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研究生:徐宗平
研究生(外文):Zong-Ping Hsu
論文名稱:玻尿酸接枝鉑類藥物結合奈米碳管之製備藉由電穿孔經皮傳輸應用於黑色素細胞瘤之治療
論文名稱(外文):Electroporation Enhanced Transdermal Drug Delivery for Melanoma Skin Cancer Therapy by Using DACH-Platinum Conjugated Hyaluronic Acid/Multi-walled Carbon Nanotube Mixtures.
指導教授:謝銘鈞謝銘鈞引用關係
口試委員:駱俊良林文澧
口試日期:2015-07-10
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學工程學研究所
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:46
中文關鍵詞:玻尿酸經皮傳輸奈米碳管電穿孔
外文關鍵詞:melanomahyaluronic acidDACHPttransdermalElectroporation
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皮膚癌為一種東方人較少的癌症病變,然而在國外則是併發率極高的癌症之一,分為基底細胞瘤,鱗狀細胞瘤,黑色素瘤.而我們的實驗則是以黑色素瘤作為實驗的細胞,主要是因為雖然他病發率低僅占皮膚癌中的百分之五左右,但因為其高轉移性導致死亡率極高,所以我們選擇以這組細胞最為實驗組.一般來說皮膚癌最常見的治療方式為手術切除,但因為黑色素瘤(melanoma)容易轉移及深入的特性,導致常常手術無法清除乾淨,所以我們希望能使用經皮傳輸將藥物以塗抹的方式進行療,如此不僅可以降低患者對於治療的恐懼,也比較不會承受那麼大的痛苦,並且接受率也較高.
  黑色素瘤細胞目前已經有許多一線藥物,例如Doxorubicin .Oxaliplatin和Cisplatin等等,其中又以鉑類藥物最常使用,所以我們選用Oxaliplatin 的active form DACHPt作為化療藥,為了增加經皮傳輸的穿透效果,且提升對於腫瘤細胞的targeting效果,我們選用了玻尿酸(Hyaluronic acid, HA)作為藥物載體不僅僅能提升穿透效果,其較高的生物相容性也可使患者不會有副作用的疑慮.
人類皮膚分為三層,分別是表皮層.真皮層.皮下組織,其中表皮層最外層的角質層排列緊緻保護著全身,導致許多藥物無法穿透,所以僅僅是利用玻尿酸本身濕潤增加通透度的能力還是不夠,所以我們利用電穿孔(Electroporation)來增加一條穿透的途徑,然而電穿孔需要較高的福特數才能使細胞產生孔洞,我們選擇搭配多壁奈米碳管(MWNT)來解決這部分的問題,因為奈米碳管極高的長徑比可使其有放大電場的效果,使我們不用用高福特數仍能達到使細胞打開孔洞的效果.
  本論文是使用玻尿酸接枝鉑類藥物後搭配奈米碳管提升電穿孔的效果對於皮膚癌進行治療,而我們也發現我們的材料對於有CD44受器的細胞有較好得毒殺效果,並且確實碳管配合電穿孔會有更好的藥物傳輸效果,因此希望將玻尿酸接枝藥物配合電穿孔的方式可以在不同的領域有貢獻,不管在治療或是醫美等等.


