Improving the efficiency of iron chelators using nanotechnology

Introduction: Significant pathology accompanies body iron accumulation in both primary and secondary iron loading disorders, so iron removal is a key therapeutic strategy. In iron loading haemoglobinopathies such as β-thalassaemia, iron is removed chemically using iron chelators. Of the three chelators in clinical use, Desferrioxamine (DFO) is the most effective iron binding compound with the most favourable safety profile, but an onerous parenteral administration regimen means that patient compliance is low. If DFO could be delivered with greater efficiency, its clinical utility could be improved. Nanotechnology approaches have greatly improved the delivery of a broad range of therapeutic agents. The aims of this study were to determine whether amphiphilic copolymer nanoparticles (NPs) can be used to deliver DFO efficiently and whether they are effective at depleting body iron. Methods: PEGylated PLGA NPs containing DFO (DFO-NP) were generated by the double emulsion method and characterized by transmission electron microscopy and dynamic light scattering. Macrophage (RAW264.7, J774) and hepatoma (HepG2, HuH7) cell lines were used to study toxicity and efficacy of iron removal in vitro. In vivo iron removal was examined by administering NPs to wild-type mice that had previously been loaded using iron dextran (0.3mg/kg, every second day, twice), as well as Hfe knockout mice and Hbb th3/+ mice. In all studies, DFO-NPs were compared to free DFO and NPs without encapsulated chelator. Results: Physical characterisation showed a uniform preparation of NPs with an average diameter of 113nm. They were stable in the pH range 4.4-7.4. When applied to the macrophage and hepatoma cell lines, DFO-NPs were much more effective at depleting cellular iron levels than free DFO, particularly at higher levels of chelators (up to 80 µM). Mice previously iron loaded with iron dextran, as well as Hfe knockout mice and Hbb th3/+ mice were treated with either saline, free DFO, empty NPs or DFO-NPs (40 mg/kg DFO) on alternate days for 4 weeks. DFO-NPs were more efficient at reducing liver and spleen iron levels than free DFO in each of the models of iron loading studied (e.g. liver: iron dextran loaded mice, 6.15, 5.12, 6.03 and 3.88 mg/g dry weight; Hbb th3/+ mice: 2.28, 2.17, 2.20 and 1.05 mg/g dry weight in the saline, free DFO, empty NPs and DFO-NP groups respectively). Western blotting for ferritin in various organs and staining for tissue iron confirmed these findings. In iron dextran loaded mice, DFO-NPs proved more efficient at promoting urinary iron excretion that free DFO (e.g. 16.0, 20.9, 13.9 and 28.9 µg of iron in 24 h for the treatment groups as specified above). In vitro, DFO-NPs were less toxic to several cell lines than free DFO, and in vivo they did not elicit any specific inflammatory response. Conclusions: Taken together, our results suggest that using a nanoformulation of DFO is potentially a highly valuable strategy for increasing its efficiency as an iron chelating agent and that it could greatly improve the therapeutic potential of this chelator in human iron overloading disorders.