title: Formation of transition metal-doxorubicin complexes inside liposomes.
abstract: Doxorubicin complexation with the transition metal manganese (Mn(2+)) has been characterized, differentiating between the formation of a doxorubicin-metal complex and doxorubicin fibrous-bundle aggregates typically generated following ion gradient-based loading procedures that rely on liposome encapsulated citrate or sulfate salts. The physical and chemical characteristics of the encapsulated drug were assessed using cryo-electron microscopy, circular dichroism (CD) and absorbance spectrophotometric analysis. In addition, in vitro and in vivo drug loading and release characteristics of the liposomal formulations were investigated. Finally, the internal pH after drug loading was measured with the aim of linking formation of the Mn(2+) complex to the presence or absence of a transmembrane pH gradient. Doxorubicin was encapsulated into either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol (Chol) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes, where the entrapped salts were citrate, MnSO(4) or MnCl(2). In response to a pH gradient or a Mn(2+) ion gradient, doxorubicin accumulated inside to achieve a drug-to-lipid ratio of approximately 0.2:1 (wt/wt). Absorbance and CD spectra of doxorubicin in the presence of Mn(2+) suggested that there are two distinct structures captured within the liposomes. In the absence of added ionophore A23187, drug loading is initiated on the basis of an established pH gradient; however, efficient drug uptake is not dependent on maintenance of the pH gradient. Drug release from DMPC/Chol is comparable regardless of whether doxorubicin is entrapped as a citrate-based aggregate or a Mn(2+) complex. However, in vivo drug release from DSPC/Chol liposomes indicate less than 5% or greater than 50% drug loss over a 24-h time course when the drug was encapsulated as an aggregate or a Mn(2+) complex, respectively. These studies define a method for entrapping drugs possessing coordination sites capable of complexing transition metals and suggest that drug release is dependent on lipid composition, internal pH, as well as the nature of the crystalline precipitate, which forms following encapsulation.
authors.affiliation: Division of Medical Oncology, Department of Advanced Therapeutics, BC Cancer Agency, 601 West 10th Ave., Vancouver, BC, Canada V5Z 1L3. shabraham@bccancer.bc.ca
authors.forename: Sheela Ann
authors.initials: SA
authors.lastname: Abraham
authors.forename: Katarina
authors.initials: K
authors.lastname: Edwards
authors.forename: Göran
authors.initials: G
authors.lastname: Karlsson
authors.forename: Scott
authors.initials: S
authors.lastname: MacIntosh
authors.forename: Lawrence D
authors.initials: LD
authors.lastname: Mayer
authors.forename: Cheryl
authors.initials: C
authors.lastname: McKenzie
authors.forename: Marcel B
authors.initials: MB
authors.lastname: Bally
mesh_terms: D000818:Animals; D002712:Chlorides / Q000737:chemistry; D002784:Cholesterol; D002942:Circular Dichroism; D020285:Cryoelectron Microscopy; D004134:Dimyristoylphosphatidylcholine; D004317:Doxorubicin / Q000032:analysis / Q000737:chemistry* / Q000493:pharmacokinetics; D016503:Drug Delivery Systems; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry* / Q000493:pharmacokinetics; D017895:Manganese Compounds / Q000737:chemistry; D008563:Membrane Lipids / Q000032:analysis; D008670:Metals / Q000737:chemistry*; D051379:Mice; D013053:Spectrophotometry; D013431:Sulfates
chemical_list: D002712:Chlorides; D008081:Liposomes; D017895:Manganese Compounds; D008563:Membrane Lipids; D008670:Metals; D013431:Sulfates; D004317:Doxorubicin; D002784:Cholesterol; C025340:manganese chloride; D004134:Dimyristoylphosphatidylcholine; C039798:manganese sulfate