Cellular Phospholipid - Accumulation Induced by Basic Drugs does not depend on Phospholipid Uptake nor Neutralization of Acidic Vesicles

Objectives : Drug induced-phospholipidosis is a pathological state caused by repeated administration of cationic amphiphilic drugs. The mechanism of drug-induced phospholipidosis is unclear. Several hypotheses have been proposed, such as inhibition of phospholipase, upregulated phospholipid synthesis, increased uptake of phospholipids, and inhibition of intracellular phospholipid traffic. Among them, two hypotheses for the mechanism were tested here: i) excessive cellular uptake of phospholipids and ii) neutralization of intralysosomal pH by drug accumulation. Methods: First, RAW264 cells were treated with inhibitors of phospholipid uptake pathways, scavenger receptor-A, LOX receptor, CD36, and pinocytosis, as well as phospholipidosis-inducing drugs. The phospholipid content in the cells were monitored by fluorescence from DiI lipophilic tracer using a flow cytometer. Second, liposomes with an acidic water phase were suspended in drug-containing solutions at neutral pH, and the accumulation of the drug into liposomes was quantitatively measured by high performance liquid chromatography/mass spectrometry. Additionally, the pH of inner phase of the liposomes were measured using fluorescent probe Lysosensor green DND-189. Results: Phospholipid content in the cells increased by treatment of phospholipidosis-inducing drugs. Increased cellular phospholipid accumulation did not decrease following treatment with the pathway inhibitors. Regardless of their phospholipidosis-inducing potential, drugs characterized by a high pKa value effectively accumulated in liposomes. This accumulation caused an upward shift of the interior pH of liposomes, which was independent of phospholipidosis-inducing potential of drugs. Conclusions : The results suggest that cellular phospholipid-accumulation deos not likely depend on increased uptake via these pathways, nor inhibition of phospholipid transport due to neutralization of acidic organelles.