以奈米金發展之建構基質金屬蛋白酶檢測平台及磁振造影與光熱治療雙功能之奈米團

Abstract

金屬奈米粒子因其高表面積體積比以及熱穩定等性質，於應用上同時兼具價廉、質輕、有效率等應用特質，因而奈米科技廣泛見於輕便性儀器開發之延用。諸多奈米粒子中，奈米金球擁有易合成之特性外、金屬表面高度修飾性以及對生物性分子的相容性，使得奈米金球成為絕佳平台選擇。於此，本研究以奈米金球為檢測藍圖建構一蛋白質酶活性檢測及其抑制性藥物篩選平台。於架構初步，為提升平台系統之檢測靈敏度，奈米金球之平台修飾過程中引入6-巰基-1-己醇（6-mercapto-1-hexanol, MCH)進行表現修飾來降低奈米金球間之排斥性。於此系統中MCH鍵結於奈米金球表面覆蓋未修飾受質之區域可避免受蛋白質酶降解之肽鏈吸附於奈米金球表面而影響奈米金球之穩定性，同時透過MCH之官能基增強奈米金球間之吸引力。本研究藉由分析吸收波長625 nm及525 nm之比值作為檢測依據執行蛋白質酶活性定量測試。應用此平台於檢測時，當MMP-2濃度為50 ng mL-1至600 ngmL-1其檢測數值與濃度亦成正相關。更進一步地，此平台亦應用於檢測不同亞型之MMPs（包含MMP-1、MMP-2以及MMP-7）和MMP抑制藥物之篩選。透過平台與酶谱法（zymography）技術比較，於MMPs活性檢測中觀察得兩方法間數值存在高度之正相關聯性。另一方面，本檢測平台亦可應用於MMP抑制藥物之篩選，藉由分析藥物對MMP抑制率建構藥物篩選平台，於此平台中以藥物抑制一半酵素活性（IC50）作為評估抑制效益。於此奈米金球篩選平台中測得Galardin對MMP-1、 MMP-2以及MMP-7之IC50分別為1.61、1.87以及16.07 nM，而相較Galardin，ONO-4817則具較差之抑制效果，其對於MMP-1無展現任何抑制效果，而於MMP-2以及MMP-7之抑制實驗中可測得ONO-4817之IC50分別為17.76 nM和40.45 M。此部分實驗以zymography技術進行確認亦可獲得相似之結果。此外，本研究以奈米金桿結合超順磁氧化鐵奈米粒子發展新式之癌症診斷及治療雙功能奈米團平台。此奈米團具備高度穩定性，在高溫加熱及強磁場環境中仍可維持奈米團之分散性，強化其在作為磁振造影診斷以及光熱療程所需要的穩定性。此平台結合聚集多顆超順磁氧化鐵奈米粒子於奈米團中，大幅增強超順磁氧化鐵奈米粒子之T2對比效果磁振造影能力及光熱治療效果，與原先超順磁氧化鐵奈米粒子和市售T2對比劑Resovist，其T2對比訊號增強1.9倍和2.2倍，透過Herceptin抗體修飾後之奈米團平台於小動物模組實驗中亦證實具高度辨識能力，其針對Her-2/neu 受體高表現之腫瘤觀測到- 70.4 ± 4.3%之訊號變弱（相較於低Her-2/neu 受體低表現之腫瘤僅- 7.5 ± 3.0%訊號差異）。此外，所建構之奈米團亦可藉由吸收近紅外光能量並轉換為熱能進而達到局部升溫效果，其於小動物模組中觀測此奈米團對腫瘤區塊有絕佳治療效果，且不影響周遭正常細胞之生長。另一方面所建構之球型奈米團相較桿狀之奈米金桿具有較低細胞毒性，同時亦改善奈米金桿不易受細胞吞噬之問題，有助於增強腫瘤細胞之累積，此奈米團於磁振造影及光熱療法應用相當具潛力，將可開發成為一新式診療載台以應臨床檢測與癌症治療的需求。

Metal nanoparticles are being extensively used in various applications due to their small size to volume ratio and extensive thermal stability. As a result, they may be applied to development possess comparable high sensitivity to conventional instruments, but with greatly reduced cost/mass/power requirements. Gold nanoparticles (AuNPs) are an obvious choice due to their amenability of synthesis and excellent biocompatibility as well as readily available conjugation chemistry at gold surfaces. In this study, we purposed to establish an optical platform by using AuNPs to assay proteinases activity and screen proteinase inhibitors. In the first part, 6-mercapto-1-hexanol (MCH) was designed to balance repulsive force among inter-particles. The results indicate that MCH enhanced the sensitivity of steric stabled AuNPs. MCH not only blocked the surface space to avoid peptide absorption on the AuNPs but also to increased the attraction among the AuNPs. The AuNPs-based platform was also applied in the detection of trypsin and matrix metelloproteinase-2 activities. For detection, the absorption ratio, A625/A525, of the reacted AuNPs solution can be used to estimate quantitatively the proteinase activity. A linear correlation has been established with matrix metelloproteinase-2 (MMP-2) activity at concentrations from 50 to 600 ng mL-1. Furthermore, a colorimetric platform, based on the surface plasmon resonance of AuNPs was applied to detect subtype of MMPs (MMP-1, MMP-2 and MMP-7) and screen MMP inhibitors. A good correlation was obtained between the results of MMP-1, MMP-2 and MMP-7 using our method and substrate zymography. In additional, the inhibitory concentration (IC50; Concentration of inhibitor that reduces enzyme activity to 50% of the activity of the native enzyme) was also estimated by calculating the A625/A525 ratio of the AuNPs/MCH-substrate. The IC50 values of ONO-4817 and Galardin were 17.76 nM and 1.87 nM for MMP-2 and 40.45 M and 16.07 nM for MMP-7. Compared with MMP-2 and MMP-7, ONO-4817 cannot inhibit MMP-1, and IC50 of Galardin toward MMP-was established to be 1.61 nM. Moreover, the efficiencies of ONO-4817 and Galardin were also analyzed by substrate zymography, and the results were similar to our proposed method. Finally, a novel dual functional theranosis platform is developed based on gold nanorods (AuNRs) and manganese magnetism-engineered iron oxide (MnMEIO) to combine magnetic resonance (MR) imaging and photothermal therapy in one nanocluster. The platform showed improved T2-weighted MR imaging and exhibited a near-infrared (NIR) induced temperature elevation due to the unique characteristics of AuNRs@MnMEIOs nanoclusters. The obtained dual functional spherical-shaped nanoclusters showed low cytotoxicity and high cellular uptake efficiency. The AuNRs@MnMEIOs nanoclusters also demonstrated a 1.9 and 2.2- fold r2 relaxivity value higher than those of monodispersed MnMEIO and Resovist. In addition, in vivo MR imaging study found that the contrast enhancements were - 70.4 ± 4.3% versus - 7.5 ± 3.0% in Her-2/neu overexpression tumors as compared to the control tumors. More importantly, NIR laser irradiation to the tumor site resulted in outstanding photothermal therapeutic efficacy and without damage to the surrounding tissue. In additional, the prepared dual functional AuNRs@MnMEIOs displayed high stability and furthermore dispersed even in the presence of external magnet, showing that AuNRs@MnMEIOs nanoclusters can be manipulated by an external magnetic field. Therefore, such nanoclusters combined MR imaging and photothermal therapeutic functionality can be developed as a promising nanosystem for effective cancer diagnosis and therapy.