(b) Development of Nanozymes for Biomedical Applications.
Engineering bio-compatible nanomaterials to mimic the cellular antioxidant machinery has assumed priority in translation research to combat oxidative stress mediated disorders. Particularly, nanomaterials with intrinsic enzyme-like activity attract significant current interest due to their ability to replace specific enzymes in enzyme-based applications. A few nanostructured materials have been shown to mimic the activity of peroxidases, oxidase, and superoxide dismutase. The main objective of our work is to understand how the surface of nanomaterials can be engineered to perform specific reactions. We believe that the enzyme-like properties of nanomaterials (nanozymes) can be fine-tuned by controlling the size, shape and morphology, surface coating and modification and composition. There appears to be a change in the enzyme-like activity when a bulk material is converted to nanoform. Recently, our group, in collaboration with D'Silva's group has conceptualized the development of a vanadium-based nanozyme that specifically exhibits GPx activity and possesses a cytoprotective function in the cell (Vernekar, A. A. et al. Nature Commun. 2014, 5, 5301, Click here for details).
The rod-shaped vanadia (V2O5) nanowires efficiently internalize into the cell by endocytic mechanisms without altering the integrity of the biological membranes. The nanowires by themselves are not toxic to the cell and exhibit remarkable antioxidant properties by reducing the accumulated peroxides within the stressed cell. Mugesh’s group competently showed that vanadia nanowires require GSH as a co-factor for its antioxidant properties both in vitro and in live cells, and mimic GPx (Figure 5). These
nanozymes could efficiently replace GPx in the cyclic GSH-GSSG cascade and function alongside with glutathione reductase and electron donor, NADPH. Like the natural enzyme, vanadia nanowires showed substrate selectivity to hydrogen peroxide, with the formation of a peroxo species on nanowire surface being the rate determining step. Although vanadia nanowires are known to function as a haloperoxidase mimic and generate a strong oxidant hypobromous acid, its antioxidant activity is not altered even in the presence of haloperoxidase substrates.
In a nano form, V2O5 is able to reduce hydrogen peroxide without changing the oxidation state of the metal, which is unusual for a metal ion. If the metal changes its oxidation state then
Figure 5. Nanovanadia - Turning cytotoxic vanadium into cytoprotective antioxidant: (a) SEM image of vanadium pentoxide nanowires (Vn). (b) and (c) TEM and HRTEM images of Vn showing the planes and inset reveals the crystallinity and orthorhombic crystal structure of Vn. (d) Glutathione peroxidase activity of Vn and GSH recycling by glutathione reductase. (e) SEM images of HEK293T cells untreated (UT) or treated with Vn. (f) Hydrogen peroxide scavenging activity of Vn in HeLa cells.
Funding for Research
vanadium can produce reactive oxygen species, which is what happens when vanadium is in a bulk form. Although the number of nanowires that get into a cell cannot be controlled, the amount of vanadia nanowires used is very small - in parts per million (ppm). Apparently, the inability to completely remove hydrogen peroxide turned out to be beneficial as optimum removal ensures that other biological functions remain unaffected by vanadia. Turning a cytotoxic vanadium into cytoprotective antioxidant by reducing the size of the material is certainly a novel concept. The vanadia nanozyme is of therapeutic importance in combating age-related degenerative diseases and as a delivery vehicle for various xenobiotics to reduce stress mediated side-effects.
Reduced Graphene Oxide (RGO) as Peroxynitrite Reductase and Isomerase:
Facile and efficient reduction of graphene oxide (GO) and novel applications of the reduced graphene oxide (RGO) based materials are of current interest. Recently, we reported a novel and facile method for the reduction of GO by using a biocompatible reducing agent dithiothreitol (DTT) (Figure 6). Stabilization of DTT by the formation of six membered ring with internal disulfide linkage upon oxidation is responsible for the reduction of GO. The reduced graphene oxide (RGO) is characterized by several spectroscopic and microscopic techniques. Dispersion of RGO in DMF remained stable for several weeks suggesting that the RGO obtained by DTT-mediated reduction is hydrophobic in nature. This method can be considered for large scale production of good quality RGO.
Figure 6. A) SEM, B) TEM, C) HRTEM, D) SAED pattern of RGO nanosheets. TEM image reveals the corrugation and scrolling of nanosheets. Few layers are seen in HRTEM image. The SAED pattern corresponds to image C. E) Schematic representation of the synthesis of RGO by the reduction using DTT. F) Inhibition of PN-mediated nitration of tyrosyl residue in BSA by inhibitors. G) Anti-nitrotyrosine immunoblotting of BSA after treatment with PN in the absence and presence of inhibitors.
Treatment of RGO with hemin afforded a functional hemin-reduced graphene oxide (H-RGO) hybrid material that exhibited remarkable protective effects against the potentially harmful peroxynitrite (PN). A detailed inhibition study on PN-mediated oxidation and nitration reactions indicates that the interaction between hemin and RGO results in a synergistic effect, which leads to an efficient reduction of PN to nitrate (Figure 6). The RGO also catalyzes the isomerization of PN to nitrate as the RGO layers facilitate the rapid recombination of .NO2 radical with Fe(IV)=O species. In the presence of reducing agents such as ascorbic acid, the Fe(IV)=O species can be reduced to Fe(III), which help maintaining PN reductase cycle. (Vernekar, A. A.; Mugesh, G. Chem. Eur. J. 2012, 18, 15122; Chem. Eur. J. 2014, 20, 11120).