Cerium oxide nanoparticles have been widely used for various applications such as catalytic converters for automobile exhaust, ultraviolet absorber, and electrolyte in fuel cells. Most recently, cerium oxide nanoparticles (nanoceria) have been employed as potent free-radical scavengers with neuroprotective, radioprotective, and anti-inflammatory properties. These properties of cerium oxide nanoparticles can open new vistas in medicine and biotechnology. The present study utilizes the water-based-wet-chemical method to synthesize biocompatible,stable and highly monodisperse polymer coated cerium oxide nanoparticles. Polymer coated cerium oxide nanoparticles possess all the characteristics of the uncoated cerium oxide nanoparticles. These nanoparticles were found to be effective as pH-dependent antioxidant giving cytoprotection to normal cell lines against hydrogen peroxide and nitric oxide radical but not to cancer cells. Moreover, cerium oxide nanoparticle also exhibits unique oxidase-like activity at acidic pH oxidizing a series of organic compound without the need of hydrogen peroxide. Based on these results, we have designed an immunoassay in which folate-conjugated cerium oxide nanoparticles provide dual functionality by binding to folate expressing cancer cells and facilitating detection by catalytic oxidation of sensitive colorimetric substrates (dyes). Finally, we have shown that the polymer coated cerium oxide nanoparticles shows distinct toxicity depending upon their subcellular localization based on uptake studies using DiI loaded cerium oxide nanoparticles. In these results, we have found that cerium oxide nanoparticles entrapped into lysosomes are more toxic as opposed to when they are localized in the cytoplasm.Overall we propose that the polymer coated cerium oxide nanoparticles displays selective antioxidant property, oxidase-like activity, and cytotoxicity to biological systems depending upon its pH environment.

Rare earth oxides with trivalent lattice dopants have been of great interest to researchers in the recent years due to its potential applications in catalysis and high temperature protective coatings. The ability to store oxygen in rare earths is the basis for catalysis because of the ability to change valence states which causes the presence of intrinsic oxygen vacancies in the crystal lattice. Although, several doped-rare earth oxide systems in micron scale have been investigated, the doping effect in cerium oxide nanoparticles with well characterized particle size has not been studied. The doping of ceria at that small size can be very beneficial to further improve its catalytic properties and alter the high temperature phases in alloy systems. Cost effective room temperature chemical methods are used in the current work to synthesize uniformly distributed undoped and doped (dopants: La, Nd, Sm, Gd, Y and Yb) rare earth oxide nanoparticles. In the present study, the variation of the properties in nanocrystalline ceria (NC) synthesized by microemulsion method is studied as a function of dopant size and its concentration. To further understand, the role of dopant (cation) size on the oxygen vacancy concentration, doped nanocrystalline oxide powders were analyzed by Raman Spectroscopy, X-ray Diffraction (XRD) and X-ray Photoelectron Spectroscopy (XPS). XRD studies showed that lattice parameter change in nanocrystalline oxide by doping trivalent rare earth elements is largely depending on size of trivalent ions. It showed that by doping larger cations (Gd3+ and Y3+) compare to Ce3+ causes lattice expansion where as smaller cations (Yb3+) leads to lattice contraction. It also showed that the lattice expansion or contraction is directly proportional to dopant concentration. The results of Raman Spectroscopy showed that the correlation length decreases resulting in increase in oxygen vacancies for larger trivalent dopants (Sm3+, Gd3+ and Y3+). However, the correlation length increases resulting in decrease in oxygen vacancies for smaller trivalent dopants (Yb3+) compare to nanocrystalline ceria. These nanostructured oxides are further applied to develop high temperature oxidation resistance coatings for austenitic steels. The present study investigates the role of oxygen vacancies in the performance of high temperature oxidation resistance as a function of various trivalent dopants and dopant concentration. NC and La3+ doped nanocrystalline ceria (LDN) particles were coated on AISI 304 stainless steels (SS) and exposed to 1243K in dry air for longer duration and subjected to cycling. The results are further compared with that of micro-ceria (MC) coatings. The coated samples showed 90% improvement in oxidation resistance compared to uncoated and MC coated steels as seen from the SEM cross-sectional studies. XRD analysis showed the presence of chromia in both NC and 20 LDN samples which is absent in uncoated steels. From SIMS depth profiles, Fe, Ni depletion zones are observed in presence of LDN coated sample indicating diffusion through the oxide layer. The role of oxygen vacancies in the nanoceria coatings on the early formation of protective chromia layer is discussed and compared to its micron counterpart. This study helps in understanding the role of oxygen vacancies to protect austenitic stainless steel at high temperature and confirms the oxygen inward diffusion rather cation outward diffusion in rare earth oxide coatings. It also gives an idea to identify the type of dopant and its concentration in nanocrystalline cerium oxide which supplies the critical oxygen partial pressure required at high temperature to form primarily impervious chromia layer.

