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Enhancement of Adriamycin Antitumor Activity by Its Binding with an Intracellular Sustained-Release Form, Polymethacrylate Nanospheres, in U-937 Cells¹

Laboratoire de Pharmacie Clinique Oncologique [A. A., B. D., M. J. F., G. B.] and Departement d'Histologie [J. F.], Chu H. Mondor et Faculté de Medecine, Paris XII, 94010 Creteil, and Laboratoire de Pharmacie Galenique et Biopharmacie-UER du medicament, 35550 Rennes [A. R., R. L.] France

We investigated the antitumor activity of Adriamycin on a monocyticlike cancer cell line U-937 after its binding on polymethacrylate nanospheres (diameter, 270–350 nm). Compared to free Adriamycin (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 µg/ml. The 90% growth inhibition concentration was 0.051 µg/ml for F-ADR and was 0.018 µg/ml for B-ADR (P < 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 < 0.001). A comparable enhancement of activity regarding the nanosphere densities was observed after a 24-h exposure to 0.02 and 0.05 µg/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 µg/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 µg/mg protein x h^-1 (P < 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.

¹ Supported in part by grants from the Université Paris-Val de Marne and the Ville de Paris.

² To whom requests for reprints should be addressed, at Laboratoire de Toxicologie-Pharmacologie, Chu H. Mondor, 51 avenue du Marechal de Lattre de Tassigny, 94010 Creteil, France.

Received 3/13/87. Revised 11/ 3/87. Accepted 12/29/87.