敏感性高分子載體常用於攜帶多種治療因子來進行有效的運送並達到最佳的協 同治療功效。然而，近年來研究中發現腫瘤自身微環境的構造與機制，例如: 緻密的細 胞外基質、不規則的血管網路分佈與異常提升的細胞間質液體壓力等原因，造成搭載 之藥物不易傳遞至固體腫瘤的核心進行治療，因而容易有癌症復發與癌轉移的風險。 為了解決上述之問題，本研究開發出以氨基酸為基底並具雙硫鍵還原性、基質酵素蛋 白酶和組織蛋白酶 B 敏感性的藥物載體，其可經由循序漸進崩解之過程以達到腫瘤重 整與治療效果。另一方面，此奈米載體分別由兩條具雙親性之氨基酸序列組成，兩者 皆包含 10 個半胱氨酸，10 個組氨酸和 10 個亮氨酸等特殊設計之氨基酸序列:半胱氨 酸上的硫醇基團形成可逆的雙硫鍵保護殼層，組氨酸展現質子化海綿效應幫助藥物逃 脫出胞內體和溶酶體，亮氨酸上的疏水基團則可使氨基酸序列具雙親性的特質並能幫 助刺蝟信號路徑抑制劑(Vismodegib)包覆於藥物載體中;主動標靶分子 tLyP-1 可用來 辨認與腫瘤相關的血管與細胞，而藥物載體的表面則以親水性的聚乙二醇修飾，避免 被網狀內皮系統吞噬和增加載體於血流的循環時間。此奈米載體的大小約為 220 奈米 左右，隨著 NRP-1 受體於細胞膜上的表現多寡，奈米載體展現高度的專一性，其中， 載體在 MDA-MB-231 乳癌細胞上的療效可匹配於單純施予抗癌藥物之組別。在立體細 胞球模型中，Vismodegib 的添加可與 Doxorubicin 展現良好的協同效果，進一步有效抑 制癌細胞增生且提升藥物在腫瘤內的穿透深度。此內源性刺激奈米載體在經過腫瘤微 環境因子層層把關後，能夠更安全地進行藥物傳遞，並期望在未來有潛力應用於各式 固態腫瘤的治療。

Stimuli-responsive polymeric nanoparticles have exhibited as effective vehicle for escorting chemotherapeutic agents with optimal synergistic effect. However, tumor microenvironment impedes deep drugs transportation due to its dense extracellular matrix, irregular vascular network and elevated interstitial fluid pressure. To address these issues, disulfide bond reductive and dual-enzyme sensitive polypeptide-based nanoparticles with programmable degradation manner are proposed in this study for solid tumor remodeling and anticancer effect. Two different amphiphilic sequences, mPEG-Peptide(LC)-Prodrug and tLyP1- Peptide(SC)-Prodrug, self-assemble into spherical nanoparticle with a diameter of 220 nm in aqueous phase. The peptide sequences are mainly composed of polyCys, polyHis and polyLeu sequences with distinct functions, including protective shell for encapsulated drugs, facilitation of chemotherapeutic agents release and hydrophobic complexation domain for Hedgehog (Hh) signaling inhibitor, respectively. tLyP-1 is selected for active-targeting ligand binding to both tumor-associated vasculature and cells, while the hydrophilic PEG at the corona of the nanoparticles is served to reduce RES system uptake and increase circulation time in blood flow. The sensitive linkages successfully secure drugs from leakage in healthy physiological condition and efficiently promote drugs release by endogenous stimuli rising in tumor site. The nanoparticles exhibited high selectivity, depending on the expression level of NRP-1 receptor. Notably, the anti-cancer effect of nanoparticles was found to be comparable with that of free doxorubicin on MDA-MB-231 breast cancer cells. Moreover, the combination of doxorubicin and vismodegib showed a desirable synergistic effect on the inhibition of cell proliferation in tumor spheroid model and enhanced drug penetration. Overall, the self-assembly polypeptide- based nanoparticles with programmable dissociation characteristic leaded to a prominent therapeutic potency on solid tumor.

