控制藥物釋放系統在全球已經引起各界高度的關注，尤其是在可受外力場刺激，以及具靶向作用之智慧型藥物釋放系統更為大家所重視。在此研究中，合成具多功能殼核型奈米藥物輸送載體，其中包載抗癌藥劑Camptothecin，並且能夠藉由外加磁場作為驅動力，有效地控制其藥物釋放的行為。油溶性的藥物被包覆在氧化鐵與高分子所形成的核中，外面包覆一層薄薄的二氧化矽，這樣的結構設計，能有效調控並防止藥物達作用前的流失。在外在磁場的刺激(非接觸性)下，核內物質發生結構上的變化，藥物能快速地由載體中釋放出來。此外，本實驗更進一步於奈米載體表面嫁接上anti-EGFR，以EGFR過度表現的癌症細胞作為靶向。令人驚喜的是，這種不含界面活性劑的奈米藥物載體擁有相當好的生物相容性，並能快速有效的進入肺癌細胞中(A549)，其進入細胞的機制主要為macropinocytosis 和clathrin-mediated endocytosis。此奈米載體除了能有效受磁場控制的特性外，在癌症細胞的治療以及作用上亦展現了多種優勢：同時具有靶向標定、熱治療、化療以及細胞顯影等功能。在此研究中，我們以非小細胞肺癌為對象，設計了具多種治療效果的靶向奈米藥物載體，並於in vitro 實驗中作進一步的探討與評估。

Controlled drug released has been received greatest attention worldwide, especially cell-specific targeted or stimuli-responsive drug delivery systems. In this study, drug-containing iron oxide/silica core–shell (SAIO@SiO2) nanocarriers were synthesized as a multifunctional drug delivery nanosystem where the drug, CPT, a highly potent anti-cancer substance, can be effectively triggered with a controllable release pattern by an external magnetic field. The hydrophobic drug (CPT) molecules were encapsulated within the iron oxide-PVA core and then further covered with a thin-layer silica shell to regulate the release pattern, where un-desirable release or leakage can be effectively inhibited. The SAIO@SiO2@AE nanocarriers were coupled with an anti-EGFR on the surface of SAIO@SiO2 nanocarriers in order to target to EGFR overexpressed cancer cells. Remote control of drug release from the SAIO@SiO2 nanocarriers was achieved successfully using an external magnetic field where the core phase being structurally disintegrated to a certain extent while subjecting to magnetic stimulus, resulting in a burst release of the encapsulated drug. Surprisingly, the surfactant-free SAIO@SiO2 and SAIO@SiO2@AE nanocarriers demonstrated excellent cytocompatibility and high uptake efficiency by the A549 cell line within short time through energy-dependent macropinocytosis and clathrin-mediated endocytosis. Together with a well-regulated controlled release design, the nanocarriers provide great advantages as an effective cell-based drug delivery nanosystem on cell-targeting, hyperthermia, chemotherapy and cellular image for anti-cancer applications. In this report, we show a stepwise study of a nanocarriers-based multifunctional targeted delivery system for in vitro therapeutic application in non small cell lung cancer.

