口腔癌細胞的人類表皮生長因子受體（EGFR）表現在對於臨床表現上、疾病的預後上以及治療策略上具有高度的相關。藉由人類表皮生長因子受體在許多腫瘤有過度表現的現象進一步設計針對特定癌細胞的表面受體標定以利癌症診斷和治療。磁振造影 (MR imaging) 由於其空間分辨率以及斷層影像能力高，因此是為一有利的非侵入性成像技術。然而，其本身的影像敏感度低，所以必須依賴發展出高效顯影劑。先前研究針對單域抗體 (EG-2) 標定人類表皮生長因子受體可成功地進行對口腔癌細胞的生物體外影像。利用單域抗體對於細胞表面受體標定是一個新穎的診斷檢測、分子成像以及進一步治療的標定工具。單域抗體的優點是具有正常IgG抗體尺寸的十分之一大小、對溫度的穩定性以及容易生產製造。樹枝狀奈米粒子 (Dendrimer) 已被證實是為一具攜帶及接合其他配體能力的奈米粒子。此研究目的以聚乙二醇 (PEG) 為中心的三代 (G3) 樹枝狀奈米粒子並且在奈米粒子表面接上四氧化三鐵 (Fe3O4) 奈米粒子再修飾上釓 (Gd) -二乙烯三胺五乙酸 (DTPA)以作為在進行動物體內磁振造影時的標定配體再進一步探討其合成方式、定性以及發展。奈米粒子中的釓及鐵離子定量狀況分別以感應耦合電漿儀(ICP-AES)、原子吸收光譜儀 (AAS) 作分析。在生物體內實驗，以四氧化三鐵 (Fe3O4) -聚乙二醇 (PEG) -樹枝狀奈米粒子- (釓-二乙烯三胺五乙酸) (GdDTPA)-單域抗體 (EG-2) 奈米粒子以尾靜脈注射到有背負腫瘤的小鼠內。所有影像獲得分別在注射前、注射15分鐘後、30分鐘、45分鐘後、1小時後、24小時及48小時候以臨床1.5特士拉強度磁振成像系統進行T1 及T2加權磁振造影。在腫瘤注射後1小時的T1 及T2加權影像相較於沒有奈米粒子注射的加權影像，分別有顯著明亮影像的促進現象及訊號下降現象。並在腫瘤的T1 及T2磁振造影信號強度明顯高於注射前信號強度。因此，我們發展出此多功能負載釓的三代樹枝狀奈米粒子接上四氧化三鐵奈米粒子並修飾接合EG-2單域抗體不僅能夠提供標定的效果，並且在生物體內的磁振造影有同步雙重T1 及T2加權顯影的潛力，而且可以進一步擴展為其他生物奈米粒子的設計運用在T1 及T2加權磁振造影上。

Expression of epidermal growth factor receptors (EGFR) in oral cancers is highly associated with clinical behavior, disease prognosis and therapeutic strategy. Up regulation of EGFR has been found in many cancer types, which provides an opportunity for designing receptor-targeted approaches for cancers detection and treatment. Magnetic resonance imaging (MR imaging) is a powerful, non-invasive imaging technique that exhibit high spatial resolution and tomographic capabilities. However, owing to its low intrinsic sensitivity, the development of highly efficient contrast agents is therefore prerequisite for the success of MR imaging. Previous studies have demonstrated that EG-2 single-domain-antibodies (SdAbs) could successfully target the EGFR of cancer cells in the in vitro imaging. As a result, SdAbs could be considered as a novel tool for receptor targeting, diagnostic sensing, molecular imaging, and therapy. SdAbs possess several advantageous features which include comparatively smaller size (only one tenth of the size of the normal IgG antibodies), appropriate thermal stability and easiness of their synthetic production. Dendrimer-based nanoparticles (NPs) were proven to be versatile carriers capable of specifically conjugating ligands. This study aimed to synthesize, characterize and develop polyethylene glycol (PEG) core generation three (G3) dendrimer conjugated with Fe3O4 and -modified with gadolinium (Gd) diethylenetriamine pentaacetic acid (DTPA) units. The surface of Fe3O4 nanoparticles bore the attachment of EG-2 SdAbs that can target EG-2 ligands for in vivo MR imaging of tumors over-expressing EGFR. In the current study, we demonstrated that the Fe3O4 nanoparticles modified with Ni-NTA could be conjugated with EG-2 SdAbs. Quantitative of gadolinium and Fe3O4 was analyzed by the inductively coupled plasma atomic emission spectroscopy (ICP-AES) and atomic absorption spectrometry (AAS). For in vivo MRI imaging, tumor-bearing mice were administered with Fe3O4-PEG dendrimer-(GdDTPA)-EG-2 SdAbs NPs via tail vein injection. T1-weighted MR imaging was performed