized CdSe QDs with a water-soluble amino acid-modified b-cyclodextrin (CD) shell to obtain a water-soluble composite material. We aimed to synthesize amino acid-modified b-CD-coated CdSe QDs with green emission and research their bioactivity. Figure 6 displays the synthesis of water-soluble and biologically compatible l-Arg-b-CD and l-Trp-b-CD-coated
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Figure 6. Schematic illustration of the preparation of amino acid-modified b-CD-coated CdSe QDs (R = l-Arg, and l-Trp residues).
green CdSe QDs. The amino acid-modified b-CD was characterized by using ESI-MS, 1H NMR spectroscopy (Figures S1, S2 in the Supporting Information), and elemental analysis. The UV/Vis spectra of the CdSe QDs with green emission and the corresponding amino acid-modified b-CDcoated CdSe QDs are shown in Figure 7. There is no difference in the position or width of the absorbance bands compared with hydrophobic CdSe QDs, which suggests that the amino acid-modified b-CD-coated CdSe QDs in phosphate buffered saline (PBS) retain the optical properties of the naked CdSe QDs. The PL spectra of CdSe QDs with green emission and the corresponding amino acid-modified b-CDcoated CdSe QDs are shown in Figure 8. Compared with naked CdSe QDs, the PL spectra of the amino acid-modi-
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Figure 9. Cell viability of CdSe QDs, l-Arg-b-CD-coated CdSe QDs, and l-Trp-b-CD-coated CdSe QDs with HeLa cells, HepG2 cells, and QSG7701 cells. Cells were incubated with samples at concentrations of 20 or 40 mg mL 1 at 37 8C for 48 h.
Figure 7. UV/Vis absorbance spectra of a) CdSe QDs with green emission in hexane, b) l-Arg-b-CD-coated CdSe QDs in PBS, and c) l-Trp-b-CDcoated CdSe QDs in PBS.
same condition. This suggested that the coating of amino acid-modified b-CD can decrease the cytotoxicity of naked CdSe QDs, that is, it can improve water solubility and biocompatibility of QDs. The cell viabilities of these QDs are shown in Figure 9. The IC50 values of these cells incubated with amino acid-modified b-CD-coated CdSe QDs are in the range of (42.3 4.2) to (95.6 5.4) mg mL 1. The IC50 values of the QDs found in the MTT assay at 48 h are shown in Table 1. Obviously, the cytotoxicity of the amino acid-modified b-CD-coated CdSe QDs in the three kinds of Table 1. Summary of the cytotoxicity activity of QDs. HeLa
Figure 8. PL spectra of a) CdSe QDs with green emissions in hexane, b) l-Arg-b-CD-coated CdSe QDs in PBS, and c) l-Trp-b-CD-coated CdSe QDs in PBS.
CdSe l-Arg-b-CD-coated CdSe l-Trp-b-CD-coated CdSe
QSG-7701
11.7 3.2 45.8 3.4 42.3 4.1
8.6 2.6 95.6 5.4 89.3 4.2
[a] The data are presented as mean values with standard deviations and cell viability was assessed after incubation for 48 h.
fied b-CD-coated CdSe QDs obviously shift to higher wavelengths from l = 539 to 550 and 552 nm. The PL spectra of the amino acid-modified b-CD-coated CdSe QDs are redshifted, which can be explained by their increased size due to ligand exchange. The QYs of l-Arg-b-CD-coated CdSe and l-Trp-b-CD-coated CdSe green QDs in PBS were measured and compared with the value of rhodamine B (QY = 89 %, EtOH) as a criterion at room temperature, and were found to be approximately 35 and 46 %, respectively. The TEM images (Figure 5) show that the sizes of the l-Arg-bCD-coated CdSe QDs and l-Trp-b-CD-coated CdSe QDs are virtually identical, which indicates that the modified particles are monodisperse and uniform in PBS. The cytotoxicity experiment was measured by using a standard methylthiazole tetrazolium (MTT) assay.[12] Cytotoxicity was examined by incubating the three cells (HeLa, HepG2, and QSG-7701 cells) with different concentrations of QDs for 48 h. After this incubation period, the CdSe QDs showed enhanced toxicity even at the lowest concentration of 10 mg mL 1, whereas the amino acid-modified bCD-coated CdSe QDs showed lower toxicity under the
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10.6 2.5 46.2 4.6 42.5 4.2
IC50 [mg mL 1][a] HepG2
