臺灣博碩士論文加值系統

多形性膠質母細胞瘤(Glioblastoma, GBM)是最致命且常見的腦癌，由於GBM的高浸潤性，因此手術時醫生難以判斷腫瘤邊界導致切除不完整而有高復發率。為了改善此問題，5-ALA被應用於GBM螢光影像引導手術，於2017年被FDA核准為醫療用藥，然而其代謝物PPIX的螢光訊號有光漂白問題，除了可能影響手術中影像穩定度外也難以搭配光動力治療與光熱治療等光療法。因此無嚴重光漂白問題且有光熱治療效果的拉曼標記有潛力取代螢光標記應用於GBM的影像引導光熱治療手術中。

此論文目的為：1.開發有機會取代螢光標記的拉曼標記以降低光漂白問題。2.利用拉曼標記的表面電漿共振特性在GBM腦癌細胞上實現專一性拉曼訊號加上光熱治療效果。

實驗室所開發的拉曼標記以核心-衛星金粒子叢集結構為基底，外部包覆二氧化矽保護殼並修飾對Epidermal growth factor receptor (EGFR)有專一性的抗體，此種核心-衛星金粒子叢級結構內部間隙能產生熱點效應將拉曼訊號有效增強約10^8倍，透過表面電漿共振能將吸收峰值波長的光能有效轉換為熱能，因此有潛力對過度表現EGFR的GBM細胞進行專一性拉曼訊號加上光熱治療。

此論文重要的成果為：1.開發出拉曼強度只差螢光約一個數量級的拉曼標記。2.相同曝光條件下拉曼標記的拉曼訊號半衰期約為螢光分子訊號的3-5倍。3.以此標記在大鼠GBM腦癌細胞與大鼠腦正常細胞共培養的環境中做出活細胞拉曼訊號專一性與光熱治療專一性。上述驗證實驗室所開發的粒子有應用到GBM拉曼訊號引導光熱治療實驗的潛力，但粒子的專一性與光熱治療效果仍有待優化。

目前尚未有團隊做出對GBM細胞與正常細胞共培養的拉曼訊號引導光熱治療實驗，因此若後續有對專一性與光熱治療效果優化，將有機會成為領域中具有突破性的研究。

Glioblastoma(GBM) is the most fatal and common brain tumor. GBM is highly infiltrative and difficult to be completely removed during surgery. The only commercial contrast agent, 5-ALA, for GBM fluorescent image guided surgery suffer from photobleaching problems which may affect the stability of image. Raman tags based on localized surface plasmon resonance (LSPR) have strong Raman signals, photo-thermal therapy effect and less photobleaching problems. Therefore, there is great potential for Raman tags to replace fluorescent tag and be developed for Raman guided photothermal therapy for GBM.

In the previous works of our lab, the EGFR-specific Raman tag based on core-satellite-assemblies(CSA) of gold nanoparticles was developed which could enhance the Raman signal to 10^8 times at the intra-nanogap theoretically .

In this work, the purposes are to optimize the functions of CSA-based Raman tags and verify if it could distinguish the GBM cells locations and treat the GBM cell through photothermal therapy. To optimize the structure stability, photothermal efficiency, Raman intensity and spectrum features, 3 generations of Raman tags were developed. The last generation of Raman tags which combined the functions from above generations has specificity of live cell Raman signal and photothermal therapy for GBM cells but there is still room for the specificity to improve.

In conclusion, the CSA-base tags with photothermal effect and strong Raman signal were developed and the potential of application of Raman guided photothermal therapy is demonstrated but there is still room for the specificity to improve.

