結合Tat和NLS胜肽於磁性奈米粒子表面對細胞分佈之研究

Abstract

奈米粒子因體積小且具有特殊物理特性而被廣泛應用於生醫研究。其中超順磁氧化鐵奈米粒子 (USPIO) 因具有強烈的順磁性，可作為核磁共振造影的顯影劑。配合磁場之使用，進一步的能夠應用在收集特定細胞和轉染研究上。除此之外， USPIO 還可以應用在癌症組織中作為非侵入性的偵測或幹細胞追蹤。但是，要能在活體中追蹤細胞，其細胞內的訊號必須夠強。因此如何讓能大量的氧化鐵奈米粒子有效率地進入細胞而又不會造成傷害，一直是科學家研究致力的目標。 在先前的研究中，已經有很多方法像是化學或是蛋白的修飾於氧化鐵奈米粒子的表面，使細胞容易吸收。然而這樣的方式通常是直接以共價鍵連接在粒子表面，步驟較複雜，所需時間也較長。為了能簡單的修飾氧化鐵奈米粒子表面，本研究以微胞體包覆氧化鐵奈米粒子。這樣的修飾方法簡單，而且只要在一個小時內即可完成。另一方面，為了有效率地將氧化鐵奈米粒子送入細胞內，吾人連接實驗室已經建立的一種穿透性胜

Nanoparticles have been widely used in biomedical research due to small size and special physical properties. One of them, ultrasmall superparamagnetic iron oxide (USPIO), has a super paramagnetic behavior, and can be used to a contrast agent in nuclear magnetic resonance imaging (MRI). By the magnetic fields, USPIO can further be used in the application of cell collection and transfection. Besides that, it can also be a noninvasive tracker in the cancer tissue or the stem cell. There has to been a stronger signal in a cell even though USPIO can be used in the cell tracking in vivo however. Recently, make large number of USPIO uptake by the cell efficiently and no toxicity has been committed to a goal in the scientific topic. Previous study, there were already many chemical or protein modifications on the surface of USPIO to enhance the cell absorbance. Neverless, most of the methods bound to the surface by covalent conjugation which is complicated and time-consuming. In order to modify the surface of the USPIO simply, I coat the surface of USPIO by the micelle formulation. The modification is not only simple, but as fast as an hour to complete. On the other hand, I would link a cell penetrating peptide fusion protein – Tat-PAST, which my lab has established by way of uptake more efficiently in the cell. In addition, I would also link a fusion protein of nuclear localization signal (NLS) – NLS-ST, to study whether NLS could help micelle-USPIO complex contact the nuclear membrane, thereby increasing access to enter the cell nucleus. In the article, it would analyze: 1. Compare the particle size, and the efficiency of cell uptake of micelle and micelle-USPIO complex between different lipid composition, 2. Confirm the protein activity and function by cell experiments after protein purification of NLS-ST and Tat-PAST, 3. when NLS-ST or Tat-PAST fusion protein bind the micelle-USPIO complex, analyze the uptake and distribution in the cell. As a result, compared different lipid composition of the micelle, adding more lipid content of DSPE-PEG 2000, the size of micelle could be smaller while inhibiting the cell uptake. When study on the function of Tat-PAST and NLS-ST fusion protein, Tat-PAST fusion protein could easily help the cell uptake of micelle-USPIO complex as expected; NLS-ST fusion protein could also help micelle-USPIO complex contact to nuclear membrane and even into the nucleus though it cannot effectively uptake by the cell alone. Finally, Tat-PAST and NLS-ST fusion protein linked micelle-USPIO complex together and enhanced the cell uptake of micelle-USPIO complex certainly, even some part of less in the nucleus though they were mainly in the cytoplasm. According to these results, it can tell that micelle-USPIO complex linking with Tat-PAST and NLS-ST fusion protein could increase the ability of cell uptake, and nuclear entry. The strategy can further improve the integration of the cell tracking and as a vector in the drug or gene therapy. In the same time, the micelle coating technology also could utilize to different nanoparticles for all kinds of modification on the surface simply. This aspect will make more application in the biomedical research.