Abstract i
摘要 iii
Table of Contents iv
Figure Index vii
Table Index ix
Chapter 1. Introduction 1
1.1 Solid tumor barriers 1
1.2 Functionalized self-assembled polypeptide drug carriers 3
1.3 Endogenous stimuli-responsive system 4
1.4 Targeting mechanism of nanoparticles 6
1.5 Motivation and purpose of this study 8
Chapter 2. Literature Review 12
2.1 Microenvironment in solid tumors 12
2.1.1 Cancer-associated components 12
2.1.2 Hedgehog signaling pathway and its inhibitors 15
2.2 Overview of smart nanoparticles for drug delivery 19
2.2.1 Development of sensitive drug carriers 20
2.2.2 Enzymatic sensitive linkages – MMP-2 and Cat-B 21
2.2.3 Reductive sensitive linkages – disulfide bond 25
2.3 Active targeting ligand (tLyP-1) in cancer therapy 27
2.4 Tailor-designed polypeptide sequence 30
2.4.1 The function of Cysteine (Cys) 30
2.4.2 The function of Histidine (His) 31
Chapter 3. Theoretical Basis 34
3.1 Improvement of drug penetration in solid tumors 34
3.2 Degradation of nanoparticles in sequential manner 35
3.3 Active targeting ligand in cancer therapy 37
Chapter 4. Materials and Methods 38
4.1 Material list 38
4.2 Experimental design 40
4.3 Synthesis of mPEG-Peptide(LC)-Prodrug 40
4.3.1 Synthesis of mPEG-Peptide(LC) 40
4.3.2 Synthesis of mPEG-Peptide(LC)-Prodrug 40
4.4 Synthesis of tLyP1-Peptide(SC)-Prodrug 41
4.4.1 Synthesis of doxorubicin-NHS ester 41
4.4.2 Synthesis of tLyP1-Peptide(SC)-Prodrug 41
4.5 Preparation of Vismodegib encapsulated nanoparticles 42
4.6 Physiochemical properties characterization 43
4.6.1 Proton nuclear magnetic resonance (1H NMR) 43
4.6.2 Fourier transform infrared spectroscopy (FTIR) 43
4.6.3 Matrix Assisted Laser Desorption/Ionization (MALDI-TOF MS) 43
4.6.4 Dynamic light scattering (DLS) 43
4.6.5 Zeta potential 43
4.6.6 Transmission electron microscopy (TEM) 43
4.6.7 Confocal laser scanning microscope 44
4.7 In vitro study (Monolayer) 44
4.7.1 Drug release profiles 44
4.7.2 Intracellular uptake study 45
4.7.3 Cell viability test of monolayer cells 45
4.8 In vitro study (tumor spheroid) 46
4.8.1 Cell viability test of tumor spheroid 46
4.8.2 Nanoparticles penetration in tumor spheroid 47
4.9 Statistical analysis 47
Chapter 5. Results 48
5.1 Characterization of mPEG-Peptide(LC)-Prodrug and tLyP1-Peptide(SC)-Prodrug sequences 48
5.1.1 Structural confirmation by 1H NMR spectrum 48
5.1.2 Chemical structure confirmation by FTIR spectrum 50
5.1.3 Molecular weight confirmation by MALDI-TOF MS 51
5.2 Characterization and physicochemical analysis of the nanoparticles 52
5.2.1 Physical characterization of nanoparticles 52
5.2.2 Dual-stimuli drug release profile 54
5.3 In vitro study (Monolayer) 55
5.3.1 Anti-cancer effect of nanoparticles 55
5.3.2 Cellular uptake and intercellular trafficking of the nanoparticles 62
5.4 In vitro study (Tumor spheroid) 63
5.4.1. Evaluation of therapeutic effect in tumor spheroid 63
5.4.2. Penetrated delivery with the enhancement of nanoparticle accumulation 66
Chapter 6. Discussion 67
6.1. Characterization of multi-sensitive nanoparticles 67
6.2. In vitro drug release trigger by pH, reductive agent and enzyme 67
6.3. In vitro anticancer effect and intracellular uptake study 68
6.4. Establishment of tumor spheroid and its corresponding inhibitory effect after the treatment of nanoparticles 69
Chapter 7. Conclusion 71
References 72

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