抗代謝藥物為臨床上重要的一類抗癌藥物，並且廣泛的使用在實體性和血液性腫瘤的治療上。這類藥物的細胞毒性是源自於它能干擾核酸新陳代謝中主要酵素的功能，使細胞週期無法進行而走向死亡。Methotrexate (MTX)是一個結構上與葉酸相似的二氫葉酸還原酶抑制劑，對許多種腫瘤具有相當明顯的抗癌效果。然而，細胞減少運送藥物的進入和增加藥物的排出，造成癌細胞對MTX產生抗藥性。此外，局部的注射水溶液形式的MTX很迅速的經由微血管吸收而進入循環系統中，以致對於注射部位的效果不彰。為能增進MTX在癌細胞內的停留時間，以及改善其藥物動力學上的性質，我們發展出一種新形式的MTX，即是將MTX結合在一種奈米材質的藥物載體上。金奈米粒子(AuNP)具有良好的生物相容性，小於五十奈米的粒徑，易於製備及定性，因此被廣泛應用在生物學的研究上。金奈米粒子的使用迄今尚未有細胞毒性的報告被提出。在這份研究中，我們將MTX與金奈米粒子結合在一起，並且在細胞和動物實驗中分析其抗癌效果。MTX能直接結合在金奈米粒子的表面上，形成MTX-AuNP錯合物，而且能從奈米粒子上釋放出來。MTX-AuNP和單獨使用MTX相較起來，能夠更快更多地累積在腫瘤細胞當中。和同樣劑量的MTX水溶液相較之下，MTX-AuNP在諸多的腫瘤細胞株中具有顯著提升的抑制細胞生長效果。在腹腔內接種的Lewis肺癌模示中，經由腹腔內施與的MTX-AuNP能抑制小鼠的腫瘤生長；然而同樣劑量下的MTX水溶液則與控制組一樣不具抑制腫瘤生長的能力。由以上結果可以推論出：將MTX結合上奈米材質的藥物載體可以有效的提升抗癌效果。

　Antimetabolites are a clinically important group of cancer drugs used in treatment of a variety of solid tumors and hematological malignancies. The cytotoxicity of antimetabolites stems from its ability to interfere with key enzymatic steps in nucleic acid metabolism. Methotrexate (MTX), structurally similar to folic acid, is a stoichiometric inhibitor of dihydrofolate reductase, displaying significant tumoricidal activity against a variety of neoplasms. However, impaired drug transport into cells and augmented drug export partially result in resistance to MTX. Furthermore, locally injected MTX in aqueous solution form that is rapidly absorbed through capillaries into the circulatory system is not effective. To retain the drug in tumor cells for a long period and alter the pharmacokinetic behavior, we developed a new formulation of MTX that is bound to the drug carrier on the nanometer scale. Colloidal gold nanoparticles (AuNPs) have been extensively used in biological applications due to their biocompatibility, dimension (<50 nm), ease of preparation and characterization, and also have a history of use without inherent cytotoxicity. In this study, we developed the MTX-AuNP complex and examined its antitumor effect in vitro and in vivo. MTX could be directly bound onto gold nanoparticles to form the MTX-AuNP complex and reversibly released from nanoparticles. The accumulation of MTX-AuNP was faster and more than that of free MTX in tumor cells. MTX-AuNP showed dramatically enhanced growth-inhibitory effects in several tumor cell lines compared to the equal dose of free MTX. The administration of MTX-AuNP suppressed tumor growth in mice of ascites LL/2 tumor model; however, the equal dose of free MTX showed no effect on suppression of the tumor growth. In conclusion, combined the nanomaterial as a drug carrier, MTX-AuNP exhibits more effective antitumoral activity than free MTX.

Assaraf, Y. G., Rothem, L., Hooijberg, J. H., Stark, M., Ifergan, I., Kathmann, I., Dijkmans, B. A., Peters, G. J., and Jansen, G. (2003). Loss of multidrug resistance protein 1 expression and folate efflux activity results in a highly concentrative folate transport in human leukemia cells. J. Biol. Chem. 278, 6680-6686.

Bertino, J. R. (1993). Karnofsky memorial lecture. Ode to methotrexate. J. Clin. Oncol. 11, 5-14.

Bhattacharya, R., Mukherjee, P., Xiong, Z., Atala, A., Soker, S., and Couvreur, P. (2004). Gold nanoparticles inhibit VEGF165-induced proliferation of HUVEC cells. Nano Lett. 4, 2479-2481.

Brigger, I., Dubernet, C., and Couvreur, P. (2002). Nanoparticles in cancer therapy and diagnosis. Adv. Drug Deliv. Rev. 54, 631-651.

Cronstein, B. N. (1996). Molecular therapeutics. Methotrexate and its mechanism of action. Arthritis Rheum. 39, 1951-1960.

