Synthesis and applications of metal based nanozymes

Abstract

Nanotechnology is the understanding and control of material properties under nanoscale dimensions (approximately 1-100 nm), where unique phenomena such as improved physicochemical and optoelectronic properties enable them to display novel applications. Among different applications, nanoparticles (NPs) mimicking the catalytic activities of natural enzymes (nanozymes) have gained immense interest over biological enzymes due to the lower cost of synthesis, better stability in harsh environmental conditions, facile synthesis methods, flexibility in composition, shape, and size control, improved shelf-life and tunability of catalytic activities. Recently, nanozymes have been utilized in biosensing, immunoassays, disease diagnosis and therapy, theranostics, cell/tissue growth/proliferation, protection of mammalian cells from oxidative stress, and removal of pollutants. Among various metallic NPs, gold NPs (AuNPs) have attracted tremendous interest due to their unique properties such as optoelectronic properties, excellent biocompatibility, and flexibility in surface modification. AuNPs have also gained a strong interest as nanozyme due to their peroxidase enzyme-like activity. The reactivity of the Au atoms at the surface of AuNPs is critical to the performance as nanozymes, yet little is known about the effect of NPs size, the effect of biomolecules, and ions on the peroxidase-like activity. To fulfill this, peroxidase-like activity was evaluated with varying the size of AuNPs at acidic pH utilizing 3,3’,5,5’ tetramethylbenzidine (TMB) as a substrate in the presence of hydrogen peroxide (H2O2). Further, the effect of nucleotide adenosine triphosphate (ATP) and other biologically relevant molecules on the peroxidase-like activity was also tested. The incorporation of ATP into artificial enzyme mimics resulted in a significant enhancement in the peroxidase-like activity of AuNPs. Further, the maximum improvement in peroxidase-like activity was observed in the case of 30 nm AuNPs compared to 15, 50 and 70 nm-sized AuNPs. The addition of ADP also improved the catalytic potential of AuNPs but was not as pronounced as it was observed in the case of ATP. Addition of ascorbic acid to the reaction mixture diminished the intrinsic peroxidase-like activity of AuNPs, even in the presence of ATP and ADP suggesting that interaction of AuNPs with H2O2 led to the generation of •OH, which was scavenged by ascorbic acid with concomitant diminished peroxidase-like activity. The surface property of AuNPs would affect the adsorption of H2O2 and the particle mediated electron transfer process. Further, in order to explore the mechanism of improved peroxidase activity of AuNPs in the presence of ATP, we also studied the effect of NPs’ charge. The surface charge of AuNPs was varied using different capping molecules such as PEG (uncharged), citrate (negatively charged), and CTAB (positively charged). Surface charge, capping molecule, and type of peroxidase substrate affected the peroxidase mimetic behaviour of AuNPs. PEG AuNPs and citrate AuNPs displayed concentration-dependent peroxidase-like activity whereas CTAB AuNPs did not elucidate any catalytic activity. PEG AuNPs demonstrated ~3.0 fold and citrate AuNPs demonstrated ~1.5 fold increase in peroxidase-like activity, respectively in the presence of ATP. Hence, ATP synergizes better with PEG AuNPs (uncharged) compared to citrate AuNPs (negatively charged). In order to understand the role of ATP, the peroxidase-like activity of PEG AuNPs, citrate AuNPs and horseradish peroxidase (HRP) enzyme was monitored by following the absorbance of oxidized TMB at 652 nm for 20 mins to 24 hrs. In the absence of ATP, as time progresses, the absorbance intensity of oxidized TMB was significantly reduced in all three test conditions. Although it was found that TMB oxidized by PEG AuNPs and citrate AuNPs show a lesser decrease in intensity than HRP. ATP provides stabilization to the oxidized TMB produced of PEG AuNPs, citrate AuNPs as well as HRP. These results provide insight into the role of ATP in stabilizing oxidized TMB. Therefore, the mechanism of boosting the effect of ATP is attributed by promoting the stabilization of oxidized TMB products via single electron transfer. A wide range of antioxidants has been generally exploited to curb and combat oxidative stress. Augmentation in reactive oxygen species (ROS) level triggers the production of prevalent antioxidant enzymes such as catalase and superoxide dismutase (SOD) in order to scavenge and eliminate the toxic species. A lower level of cellular catalase enzyme has been concomitant with several diseases such as Alzheimer's, acatalasemia, anemia and vitiligo. Despite the broad spectrum application of traditional antioxidants, natural enzyme antioxidants lack stability and are easily inactivated under non physiological conditions. While non-enzymatic antioxidants suffered from several constraints such as incapability of passing the blood-brain barrier and have low bioavailability. These limitations can be overcome by utilizing metal-based nanozymes which offer flexible operation, better stability, economic cost, mass production and easy treatment. Several attempts have been made to evaluate antioxidant nanozymes however

many aspects concerning their use in the protection of cells from oxidative stress are being unanswered. To address this, the focus has been made on designing and developing antioxidant nanozymes. However, very few attempts have been carried out to synthesized aqueous stable colloidal metallic magnesium-based NPs (MgNPs) due to the difficult synthesis process and susceptibility for metal oxide formation. The paramount interest in the synthesis of MgNPs was attributed to the abundance of Mg in nature and their role as a co-factor in many natural enzymes. Therefore, it is of tremendous interest to synthesize biocompatible Mg-based nanozymes for biomedical applications. Catalase-like activity of bovine serum albumin coated MgNPs (BSA MgNPs) was used to protect cells from oxidative stress in the human hepatocyte cell culture model (WRL-68). BSA-MgNPs led to the rapid breakdown of H2O2 and therefore, protect mammalian cells exposed to 3-amino-1,2,4-triazole (3-AT), an irreversible catalase enzyme inhibitor. Furthermore, BSA-MgNPs scavenge the free radicals produced due to the elevated H2O2 in the absence of natural catalase enzyme. BSA-MgNPs did not elicit the activation of the natural antioxidant glutathione (GSH) under elevated ROS conditions indicating the independent behavior of BSA-MgNPs as a catalase mimic. In the absence of natural catalase enzyme, the antioxidant defence mechanism gets activated and modulates the expression of proteins/genes such as catalase, thioredoxin reductase (TrxR) and peroxiredoxin6 (Prx6) involved in the H2O2 degradation pathway. The first half of the thesis describes the improvement of the peroxidase-like activity of AuNPs by ATP. The mechanistic aspect involved the effect of AuNPs size, surface charge and capping molecules on its peroxidase-like activity in the presence of ATP. The second half of the thesis discusses about the synthesis of BSA-MgNPs and the study of catalase enzyme mimetic activity. These BSA-MgNPs were also used for the protection of mammalian cells from oxidative stress. This thesis has a total of six chapters