中文摘要………………………………………………............................................i
Abstract………………………………………………………………….…………..iii
致謝………………………………………………………………………v
Contents ………………………………………………………………….…….......vi
Figures Captions………….…………………………………………….………...…ix Table Captions…………………………………………………………….……....xiii
Chapter 1 Introduction…………………………………………………………....1
1.1 Introduction ..……………………………………………………………...…….1
1.2 Motivation …………………………………………………………………….…3
Chapter 2 Literature Review and Theory………………………………..……..4
2.1 Nanomaterial in Biomedicine………………………….……………………….5
2.1.1 Nanotechnology on drug delivery system…………………….………..5
2.1.2 Nanomagnetic composites for biomedical applications……..………..7
2.1.2.1 Iron oxide………………………………………………………….7
2.1.2.2 Controlled drug release…………………………….……………8
2.1.2.3 Hyperthermia……………………………………………...…….10
2.1.2.4 MRI…………………………………………………………….…11
2.2 Drug delivery system in cancer applications………………………………..12
2.2.1 Introduction to cancer ……………………………………………...…12
2.2.2 Targeted drug delivery carrier in cancer cells…………………..…….15
2.2.2.1 Enhanced permeability and retention effect ..……………….19
2.2.3 Endocytosis mechanism ……………………………………….…….20
Chapter 3 Experiment Section ……………….………………………………23
3.1 Material and reagents ………………………………………………………...23
3.2 Apparatus…………………………………………………………………..…..25
3.3 Methods…………………………………………………………………….26
3.3.1 Synthesis of self-assemble iron oxide nanoparticles/silica core-shell (SAIO@SiO2) nanocarriers ……………………………………………26
3.3.2 Characterization .……………………………………………………. 27
3.3.3 Drug loading and release ………………………………………………28
3.3.4 Anti-EGFR modifications………………………………………………..28
3.3.4 Cell culture test ………………………………………………………….29
3.3.4.1 A549, MRC-5, MCF-7 culture and cytotoxicity detect ...……29
3.3.4.2 Determination of cellular uptake by a flow cytometer ...…...29
3.3.4.3 Cell-uptake monitored by fluorescence and confocal microscope.. 30
3.3.4.4 Cell cycle analysis.....…………………………………………. 30
3.3.4.5 Endocytosis pathway analysis.....…………………………… 31
3.3.5 Magnetic field stimulus treated ...…………………………………….…...32
Chapter 4 Results and Discussion ……………………………………………33
4.1 Characterization of SAIO@SiO2 ………………………………………….…33
4.1.1 Preparation and Formation of SAIO@SiO2 nanocarriers ....……..33
4.1.2 Characteristics…………………………………………………………..37
4.1.3 Entrapment of hydrophobic drug and drug loading efficiency……...40
4.1.4 Drug release behavior…………………………………………………..43
4.2 Interactions of drug carriers system with cells………………………..…….47
4.2.1 In-vitro cytotoxicity ……………………………………………………...47
4.2.2 Cell uptake efficiency……………………………………………………53
4.2.3 Drug release via HFMF stimulus……………………………..………..58
4.2.4 Cell cycle analysis……………………………………………………….62
4.3 Cell targeting design………………………………………………….……… 65
4.3.1 Preparation and formation of SAIO@SiO2@AE nanocarriers……65
4.3.2 Selecting cell uptake…………………………………………………. 66
4.3.3 Endocytosis pathway identification ……………………………….....72
4.3.4 Drug release from SAIO@SiO2@AE via magnetic stimulus …….78
Chapter 5 Conclusion…………………………………………………………..81
Chapter 6 References……………………………………………………………83

Allen, T. M. Cullis, P. R. Science 2004, 303, 1818.
Whitesides, G. M. Nat. Biotechnol. 2003, 21, 1161.
LaVan, D. A. McGuire, T. Langer, R. Nat. Biotechnol. 2003, 21, 1184.
Farokhzad, O. C. Karp, J. M. Langer, R. Exp. Opin. Drug Delivery 2006, 3, 311.
Zhang, L. Gu, F. X. Chan, J. M. Wang, A. Z. Langer, R. S., Farokhzad, O. C. Clin. Pharmacol.Ther. 2008, 83, 761.
Liong, M. Lu, J. Kovochich, M. Xia, T. Ruehm, S. G. Nel, A. E. Tamanoi, F. Zink, J. I. ACS Nano 2008, 2, 889.
Bangham, A. D. Standish, M. M. Watkins, J. C. J. Mol. Biol. 1965, 13, 238.
Langer, R. Folkman, J. Nature 1976, 263, 797.
Yatvin, M. B. Kreutz, W. Horwitz, B. A. Shinitzky, M. Science 1980, 210, 1253.
Leserman, L. D. Barbet, J. Kourilsky, F. Weinstein, J. N. Nature 1980, 288, 602.
Heath, T. D. Fraley, R. T. Papahdjopoulos, D. Science 1980, 210, 539.
Klibanov, A. L. Maruyama, K. Torchilin, V. P. Huang, L. FEBS Lett. 1990, 268, 235.
Gref, R. Minamitake, Y. Peracchia, M. T. Trubetskoy, V. Torchilin, V. Langer, R. Science 1994, 263, 1600.
