COMMUNICATION DOI: 10.1002/asia.201301676
A Fluorescent Polymeric Quantum Dot/Aptamer Superstructure and Its Application for Imaging of Cancer Cells Guifen Jie,* Yanbin Zhao, and Yingqiang Qin[a]
Abstract: In this work, a novel polymeric quantum dot/aptamer superstructure with a highly intense fluorescence was fabricated by a molecular engineering strategy and successfully applied to fluorescence imaging of cancer cells. The polymeric superstructure, which is composed of both multiple cell-based aptamers and a high ratio of quantum dot (QD)-labeled DNA, exploits the target recognition capability of the aptamer, an enhanced cell internalization through multivalent effects, and cellular disruption by the polymeric conjugate. Importantly, the polymeric superstructure exhibits an increasingly enhanced fluorescence with recording time and is thus suitable for long-term fluorescent cellular imaging. The unique and excellent fluorescence property of the QD superstructure paves the way for developing polymeric QD superstructures that hold promise for applications such as in vivo imaging.
Recent reports highlighted advantages of using semiconducting polymer nanoparticles in biological imaging, which are low toxicity, ultrabright photoluminescence, nonblinking property, and fast emission rates.[4–8] Aptamer-conjugated nanoparticles as sensing systems that combine the specific molecular recognition of aptamers with the strong signal transduction capacity of nanoparticles offer an alternative approach for cell labeling and imaging.[9] Consequently, aptamer-conjugated nanoparticles have been widely used as probes for disease-associated isoform discrimination,[10] virus labeling,[11] multifunctional cell imaging,[12] and cancer cell targeting and therapy.[13] However, drug toxicity and resistance still present major obstacles to the full realization of aptamer-directed cancer therapy.[14] Cellular disruption holds great potential for treating drug-resistant cancer cells if a specific uptake can be guaranteed. Therefore, an anticancer system was envisioned that obviates the drug component by utilizing the toxicity of the polymer itself after selective internalization, which is facilitated by multiple cell-based aptamers. In this paper, we report a novel polymeric quantum dot/aptamer superstructure with a highly intense fluorescence and its successful application to fluorescence imaging of cancer cells. This model multifunctional nanosystem will be fully developed specifically for preclinical applications including simultaneous in vivo imaging, cell targeting, and drug storage.[15]
Introduction One of the critical challenges in early cancer diagnosis and treatment with the use of nanotechnology is the development of multifunctional particles at the nanoscale that simultaneously serve as sensitive, cell-specific bioprobes and as agents for localized tumor treatment. Extensive efforts have been devoted to designing nanocarriers that combine cell targeting with efficient in vivo imaging, drug storage, and controlled drug-release capabilities.[1, 2] Semiconductor quantum dots (QDs) are a promising new technology with benefits in the areas of medical diagnostics and therapeutics. The use of highly fluorescent nanoparticles as labels for cellular assays and in vivo imaging is extremely promising. These nanoparticles exhibit a higher brightness and photostability, as well as lower susceptibility to cellular efflux mechanisms when compared with small molecules.[3]
Results and Discussion Characterization of the Polymeric Quantum Dot/Aptamer Superstructure The principle behind the polymeric quantum dot/aptamer superstructure is outlined in Figure 1. The conjugate was assembled by polymerization of three components: 1) A reporting element, which was prepared by conjugating acrylamide with carboxylic acid-functionalized QDs should keepconfiguration of the individual aptamers and provide a tracking signal for both targeting and internalization; this would effectively enhance the imaging capabilities for clinical versatility; 2) multiple targeting elements, which were prepared by conjugating acrylic acid with aptamers targeting cancer cells contributed greatly to cellular delivery by multivalent binding; and 3) polyacrylamide with high stability and biocompatibility was used as the backbone.[16, 17] Specifically, the polymeric aptamer conjugate has little effect on nontarget
[a] Dr. G. Jie, Dr. Y. Zhao, Dr. Y. Qin Key Laboratory of Biochemical Analysis, Ministry of Education College of Chemistry and Molecular Engineering Qingdao University of Science and Technology Qingdao 266042 (China) Fax: (+ 86) 532-84022750 E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201301676.
Chem. Asian J. 2014, 9, 1261 – 1264
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
Guifen Jie et al.
