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研究生：	施婉莉 Shih, Wan-Li
論文名稱：	發展多功能鐵鉑奈米合金粒子作為標的顯影劑及放射治療的增敏劑 Development of multifunctional FePt nanoparticle for targeted imaging agent and as a novel sensitizer for radiotherapy
指導教授：	蘇五洲 Su, Wu-Chou
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	66
中文關鍵詞：	奈米藥物、鐵鉑合金磁性奈米粒子、聚乳酸乙醇酸、神經纖毛蛋白質-1
外文關鍵詞：	Nanomedicine, FePt magnetic nanoparticle, poly(lactic-co-glycolic acid), neuropilin 1, Theranostics
相關次數：	點閱：214 下載：0
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化學工程和材料科學領域的進步推動了不同類型奈米材料應用，無論是無機或金屬的奈米粒子組成都朝診療一體的方向發展。磁性奈米粒子是主要奈米材料之一，由於其獨特磁特性使得它可作為磁共振成像造影劑。其中，鐵鉑合金奈米粒子具有優異的超順磁性和具有高穩定性X-ray吸收能力相結合，使鐵鉑奈米粒子作為MRI / CT非侵入性雙造影成像劑。在本研究中，我們開發多功能鐵鉑納米粒子既可以增強放療療效及專一辨識性，並作為MRI / CT雙造影劑。合成方法是採用奈米沉澱法合成，合成後利用動態光散射儀和原子吸收光譜儀檢測鐵鉑納米粒子，粒子大小約152.8 ± 23.9 nm而其包埋率約為97 ± 2.83％。我們利用4T1，AS2，Hep3B和CL1-5細胞株作為研究模式，並將各細胞與FePt @PLGA奈米粒子(2mg/ml)共同培養後，同時結合放射線來評估放療協同作用是否提升。在colony formation實驗中，發現細胞經由放射線照射後產生相當量的致死率，奈米粒子存在並不影響或降低4T1和AS2細胞之生長速率。本研究另一個目標是希望透過DG2胜肽的修飾來增加FePt @ PLGA奈米粒子對於其專一辨識受體蛋白 (NRP1)之鍵結，來達到腫瘤辨識的功效。由此，我們建立具穩定表達NRP-1蛋白質之細胞株(4T1-56)。在老鼠體外實驗中，發現腫瘤的冷光會受到金屬奈米粒子所產生淬滅效應(quencher effect)而影響訊號減弱，由IVIS系統中可發現，在高度表現NRP1之4T1-56細胞中，其冷光訊號於不同時間點(0、4及24小時)後會因淬滅效應而減弱，證實了FePt @ PLGA-DG2確實具有專一辨識性的功能。另外，經由組織鐵染色(iron stain)結果亦證明腫瘤組織中會有金屬鐵的存在。而比較其中神經纖毛蛋白質-1 表現的差異，在不表現的4T1腫瘤組織中染色鐵會存在於腫瘤外圍處，而4T1-56腫瘤組織則是會因為專一辨識而經由內吞噬存在於較深入腫瘤內部的區塊。總括以上結論，在本研究中我們已成功地合成出具不同功能性之FePt @ PLGA-DG2奈米粒子，並證明此奈米粒子可經由專一DG2胜肽的修飾引導而具有腫瘤辨識能力，結合其原本所具備之MRI/CT雙造影能力，使其更有機會成為未來診療一體的新型標靶奈米藥物或造影劑，而造福廣大的癌症病患。

The advancements in chemical engineering and materials science have contributed to the development of different types of nanomaterials, composed of either inorganic or polymer based nanoparticles that useful for nanotheranostics applications. Magnetic nanoparticles (MNPs) are a major class of nanomaterials. Their unique magnetic properties make them used as magnetic resonance imaging (MRI) contrast agents. Among them, FePt alloy nanoparticles (FePt NPs) have excellent super-paramagetic properties and stability combined with high X-ray absorption ability, making FePt NPs as a potential MRI/CT dual non-invasive imaging contrast agent. In this study our aim is to develop multifunctional FePt NPs for two purposes, that is they should enhance the radiotherapy efficacy and act as dual MRI/CT imaging contrast agent. Nanoprecipitation method was employed to prepare modified FePt@PLGA with synthesized particles sized 152.8 ± 23.9 nm and encapsulated efficiency was 97 ± 2.83%, detected by DLS and ICP-AES. Different cell lines 4T1, AS2, Hep3B, and CL1-5 were treated with modified 6 nmFePt@PLGA NPs (2mg/ml) for 24 hours and then combined with ionizing radiation to evaluate the synergetic effect for radiotherapy. By using clonogenic assay we have found that the 6 nm FePt@PLGA NPs exhibited no enhanced mortality of 4T1 and AS2 cells when compared to that of cells with ionizing radiation alone. Furthermore, to examine functional peptide DG2 that recognizes NRP-1 protein, FePt@PLGA NPs surface was modified to conjugate streptavidin through EDC binding ligand with biotin-DG2 for in vivo study. Therefore, Neuopilin-1 protein was over-expressed in 4T1 cells, and stably colony No. 56 (4T1-56) was selected as a model for further study. According to Western blot result that 4T1-56 cells expressed higher NRP-1 protein levels than the parental 4T1 cells. In vivo study, tumor formation induced by injected 4T1 and 4T1-56 cells in nude mice subcutaneously formed primary tumor within two weeks. FePt@PLGA-DG2 NPs (0, 4, and 24 hours later) was injected into tail vein and detected by IVIS, meanwhile tumor section was treated by iron stain to confirm the presence of Fe ions. As bioluminescent results show mice injected with NPs had lower luminescence compared to that of PBS groups due to the quencher effect. In addition, the result of iron staining indicated NRP-1 expression effect Fe present of the internal part of 4T1-56 tumor and peripheral part of 4T1 tumor. Overall, FePt@PLGA-DG2 NPs was synthesized successfully in this study, with functional surface modification of NPs that exhibited specific binding capability to both DG2 peptide and NRP-1 protein in vivo, so they have high potential as targeted agent for tumor diagnosis.

