神經幹細胞在治療神經退化性疾病或視神經損傷中具有極大的潛力。然而，在神經幹細胞的應用中，植入已初步誘導分化後的神經幹細胞可以避免非預期的細胞分化型態，也可以避免癌化的產生，將會大幅提高神經幹細胞治療效果。因此， 本研究的目的是開發一種由導電低聚物苯胺五聚體與四臂的星狀聚乳酸組成之網狀結構，以此網狀結構製備具有生物相容性與導電性質之薄膜，並混入硫酸軟骨素作為參雜劑，最終加入聚苯胺與聚乙二醇化的脂肪酸組成之奈米微粒作為光熱來源。這個導電材料可用於給予外部電場刺激控制神經幹細胞生長與分化，或是在生物組織內藉由細胞之間內源性電場的交互作用來提升組織的再生與修復速度，並藉由近紅外光的刺激塑造一個微溫環境，提升細胞的生存路徑，進而使整個修復過程更為有效率。
結果部分，我們已經成功合成苯胺五聚體與四臂的星狀聚乳酸以及他們的共聚物4a-P(D,L-lactide-co-CCAP)，我們也驗證了由這個共聚物組成之導電薄膜具有導電性質與生物可降解性。同時，我們也製備出具球形外觀及核 - 殼結構之聚苯胺為基底的光熱奈米微粒，且其具有合適的光熱轉換特性。此外，我們也證實了此導電基材的生物相容性，以及良好的細胞貼覆性，並具有誘導神經幹細胞往神經分化之能力，而加入電刺激能更進一步加速神經細胞之分化成熟。
我們預期這樣一個具有良好生物相容性及生物可降解性的導電光熱複合基材可以做為一個調控神經幹細胞命運的平台系統，應用於神經再生或是神經相關疾病的檢測。 此外，由於其導電與光熱複合之性質，提供了應用於神經電極介面、神經移植替代物、神經探針、神經系統藥物控制釋放等領域之廣泛應用。

Neural stem cells (NSCs) represent a strong potential and promise in the treatment of neurodegenerative diseases and nerve injuries. An efficient methodology or platform that can help for specifically directing stem cell fate is important and highly desirable if these cells are being used for clinical therapy. Furthermore, spontaneous differentiation into undesired lineages at the transplantation site as well as reduction in the risk of tumor formation can also be prevented.
In this study, an electrical conductive film composed of oxidative polymerized carboxyl-capped aniline pentamer (CCAP) and ring-opening polymerized tetra poly(D, L-lactide) (4a-PDLLA) was designed and developed. After doping by chondroitin sulfate (CS), nanoparticles based on polyaniline (PAni) and PEGylate fatty acid were embedded in the electroconductive film as a photothermal (PT) agent. This conductive film was suggested to act as a substrate for exogenous and endogenous electric fields transmission in tissue, resulting in the control of NSC fate as well as improvement in neural tissue regeneration. To accelerate the regeneration process and ensure implanted NSCs survival, the PAni nanoparticles triggered by near-infrared were used as a heat source to create a mild heat environment.
We have successfully synthesized and conjugated CCAP onto 4a-PDLLA to form a network structure. Besides, the electroactive film based on 4a-P(D,L-lactide-co-CCAP) was successfully prepared and showed good electrical conductivity and biodegradability. Also, photothermal nanoparticles possessing with adaptable photothermal efficiency was formulated and showed spherical appearance with core-shell structure. From the in vitro cell culture tests, biocompatibility and cell adhesion capacity on the electroactive film has been demonstrated. Besides, neuronal differentiation of NSCs can be induced by cultured cells on 4a-PLAAP, and neuronal maturation process can be facilitated by introduction of electrical stimulation. We expect this biocompatible and electroactive material with photothermal effect can serve as a medium to determine the cell fate of NSC and employ in neuroregeneration. Furthermore, its promising performance shows potential in the applications of electrode-tissue integration interfaces, coatings on neuroprostheses devices and neural probes, smart drug delivery system in neurological system, etc.