Figure 3. Energy-dispersive X-ray spectroscopy (EDX) of the polymeric quantum dot/aptamer superstructure.
Figure 1. Schematic of the synthesis of the polymeric quantum dot/aptamer superstructure. APS, ammonium persulfate; TEMED, N,N,N’,N’-tetramethylethylenediamine.
Figure 3, the results demonstrate that the polymeric superstructure contains the elements Cd, Se, Zn, S, C, O, P, etc., indicating that polymeric QD superstructure was successfully fabricated.
cells, which is more promising than traditional drug treatments. Transmission electron microscopy (TEM) was used to characterize the fabrication process of the polymeric quantum dot/aptamer superstructure. As can be observed from Figure 2 A,B), the initially prepared CdSe/ZnS QDs have an average diameter of 9–10 nm with a good monodispersity, and are uniform in size and morphology. After formation of the polymeric QD superstructure, the polymeric conjugate as a united composite became much larger, and many small particles on the surface were observed (Figure 2 C,D). In addition, energy-dispersive X-ray spectroscopy (EDX) was used to confirm the elemental composition of the polymeric quantum dot/aptamer superstructure, shown in
Figure 2. Representative TEM images (C, D) the polymeric QDs superstructure.
Chem. Asian J. 2014, 9, 1261 – 1264
of
(A, B) CdSe/ZnS
Fluorescence Imaging of the QD Superstructure and its Application for Cells Assays The prepared polymeric QD superstructure was then explored as biological fluorescent probes. As can be seen in Figure 4 A–D, a considerable fluorescent signal was observed in phosphate-buffered saline (PBS), and it was found that the fluorescent signal significantly increased with the recording time (73 s). Compared to the photobleaching properties of conventional materials, the unique fluorescent characteristic renders this class of materials an excellent bioprobe for live cell imaging. This property inspired us to fur-
Figure 4. Confocal fluorescence microscopy images of the polymeric QDs superstructure in PBS at different recording times. (A) 38 s, (B) 61 s, (C) 80 s, and (D) 111 s. Scale bars, 50 mm.
QDs,
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www.chemasianj.org
ther examine the fluorescence behavior and explore the QD superstructure for cellular imaging. Interestingly, a novel fluorescence phenomenon of the QD superstructure was observed in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 100 U mL 1 penicillin-streptomycin. As shown in Figure 5 A–H, the QD superstructure became much smaller and more uniform than that in PBS, and the fluorescence became increasingly brighter with recording time (130 s), which further highlights the strongly luminescent characteristics of the QD superstructure and its promising application in cell imaging.
Figure 6. Confocal fluorescence microscopy images of an MCF cell stained by rhodamine B (A) and MCF cells labeled with the QD bioconjugate (B–D).
Figure 5. Confocal fluorescence microscopy images of the prepared polymeric QD superstructure in cell medium with recording time. (A) 38 s, (B) 57 s, (C) 76 s, (D) 97 s, (E) 115 s, and (F) 134 s. Scale bars, 50 mm.
Aptamers possess a high recognition ability to specific targets ranging from small inorganic or organic substances to even proteins or cells,[18, 19] and have been extensively used to construct various biosensors for target detection.[20–22] In this work, aptamers can guide the internalization of the macromolecule conjugates after the aptamer binds to the cell surface, and multiple binding owing to the polymeric design facilitates the entire process. The resultant polymeric QD bioconjugates were specifically targeted to MCF cells by using aptamers for these cells. Figure 6 B–D shows that the photoluminescence of the QD bioconjugate-labeled MCF cells is intense and clearly spectrally resolved. For comparison, MCF cells stained by rhodamine B were smooth and displayed no fluorescence of QDs (Figure 6 A). More significantly, the fluorescent signals of the QD bioconjugate-labeled MCF cells was rapidly increasing in strength during the continuous observation for 86 s in our experiment (Figure 7 A–D), in good accordance with the above discussion on the excellent luminescent characteristics of QD bioconjugates. This suggests that the QD bioconjugates are particularly suitable for long-term and real-time cellular imaging due to their ultrahigh photostability. In a control experiment, the QD bioconjugates were incubated with non-targeted cells, followed by fluorescence imaging (Figure S1, Supporting Information). Obviously, the cells showed nearly no fluorescent signals, although the QD bioconjugates displayed a strong fluorescence. This result in-
Chem. Asian J. 2014, 9, 1261 – 1264
Figure 7. Confocal fluorescence microscopy images of the QDs bioconjugate-labeled target cells in cell medium with recording time. (A) 29 s, (B) 45 s, (C) 65 s, and (D) 115 s. Scale bars, 50 mm.
dicates that the cells were not targeted by the QD bioconjugates and that the QD bioconjugates displayed a good selectivity towards the target cells due to the aptamers.
