Red blood cells (RBCs) are natural carriers which can be used for targeted drug delivery. Conditions during loading and surface modification are essential for carrier-RBC preparation for specifically targeted drug delivery. Therefore, human RBCs were loaded with albumin and magnetic nanoparticles (NPs) by different hypotonic haemolysis procedures and compared based on loading efficiency and membrane damage. Samples were analysed by flow cytometry and confocal microscopy. The optimized loading procedure resulted in 90% albumin-loaded carrier-RBCs with < 4% Annexin V binding and 263 pg iron per RBC after loading with iron oxide NPs. Albumin-loaded RBCs were subsequently surface conjugated with insulin and IgG via biotin-streptavidin. Insulin-conjugated carrier-RBCs were observed to attach and to be internalized by cultured endothelial cells. Uptake was not observed for carrier-RBCs non-specifically modified with IgG. Attachment of other peptides with high specificity will open novel opportunities for targeting various cells, tissues and for crossing biological barriers.