磁性纳米粒子跨膜转运机制及生物学效应和碳纳米管对钾通道影响的研究

【作者】 徐海飞；

【导师】 曹济民；

【作者基本信息】 中国协和医科大学 ， 生理学， 2010， 博士

【摘要】 纳米生物学作为一门多学科交叉科学开启了生物学研究的新篇章。然而,纳米粒子与生物细胞具体的相互作用机制尚未明了。由于纳米粒子的大小与DNA、蛋白质等生物大分子以及病毒的尺寸相当,某些纳米粒子的生物效应可能涉及到当前尚未充分了解的生物与环境相互作用的机理,深入系统地研究纳米粒子与生物体系的相互作用,一方面不仅可以大大推动纳米科学在生物科学领域的交织发展,同时也可以为全面了解纳米粒子对人类生存环境和健康的影响提供理论依据。本研究选用了磁性纳米粒子及多壁碳纳米管两种纳米粒子,探讨了它们与细胞的相互作用及其生物学效应和机制。磁性纳米粒子已经在纳米医学等诸多领域有应用。本研究报道了吞噬细胞RAW264.7细胞摄入磁性纳米粒子有时间及剂量依赖性,摄入的磁性纳米粒子主要出现在细胞浆内,而内质网、线粒体及细胞核等细胞器内未见。而且,RAW264.7细胞摄入磁性纳米粒子的过程是能量依赖的,提示是经由内吞途径进入细胞的。通过特异性内吞抑制剂分别干扰各途径,结果发现clathrin-、caveolae介导的内吞途径,巨胞饮及清道夫受体介导的吞噬等途径均有可能参与磁性纳米粒子的入胞过程,而非某单一途径参与。同时,我们比较了三种不同吞噬能力的细胞摄入磁性纳米粒子的差异。结果发现,红细胞不能摄入磁性纳米粒子,吞噬能力强的RAW264.7细胞摄入磁性纳米粒子比弱吞噬能力的3T3L1细胞快且多,提示在不同类型细胞纳米粒子入胞途径的选择有差异。本研究还进一步探索了进入细胞内的磁性纳米粒子的生物学效应。结果表明,本研究所用的磁性纳米粒子有良好的生物相容性,对RAW264.7细胞的细胞活力、ROS生成及线粒体膜电位等无显著影响。另外,关于入胞的磁性纳米粒子的归宿提出3种可能：1)摄入的磁性纳米粒子随细胞分裂被分至子代细胞；2)摄入的磁性纳米粒子被降解并释放游离铁离子；3)磁性纳米粒子经胞吐释放至细胞外。而且,摄入的磁性纳米粒子对细胞内铁代谢相关蛋白质有影响,能使铁贮存蛋白质ferritin-L在蛋白质及mRNA水平表达均有升高,铁输出蛋白ferroportin1 mRNA水平表达有明显升高但蛋白质水平未见明显改变,而上述两种蛋白质的变化不是通过降解铁调节蛋白2来实现的。这些研究结果为磁性纳米粒子作为造影剂及药物载体等的应用研究提供了理论依据。作为最有应用前景的纳米材料之一,碳纳米管已经成为了纳米生物技术研究领域里的热点。但是,碳纳米管与生物体细胞相互作用的性质及其作用机制尚不明了,尤其对于细胞膜上离子通道的影响。离子通道负责机体可兴奋细胞的信号传导,对正常生理活动起至关重要的作用。以钾通道为例,它参与可兴奋细胞动作电位的复极化。钾通道功能异常会导致动作电位复极化失调、静息电位不能正确复位,最终导致机体功能障碍。在前期工作中我们发现末端羧基化的多壁碳纳米管(长300-800nm,内径10-20nm,外径40-50nm)可抑制未分化的嗜铬细胞瘤(PC12)细胞上3种钾通道(Ito、IK、IKl)电流。在此实验结果的基础上,我们对多壁碳纳米管抑制钾通道的可能机制进行了探讨。多数内外源性有害物质都是经由氧化应激的信号通路引起细胞毒性,因此,我们推测多壁碳纳米管可能通过诱导PC12细胞的氧化应激,进而抑制了3种钾电流。然而,结果表明,多壁碳纳米管未能引起PC12细胞内ROS生成增多、线粒体膜电位降低及细胞内钙水平升高。此结果提示我们多壁碳纳米管不是通过氧化应激的信号通路抑制钾通道的。尽管如此,将多壁碳纳米管用于纳米医学领域时,应该考虑其可能会抑制可兴奋细胞上的钾通道。更多还原

