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研究生：	施宣宇 Shi, Xian-Yu
論文名稱：	具選擇性之超順磁核心-外鞘型奈米為基礎之新穎口腔癌治療策略 A novel oral cancer therapeutic strategy based on superparamagnetic core-shell nanoparticle with selective anti-cancer effect
指導教授：	謝達斌 Shieh, Dar-Bin
學位類別：	碩士 Master
系所名稱：	醫學院 - 口腔醫學研究所 Institute of Oral Medicine
論文出版年：	2004
畢業學年度：	92
語文別：	英文
論文頁數：	77
中文關鍵詞：	奈米、口腔癌、治療
外文關鍵詞：	oral cancer, therapy, nanotechnology
相關次數：	點閱：100 下載：4
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　　據統計，在台灣男性的癌症十大死因中，口腔癌居第四位，而嚼食檳
榔則為其主要原因。奈米科技是一研究奈米尺度層級材料的新興科學，
奈米材料的物化學特性往往異於巨觀態因而得以應用於許多不同的領
域，然而其再生醫界的應用仍在萌芽的階段。在本研究中，我們合成一
新型超順磁核心(鐵)-外鞘(金)型奈米顆粒(Fe@Au)並分析評估單獨或結
合抗癌藥劑後之奈米粒子對於口腔癌的治療效果。本研究中利用的奈米
粒徑有9 nm 與22 nm 二種且具有可被磁場導引之特質。利用表面交聯
的方式，我們將抗癌藥物methotrexate(MTX) 銜接於22 nm 的Fe@Au
奈米表面(Fe@Au-MTX)並比較Fe@Au、MTX 本身及同時給予Fe@Au
和MTX 之癌細胞毒殺性及其對正常細胞之影響。由細胞毒殺測試的實
驗結果發現在口腔癌細胞OECM1 中，Fe@Au-MTX 造成的細胞毒殺效
果遠比Fe@Au 或MTX 單獨給予來得差。但同時給予Fe@Au 和自由態
之MTX 所造成的毒殺效果則有加成作用。而正常的口腔上皮細胞則對
四種處置皆有明顯之抗性或僅表現些微毒殺性。此外，我們發現當奈米
粒子保存在一般空氣或水溶液中，會在約二週間漸漸失去其毒殺效果。
由於奈米殼層僅約三層原子厚度或可使內部之鐵元素有機會轉移至外
部，而過量之三價鐵離子具有細胞毒殺性，因此我們比較了三價鐵離子
對於正常細胞與口腔癌細胞的毒殺效果。結果發現正常細胞與OECM1
對三價鐵離子的抵抗力卻沒有顯著差異。然而轉移癌細胞株HSC-3 具較
強的三價鐵離子抵抗力，其對Fe@Au 奈米粒子的毒殺抗性也相對較
高。在粒線體膜電位變化的實驗裡，我們發現Fe@Au 可以明顯地增加
OECM1 粒線體膜的通透性，而在正常細胞的影響則不明顯。另Fe@Au
明顯地促進粒線體膜電位喪失，正常細胞可在24 小時後漸行復原，但
癌細胞卻無法。本研究初步開發了一種具有選擇性毒殺癌細胞、可磁場
導引、可自我定時轉化為無毒性物質的新穎抗癌藥物。並於體外模型研
究中證實其選擇性毒殺口腔癌的成效。奈米粒子造成癌細胞細胞毒殺機
制可能包括了影響粒線體膜的通透性、三價鐵離子在局部的釋放與細胞
毒性、細胞內氧化還原酵素的干擾等。然而更詳細的毒殺機制仍待進一
步的研究釐清。