Clostridioides difficile is a Gram positive, spore-producing, anaerobic bacterium. It is considered a nosocomial pathogen due to high incidence rates of C. difficile infections (CDI) in hospitals. However, research reveals an increase in community-associated cases. CDI is most common in the elderly, immunocompromised, or those taking a course of antibiotics. These individuals are more vulnerable to experiencing gut dysbiosis, allowing C. difficile to colonize the colon. CDI are an urgent threat due to their ability to sporulate. Spores are hardy and not eradicated by common disinfectants. They persist on surfaces the patient may have contact with during CDI. Current decontamination methods include the utilization of bleach-based disinfectants followed by ultraviolet (UV) light and adherence to a strict quality control protocol. However, spores may remain in the environment even after this process, thus allowing the pathogen to spread through surface contact. Another problem is the cost incurred by the hospital over time in terms of inpatient and equipment turnover related costs. These reasons make it imperative a better means of disinfection is developed. Silver is a known antimicrobial agent and utilized in clinical settings for burn treatment. Cerium oxide nanoparticles (CNP) possess antioxidant properties and can be used for drug delivery. Preliminary studies show silver-modified CNP (AgCNP) possess antiviral activity against COVID-19 and rhinovirus. To study the antimicrobial effects of the AgCNP against vegetative cells, a three-day time-kill assay was performed. Two strains of C. difficile, R20291 and NAP1, were cultured in BHIS (brain heart infusion, supplemented) and grown overnight. Glass coupons coated with AgCNP1 or AgCNP3, CNP1 or CNP3, or left uncoated were used in this study. They were incubated with cell culture at a variety of time points. The results indicate AgCNP3 may possess bactericidal activity. Further research should be conducted to determine the extent of this activity.

Cerium oxide nanoparticles (nanoceria) have shown great potential as antioxidant and radioprotective agents for applications in cancer therapy. Recently, various polymer-coated nanoceria preparations have been developed to improve their aqueous solubility and allow for surface functionalization of these nanoparticles. However, the interaction of polymer-coated nanoceria with cells, their uptake mechanism, and subcellular localization are poorly understood. Herein, we engineered polymer-coated cerium oxide nanoparticles with different surface charges (positive, negative, and neutral) and studied their internalization and toxicity in normal and cancer cell lines. The results showed that nanoceria with a positive or neutral charge enters most of the cell lines studied, while nanoceria with a negative charge internalizes mostly in the cancer cell lines. Moreover, upon entry into the cells, nanoceria is localized to different cell compartments (e.g., cytoplasm and lysosomes) depending on the nanoparticles surface charge. The internalization and subcellular localization of nanoceria plays a key role in the nanoparticles cytotoxicity profile, exhibiting significant toxicity when they localize in the lysosomes of the cancer cells. In contrast, minimal toxicity is observed when they localize into the cytoplasm or do not enter the cells. Taken together, these results indicate that the differential surface-charge-dependent localization of nanoceria in normal and cancer cells plays a critical role in the nanoparticles toxicity profile.

Cerium oxide has been used extensively for various applications over the past two decades. The use of cerium oxide nanoparticles is beneficial in present applications and can open avenues for future applications. The present study utilizes the microemulsion technique to synthesize uniformly distributed cerium oxide nanoparticles. The same technique was also used to synthesize cerium oxide nanoparticles doped with trivalent elements (La and Nd). The fundamental study of cerium oxide nanoparticles identified variations in properties as a function of particle size and also due to doping with trivalent elements (La and Nd). It was found that the lattice parameter of cerium oxide nanoparticles increases with decrease in particle size. Also Raman allowed mode shift to lower energies and the peak at 464 cm-1 becomes broader and asymmetric. The size dependent changes in cerium oxide were correlated to increase in oxygen vacancy concentration in the cerium oxide lattice. The doping of cerium oxide nanoparticles with trivalent elements introduces more oxygen vacancies and expands the cerium oxide lattice further (in addition to the lattice expansion due to the size effect). The lattice expansion is greater for La-doped cerium oxide nanoparticles compared to Nd-doping due to the larger ionic radius of La compared to Nd, the lattice expansion is directly proportional to the dopant concentration. The synthesized cerium oxide nanoparticles were used to develop an electrochemical biosensor of hydrogen peroxide (H2O2). The sensor was useful to detect H2O2 concentrations as low as 1µM in water. Also the preliminary testing of the sensor on tomato stem and leaf extracts indicated that the sensor can be used in practical applications such as plant physiological studies etc. The nanomolar concentrations of cerium oxide nanoparticles were also found to be useful in decreasing ROS (reactive oxygen species) mediated cellular damages in various in vitro cell cultures. Cerium oxide nanoparticles reduced the cellular damages to the normal breast epithelial cell line (CRL 8798) induced by X-rays and to the Keratinocyte cell line induced by UV irradiation. Cerium oxide nanoparticles were also found to be neuroprotective to adult rat spinal cord and retinal neurons. We propose that cerium oxide nanoparticles act as free radical scavenger (via redox reactions on its surface) to decrease the ROS induced cellular damages. Additionally, UV-visible spectroscopic studies indicated that cerium oxide nanoparticles possess auto-regenerative property by switching its oxidation state between Ce3+ and Ce4+. The auto-regenerative antioxidant property of these nanoparticles appears to be a key component in all the biological applications discussed in the present study.

