EDTA-Gradient Loading of Doxorubicin into Ferrocene-Containing Liposomes: Effect of Lipid Composition and Visualization of Triggered Release by Cryo-TEM

Efficient delivery of therapeutic compounds to their sites of action has been a ubiquitous concern throughout the history of human medicine. The tumor microenvironment offers a variety of endogenous stimuli that may be exploited by a responsive nanocarrier, including heterogeneities in redox potential. In the early stages of the design of such responsive delivery systems, it is necessary to develop a comprehensive understanding of the biophysical mechanism by which the stimulus response occurs, as well as how the response may change from the inclusion of cargo compounds. We describe the optimization of lipid compositions for liposomes containing synthetic ferrocene-appended lipids to achieve highly efficient loading of doxorubicin via an ethylenediaminetetraacetic acid (EDTA) gradient. Liposomes containing ferrocenylated phospholipid are shown to be unstable to the loading conditions, while those including a ferrocenylated alkylammonium amphiphile obtain a near-quantitative loading efficiency. Calorimetric studies demonstrate that this instability is the consequence of the relative degree of lipid hydrolysis that occurs under the acidic loading conditions. Drug-loaded liposomes of the optimized composition are studied by cryo-TEM; the presence of doxorubicin aggregates is observed inside vesicles, and doxorubicin release, as well as the changes in membrane structure resulting from oxidant treatment, is also observed by cryogenic transmission electron microscopy (cryo-TEM). These results further demonstrate the potential of ferrocene lipids in the design of redox-responsive nanocarriers and begin to explore their possible role as probes of membrane dynamics.