利用二氧化矽奈米酵素載體進行細胞內活性氧物質偵測

Abstract

活性氧物質(ROS) 在細胞生理反應中扮演著細胞內訊息傳遞者的角色。在正常情況下，細胞內有足夠的抗氧化機制與活性氧物質達成平衡，但當細胞受到特定刺激時，細胞內過量的活性氧物質造成細胞產生氧化壓力，導致細胞內蛋白質、脂質、遺傳物質不可逆的破壞，造成細胞凋亡，甚至老化、糖尿病、癌症等疾病的發生。因此，若能夠在早期即有效的監控細胞內活性氧物質的水平，能夠及早預防疾病的產生。 可穿透細胞的二氧化矽空心球酵素載體搭配活性氧物質螢光試劑的組合非常適合應用在細胞內氧化壓力的偵測。在這份研究中，我們將辣根過氧化酶(HRP)以溫和的製程包覆在奈米空心球內部，稱之為HRP@HSN。此酵素載體具有好的催化活性、高穩定性、細胞膜穿透性、生物相容性、能夠保護酵素避免受到蛋白酶的分解，並且具有核內體 (endosome) 脫離的機制。當HRP@HSN 被細胞吞噬後，這些酵素載體內的HRP 能夠與活性氧物質反應，進一步與能通透細胞膜的DHR123 試劑作用，在波長530 nm 偵測其螢光值。在我們的系統中，我們以濃度為每毫升 100 微克的酵素載體在1 小時內即可進行細胞內活性氧物質的偵測。並且，利用此具有高靈敏度的系統，我們能夠在細胞發炎反應發生時進行偵測以及定量細胞所產生的活性氧物質。

Reactive oxygen species (ROS) which served as intracellular messengers are associated with cellular functions through affecting cellular signaling. Under certain stimulations, ROS would be overproduced and further caused oxidative stress resulting in the cell irreversible damage or death in many diseases. Therefore, it is useful to monitor the cellular level of ROS for prevention of cell damage at the early stage as well as indication of the staging of disease. ROS sensitive chromosphere agent combining with transmembrane nanoparticles was considered to apply for intracellular reporter signal. In this study, we developed a mild synthesis protocol to load horseradish peroxidase (HRP) into the interior cavity of hollow silica nanospheres (HSN) to form the HRP encapsulated HSNs, named HRP@HSN. This particle shares several advantages such as effectual catalytic activity, high stability, membrane permeability, cell biocompatibility, the ability to escape from endosome, and enzyme protection from protease. When HRP@HSNs were introduced to HeLa cells and RAW264.7 macrophage cell line, the particles were able to convert ROS into hydroxyl radical which reacting with dihydrorhodamine 123, a cell-permeable dye. In our system, we could detect the cellular ROS within 1 hour at a dose limitation as low as 100 μg/mL, and illuminate at emission wavelength 530 nm. We exploited this system with high sensitivity and also demonstrated the cellular example at ROS-associated inflammatory status.