using a clinical 1.5 T MR imaging system, and acquisition of the results was respectively done at 15 min, 30 min, 45 min, 1 hr, 24 hr, and 48 hr post injections. Compared with the image without nanoparticles injection, both T1-weighted and T2-weighted MR imaging of tumor 1 hr post injection showed significant contrast enhancement effect and signal decreasing, respectively. The intensity of T1-weighted and T2-weighted MR imaging in tumor of the treated mice were higher than that found in its negative control. In conclusion, we successfully developed a multifunctional Ni-NTA-modified, EG-2 SdAbs-conjugated Fe3O4-G3 dendrimer-GdDTPA, which not only harbored specific targeting efficacy but also had great potential for the in vivo dual contrast MR imaging. The technology developed in this study could be extended for fabricating other biologically active NPs used in dual contrast T1- and T2-weighted MR imaging.

1.Wanga, M. & Thanou, M. Targeting nanoparticles to cancer. Pharmacological Research 2, 90-99 (2010)
2.Ferrari, M., et al. Cancer nanotechnology: opportunities and challenges. Nat Rev Cancer 5, 161-171 (2005)
3.Ebbesen, M. & Jensen, T.G. Nanomedicine: Techniques, Potentials, and Ethical Implications. Journal of Biomedicine and Biotechnology 5, 1-11 (2006)
4.Williams, D. The risks of nanotechnology. Medical device technology 16, 9-10 (2005)
5.Williams, D. Nanotechnology: a new look. Medical device technology 15, 9-10 (2004)
6.Zhang, Y., Li, H., Luo, Y., Shi, X., Tian, J. & Sun, X. Poly (m-phenylenediamine) nanospheres and nanorods: selective synthesis and their application for multiplex nucleic acid detection. PloS one 6, 1-11 (2011)
7.Ramanathan, S., Patibandla, S., et al. Fluorescence and infrared spectroscopy of electrochemically self assembled ZnO nanowires: evidence of the quantum confined Stark effect. J Mater Sci: Mater Electron 17, 651-655 (2006)
8. Teja, Amyn, S., Pei-Yoong, Koh. Synthesis, properties, and applications of magnetic iron oxide nanoparticles. Progress in Crystal Growth and Characterization of Materials 55, 22-45 (2009)
9. Laurent, S., Forge, D., et al. Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chem Rev. 108, 2064-2110 (2008)
10. Wu, W., He, Q., Jiang, C. Magnetic iron oxide nanoparticles: synthesis and surface functionalization strategies. Nanoscale Res Lett. 3, 397-415 (2008)
11. Astruc, D., Boisselier, E., Ornelas, C. Dendrimers Designed for Functions: From Physical, Photophysical, and Supramolecular Properties to Applications in Sensing, Catalysis, Molecular Electronics, and Nanomedicine. Chem. Rev. 110, 1857-1959 (2010)
12. Nanjwade, BK., Bechra, HM., et al. Dendrimers: emerging polymers for drug-delivery systems. Eur J Pharm Sci. 38, 185-196 (2009)
13. Prajapati, RN., Tekade, RK., et al. Dendimer-Mediated Solubilization, Formulation Development and in Vitro-in Vivo Assessment of Piroxicam. Synthesis 6, 940-950 (2009)
14. Chen, WT., Thirumalai, D., et al. Dynamic contrast-enhanced folate-receptor-targeted MR imaging using a Gd-loaded PEG-dendrimer-folate conjugate in a mouse xenograft tumor model. Mol Imaging Biol. 12, 145-154 (2010)