Conclusions In summary, we fabricated a highly fluorescent polymeric quantum dot/aptamer superstructure and successfully applied it to fluorescence imaging of cancer cells. The polymeric superstructure, which is composed of both multiple cellbased aptamers and a high ratio of quantum dot (QD)-labeled DNA, can specifically bind target cells and shows
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Guifen Jie et al.
www.chemasianj.org
Acknowledgements
highly intense fluorescence. The polymeric backbone design facilitates multiple binding and uptake, and should be cytotoxic after selective internalization. Importantly, the polymeric superstructure exhibits an increasingly enhanced fluorescence with recording time and is suitable for long-term fluorescent cellular imaging. Thus, this study paves the way for developing polymeric QD superstructures that hold promise for in vivo imaging applications. Furthermore, our approach might find potential applications in drug development, improvement of existing drugs, and drug delivery for the therapy of cancer.
This work was supported by the National Natural Science Foundation of China (Nos. 21175078), the Natural Science Foundation of Shandong province (No. ZR2010BZ003), and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT).
Keywords: aptamers · cancer · cell imaging · fluorescence · polymeric superstructure · quantum dots
[1] K. C. Wood, S. M. Azarin, W. Arap, R. Pasqualini, R. Langer, P. T. Hammond, Bioconjugate Chem. 2008, 19, 403 – 405. [2] Y. Inoue, K. Izawa, K. Yoshikawa, H. Yamada, A. Tojo, K. Ohtomo, Eur. J. Nucl. Med. Mol. Imaging 2007, 34, 2048 – 2056. [3] U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, T. Nann, Nat. Methods 2008, 5, 763 – 775. [4] J. Pecher, S. Mecking, Chem. Rev. 2010, 110, 6260 – 6279. [5] C. Wu, Y. Jin, T. Schneider, D. R. Burnham, P. B. Smith, D. T. Chiu, Angew. Chem. Int. Ed. 2010, 49, 9436 – 9440; Angew. Chem. 2010, 122, 9626 – 9630. [6] C. Wu, T. Schneider, M. Zeigler, J. Yu, P. G. Schiro, D. R. Burnham, J. D. McNeill, D. T. Chiu, J. Am. Chem. Soc. 2010, 132, 15410 – 15417. [7] C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. McNeil, J. M. Olson, D. T. Chiu, Angew. Chem. Int. Ed. 2011, 50, 3430 – 3434; Angew. Chem. 2011, 123, 3492 – 3496. [8] K. Petkau, A. Kaeser, I. Fischer, L. Brunsveld, A. P. H. J. Schenning, J. Am. Chem. Soc. 2011, 133, 17063 – 17071. [9] R.-M. Kong, X.-B. Zhang, Z. Chen, W. Tan, Small 2011, 7, 2428 – 2436. [10] S. J. Xiao, P. P. Hu, X. D. Wu, Y. L. Zou, L. Q. Chen, L. Peng, J. Ling, S. J. Zhen, L. Zhan, Y. F. Li, C. Z. Huang, Anal. Chem. 2010, 82, 9736 – 9742. [11] Z.-Q. Cui, Q. Ren, H.-P. Wei, Z. Chen, J.-Y. Deng, Z.-P. Zhang, X.E. Zhang, Nanoscale 2011, 3, 2454 – 2457. [12] W. J. Kang, J. R. Chae, Y. L. Cho, J. D. Lee, S. Kim, Small 2009, 5, 2519 – 2522. [13] C. D. Medley, S. Bamrungsap, W. Tan, J. E. Smith, Anal. Chem. 2011, 83, 727 – 734. [14] D. S. Shewach, R. D. Kuchta, Chem. Rev. 2009, 109, 2859 – 2861. [15] M. C. Franchini, G. Baldi, D. Bonacchi, D. Gentili, G. Giudetti, A. Lasciafari, M. Corti, P. Marmorato, J. Ponti, E. Micotti, U. Guerrini, L. Sironi, P. Gelosa, C. Ravagli, A. Ricci, Small 2010, 6, 366 – 370. [16] T. H. Yang, Recent Pat. Mater. Sci. 2008, 1, 29 – 40. [17] M. J. Caulfield, G. G. Hao, X. J. Qiao, D. H. Solomon, Polymer 2003, 44, 1331 – 1337. [18] M. Famulok, G. Mayer, M. Blind, Acc. Chem. Res. 2000, 33, 591 – 599. [19] S. Tombelli, A. Minunni, A. Mascini, Biosens. Bioelectron. 