　　Oral cancer ranked the forth-leading cause of cancer deaths in
Taiwanese male population owing to the prevalent betel quid chewing habits.
Nanotechnology holds great potential for the development of new
therapeutic strategies as nanoscale sized material may present distinct
physical and chemical properties than their bulk status. In this study, we
synthesized a novel Fe-core-Au-shell (Fe@Au) nanoparticle that exhibited
superparamagnetic property for magnetic force guided targeting. The
nanoparticles with the particle size of 9 or 22 nm presented enhanced
cytotoxic effect to the oral cancer cells compared to the normal oral
keratinocytes. The cross-linking strategy for the Au-shell was established
to carry the anti-cancer drug – methotrexate (Fe@Au-MTX). The LD50 in
normal oral keratinocyte, dysplasia, and oral cancer cells was evaluated and
the results showed that Fe@Au-MTX has significantly decreased cytotoxic
effect to oral cancer cells compared to the Fe@Au alone, while Fe@Au and
free MTX presented synergic cytotoxic effect to the cancer cell. Besides,
the Fe@Au nanoparticle lost their cytotoxic effect spontaneously after two
weeks exposure in air or in the aqueous solutions. As the Au shell layer
has only about three atoms thickness and the inner iron may get access to the
surface, the released iron may play certain role in the observed cytotoxic
response as the accumulation of excess ferric ions has been reported to
induce cell death. To evaluate this potential pathway, the cytotoxic effect
of free ferric ion (III) to the normal versus oral cancer cells was evaluated.
The results failed to show significant differences in the tolerance of ferric
ion challenge between cancer and normal oral keratinocytes except one
metastatic cancer line that presented high resistance to both ferric ion and
the nanoparticle treatments. In addition, inhibition of endosome activity by
chloroquinone, which may prevents the ferric ion (III) releasing from the
nanoparticle core, abate the nanoparticle derived cytotoxicity effect. In the
study of mitochondrial membrane potential alterations in response to the
Fe@Au treatment, significantly enhanced mitochondrial membrane
permeability and lost of membrane potential was observed, while only
normal cells could reverse the membrane potential aftyer 24 hrs of treatment.
In summary, this study demonstrated the potential development of a novel
class of anti-cancer drug with selective cytotoxicity toward cancer cells,
magnetic field guiding property and self-timer controlled transformation into
non-toxic form for safely metabolic excretion. The execution of its
differential cytotoxic effect on cancer cells may involve multiple pathways
including the mitochondrial membrane alterations, local releasing of ferric
ions and the interference with the cellular REDOX enzyme activity.
However, the underlying molecular mechanisms for this selective reponse
remained to be elucidated.

1
中文摘要 6
Abstract .. 8
I. INTRODUCTION.......................................................................................................... 10
1. Oral cancer ...................................................................................................................... 10
1.1 Epidemiology ...................................................................................................... 10
1.2 Classification and staging of OSCC.................................................................... 11
1.3 Disease survival and recent development in OSCC therapeutic strategy ........... 13
2. Nanotechnology................................................................................................................ 15
2.1 Nano era .............................................................................................................15
2.2 Characteristics of nanoparticles .......................................................................... 18
2.2.1 The surface effect ..................................................................................... 18
2.2.2 The size effect36, 38.................................................................................... 19
2.2.3 Particle to particle interactions 39 ......................................................... 20
2.3 Metal nanoparticles ............................................................................................. 21
2.3.1 Magnetic property and Superparamagnetism........................................... 21
2.3.2 Catalytic property..................................................................................... 22
2.3.3 Optics property39 ...................................................................................... 23
2.3 Nanotechnology and cancer therapy .................................................................. 23
II. Material and Methods..................................................................................................... 26
Culture of normal oral keratinocytes.................................................................... 26
Culture of oral cancer lines .................................................................................. 26
Preparation of the superparamagnetic nanoparticles and associated reagents ..... 28
Cell viability assay ............................................................................................... 29
Mitochondria membrane potential assay by JC-1 dye ......................................... 31
Transmission electron microscopy (TEM) observation ....................................... 34
Statistic analysis ................................................................................................... 34
III. Results ............................................................................................................................ 36
Cytotoxicity of 22 nm Fe@Au, MTX, Fe@Au-MTX, Fe@Au combined MTX
in oral cancer cell lines and normal oral keratinocytes ........................................ 36
Cytotoxicity of 9 nm Fe@Au nanoparticles in the normal oral keratinocyte and
cancer cell lines .................................................................................................... 37
The aging effect on the cytotoxicity effect of nanoparticle.................................. 39
The cytotoxic effect of Fe3+ on normal and cancer cells...................................... 40
The mitochondrial membrane potential alterations after treatment with Fe@Au
nanoparticle .......................................................................................................... 41
IV. Discussion ...................................................................................................................... 60
V. References ...................................................................................................................... 66
VI. Appendix ........................................................................................................................ 70
Characteristics of Fe@Au and Fe@Au-MTX...................................................... 70
VII. About the author................................................................................................... 77

                                    

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校內：2006-08-30公開
校外：2007-08-30公開

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