壳聚糖纳米粒与细胞相互作用机制研究.docVIP

壳聚糖纳米粒与细胞相互作用的机制研究 中文摘要：目的：探讨壳聚糖纳米粒与细胞间相互作用的机制，并阐明其与壳聚糖分子作用机制的异同。方法：以Caco-2细胞为细胞模型，考察壳聚糖纳米粒的细胞摄取机制采用激光共聚焦技术观察骨架蛋白F-actin及紧密连接蛋白Zo-1的结构变化壳聚糖纳米粒细胞膜流动性的实时检测纳米粒与细胞的相互作用。壳聚糖纳米粒经Caco-2细胞的摄取与壳聚糖的分子量和脱乙酰度密切相关，即壳聚糖的分子量越大，脱乙酰度越高，则壳聚糖纳米粒经细胞的摄取量越大。壳聚糖分子经Caco-2细胞的摄取仅与壳聚糖的脱乙酰度有关，而壳聚糖的分子量不能影响其摄取激光共聚焦壳聚糖纳米粒能够与细胞发生相互作用，纳米粒通过穿细胞途径进入细胞。此外，壳聚糖纳米粒主要存在于Caco-2细胞单层的顶端，难以到达基底端壳聚糖分子仅吸附于绒毛层，不能向壳聚糖纳米粒那样插入膜内。壳聚糖及壳聚糖纳米粒能够造成F-actin环的缺失，使得F-actin环显示不连续，并能使紧密连接上的ZO-1蛋白解散。傅立叶红外光谱法（FTIR），激光拉曼光谱法（Raman）和荧光偏振法壳聚糖纳米粒能增加膜脂的流动性，降低膜的微粘度，改变膜蛋白构象，这些作用都有利于药物的跨细胞转运。壳聚糖纳米粒或壳聚糖分子C6细胞在作用30min内，SPR信号升高并且具有浓度依赖性。Nanoparticles and Live Cells ABSTRACT Objective: Study on the mechanism of interaction between chitosan nanoparticles(CS-NPs) and cells, also to elucidate the similar and difference between CS-NPs and chitosan (CS) molecules. Methods: Caco-2 monolayer was selected as model cell, cellular uptakes of CS-NPs and CS molecules were carried out in order to evaluate the effects of molecular weight and degree of deacecylation. In laser confocal scanning microscope (LCSM) study, the effects of CS-NPs or chitosan molecules on the cell skeleton protein (F-actin) of Caco-2 cells were carried out. Some physicochemical methods were used to explore the mechanism of CS-NPs acting on Caco-2 cells, including Fourier transformed infrared spectroscopy (FTIR), Raman spectrum and fluorescence polarization. For further study, surface plasmon resonance (SPR) was also employed to monitor the interaction between molecules and cells online. Results: Uptake of CS-NPs and CS by Caco-2 cells was quantified by fluorometry. Resluts indicated nanoparticles uptakes was a saturable event for all samples, with the binding affinity and uptake capacity decreasing with decreasing polymer molecular weight and degree of deacecylation. While uptake of CS molecules did not exhibit saturation kinecits and was less dependent on the molecular weight and degree of deacetylation. CS-NPs and CS molecules are able to open the tight junction. Furthermor