設計與合成化學感測分子及其於細胞顯影之應用與 孔洞二氧化矽奈米粒子應用於氧化還原反應啟動釋放藥物的標靶藥物 傳輸之奈米載體

Design and synthesis of chemosensors for applications in bio-imaging and redox stimuli-responsive nanocarriers based on MSNPs for targeted drug delivery in cancer therapy

潘華森(P. Venkatesan)

指導教授 ： 吳淑褓

國立交通大學/理學院/應用化學系所/博士(2019年)

摘要

本論文的目標有二，第一是設計並合成出化學感測分子與其於細胞顯影之應用，第二是以孔洞二氧化矽奈米粒子 (MSNPs) 作為標靶藥物傳輸之奈米載體，針對氧化還原反應啟動釋放藥物之應用

第一章 探討化學感測分子的重要性以及螢光訊號傳遞之機制並解釋有機螢光感測分子對不同金屬離子、過氧化物和過氮化物之偵測
第二章 化合物 HCTe 利用次氯酸誘發碲化二苯之氧化反應而能快速偵測次氯酸分子，並伴隨強烈的螢光放射(? = 0.75)。感測分子 HCTe 在與次氯酸分子反應後，會抑制碲化二苯對 BODIPY 之光誘導電子轉移效應 (Photoinduced Electron Transfer, PET) ，阻斷螢光淬熄的機制進而放出BODIPY 的螢光，並有82倍的螢光增強。化合物 HCTe 對於次氯酸分子有良好之線性關係 (1-10 ?M) ，且偵測極限可達到 41.3 nM (S/N = 3)。此外，在 RAW 264.7 cell 的螢光顯影實驗中，成功觀察到化合物 HCTe對次氯酸的偵測能力。
第三章 化合物 PHP 以 pyrene 為基底，在偵測到銅離子後藉由抑制 PET 效應的方式放出螢光。化合物 PHP 在與其他金屬離子反應後不產生螢光放射，顯示其對銅離子之高度選擇性，並能在 pH 5.0~10.0 範圍偵測銅離子。化合物 PHP 對銅離子之結合常數為 1.00 X 104 M-1。由 job plot 得知 PHP-Cu2+ 以1:1比例錯合。此外，錯合物 PHP-Cu2+ 與螯合劑EDTA之反應顯示其具有可逆性。其更可進一步應用於細胞實驗，在 RAW 264.7 cell 的螢光顯影實驗中，成功觀察到 PHP 對銅離子之偵測能力。
第四章 化合物 RhoSe 以 rhodamine-B 為基底，對汞離子具有絕佳的選擇性與靈敏度，並可快速偵測汞離子。由 job plot 得知 RhoSe-Hg2+ 以1:1比例錯合，並能在 pH 4.0~10.0 範圍偵測汞離子。此外，錯合物 RhoSe-Hg2+ 與 Na2S 之反應顯示其具有可逆性。化合物 RhoSe 可用於條帶法偵測銅離子，其更可進一步應用於細胞實驗，在共軛焦顯微鏡之螢光顯影實驗中，化合物 RhoSe 可於生物體內及體外偵測銅離子之存在。
第五章 化合物 RhoTe 是在發光團 rhodamine-B 上修飾 2-(phenyltellanyl) -benzaldehyde 基團，以此做為汞離子辨識端，當環境中存在汞離子時會觸發螺內?胺開環反應與碲化物氧化反應，並放出螢光放射。化合物 RhoTe 對汞離子具有良好的選擇性，可於不同的金屬離子與過氧化物中專一的偵測汞離子。此外，在 HeLa cells 與斑馬魚的螢光顯影實驗中，成功觀察到 RhoTe 可於活體細胞及生物體內偵測汞離子之存在。據我們所知，這是第一個設計出以 rhodamine 為基底且用於偵測汞離子之“雙鎖”螢光化學感測分子。
第六章 孔洞二氧化矽奈米粒子 (MSNPs) 是作為標靶藥物傳輸之奈米載體，其可應用於癌症治療之藥物釋放。轉鐵蛋白以共價的方式鍵接在 MSNPs 的表面，利用雙硫鍵作為穀胱甘?誘導細胞內藥物釋放之鍵橋。作為腫瘤標靶劑之轉鐵蛋白可增強腫瘤部位的藥物積累，以奈米閥的角色控製藥物釋放。我們分別利用BET / BJH 、 TEM 、 TGA 、 NMR 、和 FT-IR 確認其藥物釋放之應用。此外，我們也證實，一旦奈米粒子進入癌細胞後，癌細胞內生性之穀胱甘?會使雙硫鍵斷鍵，打開奈米閥，使奈米載體釋放藥物。

關鍵字

化學感測分子 ； Bodipy ； 細胞顯影 ； 體內偵測 ； 藥物釋放

並列摘要

The prime objective of this thesis is the design and synthesis of chemosensor probes for applications in bio-imaging and redox stimuli-responsive nanocarriers based on MSNPs for targeted drug delivery in cancer therapy.