2005, 20, 2424 – 2434. [20] C. A. Kellenberger, S. C. Wilson, J. S. Lee, M. C. Hammond, J. Am. Chem. Soc. 2013, 135, 4906 – 4909. [21] L. Li, P. Ge, P. R. Selvin, Y. Lu, Anal. Chem. 2012, 84, 7852 – 7856. [22] Y. Xiang, Yi. Lu, Anal. Chem. 2012, 84, 9981 – 9987.
Experimental Section Preparation of the reporting element The reporting element was prepared by conjugating acrylamide with QDs. Acrylamide (3.0 g) was first dissolved in water (2 mL) to obtain the acrylamide solution. Next, EDC (0.1 m, 10 mL) and NHS (0.025 m, 10 mL) were added to a solution of CdSe/ZnS QDs (200 mL) for 30 min, and then acrylic acid (200 mL) was added to the above activated QD solution and reacted at room temperature overnight under gentle shaking. Preparation of the targeting element The targeting element was prepared by conjugating acrylic acid with aptamers for Ramos cells (aptamer sequence: 5-NH2-TACAGAACACCGGGAGGATAGTTCGGTGGCTGTTCAGGGTCTCCTCCCGGTG-3). First, EDC (0.1 m, 10 mL) and NHS (0.025 m, 10 mL) were added to acrylic acid (200 mL) for 30 min for activation. Subsequently, 80 mL of the activated acrylic acid solution were mixed with 20 mL of 10 5 m aptamers and reacted at 37 8C overnight under gentle shaking. Preparation of the QD-containing probe for cell imaging Initiator and catalyst solutions were freshly prepared by adding ammonium persulfate (0.05 g) and tetramethylethylenediamine (TEMED, 25 mL), respectively, to 0.5 mL of H2O. Next, the acrylamide/QD composite (400 mL) was mixed with the acrylic acid/aptamer conjugate (400 mL), followed by addition of 3 % initiator and catalyst. The mixture was then kept at room temperature in the dark under gentle shaking for 40 min. Subsequently, it was centrifuged at 10 000 rpm for 30 min to remove unbound reactants, and the precipitate was redispersed in 0.01 m PBS (800 mL) to obtain the QD-containing probe for cell imaging. Application of the QD-containing probe for cells imaging MCF cells (~ 1 106, 1 mL) were centrifuged at 3000 rpm for 3 min and redispersed in 100 mL of RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 100 U mL 1 penicillin-streptomycin. Then, 500 mL of the QD-containing probe was washed three times and suspended in 10 mm PBS containing 0.1 m NaCl. Subsequently, the QD-containing probe was added to the cells and incubated at 37 8C for 30 min under gentle shaking. The mixture was then centrifuged at 3000 rpm for 3 min, washed with 10 mm PBS containing 0.1 m NaCl, and resuspended in 500 mL of 10 mm PBS containing 0.1 m NaCl. For confocal microscopy imaging, an aliquot of sample solution (10 mL) was deposited onto a microscope slide and covered with a standard microscope slide. The fluorescent QDs and labeled cancer cells were observed with a Leica TCS SP5 II apparatus equipped with a HCX PL APO 40/0.85 objective using a 488 nm laser beam (Argon ion laser).