cells was lower in our studies. Interestingly, the amino acidmodified b-CD-coated CdSe QDs showed less toxicity towards the normal QSG-7701 cells compared with other two types of cancer cells. Our data indicated that the release of Cd2 + from the QDs surface can be reduced by employing particles coated with biomolecules. Because the amino acid-modified b-CD-coated CdSe QDs showed good optical properties and minimal toxicity, their specific binding to the surface of living cells was investigated by using HepG2 cells in vitro. Figure 10 shows the flow cytometry histograms of HepG2 cells incubated with l-Arg-bCD-coated CdSe QDs. On the basis of the average fluorescence intensity of cells, it was apparent that a substantial intracellular uptake of l-Arg-b-CD-coated CdSe QDs took place in HepG2 cancer cells. The average fluorescence intensity of HepG2 cells was increased 11 times compared with the control ( 73.9 to 6.4). The results provide strong evidence for the high uptake of amino acid-modified b-CD-
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Conclusion In summary, good-quality CdSe QDs with multicolor emission were obtained by using a simple and environmentally friendly method, and the green CdSe QDs synthesized with higher QYs were coated with amino acids by using a sonochemical method in which l-Arg and l-Trp acted as surface ligands. The shape- and the size-dependent optical and magnetic properties of the CdSe QDs were studied, and these QDs presented good dispersion, uniformity, and excellent fluorescent properties. Compared with naked CdSe QDs, the PL spectra of amino acid-modified b-CD-coated green CdSe QDs with a large size distribution presented an obvious redshift. l-Arg-b-CD-coated green CdSe QDs incubated with HepG2 cells were capable of locating to the cytoplasm of cells, which indicated that the QDs could be useful for tracking cells and intracellular processes. Therefore, biomolecule-coated QDs are expected to be used in biolabeling, bioimaging, or other applications in vivo.
Figure 10. Flow cytometry data of l-Arg-b-CD-coated CdSe QDs in HepG2 cells.
coated CdSe QDs in cells. The possible mechanism of intracellular uptake of amino acid-modified QDs is that both clathrin-mediated endocytosis and the electrostatic interaction between the positive charge of the amino acids and the negatively charged plasma membrane played important roles in the intracellular delivery of QD.[13] To further confirm the cell labeling of amino acid-modified b-CD-coated CdSe QDs, HepG2 cells were treated with l-Arg-b-CD-coated CdSe QDs. Confocal microscopy was used to investigate cell labeling by the green QDs and observe their fluorescent emission. The green QD samples were excited by using a green laser (l = 480 nm), and the nuclei, stained with Hoechst 33342 solution, were excited by using a blue laser (l = 405 nm). In Figure 11, many green
Experimental Section Materials and Reagents Selenium powder (99.99 %), Na2SO3 (98.0 %), oleic acid (OA, 90 %), and [Cd(Ac)2]·2 H2O were purchased from Alfa Aesar (Karlsruhe, Germany). The selenium precursor solution of Na2SeSO3 was prepared by heating selenium powder (5 mmol) and Na2SO3 (6 mmol) in of distilled water (50 mL) at reflux for 3 h. b-Cyclodextrin (b-CD), triethanolamine, p-toluenesulfonyl chloride, l-tryptophan (l-Trp), l-arginine (l-Arg), ethanol, NaOH, and n-hexane were obtained from Shanghai Chemical Factory, China. b-CD was recrystallized three times from distilled water and dried under vacuum for 12 h at 95 8C before use. Fetal bovine serum (FBS) was purchased from Gibco (Life Technologies AG, Switzerland). Dulbecco minimum essential medium (DMEM) was from Invitrogen Corporation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst 33342 were from Sigma. Other organic solvents were purchased from EM Sciences. Characterization Powder X-ray diffraction (XRD) of the products were carried out by using a Shimadzu XRD-6000 X-ray diffractometer equipped with CuKa radiation (l = 0.154060 nm) and recorded at a scanning rate of 0.02 s 1; 2q ranged from 15–708. The transmission electron micrograph (TEM) measurements were performed by using a JEOL JEM-200CX microscope. UV/Vis absorption spectra were acquired by using a PerkinElmer Lambda-850 UV/Vis spectrometer. Fluorescence spectra were acquired by using a PerkinElmer Ls55 fluorescence spectrometer with excitation at l = 480 nm. QYs for TOPO-coated CdSe in hexane and amino acidmodified b-CD-coated CdSe in PBS were evaluated against rhodamine B (RhB). Confocal microscopy images were obtained by using a confocal laser scanning fluorescence microscopy equipped with a laser at l = 480 nm.
