螢光奈米鑽石之生醫研究應用

Fluorescent Nanodiamonds for Biomedical Applications: In-vitro and In-vivo Studies

蘇隆畯(Long-Jyun Su)

指導教授 ： 陳逸聰

共同指導教授 ： 張煥正(Huan-Cheng Chang)

國立臺灣大學/理學院/化學研究所/博士(2018年)

https://doi.org/10.6342/NTU201800966

摘要

螢光奈米鑽石(Fluorescent Nanodiamond, FND)近年來倍受矚目，乃是由於該材料中具有氮-空缺中心(N-V)的結構，此單一電荷的氮-空缺中心(N-V-)經黃綠光雷射光激發後所放出近紅外光譜的螢光，具有高穩定性且不會有螢光閃爍與光漂白的現象產生，因此適合作為長時間生物追蹤的工具。螢光奈米鑽石具有較長的 螢光生命週期(~20 奈秒)且這個碳基材的奈米材料同時具有無毒性且具有良好的 生物相容性，也很容易與蛋白質結合，而且容易標定細胞，總合以上這些特點，螢光奈米鑽石將成為最具前瞻性的螢光探針來作為生醫領域之應用。
在本篇的研究裡面，我們展現了這個新穎材料在活體外以及活體外的應用，其中包含了利用 Intensified Charge Coupled Device (ICCD)來作為即時的廣場視野螢光影像以及流式細胞儀的分析，除了在血液以及流式細胞儀中可以觀察到一顆鑽石標定的細胞外，在活體的老鼠耳朵內的血管也能達到單一鑽石標定細胞的偵測， 我們也利用鑽石的螢光可以被磁場調控的特性，並以小鼠以及豬來作為我們的實驗動物，在活體內，利用螢光奈米鑽石作為細胞的標定，活體內細胞的追蹤，複雜情況下達到無背景值干擾的偵測以及活體內定量追蹤人類間質幹細胞，同時也 證實了螢光奈米鑽石的標定，並不會影響到細胞活性及功能，因此螢光奈米鑽石標定技術，可以在動物體內追蹤幹細胞的生物分佈(biodistribution)以及幹細胞藥物動力(pharmacokinetics)的分析，我們更利用螢光奈米鑽石結合螢光素酶(luciferase) 建立一個快速且高效率與細胞能均勻表現冷光的平台，並且可以在活體內/外用於追蹤幹細胞，此標定技術具有優良的靈敏度可達到 10 顆細胞的檢測。仰賴上述的鑽石標定技術，我們不僅僅可以精準的定位以及定量移植到動物體內的細胞，更可即時在活體內觀察這些細胞的運動狀態，也可以結合冷光的顯影技術，提供 一個快速既有效率地且具有高靈敏度的細胞標定檢驗平台，這項鑽石標定技術，並不會改變細胞的狀態以及功能，且適用於少量且取得不易的幹細胞或神經細胞， 這將在未來的生醫研究領域中扮演一項重要的角色。

關鍵字

螢光奈米鑽石 ； 氮-空缺中心 ； 生物分佈 ； 無背景值干擾顯影 ； 螢光素酶-螢光奈米鑽石

並列摘要

Fluorescent nanodiamond (FND) has drawn much attention in recent years, because the negatively charged nitrogen-vacancy centers (NV–) within FNDs can emit a highly photostable far-red fluorescence emission (~700 nm). Since it does not photobleach or photoblink, it can be available for long term cell tracking. In addition, the FNDs has a relatively long fluorescence lifetime (~20 ns) and this carbon-based nanomaterial is non-toxic, biocompatible, easy interact with proteins by hydrophobic forces or physical adsorption and can be easily taken up by cells. All these characteristics make FND to be a powerful fluorescent probe for biological applications. Our studies have demonstrated that fabrication of high density ensembles of NV– centers in nitrogen-rich type Ib nanodiamonds can be used to produce brighter and smaller fluorescent nanodiamonds for bio-applications. Then we have applied this novel nanomaterial for in vivo real-time wide-field time-gated fluorescence imaging and flow cytometric analysis with a nanosecond intensified charge-coupled device (ICCD). We have also demonstrated the application of FNDs for quantitative tracking of human mesenchymal stem cell in animals by magnetic modulation of FND emission. The fluorescence emission of the FNDs decreases in the presence of a magnetic field and returns to its maximum intensity when the field is eliminated. This unique magneto- optical property of FND provides a new detection method of FNDs to improve the signal-to-background ratio for complex environment. We have also presented a new platform by using luciferase conjugated FND (Luci-FNDs) as dual-functional nanocarriers providing a rapid, highly efficient and homogeneous luciferase enzyme expression in human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs). Transplanted pcMSCs with Luci-FNDs can also be applicable for tracing and tracking in-vivo. Luci-FND provides extreme sensitivity of luminescence detection down to 10 cells. The ideal luciferase and FND combination (Luci-FND) is capable of generating fluorescence without quenching, and luminescence which can achieve optimal sensitivity and provide an easy analytical procedure and reliable results.
The combined techniques have enabled us (1) to detect individual FND-labeled cells in human blood flowing through a microfluidic device, (2) to monitor FND-labeled cells migration or extravasation in the mouse ear, (3) to precisely determine the number of the transplanted FND-labeled cells after intravenous administration, and (4) express a functional enzyme in primary cells with a rapid, highly efficient and homogeneous express functional enzyme in primary cells. In conclusion, our results not only have shown promising applications of FND labelling technique for background-free images, but also provided a new reliable platform to assess the biodistribution and pharmacokinetics of human stem cells in rodents and swine as well. Our Luci-FND labelling technique particularly suited the primary cell study with limited cell number and provides a novel and reliable platform for a functional enzyme expression in primary cells with a rapid, highly efficient and homogeneous way.

並列關鍵字

Fluorescnet nanodiamond ； nitrogen-vacancy centers ； biodistribution ； background-free images ； luciferase conjugated FND

參考文獻

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