隱形生物屏障:探測細胞培養液中半胱胺酸之存在用於干擾聚乙二醇化金奈米粒子之穩定性

Probing the Invisible Barrier of Cysteine Interfering the Stability of Pegylated Gold Nanoparticles in the Culture Medium

10.6840/cycu201700904

王駿偉

半胱胺酸；金奈米粒子；巨噬細胞；金硫鍵結；奈米醫學 ； Cysteine；Macrophage；Gold nanoparticle；Au-S bond；Nanomedicine

中原大學奈米科技碩士學位學程學位論文

2017年

碩士

林政鞍

繁體中文

近年來金奈米粒子在癌症醫學中扮演著診斷與治療之材料，因生物相容性高、良好光學特性、延展性佳，常於生物醫學應用，但血液中蛋白會黏附在奈米材料表面形成蛋白暈狀體(protein corona)，進而被免疫系統中單核吞噬細胞系統(Mononuclear phagocyte system, MPS)清除，造成奈米材料到達目的前的生物屏障。本研究提出血液中小分子半胱胺是影響金奈米粒子穩定性之重要因子，巨噬細胞則扮演影響半胱胺酸恆定之重要角色，成為金奈米材料應用於體內時之重要隱形生物屏障。本研究論文首先利用RAW264.7巨噬細胞與金奈米粒子共培養模式，以暗視野顯微系統觀測血清蛋白與巨噬細胞辨識、清除奈米材料之現象，並驗證聚乙二醇化金奈米粒子可逃脫生物辨識之過程，但聚乙二醇僅提供24小時穩定性，推究其原因，我們透過培養液中胱胺酸添加之有無，以DTNB(Ellman’s reagent)與螢光金奈米團簇(Au@GSH)檢測巨噬細胞培養液中半胱胺酸生成之含量，並探討其影響聚乙二醇化金奈米粒子長期穩定性之關係。本研究發現巨噬細胞的確可代謝胱胺酸，並維持培養液中半胱胺酸之恆定，在無胱胺酸添加組別中則無法提升半胱胺酸恆定濃度，故可推斷血液中半胱胺酸之恆定關係，應對於金奈米粒子穩定性扮演舉足輕重的角色，未來在奈米探針設計上也因避免小分子半胱胺酸之攻擊，提升奈米材料用於精準治療之效益。

Gold nanoparticles are a material of diagnosis and therapy in cancer medicine in recent years. Because of its high biocompatibility, excellent optical properties and extension, gold nanoparticles are usually utilized to biomedicine. Mononuclear phagocyte system (MPS) clears protein corona which is formed by attaching protein in blood to the nanomaterial and further becomes a biological barrier before the nanomaterial reaches the target. In this study, cysteine of small molecule in blood is a key to influence stability of gold nanoparticles. Macrophage plays an important role in constant cysteine and further becomes an important and invisible biological barrier of gold nanoparticles applications in vivo. First of all, RAW264.7 macrophage is utilized to co-culture with gold nanoparticles, serum protein, macrophage identification and nanomaterial clearance are observed by the dark-field microscope and further verify that PEGylated gold nanoparticles are able to escape from biological identification. Because polyethylene glycol has only 24hrs stability, DTNB(Ellman’s reagent) and fluorescent gold nanocluster are utilized to inspect amount of cysteine generated in macrophage medium through addition of cystine in medium or not. Therefore, long-term stability of PEGylated gold nanoparticles is explored by cystine. This study found that macrophage metabolizes additional cystine into cysteine in culture medium but could not be elevated without cystine addition. Constant cysteine in blood plays an important role to interfere the stability of gold nanoparticles. Proper nanoprobes design can enhance efficiency of nanomaterial applied for precision therapy when avoiding attacks of small molecule cysteine.

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