Chem. Asian J. 2014, 9, 1261 – 1264
Received: December 18, 2013 Published online: March 11, 2014
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
A Fluorescent Polymeric Quantum Dot/Aptamer Superstructure and Its Application for Imaging of Cancer Cells Guifen Jie,* Yanbin Zhao, and Yingqiang Qin[a]
Abstract: In this work, a novel polymeric quantum dot/aptamer superstructure with a highly intense fluorescence was fabricated by a molecular engineering strategy and successfully applied to fluorescence imaging of cancer cells. The polymeric superstructure, which is composed of both multiple cell-based aptamers and a high ratio of quantum dot (QD)-labeled DNA, exploits the target recognition capability of the aptamer, an enhanced cell internalization through multivalent effects, and cellular disruption by the polymeric conjugate. Importantly, the polymeric superstructure exhibits an increasingly enhanced fluorescence with recording time and is thus suitable for long-term fluorescent cellular imaging. The unique and excellent fluorescence property of the QD superstructure paves the way for developing polymeric QD superstructures that hold promise for applications such as in vivo imaging.
Recent reports highlighted advantages of using semiconducting polymer nanoparticles in biological imaging, which are low toxicity, ultrabright photoluminescence, nonblinking property, and fast emission rates.[4–8] Aptamer-conjugated nanoparticles as sensing systems that combine the specific molecular recognition of aptamers with the strong signal transduction capacity of nanoparticles offer an alternative approach for cell labeling and imaging.[9] Consequently, aptamer-conjugated nanoparticles have been widely used as probes for disease-associated isoform discrimination,[10] virus labeling,[11] multifunctional cell imaging,[12] and cancer cell targeting and therapy.[13] However, drug toxicity and resistance still present major obstacles to the full realization of aptamer-directed cancer therapy.[14] Cellular disruption holds great potential for treating drug-resistant cancer cells if a specific uptake can be guaranteed. Therefore, an anticancer system was envisioned that obviates the drug component by utilizing the toxicity of the polymer itself after selective internalization, which is facilitated by multiple cell-based aptamers. In this paper, we report a novel polymeric quantum dot/aptamer superstructure with a highly intense fluorescence and its successful application to fluorescence imaging of cancer cells. This model multifunctional nanosystem will be fully developed specifically for preclinical applications including simultaneous in vivo imaging, cell targeting, and drug storage.[15]
Introduction One of the critical challenges in early cancer diagnosis and treatment with the use of nanotechnology is the development of multifunctional particles at the nanoscale that simultaneously serve as sensitive, cell-specific bioprobes and as agents for localized tumor treatment. Extensive efforts have been devoted to designing nanocarriers that combine cell targeting with efficient in vivo imaging, drug storage, and controlled drug-release capabilities.[1, 2] Semiconductor quantum dots (QDs) are a promising new technology with benefits in the areas of medical diagnostics and therapeutics. The use of highly fluorescent nanoparticles as labels for cellular assays and in vivo imaging is extremely promising. These nanoparticles exhibit a higher brightness and photostability, as well as lower susceptibility to cellular efflux mechanisms when compared with small molecules.[3]
Results and Discussion Characterization of the Polymeric Quantum Dot/Aptamer Superstructure The principle behind the polymeric quantum dot/aptamer superstructure is outlined in Figure 1. The conjugate was assembled by polymerization of three components: 1) A reporting element, which was prepared by conjugating acrylamide with carboxylic acid-functionalized QDs should keepconfiguration of the individual aptamers and provide a tracking signal for both targeting and internalization; this would effectively enhance the imaging capabilities for clinical versatility; 2) multiple targeting elements, which were prepared by conjugating acrylic acid with aptamers targeting cancer cells contributed greatly to cellular delivery by multivalent binding; and 3) polyacrylamide with high stability and biocompatibility was used as the backbone.[16, 17] Specifically, the polymeric aptamer conjugate has little effect on nontarget
[a] Dr. G. Jie, Dr. Y. Zhao, Dr. Y. Qin Key Laboratory of Biochemical Analysis, Ministry of Education College of Chemistry and Molecular Engineering Qingdao University of Science and Technology Qingdao 266042 (China) Fax: (+ 86) 532-84022750 E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201301676.
Chem. Asian J. 2014, 9, 1261 – 1264
1261
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
Guifen Jie et al.
Figure 3. Energy-dispersive X-ray spectroscopy (EDX) of the polymeric quantum dot/aptamer superstructure.
