Erythrocyte Exposure to Silica Nanoparticles

The erythrocytes were prepared using a previously described method [36 (link),68 (link)]. Briefly, collected rat blood was mixed by gentle inversion of the tube and centrifuged at 1200× g for 15 min. The plasma supernatant was discarded, and the erythrocytes were washed 4 times by suspending them in 0.9% NaCl followed by centrifugation at 1200× g for 10 min. The final suspension consisted of 5% by volume of erythrocyte in saline. Flat bottom 12 well plates were used to incubate 900 µL of erythrocyte with 100 µL of SiNPs (0.2, 1, 5, and 25 µg/mL). The plate was positioned in an MS 3 digital microtiter shaker (IKA WER KE GmbH & CO, Staufen, Germany) and rotated at 300 rpm for 30 min at room temperature, under shaded light. After incubation, the samples were transferred to a 1.5 mL Eppendorf tube and were centrifuged at 1200× g for 5 min. The resulting supernatant was collected for hemolysis assay, oxidative markers assays, intracellular calcium, Annexin V binding, ATPase activity, and calpain activity. The deposited erythrocytes were fixed with Karnovsky’s fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer at 7.2 pH) for transmission electron microscopy (TEM). The deformed erythrocytes were quantitated and expressed as the number of RBCs per field at a magnification of 11,100.