Abstract
Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier because it can be naturally internalized into mammalian cells, due to its nano-size dimension. Therefore, deciphering the mechanisms of sepiolite cell internalization constitutes a question interesting biotechnology, for the use of sepiolite as nanocarrier, as well as environmental and public health concerns. Though it is low, the perfectly stable and natural intrinsic fluorescence of sepiolite nanofibers allows to follow their fate into cells by specifically sensitive technics. By combining fluorescence microscopy (including confocal analysis), time-lapse video microscopy, fluorescence activated cell sorting and transmission electron microscopy, we show that sepiolite can be spontaneously internalized into mammalian cells through both non-endocytic and endocytic pathways, macropinocytosis being one of the main pathways. Interestingly, exposure of the cells to endocytosis inhibitors, such as chloroquine, two-fold increase the efficiency of sepiolite-mediated gene transfer, in addition to the 100-fold increased resulting from sepiolite sonomechanical treatment. As sepiolite is able to bind various biological molecules, this nanoparticulate silicate could be a good candidate as a nanocarrier for simultaneous vectorization of diverse biological molecules.
Highlights
Sepiolite is a natural and abundant magnesium silicate belonging to the clay mineral family, which constitutes a potential promising nanocarrier for non-viral transfer of biomolecules
Using transmission electron microscopy (TEM) analysis, we have previously show that sepiolite fibers from Vallecas-Vicalvaro deposits near Madrid exhibit a mean width of 15 nm, and that 80% of fibers were between 200 and 400 nm long, with a maximal length of 800 nm[23]
TEM, time-lapse video-microscopy and fluorescence-activated cell sorting analyses, we show here that sepiolite can be spontaneously internalized and externalized into mammalian cells through both endocytic and non-endocytic pathways
Summary
Spontaneous internalization of sepiolite into mammalian cells. In this work was used a commercial sepiolite obtained from the Vallecas-Vicalvaro deposits near Madrid, Spain (see Experimental Section). 10 μM of chloroquine reduced the internalization of sepiolite into V79 cells by only 20%, whereas 100 μM amiloride caused a 50% reduction These data show that sepiolite internalization into cells more efficiently results from macropinocytosis, as shown with the TEM observations (see Fig. 4E,F). Under the present conditions, it is clear that chloroquine promotes endosomal escape more efficiently than it inhibits internalization, while the impact on internalization is moderate, consistently with the FACS analysis (see above)
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