Figure 1. Schematic of the synthesis of the polymeric quantum dot/aptamer superstructure. APS, ammonium persulfate; TEMED, N,N,N’,N’-tetramethylethylenediamine.
Figure 3, the results demonstrate that the polymeric superstructure contains the elements Cd, Se, Zn, S, C, O, P, etc., indicating that polymeric QD superstructure was successfully fabricated.
cells, which is more promising than traditional drug treatments. Transmission electron microscopy (TEM) was used to characterize the fabrication process of the polymeric quantum dot/aptamer superstructure. As can be observed from Figure 2 A,B), the initially prepared CdSe/ZnS QDs have an average diameter of 9–10 nm with a good monodispersity, and are uniform in size and morphology. After formation of the polymeric QD superstructure, the polymeric conjugate as a united composite became much larger, and many small particles on the surface were observed (Figure 2 C,D). In addition, energy-dispersive X-ray spectroscopy (EDX) was used to confirm the elemental composition of the polymeric quantum dot/aptamer superstructure, shown in
Figure 2. Representative TEM images (C, D) the polymeric QDs superstructure.
Chem. Asian J. 2014, 9, 1261 – 1264
of
(A, B) CdSe/ZnS
Fluorescence Imaging of the QD Superstructure and its Application for Cells Assays The prepared polymeric QD superstructure was then explored as biological fluorescent probes. As can be seen in Figure 4 A–D, a considerable fluorescent signal was observed in phosphate-buffered saline (PBS), and it was found that the fluorescent signal significantly increased with the recording time (73 s). Compared to the photobleaching properties of conventional materials, the unique fluorescent characteristic renders this class of materials an excellent bioprobe for live cell imaging. This property inspired us to fur-
Figure 4. Confocal fluorescence microscopy images of the polymeric QDs superstructure in PBS at different recording times. (A) 38 s, (B) 61 s, (C) 80 s, and (D) 111 s. Scale bars, 50 mm.
QDs,
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Guifen Jie et al.
www.chemasianj.org
ther examine the fluorescence behavior and explore the QD superstructure for cellular imaging. Interestingly, a novel fluorescence phenomenon of the QD superstructure was observed in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 100 U mL 1 penicillin-streptomycin. As shown in Figure 5 A–H, the QD superstructure became much smaller and more uniform than that in PBS, and the fluorescence became increasingly brighter with recording time (130 s), which further highlights the strongly luminescent characteristics of the QD superstructure and its promising application in cell imaging.
Figure 6. Confocal fluorescence microscopy images of an MCF cell stained by rhodamine B (A) and MCF cells labeled with the QD bioconjugate (B–D).
Figure 5. Confocal fluorescence microscopy images of the prepared polymeric QD superstructure in cell medium with recording time. (A) 38 s, (B) 57 s, (C) 76 s, (D) 97 s, (E) 115 s, and (F) 134 s. Scale bars, 50 mm.
Aptamers possess a high recognition ability to specific targets ranging from small inorganic or organic substances to even proteins or cells,[18, 19] and have been extensively used to construct various biosensors for target detection.[20–22] In this work, aptamers can guide the internalization of the macromolecule conjugates after the aptamer binds to the cell surface, and multiple binding owing to the polymeric design facilitates the entire process. The resultant polymeric QD bioconjugates were specifically targeted to MCF cells by using aptamers for these cells. Figure 6 B–D shows that the photoluminescence of the QD bioconjugate-labeled MCF cells is intense and clearly spectrally resolved. For comparison, MCF cells stained by rhodamine B were smooth and displayed no fluorescence of QDs (Figure 6 A). More significantly, the fluorescent signals of the QD bioconjugate-labeled MCF cells was rapidly increasing in strength during the continuous observation for 86 s in our experiment (Figure 7 A–D), in good accordance with the above discussion on the excellent luminescent characteristics of QD bioconjugates. This suggests that the QD bioconjugates are particularly suitable for long-term and real-time cellular imaging due to their ultrahigh photostability. In a control experiment, the QD bioconjugates were incubated with non-targeted cells, followed by fluorescence imaging (Figure S1, Supporting Information). Obviously, the cells showed nearly no fluorescent signals, although the QD bioconjugates displayed a strong fluorescence. This result in-
Chem. Asian J. 2014, 9, 1261 – 1264
Figure 7. Confocal fluorescence microscopy images of the QDs bioconjugate-labeled target cells in cell medium with recording time. (A) 29 s, (B) 45 s, (C) 65 s, and (D) 115 s. Scale bars, 50 mm.
dicates that the cells were not targeted by the QD bioconjugates and that the QD bioconjugates displayed a good selectivity towards the target cells due to the aptamers.
