REGULATION OF THE COMPLEMENT-MEDIATED ELIMINATION OF RED-BLOOD-CELLS MODIFIED WITH BIOTIN AND STREPTAVIDIN

Red blood cells (RBC) modified with biotin and streptavidin (SA) prese nt an interesting potential drug delivery system. Biotinylation and SA attachment, however, alter the biocompatibility of RBC. We have repor ted that polyvalent SA attachment induces lysis of biotinylated RBC (b -RBC) by homologous complement via the alternative pathway. Lysis occu rs due to inactivation of the membrane regulators of complement, DAF a nd CD59, cross-linked by SA. However, monovalent SA attachment does no t induce lysis. On the basis of these findings we hypothesized that re duction of the biotin surface density on b-RBC would allow for monoval ent SA attachment to b-RBC and that such SA/b-RBC should then be stabl e in the circulation. In the present work we injected into rats severa l different radiolabeled RBC probes: rat RBC biotinylated to varying d egrees (b(n)-RBC, where b(n) represents the input micromolar concentra tion of biotinylating agent), as well as SA/b(n)-RBC. Extensively biot inylated rat RBC (b(700)-RBC, stable in serum in vitro) were rapidly c leared from the bloodstream. We further found that extensively biotiny lated human b(1000)-RBC bound C3b from serum in vitro without detectab le lysis, and that rat b(700)-RBC bound to isolated macrophages in a c omplement-dependent fashion. Therefore, nonlytic C3b fixation and upta ke of C3b-carrying b(700)-RBC by macrophages appears to be the mechani sm leading to clearance of b(700)-RBC in vivo. Moderately biotinylated RBC (b(70)-RBC and b(240)-RBC) were stable in serum in vitro. SA atta chment to b(240)-RBC led to their rapid lysis in serum in vitro, lysis in the bloodstream, and clearance by the Liver and spleen. SA attachm ent to b(70)-RBC led to fast elimination of SA/b(70)-RBC from the bloo dstream, while in vitro SA/b(70)-RBC were stable in serum. Modestly bi otinylated RBC (b(23)-RBC) demonstrated only marginally decreased 60-m in survival in the bloodstream regardless of SA attachment. Our in vit ro studies indicate that b(23)-RBC bound approximately 10(5) SA molecu les per cell, and the resulting SA/b(23)-RBC bound 5 x 10(4) molecules of biotinylated IgG (b-IgG) per cell. About 60% of the injected dose of b-IgG/SA/b(23)-RBC labeled with Cr-51 was detected in the rat blood cells 1 day after iv injection. To assess whether b-IgG/SA/b(23)-RBC circulate in the bloodstream as a stable complex, we have injected I-1 25-labeled b-IgG/ SA/Cr-51-labeled b(23)-RBC in rats. Up to 60 min aft er injection, both radiolabels display similar level in bloodstream. U p to 3 h after injection, about 70% of I-125 was detected in the blood cells. In contrast, 100% of I-125 was detected in plasma after inject ion of nonconjugated I-125-labeled b-IgG. Thus, major portion of SA/b( 23)-RBC-attached b-IgG circulates as a complex with RBC. About 30% of RBC-bound b-IgG undergoes detachment from the carrier b-RBC, probably in the pulmonary capillaries, because lung level of I-125 was twice as high as that of Cr-51. Therefore, the surface density of biotin on th e b-RBC membrane appears to play a key role in regulating complement-m ediated clearance of b(n)-RBC and SA/b(n)-RBC from the bloodstream. Mo dest biotinylation generates b-IgG/SA/b(23)-RBC circulating for severa l hours as stable immunoerythrocytes without detectable lysis or marke d elimination, and it may be possible to use these RBC in a drug deliv ery system. (C) 1996 Academic Press, Inc.