Quantum dot-Insect Neuropeptide Conjugates for Fluorescence

Aug 24, 2007 - Nano-bioanalysis Team and Glycolipid Function Analysis Team, Health Technology Research Center, National Institute of Advanced Industri...
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Quantum dot-Insect Neuropeptide Conjugates for Fluorescence Imaging, Transfection, and Nucleus Targeting of Living Cells Vasudevanpillai Biju,*,†,§ Damodaran Muraleedharan,†,§ Ken-ichi Nakayama,‡ Yasuo Shinohara,| Tamitake Itoh,† Yoshinobu Baba,†,⊥ and Mitsuru Ishikawa†,§ Nano-bioanalysis Team and Glycolipid Function Analysis Team, Health Technology Research Center, National Institute of AdVanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, Japan, Center for Arthropod Bioresources and Biotechnology (CABB), Kerala UniVersity, KariaVattom, TriVandrum 695 583, India, DiVision of Gene Expression, Institute for Genome Research, The UniVersity of Tokushima, Kuramoto-cho 3-18, Tokushima 770-8503, Japan, and Department of Applied Chemistry, Graduate School of Engineering, Nagoya UniVersity, Furo-cho, Nagoya 464-8603, Japan ReceiVed May 1, 2007. In Final Form: July 13, 2007 We identified an insect neuropeptide, namely, allatostatin 1 from Drosophila melanogaster, that transfects living NIH 3T3 and A431 human epidermoid carcinoma cells and transports quantum dots (QDs) inside the cytoplasm and even the nucleus of the cells. QD-conjugated biomolecules are valuable resources for visualizing the structures and functions of biological systems both in vivo and in vitro. Here, we selected allatostatin 1, Ala-Pro-Ser-Gly-AlaGln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2, conjugated to streptavidin-coated CdSe-ZnS QDs. This was followed by investigating the transfection of live mammalian cells with QD-allatostatin conjugates, the transport of QDs by allatostatin inside the nucleus, and the proliferation of cells in the presence of allatostatin. Also, on the basis of dose-dependent proliferation of cells in the presence of allatostatin we identified that allatostatin is not cytotoxic when applied at nanomolar levels. Considering the sequence similarity between the receptors of allatostatin in D. melanogaster and somatostatin/galanin in mammalian cells, we expected interactions and localization of allatostatin to somatostatin/ galanin receptors on the membranes of 3T3 and A431 cells. However, with QD conjugation we identified that the peptide was delivered inside the cells and localized mainly to the cytoplasm, microtubules, and nucleus. These results indicate that allatostatin is a promising candidate for high-efficiency cell transfection and nucleus-specific cell labeling. Also, the transport property of allatostatin is promising with respect to label/drug/gene delivery and high contrast imaging of live cells and cell organelles. Another promising application of allatostatin is that the transport of QDs inside the nucleus would lift the limit of general photodynamic therapy to nucleus-specific photodynamic therapy, which is expected to be more efficient than photosensitization at the cell membrane or in the cytoplasm as a result of the short lifetime of singlet oxygen.

Introduction Insects not only contribute to more than two-thirds of the biodiversity on earth but also play imperative roles in the economy and health by functioning as pollination agents, pests, and vectors of several infectious diseases. Despite these general aspects, it would be valuable to consider insects to be perpetual resources of peptides and hormones to supplement biological research for which a barrier is a poor interface between invertebrate bioresources, bioconjugate chemistry, and nanoscale materials. An interface between biology, bioconjugation, and nanoscale materials is attractive because of emerging applications in bioanalytical and biomedical detections. As a typical example, the significance of nanoscale materials such as quantum dots (QDs)1-13 and bioconjugated QDs5,6,14-25 and the application of QDs to the visualization of the structure and function of live * Corresponding author. E-mail: [email protected]. Phone: +81-87-8693558. Fax: +81-87-869-4113. † Nano-bioanalysis Team, National Institute of Advanced Industrial Science and Technology. ‡ Glycolipid Function Analysis Team, National Institute of Advanced Industrial Science and Technology (AIST). § Kerala University. | The University of Tokushima. ⊥ Nagoya University. (1) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993, 115, 8706-8715. (2) Norris, D. J.; Sacra, A.; Murray, C. B.; Bawendi, M. G. Phys. ReV. Lett. 1994, 72, 2612-2615.