Figure 11. Confocal microscopy imaging of l-Arg-b-CD-coated CdSe QDs in HepG2 cells. a) Fluorescent images of HepG2 cells with l-Arg-bCD-coated CdSe QDs, b) fluorescent images of Hoechst 33342-stained HepG2 cells, c) an overlay of fluorescent images of the l-Arg-b-CDcoated CdSe QDs labeled HepG2 cells. (HepG2 cells were treated with 40 mg mL 1 of l-Arg-b-CD-coated CdSe QDs).
fluorescent dots are present in the cytoplasm of the cells, which shows that the green l-Arg-b-CD-coated CdSe QDs can permeate the cell membrane and enter the cells. Notably, no obvious heavy aggregates settled in the cytoplasm. Therefore, the high brightness of the QDs made it possible to record fluorescent images for cells or in vivo and leads to the observation of QD localization. These results indicate the potential of using biomaterial-coated QDs systems for cell fluorescent labeling.
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Synthesis of l-Amino Acid-Modified b-Cyclodextrin l-Amino acid-modified b-cyclodextrin was synthesized based on a previous method with slight modification.[14] Briefly, monoACHTUNGRE[6-O-(p-toluene-sulfonyl)]-b-cyclodextrin (0.77 mmol) and amino acid (l-Trp and l-Arg; 2.3 mmol) were dissolved in water (30 mL) that contained triethanolamine (20 mL) at 85 8C with stirring for 24 h under N2. After evaporation of most of the solvent under reduced pressure, the resulting solution was poured into vigorously stirred anhydrous ethanol (500 mL), and the resultant mixture was stored in a refrigerator 18 h to produce a pale yellow precipitate. The solid product was collected by centrifugation and then
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purified by chromatography on a Sephadex G-25 column with deionizated water as the eluent, followed by repeated recrystallization from water and ethanol to give pale yellow product. Elemental analysis calcd (%) for l-Arg-b-CD complex (C48H82O36N4·10 H2O): C 39.18, H 6.99, N 3.81; found: C 39.32, H 6.65, N 3.81; elemental analysis calcd (%) for l-Trp-bCD complex (C53H80O36N2·6 H2O): C 44.54, H 6.49, N 1.96; found: C 44.28, H 6.28, N 1.99.
laser scanning fluorescence microscopy was used to detect the fluorescence in the cells by using l = 488 nm laser to excite the QDs.
Author Contributions M. X. Zhao, Y. Li and E.-Z. Zeng performed the experiments. M. X. Zhao, Y. Li, E.-Z. Zeng, and C.-J. Wang reviewed, analyzed, and interpreted the data. M. X. Zhao and C.-J. Wang wrote the paper. All authors discussed the results and commented on the manuscript.
Preparation of CdSe QDs QDs with different emission wavelengths were prepared according to a modified literature approach.[15] Typically, [Cd(Ac)2]·2 H2O (0.133 g) dissolved in deionized water (5 mL) was added with stirring to a solution of OA (4 mL) and NaOH (0.35 g) in C2H5OH/deionized water (20 mL, 15:5 v/v). Na2SeSO3 solution (5 mL, 0.1 m) was added to the mixture. After reacting for about 5 min, the mixture was transferred to several autoclaves and sealed. These autoclaves were held at the designed temperature (50, 80, 100, 120, and 150 8C) for 10 h. When the reaction was complete, the products deposited in the autoclaves were dissolved in nhexane, then the raw products were separated by ethanol precipitation followed by centrifugation. Afterwards, the precipitation was repeatedly purified from n-hexane and ethanol. Finally, the final CdSe quantum dot products were redissolved in n-hexane and used in the following measurements.
Acknowledgements This work was supported by the National Natural Science Foundation of China (U1204201), the Postdoctoral Science Surface Foundation of China (no. 2012M511570), and the Department of Education Science and Technology Research Key Projects of Henan (13A150063).
