現今在癌症治療與再生醫學廣泛應用的基因治療與幹細胞療法，相較於傳統臨床治療方式，不僅展現極佳的療效與潛力，在全球的學界與業界亦引起高度關注。鑑於目前主要的基因傳遞方法為高效率病毒載體，其具有生產成本過高、難以大量生產、核酸嵌入染色體與恐造成嚴重免疫反應等缺點，而大幅限制其臨床應用性。因此，本論文研究目的即透過開發生醫高分子修飾之氧化鐵奈米粒子，作為高效率非病毒基因傳遞載體，並可建構具治療性之基因改造間質幹細胞，期以最終達成癌症治療與再生醫學之應用。
本論文研究依開發進程區分為三大部份。磁轉染，即結合磁性奈米粒子與基因傳遞載體，藉由外加磁場作用從而提升基因傳遞效率的策略，其中高分子量之聚乙烯亞胺(HMW-PEI)具有良好的轉染效率，因而被廣泛用於修飾磁性奈米粒子進行磁轉染，但其不可降解性將造成嚴重細胞毒性，因此，本論文研究第一部分，即開發生物可分解聚乙烯亞胺，改善HMW-PEI之毒性，並用以修飾超順磁氧化鐵奈米粒子(SPIO)，形成PSPIO之磁轉染載體。PSPIO可有效攜載核酸，並保護核酸不被酵素降解與提升其於血清環境中的穩定性。另外，在外加磁場作用下進行細胞試驗，也顯見其有效提升對於人類癌細胞株之基因轉染效率，以及核磁共振T2影像對比偵測之可行性。然而，此階段之PSPIO對於建構基因改造幹細胞之效率仍稍嫌不足。為了強化前述PSPIO於癌症基因治療之應用潛力，在本論文第二部分，便著眼開發新穎性磁性基因傳遞材料，以針對間質幹細胞進行安全且高效率之基因傳遞。為了增進間質幹細胞對於傳遞材料之攝取效率，具間質幹細胞表面受器CD44標靶性之透明質酸將用於修飾至SPIO表面，並結合對間質幹細胞具良好轉染效率之poly β-amino ester (PAE)與欲遞送之質體DNA，形成磁性奈米複合體(MTN)。相較於單純PAE/DNA轉染，MTN可在外加磁場與CD44受器雙標靶作用下，大幅增進三倍的轉染效率，且不影響間質幹細胞自身對於腫瘤的遷移與滲透能力。後續便以MTN磁轉染建構可表達具誘發腫瘤凋亡之蛋白(tumor necrosis factor-related apoptosis-inducing ligand, TRAIL)間質幹細胞，同時在原位人類神經膠質瘤之裸鼠模型中，試驗出可有效抑制腫瘤生長並延長其存活率之結果。而間質幹細胞除了應用於腫瘤治療外，其自身的分化能力與營養效應(Trophic effect)，對於應用創傷性腦損傷(Traumatic brain injury, TBI)之修復亦備受注目。因此在本論文第三部分之研究，便應用MTN磁轉染技術，使間質幹細胞(MSC)表達可促進神經再生之鹼性纖維母細胞生長因子(bFGF)，以提升修復TBI之療效。同時，直接注入幹細胞於TBI受損處，並不利於細胞滯留，也容易因損傷處缺氧或高度發炎等惡劣因子，降低植入細胞存活率，進而影響修復效果。因此，bFGF表達之間質幹細胞(MTN/bFGFMSC)將包裹於實驗室已開發之自癒性可注射水凝膠(sfADA-GEL)，再植入TBI受損處，藉此提升幹細胞存活率與修復效果。sfADA-GEL由海藻酸鈉與明膠組成，其兼具良好生物相容性，降解速率與機械強度亦可藉由不同高分子濃度調整，以相符腦內組織強度(約100 Pa)等特質，此外sfADA-GEL包覆之MTN/bFGFMSC可有效分泌bFGF並擴散至膠體外，且隨著膠體降解亦可遷移至膠體外。為了進行神經再生與修復TBI之療效評估，本研究後期，將建構大鼠TBI動物模型，植入sfADA-GEL包覆MTN/bFGFMSC進行治療，並以神經缺損行為評分(Neurological Severity Score, NSS)與TBI缺損處之免疫螢光切片染色評估療效。
總結上述研究成果，本論文研究所開發之磁性奈米基因傳遞材料，不僅能成功構築高效率基因改造間質幹細胞，並於腦瘤治療與創傷性腦損傷修復中，展現極佳的應用潛力，同時也是MTN得攜載更多樣化基因的先驅示範，預期其未來尚可嘗試磁轉染它種幹細胞，以提升其生醫應用之研究價值。