cells and tissues has been recognized recently.19,20,26-53 Here, we consider that invertebrate bioresources would be valuable if they are interfaced with the advantages of bioconjugated QDs. QDs are semiconductor nanocrystals in which excitons are threedimensionally confined (quantum confinement). The quantum confinement effect provides unique optical properties including size-dependent photoluminescence (PL) color, bright and stable PL, and narrow PL and broad absorption bands to QDs.1-4,27,46 The unique properties made QDs ideal candidates to complement (3) Dabbousi, B. O.; RodriguezViejo, J.; Mikulec, F. V.; Heine, J. R.; Mattoussi, H.; Ober, R.; Jensen, K. F.; Bawendi, M. G. J. Phys. Chem. B 1997, 101, 94639475. (4) Kuno, M.; Lee, J. K.; Dabbousi, B. O.; Mikulec, F. V.; Bawendi, M. G. J. Chem. Phys. 1997, 106, 9869-9882. (5) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (6) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016-2018. (7) Empedocles, S.; Bawendi, M. Acc. Chem. Res. 1999, 32, 389-396. (8) Kuno, M.; Fromm, D. P.; Hamann, H. F.; Gallagher, A.; Nesbitt, D. J. J. Chem. Phys. 2001, 115, 1028-1040. (9) Willard, D. M.; Van Orden, A. Nat. Mater. 2003, 2, 575-576. (10) Robel, I.; Subramanian, V.; Kuno, M.; Kamat, P. V. J. Am. Chem. Soc. 2006, 128, 2385-2393. (11) Willard, D. M.; Mutschler, T.; Yu, M.; Jung, J.; Van Orden, A. Anal. Bioanal. Chem. 2006, 384, 564-571. (12) Biju, V.; Makita, Y.; Sonoda, A.; Yokoyama, H.; Baba, Y.; Ishikawa, M. J. Phys. Chem. B 2005, 109, 13899-13905. (13) Tomasulo, M.; Yildiz, I.; Kaanumalle, S. L.; Raymo, F. M. Langmuir 2006, 22, 10284-10290. (14) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 1214212150.

10.1021/la7012705 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/24/2007

Quantum Dot Insect Neuropeptide Conjugates

several drawbacks of conventional organic fluorescent dye molecules including narrow excitation bands, tailing of fluorescence in the red, and, more importantly, low photobleaching thresholds. The superior properties of QDs make them attractive candidates not only in the above biological applications but also in materials science.3,4,8,10-13,24,54-60 In the current work, we initiate a novel interface among invertebrate bioresources, mammalian cells, and nanoscale materials by investigating the efficiency of transfecting and labeling mammalian cells with insect neuropeptide-conjugated QDs and the effect of the neuropeptide on the proliferation of mammalian cells. Modulation of the surface chemistry of QDs considerably helped to design several water-soluble and targeted QD-conjugates for bioanalyses and in vivo and in vitro visualizations of the structures and functions of biological systems.19,20,26-53 The conjugation of hydrophilic moieties such as carboxylic acids, sugars, amino acids, alcohols, silanols, amines, nucleic acids, proteins, and amphiphilic polymers to the surfaces of quantum dots was employed in those investigations. The various ligands for the surface modification of QDs and specific applications of the surface-modified QDs are reviewed recently.21,23,36,37 Despite these advancements, there are several limitations to transfecting live cells with QDs. Limitations include the aggregation of QDs on the cell membrane due to a barrier provided by the membrane and aggregation inside the cytoplasm due to trapping by liophilic (15) Willard, D. M.; Carillo, L. L.; Jung, J.; Van Orden, A. Nano Lett. 2001, 1, 469-474. (16) Goldman, E. R.; Anderson, G. P.; Tran, P. T.; Mattoussi, H.; Charles, P. T.; Mauro, J. M. Anal. Chem. 2002, 74, 841-847. (17) Clapp, A. R.; Medintz, I. L.; Mauro, J. M.; Fisher, B. R.; Bawendi, M. G.; Mattoussi, H. J. Am. Chem. Soc. 2004, 126, 301-310. (18) Goldman, E. R.; Medintz, I. L.; Whitley, J. L.; Hayhurst, A.; Clapp, A. R.; Uyeda, H. T.; Deschamps, J. R.; Lassman, M. E.; Mattoussi, H. J. Am. Chem. Soc. 2005, 127, 6744-6751. (19) Howarth, M.; Takao, K.; Hayashi, Y.; Ting, A. Y. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7583-7588. (20) Zhou, M.; Ghosh, I. Biopolymers 2007, 88, 325-339. (21) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435-446. (22) Goldman, E. R.; Medintz, I. L.; Mattoussi, H. Anal. Bioanal. Chem. 2006, 384, 560-563. (23) Klostranec, J. M.; Chan, W. C. W. AdV. Mater. 2006, 18, 1953-1964. (24) Liu, T. C.; Huang, Z. L.; Wang, H. Q.; Wang, J. H.; Li, X. Q.; Zhao, Y. D.; Luo, Q. M. Anal. Chim. Acta 2006, 559, 120-123. (25) Tomlinson, I. D.; Mason, J. N.; Blakely, R. D.; Rosenthal, S. J. Bioorg. Med. Chem. Lett. 2006, 16, 4664-4667. (26) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759-1762. (27) Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Nat. Biotechnol. 2003, 21, 47-51. (28) Gao, X. H.; Cui, Y. Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. M. Nat. Biotechnol. 2004, 22, 969-976. (29) Jaiswal, J. K.; Goldman, E. R.; Mattoussi, H.; Simon, S. M. Nat. Methods 2004, 1, 73-78. (30) Pinaud, F.; King, D.; Moore, H. P.; Weiss, S. J. Am. Chem. Soc. 2004, 126, 6115-6123. (31) Alivisatos, A. P.; Gu, W. W.; Larabell, C. Annu. ReV. Biomed. Eng. 2005, 7, 55-76. (32) Bentzen, E. L.; Tomlinson, I. D.; Mason, J.; Gresch, P.; Warnement, M. R.; Wright, D.; Sanders-Bush, E.; Blakely, R.; Rosenthal, S. J. Bioconjugate Chem. 2005, 16, 1488-1494. (33) Gao, X. H.; Yang, L. L.; Petros, J. A.; Marshal, F. F.; Simons, J. W.; Nie, S. M. Curr. Opin. Biotechnol. 2005, 16, 63-72. (34) Hasegawa, U.; Nomura, S. I. M.; Kaul, S. C.; Hirano, T.; Akiyoshi, K. Biochem. Biophys. Res. Commun. 2005, 331, 917-921. (35) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538-544. (36) Parak, W. J.; Pellegrino, T.; Plank, C. Nanotechnology 2005, 16, R9R25. (37) Weissleder, R.; Kelly, K.; Sun, E. Y.; Shtatland, T.; Josephson, L. Nat. Biotechnol. 2005, 23, 1418-1423. (38) Anikeeva, N.; Lebedeva, T.; Clapp, A. R.; Goldman, E. R.; Dustin, M. L.; Mattoussi, H. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16846-16851. (39) Byrne, S. J.; Corr, S. A.; Rakovich, T. Y.; Gun’ko, Y. K.; Rakovich, Y. P.; Donegan, J. F.; Mitchell, S.; Volkov, Y. J. Mater. Chem. 2006, 16, 28962902. (40) Cai, W. B.; Shin, D. W.; Chen, K.; Gheysens, O.; Cao, Q. Z.; Wang, S. X.; Gambhir, S. S.; Chen, X. Y. Nano Lett. 2006, 6, 669-676.