Conclusions In summary, we fabricated a highly fluorescent polymeric quantum dot/aptamer superstructure and successfully applied it to fluorescence imaging of cancer cells. The polymeric superstructure, which is composed of both multiple cellbased aptamers and a high ratio of quantum dot (QD)-labeled DNA, can specifically bind target cells and shows
1263
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Guifen Jie et al.
www.chemasianj.org
Acknowledgements
highly intense fluorescence. The polymeric backbone design facilitates multiple binding and uptake, and should be cytotoxic after selective internalization. Importantly, the polymeric superstructure exhibits an increasingly enhanced fluorescence with recording time and is suitable for long-term fluorescent cellular imaging. Thus, this study paves the way for developing polymeric QD superstructures that hold promise for in vivo imaging applications. Furthermore, our approach might find potential applications in drug development, improvement of existing drugs, and drug delivery for the therapy of cancer.
This work was supported by the National Natural Science Foundation of China (Nos. 21175078), the Natural Science Foundation of Shandong province (No. ZR2010BZ003), and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT).
Keywords: aptamers · cancer · cell imaging · fluorescence · polymeric superstructure · quantum dots
[1] K. C. Wood, S. M. Azarin, W. Arap, R. Pasqualini, R. Langer, P. T. Hammond, Bioconjugate Chem. 2008, 19, 403 – 405. [2] Y. Inoue, K. Izawa, K. Yoshikawa, H. Yamada, A. Tojo, K. Ohtomo, Eur. J. Nucl. Med. Mol. Imaging 2007, 34, 2048 – 2056. [3] U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, T. Nann, Nat. Methods 2008, 5, 763 – 775. [4] J. Pecher, S. Mecking, Chem. Rev. 2010, 110, 6260 – 6279. [5] C. Wu, Y. Jin, T. Schneider, D. R. Burnham, P. B. Smith, D. T. Chiu, Angew. Chem. Int. Ed. 2010, 49, 9436 – 9440; Angew. Chem. 2010, 122, 9626 – 9630. [6] C. Wu, T. Schneider, M. Zeigler, J. Yu, P. G. Schiro, D. R. Burnham, J. D. McNeill, D. T. Chiu, J. Am. Chem. Soc. 2010, 132, 15410 – 15417. [7] C. Wu, S. J. Hansen, Q. Hou, J. Yu, M. Zeigler, Y. Jin, D. R. Burnham, J. McNeil, J. M. Olson, D. T. Chiu, Angew. Chem. Int. Ed. 2011, 50, 3430 – 3434; Angew. Chem. 2011, 123, 3492 – 3496. [8] K. Petkau, A. Kaeser, I. Fischer, L. Brunsveld, A. P. H. J. Schenning, J. Am. Chem. Soc. 2011, 133, 17063 – 17071. [9] R.-M. Kong, X.-B. Zhang, Z. Chen, W. Tan, Small 2011, 7, 2428 – 2436. [10] S. J. Xiao, P. P. Hu, X. D. Wu, Y. L. Zou, L. Q. Chen, L. Peng, J. Ling, S. J. Zhen, L. Zhan, Y. F. Li, C. Z. Huang, Anal. Chem. 2010, 82, 9736 – 9742. [11] Z.-Q. Cui, Q. Ren, H.-P. Wei, Z. Chen, J.-Y. Deng, Z.-P. Zhang, X.E. Zhang, Nanoscale 2011, 3, 2454 – 2457. [12] W. J. Kang, J. R. Chae, Y. L. Cho, J. D. Lee, S. Kim, Small 2009, 5, 2519 – 2522. [13] C. D. Medley, S. Bamrungsap, W. Tan, J. E. Smith, Anal. Chem. 2011, 83, 727 – 734. [14] D. S. Shewach, R. D. Kuchta, Chem. Rev. 2009, 109, 2859 – 2861. [15] M. C. Franchini, G. Baldi, D. Bonacchi, D. Gentili, G. Giudetti, A. Lasciafari, M. Corti, P. Marmorato, J. Ponti, E. Micotti, U. Guerrini, L. Sironi, P. Gelosa, C. Ravagli, A. Ricci, Small 2010, 6, 366 – 370. [16] T. H. Yang, Recent Pat. Mater. Sci. 2008, 1, 29 – 40. [17] M. J. Caulfield, G. G. Hao, X. J. Qiao, D. H. Solomon, Polymer 2003, 44, 1331 – 1337. [18] M. Famulok, G. Mayer, M. Blind, Acc. Chem. Res. 2000, 33, 591 – 599. [19] S. Tombelli, A. Minunni, A. Mascini, Biosens. Bioelectron. 