We investigated the antitumor activity of Adriamycin on a monocytic-like cancer cell line U-937 after its binding on polymethacrylate nanospheres (diameter, 270-350 nm). Compared to free Adramycin (F-ADR), nanosphere-bound Adriamycin (B-ADR) exhibits a 3-fold enhancement of cytotoxicity, as determined by cell growth inhibition and DNA synthesis, after continuous exposure to 0.02 and 0.04 microgram/ml. The 90% growth inhibition concentration was 0.051 microgram/ml for F-ADR and was 0.018 microgram/ml for B-ADR (P less than 0.001). Furthermore, the nanosphere densities per cell play an important role since for the same drug concentration the higher the density increases, the better the activity is. Indeed, after 4 days of incubation in a medium containing 160 nanospheres at 0.5 fg/cell, the cell counts were 62.8 +/- 12.8% (SD) of the initial inoculum and they were only 16.1 +/- 0.1% after incubation in a medium containing 800 nanospheres at 0.1 fg/cell (P less than 0.001). A comparable enhancement of activity regarding the nanosphere densities was observed after a 24-h exposure to 0.02 and 0.05 microgram/ml. Short-term uptake studies showed that B-ADR accumulation was higher with B-ADR than with F-ADR. In addition, the efflux kinetics was modified. For cells exposed to F-ADR for 4 h, the efflux half-life was 23.7 +/- 7.7 h and the area to infinity under the efflux curve was 8.6 +/- 2.8 micrograms/mg protein x h-1. For cells exposed to B-ADR, the efflux half-life increased to 85.9 +/- 19.2 h and the area to infinity under the efflux curve to 29.6 +/- 6.6 micrograms/mg protein x h-1 (P less than 0.001). Electron transmission microscopy and previous findings have revealed that B-ADR was well internalized into cells. Our data support the hypothesis that B-ADR acts as an intracellular drug release complex after endocytosis. The findings regarding the number of nanospheres per cell and dose-effect relationships are consistent with mechanisms of drug actions extending to membrane domains.