Wagner, V. Dullaart, A. Bock, A. K. Zweck, A. Nat. Biotechnol. 2006, 24, 1211.
Park K. J. of Controlled Release 2007, 20, 1.
Schwertmann, U. Cornell, R.M. Weinheim, Cambridge: VCH; 1991.
Cornelis, K. Hurlburt, C. S. Manual of Mineralogy. New York: Wiley; 1977.
Goya, G. F. Berquo, T. S. Fonseca, F. C. J Appl Phys. 2003, 94, 3520.
Tartaj, P. Morales, M. P. Veintemillas, V. S, Gonza’ lez, C. T, Serna, C. J. J Phys D: Appl Phys 2003, 36, R182.
Chatterjee, J. Haik, Y. Chen, C. J. J Magn Magn Mater. 2003, 257, 113.
Pratsinis, S. E. Vemury, S. Powder Technol 1996, 88, 267.
Nasongkla, N. Bey, E. Ren, J. Ai, H. Khemtong, C. Guthi, J. S. Chin, S. F. Sherry, A. D. Boothman, D. A. Gao, J. Nano Lett. 2006, 6, 2427.
Kohler, N. Sun, C. Wang, J. Zhang, M. Langmuir 2005, 21, 8858.
Doyle, P. S. Bibette, J. Bancaud, A. Viovy, J. L. Science 2002, 295, 2237.
Koh, I. Wang, X. Varughese, B. Isaacs, L. Ehrman, S. H. English, D. S. J. Phys. Chem. B 2006, 110, 1553.
Lu, C. W. Hung, Y. J. Hsiao, K. Yao, M. Chung, T. H. Lin, Y. S. et al. Nano Lett. 2007, 7, 149.
Seo, W. S. Lee, J. H. Sun, X. Suzuki, Y. Mann, D. Liu, Z. et al. Nat. Mater. 2006, 5, 971.
Veiseh, O. Sun, C. Gunn, J. Kohler, N. Gabikian, P. Lee, D. et al. Nano Lett. 2005, 5, 1003.
Jordan, A. Scholz, R. Wust, P. Schirra, H. Schiestel, T. Schmidt, H. Felix, R. J. Magn. Magn. Mater. 1999, 194, 185.
Grayson, A. C. R. Choi, I. S. Tyler, B. M. Wang, P. P. Brem, H. Cima, M. J. Langer, R. Nat. Mater. 2003, 2, 767.
Hu, Z. Xia, X. Adv. Mater. 2004, 16, 305.
Zhang, X. Z. Wu, D. Q. Chu, C. C. Biomaterials 2004, 25, 3793.
Das, M. Mardyani, S. Chan, W. C. W. Kumacheva, E. Adv. Mater. 2006, 18, 80.
Zhu, Y. Shi, J. Shen, W. Dong, X. Feng, J. Ruan, M. Li, Y. Angew. Chem. Int. Ed. 2005, 44, 5083.
Abidian, M. R. Kim, D. H. Martin, D. C. Adv. Mater. 2006, 18, 405.
Lee, K. Y. Peters, M. C. Mooney, D. J. Adv. Mater. 2001, 13, 837.
De Geest, B. G. Skirtach, A. G. Mamedov, A. A. Antipov, A. A. Kotov, N. A. De Smedt, S. C. Sukhorukov, G. B. Small 2007, 3, 804.
Saslawski, O. Weingarten, C. Benoit, J. P. Couvreur, P. Life Sci. 1988, 42, 1521.
Kost, J. Wolfrum, J. Langer, R. Biomed, J. Mater. Res. 1987, 21, 1367.
Kost, J. Noecker, R. Kunica, E. Langer, R. J. Biomed. Mater. Res. 1985, 19, 935.
Kost, J. Wolfrum, J. Langer, R. J. Biomed. Mater. Res. 1987, 21, 1367.
Edelman, E. R. Kost, J. Bobeck, H. Langer, R. J. Biomed. Mater. Res. 1985, 19, 67.
Saslawski, O. Weingarten, C. Benoit, J. P. Couvreur, P. Life Sci. 1988, 42, 1521.
