Macrophages loaded with doxorubicin by ATP-mediated permeabilization: potential carriers for antitumor therapy

In a number of cell types extracellular ATP (ATPe) is known to cause reversible plasma membrane permeabilization to low molecular weight water soluble compounds (Mr<900 D). In the present report we have exploited this technique to charge with the anticancer drug doxorubicin the cytoplasm of J774 and L929 cells. These two mouse cell lines were previously characterized for the presence of the ATPe-gated pore, J774 macrophages and tumor necrosis factor (TNF)-resistant L929 fibroblasts. ATPe-mediated reversible permeabilization allowed an at least a fourfold increase of doxorubicin intracellular trapping with respect to passively loaded cells (2 pg/cell versus 0.5 pg/cell respectively). The analysis of the doxorubicin release kinetic at 37°C from loaded cells showed that about 40% of total intracellular doxorubicin was released during the first hour from both ATPe-permeabilized and passively-loaded cells; little further release (of less than 10% of the total loaded doxorubicin) was observed during the following 4 hours. It is to be underlined that although the doxorubicin relative release profiles were, in both cases similar, the absolute doxorubicin release was higher in the case of ATPe-permeabilized respect to passively-loaded cells. ATPe-permeabilized, doxorubicin-loaded cells strongly inhibited proliferation of K562 tumor cells in co-culture experiments. Taken together these results indicate that ATPe-mediated reversible plasma membrane permeabilization can be efficiently employed to load cells of different histotypes with high concentrations of antitumor agents. This drug delivery approach could be proposed as a new strategy for the experimental treatment of neoplastic diseases, delivering high doses of drug to tumor sites, limiting the cytotoxic effects towards normal tissues