ACCUMULATION OF DOXORUBICIN AND OTHER LIPOPHILIC AMINES INTO LARGE UNILAMELLAR VESICLES IN RESPONSE TO TRANSMEMBRANE PH GRADIENTS

The uptake of the anticancer agent doxorubicin into large unilamellar vesicles (LUVs) exhibiting a transmembrane pH gradient (inside acidic) has been investigated using both kinetic and equilibrium approaches. It is shown that doxorubicin uptake into the vesicles proceeds via per meation of the neutral form and that uptake of the drug into LUVs with an acidic interior is associated with high activation energies (E(a)) which are markedly sensitive to lipid composition. Doxorubicin uptake into egg-yolk phosphatidylcholine (EPC) LUVs exhibited an activation energy of 28 kcal/mol, whereas for uptake into EPC/cholesterol (55:45, mol/mol) LUVs E(a) = 38 kcal/mol. The equilibrium uptake results obta ined are analyzed in terms of a model which includes the buffering cap acity of the interior medium and the effects of drug partitioning into the interior monolayer. From the equilibrium uptake behaviour, a doxo rubicin partition coefficient of 70 can be estimated for EPC/cholester ol bilayers. For a 100 nm diameter LUV, this indicates that more than 95% of encapsulated doxorubicin is partitioned into the inner monolaye r, presumably located at the lipid/water interface. This is consistent with C-13-NMR behaviour as a large proportion of the drug appears mem brane associated after accumulation as reflected by a broadening beyon d detection of the C-13-NMR spectrum. The equilibrium accumulation beh aviour of a variety of other lipophilic amines is also examined in ter ms of the partitioning model.