Center for Environmental Health Sciences, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, United States of America.
Department of Chemistry and Biochemistry, University of Montana, Missoula, United States of America.
Methods Appl Fluoresc. 2021 May 7;9(3). doi: 10.1088/2050-6120/abf424.
With the use of engineered nano-materials (ENM) becoming more prevalent, it is essential to determine potential human health impacts. Specifically, the effects on biological lipid membranes will be important for determining molecular events that may contribute to both toxicity and suitable biomedical applications. To better understand the mechanisms of ENM-induced hemolysis and membrane permeability, fluorescence lifetime imaging microscopy (FLIM) was performed on human red blood cells (RBC) exposed to titanium dioxide ENM, zinc oxide ENM, or micron-sized crystalline silica. In the FLIM images, changes in the intensity-weighted fluorescence lifetime of the lipophilic fluorescence probe Di-4-ANEPPDHQ were used to identify localized changes to membrane. Time-resolved fluorescence anisotropy and FLIM of RBC treated with methyl--cyclodextrin was performed to aid in interpreting how changes to membrane order influence changes in the fluorescence lifetime of the probe. Treatment of RBC with methyl--cyclodextrin caused an increase in the wobble-in-a-cone angle and shorter fluorescence lifetimes of di-4-ANEPPDHQ. Treatment of RBC with titanium dioxide caused a significant increase in fluorescence lifetime compared to non-treated samples, indicating increased membrane order. Crystalline silica also increased the fluorescence lifetime compared to control levels. In contrast, zinc oxide decreased the fluorescence lifetime, representing decreased membrane order. However, treatment with soluble zinc sulfate resulted in no significant change in fluorescence lifetime, indicating that the decrease in order of the RBC membranes caused by zinc oxide ENM was not due to zinc ions formed during potential dissolution of the nanoparticles. These results give insight into mechanisms for how these three materials might disrupt RBC membranes and membranes of other cells. The results also provide evidence for a direct correlation between the size, interaction-available surface area of the nano-material and cell membrane disruption.
随着工程纳米材料（ENM）的应用越来越普遍，确定其对人类健康的潜在影响至关重要。特别是，生物脂质膜的影响对于确定可能导致毒性和合适的生物医学应用的分子事件非常重要。为了更好地理解 ENM 诱导溶血和膜通透性的机制，对暴露于二氧化钛 ENM、氧化锌 ENM 或微米尺寸结晶二氧化硅的人红细胞（RBC）进行了荧光寿命成像显微镜（FLIM）。在 FLIM 图像中，使用亲脂性荧光探针 Di-4-ANEPPDHQ 的强度加权荧光寿命变化来识别膜的局部变化。用甲基--环糊精处理 RBC 的时间分辨荧光各向异性和 FLIM 用于辅助解释膜有序性的变化如何影响探针的荧光寿命变化。用甲基--环糊精处理 RBC 会导致在锥体内的摆动角度增大和 Di-4-ANEPPDHQ 的荧光寿命缩短。与未处理样品相比，用二氧化钛处理 RBC 会导致荧光寿命显著增加，表明膜有序性增加。与对照水平相比，结晶硅也增加了荧光寿命。相比之下，氧化锌降低了荧光寿命，代表膜有序性降低。然而，用可溶性硫酸锌处理并没有导致荧光寿命发生显著变化，这表明氧化锌 ENM 引起的 RBC 膜有序性降低不是由于纳米颗粒潜在溶解形成的锌离子所致。这些结果深入了解了这三种材料如何破坏 RBC 膜和其他细胞的膜的机制。结果还为纳米材料的尺寸、与细胞膜相互作用的可用表面积与细胞膜破坏之间的直接相关性提供了证据。
