Dermatology Clinic, University Medical Center Mainz, Langenbeckstraße 1, 55131 Mainz, Germany; Max Planck Institute for Polymer Research (MPIP), Ackermannweg 10, 55128 Mainz, Germany.
Max Planck Institute for Polymer Research (MPIP), Ackermannweg 10, 55128 Mainz, Germany.
Acta Biomater. 2018 Apr 15;71:432-443. doi: 10.1016/j.actbio.2018.03.006. Epub 2018 Mar 10.
The transport of nanocarriers through barriers like the gut in a living organism involves the transcytosis of these nanocarriers through the cell layer dividing two compartments. Understanding how this process works is not only essential to further developing strategies for a more effective nanocarrier transport system but also for providing fundamental insights into the barrier function as a means of protection against micro- and nanoplastics in the food chain. We therefore set out to investigate the different uptake mechanisms, intracellular trafficking and the routes for exocytosis for small polystyrene nanoparticles (PS-NPs ca. 100 nm) as mimicking nanocarriers in a Caco-2 cell model for gut-blood transition. We used label-free, quantitative mass spectrometry (MS) for determining the proteome that adhered to transversed nanoparticles. From this rich proteomics dataset, as well as previous studies, we generated stable-transfected Caco-2 cell lines carrying the green fluorescent protein (GFP) coupled to proteins of interest for uptake, early, late and exocytotic endosomes. We detected the spatial and temporal overlap of such marked endosomes with the nanocarrier signal in confocal laser scanning and super-resolution microscopy. There was a clear distinction in the time course of nanoparticle trafficking between groups of proteins for endocytosis, intracellular storage and putatively transcytosis and we identified several key transcytotic markers like Rab3 and Copine1. Moreover, we postulate the necessity of a certain protein composition on endosomes for successful transcytosis of nanocarriers. Finally, we define the two-sided impasse of the lysosome as a dead end for nano-plastic and the limit of nanocarriers in the 100 nm range.
Here we focus on mechanisms of transcytosis and how we can follow these with methods not used before. First, we use mass spectrometry of transcytosed nanoparticles to pick proteins of the transcytosis machinery describing key proteins involved. We can detect the complex mixtures of proteins. As this is a dynamic process involving whole families of proteins interacting with each other and as this is an orchestrated process we coined the term protein machineries for this active interplay. By genetically modifying the proteins attaching GFP we are able to follow the transcytosis pathway. We evaluate the process in a quantitative manner over time. This reveals that the most obvious obstacle to transcytosis is a routing of the nanocarriers to the lysosomes.
纳米载体在活体生物中的运输，如穿过肠道等屏障，涉及这些纳米载体通过分隔两个隔室的细胞层的胞吞作用。了解这个过程是如何运作的，不仅对于进一步开发更有效的纳米载体运输系统至关重要，而且对于深入了解屏障功能作为防止食物链中微纳米塑料进入的一种手段也至关重要。因此，我们着手研究小聚苯乙烯纳米颗粒（PS-NP，约 100nm）的不同摄取机制、细胞内运输和胞吐途径，这些纳米颗粒在 Caco-2 细胞模型中模拟了肠道-血液转化中的纳米载体。我们使用无标记、定量质谱（MS）来确定穿过的纳米颗粒附着的蛋白质组。从这个丰富的蛋白质组学数据集中，以及以前的研究中，我们生成了稳定转染的 Caco-2 细胞系，这些细胞系携带绿色荧光蛋白（GFP）与感兴趣的摄取、早期、晚期和胞吐作用的内体蛋白偶联。我们在共焦激光扫描和超分辨率显微镜中检测到这些标记的内体与纳米载体信号的空间和时间重叠。在纳米颗粒运输的时间过程中，我们可以清楚地区分用于内吞作用、细胞内储存和推测的胞吞作用的蛋白质组，我们确定了几个关键的胞吞作用标记物，如 Rab3 和 Copine1。此外，我们假设内体上一定的蛋白质组成对于成功的纳米载体胞吞作用是必要的。最后，我们将溶酶体的两面僵局定义为纳米塑料的死胡同和纳米载体在 100nm 范围内的限制。
在这里，我们专注于胞吞作用的机制，以及我们如何以前所未有的方法来跟踪这些机制。首先，我们使用穿过的纳米颗粒的质谱法来挑选参与其中的转导机制蛋白，描述涉及的关键蛋白。我们可以检测到蛋白质的复杂混合物。由于这是一个涉及相互作用的整个蛋白质家族的动态过程，并且这是一个协调的过程，我们将这个术语称为蛋白质机器，用于这种活跃的相互作用。通过遗传修饰附着 GFP 的蛋白质，我们能够跟踪转导途径。我们在时间上以定量的方式评估这个过程。这表明，纳米载体转导的最明显障碍是将纳米载体路由到溶酶体。