Liu, T. Y. Hu, S. H. Liu, K. H. Liu, D. M. Chen. S. Y. J Magn Magn Mater. 2006, 304, 397.
Liu, T .Y. Hu, S. H. Liu, D. M. Chen, S.Y. Langmuir 2006, 22, 5977.
Lu, Z. Prouty, M. D. Guo, Z. Golub, V. O. Kumar, C. S. S. R. Lvov, Y. M. Langmuir 2005, 21, 2042.
Yoon, T. J. Kim, J. S. Kim, B. G. Yu, K. N. Cho, M. H. Lee, J. K. Angew. Chem. Int. Ed. 2005, 44, 1068.
Yanase, M. Shinkai, M. Honda, H. Yoshida, J. Wakabayashi, T. Kobayashi, T. Jpn J Chem Res. 1998, 89, 463.
Nielsen, O. S. Horsman, M. Overgaard, J. Eur J Cancer 2001, 37, 1587.
Luderer, A. A. Borrelli, N. F. Panzarino, J. N. Mansfield, G. R. Hess, D. M. Brown, J. L. Barnett, E. H. Radiat Res. 1983, 94, 190.
Chan, D. C. F. Kirpotin, D. B. Bunn, P. A. J Magn Magn Mater. 1993, 122, 374.
Sneed, P. K. Stea, B. Thermoradiotherapy and thermochemotherapy 1996, 2.
Bulte, J. W. M. Brooks, R. A. Plenum, New York London, 1997, 527.
Sorensen CM. In: Klabunde KJ, editor. New York: Wiley 2001, 169.
Johnson, G.A. Mag Reson 1993,Q9, 1.
Yeh, T. C. Zhang, W. G. Ildstad, S. T. Ho, C. Magn Reson Med 1995, 33, 200.
Suzuki, M. Honda, H. Kabayashi, T. Yoshida, J. Takahashi, M. Brain Tumor Pathol. 1996, 13, 127.
Robert A. Weinberg, The Biology of Cancer, First Edition, Copyright © Garland Science 2007.
Gu, F. Zhang, L. Teply, B. A. Mann, N. Wang, A. Radovic-Moreno, A. F. Langer, R. Farokhzad, O. C. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2586.
Decuzzi, P. Pasqualini, R. Arap, W. Ferrari, M. Pharm. Res. 2009, 1, 235.
Patil, Y. B. Stoti, U. S. Khdair, A. Ma, L. Panyam, J. Biomaterials 2008, 30, 859.
Kim, B.S. Park, S. W. Hammond, P. T. ACS Nano 2008, 2, 386.
Groneberg, D. A. Rabe, K. F. Fischer, A. Eu J Pharmacology 2006, 533, 182.
Gloria J. K. Shuming, Nie. Nanotoday 2005, 28.
Farokhzad, O. C. Langer, R. ACSNANO 2009, 3, 16.
Fahmy, T. M. Fong,P. M. Goyal, A. Saltzman ,W. M. Nanotoday 2005, 18.
Jain, R. K. Annu. Rev Biomed Eng. 1999, 1, 241.
Teicher, B. A. Drug Resist. Update 2000, 3, 67.
Duncan, R. Nat. Rev. Drug Discov. 2003, 2, 347.
Matsumura, Y. Maeda, H. Cancer Res. 1986, 6, 6397.
Moghimi, S. M. Pharmacol. Rev. 2001, 53, 283.
Juliano, R. L. Stamp, D. Biochem. Biophys. Res. Commun.1975, 63, 651.
Dellian, M. Br. J. Cancer 2000, 82, 1513.
Berry, C. C. J. Mater. Chem. 2005, 15, 543.
Kam, N. W. Liu, Z. Dai, H. Angew. Chem. Int. Ed. 2006, 45, 577.
Stuart, A. D. Brown, T. D. J. Virol. 2006, 80, 7500.
Patel, L. N. Zaro, J. L. Shen, W. C. Pharm. Res. 2007, 24, 1977.
Swanson, J. A. Watts, C. Trends Cell Biol. 1995, 5, 424.
Takei, K. Haucke, V. Trends Cell Biol. 2001, 11, 385.
Khalil, I. A. Kogure, K. Akita, H. Harashima, H. Pharmacol. ReV. 2006, 58, 32.