摘要 i
ABSTRACT ii
致謝 x
圖目錄 xv
表目錄 xviii
縮寫解釋 xix
第 1 章 序論 1
1-1 前言 1
1-2 研究動機與目的 1
1-3 文獻回顧 1
1-4 論文架構 6
第 2 章 研究方法 7
2-1 原理： 7
2-1-1 拉曼散射 7
2-1-2 表面增強拉曼散射(Surface enhanced Raman scattering, SERS) 9
2-1-3 奈米金屬粒子區域性表面電漿共振 9
2-1-4 複合粒子表面電漿共振特性 10
2-1-5 光熱轉換 11
2-1-6 光熱治療原理 12
2-1-7 抗體-抗原結合原理 13
2-2 儀器 14
2-2-1 掃描式電子顯微鏡(Scanning electron microscope, SEM) 14
2-2-2 穿透式電子顯微鏡(Transmission electron microscope, TEM) 14
2-2-3 動態光散射儀(Dynamic light scattering, DLS) 14
2-2-4 傅立葉轉換紅外光譜儀與ATR量測模式 15
2-2-5 UV-Vis穿透光譜儀 16
2-2-6 COMSOL模擬軟體 17
2-2-7 拉曼光譜儀(正立、倒立) 17
2-2-8 雷射切割機 17
2-3 藥品 18
2-4 實驗手法 20
2-4-1 FEM模擬CSA的SERS增強效果並與其他SERS標記比較 20
2-4-2 粒子製程 22
2-4-3 粒子製程檢測方法 25
2-4-4 粒子水溶液樣品拉曼光譜與Cy5螢光光譜量測 25
2-4-5 細胞培養與固定手法 25
2-4-6 光熱治療系統架設與雷射照射細胞手法簡介 26
2-4-7 光熱系統架設手法 26
2-4-8 光熱系統校正手法 27
2-4-9 96孔盤中執行光熱治療實驗步驟 28
2-4-10 ITO玻璃矽膠隔間基板 28
2-4-11 SEM影像分析細胞上粒子吸附效果 28
2-4-12 基板上固定細胞的Cy5-CSAG1@Ab拉曼訊號拼接影像 29
2-4-13 96孔盤隔間作法 30
2-4-14 96孔盤隔間種細胞手法 30
2-4-15 96孔盤隔間加入粒子與細胞反應手法 30
2-4-16 隔間系統中光熱治療雷射照射方法 31
2-4-17 拉曼加上光熱治療實驗 31
2-5 分析與計算 32
2-5-1 Trypan blue死亡率計算 32
2-5-2 Tumor to normal ratio (TNR)計算 32
第 3 章 實驗結果 33
3-1 FEM網格大小對模擬光譜的影響 33
3-2 由模擬光譜推測CSA的內部間隙大小 33
3-3 FEM模擬CSA結構拉曼增強因子，並與其他常見結構比較 34
3-4 CSAG1間隙中加入Cy5後的拉曼光譜與Cy5螢光強度比較 36
3-5 Cy5-CSAG1、Cy5-CSAG1@SiO2吸收光譜與水溶液樣品拉曼光譜 37
3-6 Cy5-CSAG1 @Ab對(CNS-1 / HA)拉曼影像TNR計算 38
3-7 Cy5-CSAG1 @Ab衰減情形 39
3-8 光熱系統架設誤差分析 40
3-9 CSAG1@COO-光熱治療效果 41
3-10 CSAG1@Ab光熱治療專一性 42
3-11 優化CSAG1粒子複合製程與反應後清洗手法效果 44
3-11-1 CSAG2@Ab修飾抗體各步驟效果分析 46
3-11-2 CSAG2@Ab光熱治療專一性 48
3-11-3 光熱治療粒子專一性SEM吸附效果分析 51
3-12 CSAG2@Ab對CNS-1與RA細胞光熱治療專一性 52
3-12-1 CSAG2@Ab提高粒子數、濃度與縮短反應時間後光熱治療專一性 54
3-13 96孔盤隔間模擬腫瘤邊界CSAG2@Ab光熱治療實驗 55
3-14 優化Cy5-CSAG2的結果 59
3-14-1 測試Cy5-CSAG2@Ab光熱治療效果 60
3-14-2 提高反應時間測試Cy5-CSAG2@Ab光熱治療與拉曼訊號專一性 61
3-15 優化Cy5-CSAG3@Ab的結果 66
3-16 Cy5-CSAG3@Ab拉曼加上光熱治療 68
3-17 其他：96孔盤隔間開發結果 74
3-17-1 製程品質分析 74
3-17-2 隔間模擬腫瘤邊界效果 75
3-17-3 隔間左右面積 76
3-17-4 隔間細胞阻漏率 77
3-17-5 開發隔間中遇到的問題與解決方法 77
3-18 其他：細胞培養相關研究 77
3-19 結論 78
第 4 章 討論 80
4-1 Cy5-CSAG3@Ab拉曼訊號專一性與光熱專一性無法同時顯著的原因 80
4-2 以Cy5-CSAG3@Ab在動物層級執行拉曼訊號引導手術的可行性 80
4-3 SERRS的優缺點 80
4-4 有無機會以CSAG2@Ab透過光熱治療做到GBM完全切除? 81
4-5 Cy5-CSA@Ab粒子與(固定細胞/活細胞)反應的TNR差異 81
4-6 SEM評分細胞上粒子吸附效果的問題與延伸 81
參考文獻 82

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