[1] a) D. Zhang, G. Li, H. Li, Y. Lu, Chem. Asian J. 2013, 8, 26 – 40; b) P. Wu, X.-P. Yan, Chem. Soc. Rev. 2013, 42, 5489 – 5521; c) F. Zhao, Y. Zhao, Y. Liu, X. Chang, C. Chen, Y. Zhao, Small 2011, 7, 1322 – 1337; d) V. Biju, T. Itoh, A. Anas, A. Sujith, M. Ishikawa, Anal. Bioanal. Chem. 2008, 391, 2469 – 2495. [2] a) V. Biju, Chem. Soc. Rev. 2014, 43, 744 – 764; b) V. Biju, T. Itoh, M. Ishikawa, Chem. Soc. Rev. 2010, 39, 3031 – 3056. [3] a) W. R. Siah, A. P. LaGrow, M. J. Banholzer, R. D. Tilley, Cryst. Growth Des. 2013, 13, 2486 – 2492; b) M. M. Moghaddam, M. Baghbanzadeh, A. Sadeghpour, O. Glatter, C. O. Kappe, Chem. Eur. J. 2013, 19, 11 629 – 11 636; c) M. M. Moghaddam, M. Baghbanzadeh, A. Keilbach, C. O. Kappe, Nanoscale 2012, 4, 7435 – 7442; d) S. Taniguchi, M. Green, S. B. Rizvi, A. Seifalian, J. Mater. Chem. 2011, 21, 2877 – 2882; e) X. Jiang, M. Ahmed, Z. Deng, R. Narain, Bioconjugate Chem. 2009, 20, 994 – 1001; f) L. Qu, X. Peng, J. Am. Chem. Soc. 2002, 124, 2049 – 2055. [4] a) A. J. Morris-Cohen, M. T. Frederick, G. D. Lilly, E. A. McArthur, E. A. Weiss, J. Phys. Chem. Lett. 2010, 1, 1078 – 1081; b) Z. A. Peng, X. G. Peng, J. Am. Chem. Soc. 2001, 123, 183 – 184; c) C. B. Murray, D. J. Norris, M. G. Bawendi, J. Am. Chem. Soc. 1993, 115, 8706 – 8715. [5] a) Y. A. Yang, H. Wu, K. R. Williams, Y. C. Cao, Angew. Chem. Int. Ed. 2005, 44, 6712 – 6715; Angew. Chem. 2005, 117, 6870 – 6873; b) L. H. Qu, W. W. Yu, X. G. Peng, Nano Lett. 2004, 4, 465 – 469. [6] a) W. Xiong, D. D. Hickstein, K. J. Schnitzenbaumer, J. L. Ellis, B. B. Palm, K. E. Keister, C. Ding, L. Miaja-Avila, G. Dukovic, J. L. Jimenez, M. M. Murnane, H. C. Kapteyn, Nano Lett. 2013, 13, 2924 – 2930; b) J. Chen, H. Jiao, W. Li, D. Liao, H. Zhou, C. Yu, Chem. Asian J. 2013, 8, 276 – 281; c) Y. Li, Y. Zhou, H.-Y. Wang, S. Perrett, Y. Zhao, Z. Tang, G. Nie, Angew. Chem. Int. Ed. 2011, 50, 5860 – 5864; Angew. Chem. 2011, 123, 5982 – 5986; d) R. Viswanatha, D. D. Sarma, Chem. Asian J. 2009, 4, 904 – 909; e) X. Gao, L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, S. Nie, Curr. Opin. Biotechnol. 2005, 16, 63 – 72. [7] a) C.-P. Huang, C.-F. Chao, M.-Y. Shen, T.-M. Chen, Y.-K. Li, Chem. Asian J. 2012, 7, 2848 – 2853; b) N. Varghese, K. Biswas, C. N. Rao, Chem. Asian J. 2008, 3, 1435 – 1442; c) X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, S. Weiss, Science 2005, 307, 538 – 544; d) K. Narayanan, S. K. Yen, Q. Dou, P. Padmanabhan, T. Sudhaharan, S. Ahmed, J. Y. Ying, S. T. Selvan, Sci. Rep. 2013, 3, 2184, DOI: 10.1038/srep02184; e) S. T. Selvan, Biointerphases 2010, 5, FA110 – FA115; f) F. Pinaud, D. King, H.-P. Moore, S. Weiss, J. Am. Chem. Soc. 2004, 126, 6115 – 6123; g) T. Pellegrino, L. Manna, S. Kudera, T. Liedl, D. Koktysh, A. L. Rogach, S. Keller, J. Rdler, G. Natile, W. J. Parak, Nano Lett. 2004, 4, 703 – 707. [8] a) F. A. Esteve-Turrillas, A. Abad-Fuentes, Biosens. Bioelectron. 2013, 41, 12 – 29; b) D. Painuly, A. Bhatt, V. K. Krishnan, J. Biomed.
