Institute for Innovation and Biomedical Research (IRIB), CNR, Palermo, 90146, Italy.
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), Sect. Chemistry, University of Palermo, Palermo, 90128, Italy.
Int J Nanomedicine. 2021 Jul 12;16:4755-4768. doi: 10.2147/IJN.S303816. eCollection 2021.
Halloysite nanotubes (HNTs) are a natural aluminosilicate clay with a chemical formula of AlSiO(OH)×nHO and a hollow tubular structure. Due to their peculiar structure, HNTs can play an important role as a drug carrier system. Currently, the mechanism by which HNTs are internalized into living cells, and what is the transport pathway, is still unclear. Therefore, this study aimed at establishing the in vitro mechanism by which halloysite nanotubes could be internalized, using phagocytic and non-phagocytic cell lines as models.
The HNT/CURBO hybrid system, where a fluorescent probe (CURBO) is confined in the HNT lumen, has been used as a model to study the transport pathway mechanisms of HNTs. The cytocompatibility of HNT/CURBO on cell lines model was investigated by MTS assay. In order to identify the internalization pathway involved in the cellular uptake, we performed various endocytosis-inhibiting studies, and we used fluorescence microscopy to verify the nanomaterial internalization by cells. We evaluated the haemolytic effect of HNT/CURBO placed in contact with human red blood cells (HRBCs), by reading the absorbance value of the supernatant at 570 nm.
The HNT/CURBO is highly biocompatible and does not have an appreciable haemolytic effect. The results of the inhibition tests have shown that the internalization process of nanotubes occurs in an energy-dependent manner in both the investigated cell lines, although they have different characteristics. In particular, in non-phagocytic cells, clathrin-dependent and independent endocytosis are involved. In phagocytic cells, in addition to phagocytosis and clathrin-dependent endocytosis, microtubules also participate in the halloysite cellular trafficking. Upon internalization by cells, HNT/CURBO is localized in the cytoplasmic area, particularly in the perinuclear region.
Understanding the cellular transport pathways of HNTs can help in the rational design of novel drug delivery systems and can be of great value for their applications in biotechnology.
埃洛石纳米管（HNTs）是一种天然的铝硅酸盐粘土，化学式为 AlSiO(OH)×nHO，具有中空管状结构。由于其特殊的结构，HNTs 可以作为药物载体系统发挥重要作用。目前，HNTs 进入活细胞的机制以及运输途径尚不清楚。因此，本研究旨在建立 HNTs 内化的体外机制，以吞噬和非吞噬细胞系为模型。
使用荧光探针（CURBO）被限制在 HNT 管腔中的 HNT/CURBO 混合系统作为模型，研究 HNTs 运输途径机制。通过 MTS 测定法研究 HNT/CURBO 对细胞系模型的细胞相容性。为了确定涉及细胞摄取的内化途径，我们进行了各种内吞作用抑制研究，并使用荧光显微镜验证细胞内纳米材料的内化。我们通过读取 570nm 处上清液的吸光度值来评估与人类红细胞（HRBC）接触的 HNT/CURBO 的溶血作用。
HNT/CURBO 具有高度的生物相容性，并且没有明显的溶血作用。抑制试验的结果表明，两种细胞系中的纳米管内化过程都是能量依赖的，尽管它们具有不同的特征。特别是在非吞噬细胞中，涉及网格蛋白依赖性和非依赖性内吞作用。在吞噬细胞中，除了吞噬作用和网格蛋白依赖性内吞作用外，微管也参与了埃洛石的细胞运输。进入细胞后，HNT/CURBO 定位于细胞质区域，特别是核周区域。
了解 HNTs 的细胞转运途径有助于合理设计新型药物传递系统，并为其在生物技术中的应用提供重要价值。
