臺灣博碩士論文加值系統

大腸直腸癌為惡性腫瘤耗發率前三名，而常規的化學或放射治療方式仍會因腫瘤復發而失敗，其中促進腫瘤再生-癌症幹細胞扮演重要的角色，癌症幹細胞會促進腫瘤的生長，並且在治療後會有抗藥性產生，若癌症幹細胞未被完全清除，可能會造成腫瘤對藥物有抵抗性且造成復發的可能，而使治療更加困難。為了實現更良好的大腸直腸癌治療，高分子奈米藥物載體已被廣泛運用於藥物傳輸系統來治療癌症，因奈米藥物載體粒徑極小之特性可透過腫瘤延遲效應(EPR effect)而進入腫瘤微環境中，並且可包覆溶解低、毒性高藥物用以減少對正常細胞的毒性，奈米藥物載體具有增強藥物於腫瘤累積與減少正常細胞傷害之優點。
　　大腸直腸癌在臨床上使用CPT-11為第一線藥物，而SN38則為CPT的活性代謝物，其活性優於CPT-11的100-1000倍，而因SN38為極疏水性藥物不能直接打入活體，故可利用奈米載體藥物之特性將SN38包覆於其中用以毒殺大腸直腸癌細胞。而標靶治療是現今治療癌症的方法之一，對於特定癌細胞有顯著抑制生長的效果，將標靶抗體修飾於奈米藥物可增加癌細胞對藥物攝取之專一性進而增強毒殺腫瘤的能力。
本論文研究利用生物相容性高分子mPEG-PCL及mal-PEG-PCL形成奈米微胞作為化療藥物SN38的載體，此外，被視為大腸癌幹細胞標記的CD133蛋白質(prominin-1)，扮演促進腫瘤生長、增加復發率與高度轉移性的角色，故為了增加毒殺癌幹細胞之效果，我們於微胞外圍修飾CD133抗體，希望藉由標靶的方式增加癌幹細胞的死亡並減少腫瘤復發的可能性。
　　　在物理性質方面，CD133抗體奈米微胞粒徑大小位於160-170nm左右，藥物包覆率可達80%以上且穩定性高，CD133抗體接枝率為70%左右。在細胞攝取量及細胞毒性等觀察，可發現相較於未接枝抗體之奈米藥物其外圍修飾CD133抗體之載體有較佳的標靶能力。在細胞毒殺及抑制其形成球體方面，也有良好的效果。除此之外，動物實驗方面，在給予包覆SN38之CD133抗體奈米載體有抑制腫瘤生長且減緩復發的趨勢，而在切片的結果發現在處理CD133標靶型奈米藥物後,其CD133高度表現細胞有明顯減少的現象，因此希望能將這奈米藥物載體應用於大腸直腸癌治療，並希望對生物醫學領域有些許的貢獻。

Cancer stem-like cells play key role in tumor development and these cells is relevant to the failure of conventional chemotherapy. To achieve a favorable therapy of colorectal cancer, amphiphilic polymer ethylene-glycol-ε-caprolactone (mPEG-PCL) and maleimide-ethylene-glycol-ε-caprolactone (mal-PEG-PCL) is fabricated and may self-assemble to form micelle which possesses well biological compatibility as a topoisomerases inhibitor, SN38 nanocarrier. For cancer stem cell therapy, CD133 (prominin-1) is a putative cancer stem-like cells (CSLCs) maker for colorectal cancer and is suggested to be a therapeutic target. Overexpression of CD133 has demonstrated enhanced tumor initiating ability and tumor relapse probability. To resolve the problems of chemotherapy failure, SN38 loaded micelles was conjugated CD133 antibody for targeting CD133+ cells. In this study, CD133 antibody-conjugated SN38 loaded nanoparticles (CD133Ab-NPs-SN38) were efficiently binding to HCT116 cells, which overexpression of CD133 glycoprotein. In cytotoxic effect, CD133Ab-NPs- SN38 was more than non-targeted mixed nanoparticles (NPs-SN38) in HCT116 cells. Furthermore, CD133Ab-NPs-SN38 could target to CD133+ cells and inhibits colony formation compare to NPs-SN38. In vivo studies in HCT116 xenograft model revealed that CD133Ab-NPs-SN38 depresses tumor growth and retard recurrence. Immuno- histochemistry image showed that reduction of CD133 expression when treated with CD133Ab-NPs-SN38. Therefore, this CD133-targeted nanoparticles delivery system could killing CD133 positive cells and has potential to offer cancer stem cell therapy.

