多功能中孔洞複合性奈米矽球於癌症同位診斷治療之應用

近幾十年來，中孔洞奈米矽球作為藥物載體已在生物醫學上的應用領域越來越受到重視。中孔洞奈米矽球所具有的高表面積、均一的孔洞大小、易調控的結構及高度的生物相容性，使其在癌症治療及診斷領域成為極具潛力的奈米藥物平台。中孔洞奈米矽球與其他有機及無機材料的組合，更拓展了此材料在治療診斷上的許多協同功能性和應用多樣性。本研究主要探討中孔洞奈米矽球為基底的奈米材料於醫學診斷治療上之應用，另外，也利用了螢光生命期之變化探究藥物在離開奈米載體後，於細胞內的機制和命運。以三大主題分別探討: (1) 雙光子雷射激發所造成的電漿共振能量轉移於中孔洞矽球包覆之金奈米棒於光動力療法的應用;(2) 凝集素修飾之中孔洞奈米矽球於大腸癌症的診斷;以及(3) 利用螢光生命期影像顯微系統探討doxorubicin與DNA在細胞凋亡初期之結合和機制。主題(1): 以中孔洞矽球包覆之金奈米棒為平台，附載光敏劑-鈀紫質，藉由粒子內所造成的電漿共振能量轉移而達到雙光子活化之光動力療法。金奈米棒於雙光子激發的影像診斷及熱治療一直是許多臨床癌症研究的重要方向，然而，金奈米棒於持續的雙光子雷射激發下的不穩定性一直是研究者欲於克服的課題，本研究中使用的中孔洞矽球包覆之金奈米棒，可達到 (i) 高度穩定金奈米棒在雙光子雷射照射下的穩定度 (ii) 提升金奈米棒釋放之雙光子冷光的強度和影像生命期 (iii)提供大量的表面積可附載鈀紫質。在雙光子雷射的激發下，藉由粒子內的能量轉移，活化鈀紫質而產生單氧，藉此殺死癌細胞。此一複合性的奈米材料不僅能做為影像診斷，並利用雙光子雷射對於組織的高穿透度，拓展了光動力療法於深層組織方面的應用，將為癌症研究帶來新的契機。主題(2): 大腸癌為國人最常罹患之三大癌症種類之一，然而，在大腸癌症的診斷中，仍缺乏有效的顯影標記。本研究以中孔洞奈米矽球為基底，於骨架內修飾螢光分子並在矽球外表面修飾凝集素，與內視鏡系統結合，作為大腸癌症診斷的螢光探針。由體外細胞結合測試及體外及體內動物試驗，均證明凝集素修飾之中孔洞奈米矽球可專一性的結合於α-L-fucose表現之細胞和動物大腸癌組織，而競爭性測試可更進一步的確定其專一性。因此，此中孔洞奈米矽球為基底的螢光探針，可做為未來大腸癌症診斷之工具。主題(3): 為了解藥物於奈米載體中釋放後的機制，本研究利用螢光生命期影像顯微系統，動態的觀察藥物於細胞核內與DNA結合，於早期細胞凋亡發生時所造成的螢光生命期改變，所反映的微環境變化。總結，本研究利用多功能性的中孔洞奈米材料，結合金奈米棒或修飾外表面的標靶物質，達成此奈米材料於醫學診斷治療上的應用。未來，此奈米系統於生物醫學之應用應即具潛力。

