Complexation of Internalized Doxorubicin into Fiber Bundles Affects its Release Rate from Liposomes

Doxorubicin loads quite efficiently into preformed citrate containing liposomes (<200 nm) in response to an inside-acidic pH gradient: >95% loading for drug/lipid as high as 1:1 w/w. In fact, doxorubicin's loading was found to exceed theoretical predictions because it forms an insoluble complex with citrate. In this report, we investigated to what extent doxorubicin's interaction with citrate affected its release from these ΔpH-loaded liposomes. Doxorubicin leak rates were found to decrease significantly as doxorubicin concentration (i.e., drug/lipid) was increased. While the solubility limit of doxorubicin in 300 mM citrate (pH 4) was found to be approximately 1.4 mM, the internal doxorubicin concentration above which its leak rate started to decline was estimated to be –26 mM. As doxorubicin loads into liposomes at low concentrations it begins to stack into fibers and as the concentration continues to increase the fibers, crosslinked by citrate, pack into bundles. We found from circular dichroism (CD) spectroscopy and electron cryo-micros-copy (ECM) that the concentration of –26 mM was the value above which predominantly bundles existed. One explanation why bundling might reduce leak rate is that doxorubicin located in interior fibers is unavailable and the fraction of doxorubicin in this population increases as the number of fibers per bundle (concentration) increases. By modeling the expected flux as a function of the number of fibers per bundle with only 'surface' doxorubicin available for exchange, we noted a trend that could account only in part for the experimental results indicating that other factors are involved and that doxorubicin exchange from outer fibers might also be restricted. Although the exact mechanistic details remain unclear, the crosslinking of stacked doxorubicin libers by citrate was found to be rate limiting to doxorubicin's release and thus likely accounts for much of the reduction in toxicity afforded this liposomal formulation and its clinical success.