口試委員會審定書 i
致謝 ii
中文摘要 iii
ABSTRACT iv
CONTENTS vi
LIST OF SCHEMES ix
LIST OF TABLES x
LIST OF FIGURES xi
Chapter1 Introduction 1
1.1 Cancer therapy-active targeting system 1
1-2 Cancer stem-like cells 2
1-3 CD133 (prominin-1) 3
1-4 CPT-11(7-ethyl-10-[4-(1-piperidino)-1-piperidono]
carbonyloxycamptothecin)
and SN38(7-ethyl- 10-hydroxy-camptothecin) 4
1-5 Aim 5
Chapter 2 Materials and Methods 6
2.1 Materials 7
2.2 Synthesis amphiphilic of mPEG-PCL 7
2.3 Synthesis amphiphilic of mal-PEG-PCL 8
2.4 Critical micelle concentration 8
2.5 Preparation of SN38-loaded nanoparticles 9
2.6 Characterization of SN38-loaded nanoparticles 9
2.7 Stability of SN38-micelles 10
2.8 Drug release profile of SN38 10
2.9 Thiolation, preparation and observation of CD133
antibody conjugations / Preparation and observation
of CD133 peptide conjugations 11
2.10 Static light scattering 12
2.11 Cell culture 13
2.12 Colonosphere culture 14
2.13 Expression of CD133+ cells in colon cancer
cells 14
2.14 Binding affinity of CD133 probe targeted
nanoparticles 15
2.15 Cellular uptake 16
2.16 In vitro cytotoxicity 17
2.17 Colony formation assay 18
2.18 Percentage of CD133+ cells in HCT116 spheres after
treated with target and non-target SN38 loaded
micelles 18
2.19 In vivo, Growth inhibition studies of tumors 19
2.20 Cancer specimens by immunohistochemistry 19
2.21 Biodistribution 20
2.22 Statistical Analysis 21
Chapter 3 Results 22
3.1 Synthesis of SN38-loaded micelles 22
3.2 Characterization of SN38-loaded micelles 22
3.3 Drug release profiles of SN38 loaded micelles in
different PH values 24
3.4 Conjugation of CD133 antibody or peptide to SN38-
loaded micelles 24
3.5 Compositions of CD133 probe-conjugated SN38 loaded
SN38 mixed micelles 25
3.6 Expression level of CD133 surface marker on
colorectal cancer cells 26
3.7 Sphere-forming abilities of HCT116 CD133+ and CD133-
cells 27
3.8 Analysis of different CD133 peptide targeting
efficiency 27
3.9 The binding affinity of CD133 probe-conjugated
nanoparticles 28
3.10 Cellular uptake of CD133 probe-conjugated FITC
micelles 29
3.11 In vitro cytotoxicity 29
3.12 Colony formation assay 30
3.13 Percentage of CD133+ cells in HCT116 spheres after
treated with target and non-target SN38 loaded
micelles 31
3.14 Cytotoxicity of SN38 loaded micelles in HCT116
spheres 31
3.15 In vivo, recurrence inhibition studies of xenograft
HCT116 tumor 32
3.16 In vivo, growth inhibition studies of xenograft
HCT116 tumor 33
3.17 In vivo, HCT116 cancer specimens by
immunohistochemistry 34
3.18 In vivo, biodistribution of CD133Ab-NPs-SN38 and
NPs-SN38 35
Chapter4 Conclusions and Discussions 36
4-1 Conclusions 36
4-2 Discussions 37
REFERENCE 40
SCHEME 44
TABLE 47
FIGURE 51

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