AN EFFICIENT METHOD FOR INTRODUCING MACROMOLECULES INTO LIVING CELLS

The hemagglutinin (HA) of influenza virus was used to obtain efficent and rapid bulk delivery of antibodies and horseradish peroxidase (HRP) into the cytoplasm of living tissue culture cells. By exploiting HA efficient cell surface expression, its high affinity for erythrocytes and its acid-dependent membrane fusion activity, a novel delivery method was developed. The approach is unique in that mediator of both binding and fusion (the HA) is present on the surfaces of the target cells. A recently developed 3T3 cell line which permanently expresses HA, Madin-Darby canine kidney cells infected with influenza virus, and CV-1 cells infected with a SV40 vector carrying the HA gene were used as recipient cells. Protein-loaded erythrocytes were bound to the HA on the cell surface and a brief drop in pH to 5.0 was used to trigger HA fusion activity and hence delivery. About 3-8 erythrocytes fused per 3T3 and CV-1 cell, respectively, and 75-95% of the cells received IgG or HRP. Quantitative analysis showed that 1.8 .times. 108 molecules of HRP and 1.4 .times. 107 IgG molecules were delivered per CV-1 cell and 6.2 .times. 107 HRP molecules per 3T3 cell. Cell viability, as judged by Met incorporation into protein and cell growth and division, was not impaired. EM and fluorescence microscopy showed that the fused erythrocyte membranes remained as discrete domains in the cell''s plasma membrane. The method is simple, reliable and nonlytic. The ability to simultaneously and rapidly deliver impermeable substances into large numbers of cells will permit biochemical analysis of the fate and effect of a variety of delivered molecules.