15. Zhang, L., F. X. Gu, et al. Nanoparticles in Medicine: Therapeutic Applications and Developments. Clin Pharmacol Ther 83, 761-769 (2007)
16. Fonseca, M. J., J. C. Jagtenberg, et al. Liposome-mediated targeting of enzymes to cancer cells for site-specific activation of prodrugs: comparison with the corresponding antibody-enzyme conjugate. Pharm. Res 20, 423-428 (2003)
17. Bertrand, N., C. l. Bouvet, et al. Transmembrane pH-Gradient Liposomes To Treat Cardiovascular Drug Intoxication. ACS Nano 4, 7552-7558 (2010)
18. Astete, C. E. and C. M. Sabliov. Synthesis and characterization of PLGA nanoparticles. Journal of Biomaterials Science, Polymer Edition 17, 247-289 (2006)
19. Nair K, L., Jagadeeshan, S., et al. Biological evaluation of 5-fluorouracil nanoparticles for cancer chemotherapy and its dependence on the carrier, PLGA. Int J Nanomedicine 6, 1685-1697 (2011)
20. Li, F., J. Sun, et al. Preparation and characterization novel polymer-coated magnetic nanoparticles as carriers for doxorubicin. Colloids and Surfaces B: Biointerfaces 88, 58-62 (2011)
21. Hu, M., et al. Gold nanoparticle-protein arrays improve resolution forcryo-electron microscopy. Journal of structural biology 161, 83-91 (2008).
22. Anker, J.N., et al. Biosensing with plasmonic nanosensors. Naturematerials 7, 442-453 (2008)
23. Li, S., Wang, A., Jiang, W. & Guan, Z. Pharmacokinetic characteristicsand anticancer effects of 5-fluorouracil loaded nanoparticles. BMC cancer 8, 103 (2008).
24. Zhou, W., et al. Drug-loaded, magnetic, hollow silica nanocomposites for nanomedicine. Nanomedicine 1, 233-237 (2005).
25. Boulikas, T. & Vougiouka, M. Recent clinical trials using cisplatin, carboplatin and their combination chemotherapy drugs (review). Oncology reports 11, 559-595 (2004).
26. Liu, J., et al. Nanoparticles as image enhancing agents for ultrasonography. PHYSICS IN MEDICINE AND BIOLOGY 51, 2179-2189 (2006).
27. Kuo, P.H. Gadolinium-containing MRI contrast agents: important variations on a theme for NSF. J Am Coll Radiol 5, 29-35 (2008).
28. Weinmann, H.J., Brasch, R.C., Press, W.R. & Wesbey, G.E. Characteristics of gadolinium-DTPA complex: a potential NMR contrast agent. Ajr 142, 619-624 (1984).
29. Yoo, B. & Pagel, M.D. An overview of responsive MRI contrast agents for molecular imaging. Front Biosci 13, 1733-1752 (2008).
30. Harvey, B.H., McEwen, B.S. & Stein, D.J. Neurobiology of antidepressant withdrawal: implications for the longitudinal outcome of depression. Biological psychiatry 54, 1105-1117 (2003).
31. Pushkar, S., Philip, A., Pathak, K. and Pathak, D. Dendrimers: Nanotechnology Derived Novel Polymers in Drug Delivery. Indian J. Pharm. Educ. Res 40, 153-158 (2006)
32. Sakthivel, T and Florence, A.T. Adsorption of Amphipathic Dendrons on Polystyrene Nanoparticles. Int. J. Pharm. 254, 23-26 (2003)
33. Jevprasesphant, R., Penny, J., et al. The influence of surface modification on the cytotoxicity of PAMAM dendrimers. Int. J. Pharm. 252, 263- 266(2003).