Thomsen, P. Roepstorff, K. Stahlhut, M. Van Deurs, B. Mol. Biol. Cell. 2002, 13, 238.
Parton, R. G. Richards, A. A. Traffic 2003, 4, 724.
Sun, S. Zeng, H. Robinson, D. B. Raoux, S. Rice, P.M. Wang, S. X. Li, G. J. Am. Chem. Soc. 2004, 126, 273.
Roy, I. Ohulchanskyy, T. Y. Pudavar, H. E. Bergey, E. J. Oseroff, A. R. Morgan, J. et al. J. Am.Chem. Soc. 2003, 125, 7860.
Kim, J. S. Yoon, Tae. J. Yu, K. N. Noh, M. S. Woo, M. Kim, B. G. Lee, K. H. et al. J. Vet. Sci. 2006, 7, 321.
Lin, Y. S. Tsai, C. P. Huang, H. Y. Kuo, C. T. Hung, Y. Huang, D. M. Chen, Y. C. Mou, C. Y. Chem. Mater. 2005, 17, 4570.
Long, J. W. Logan, M. S. Rhodes, C. P. Carpenter, E. E. Stroud, R. M. Rolison, D. R. J. Am. Chem. Soc. 2004, 126, 16879.
Li, C. Adv. Drug Delivery Rev. 2002, 54, 695.
Na, K. Park, K. H. Kim, S. W. Bae, Y. H. J. Controlled Release 2001, 69, 225.
Saul, J. M. Annapragada, A. Natarajan, J. V. Bellamkonda, R. V. J. Controlled Release 2003, 92, 49.
Liang, H. C. Chang, W. H. Lin, K. J. Sung, H. W. J. Biomed. Mater. Res. 2003, 65A, 271.
Na, K. Lee, T. B. Park, K. G. Shin, E. K. Lee, Y. B. Choi, H. K. Eur. J. Pharm. Sci. 2003, 18, 165.
Giovanella, B. C. Stehlin, J. S. Wall, M. E. Wani, M. C. Nicholas, A. W. Liu, L. F. Silber, R. Potmesil, M. Science 1989, 246, 1046.
Pommier, Y. Pourquier, P. Fan, Y. Strumberg, D. Biochim. Biophys. Acta 1998 ,1400, 83.
Goldwasser, F. Shimizu, T. Jackman, J. Hoki, Y. O’Connor, P. M. Kohn, K. W. Pommier, Y. Cancer Res. 1996, 56, 4430.
Akimoto, K. Kawai, A. Ohya, K. Chem. Pharm. Bull. 1994, 42, 2135.
Opanasopit, P. Yokoyama, M. Watanabe, W. Kawano, K. Maitani, Y. Okano, T. J. Control. Release 2005, 104, 313.
Kim, R. Emi, M. Tanabe, K. Cancer Biol. Ther. 2006, 5, 1429.
Gibson, S. Tu, S. Oyer, R. Anderson, S. M. Johnson, G. L. J. Biol. Chem. 1999, 274, 17612.
Jorissen, R. N. Walker, F. Pouliot, N. Garrett, T. P. Ward, C. W. Burgess, A. W. Exp. Cell Res. 2003, 284, 31.
Roskoski, R. Jr. Biochem. Biophys. Res. Commun. 2004, 319, 1.
Salomon, D. S. Brandt, R. Ciardiello, F. Normanno, N. Crit. Rev. Oncol. Hematol. 1995, 19, 183.
Gorden, P. Carpentier, J. L. Cohen, S. Orci, L. Proc.Nat. Acad. Sci. U. S. A. 1978, 75, 5025.
Hanover, J. A. Willingham, M. C. Pastan, I. Cell 1984, 39, 283.
Johannessen, L. E. Pedersen, N. M. Pedersen, K. W. Madshus, I. H. Stang, E. Mol. Cell Biol. 2006, 26, 389.
Kam, N. W. S. Liu, Z. Dai, H. Angew. Chem. Int. Ed. 2006, 45, 577.
Letoha, T. Gaa´l, S. Somlai, C. Czajlik, A. Perczel, A. Penke, B. J. Mol. Recognit. 2003, 16, 272.