整合型奈米基因藥物載體結合了治療與診斷兩大功能，可協助研究與醫療人員即時觀測追蹤藥物載體，同時觀察治療成效。在整合型奈米藥物研究中，多將現有之醫療診斷影像分子，例如:核磁共振顯影劑(MRI contrast agent)、生物螢光染劑(fluorescent dyes)、量子點(quantum dots)等，與具治療性的化學藥物、放射性分子或是核甘酸序列(nucleic acid sequence)結合，以達成治療與診斷結合之目的。本研究中，應用近年來廣受研究的碳量子點(carbon dots)於診斷病理現象，其可隨量子點大小調控的光致發螢光特性(photoluminescence)、環保的製作過程(environmental-friendly)、高度生物相容性(biocompatibility)以及抗光漂白作用(photobleaching resistance)，使碳量子點較其他類型的診斷分子更適合應用於生物顯像領域中。
治療部分，基因靜默(gene silencing)為本研究在治療層面的主軸。利用轉殖小干擾RNA (small interfering RNA)與癌細胞中特定致癌化基因(ontogenetic gene)的專一性，抑制致癌化基因的表現，進而控制癌細胞增生與腫瘤生長。另外，為提高此整合型奈米基因藥物載體被癌細胞專一性胞吞(endocytosis)之比例，同時降低治療的副作用，我們在載體表面嵌合上癌症標靶性葉酸(folate)分子。利用肺癌細胞表面異常增生的葉酸受體(folate receptor)與載體表面的葉酸分子結合，可有效提升載體轉染肺癌細胞的機率。
本研究於碳量子點的表面包覆以雙硫鍵串聯之聚乙烯亞胺(polyethylenimine)分子，再於聚乙烯亞胺上接枝具標靶性的葉酸分子，透過靜電吸引方式與小干擾RNA結合後，形成結合治療與診斷的奈米藥物載體-fc-rPEI-Cdots。此奈米藥物載體會藉由葉酸引發之胞吞作用進入肺癌細胞中，再藉由雙硫鍵於核內體(endosome)的可被降解性以及聚乙烯亞胺的質子海綿效應，釋放出具療效的小干擾RNA，達到更好的治療效果並降低聚乙烯亞胺的細胞毒性。核磁共振光譜分析中證實成功合成多功能奈米基因藥物載體(fc-rPEI-Cdots)。此載體也具有光致發螢光特性，可接受360nm光照放出可見光(460nm)。同時載體透過靜電吸附具治療性核醣核酸，並在還原劑作用下裂解雙硫鍵放出治療性核醣核酸分子，達到治療效果。細胞實驗中證實此奈米基因藥物載體具有肺癌細胞標靶功能，同時也能透過釋放治療性的核醣核酸抑制基因表現，達到針對癌細胞毒殺的治療效果。

