During inhalation, airborne particles such as particulate matter ≤ 2.5 μm (PM2.5), can deposit and accumulate on the alveolar epithelial tissue. In vivo studies have shown that fractions of PM2.5 can cross the alveolar epithelium to blood circulation, reaching secondary organs beyond the lungs. However, approaches to quantify the translocation of particles across the alveolar epithelium in vivo and in vitro are still not well established. In this study, methods to assess the translocation of standard diesel exhaust particles (DEPs) across permeable polyethylene terephthalate (PET) inserts at 0.4, 1, and 3 μm pore sizes were first optimized with transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-VIS), and lock-in thermography (LIT), which were then applied to study the translocation of DEPs across human alveolar epithelial type II (A549) cells. A549 cells that grew on the membrane (pore size: 3 μm) in inserts were exposed to DEPs at different concentrations from 0 to 80 µg.mL− 1 ( 0 to 44 µg.cm− 2) for 24 h. After exposure, the basal fraction was collected and then analyzed by combining qualitative (TEM) and quantitative (UV-VIS and LIT) techniques to assess the translocated fraction of the DEPs across the alveolar epithelium in vitro. We could detect the translocated fraction of DEPs across the PET membranes with 3 μm pore sizes and without cells by TEM analysis, and determine the percentage of translocation at approximatively 37% by UV-VIS (LOD: 1.92 µg.mL− 1) and 75% by LIT (LOD: 0.20 µg.cm− 2). In the presence of cells, the percentage of DEPs translocation across the alveolar tissue was determined around 1% at 20 and 40 µg.mL− 1 (11 and 22 µg.cm− 2), and no particles were detected at higher and lower concentrations. Interestingly, simultaneous exposure of A549 cells to DEPs and EDTA can increase the translocation of DEPs in the basal fraction. We propose a combination of analytical techniques to assess the translocation of DEPs across lung tissues. Our results reveal a low percentage of translocation of DEPs across alveolar epithelial tissue in vitro and they correspond to in vivo findings. The combination approach can be applied to any traffic-generated particles, thus enabling us to understand their involvement in public health.

中文翻译：

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结合分析技术评估柴油颗粒在体外穿过肺泡组织屏障的易位


吸入过程中，空气中的颗粒物，例如≤2.5μm的颗粒物（PM2.5），可以沉积并积聚在肺泡上皮组织上。体内研究表明，部分 PM2.5 可以穿过肺泡上皮进入血液循环，到达肺部以外的次要器官。然而，在体内和体外量化颗粒穿过肺泡上皮的易位的方法仍然没有很好地建立。在本研究中，首先使用透射电子显微镜 (TEM)、紫外-可见光谱法优化了评估标准柴油机尾气颗粒 (DEP) 在孔径为 0.4、1 和 3 μm 的渗透性聚对苯二甲酸乙二醇酯 (PET) 插入物中易位的方法(UV-VIS) 和锁定热成像 (LIT)，然后将其用于研究 DEP 在人肺泡上皮 II 型 (A549) 细胞中的易位。将在插入物中的膜（孔径：3 μm）上生长的 A549 细胞暴露于 0 至 80 µg.mL− 1（0 至 44 µg.cm− 2）不同浓度的 DEP 中 24 小时。暴露后，收集基础部分，然后通过结合定性 (TEM) 和定量（UV-VIS 和 LIT）技术进行分析，以评估体外 DEP 跨肺泡上皮的易位部分。我们可以通过 TEM 分析检测 DEP 穿过孔径为 3 μm 且无细胞的 PET 膜的易位分数，并通过 UV-VIS 确定约 37% 的易位百分比（LOD：1.92 µg.mL− 1）和 75 % LIT（LOD：0.20 µg.cm− 2）。在细胞存在的情况下，在 20 和 40 µg.mL− 1（11 和 22 µg.cm− 2）浓度下，DEP 穿过肺泡组织的易位百分比约为 1%，并且在较高和较低浓度下均未检测到颗粒。 有趣的是，A549 细胞同时暴露于 DEP 和 EDTA 可以增加基础部分中 DEP 的易位。我们提出了结合分析技术来评估 DEP 在肺组织中的易位。我们的结果表明，体外 DEP 穿过肺泡上皮组织的易位百分比较低，并且与体内研究结果相符。这种组合方法可以应用于任何交通产生的颗粒，从而使我们能够了解它们对公共卫生的参与。