We designed and tested an antioxidant nanoreactor based on encapsulation of Cu,Zn superoxide dismutase in amphiphilic copolymer nanovesicles, the membranes of which are oxygen permeable. The nanovesicles, made of poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline), successfully encapsulated the protein during their self-assembling process, as proved by confocal laser-scanning microscopy and fluorescence-correlation spectroscopy. Electron paramagnetic resonance spectroscopy and circular dichroism analyses showed that no structural changes appeared in the protein molecules once inside the inner space of the nanovesicles. The function of this antioxidant nanoreactor was tested by pulse radiolysis, which demonstrated that superoxide dismutase remains active inside the nanovesicles and detoxifies the superoxide radical in situ. The membrane of our triblock copolymer nanovesicles plays a double role, both to shield the sensitive protein and to selectively let superoxide and dioxygen penetrate to its inner space. This simple and robust hybrid system provides a selective shielding of sensitive enzymes from proteolytic attack and therefore a new direction for developing drug delivery applications.

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SummaryThe VirE2 protein is crucial for the transfer of singlestranded DNA (ssDNA) from Agrobacterium tumefaciens to the nucleus of the plant host cell because of its ssDNA binding activity, assistance in nuclear import and putative ssDNA channel activity. The native form of VirE2 in Agrobacterium 's cytoplasm is in complex with its specific chaperone, VirE1. Here, we describe the ability of the VirE1VirE2 complex to both bind ssDNA and form channels. The affinity of the VirE1VirE2 complex for ssDNA is slightly reduced compared with VirE2, but the kinetics of binding to ssDNA are unaffected by the presence of VirE1. Upon binding of VirE1VirE2 to ssDNA, similar helical structures to those reported for the VirE2-ssDNA complex were observed by electron microscopy. The VirE1VirE2 complex can release VirE1 once the VirE2-ssDNA complexes assembled. VirE2 exhibits a low affinity for small unilamellar vesicles composed of bacterial lipids and a high affinity for lipid vesicles containing sterols and sphingolipids, typical components of animal and plant membranes. In contrast, the VirE1VirE2 complex associated similarly with all kind of lipids. Finally, black lipid membrane experiments revealed the ability of the VirE1VirE2 complex to form channels. However, the majority of the channels displayed a conductance that was a third of the conductance of VirE2 channels. Our results demonstrate that the binding of VirE1 to VirE2 does not inhibit VirE2 functions and that the effector-chaperone complex is multifunctional.

A series of block copolymers containing a dendronised cationic block for efficient DNA binding and a poly(ethylene glycol) block for encapsulation of the complex were synthesised in a modular fashion using a combination of click chemistry and ring-opening metathesis polymerisation. DNA binding experiments, investigated using gel electrophoresis, dynamic light scattering and transmission electron microscopy, showed that all polymers prepared in this study strongly complex DNA and self-assemble into polyion complex micelles with apparent hydrodynamic radii ranging from 20-120 nm at physiological pH (7.4). The in vitro transfection efficiency and toxicity of these potential non-viral vectors were also evaluated in HeLadouble dagger cells using plasmid DNA encoding for green fluorescent protein as the reporter gene.

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