Enzyme mimic to develop antioxidant nanoreactors : from synthesis to application

Reactive oxygen species have been implicated in various diseases, but attempts to find efficient
antioxidant treatments for such conditions have met with only limited success. We have demonstrated the first successful global structure–activity relationship based on statistical analysis of all classes of copper complexes proposed to have a SOD like activity. We took into account all copper-based SODm with electronic and EPR parameters that have been published, and established the specificity of the geometry around the metal as an essential criterion for a highly active SODm. The model we obtained for highly active SODm is that a slightly distorted square planar geometry seems to favour high catalytic activity for 4-coordination sphere in agreement with the geometry of native SOD and for 5-coordinated SODm is square pyramidal, with a slightly distorted basal plane.
We have successfully synthesized a highly active enzyme mimics (CuIIENZm). The detailed structure of CuIIENZm in solid state as well as in solution was studied by X-ray crystallography and Electron paramagnetic resonance. CuIIENZm is shown to a change from a di-copper complex in the solid state to a mono-copper complex in solution and obtained a square-planar conformation. Upon a weak ligation of the solvent molecule, it preserves a pyramidal structure, as established by the combined pulsed EPR and DFT analysis. In this respect, the flexible solvent coordination site of CuIIENZm can be related to its high SOD activity. The presence of a solvent molecule in an equatorial position may be related to the labile bridge present in native SOD between Cu and Zn centers, which breaks to allow the geometric change during the catalytic reaction. Therefore, the structural geometry of CuIIENZm can be related to the obtained structural model for highly active SODm based on SAR analysis.
We developed a robust antioxidant nanoreactor based on the encapsulation of highly active CuIIENZm that permits higher encapsulation efficiency as compared to a nanoreactor containing the enzymes themselves. This is attributable to intrinsic properties of the CuIIENZm, such as improved solubility and elimination of the severe constraints required to avoid enzyme denaturation. CuIIENZm was encapsulated inside the aqueous cavity of polymeric nanovesicles generated by the self-assembly of PMOXA-PDMS-PMOXA amphiphilic copolymers. The mild, structure-preserving procedure used to encapsulate CuIIENZm had no affect on the size/shape of the vesicles, as indicated by light scattering and TEM, or on the metal-coordination sphere that served as a center for catalytic activity, as demonstrated by EPR and UV-Vis spectroscopy.
The cellular interaction of CuIIENZm containing nanoreactors was studied with THP-1 cells such as internalization, cytotoxicity, and in vitro activity. Approximately 11 % of cells contained internalized nanoreactors after 24 h incubation even without a functionalized vesicle surface to support a targeted approach. The antioxidant nanoreactors showed minimal cytotoxicity after 24 h incubation compared to free CuIIENZm. The population of THP-1 cells containing the antioxidant nanoreactors was significantly increased after 48 h incubation, and a protective antioxidant effect (23 %) against oxidative stress by paraquat was established. As CuIIENZm is both a mimic of SOD and of catalase, it serves to detoxify superoxide radicals and related H2O2 inside vesicles, preventing the generation of ROS as a side effect. The well pronounced antioxidant activity of nanoreactors in cells clearly points to a unique method to provide enzyme- mimics based antioxidant therapy, whereby encapsulating mimics in polymeric nanoreactors avoids mimic degradation within biological compartments and simultaneously allows CuIIENZm to act in situ when concentrated inside nanometer-range polymer cavities.
Compared with conventional drug nanocarriers made of liposomes or polymers, our system is more stable and combines the advantages of a polymer shield with an in situ active antioxidant compound. This type of nanoreactors is proposed to function as versatile antioxidants, able to escape the immune system, for medical applications. Development of this simple, robust antioxidant nanoreactor represents a new direction in fighting oxidative stress efficiently.