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bodies (endosomes and lysosomes).38,47,49,61,62 Although QDs possess a high photobleaching threshold that is advantageous for prolonged photodynamic therapy and the visualization of biochemical and biophysical processes in labeled cells, one of the difficulties of direct application is the targeted delivery of QDs. Transfection of live cells with QD-bioconjugates and organelle-specific targeting are demanding requirements to understand complex processes involved in drug/gene delivery and signal transduction. The difficulty of transfecting live cells with nanoparticles was resolved to a certain degree by conjugating or coating grafted polyamine polymers,49 polyarginine,62 ligands of growth factor63 and glucose-regulated protein,42 nuclear localization signal peptide,61 protease,47 TAT peptide,64 and so forth on the surface of QDs. To date, peptides constitute one of the most promising classes of ligands used in transfecting live cells with QDs.38,40,42,51,53,61,62,64-66 Specifically, viral peptide complexes, TAT peptide, and peptides specific to glucoseregulated protein efficiently label live cells. Despite the general applications of peptides as cell-targeting and cell-penetrating agents, it would be valuable to consider that several peptides are promising candidates in cancer treatments with minimal undesired side effects. The cell-penetrating ability of peptides, in addition to antibodies, is promising for selective in vivo targeting of nanoparticles, drugs, and genes to the cytoplasm and nucleus of cells that are otherwise practically challenging. The transfection of cells with QDs would lift the limit of photodynamic therapy of cancer that was first envisaged and investigated by Burda and co-workers in 2003.67 However, it would be valuable to consider (41) Courty, S.; Luccardini, C.; Bellaiche, Y.; Cappello, G.; Dahan, M. Nano Lett. 2006, 6, 1491-1495. (42) Kim, Y.; Lillo, A. M.; Steiniger, S. C. J.; Liu, Y.; Ballatore, C.; Anichini, A.; Mortarini, R.; Kaufmann, G. F.; Zhou, B.; Habermann, B. F.; Janda, K. D. Biochemistry 2006, 45, 9434-9444. (43) Le Gac, S.; Vermes, I.; van den Berg, A. Nano Lett. 2006, 6, 1863-1869. (44) Rajan, S. S.; Vu, T. Q. Nano Lett. 2006, 6, 2049-2059. (45) Smith, A. M.; Dave, S.; Nie, S. M.; True, L.; Gao, X. H. Expert ReV. Mol. Diagn. 2006, 6, 231-244. (46) Sun, Y. H.; Liu, Y. S.; Vernier, P. T.; Liang, C. H.; Chong, S. Y.; Marcu, L.; Gundersen, M. A. Nanotechnology 2006, 17, 4469-4476. (47) Zhang, Y.; So, M. K.; Rao, J. H. Nano Lett. 2006, 6, 1988-1992. (48) Zimmer, J. P.; Kim, S. W.; Ohnishi, S.; Tanaka, E.; Frangioni, J. V.; Bawendi, M. G. J. Am. Chem. Soc. 2006, 128, 2526-2527. (49) Duan, H.; Nie, S. J. Am. Chem. Soc. 2007, 129, 3333-3338. (50) Liu, Y. S.; Sun, Y. H.; Vernier, P. T.; Liang, C. H.; Chong, S. Y. C.; Gundersen, M. A. J. Phys. Chem. C 2007, 111, 2872-2878. (51) Xue, F. L.; Chen, J. Y.; Guo, J.; Wang, C. C.; Yang, W. L.; Wang, P. N.; Lu, D. R. J. Fluoresc. 2007, 17, 149-154. (52) Zheng, Y. G.; Gao, S. J.; Ying, J. Y. AdV. Mater. 2007, 19, 376-380. (53) Zhou, M.; Nakatani, E.; Gronenberg, L. S.; Tokimoto, T.; Wirth, M. J.; Hruby, V. J.; Roberts, A.; Lynch, R. M.; Ghosh, I. Bioconjugate Chem. 2007, 18, 323-332. (54) Biju, V.; Itoh, T.; Baba, Y.; Ishikawa, M. J. Phys. Chem. B 2006, 110, 26068-26074. (55) Biju, V.; Itoh, T.; Makita, Y.; Ishikawa, M. J. Photochem. Photobiol., A 2006, 183, 315-321. (56) Biju, V.; Kanemoto, R.; Matsumoto, Y.; Ishii, S.; Nakanishi, S.; Itoh, T.; Baba, Y.; Ishikawa, M. J. Phys. Chem. C 2007, 111, 7924-7932. (57) Biju, V.; Makita, Y.; Nagase, T.; Yamaoka, Y.; Yokoyama, H.; Baba, Y.; Ishikawa, M. J. Phys. Chem. B 2005, 109, 14350-14355. (58) Grebinski, J. W.; Hull, K. L.; Zhang, J.; Kosel, T. H.; Kuno, M. Chem. Mater. 2004, 16, 5260-5272. (59) Ishii, S.; Ueji, R.; Nakanishi, S.; Yoshida, Y.; Nagata, H.; Itoh, T.; Ishikawa, M.; Biju, V. J. Photochem. Photobiol., A 2006, 183, 285-291. (60) Robel, I.; Bunker, B. A.; Kamat, P. V.; Kuno, M. Nano Lett. 2006, 6, 1344-1349. (61) Chen, F.; Gerion, D. Nano Lett. 2004, 4, 1827-1832. (62) Lagerholm, B. C.; Wang, M.; Ernst, L. A.; Ly, D. H.; Liu, H.; Bruchez, M. P.; Waggoner, A. S. Nano Lett. 2004, 4, 2019-2022. (63) Lidke, D. S.; Nagy, P.; Heintzmann, R.; Arndt-Jovin, D. J.; N., P. J.; Grecco, H. E.; Jares-Erijman, E. A.; Jovin, T. M. Nat. Biotechnol. 2004, 22, 198-203. (64) Delehanty, J. B.; Medintz, I. L.; Pons, T.; Brunel, F. M.; Dawson, P. E.; Mattoussi, H. Bioconjugate Chem. 2006, 17, 920-927. (65) Medintz, I. L.; Sapsford, K. E.; Clapp, A. R.; Pons, T.; Higashiya, S.; Welch, J. T.; Mattoussi, H. J. Phys. Chem. B 2006, 110, 10683-10690. (66) Vu, T. Q.; Maddipati, R.; Blute, T. A.; Nehilla, B. J.; Nusblat, L.; Desai, T. A. Nano Lett. 2005, 5, 603-607.