Synthesis of Amino Acid-Modified b-CD-Coated CdSe Amino acid-modified b-CD-coated CdSe QDs were formed by surface modification of the QDs. The synthesis was based on our previous method with slight modification.[16] A mixture of purified CdSe QDs (0.016 m) in chloroform (2.0 mL) and amino acid-modified b-CD (10 mg mL 1) in water (2.0 mL) was sonicated for 4 h. Triethanolamine (25 %, w/v) was added to raise the pH of the mixture above 10.0 to initiate surfactant exchange. Then ethyl acetate was added to precipitate the nanocrystal complexes and purify the nanocrystals from side products and unreacted precursors. Cell Culture HepG2 (human hepatocellular liver carcinoma cell), HeLa, and QSG7701 cells were maintained in DMEM medium supplemented with 10 % (v/v) heat-inactivated FBS and 1 % (v/v) penicillin–streptomycin (100 U mL 1 penicillin and 100 mg mL 1 streptomycin), and cultured in a 5 % CO2 atmosphere at 37 8C. Thereafter, the medium was replaced every third day. The adherent cells were allowed to reach 80 % confluence (7–10 days for the first passage). Cells were passaged in culture, and passage 3 cells (P3) were used for experiments. Cell Viability Assay The cytotoxicity of the samples was assayed by using a modified 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, according to a previous method.[17] Briefly, cells were seeded into 96-well plates at 5 104 cells per well. After 24 h, various concentrations (5, 10, 20, 50, 100, and 200 mg mL 1) of QDs were subsequently added and incubated for 48 h. MTT (20 mL, 2.5 mg mL 1) was added to each well and the plates were incubated for 4 h at 37 8C under 5 % CO2. After the medium was removed, DMSO (100 mL) was added to each well. The OD570 of QDs was measured at l = 690 nm by using an Infinite M200 monochromator-based multifunction microplate reader. Cell Labeling Fluorescent images were obtained from l-Arg-b-CD-conjugated CdSe QDs in HepG2 cells and were recorded by using confocal laser scanning fluorescence microscopy. HepG2 cells were seeded in culture medium that contained 10 % FBS in a utensil (35 mm diameter) that contained glass cover-slips (15 mm diameter) and incubated for 24 h at 37 8C and 5 % CO2. Pristine l-Arg-b-CD-conjugated CdSe QD probes containing medium solution were then added to the cells at a concentration of 40 mg mL 1. The cells were incubated at 37 8C in 5 % CO2 for 6 h, and then washed with PBS. After washing, the cell nuclei were stained by using Hoechst 33342 solution and mounted in glycerol buffer. Confocal
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[9] [10]
[11] [12]
[13] A. Anas, T. Okuda, N. Kawashima, K. Nakayama, T. Itoh, M. Ishikawa, V. Biju, ACS Nano 2009, 3, 2419 – 2429. [14] a) M.-X. Zhao, Q. Xia, X.-D. Feng, X.-H. Zhu, Z.-W. Mao, L.-N. Ji, K. Wang, Biomaterials 2010, 31, 4401 – 4408; b) Y. Liu, B. H. Han, S. X. Sun, T. Wada, Y. Inoue, J. Org. Chem. 1999, 64, 1487 – 1493. [15] P. Yang, M. Ando, N. Murase, Colloids Surf. A 2011, 390, 207 – 211. [16] M.-X. Zhao, H.-F. Huang, Q. Xia, L.-N. Ji, Z.-W. Mao, J. Mater. Chem. 2011, 21, 10290 – 10297. [17] H. Z. Wang, C. H. Chang, C. P. Lin, M. C. Tsai, J. Ocul. Pharmacol. Ther. 1996, 12, 35 – 43. Received: December 20, 2013 Published online: February 24, 2014
Nanotechnol. 2013, 9, 257 – 266; c) P. Babu, S. Sinha, A. Surolia, Bioconjugate Chem. 2007, 18, 146 – 151. F. Dubois, B. Mahler, B. Dubertret, E. Doris, C. Mioskowski, J. Am. Chem. Soc. 2007, 129, 482 – 483. a) S. B. Lowe, J. A. G. Dick, B. E. Cohen, M. M. Stevens, ACS Nano 2012, 6, 851 – 857; b) V. Biju, S. Mundayoor, R. V. Omkumar, A. Anas, M. Ishikawa, Biotechnol. Adv. 2010, 28, 199 – 213. J. Rodriguez-Viejo, K. F. Jensen, H. Mattoussi, J. Michel, B. O. Dabbousi, M. G. Bawendi, Appl. Phys. Lett. 1997, 70, 2132 – 2134. H. S. Baek, J. Y. Yoo, D. K. Rah, D.-W. Han, D. H. Lee, O.-H. Kwon, J.-C. Park, Yonsei Med. J. 2005, 46, 579 – 583.
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