2005, 20, 2424 – 2434. [20] C. A. Kellenberger, S. C. Wilson, J. S. Lee, M. C. Hammond, J. Am. Chem. Soc. 2013, 135, 4906 – 4909. [21] L. Li, P. Ge, P. R. Selvin, Y. Lu, Anal. Chem. 2012, 84, 7852 – 7856. [22] Y. Xiang, Yi. Lu, Anal. Chem. 2012, 84, 9981 – 9987.
Experimental Section Preparation of the reporting element The reporting element was prepared by conjugating acrylamide with QDs. Acrylamide (3.0 g) was first dissolved in water (2 mL) to obtain the acrylamide solution. Next, EDC (0.1 m, 10 mL) and NHS (0.025 m, 10 mL) were added to a solution of CdSe/ZnS QDs (200 mL) for 30 min, and then acrylic acid (200 mL) was added to the above activated QD solution and reacted at room temperature overnight under gentle shaking. Preparation of the targeting element The targeting element was prepared by conjugating acrylic acid with aptamers for Ramos cells (aptamer sequence: 5-NH2-TACAGAACACCGGGAGGATAGTTCGGTGGCTGTTCAGGGTCTCCTCCCGGTG-3). First, EDC (0.1 m, 10 mL) and NHS (0.025 m, 10 mL) were added to acrylic acid (200 mL) for 30 min for activation. Subsequently, 80 mL of the activated acrylic acid solution were mixed with 20 mL of 10 5 m aptamers and reacted at 37 8C overnight under gentle shaking. Preparation of the QD-containing probe for cell imaging Initiator and catalyst solutions were freshly prepared by adding ammonium persulfate (0.05 g) and tetramethylethylenediamine (TEMED, 25 mL), respectively, to 0.5 mL of H2O. Next, the acrylamide/QD composite (400 mL) was mixed with the acrylic acid/aptamer conjugate (400 mL), followed by addition of 3 % initiator and catalyst. The mixture was then kept at room temperature in the dark under gentle shaking for 40 min. Subsequently, it was centrifuged at 10 000 rpm for 30 min to remove unbound reactants, and the precipitate was redispersed in 0.01 m PBS (800 mL) to obtain the QD-containing probe for cell imaging. Application of the QD-containing probe for cells imaging MCF cells (~ 1 106, 1 mL) were centrifuged at 3000 rpm for 3 min and redispersed in 100 mL of RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 100 U mL 1 penicillin-streptomycin. Then, 500 mL of the QD-containing probe was washed three times and suspended in 10 mm PBS containing 0.1 m NaCl. Subsequently, the QD-containing probe was added to the cells and incubated at 37 8C for 30 min under gentle shaking. The mixture was then centrifuged at 3000 rpm for 3 min, washed with 10 mm PBS containing 0.1 m NaCl, and resuspended in 500 mL of 10 mm PBS containing 0.1 m NaCl. For confocal microscopy imaging, an aliquot of sample solution (10 mL) was deposited onto a microscope slide and covered with a standard microscope slide. The fluorescent QDs and labeled cancer cells were observed with a Leica TCS SP5 II apparatus equipped with a HCX PL APO 40/0.85 objective using a 488 nm laser beam (Argon ion laser).
Chem. Asian J. 2014, 9, 1261 – 1264
Received: December 18, 2013 Published online: March 11, 2014
1264
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
