鑽石奈米顆粒的分子胞飲機轉、追蹤、分離與應用：在人類癌細胞及老鼠異體移植腫瘤模式

Abstract

奈米顆粒的開發提供生物創新的應用，包括生物標定、影像及檢測。奈米鑽石是一種 由碳組成的奈米顆粒，我們發現奈米鑽石會進入人類肺癌細胞，卻不會影響細胞的存活及 生長，奈米鑽石顆粒會隨著細胞分裂分配到子代細胞，然而奈米鑽石進入細胞的分子胞飲 機轉、在細胞及動物體的分佈及追蹤、以及可能的生物應用，值得深入研究，因此本計畫 目的包括探究: (一)奈米鑽石的分子胞飲機轉、(二)奈米鑽石在體外及體內的分佈與追蹤、 (三)攜帶奈米鑽石細胞株的分離與應用。本計畫將建立人類肺癌細胞之體外培養及老鼠異體 移植人類肺癌之動物體內模式，比較不同尺寸(包括5 到100 奈米尺寸等)和修飾過(包括羧 基化及磁化等)的奈米鑽石，對胞飲作用的影響及可能的分子胞飲路徑。將使用流式細胞儀 分析量化奈米鑽石進入細胞的能力與在細胞中的顆粒複雜度，並以活細胞影像顯微攝影、 共軛焦顯微鏡、生物原子力顯微鏡及穿透式電子顯微鏡等，觀察奈米鑽石的胞飲路徑(包括 macropinocytosis、依賴clathrin 及caveolae 等)，並利用不同的胞飲路徑抑制劑與基因阻斷 方式，研究相關胞飲蛋白(包括clathrin 及caveolin 等)調控奈米鑽石進入細胞的機轉。此外， 進一步長期追蹤奈米鑽石在肺癌細胞中的分布與對細胞功能的影響，並研究奈米鑽石對肺 癌細胞形成腫瘤的影響及追蹤奈米鑽石在老鼠體內的分布。最後，我們將建立攜帶螢光及 磁性奈米鑽石的細胞株，分別以流式細胞儀及磁性裝置分離帶有奈米鑽石的肺癌細胞，並 研究攜帶奈米鑽石的細胞與原始細胞間的特性差異(包括基因和蛋白表現、細胞生長及腫瘤 形成能力等)。本計畫最終目標為探究奈米鑽石之分子胞飲機轉的基礎研究，並創新開發攜 帶奈米鑽石細胞株，提供作為癌細胞的標定及追蹤等應用。

Development of nanoparticles can provide novel bio-applications including bio-labeling, -imaging, and -detection. Diamond nanoparticle (nanodiamond, ND) is composed of carbon nanomaterial. We have found that NDs were taken into human lung cancer cells but did not alter cell survival and growth. ND particles can be separated into daughter cells during cell division. However, the molecular endocytic mechanisms, the distribution and chase in cell and animal body, and the possible bio-applications of NDs are valuable to further investigations. Thus, the purposes of this project are focused on: (1) To investigate the molecular endocytic mechanisms of NDs; (2) To investigate the distribution and chase of NDs in vitro and in vivo; (3) To investigate the separation and applications of ND-bearing cells. We will study both the models of lung cancer cell culture in vitro and the xenograft lung tumor of SCID mice in vivo. Various sizes (including 5-100 nm, etc.) and modifications (including carboxylated, magnetic, etc.) of NDs will be compared on the effects of uptake and their endocytic mechanisms. The uptake ability and particle complexity of NDs in cells will be analyzed by flow cytometer. Living-cell imaging microscope, confocal microscope, bio-atomic force microscope and transmission electron microscope will provide to observe endocytic pathways (including macropinocytosis, clathrin and caveolae pathways, etc.) and distribution of NDs. We will further investigate the effects of blocking endocytic regulated proteins (including clathrin, caveolin, etc.) by endocytosis inhibitors and specific gene knockdown strategy on the regulation of ND’s uptake. In addition, we will examine the long-term chasing on the distribution and cellular functions following treatment with NDs. Moreover, we will study the effects of NDs on tumorigenesis and distribution in SCID mice. Finally, we will create a fluorescent and magnetic ND-bearing cell line, which is separated by flow cytometer and magnetic device, respectively. The separated ND-bearing cells will be compared with the parental cells on gene and protein expression profiles, cell growth, tumor formation ability, etc. The final goal of this project is basically to illustrate the molecular endocytic mechanisms of NDs and develops a novel ND-bearing cell line for bio-applications including labeling and chasing of cancer cells.