螢光奈米鑽石對於細胞膜通道結構之追蹤與奈米溫度量測

Single Particle Tracking and Nanothermometry of Fluorescent Nanodiamonds in Membrane Nanotubes

艾培仁(Chandra Prakash Epperla)

指導教授 ： 張煥正 ； 倪其焜

國立清華大學/理學院/化學系所/博士(2016年)

摘要

螢光奈米鑽石(FND)材料近年來受到矚目，起因於該材料具有氮-空缺中心(N-V) 的結構。由於單一電荷的氮-空缺中心(NV-)經雷射光激發後所放出近紅外光譜與光強度具有高穩定性且不會有螢光閃爍與光漂白的現象產生，因此適合當成長時間生物追蹤的材料◦ 另一方面，螢光奈米鑽石存在著生物相容性與材料表面多功能修飾鍵結的能力，因此常用於環境變異的偵測，如偵測溫度、電場、磁場的變化等。
生物組織成長發展中，細胞之間的溝通有其重要性。最近發現細胞間存在細胞膜奈米管(MNTs)的結構，該結構提供細胞間溝通管道與交換傳遞物質，不同種類的細胞其細胞膜奈米管結構差異性相當大。常見的疾病，如阿玆海默症、帕金森症、HIV病毒皆存在著細胞膜奈米管結構，因而針對此結構做深入的研究將有助於運送藥物治療上述疾病的應用。
本論文使用螢光奈米鑽石的特性對細胞膜奈米管做詳盡的探討。首先利用BSA與GFP分別包覆100奈米大小的螢光奈米鑽石形成蛋白質載體，當成生物追蹤的材料，我們運用共軛焦螢光顯微鏡，追蹤這些蛋白質載體於細胞膜奈米管內的移動與速率分佈解析，並得到幾種載體運送模式，這些成果都來自於單一螢光奈米鑽石的追蹤結果。另一方面，我們研發出奈米金棒吸附螢光奈米鑽石的多功能奈米複合材料，該複合材料可測量奈米尺度的溫度變化並可提供加熱的功用，我們針對癌細胞膜與癌細胞膜奈米管的熱穩定性做深入研究，並提供局部高熱殺死癌細胞的溫度資訊,，而且此治療過程具有選擇性，不會影響正常細胞。
綜合言之，我們的成果提供利用此單一螢光奈米鑽石可了解細胞間溝通與交換傳遞物質的過程，並提供局部高熱殺死癌細胞的研究與應用。

關鍵字

Fluorescent nanodiamond

並列摘要

Fluorescent nanodiamond (FND) is a novel carbon based material that has drawn much attention in recent years due to its uniquely embedded defect centers named nitrogen-vacancy
(N-V) centers. Most notable is the negatively charged nitrogen-vacancy (NV ̶ ) color center which emits a highly photostable far-red fluorescence emission. Since it does not photobleach or photoblink, it can be used to track for longer times. The FND also exhibits a very good biocompatibility and its surface can be easily functionalized through covalent or non-covalent interactions with biomolecules. The NV ̶ center has been used to sense environmental variables such as temperature and electric or magnetic fields by studying the shifts in their electronic transitions or spin transitions at electronic ground state. All these characteristics make FND a promising fluorescent probe for biological applications.
Cell-to-cell communication is essential for the development and maintenance of multicellular organisms. Recently discovered membrane nanotubes (MNTs) are capable of creating intercellular communication pathways through which transport of proteins and other cytoplasmic components occurs. These cellular connections are very heterogeneous in both structure, function, and have been found to be formed in numerous cell types. MNTs are also known to participate in pathogenesis of many diseases such as Alzheimer’s, Parkinson’s and HIV. Hence, it is important to understand the dynamics of transport along these nanotubes and to explore the potential of MNTs as drug delivery channels.
This doctoral thesis presents several applications of variously functionalized FNDs in membrane nanotubes. We applied protein functionalized FNDs as a photostable tracker, as well as a protein carrier, to illustrate the transport events in MNTs of human cells. Proteins, including bovine serum albumin and green fluorescent protein, were coated on 100-nm FNDs by physical adsorption. Then single-particle tracking of the bio-conjugates in the transient membrane connections was carried out by fluorescence microscopy. We observed different types of motions and velocity distribution of cargos that took takes place inside the MNTs. Our results demonstrate the promising applications of this novel carbon-based nanomaterial for intercellular delivery of biomolecular cargo down to the single-particle level. Further, we have studied the thermostability of both MNTs and cell membrane. We have developed gold nanorods (GNR) functionalized FNDs as a two-in-one optical nanodevice that can heat and sense the temperature simultaneously. We used all-optical method to study the nanothermometry of GNR-FNDs. We also demonstrated the photoporation on MNTs using GNR-FND nanohybrids to selectively deliver drugs to cytoplasm. Finally, we performed hyperlocalized hyperthermia on cell membrane for the treatment of cancer cell. During such a treatment, cancer cells can be killed selectively, while healthy cells remain unaffected.
Our results demonstrate promising applications of this novel carbon-based nanomaterial for intercellular delivery of biomolecular cargo down to the single-particle level and a new paradigm for hyperthermia research and application.

並列關鍵字

Fluorescent nanodiamond

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