中文摘要 I
Abstract II
List of content IV
Table index VIII
Figure index IX
Scheme index XII
Chapter 1. Introduction 1
1-1 Neural stem cells (NSCs) 1
1-1.1 Neural stem cells fate in neuroregeneration 2
1-2 Regulating factors of cell behaviors 3
1-2.2 Cellular responses to electrical stimulation 3
1-2.3 Cellular responses to thermal modulation 5
1-3 Conductive polymers (CPs) 7
1-3.1 Aniline polymer and oligomer 9
1-4 Purpose of this study 9
Chapter 2. Literature reviews 12
2-1 Cell behavior of neural stem cells on electrical conducting materials 12
2-2 Controlling cell behaviors of neural stem cells electrically 13
2-3 NSCs response to thermal modulation and role of heat shock proteins (Hsps) in neuroregeneration 15
2-4 Properties of Aniline derivative 18
2-4.1 Biodegradability of aniline pentamer composites 18
2-4.2 Biocompatibility of aniline pentamer composites 20
2-4.3 Photothermal effect of polyaniline nanoparticles 21
Chapter 3. Theoretical basis 23
3-1 Mechanism of electrical conduction in aniline pentamer 23
3-2 Mechanism of photothermal effect in PAni 26
3-3 Possible mechanisms of thermal modulation in NSC behaviors and neuroregeneration 26
3-3.1 The protective effects of HSPs: HSP interaction with cell death signaling 27
3-3.2 Protein aggregation and disaggregation by HSPs in neurodegenerative disease 28
Chapter 4. Materials and methods 31
4-1 Materials 31
4-2 Methods 34
4-2.1 Material preparation of electroactive network and stimulation device 34
4-2.1.1 Synthesis of CCAP for network construction 34
4-2.1.2 Synthesis of 4a-PDLLA for network construction 35
4-2.1.3 Construction of electrical conductive network 36
4-2.2 Material preparation of photothermal nanoparticles 37
4-2.2.1 Synthesis of PAni 37
4-2.2.2 Photothermal nanoparticles 37
4-2.2.3 Assembly of 4a-PLAAP stimulation device 38
4-2.3 Physiochemical properties characterization 38
4-2.3.1 Chemical structure analysis 38
4-2.3.2 Molecular weight characterization of PAni by gel permeation chromatography (GPC) 38
4-2.3.3 Molecular weight characterization of 4a-PDLLA by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) 39
4-2.3.4 Electrochemical properties of CCAP and 4a-PLAAP thin film 39
4-2.3.5 Charaterization of Phototehrmal nanoparticles 39
4-2.4 Cell culture 40
4-2.4.1 Culture of neural human fibroblast (3T3) 40
4-2.4.2 Culture of neural stem cells (HCN-A94-2) 40
4-2.4.3 Cytotoxicity of electrical conductive film 40
4-2.4.4 Cell adhesion to electrical conductive film 41
4-2.4.5 Cell viability of electrical conductive film 41
4-2.4.6 Manipulation of NSC behavior via electrical stimulation 41
4-2.4.7 Gene quantification by PCR 42
4-2.5 Statistical Analysis 43
Chapter 5. Results and Discussion 44
5-1 Results 44
5-1.1 Structural characterization and molecular weight of CCAP 44
5-1.2 Electrochemical characterization of CCAP with different oxidation state and doping level 47
5-1.2.1 Cyclic voltammetry (CV) 47
5-1.3 Structural characterization and molecular weight of 4a-PDLLA 50
5-1.4 Structural characterization of 4a-Poly(D,L-lactide-co-CCAP) (4a-PLAAP) 53
5-1.5 Electrochemical property of 4a-PLAAP 53
5-1.6 Electrical conductivity of 4a-PLAAP 55
5-1.7 Degradation profile of eletroconductive film 59
5-1.8 Biocompatibility and cell adhesion property of electrical conducting film 59
5-1.9 Gene expression of NSC under electrical stimulation 62
5-1 Discussion 65
5-2.1 Physiochemical Characterization 65
5-2.2 Electrochemical properties and electrical conductivity 65
5-2.3 Degradation 67
5-2.4 Characterization of PAni NPs 67
5-2.5 In vitro biocompatibility test and cell adhesion of electrical conducting film 68
5-1.10 Gene expression of NSC under electrical stimulation 68
Chapter 6. Conclusion 69
Reference 70
Appendix 75
A-1. Molecular weight determination of EM PAni for photothermal nanoparticles as well as particle size and morphology of PAni based-photothermal nanoparticles 75
A-2. Photothermal efficiency of PAni based-photothermal nanoparticles 76

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