Molecular and Cellular Biochemistry 144: 53-59, 1995. 9 1994 Kluwer Academic Publishers. Printed in the Netherlands. Modulated red blood cell survival by membrane protein clustering Laura Chiarantini, Luigia Rossi, Alessandra Fraternale and Mauro Magnani Institute of Biochemistry 'Giorgio Fornaini ', University of Urbino, Urbino, (PS) Italy Received 28 July 1994; accepted 31 October 1994 Abstract Human and murine blood cells treated with ZnC12 and bis(sulfosuccinimidyl)suberate (BS 3) (a cross linking agent) undergo band 3 clustering and binding of hemoglobin to red blood cell membrane proteins. These clusters induce autologous IgG bind- ing and complement fixation, thus favouring the phagocytosis of ZnCIJBS 3 treated cells by macrophages. The extension of red blood cell opsonization can be easily modulated by changing the ZnC12 concentration in the 0.1-1.0 mM range thus pro- viding an effective way to affect blood cell recognition by macrophages. In fact, murine erythrocytes treated with increasing ZnC12 concentrations have proportionally reduced survivals when reinjected into the animal. Furthermore, the organ seques- tration of ZnClz/BS3 treated cells strongly resembles the typical distribution of the senescent cells. Since the ZnCIz/BS3 treat- ment can also be performed on red blood cells loaded with drugs or other substances, this procedure is an effective drug-targeting system to be used for the delivery of molecules to peritoneal, liver and spleen macrophages. (Mol Cell Bioehem 144: 53-59, 1995) Key words: band 3 clustering, red blood cell survival, IgG binding, drug delivery system Introduction An antigenic site that appears on the surface of aged or dam- aged erythrocytes (known as 'senescent antigen') leads to autologous IgG binding and promotes the removal of senes- cent cells by macrophages [1]. The nature of the 'senescent antigen' has not yet been clarified but several reports sug- gest that it can be derived by band 3 proteolytic degradation [2], dimerization [3], polymerization [4] or deglycosylation [5]. In vitro it has been shown that several agents able to in- duce band 3 clustering also promote autologous IgG bind- ing [6, 7] and complement deposition [8] which in turn promote phagocytosis of treated cells. ZnC12 is one of those agents that induces the reversible clusters of integral mem- brane proteins. Addition of cross-linking agents (i.e. BS 3) stabilizes the clusters and make these irreversible. We hypoth- esized that if the amount of band 3 clusters and IgG binding can be controlled in vitro, this procedure could be useful for modifying the in vivo survival of the red blood cell (RBC). This procedure could be helpful not only to understand the base of senescent cell recognition by macrophages but also to target drug-loaded red blood cells to macrophages for spe- cific therapeutic purposes. The duration of action of an intravenously administered drug is often very short because the drug is rapidly distrib- uted to the tissue and immediately available for elimination. In theory, a slow released intravenous preparation would overcome this disadvantage. Red blood cells can be easily transformed into carrier cells. When erythrocytes are treated by hypo-osmotic dialysis, they react by swelling. Although red cells are leaky, it is possible to load them with drugs or enzymes before resealing the membrane holes restoring the iso-osmotic conditions. Resealed erythrocytes are heteroge- neous in size and hemoglobin content but can function and circulate normally even if they are injected intraperitoneally [9, 10]. In our laboratory we have evaluated the possible use of erythrocytes as carriers of drugs [11] or enzymes [12]. Encapsulation in carrier erythrocytes has been proposed to Address for offprints: L. Chiarantini, Institute of Biochemistry 'Giorgio Fomaini', Via Saffi, 2 61029 Urbino (PS) Italy