Chapter 1 describes the significance of chemosensors and mechanism of signal transduction of several organic fluorescent probes for metal ions and ROS & RNS.

Chapter 2 discusses a ?uorescent probe HCTe for rapid detection of hypochlorous acid based on the speci?c HOCl-promoted oxidation of diphenyl telluride. The reaction is accompanied by an 82-fold increase in the ?uorescence quantum yield (from 0.009 to 0.75). The ?uorescence turn-on mechanism is achieved by the suppression of photoinduced electron transfer (PET) from the diphenyl telluride group to BODIPY. The ?uorescence intensity of the reaction between HOCl and HCTe is linear in the HOCl concentration range of 1 to 10 μM with a detection limit of 41.3 nM (S/N = 3). In addition, confocal ?uorescence microscopy imaging using RAW264.7 macrophages demonstrated that HCTe could be an e?cient ?uorescent probe for HOCl detection in living cells.

Chapter 3 describes the pyrene-based fluorescent sensor for Cu(II) detection. It demonstrated high selectivity towards Cu2+ ions via PET based fluorescence enhancement. However, the metal ions Ag+, Ca2+, Co2+, Cr3+, Fe2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+ produced no changes in the fluorescence emission of the system. The binding constant (Ka) of Cu2+ binding to PHP was found to be 1.00 X 104 M-1. The 1: 1 binding ratio of PHP-Cu2+ complex was determined from the Job plot. The maximum fluorescence enhancement caused by Cu2+ was observed between the pH ranges 5.0–10. Additionally, the PHP-Cu2+ complex reversibility with addition of EDTA was observed. Confocal fluorescence microscopy imaging using RAW264.7 cells showed that PHP can be used as an effective fluorescent probe for detecting Cu2+ in living cells.

Chapter 4 describes the probe RhoSe for selective detection of mercury ions (Hg2+). RhoSe shows colorimetric and fluorescent turn-on responses towards Hg2+. The sensor probe RhoSe exhibited a fast response for Hg2+ with excellent sensitivity and the detection limits was found to be 12nM. The binding ratio of RhoSe-Hg2+ was determined by Job plots as a 1:1 ratio, and the effective pH range for Hg2+ detection was 4.0–10. Importantly, the reversibility of the RhoSe-Hg2+ complex was observed through the addition of Na2S. For practical applications, the strip method was utilized to detect Hg2+ in water. In addition, cell imaging experiments demonstrated that RhoSe is an effective fluorescent probe for Hg2+ detection in vitro and in vivo.

Chapter 5 reports, a rhodamine based fluorescent probe RhoTe for Hg2+ detection. The fluorescent probe RhoTe displayed a selective response to mercury ions over other metal ions and ROS. Additionally, experiments with confocal fluorescence microscopy imaging using Hela cells and zebrafish showed that RhoTe can be used as an effective fluorescent probe for detecting Hg2+ in living cells and organism. To the best our knowledge, we first designed an incorporating of ‘dual lock’ rhodamine based fluorescent probe RhoTe receptors provides an alternative route for Hg2+ sensing.

Chapter 6 discusses the mesoporous silica nanoparticle (MSNPs) for tumor-triggered targeting drug delivery to cancerous cells. Transferrin was covalently anchored on the surface of mesoporous silica nanoparticles via disul?de linking for glutathione-induced intracellular drug release. In this case, tumor-targeting agent enhances drug accumulation at the tumor site and transferrin functions as the gatekeeper to control the drug release. The successful functionalization of redox responsive MSNPs was confirmed by using BET / BJH, TEM, TGA, NMR and FT-IR respectively. More importantly, we demonstrated that the nanoparticles enter the cancer cell through the recognition of Tf receptor and the Tf gatekeeper is removed by the cleavage of the disulfide bond using an endogenous glutathione stimulus.

並列關鍵字

Chemosensor ； Bodipy ； Bio-imaging ； In vivo ； Drug delivery

參考文獻

1. Bargossi, C.; Fiorini, M. C.; Montalti, M.; Prodi, L.; Zaccheroni, N., Recent developments in transition metal ion detection by luminescent chemosensors. Coord. Chem. Rev., 2000, 208, 17-32.

2. Wu, J.; Kwon, B.; Liu, W.; Anslyn, E. V.; Wang, P.; Kim, J. S., Chromogenic/Fluorogenic Ensemble Chemosensing Systems. Chem. Rev., 2015, 115, 7893-7943.

3. Lou, X.; Ou, D.; Li, Q.; Li, Z., An indirect approach for anion detection: the displacement strategy and its application. Chem. Commun., 2012, 48, 8462-8477.

4. Yang, Y.; Zhao, Q.; Feng, W.; Li, F., Luminescent Chemodosimeters for Bioimaging. Chem. Rev., 2013, 113, 192-270.

5. Boens, N.; Leen, V.; Dehaen, W., Fluorescent indicators based on BODIPY. Chem. Soc. Rev., 2012, 41, 1130-1172.