Skin cancer is a cancer lesion that Oriental less, but in foreign countries is complicated by high rates of cancer, into basal cell tumor, squamous cell tumors, melanoma. Our experiment is based on the melanoma cells as experimental, mainly because the disease is low, although he was only five percent of skin cancer in the left and right, but because of its high metastatic cause high mortality rate, so we choose in this group were the most experimental groups.
Melanoma cells has many first-line drugs, such as Doxorubicin .Oxaliplatin and Cisplatin, etc., among which the most commonly used platinum-based drugs, so we use the active form DACHPt Oxaliplatin as chemotherapy drugs, in order to increase the penetration effect of percutaneous transmission and to enhance the effect for targeting tumor cells, we use hyaluronic acid (Hyaluronic acid, HA) not only as a drug carrier can enhance the penetration effect, its high biocompatibility also allows patients will not have adverse effects concerns.
Human skin is divided into three layers, namely the epidermis. Dermis subcutaneous tissue, wherein the outermost layer of the epidermis cuticle compact arrangement protects the body, causing many drugs can not penetrate, so just increase the use of hyaluronic acid itself moist transparent ability degree is not enough, so we use electroporation (Electroporation) a way to increase the penetration, however electroporation Ford needs a higher number of cells in order to make the holes, we choose with multi-walled carbon nanotubes (MWNT) to this part of the problem, because the high aspect ratio of carbon nanotubes can amplify it has the effect of an electric field, so that we do not use a high number of Ford still achieve the effect of the cells to open pores.
This paper is the use of hyaluronic acid after graft carbon nanotubes with platinum drugs to enhance the effect of electroporation treatment for skin cancer, and we have found our material for CD44 by the cells had better get toxic effect, and indeed carbon tubes with electroporation have better drug delivery effect, it is desirable to coordinate the way electroporation grafted hyaluronic acid drugs can contribute in different areas, whether in health or beauty treatment and so on.


CONTENT
ABSTRACT II
CONTENT IV
LIST of SCHEMES VI
LIST of FIGURES VII
Chapter 1 Introduction 1
Chapter 2 Materials and methods 4
Experiments procedure 4
2.1 Material 4
2.2 Synthesis of HA-DACHPt 5
2.3 Synthesis of HA-FITC and HA-Cy5.5 5
2.4 Oxidation of Carbon nanotube(CNT) 6
2.5. In vitro serum stability test of HA-DACHPt conjugate 6
2.6 In vitro drug release profile of DACHPt from HA-DACHPt with and without electroporation 6
2.7 In vitro cellular uptake 7
2.8 In vitro cytotoxicity 7
2.9 In vitro transdermal delivery of drug 8
2.10 animals and tumor model 9
2.11 In vivo tumor accumulation and imaging 10
2.12 In vivo and In vitro application of electroporation treatment 10
Chapter 3 Result and discussions 11
3.1 Synthesis and characterization of HA-DACHPt and HA-FITC 11
3.2 Oxidation of multiwall carbon nanotube(MWNT) 13
3.3 Expression level of CD44 surface marker on melanoma cells 14
3.4 Cellular uptake of HA-DACHPt 15
3.5 In vitro cytotoxicity 15
3.6 In vitro penetration of skin 16
3.7 HA penetration through the stratum corneum 17
3.8 In Vivo Transdermal Therapy of Skin Cancer 18
Chapter 4 Conclusions 20
REFERENCE 21
SCHEME 25
FIGURE 29

LIST of SCHEMES
Scheme 1. Mechanism 24
Scheme 2 .Synthesis of HA-DACHPt 25
Scheme 3. Synthesis of HA-FITC 26
Scheme 4. Spectrum of HA-FITC 27
LIST of FIGURES
Figure1. HA-DACHPt , L.E.&D.C. 28
Figure2. L.E. and D.C.. of HA-DACHPt 29
Figure3. TEM of MWNT , Oxidation for (a)7h(b)14h(c)24H 30
Figure4. Flow cytometer & Western blot 31
Figure5. Binding test 32
Figure6. Cellular uptake of HA-FITC 33
Figure7. Cellular uptake of HA-FITC & HA-DACHPt 34
Figure8. Cell viability
(a).(b) pure HA treat B16-F10 , B16-F10(K.O.) 35
(c),(d) DACHPt treat B16-F10 , B16-F10(K.O.) 36
(e),(f) Oxaliplatin treat B16-F10 , B16-F10(K.O.) 37
(g),(h) HA-DACHPt treat B16-F10 , B16-F10(K.O.) 38
Figure9. LD50 39
Figure10. Ex vivo penetration 40
Figure11. Ex vivo transdermal penetration 41
Figure12. Animal model schedule 42
Figure13. Tumor 43
Figure14. HA-EDA conjugation ratio -44
Figure15. ICP-MS 45




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