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that the efficiency of singlet oxygen in damaging the cell nucleus and inducing apoptosis depends on the distance of the sensitizer from the nucleus. Therefore, although QDs are powerful agents for prolonged photosensitization, tethering onto the cell membrane and delivery inside the cytoplasm are less efficient compared to nucleus specific photosensitization. In this regard, as observed in the current work, transportation of QDs inside the nucleus by allatostatin is expected to bring radical changes in photodynamic therapy. Here, we selected allatostatin 1 and investigated its efficiency to transfect live mammalian cells when conjugated to QDs and cytotoxicity to the cells. These investigations were considered because of the amino acid sequence similarity between the receptors of allatostatin and somatostatin/galanin,68 growth regulatory receptors distributed in the central nervous system and peripheral organs of all vertebrates. Allatostatins are a ubiquitous family of peptide hormones present in invertebrates and function to inhibit the biosynthesis of juvenile hormones (JH), activate invertase and R-amylase reactions, and block muscle contractions.69 Also, we were motivated by anticancer drugs based on somatostatin analogues that target G-protein-coupled somatostatin receptors (SSTRs), antagonize mitogenic functions of growth factors, and stimulate tyrosine phosphatases; stimulations of SSTRs have been known to induce apoptosis and control cell proliferation via activation of calcineurin and serine threonine and depletion of cAMP and Ca2+ levels.70-72 However, several somatostatin analogues are susceptible to in vivo enzymatic degradation.73 Furthermore, the response of A431 cells toward somatostatin itself is proliferative,74 for which the mechanism is largely unknown. This demands a search for stable and effective anticancer and apoptotic agents through synthetic routes and natural resources to target SSTR. Here, we identified not only that the conjugation of allatostatin to QDs promoted the transfection of the cells and the targeting of cytoplasm, microtubules, and the nucleii of living cells but also that the proliferation of A431 cells was slightly down regulated by allatostatin under selected concentrations (>100 nM). Experimental Section Culturing of Cells in the Presence and Absence of Allatostatin 1. NIH 3T3 and A431 cells were inoculated and subcultured for 4 days in Dulbecco’s modified Eagle’s medium (DMEM), which was supplemented with 5% fetal bovine serum (FBS). The cells were split into nine dishes each containing 5 mL of DMEM-FBS solution. Each dish was partitioned into 4 such that 36 samples are involved in the measurements of each cell type. The cells are allowed to attach to the bottom of the dishes over 1 h and allatostatin 1 solutions prepared in phosphate-buffered saline (PBS) were added. The volume of the PBS solution of allatostatin was below 25 µL in all of the dishes. Allatostatin 1 was obtained from Aldrich, and the concentrations of allatostatin solutions used were 0.038, 0.15, 0.61, (67) Samia, A. C. S.; Chen, X.; Burda, C. J. Am. Chem. Soc. 2003, 125, 15736-15737. (68) Lenz, C.; Sondergaard, L.; Grimmelikhuijzen, C. J. P. Biochem. Biophys. Res. Commun. 2000, 269, 91-96. (69) Weaver, R. J.; Edward, J. P.; Bendena, W. G.; Tobe, S. S. Recent AdVances in Arthropod Endocrinology; Coast, G. M., Webster, S. G., Eds.; Cambridge University Press: Cambridge, U.K., 1998; p 2. (70) Buscail, L.; Esteve, J. P.; Saintlaurent, N.; Bertrand, V.; Reisine, T.; Ocarroll, A. M.; Bell, G. I.; Schally, A. V.; Vaysse, N.; Susini, C. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1580-1584. (71) Kaupmann, K.; Bruns, C.; Raulf, F.; Weber, H. P.; Mattes, H.; Lubbert, H. EMBO J. 1995, 14, 727-735. (72) Liebow, C.; Reilly, C.; Serrano, M.; Schally, A. V. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 2003-2007. (73) Lamberts, S. W. J.; Krenning, E. P.; Reubi, J. C. Endocr. ReV. 1991, 12, 450-482. (74) Kamiya, Y.; Ito, J.; Fujii, T.; Horie, K.; Kawaguchi, M.; Hori, R.; Sakuma, N.; Fujinami, T. Horm. Metab. Res. 1995, 27, 179-181.