【Abstract】 Nanobiology as a newly emerging multidisciplinary fused science has introduced a new dimension to research endeavor and a range of technologies in biology. However, the mechanisms underlying cell uptake of nanoparticles remain obscure. The characteristic size in the nanoscale, which is equivalent to the size of some biological macromolecules (such as DNA, proteins, etc.), nanoparticles would possess some unknown biological effects on the biological systems. Extensive studies need to be carried out to detect the interaction between nanoparticles and biological systems, providing robust evidence for the application of nanoparticles in life science. In our study, superparamagnetic iron oxide nanoparticles (SPION) and multi-walled carbon nanotubes had been chosen to study, the cellular influences and the potential molecular mechanisms were investigated.As one of the prospective nanoparticles candidates, SPION have been focused on in several nanomedical fields. Here, we reported that the internalization of SPION into a macrophage-like cell line RAW264.7 was in a time-and concentration-dependent manner to some degree. The internalized SPION were mainly found in the cytoplasmic vesicles, with no localization in the endoplasmic reticulum, mitochondria and nucleus. Moreover, the RAW264.7 cellular uptake was via an energy-dependent process that was suggestive of endocytosis. By blocking various endocytic pathways, we demonstrated that multiple endocytic processes were involved including clathrin-and caveolae-mediated endocytosis, macropinocytosis and scavenger receptor-mediated phagocytosis. Our data showed that nanoparticle endocytosis is therefore not mediated by a unique signaling pathway in a giving cell type.Meanwhile, the present study investigated the internalization of SPION in three cell models with different phagocytic capacity using transmission electron microscopy and energy dispersive spectrometer analysis. The results showed that the iron element was the nanoparticles composition in the cytoplasm of RAW264.7 cells but not in the red blood cells. SPION could be uptaken by RAW264.7 (with strong phagocytic capacity) and the 3T3-L1 cell (with weak phagocytic capacity), but not by red blood cells (with no phagocytic capacity), suggesting that different internalization pathways could be chosen for the nanoparticles cellular uptake in different cell types. The internalization occurred much more quickly in RAW264.7 cells than 3T3-L1 cells.The potential applications of SPION in several nanomedical fields have attracted intense interests, but the intracellular trail, the final fate and the biological effect of the internalized iron oxide nanoparticles have not been clearly elucidated. Here we showed that the internalized SPION had no effect on the cell viability, ROS induction and mitochondrial membrane potential dysfunction, possessing well biocompatible characteristics in RAW264.7 cells. Moreover, three kinds of possible metabolic fate for the internalized SPION in RAW264.7 cells were speculated:first, the internalized SPION are distributed to daughter cells; second, the internalized SPION are degraded in the lysosome and free iron was released into the intracellular iron metabolic pool; third, the intact iron oxide nanoparticles could be exocytosed out of cells. In addition, the internalized SPION indeed affected the intracellular iron metabolism in RAW264.7 cell, inducing an up-regulation of ferritin light chain at both protein and mRNA level, and ferroportinl at the mRNA level. The central player in the intracellular iron metabolism iron regulatory protein 2 has no degradation opposite to the hypothesis. The results in the present study provided evidences for the consideration of biological safety of SPION administered as contrast agents or drug delivery tools.Intense interest in the applications of nanomaterials for drug delivery, diagnostic or imaging tests and regenerative medicine has been generated because of the nanoscale size of such materials. Among the nanomaterials, carbon nanotube has been the focus of extensive researches as a potential player in nanobiotechnology. However, the nature or mechanism by which carbon nanotubes interact with cells or specifically ion channels is largely unknown. Ion channels transduce electrical signals in excitable cells and they therefore play critical roles in many physiological systems. A notable example of ion channels that plays an essential role in physiology is potassium channels that are involved in the repolarization of action potentials in excitable cells. Functional abnormality of these K+channels contribute to disturbance of action potential repolarization and inability to reset the resting potential properly and thus leading to dysfunction of an organ.In our previous study, we observed that carboxyl-terminated multi-walled carbon nanotubes (MWCNTs, length 300-800nm, inner diameter 10-20nm and outer diameter 40-50nm) act as antagonists of three types of potassium channels as assessed by whole-cell patch clamp electrophysiology on undifferentiated pheochromocytoma (PC 12) cells. Moreover, the possible signal pathway through which the MWCNTs have impact on the K+ channels was further explored. Oxidative stress could develop as a cellular response to hazardous materials or endogenous adverse metabolite and could also be a potential downstream effect pathway by which nanotubes affect ion channels. The physiological effects of oxidative stress include the production of reactive oxygen species (ROS), increasing of [Ca2+]i and decreasing of the mitochondrial membrane potential (⊿Ψm). However, MWCNTs did not significantly change the expression levels of ROS and did not alter the⊿Ψm in PC 12 cells. MWCNTs also did not significantly change the level of intracellular free calcium. These results suggest that oxidative stress was not involved in MWCNTs suppression of Ito, IK and IK1 current densities. Nonetheless, the suppression of potassium currents by MWCNTs will impact on electrical signaling of excitable cells such as neurons and muscles. As such, the potential side effects of nanotubes on potassium channels should be examined before they are used as a delivery tool in vivo.更多还原