Doxorubicin physical state in solution and inside liposomes loaded via a pH gradient

We have examined doxorubicin's (DOX) physical state in solution and inside EPC/cholesterol liposomes that were loaded via a transmembrane pH gradient. Using cryogenic electron microscopy (cryo-EM) we noted that DOX loaded to 200-300 mM internal concentrations in citrate containing liposomes formed l inear, curved, and circular bundles of fibers with no significant interacti on/perturbation of the vesicle membrane. The individual DOX fibers are puta tively comprised of stacked DOX molecules. From end-on views of bundles of fibers it appeared that they are aligned longitudinally in a hexagonal arra y with a separation between fibers of approx. 3-3.5 nm. Two distinct small angle X-ray diffraction patterns (oblique and simple hexagonal) were observ ed for DOX-citrate fiber aggregates that had been concentrated from solutio n at either pH 4 or 5. The doxorubicin fibers were also present in citrate liposomes loaded with only one-tenth the amount of doxorubicin used above ( approx. 20 mM internal DOX concentration) indicating that the threshold con centration at which these structures form is relatively low. In fact, from cryo-EM and circular dichroism spectra, we estimate that the DOX-citrate fi ber bundles can account for the vast majority (> 99%) of DOX loaded via a p H gradient into citrate buffered liposomes. DOX loaded into liposomes conta ining lactobionic acid (LBA), a monoanionic buffer to control the internal pH, remained disaggregated at internal DOX concentrations of approx. 20 mM but formed uncondensed fibers (no bundles) when the internal DOX concentrat ion was approx. 200 mM. This finding suggests that in the citrate containin g liposomes the citrate multianion electrostatically bridged adjacent fiber s to form the observed bundles. C-13-NMR measurements of [1,5-C-13]citrate inside liposomes suggested that citrate 'bound' to the DOX complex and 'fre e' citrate rapidly exchange indicating that the citrate-DOX interaction is quite dynamic. DOX release into buffer was relatively slow (< 4% at 1 h) fr om liposomes containing DOX fibers (in citrate loaded to a low or high DOX concentration or in LBA liposomes loaded to a high internal DOX concentrati on). LBA containing liposomes loaded with disaggregated DOX, where the inte rnal DOX concentration was only approx. 20 mM, experienced an osmotic stres s induced vesicle rupture with as much as 18% DOX leakage in less than 10 m in. The possible implications for this in vivo are discussed. (C) 1998 Else vier Science B.V. All rights reserved.