Gene delivery and stem cell-based therapy, with unique functions and therapeutic efficacy improvement, have received great attention worldwide in cancer treatment and regenerative medicine. Currently, the efficient viral transduction is the major strategy for gene delivery; however, the high cost and difficulty in large production, DNA integration and the possibility of inducing immune response limit the usage in clinical applications. In this thesis, we proposed biopolymer-modified iron oxide nanoparticles as efficient non-viral gene delivery carriers, which can be used to construct genetically-engineered mesenchymal stem cells for cancer therapy and regenerative medicine.

Magnetofection is a strategy to enhance gene transfection efficiency by applying an external magnetic field (EMF) on gene-loaded magnetic nanoparticles (MNP). High molecular weight polyethylenimines (HMW-PEI) with great transfection efficiency is widely used for non-viral MNP-mediated magnetofection. However, the non-degradability of HMW-PEI also induces significant cytotoxicity. To tackle the aforementioned challenges, we combined bioreducible polyethylenimines with the superparamagnetic iron oxide (SPIO) to form a magnetofection agent (PSPIO). PSPIO could condense the nucleic acids efficiently, protect them from enzymatic degradation, and preserve colloidal stability in serum. Under the EMF, PSPIO exhibited efficient in vitro transfection on human cancer cells. In addition, cells that internalized the PSPIO could be detected by T2-weighted magnetic resonance imaging. Despite the great potential of using PSPIO on cancer gene therapy, their transfection efficiency on stem cells was not satisfied. Consecutively, we proposed novel magnetic ternary nanohybrids (MTN) for safe and efficient gene delivery on human mesenchymal stem cells (MSCs). MTN was composed of hyaluronic acid (HA) modified SPIO (HA-SPIO) and poly β-amino ester (PAE)/DNA polyplex. The HA could target the CD44 which was overexpressed on MSC while the poly β-amino ester (PAE) was reported to present high transfection efficiency on MSC. As the CD44-/magneto- dual targeting effects, MTN showed three times enhancement of transfection efficiency on MSC without causing any detrimental effect on their tumor migration and penetration capabilities. Afterward, MSC (TRAILMSC) expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was constructed by MTN-mediated TRAIL gene delivery. In an orthotopic xenograft glioma cancer model, the TRAILMSC significantly suppressed the tumor growth and prolonged animal survival. Lastly, we proposed MTN-mediated genetically-engineered MSC for expressing basic fibroblast growth factor (bFGF), which could promote the neurogenesis, to improve the therapeutic efficacy on TBI repairing. MSCs with inherent differentiated capability and trophic effects are beneficial for tissue repairing. Thus, utilizing MSC to treat traumatic brain injury (TBI) has gained increasing attentions in recent years. However, the cell survival of implanted MSC was greatly decreased by the harsh environmental factors (i.e. hypoxia, free radical, immune factors) at the damaged area. To improve the cell survival and retention, MTN-mediated gene delivery was utilized to construct bFGF-expressed MSCs (bFGFMSCs). The bFGFMSCs were encapsulated into the self-healing and injectable hydrogel (sfADA-GEL) which were composed of biocompatible alginate and gelatin. The degradation rate and stiffness of sfADA-GEL were controllable by adjusting the polymer composition to meet the requirement of brain implant (~100 Pa). bFGFMSCs were capable of migrating from the sfADA-GEL during their degradation, and the secreted bFGF could also diffuse from the hydrogel. In future works, the TBI animal model will be established to evaluate the neurogenesis and repairing efficiency of bFGFMSC-laden sfADA-GEL.