34. Donald, A., Tomalia., et al. Dense star polymers having core, core branches, terminal groups. U.S. Patent 466 (1983)
35. Goldsmith, M., Koutcher, J. A., and Damadian, R. Nuclear magnetic resonance in cancer, XII: Application of NMR malignancy index to human lung tumours. Br J Cancer 36, 235-242 (1977)
36. Turner, DA. Nuclear magnetic resonance in oncology. Semin Nucl Med. 15, 210-223 (1985).
37. Botnar, R.M. & Nagel, E. Structural and functional imaging by MRI. Basic research in cardiology 103, 152-160 (2008).
38. Bergstrom, K., Osterberg, E., Holmberg, K., et al. Effects of branching and molecular weight of surface-bound poly(ethylene oxide) on protein rejection. J Biomater Sci Polym Ed 6, 123-132 (1994)
39. Tomalia, D.A., Uppuluri, S., Swanson, D.R., et al. Dendritic macromolecules: a fourth major class of polymer architecture—new properties driven by architecture. Mater Res Soc Symp P 543, 289-298 (1999)
40. Wiener, E.C., Brechbiel, M.W., Brothers, H., et al. Dendrimer-based metal chelates: a new class of magnetic resonance imaging contrast agents. Magn Reson Med 31, 1-8 (1994)
41. Elbayoumi, T.A., Torchilin, V.P., Enhanced accumulation of long-circulating liposomes modified with the nucleosome-specific monoclonal antibody 2C5 in various tumours in mice: gamma-imaging studies. Eur J Nucl Med Mol Imag 33, 1196-1205(2006)
42. Gunasekera, U.A., Pankhurst, Q.A., Douek, M. Imaging applications of nanotechnology in cancer. Target Oncol 4, 169-181 (2009)
43. Peng, X.H., Qian, X., Mao, H., et al. Targeted magnetic iron oxide nanoparticles for tumor imaging and therapy. Int J Nanomed 3 311-321(2008)
44. Yu, M.K., et al. Drug-Loaded Superparamagnetic Iron Oxide Nanoparticles for Combined Cancer Imaging and Therapy In Vivo. Angewandte Chemie (International ed (2008).
45. Thomas, T.P., et al. Investigation of tumor cell targeting of a dendrimer nanoparticle using a double-clad optical fiber probe. Journal of biomedical optics 13, 014024 (2008).
46. Zhang, J., et al. A pentavalent single-domain antibody approach to tumor antigen discovery and the development of novel proteomics reagents. Journal of molecular biology 341, 161-169 (2004).
47. Caravan, P., Ellison, J.J., McMurry, T.J., Lauffer, R.B. Gadolinium(III) chelates as MRI contrast agents: Structure, dynamics, and applications. Chemical Reviews 99, 2293-2352(1999)
48. Ma, P., Liu, S., et al. Lactose mediated liver-targeting effect observed by ex vivo imaging technology. Biomaterials 31, 2646-2654 (2010).
49. Lin, J.J., Chen, J.S., et al. Folic acid-Pluronic F127 magnetic nanoparticle clusters for combined targeting, diagnosis, and therapy applications. Biomaterials 30, 5114-5124 (2009).
50. Langereis, S., Dirksen, A., et al. Dendrimers and magnetic resonance imaging. New Journal of Chemistry 31, 1152-1160 (2007)
51. Tomalia, D.A., Baker, H., et al. A New Class of Polymers: Starburst-Dendritic Macromolecules. Polymer Journa. 17, 117-132 (1985)
52. Kobayashi, H., Kawamoto, S., et al. Novel liver macromolecular MR contrast agent with a polypropylenimine diaminobutyl dendrimer core: Comparison to the vascular MR contrast agent with the polyamidoamine dendrimer core. Magnetic Resonance in Medicine 46, 795-802 (2001)
53. Cheng, Y., Zhao, L., et al. Design of biocompatible dendrimers for cancer diagnosis and therapy: current status and future perspectives. Chemical Society Reviews 40, 2673-2703 (2011)
54. Wang, M., Thanou, M.. Targeting nanoparticles to cancer. Pharmacological Research 62, 90-99 (2010)
55. Hwang, S.H., Moorefield, C.N., Newkome, G.R. Dendritic macromolecules for organic light-emitting diodes. Chemical Society Reviews 37, 2543-2557 (2008)