Biju et al. 2.43, 9.73, 38.91, and 155.65 nM. Control dishes without allatostatin, two each for 3T3 and A431 cells, were also partitioned for 16 control measurements. The samples were incubated at 36 °C in an atmosphere of 5% CO2 gas, and the cells were counted at 24 h intervals for 4 days. Counting was performed after taking microphotographs (Figure S1) of the cells in premarked areas. Rates of proliferation were estimated from analyzing 864 microphotographs, each containing a few tens of cells (first day) to a few hundred cells (fourth day). The number of cells varied from the 3T3 to A431 cell line. Preparation of QD-Allatostatin Conjugates. For the transfection of the cells with QDs and the localization of allatostatin inside the cells, allatostatin was conjugated to streptavidin-coated CdSe-ZnS QDs. The conjugation was carried out by a heterobifunctional cross-linking reaction. Steps involved in the conjugation reaction are shown in Figure 1A. In a typical reaction a mixture of allatostatin 1 (40 nM solution in PBS buffer) and biotin-Nhydroxysuccinimide ester (biotin-NHS ester, 40 nM solution in PBS buffer) was incubated at room temperature (25 °C) for 1 h. The arginine residue in allatostatin 1 provides a reactive amino group for coupling with the NHS-ester, and the reaction proceeds by replacing the succinimide moiety with allatostatin at the arginine center. Reaction at the N terminus of the peptide is not completely ruled out. This reaction provided biotinylated allatostatin. Excess biotin-NHS ester, if present, was removed by a sephadex G25 column using PBS buffer as the eluent, and the purified biotinylated allatostatin was reacted at room temperature for 30 min with streptavidin-coated QDs having red fluorescence (Emλmax ≈ 605 nm). Biotin-NHS ester was obtained from Sigma, and QDstreptavidin conjugates were obtained from Invitrogen Corporation. Concentrations of biotinylated allatostatin and QD-streptavidin conjugates were selected to be 1:1 to minimize multiple allatostatin binding to QDs; the presence of several streptavidin moieties on the QD surface, as specified by the manufacturer, each with multiple free pockets for biotin, can bind several biotinylated allatostatin molecules if biotinylated allatostatin is used in excess. However, 1:1 biotinylated allatostatin/QD-streptavidin selected in the current work probably avoided the formation of QD-streptavidin with multiple allatostatin. The conjugation reaction was carried out in a specified buffer supplied with the QD sample. The avidin-biotin coupling provided QD-allatostatin conjugates that were purified using a sephadex G25 column using the PBS buffer as the eluent. Instruments Used. Absorption and PL spectra of QD samples were recorded using a Hitachi-4100 spectrophotometer and a Hitachi4500 spectrofluorometer, respectively. Microphotographs were recorded with an inverted optical microscope (Olympus IX70) equipped with a 10× or 60× objective lens and a zoom-in digital CCD camera (Olympus). Fluorescence images were recorded by exciting the labeled cells with either a 488 nm (sapphire 488-25 CDRH, Coherent) or a 532 nm (Nd:YVO4, Spectra Physics Millennia IIs) CW laser in an inverted optical microscope (Olympus IX70) that was equipped with an image intensifier (C8600, Hamamatsu), a digital CCD camera (Olympus), and a time-gated CCD camera (C5985, Hamamatsu). Fluorescence images were collected through either a 10× (UPlanFl, Olympus) or a 60× (PUlanApo, Olympus) objective lens and a long-pass filter for red fluorescence.

