Uptake and binding of liposomal 2',3'-dideoxycytidine by RAW 264.7 cells : a three-step process

It was recently reported that the sequestration of virus by macrophages in reticuloendothelial system organs, such as lymph nodes, is possibly responsible for the clinical latency of disease in asymptomatic HIV-infected patients. Since macrophages may sequester HIV after phagocytosis, and because phagocytosis is a specialized function of any mammalian macrophage, a mouse-macrophage cell line (RAW 264.7) was used as a macrophage model to evaluate the uptake and binding of 2',3'-dideoxycytidine (ddC) encapsulated in liposomes of an average size of 300 nm containing 350 mumols 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 liposomes. Under these conditions, we found an apparent Michaelis-Menten constant (Km) of 40 microM and an initial velocity of 0.12 nmol ddC/mg protein/min for the saturable component and a constant rate of accumulation (KN) of 0.017/min for the nonsaturable component. The inhibition of uptake of ddC from L-ddC in the presence of phagocytosis inhibitors (deoxyglucose plus sodium azide) and nucleoside transport inhibitors (dipyridamole or nitrobenzylthioinosine) also confirmed the existence of several mechanisms in the liposome-mediated accumulation process of ddC into macrophages. Furthermore, studies of efflux of ddC in drug-free medium from cells preloaded with L-ddC or ddC established longer retention of ddC in cells preloaded with L-ddC than with ddC.