EA4483, IMPRT, IFR 114, Université de Lille Nord de France, 1 place Verdun, 59000 Lille, France.
Biomaterials. 2012 Dec;33(35):9117-26. doi: 10.1016/j.biomaterials.2012.08.064. Epub 2012 Sep 14.
The delivery of protein in the airway using nanoparticles (NP) is an emerging strategy that shows encouraging results in vivo for several applications. However, the mechanisms by which NP deliver proteins to the inside of cells remain poorly understood. In this study, we investigated the intracellular delivery of ovalbumin (OVA) in human airway cells by two porous cationic polysaccharides nanoparticles. These NP have the same surface charge density but differ in that their inner core contains either cationic or anionic charges (respectively: NP(+) and DGNP(+)). Confocal microscopy showed a rapid uptake of both NP by human airway cells, followed by a significant accumulation in clathrin vesicles and early endosomes. Both NP were found to associate OVA in a quantitative manner, and this association was stable even in presence of serum proteins. We observed that the two NP greatly increased OVA uptake by human airway cells, meanwhile FRET studies using FITC-labelled NP and TRITC-labelled OVA showed a gradual release of OVA from NP within cells, and this was much faster with DGNP(+) than NP(+). These results were confirmed using OVA-DQ to follow OVA degradation fragments within cells. Both NP increased intracellular proteolysis of OVA, however DGNP(+) facilitated OVA escape from endosomes. Studies with trypsin and pepsin at different pH strongly suggested that both NP can protect (in the extracellular medium) or promote (in acidic endosomes) protein proteolysis, depending on the environment. Interestingly, the mechanisms involved could be explained as a function of protein global charge at different pH. All these results confirm the importance of not only the surface charge but also the inner composition of NP in determining their efficacy as tools for the delivery of proteins to different cellular compartments.
利用纳米颗粒（NP）在气道中输送蛋白质是一种新兴策略，在多种应用中在体内显示出令人鼓舞的结果。然而，NP 将蛋白质递送到细胞内部的机制仍知之甚少。在这项研究中，我们研究了两种多孔阳离子多糖纳米颗粒（NP）在人呼吸道细胞中卵清蛋白（OVA）的细胞内递呈。这些 NP 具有相同的表面电荷密度，但不同之处在于它们的内核分别含有阳离子或阴离子电荷（分别为：NP(+)和 DGNP(+)）。共焦显微镜显示两种 NP 均迅速被人呼吸道细胞摄取，随后在网格蛋白小泡和早期内体中大量积累。两种 NP 均以定量的方式与 OVA 结合，并且这种结合即使在存在血清蛋白的情况下也是稳定的。我们观察到两种 NP 均可显著增加 OVA 被人呼吸道细胞摄取，而使用 FITC 标记的 NP 和 TRITC 标记的 OVA 的 FRET 研究表明，OVA 从 NP 内逐渐释放，DGNP(+)比 NP(+)更快。使用 OVA-DQ 跟踪细胞内 OVA 降解片段证实了这些结果。两种 NP 均增加了 OVA 的细胞内蛋白水解，但 DGNP(+)促进了 OVA 从内体逃逸。在不同 pH 值下用胰蛋白酶和胃蛋白酶进行的研究强烈表明，两种 NP 都可以保护（在细胞外介质中）或促进（在酸性内体中）蛋白质水解，具体取决于环境。有趣的是，涉及的机制可以解释为不同 pH 值下蛋白质整体电荷的函数。所有这些结果都证实了不仅表面电荷，而且 NP 的内部组成在确定它们作为将蛋白质递送到不同细胞区室的工具的功效方面的重要性。
