UPTAKE AND BINDING OF LIPOSOMAL 2',3'-DIDEOXYCYTIDINE BY RAW-264.7 CELLS - A 3-STEP PROCESS

It was recently reported that the sequestration of virus by macrophage s in reticuloendothelial system organs, such as lymph nodes, is possib ly responsible for the clinical latency of disease in asymptomatic HIV -infected patients. Since macrophages may sequester HIV after phagocyt osis, and because phagocytosis is a specialized function of any mammal ian macrophage, a mouse-macrophage cell line (RAW 264.7) was used as a macrophage model to evaluate the uptake and binding of 2',3'-dideoxyc ytidine (ddC) encapsulated in liposomes of an average size of 300 nm c ontaining 350 mu mol of ddC per mmol of lipids. Liposomal ddC (L-ddC) was rapidly taken up by macrophages. In contrast, its free form (ddC) accumulated slowly in these cells. The accumulation of ddC from L-ddC into cells seemed to consist of two components: a saturable one, which fitted with the Michaelis-Menten model, and a nonsaturable one, which proceeded linearly in the presence of an excess amount of unlabeled l iposomes. Under these conditions, we found an apparent Michaelis-Mente n constant (K-m) of 40 mu M and an initial velocity of 0.12 nmol ddC/m g protein/min for the saturable component and a constant rate of accum ulation (K-N) of 0.017/min for the nonsaturable component. The inhibit ion of uptake of ddC from L-ddC in the presence of phagocytosis inhibi tors (deoxyglucose plus sodium azide) and nucleoside transport inhibit ors (dipyridamole or nitrobenzylthioinosine) also confirmed the existe nce of several mechanisms in the liposome-mediated accumulation proces s of ddC into macrophages. Furthermore, studies of efflux of ddC in dr ug-free medium from cells preloaded with L-ddC or ddC established long er retention of ddC in cells preloaded with L-ddC than with ddC. These results clearly show that the enhancement of accumulation of 2',3'-di deoxycytidine in macrophages by liposomes proceeds by more than one pr ocess and also suggest that liposome encapsulation enhances the retent ion of 2',3'-dideoxycytidine into macrophages, which have been known t o be long-term reservoirs for dissemination of human immunodeficiency virus to other cell types. A three-step process by which ddC from L-dd C could accumulate in cells is proposed.