Goodman, C. M., McCusker, C. D., Yilmaz, T., and Rotello, V. M. (2004). Toxicity of gold nanoparticles functionalized with cationic and anionic side chains. Bioconjug. Chem. 15, 897-900.

Hainfeld, J. F., Slatkin, D. N., and Smilowitz, H. M. (2004). The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol. 49, N309-N315.

Halbert, G. W. and Florence, A. T. (1989). Physiochemical characterization of methotrexate-bovine serum albumin conjugates. J. Pharm. Pharmacol. 41, 222-226.

Keefe, D. A., Capizzi, R. L., and Rudnick, S. A. (1982). Methotrexate cytotoxicity for L5178Y/Asn- lymphoblasts: relationship of dose and duration of exposure to tumor cell viability. Cancer Res. 42, 1641-1645.

Kojima, C., Kono, K., Maruyama, K., and Takagishi, T. (2000). Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate anticancer drugs. Bioconjug. Chem. 11, 910-917.

Liu, J. and Lu, Y. (2003). A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles. J. Am. Chem. Soc. 125, 6642-6643.

Liu, J. and Lu, Y. (2004). Accelerated color change of gold nanoparticles assembled by DNAzymes for simple and fast colorimetric Pb2+ detection. J. Am. Chem. Soc. 126, 12298-12305.

Matherly, L. H. and Goldman, D. I. (2003). Membrane transport of folates. Vitam. Horm. 66, 403-456.

Mirkin, C. A., Letsinger, R. L., Mucic, R. C., and Storhoff, J. J. (1996). A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 382, 607-609.

Mukherjee, P., Bhattacharya, R., Wang, P., Wang, L., Basu, S., Nagy, J. A., Atala, A., Mukhopadhyay, D., and Soker, S. (2005). Antiangiogenic properties of gold nanoparticles. Clin. Cancer Res. 11, 3530-3534.

Nakase, Y., Hagiwara, A., Kin, S., Fukuda, K., Ito, T., Takagi, T., Fujiyama, J., Sakakura, C., Otsuji, E., and Yamagishi, H. (2004). Intratumoral administration of methotrexate bound to activated carbon particles: antitumor effectiveness against human colon carcinoma xenografts and acute toxicity in mice. J. Pharmacol. Exp. Ther. 311, 382-387.

Niidome, T., Nakashima, K., Takahashi, H., and Niidome, Y. (2004). Preparation of primary amine-modified gold nanoparticles and their transfection ability into cultivated cells. Chem. Commun. (Camb.) 1978-1979.

Ow Sullivan, M. M., Green, J. J., and Przybycien, T. M. (2003). Development of a novel gene delivery scaffold utilizing colloidal gold-polyethylenimine conjugates for DNA condensation. Gene Ther. 10, 1882-1890.

Pignatello, R., Puleo, A., Puglisi, G., Vicari, L., and Messina, A. (2003). Effect of liposomal delivery on in vitro antitumor activity of lipophilic conjugates of methotrexate with lipoamino acids. Drug Deliv. 10, 95-100.

Porter, L. A., Jr., Ji, D., Westcott, S. L., Graupe, M., Czernuszewicz, R. S., Halas, N. J., and Lee, T. R. (1998). Gold and silver nanoparticles functionalized by the adsorption of dialkyl disulfides. Langmuir 14, 7378-7386.

Sandstrom, P. and Akerman, B. (2004). Electrophoretic properties of DNA-modified colloidal gold nanoparticles. Langmuir 20, 4182-4186.

Tsai, C. Y., Shiau, A. L., Cheng, P. C., Shieh, D. B., Chen, D. H., Chou, C. H., Yeh, C. S., and Wu, C. L. (2004). A biological strategy for fabrication of Au:EGFP nanoparticleconjugates retaining bioactivity. Nano Lett. 4, 1209-1212.

Utreja, S., Khopade, A. J., and Jain, N. K. (1999). Lipoprotein-mimicking biovectorized systems for methotrexate delivery. Pharm. Acta Helv. 73, 275-279.

Wosikowski, K., Biedermann, E., Rattel, B., Breiter, N., Jank, P., Loser, R., Jansen, G., and Peters, G. J. (2003). In vitro and in vivo antitumor activity of methotrexate conjugated to human serum albumin in human cancer cells. Clin. Cancer Res. 9, 1917-1926.

Zhao, R., Chattopadhyay, S., Hanscom, M., and Goldman, I. D. (2004). Antifolate resistance in a HeLa cell line associated with impaired transport independent of the reduced folate carrier. Clin. Cancer Res. 10, 8735-8742.

Zhao, R. and Goldman, I. D. (2003). Resistance to antifolates. Oncogene 22, 7431-7457.

蔡秀雯, (2004) 利用反轉錄病毒攜帶受缺氧調控之endostatin基因治療肺腺癌, 國立成功大學生物化學暨分子生物學研究所碩士論文