Results and Discussion We examined the effect of the conjugation of allatostatin to QDs on the optical properties of QDs. Figure 1 shows the absorption and fluorescence spectra of QDs (optical densities at the excitation wavelength, 488 nm, were matched) before and after allatostatin conjugation. We identified that spectral characteristics such as absorption and fluorescence emission maxima of QDs are not essentially affected by peptide conjugation. This is consistent with previous reports where surface modifications of QDs with various ligands did not considerably alter the ground- and excited-state energies of QDs. However, we observed a slight (6%) decrease in the fluorescence emission intensity of

Quantum Dot Insect Neuropeptide Conjugates

Figure 1. (A) Schematic presentation of the preparation of QDallatostatin conjugates and (B) absorption (a, b) and fluorescence (c, d) spectra of QDs before (b, c) and after (a, d) allatostatin conjugation. The fluorescence spectra were recorded under excitation at 488 nm.

QDs after conjugation to allatostatin. This decrease in the emission intensity is negligible when compared to considerable decreases observed in previous reports where ligands and media were exchanged.75,76 The activation of defect states present in the band gap of QDs, which are otherwise removed by passivating ligand molecules, are considered to be one of the reasons for the

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decrease in the fluorescence intensity during the ligand-exchange reactions and purification steps. In the current work, the presence of protecting polymer shells on the commercial QDs probably prevented the activation of trap states during allatostatin conjugation, and this considerably preserved the band gap energy and PL quantum efficiency of QDs. Although a definitive reason for the slight decrease in PL intensity of QDs is unknown, we propose that a change in the surface charge density of QDs due to the conjugation of the positively charged peptide contributed to the decrease in fluorescence intensity. Also, we examined the stability of the peptide-conjugated QDs by monitoring the absorption and emission spectra as a function of time and identified that the spectral properties remained unaffected for 2 weeks or more. The transfection of 3T3 and A431 cells with QDs and QDallatostatin conjugates was examined as a function of time under incubation. For this, 3T3 and A431 cells were cultured in DMEM-FBS medium for 4 days in the absence of allatostatin. This was followed by washing the cells with PBS, and the medium was exchanged with PBS. The transfection of 3T3 and A431 cells and labeling with QDs were carried out by incubating the cells with 2 nM QD-allatostatin conjugate solutions. QDallatostatin conjugate solutions were added to the 3T3 and A431 cells (the final concentration of QD-allatostatin conjugate solution in PBS was adjusted to be 2 nM) and incubated for 30 min to 1 h. This was followed by washing the cells with PBS to remove excess QD-allatostatin conjugates, and the medium was exchanged with PBS. The QD-allatostatin-labeled cells were excited at 488 or 532 nm with a cw laser beam in an inverted optical microscope, and fluorescence images were recorded. Fluorescence images of 3T3 cells recorded after 30 min and 1 h incubations with QD-allatostatin conjugates are shown in panels Aa and Ab in Figure 2. We identified a gradual distribution of QD-allatostatin conjugates inside the cells; the intensity of the fluorescence images of labeled cells recorded after 30 min of incubation was around the cell membrane and in the perinuclear area (Figure 2Aa). Interestingly, the fluorescence intensity inside the nucleus increased with time under incubation. A fluorescence image recorded after 1 h of incubation is shown in Figure 2Ab; the fluorescence intensity inside the nucleus dramatically increased over 1 h. High-resolution images indicating the distribution of QDs inside the nucleus of 3T3 and A431 cells are shown in Figure 4 and are discussed later in this section. This observation demonstrates that QD-allatostatin conjugates are gradually transported inside the cytoplasm and eventually to the nucleus. Also, we confirmed these results on the basis of fluorescence images of A431 cells incubated with QDallatostatin conjugates. Typical transmission and fluorescence images of A431 cells incubated with QD-allatostatin conjugates for 30 min and 1h are shown in Figures 3A and 3B, respectively. Overlaid transmission and fluorescence images are presented in panels Ac and Bc in Figure 3, which were useful to confirm the localization of QD-allatostatin conjugates inside the cells. As in the case of 3T3 cells, QD-allatostatin conjugates were initially localized to the cell membrane and perinuclear region (Figure 3Ab) of A431 cells, and after 1 h, the fluorescence intensity inside the nucleus was considerably increased (Figure 3Bb). On the basis of these fluorescence images, we consider that 3T3 and A431 cells are efficiently transfected with QD-allatostatin conjugates and allatostatin transported QDs inside the cytoplasm and nucleus of living cells. (75) Duan, H. W.; Nie, S. M. J. Am. Chem. Soc. 2007, 129, 3333-3338. (76) Uyeda, H. T.; Medintz, I. L.; Jaiswal, J. K.; Simon, S. M.; Mattoussi, H. J. Am. Chem. Soc. 2005, 127, 3870-3878.

