ENZYME THERAPY .14. COMPARISON OF METHODS FOR ENZYME ENTRAPMENT IN HUMAN-ERYTHROCYTES

Abstract

Seven methods of human erythrocyte entrapment, 6 by hypotonic exchange loading and 1 by chlorpromazine-induced endocytosis, were evaluated for efficiency of incorporation of .beta.-glucuronidase, inulin and glucose; presence of .beta.-glucuronidase on the erythrocyte surface, as detected by hemagglutination or complement-dependent lysis in the presence of antibody to .beta.-glucuronidase; in vitro leakage of entrapped markers; and morphologic alterations by scanning electron microscopy. Dependent on the method of entrapment, the incorporation of markers ranged from 0.7-6.2% for .beta.-glucuronidase, 3.4-31% for inulin and 1.2-12.2% for glucose. Maximal incorporation by hypotonic exchange loading occurred when the initial concentration of NaCl (150 mM) was reduced to 50 or 75 mM. .beta.-Glucuronidase was detected on the erythrocyte surface by hemagglutination for these methods and a dialysis method of loading. Essentially no leakage of entrapped enzyme was detected (< 1%) for all methods, but up to 11% of entrapped glucose was released during a 3 h incubation at 37.degree. C in buffered whole blood. Finally, entrapment methods requiring the greatest reduction in salt concentration caused the formation of echinocytes, but stomatocytes were observed after entrapment by methods requiring lesser salt dilutions. The efficiency of entrapment and relative integrity of erythrocytes after various loading procedures are demonstrated; in vitro assessment may provide a useful predictor of the immunogenicity and in vivo fate of erythrocyte-entrapped enzymes or other therapeutic agents.