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研究生:程鈺媚
研究生(外文):Yu-Mei Cheng
論文名稱:電解沉積阿黴素幾丁聚醣複合塗層於已披覆氫氧基磷灰石鈦合金之研究
論文名稱(外文):Electrolytic deposition of doxorubicin-chitosan composites on post hydroxyapatite coated titanium alloy
指導教授:顏秀崗顏秀崗引用關係
口試委員:許學全郭明智
口試日期:2016-07-04
學位類別:碩士
校院名稱:國立中興大學
系所名稱:材料科學與工程學系所
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:42
中文關鍵詞:阿黴素幾丁聚醣氫氧基磷灰石鈦合金
外文關鍵詞:DoxorubicinChitosanHydroxyapatiteTi alloy
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癌症為目前四大非傳染性疾病之一,為了減少化療造成的副作用,各種標靶治療方式與載藥系統逐漸被研究。本研究將用化療藥物阿黴素(Doxorubicin)與幾丁聚醣(Chitosan)複合材料沉積在已披覆氫氧基磷灰石(Hydroxyapatite)之鈦合金(Ti6Al4V)上,企圖應用於腫瘤附近之血管支架,藉由局部的藥物釋放抑制癌細胞分化生長,使細胞凋亡,提高局部療效與減少全身副作用。實驗中利用陰極曲線探討阿黴素沉積方式,而後以分光光度計(UV visible spectrometer)做阿黴素全圖譜、釋放曲線與量測載藥量; 以場發射式掃描電子顯微鏡(FESEM) 觀察表面形貌、傅立葉紅外線光譜儀(FTIR) 分析成分化學鍵結、X光繞射儀(XRD)鑑定晶體結構; 以細胞存活率分析(MTT Assay)觀察藥物抑制細胞分化的效果。結果顯示阿黴素/幾丁聚醣複合材料能成功地藉由電化學方法沉積於鈦合金上,而已披覆之氫氧基磷灰石(Hydroxyapatite)之鈦合金的多孔性質更可提高阿黴素載藥量至37.46 μg /cm2並呈現完整的藥量釋放。同時電化學的沉積製程並不會改變阿黴素的化學鍵結與阻止細胞增殖的藥效,而呈現本方法的實用性。

Cancer is one of the four major non-communicable diseases. In order to reduce the side effects caused by chemotherapy, various target treatments and drug-delivery system have being studied. In this study, the doxorubicin-chitosan composite is deposited on the post hydroxyapatite Ti alloy applied to vascular stents for the tumor treatment by sustaining release of drug at local location to achieve the inhibition of cancer or apoptosis of cancer. In the experiment, cathodic polarization tests coupled with electrochemical reactions were analyzed to speculate the deposition mechanism of doxorubicin, spectrophotometer (UV visible spectrometer) to measure doxorubicin loading and release, field emission scanning electron microscope (FESEM) to observe surface morphology, Fourier transform infrared spectroscopy (FTIR) for chemical bonding of composites, and X-ray diffractometry (XRD) for crystal structure. The MTT Assay was carried out to analyze cell viability for drug efficacy. It is concluded that doxorubicin-chitosan composites can be successfully deposited on the titanium alloy by electrochemical method and the post hydroxyapatite coated specimen with high porosity can enhance the drug loading to 66 μg /cm2 and reveal the complete drug release. Besides, the electrochemical deposition does not vary any chemical bonding or destroy any medical function of doxorubicin, revealing a practical method for drug loading.

Contents
摘要...i
Abstract...ii
Contents...iii
Figure Contents...v
Table Contents...viii
1.Introduction...1
2. Materials and methods...7
2-1 Specimen preparation...7
2-2 Cathodic polarization tests and deposition...7
2-2-2 Cathodic polarization tests in Dox, Dox/Chi mixed solution....7
2-2-3 Deposition of Dox/Chi composite...8
2-3 Coating characterization...8
2-4 Drug loading and release...8
2-5 Cell culture...9
2-5-1. Mediums collected from immersion test...9
2-5-2. Cell culture and MTT test...9
3.Results and discussion...11
3-1 Polarization...11
3-2 Coating characterization...18
3-2-1 FTIR...18
3-2-2 X-Ray diffraction...21
3-3 UV-vis. Spectrum for weight gain & drug loading...26
3-6 MTT tests...32
4.Summary & Conclusions...39
5.References...40