Theranostic nanoagent is defined as a carrier with combination of therapeutic and diagnostic application. In these agents, therapeutic strategies such as gene therapy, chemotherapy, photodynamic and radiation therapy are coupled with diagnostic segment, including MRI contract agents, fluorescent dyes, or quantum dots. While most contrast agents are radioactive or environmental-unfriendly, carbon dots have uniqueness to function as bioimaging molecules with low adverse effects for organism and the environment. Their good biocompatibility, special optical property, and excellent water solubility also make carbon dots as promising diagnostic bioimaging agents.
Gene silencing, the therapeutic strategy used in this study, knocks down cellular oncogenic signaling pathway. Specificity of siRNA to oncogenic mRNA regulates tumor growth with lowered affection to surrounding normal tissue. It is well known that enhanced expression of folate receptors (FR) appears on nearly all cancer cellular membranes, while FRs are nearly absent in normal tissue. Therefore, folate molecules are promising targeting motifs in cancer treatment.
Combining gene silencing strategy, bioimaging property of carbon dots, and targeting motif, our theranostic agents will benefit clinicians modifying therapeutic strategy in time by monitoring the development of carcinomatous tissues during treatment.
In this study, a novel theranostic nanoagent is developed and characterized. The reducible disulfide linkage between polyethylenimine (PEI) molecules in fc-rPEI-Cdots (folate conjugated-reducible PEI-carbon dots) is designed so as to be cleaved in cytoplasmic endosomal reductive environment; thus, enhancing gene delivery efficiency and decreasing cytotoxicity. From the results of 1H NMR, we have successfully conjugated PEI molecules and folate onto Cdots through disulfide linkage. The absorption spectrum of fc-rPEI-Cdots indicated the maximum excitation peak was shown under 360nm wavelength, while the highest intensity of the photoluminescence was observed at 460nm wavelength with blue emission light. The fabricated fc-rPEI-Cdots could form stable polyplex with nucleic acids in the weight ratio (fc-rPEI-Cdots/pooled siRNAs) higher than 15. A light band in agarose gel could be observed after treated with dithiothreitol (DTT) at 37℃, suggesting the redox property of fc-rPEI-Cdots. In vitro cell culture study demonstrated that fc-rPEI-Cdots is a highly biocompatible material and an excellent siRNA carrier for lung cancer treatment. Moreover, fc-rPEI-Cdots/pooled siRNAs can be selectively accumulated in lung cancer cells through receptor mediated endocytosis, resulting in better gene silencing effect in lung cancer cells.

Table of content
摘要 I
Abstract III
Table of content V
Table index VIII
Figure index IX
Chapter 1. Introduction 11
1.1 Theranostic nanoagents 11
1.1.1 Diagnostic compartments 11
1.1.2 Therapeutic segments 14
1.2 Carbon dots 18
1.3 Gene therapy for lung cancer treatment 19
1.4 Materials in this study 21
1.5 Motivation and purpose of study 23
Chapter 2. Literature Review 26
2.1 Carbon nanodots 26
2.1.1 Passivation 28
2.2. Degradable linkages in gene delivery 30
2.3 Folate targeting in cancer research47 32
2.4 Gene therapy in lung cancer treatment 33
Chapter 3. Theoretical Basis 37
3.1 Photoluminescence of carbon dots 37
3.2. RNA interference 37
3.3. Delivery of siRNA 38
3.3.1 siRNA protection 38
3.3.2 Receptor mediated endocytosis 39
3.3.3 Endosomal escape 39
3.3.4 Reduction of disulfide bond in cytosol 40
3.4 Tumor targeting 41
Chapter 4. Materials and Methods 43
4.1. Materials 43
4.2 Experimental Design 44
4.3 Synthesis of fc-rPEI-Cdots 44
4.3.1. Synthesis of PEI-Cdots 44
4.3.2. Synthesis of reducible PEI-Cdots 44
4.3.3. Folic acid conjugation 45
4.4. Physiochemical properties characterization 45
4.5. Optical property examination 45
4.6. Nucleic acid encapsulating ability and redox property 45
4.7. In vitro study 46
4.7.1. Biocompatibility and cellular uptake of fc-rPEI-Cdots. 46
4.7.2. Intracellular uptake of fc-rPEI-Cdots/siRNA 46
4.7.3. Gene silencing effect of fc-rPEI-Cdots/siRNA 47
Chapter 5. Results 49
5.1. Characterization of synthesized fc-rPEI-Cdots compounds. 49
5.2. Diagnostic potential of fc-rPEI-Cdots 54
5.3. Therapeutic siRNA loading 55
5.4. In vitro study 56
5.4.1 Biocompatibility of fc-rPEI-Cdots 56
5.4.2 Diagnostic and lung cancer-targeting effect of fc-rPEI-Cdots 57
5.4.3. Therapeutic effect of fc-rPEI-Cdots/pooled siRNA 59
Chapter 6. Discussion 62
Chapter 7. Conclusion 68
References 69

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