Study of uptake mechanisms of halloysite nanotubes in different cell lines

Purpose: Halloysite nanotubes (HNTs) are a natural aluminosilicate clay with a chemical formula of Al2Si2O5(OH)4×nH2O 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. Methods: 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. Results: 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. Conclusion: 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