關鍵字

中孔洞；奈米矽球；癌症治療

並列摘要

During the last decade, mesoporous silica nanoparticles (MSNs) have garnered great interest for potential use as nanocarriers of anticancer drugs. Their unique chemical and structural properties – their tunable size and pore diameter, excellent biocompatibility, intrinsically large surface area, and topologically distinct domains that can be easily and individually functionalized – make MSNs especially well-suited to efficient conveyance of a wide variety of therapeutic payloads. When combined with other organic/inorganic nanomaterials, the resultant MSN composites demonstrate novel synergies and even greater versatility than those of their constituents. In this dissertation we examine, in depth, the design, synthesis, and evaluation of MSNs and MSN composites as cancer nanotheranostics: nanomaterials that simultaneously serve as both diagnostic and therapeutic agents. To accomplish this task we focus on three very different nanotheranostic constructs with three very different mechanisms of action: (1) Mesoporous silica-encased gold nanorods, for two-photon activated photodynamic therapy (TPA-PDT) via plasmonic resonance energy transfer; (2) Lectin-functionalized fluorescent MSNs, for in situ diagnostic imaging of nascent colorectal cancers using confocal fluorescence endoscopy; and (3) pH-sensitive controlled-release MSNs, for real-time fluorescence lifetime imaging microscopy (FLIM) of doxorubicin-DNA intercalation dynamics during the initial activation of apoptosis. We begin with a description of the design, synthesis, and evaluation of mesoporous silica-coated gold nanorods (MS-GNRs) that incorporate photosensitizers for TPA-PDT. Enveloping gold nanorods with mesoporous silica confers a number of advantages on bare GNRs that include: (i) stabilization of the gold nanorods’ shape against thermal deformation following repeated laser pulse irradiation; (ii) enhanced two-photon luminescence intensity and nanoplatform longevity, and (iii) increased surface area for conjugation of additional photosensitizers. Using a 2-step sequential intra-particle plasmonic energy transfer post two-photon excitation, we observe substantial cytotoxicity both in vitro and in vivo, using human breast carcinoma cells and a murine model of human breast cancer. Next we discuss our development and evaluation of MSNs that target dysplastic adenomas and whose silica frameworks contain the fluorophore fluorescein isothiocyanate (FITC), for potential use as endoscopic contrast agents of nascent colon cancer. The exteriors of these fluorescent MSNs were labeled with the lectin Ulex europaeus agglutinin 1 (UEA-1), to target the α-L-fucose that is expressed on the luminal surfaces of glycoproteins of colorectal dysplastic adenomas and nascent cancers. In vitro affinity assays, as well as in vivo endoscopy and ex vivo histopathological analyses of tissues harvested from murine models of human colorectal cancer, demonstrate significant binding specificity to α-L-fucose of these UEA-1-FITC-MSN constructs. Lastly, we describe our use of (a) pH-sensitive MSNs, to target and controllably release the chemotherapeutic drug doxorubicin, and (b) FLIM, to dynamically monitor drug release, migration, and DNA intercalation during the early apoptosis – as a prototypical analysis of nanotheranostic function. In these studies we discovered that monitoring the rate of change of doxorubicin fluorescence lifetime within cell nuclei enables the detection of very subtle changes in chromatin structure, well in advance of the commencement of DNA fragmentation. Taken together, these studies, and the endless permutations of form/function such nanocomposites afford, provide a compelling argument for the clinical translation of multifunctional MSNs and MSN composites as cancer nanotheranostic compounds.

並列關鍵字

Mesoporous Silica Nanoparticles(MSN) ； Nanocomposite ； Theranostic ； Doxorubicin ； Fluorescence Lifetime Imaging Microscopy (FLIM) ； Gold nanorod ； Photoluminescence

參考文獻

Chapter 1:

1. Kresge, C. T.; Leonowicz, M. E.; Roth, W. J.; Vartuli, J. C. and Beck, J. S. Ordered mesoporous molecular sieves synthesized by a liquid-crystal template mechanism. Nature, 1992, 359, 710-712.

3. Lee, C. H; Cheng, S. H.; Huang, I. P.; Souris, J. S.; Yang, C. S.; Mou, C. Y.; Lo, L.W., Intracellular pH-responsive mesoporous silica nanoparticles for the controlled release of anticancer chemotherapeutics, Angew Chem Int Ed Engl. 2010 , 49 (44), 8214-8219.

4. Trewyn, B. G.; Slowing, I.I.; Giri, S.; Chen, H.T.; Lin, V. S.Y. Synthesis and Functionalization of a Mesoporous Silica Nanoparticle Based on the Sol–Gel Process and Applications in Controlled Release. Accounts of Chemical Research 2007, 40 (9), 846–853.

5. Xu, C. Y.; Tang, R. L.; Hua, Y. X.; Zhang, P. X. Mesoporous Silica Materials Synthesized via Sol-Gel Methods Modified with Ionic Liquid and Surfactant Molecules Chin. J. Chem. Phys. 2008, 21, 596

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多功能中孔洞複合性奈米矽球於癌症同位診斷治療之應用

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