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Figure 2. Optical microscopy images of 3T3 cells labeled with QD-allatostatin and QD-streptavidin conjugates: (Aa) fluorescence image recorded after 30 min of incubation with QD-allatostatin conjugates; (Ab) fluorescence image recorded after 1 h of incubation with QDallatostatin conjugates; (Ba) transmission optical image recorded after 1 h of incubation with QD-streptavidin conjugates; (Bb) fluorescence image of the cells imaged in panel Ba, recorded after 1 h of incubation with QD-streptavidin conjugates; and (Bc) an overlay of the images in panels Ba and Bb showing that QDs without allatostatin conjugation were localized to the cell membrane. The cells were excited at 488 nm, and the fluorescence images were collected through a 570 nm long-pass filter.

We examined the endocytosis efficiency of QD-allatostatin conjugates by measuring the optical densities of QD-allatostatin solutions before and after incubation with a cell culture. For this, the optical density of a 2 nM QD-allatostatin conjugate solution (in PBS) was measured at 450 nm. This was followed by replacing the medium of 3T3 cells in a 60 mm culture dish (containing approximately (4-5) × 105 cells in confluence) with the QDallatostatin solution (1 mL) and incubating. After 1 h of incubation, the QD-allatostatin solution was gently pipetted out, and the optical density was measured again. Care was taken to avoid dilution due to the presence of trace amounts of PBS left in the culture dish before the addition of the QD-allatostatin conjugate solution. For this, the cells were rinsed twice with the QDallatostatin solution. On the basis of a decrease in the optical density, we identified that 1 h with QD-allatostatin conjugates. Prolonged incubation of the cells resulted in considerable transportation of QD-allatostatin conjugates inside the cytoplasm and nucleus of the cells. The cells were excited at 532 nm, and the fluorescence images were collected through a 570 nm long-pass filter.

cytoplasm.19,26,27,38,42,43,63,75 However, passive diffusion after cell division,26 and incorporation of an antinuclear antigen and a nuclear localization signaling moiety were required to target QDs to the cell nucleus.61 For highly efficient and specific transportation of QDs to the nucleus of a living cell, which is challenging, it is important that the surface chemistry of QDs allows their escape from endosomes/lysosomes, transportation by nuclear trafficking proteins, and interaction with nuclear pore complexes before entering the nucleus. Again, the size of nuclear pore complexes (20-50 nm78) is another limitation. The commercial QDs used in the current work have an average diameter of ∼15 nm and were conjugated with allatostatin molecules (a trideca peptides). Therefore, the size of a QDallatostatin conjugate is expected to be within the size of nuclear pore complexes. At the same time, the presence of multiple amino groups in allatostatin probably enabled the escape of QDallatostatin conjugates from endosomes/lysosomes. This possibility, the size of a QD-allatostatin conjugate, and the presence of QD-allatostatin conjugates inside the nucleus of 3T3 and A431 cells (Figure 4) show that allatostatin signals with the (78) Fahrenkrog, B.; Aebi, U. Nat. ReV. Mol. Cell Biol. 2003, 4, 757-766.

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Figure 5. Histograms of the dose-dependent proliferation of (A) 3T3 and (B) A431 in the presence of allatostatin. The histograms were constructed on the basis of the number of 3T3 and A431 cells in 864 microphotographs recorded at 24 h intervals for 4 days.

nuclear trafficking proteins. This possibility is also supported by the transport of TT-232 molecules, structural analogues of somatostatin that are known to localize inside the nucleus and induce apoptosis to A431 cells.79 We consider the transportation of QDs inside the cytoplasm and nucleus by allatostatin conjugation provide a new QD-bioconjugate for the visualization of complex biophysical processes including intracellular trafficking and to execute label/drug/gene delivery and high contrast imaging of live cells and cell organelles. Furthermore, we identified that QDs can be efficiently delivered inside the nucleus of living cells with the help of allatostatin, which we expect to lift the limit of general photodynamic therapy to nucleus-specific photodynamic therapy. The transportation of photosensitizers inside the nucleus and nucleus-specific photoactivation would be highly efficient in next-generation photodynamic therapy because singlet oxygen formed at the cell membrane and inside the cytoplasm is less efficient to induce apoptosis due to its short lifetime. Despite the advantage of allatostatin as an agent for transporting QDs and other materials/molecules inside the cytoplasm and nucleus of living cells, it is important to understand the interference (79) Keri, G.; Erchegyi, J.; Horvath, A.; Mezo, I.; Idei, M.; Vantus, T.; Balogh, A.; Vadasz, Z.; Bokonyi, G.; Seprodi, J.; Teplan, I.; Csuka, O.; Tejeda, M.; Gaal, D.; Szegedi, Z.; Szende, B.; Roze, C.; Kalthoff, H.; Ullrich, A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12513-12518.

