CELLULAR RETENTION OF LIPOSOME-DELIVERED ANIONIC COMPOUNDS MODULATED BY A PROBENECID-SENSITIVE ANION TRANSPORTER

Purpose. Drug carriers such as liposomes may enhance the intracellular delivery of therapeutic agents for infectious or neoplastic diseases. However, the mechanisms affecting cellular retention of liposome cont ents are understood poorly. We tested the hypothesis that retention of anionic compounds may be modulated by a nonspecific probenecid-sensit ive anion transport mechanism, and that liposome composition may deter mine the impact of such transporters on drug retention by cells. Metho ds. The fluorescent anionic dye hydroxy-pyrene-[1,3,6]-trisulfonate (H PTS) was transferred to the cytoplasm of cultured CV-I or J774 cells b y direct needle-microinjection or by ATP-induced permeabilization of t he plasma membrane, respectively, to investigate whether the cells hav e anion transport mechanisms capable of extruding HPTS from the cytopl asm. Cellular retention of dye was monitored in the presence and absen ce of the anion transport inhibitors probenecid or sulfinpyrazone. Lip osomes containing HPTS were co-labeled with tetramethylrhodamine-label ed phosphatidylethanolamine (Rho-PE) as a marker of liposome membrane fate, and uptake was investigated using 5774 cells. Results. Needle-in jected HPTS underwent both sequestration in early endocytic vesicles a nd rapid extrusion into the extracellular medium. Probenecid or sulfin pyrazone reduced the extrusion of HPTS. Thus HPTS is a substrate for a probenecid-sensitive anion transporter in 5774 and CV1 cells. After d elivery via fluid liposomes composed of osphatidylglycerol:phosphatidy ldholine:cholesterol (3:7:5 mole ratio) and co-labeled with Rho-PE, ce ll-associated HPTS declined more rapidly than did Rho-PE. Exposure of cells to 5 mM probenecid doubled the quantity of HPTS retained by cell s, without changing the retention of the Rho-PE membrane marker. In co ntrast, the effect of probenecid was negligible when gel-phase liposom es of distearoylphosphatidyl-glycerol:cholesterol (10:5 mole ratio) we re used. Conclusions. Probenecid-sensitive nonspecific anion transport ers can mediate the extrusion of model anions delivered via liposomes. However, liposome composition modulates the amount of material subjec t to extrusion from cells, possibly by altering the endocytic compartm ent in which liposomes release their contents.