Table of Contents
Abstract	I
摘要	III
致謝	V
Table of Contents	1
Abbreviation list	7
Introduction	9
1.1 Nanotechnology	11
1.1.1 Nanomedicine	12
1.2 Magnetic nanoparticles	14
1.2.1 Magnetic Nanoparticles for imaging agents and drug delivery	16
1.3 Neuropilin signaling pathways	17
1.4 The role of DG2 peptide in the neuropilin signaling pathways	18
1.5 Rationales	18
Hypothesis	19
Specific aims	19
Materials and Methods	20
2.1.1 Cell culture	21
2.1.2 Cell lines	21
2.2.1 Synthesis of 6 nm FePt Nanoparticles	21
2.2.2 Synthesis of 12 nm FePt Nanoparticles	22
2.3 Ligand Exchange of 6 nm FePt Nanoparticles	22
2.4 Hydrodynamic size of the FePt@PLGA nanoparticles	23
2.5 Plasmid	23
2.6 Transfection by electroporation	24
2.7 Transfection cell selection	24
2.8 Cell count	25
2.9 Collection of whole cell lysates	25
2.10.1 Western blot	26
2.10.2 Antibodies	26
2.11 Proteomic approaches	27
2.11.1 One-dimensional electrophoresis	27
2.11.2 Commassie brilliant blue staining	27
2.11.3 Destain of the gels	27
2.12 Colony Formation Assay	27
2.13 Subcutaneous Animal Tumor Models	28
2.14 Iron stain	28
2.15 Haematoxylin and eosin staining	29
2.16 Statistical analysis	29
Results	30
3.1 The synthesis of FePt@PLGA NPs	31
3.1.1 Physical Characteristics of FePt@PLGA NPs	31
3.1.2 Magnetic Characteristics of FePt@PLGA NPs	31
3.2 The cytotoxicity of modifyied NPs	32
3.2.1 The cytotoxicity of 6-nm-FePt@PLGA NPs	32
3.2.2 The cytotoxicity of 6-nm-FePt@PLGA-DG2 NPs	33
3.3 The cellular uptake of 6nm-FePt@PLGA NPs in different cell lines	33
3.4 The efficacy of modified NPs combined with radiation	34
3.4.1 The enhanced efficacy of 6nm-FePt@PLGA NPs combined with radiation in 4T1 cell	34
3.4.2 The enhanced efficacy of different modified NPs combined with radiation in AS2 cell	35
3.5 The effect of FePt@PLGA-DG2 nanoparticles on imaging	35
3.5.1 The presence of iron accumulation in tumor	36
Discussion and Conclusion	38
4. Discussion	39
5. Conclusions	41
Tables and Figures	43
Table 3. Characterizations of FePt@PLGA NPs	44
Table 4. The cellular uptake of Fe concentration of different	44
Schematic 1. The synthesis process of FePt@PLGA-DG2 showed in schematic diagram.	45
Figure 1. TEM images of the FePt and FePt@PLGA NPs.	47
Figure 2. Micro-CT images and magnetic hysteresis loop of the FePt and FePt@PLGA NPs.	48
Figure 3. The effects of FePt@PLGA nanoparticles on the colony formation assay of 4T1/4T1-56 cell.	49
Figure 4. The cellular uptake of 6-nm-FePt@PLGA NPs in different cell lines.	51
Figure 5. The survival curve of 4T1 cell with radiation for cell proliferation.	52
Figure 6. The effects of different modified FePt NPs with radiation for cell survival.	55
Figure 7.The protein expression of Neuropilin-1 (NRP1) in cell lines.	56
Figure 8. The effects of mice 4T1 tumors from 0 to 24 hours after nanoparticles were injected.	57
Figure 9. The effects of mice with 4T1-56 tumors from 0 to 24 hours after nanoparticles were injected.	58
Figure 10. Histopathology of 4T1 and 4T1-56 tumor cells after FePt@PLGA-DG2 NPs injected for 24 hour.	61
Reference	62
                                    

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