氧化鐵奈米粒子於巨噬細胞及幹細胞之標示：標示效能、攝取機制及細胞生理功能影響之探討

Abstract

在應用於磁振造影的對比劑當中，超順磁性氧化鐵奈米粒子(SPIO)是目前最常使用的超順磁性粒子，而釓(Gd)是目前最常用的順磁性粒子。巨噬細胞(macrophage)是一種具有吞噬能力(phagocytosis)的特殊細胞，因此會主動吞噬SPIO而在可在磁振造影中顯影。若要標示其他無吞噬能力的細胞（如幹細胞），為使標示效率提高及避免高濃度的培養所帶來的細胞毒性，大部份的研究會使用轉染試劑(transfection agent)來幫助標示，不過這個做法可能會帶來新的細胞毒性或對細胞功能產生影響。此論文中是在利用直接細胞標示方法下，氧化鐵奈米粒子在巨噬細胞及間質性幹細胞之標示效能、攝取機制與其對細胞生理功能影響之探討。 在巨噬細胞吞噬含氧化鐵奈米粒子(Ferucarbotran)的實驗中，我們在分光光譜儀及流式細胞儀分析的結果發現Ferucarbotran培養濃度與吞噬量及細胞顆粒性(granularity)成正相關。細胞內的鐵含量與側面散射特性呈現線性關係(p <0.001, R2 = 0. 8048)。在經過Ferucarbotran標示後，我們觀察到巨噬細胞在細胞活性與活性氧分子群的增加，及粒線體電位差的變化，這表示巨噬細胞的活性發生了變化。隨著顆粒的大小及特性不同，巨噬細胞會由不同的內噬路徑吞噬顆粒。了解巨噬細胞是經由何種內噬路徑吞噬SPIO，將有助於對比劑的發展及對細胞生理的了解。在添加不同的內噬路徑抑制劑後，我們在流式細胞儀及磁振造影影像的證據中發現在抑制Clathrin路徑後，培養於Ferucarbotran的巨噬細胞內的鐵含量相較之下大幅減小，由此可見巨噬細胞是經由Clathrin路徑來吞噬 Ferucarbotran。 由於間質幹細胞具有組織修補及器官移殖等細胞療法之用途，標示間質幹細胞的細胞影像可以在非侵入性的方式之下幫助我們了解細胞療法之成效。因此我們對於Ferucarbotran的標示對於間質幹細胞的毒性及細胞生理影響也很有興趣。在此篇論文中我們利用了光學顯微鏡、流式細胞儀分析在24小時及72小時使用直接標示Ferucarbotran的間質幹細胞，其細胞的巨觀型態，大小，繁殖數量，顆粒性，活性及活性氧分子群的產生均與控制組的細胞沒有顯著相關，而且其標示效率皆達到可被磁振造影偵測的程度。過去的文獻中已經知道間質幹細胞在直接標示Ferucarbotran後的骨骼以及脂肪組織分化能力依然存在，可是軟骨組織分化能力一直都還沒得到證實。在這個研究中，我們成功地讓直接標示後的間質幹細胞分化成軟骨組織，並以染色及組織細胞化學方法加以驗證。因此我們可以得到的結論是直接標示法在可被磁振造影偵測的特定濃度下不會對間質幹細胞的細胞活性及生理功能產生明顯的影響，也保留了間質幹細胞分化能力的完備性。我們也發現在間質幹細胞吸收Ferucarbotran之後，在螢光染色的資料證實這些氧化鐵奈米粒子分佈在溶小體中，這個結果與巨噬細胞吞噬Ferucarbotran後的結果相仿，也跟其他研究文獻的結果相符。在此論文中也比較使用含氧化鐵奈米粒子(Ferucarbotran)與含釓(Gadodiamide)兩種臨床上使用中之對比劑在標示間質幹細胞時，測定其標示效率，靈敏度與磁 振造影偵測之可行性。我們發現在不影響細胞功能的濃度下，間質幹細胞可在沒有轉染試劑的幫助下成功地使用這兩種對比劑標示並於磁振造影中顯影。我們也分別測定了在磁振造影中體外能夠顯影之最少細胞數目量，這些閾值可供日後實驗設計或臨床運用之參考。在小動物實驗中，Ferucarbotran的磁振造影靈敏度可以達到Gadodiamide的八倍，不過因為磁敏感度(magnetic susceptibility)強，Ferucarbotran所產生之訊號可能會扭曲或遮蔽周圍的解剖構造。 這篇論文提供了將細胞直接標示超順磁性氧化鐵奈米粒子在磁振造影偵測可行性之論證。間質幹細胞的生理活性與分化功能在此論文提供的分法下直接標示後可以不受影響。而巨噬細胞在吞噬足以在磁振造影顯影的氧化鐵奈米粒子數量下，是經由Clathrin-mediated路徑，並會發生一些細胞生理上的變化。這些發現有助於幹細胞生物學，組織工程，細胞影像及細胞療法的研究及發展。