In conclusion, we have successfully developed a safe and efficient magnetic gene delivery materials for construction of genetically-engineered MSCs. And these MSCs exhibited great potential on glioma therapy and the treatment of TBI. For future perspective, the MTN is worthy of verifying their transfection efficiency on different types of stem cells with other functional genes for versatile biomedical applications.

摘要 i
Abstract iii
致謝 v
Table of Contents vi
Figure Captions viii
Table Captions xiii
List of Abbreviation xiv

Chapter 1 Introduction 1
Motivation and Hypothesis 3

Chapter 2 Literature Review: Magnetic Nanocomplexes for Gene Delivery Applications 8
2.1 Introduction 8
2.2 Principle and benefits of utilizing MNP on gene therapy 9
2.2.1 The formulation of MNP-based gene carriers 11
2.2.2 The mechanism behind the magnetofection 12
2.2.3 The cytotoxic and biological toxicity of MNP 14
2.3 MNP incorporated with the virus for gene delivery 15
2.4 MNP mediated non-viral gene delivery for cancer therapy 17
2.4.1 Cationic polymer modified MNP gene carriers 18
2.4.2 Lipid modified MNP gene carriers 19
2.4.3 Non-ionic based MNP gene carriers 22
2.5 MNP mediated genetically-engineered cell therapy 23
2.5.1 MNP mediated genetically-engineered cells for cancer therapy 23
2.5.2 MNP mediated genetically-engineered cells for regeneration medicine 24
2.6 Conclusion and future perspective 27

Chapter 3 Redox-Sensitive Polymer/SPIO Nanocomplexes for Efficient Magnetofection and MR Imaging of Human Cancer Cells 28
3.1 Abstract 29
3.2 Introduction 30
3.3 Experimental section 31
3.4 Result and Discussion 37
3.4.1 Synthesis and characterizations of PSPIO 37
3.4.2 Functional assay of PSPIO/DNA Nanocomplex 38
3.4.3 Cellular uptake of PSPIO/pDNA nanocomplexes 40
3.4.4 In vitro T2-weighted cell imaging 41
3.4.5 In vitro gene transfection 41
3.5 Conclusion 42
3.6 Associated Content 42

Chapter 4 Magnetic Ternary Nanohybrids for Nonviral Gene Delivery of Stem Cells and Applications on Cancer Therapy 57
4.1 Abstract 58
4.2 Introduction 59
4.3 Experimental section 62
4.4 Result and discussion 67
4.4.1 Synthesis and characterization of HA-SPIO 67
4.4.2 Magnetofection and cellular uptake mechanism 68
4.4.3 In vitro antineoplastic effect and tumor tropism behavior of hMSCs 70
4.4.4 In vitro and in vivo imaging of MTNhMSCs 71
4.4.5 In vivo anti-cancer effect 72
4.5 Conclusion 74
4.6 Associated Content 75

Chapter 5 Injectable Hydrogel-Based Delivery of Nonviral Genetically-Engineered Stem Cells for Enhancing Neurogenesis on Traumatic Brain Injury Model 96
5.1 Abstract 96
5.2 Introduction 97
5.3 Experimental section 99
5.4 Result and discussion 102
5.4.1 Preparation and characterization of sfADA-GEL hydrogel 102
5.4.2 Cell proliferation in sfADA-GEL injectable hydrogel 102
5.4.3 Construction and characterization of bFGFhMSCs 103
5.4.4 In vivo therapeutic efficacy of bFGFhMSCs-sfADA-GEL on TBI 104
5.5 Conclusion 105

Chapter 6 Conclusion and Future Perspectives 110
Reference 112

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