Methotrexate loading of red cell carriers by osmotic stress and electric-pulse methods: ultrastructural observations

Transmission and scanning electron microscopy (EM) of osmotically stressed and electrically pulsed human erythrocyte carriers indicated that the mode of uptake of methotrexate (MTX) varied depending on the method of carrier production. The preswell and electroporation loading methods, apparently by facilitating passive diffusion across the cell membrane, promoted incorporation of the MTX directly into the cytoplasm of the carriers, as evidenced by their variable electron densities. The preswell method produced carriers displaying leptocytic characteristics, whereas the electroporation method produced carriers exhibiting sphero- and stomatocytic transformation. Hypotonic dialysis-prepared carriers took up MTX primarily by endocytosis and secondarily by passive diffusion. Endocytotic activity was not induced by the MTX. Scanning EM revealed that most of the dialysis-prepared carriers exhibited prominent invaginations of the cell surface. Transmission EM of serial thin sections through these carriers revealed numerous fully internalized, membrane-bound vesicles. Endocytotic activity caused progressive loss of membrane and resulted in the sphero- and stomatocytic transformation of the carriers.