Various magnetic resonance (MR) contrast agents, including paramagnetic and superparamagnetic substances, have been used for cell labeling in cellular imaging. Superparamagnetic iron oxide (SPIO) nanoparticles are the most commonly used superparamagnetic contrast agents used for magnetic resonance imaging while Gadolinium (Gd) is the most common clinically used paramagnetic contrast agents. Macrophages, one kind of specialized blood cells that are capable of phagocytosis, would ingest SPIOs and produces signals on MR imaging. To improve the labeling efficacy on non-phagocytotic cells, for example, stem cells, and to prevent the toxicity of high incubation concentration, most of the labeling were performed with the aids of various transfection agents, which may introduce to other cellular toxicity or influence of cellular function. Therefore direct labeling method without transfection agent is used in this thesis. The objectives of this thesis are to examine the impact of the direct SPIO labeling on macrophages and mesenchymal stem cells, especially the labeling efficacy, mechanism of cellular uptake and impact on cellular physiology. The murine macrophage cell line Raw 264.7 and a clinical SPIO contrast agent (Ferucarbotran, carboxydextran-coated SPIO with a diameter of about 45–60 nm) were used in this thesis. We observed a dose-dependent uptake of these SPIO particles by spectrophotometer analysis and also a dose-dependent increase in the granularity of the macrophages as determined by flow cytometry. There was a linear correlation between the side scattering mean value and iron content (P <0.001, R2 = 0. 8048). For evaluation of the endocytotic pathway of these ingested SPIO particles, different inhibitors of the endocytotic pathways were employed. Significant decrease of side scattering counts and change in signal intensity on magnetic resonance image were observed only in the phenylarsine oxide-treated macrophages, which suggested the clathrin-mediated pathway plays a major role in the endocytosis of the Ferucarbotran. After labeling with SPIO particles, the macrophages showed an increase in the production of reactive oxygen species (ROS) at 2, 24, and 48 h; a decrease in mitochondrial membrane potential (MMP) at 24 h; and an increase in cell proliferation at 24 h. The results suggested an alteration of the cellular physiological function of the macrophage after direct labeling of Ferucarbotran. This thesis also describes the evaluation the long-term cellular toxicity, labeling efficiency, chondrogenic differentiation capacity, and intracellular distribution following direct SPIO nanoparticle labeling of human MSCs (hMSCs) in the absence of transfection agents. hMSCs were incubated with Ferucarbotran at concentrations of 0, 1, 10, and 100 μg Fe/ml for 24 or 72 h. The cell granularity and size change, ROS generation, and MMP change were measured by flow cytometry. After induction of chondrogenesis, the differentiation capacity of the cells into chondrocytes was determined by Alcian blue and Safranin-O staining, immunocytochemical analysis, and RT-PCR. The intracellular distribution of the internalized particles was visualized via confocal microscopy. No significant difference was found in the toxicity of labeled cells relative to controls until the twelfth day. Successful chondrogenesis of Ferucarbotran-labeled hMSCs was confirmed. The intracellular SPIO nanoparticles were located within the lysosomes. This thesis also compared the efficacy, sensitivity and feasibility between paramagnetic and superparamagnetic substances labeling in hMSCs. We used Ferucarbotran and Gadodiamide (Gd chelate [Gd-DTPA-BMA]) for comparison. Without the aid of transfection agent, human mesenchymal stem cells were labeled with each agent separately in different concentration and the optimized concentration was determined by maintaining same cell viability as unlabeled cells. Iron oxide nanoparticle labeling has a detecting threshold of 12,500 cells in vitro, while gadolinium chelates labeling could be detected for at least 50,000 cells. In life animal study, there is an 8-fold sensitivity in cells labeled with iron oxide superparamagnetic nanoparticles; however, the magnetic susceptibility artifact would obscure the detail of adjacent anatomical structures. In conclusion, the direct labeling method of SPIO on macrophages and hMSCs is feasible for cellular imaging. The alteration of ROS and MMP raised a concern of biosafety while SPIO labeling of macrophages. The cellular physiological functions of the labeled hMSCs were not altered in a long-term follow up and the chondrogenic differentiation capacity was preserved. The endocytotic mechanism of SPIO uptake into macrophage was via the clathrin-mediated pathway. Further development of nanoparticles targeting the clathrin may increase the efficiency of this cell labeling method. The minimum thresholds for MRI detection of SPIO and Gd were determined and there is an 8-fold sensitivity in cells labeled with SPIO compared to Gd labeling in the life animal study.