Quantum Dot Insect Neuropeptide Conjugates

of allatostatin with the physiology of cells. In the current work, we examined the cytotoxicity of allatostatin and not QDallatostatin conjugates by considering that the cytotoxicity of quantum dots was addressed in previous reports. According to reports on cytotoxicity, QDs with bare surface are cytotoxic,80,81 and QDs with surface coverage are less cytotoxic.82,83 From the selection of commercial quantum dots with ZnS and polymerprotecting shells, we expect a relatively low cytotoxicity of QDs in the current work. We investigated the cytotoxicity of allatostatin on the basis of dose-dependent proliferation of 3T3 and A431 cells. Interestingly, allatostatin down regulated the proliferation of A431 cells when applied in a high dose (>100 nM), which is promising for anticancer applications. The proliferation of 3T3 cells was down regulated at a moderate dose (∼2 nM) of allatostatin; however, at higher doses, the effect on 3T3 cells was slightly proliferative, for which the mechanism is under investigation. For the cytotoxicity tests, 3T3 and A431 cells were cultured for 4 days in the absence and presence of 0.038, 0.15, 0.61, 2.43, 9.73, 38.91, and 155.65 nM allatostatin solutions in DMEM-FBS media. The rates of proliferations were estimated on the basis of the number of cells within marked areas in microphotographs of the cells. We identified the lowest proliferation (92%) of 3T3 cells in the presence of ∼2.4 nM allatostatin. The dose-dependent proliferation of 3T3 cells is shown in Figure 5A. The proliferation of A431 cells was suppressed to 88% in the presence of 156 nM allatostatin after 4 days. A histogram of the dose-dependent proliferation of A431 cells for 4 days is shown in Figure 5B. The trend of the antiproliferation of the A431 cell line at a high allatostatin dose was observed from the first day and continued until confluence. The suppression of proliferation to 88% in the case of A431 is considerable compared to that of 3T3 cells for a similar dose of allatostatin. It may be noted that the labeling of 3T3 and A431 cells was carried out with 2 nM QD-allatostatin conjugate solutions. Also, from Figures 5A and 5B, the effects of ∼2 nM allatostatin concentration on A431 and 3T3 cells are not proliferative. Therefore, allatostatin may be considered to be a carrier peptide to transport guest molecules and materials to the (80) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2004, 4, 11-18. (81) Tsay, J. M.; Michalet, X. Chem. Biol. 2005, 12, 1159-1161. (82) Guo, G. N.; Liu, W.; Liang, J. G.; He, Z. K.; Xu, H. B.; Yang, X. L. Mater. Lett. 2007, 61, 1641-1644. (83) Ryman-Rasmussen, J. P.; Riviere, J. E.; Monteiro-Riviere, N. A. J. InVest. Dermatol. 2007, 127, 143-153.

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cytoplasm and nucleus of living cells without adversely affecting the physiology of the cell.

Conclusions The conjugation of allatostatin to QDs enabled highly efficient transfection of 3T3 and A431 cells with QDs. Also, QDallatostatin conjugates were initially distributed in the perinuclear region and were gradually delivered inside the cell nucleus. The localization of QD-allatostatin conjugates to the nucleus is similar to that of TT-232 molecules, structural analogues of somatostatin that is known to induce apoptosis in A431 cells.79 Therefore, as in the case of TT-232, the function of allatostatin is intracellular, for which the mechanism is under investigation, and is probably unrelated to the recognition of extracellular domains of somatostatin/galanin receptors or the inhibition of tyrosine phosphatase. Besides the significance of this investigation to the relation between insect bioresources and mammalian cells, the role of allatostatin in carrying QDs to the cytoplasm and cell nucleus shows the possibility to explore insect peptides/hormones as carrier molecules in drug/gene delivery and cancer research. The current work shows that the coating or conjugation of nanoparticles with allatostatin is a promising strategy for achieving high transfection efficiency, which may also be applied to plasmid DNA in gene delivery. Also, the labeling of microtubules and the nucleus of the cells by QD-allatostatin conjugates shows that dye-allatostatin conjugates would also be promising cell labels. Yet another promising application of allatostatin would be nucleus-targeted photodynamic therapy of cancer in which the generation of reactive oxygen species inside the nucleus induces apoptosis more efficiently than cell membrane- and cytoplasm-specific photosensitization. Acknowledgment. V.B. and M.I. are thankful to the Special Coordination Funds for Promoting Science and Technology of the Ministry of Education, Culture, Sports, Science, and Technology, the Japanese Government. Supporting Information Available: Microphotographs of 3T3 and A431 cells recorded at different periods under culture in the presence of allatostatin 1. This material is available free of charge via the Internet at http://pubs.acs.org. LA7012705