Figure Contents
Fig. 1 The side effect caused ultimately congestive heart failure.【9】...2
Fig. 2 The structural formula of doxorubicin hydrochloride.【11】...3
Fig. 3 The Cell cycle.【12】...3
Fig. 4 Keto-enol tautomerization and deprotonation of doxorubicin.【13】...4
Fig. 5 LogK''- pH profiles for doxorubicin (○) and daunorubicin (●) degradation at 50℃, corrected for buffer and ionic strength influences.【13】...4
Fig. 6 The proposed degradation scheme for the conversion of doxorubicin into II. S refers to the daunosamine sugar moiety.【13】...5
Fig. 7 The procedure of indirect contact MTT tests....10
Fig. 8 Cathodic polarization curves of Ti specimens in 20 ppm Dox, 20 ppm Dox (de-aerated with N2), 20 ppm Dox in acetic acid and 20 ppm Dox in HCl....12
Fig. 9 The cathodic polarization of Ti specimens in Chi, Chi de-aerated, Chi-40 ppm Dox solutions....14
Fig. 10 The color various in (a) Dox (orange-red), pH 6.29,(b) Dox solution (purple), pH 11.23, during the deposition in (c) Dox solution (purple on the cathode) and (d) Chi-Dox solution (orange-red)....15
Fig. 11 The cathodic polarization of HAp/Ti specimens in 40 ppm Dox, 40 ppm Chi-Dox....17
Fig. 12 The functional groups of Dox structure....19
Fig. 13 FTIR (ATR) spectra of Dox powder mixed with KBr....19
Fig. 14 FTIR (ATR) spectra of (a) Dox power, (b) HAp coated, (c) Chi-Dox/HAp coated, (d) -0.7 V Chi, (e) -0.7 V Chi-Dox, (f) -1.4 V Chi and (g) -1.4 V Chi-Dox coated specimens....20
Fig. 15 The XRD pattern of (a)HAp coated, (b)Chi-Dox coated and (c) Chi-Dox/HAp coated on Ti specimens....22
Fig. 16 The grazing angle XRD pattern of (a) Chi-Dox coated and (b) Chi-Dox/HAp coated on Ti specimens....22
Fig. 17 FESEM images of HAp coated (a, b, c), Chi coated (d, e, f, g), Chi-Dox coated (h, i, j, k) and Chi-Dox/HAp coated (l, m, n) Ti specimens....25
Fig. 18 The UV visible spectra of Dox solution....27
Fig. 19 The calibration curve of the absorbance at 480 nm vs. the concentration of Dox in 0.1M HCl solution....27
Fig. 20 The UV spectrum of Chi coated at -1 V, Chi-Dox coated at -1 V, Chi-Dox coated at -1.6 V, Chi-Dox/HAp coated at -1 V and Chi-Dox/HAp coated at -1.6 V on Ti specimens, subsequently dissolved in HCl....28
Fig. 21 (a) Dox release profiles of Dox-Chi coated and Dox-Chi/HAp coated Ti specimens in 24 hours, and (b) Dox release profiles of Dox-Chi coated and Dox-Chi/HAp coated Ti specimens in 4 weeks....31
Fig. 22 G-292 cell viability after culture with control (PS), Dox powder, Ti specimen, Chi coating, Chi-Dox coated specimens immersion for (A)1, (B)3, (C)5 and (D)7 day....33
Fig. 23 G-292 cell morphology is evaluated under light microscopy and photos taken at 400 x after cultured in (a) control (PS) and mediums extracted from (b) Dox powder, (c) Ti specimen, (d) Chi coating and (e) Chi-Dox coated specimens immersed for 1 day....34
Fig. 24 G-292 cell morphology is evaluated under light microscopy and photos taken at (left)100 x, (mid)200 x and (right)400 x lens after culture with (1-3a) control (PS), and mediums extracted from (1-3b) Dox powder, (1-3c) Ti specimen, (1-3d) Chi coating and (1-3e) Chi-Dox coated specimens immersed for 3 days....35
Fig. 25 G-292 cell morphology is evaluated under light microscopy and photos taken at (left)100 x, (mid)200 x and (right)400 x lens after culture with (1-3a) control (PS), and mediums extracted from (1-3b) Dox powder, (1-3c) Ti specimen, (1-3d) Chi coating and (1-3e) Chi-Dox coated specimens immersed for 5 days....36
Fig. 26 G-292 cell morphology is evaluated under light microscopy and photos taken at (left)100 x, (mid)200 x and (right)400 x lens after culture with (1-3a) control (PS), and mediums extracted from (1-3b) Dox powder, (1-3c) Ti specimen, (1-3d) Chi coating and (1-3e) Chi-Dox coated specimens immersed for 7 days....37
Fig. 27 Optical density of G-292 cell after culture in control (PS) and extracted mediums from Dox powder, Ti specimen, Chi coating and Chi-Dox coated specimens immersion for 1, 3, 5, 7 days....38

Table Contents
Table 1 Estimated Numbers of US Cancer Survivors by Site in 2014【1】....1
Table 2 The measured pH values, O2 concentrations and conductivities of electrolytes....12
Table 3 The measured pH values, O2 concentrations and conductivities of electrolytes....14
Table 4 The measured pH values, O2 concentrations and conductivities of electrolytes....17
Table 5 The drug loadings of Chi-Dox coated and Chi-Dox/HAp coated at various applied voltages and time. (The concentration of Chi-Dox/HAp* solution: 100ppm Dox+2.5wt% Chi. )...29
Table 6 The weight gain and percentage of drug content at -1.6 V of Chi-Dox coated and Chi-Dox/HAp coated Ti specimens....29
Table 7 The percentage of total drug release of Dox in day 1, 2, 3, 4, 14, 21, 28....31


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