In vitro binding and erythrophagocytosis of RGD-modified erythrocytes by endothelial cells. (A) Structural formula of cyclic RGD-peptide coupled to erythrocyte transmembrane proteins via a sulfoEMCS heterobifunctional cross-linker. (B) SDS-PAGE gel (i) and anti-RGD antiserum-stained immunoblot (ii) of control untreated erythrocytes (lane 2), erythrocytes with sulfoEMCS linker (lane 3), erythrocytes with RGD (lane 4), and erythrocytes with sulfoEMCS linker and RGD (lane 5) lysates. The sizes of the bands of the PageRuler protein ladder (lane 1) are shown next to the SDS-PAGE gel. The corresponding immunoblot shows association of RGD-peptides with multiple transmembrane proteins only for the erythrocytes that contained both the sulfoEMCS linker and the RGD-peptides. (C) Scanning electron microscopy image of extensively bound RGD-erythrocytes to the endothelial cell membrane after 1-hour incubation. Scale bar represents 10 μm. (D) Retracted endothelial cell with bound RGD-erythrocytes, showing extensive blebbing as a sign of cytotoxicity that can already be observed after 30 minutes. Scale bar represents 5 μm. (E) Confocal microscopy image of autofluorescence of RGD-erythrocytes taken up by an endothelial cell after 4-hour incubation, demonstrating that cell binding is followed by erythrophagocytosis. Scale bar represents 10 μm. (F) Transmission electron microscopy (TEM) image of phagocytosed RGD-erythrocytes () in an endothelial cell after 1-hour incubation. Scale bar represents 2 μm. (G) Endothelial cell association of RAD- and RGD-modified erythrocytes. RGD-erythrocytes displayed enhanced binding at all time points compared with control RAD-erythrocytes (values ± SEM). Inserts show representative light microscopy images of erythrocyte-association at 4-hour incubation.