Doxorubicin-loaded casein microspheres: protein nature of drug incorporation.

We have studied incorporation of [14C]doxorubicin within protease-sensitive casein microspheres both by 14C-activity, measuring total drug, and HPLC, measuring free drug only. It was found that total drug content (27.7 micrograms mg-1) exceeded free drug content (3.2 micrograms mg-1) suggesting that the major portion of doxorubicin was incorporated via a covalent linkage to matrix protein. In-vivo drug disposition and activity studies suggested that this fraction of doxorubicin was the major species within tumour tissue (total vs free: 5 min, 14.3 micrograms g-1 vs 0.7 micrograms g-1; 24 h, 11.7 micrograms g-1 vs 1.1 micrograms g-1; 48 h, 11.2 micrograms g-1 vs 1.2 micrograms g-1; 72 h, 10.0 micrograms g-1 vs 0.8 micrograms g-1), did not exhibit a 'burst' effect, was slowly cleared (30% loss over 3 days), and was equiactive (growth delay = 12 days) compared with drug in solution (growth delay = 10 days). This work clearly implicates in-vivo microsphere matrix biodegradation in drug release and subsequent disposition and activity.