Peuschel Henrike, Ruckelshausen Thomas, Kiefer Silke, Silina Yuliya, Kraegeloh Annette
INM - Leibniz Institute for New Materials, Campus D2 2, 66123, Saarbrücken, Germany.
J Nanobiotechnology. 2016 Sep 26;14(1):70. doi: 10.1186/s12951-016-0222-9.
Quantum dots (QDs) have great potential as fluorescent labels but cytotoxicity relating to extra- and intracellular degradation in biological systems has to be addressed prior to biomedical applications. In this study, human intestinal cells (Caco-2) grown on transwell membranes were used to study penetration depth, intracellular localization, translocation and cytotoxicity of CdSe/ZnS QDs with amino and carboxyl surface modifications. The focus of this study was to compare the penetration depth of QDs in differentiated vs undifferentiated cells using confocal microscopy and image processing.
Caco-2 cells were exposed to QDs with amino (NH) and carboxyl (COOH) surface groups for 3 days using a concentration of 45 µg cadmium ml. Image analysis of confocal/multiphoton microscopy z-stacks revealed no penetration of QDs into the cell lumen of differentiated Caco-2 cells. Interestingly, translocation of cadmium ions onto the basolateral side of differentiated monolayers was observed using high resolution inductively coupled plasma mass spectrometry (ICP-MS). Membrane damage was neither detected after short nor long term incubation in Caco-2 cells. On the other hand, intracellular localization of QDs after exposure to undifferentiated cells was observed and QDs were partially located within lysosomes.
In differentiated Caco-2 monolayers, representing a model for small intestinal enterocytes, no penetration of amino and carboxyl functionalized CdSe/ZnS QDs into the cell lumen was detected using microscopy analysis and image processing. In contrast, translocation of cadmium ions onto the basolateral side could be detected using ICP-MS. However, even after long term incubation, the integrity of the cell monolayer was not impaired and no cytotoxic effects could be detected. In undifferentiated Caco-2 cells, both QD modifications could be found in the cell lumen. Only to some extend, QDs were localized in endosomes or lysosomes in these cells. The results indicate that the differentiation status of Caco-2 cells is an important factor in internalization and localization studies using Caco-2 cells. Furthermore, a combination of microscopy analysis and sensitive detection techniques like ICP-MS are necessary for studying the interaction of cadmium containing QDs with cells.
量子点（QDs）作为荧光标记物具有巨大潜力，但在生物医学应用之前，必须解决与生物系统中细胞外和细胞内降解相关的细胞毒性问题。在本研究中，使用生长在Transwell膜上的人肠道细胞（Caco-2）来研究具有氨基和羧基表面修饰的CdSe/ZnS量子点的穿透深度、细胞内定位、转运和细胞毒性。本研究的重点是使用共聚焦显微镜和图像处理比较量子点在分化细胞与未分化细胞中的穿透深度。
将Caco-2细胞暴露于具有氨基（NH）和羧基（COOH）表面基团的量子点中，浓度为每毫升45微克镉，持续3天。共聚焦/多光子显微镜z轴堆叠的图像分析显示，量子点未穿透分化的Caco-2细胞的细胞腔。有趣的是，使用高分辨率电感耦合等离子体质谱（ICP-MS）观察到镉离子转运到分化单层的基底外侧。在Caco-2细胞中，短期和长期孵育后均未检测到膜损伤。另一方面，观察到未分化细胞暴露后量子点的细胞内定位，且量子点部分位于溶酶体内。
在代表小肠肠上皮细胞模型的分化Caco-2单层中，使用显微镜分析和图像处理未检测到氨基和羧基功能化的CdSe/ZnS量子点穿透细胞腔。相比之下，使用ICP-MS可以检测到镉离子转运到基底外侧。然而，即使长期孵育后，细胞单层的完整性也未受损，且未检测到细胞毒性作用。在未分化的Caco-2细胞中，细胞腔内可发现两种量子点修饰。在这些细胞中，量子点仅部分定位于内体或溶酶体中。结果表明，Caco-2细胞的分化状态是使用Caco-2细胞进行内化和定位研究的重要因素。此外，显微镜分析和ICP-MS等灵敏检测技术的结合对于研究含镉量子点与细胞的相互作用是必要的。
