Cellular entry fashion of hollow milk protein spheres

Cellular uptake of hollow casein nanospheres was investigated by confocal microscopy, flow cytometry and transmission electron microscopy. It was found that the casein spheres could enter cellsvia a temperature- or energy-independent mechanism. Confocal microscopy and flow cytometry showed that the cellular uptake amount and velocity of the casein spheres at 4 °C were almost the same as those at 37 °C. Moreover, cellular uptake of casein spheres could not be affected by endocytosis inhibitors, suggesting that a large fraction of casein spheres enter cells in a non-endocytosis fashion. In addition, real-time confocal laser scanning microscopy observation found that the casein spheres first absorbed onto the cell membrane, and then were internalized into the cells mainly through an endocytosis-independent mechanism. Transmission electron microscopy analysis also demonstrated that the casein spheres internalized by cells were mainly distributed in the cytoplasm either at 37 or 4 °C. These findings extend the current understanding of the interactions between milk proteins and biologic systems, and offer a drug nanocarrier which itself has an extraordinary capability to penetrate cell barriers in an energy-independent fashion.