The ability of engineered cerium oxide nanoparticles to confer radioprotection was examined. Rat astrocytes were treated with cerium oxide nanoparticles to a final concentration of 10 nanomolar, irradiated with a single 10 Gy dose of ionizing radiation and cell death was evaluated by propidium iodine uptake at 24 and 48 hours after radiation insult. Treatment of rat astrocytes with nanoceria resulted in an approximate 3-fold decrease in radiation induced death. These results suggest that the nanoceria are conferring protection from radiation induced cell death. Further experiments with human cells were conducted. Human normal and tumor cells (MCF-7 and CRL8798) were treated with the same dosage of cerium oxide nanoparticles, irradiated and evaluated for cell survival. Treatment of normal cells (MCF-7) conferred nearly 99% protection from radiation-induced cell death while the same concentration of nanoceria showed almost no protection in tumor cells (CRL8798). TUNEL analysis results of similarly treated cells demonstrated that nanoceria reduced radiation-induced cell death by 3-fold in normal breast cells but not in MCF-7 tumor cell lines when cultured under the same conditions. We concluded that cerium oxide nanoparticles confer radioprotection in a normal human breast line (CRL 8798) but not in a human breast tumor line (MCF-7). It is hoped that the outcome of this study will guide future endeavors toward a better elucidation of the molecular pathways involved in the protection of cells with nanoceria against radiation-induced cell death, as well as the minimization of the bystander effect in radiation therapy.

Cerium is the most abundant of the rare earth metals, found on average at a level of 66 parts per million in the earth's crust. The unique redox properties of cerium and cerium oxide nanoparticles have led to its use in a wide variety of industrial and commercial uses such as oxygen sensors, fertilizers and as a catalyst to remove toxic gases in automobile exhaust. The use of cerium has also garnered interest in the nanotechnology field. Nanoceria has been generated in its oxide form as nanoparticles and nanorods. Recently, nanoceria has been shown to protect against oxidative stress in both animal and cell culture models. Although not fully understood, this observed protective effect of nanoceria is believed to be the result of recently identified SOD mimetic activity. Currently there is little understanding as to how nanoceria is capable of scavenging radicals or what properties makes nanoceria an effective SOD mimetic. Our data shows strong evidence that the oxidation state of nanoceria is directly related to its reported SOD mimetic activity. As such, future studies of nanoceria should be mindful of the oxidation state of nanoceria preparations as only nanoceria with a high concentration of cerium (III) have shown effective SOD mimetic activity. In addition to the characterization of nanoceria and its SOD mimetic activity, we have evidence that free cerium is capable of generating radicals and damaging DNA in vitro in the presence of hydrogen peroxide. These data strongly suggests that the rare earth inner-transition metal cerium is capable of generating hydroxyl radicals via a Fenton-like reaction. Based on these results the use of free cerium salts should be monitored to limit environmental exposure to cerium. Altogether our data would suggest that cerium by virtue of its unique redox chemistry is quite capable of accepting and donating electrons from its surroundings. In its free form cerium is able to redox cycle easily and can generate radicals. However, paradoxically nanoceria may not easily redox cycle due to the bound lattice structure of the particle. The unique nature of nanoceria and cerium leads to a unique circumstance where nanoceria is a radical scavenger while free cerium generates radicals. As such, further investigation is needed to insure that leeching or cerium from nanoceria does not abrogate any potential benefit nanoceria may provide.

Research on the cure and early detection of diseases such as diabetes, Alzheimer's, and Parkinson's is becoming of great interest due to the increasing number of people affected by them every year. An accurate and quick detection of various damaging species is highly critical in treatments of such diseases not only for exploring possible cures but also for early detection. If these diseases are detected during the initial stages than the possibility of curing them is much higher. Motivated by this, many researchers today have developed numerous types of sensing devices that can detect various physiological and biological compounds. However, most of these sensors are enzyme based. They have several setbacks such as the lack of sensitivity, restricted selectivity, short shelf life, and biological fouling. To overcome these obstacles, we examine the use of nanoceria modified Pt and Au electrodes for the detection of glucose and reactive oxygen species such as hydrogen peroxide. Amperometric detection of glucose and hydrogen peroxide is critical for biological applications for diabetes and possible Alzheimer's and Parkinson's patients. This dissertation focuses on the exploration of non-enzymatic detection of glucose and reactive oxygen species which has the prospective to be used for biological applications, in addition to an investigation of an odor control technology that uses these reactive oxygen species for the treatment of wastewater plants. The combination of bi-metallic composites with nanoceria showed increased oxidation ability towards glucose and hydrogen peroxide. The following dissertation expands on the relationship between bi-metallic nanoceria composite materials and its electro-oxidation of glucose and hydrogen peroxide towards biological sensing along with an investigation of an odor control technology that utilizes generates hydroxyl radical fine particle mist for the degradation of hydrogen sulfide odor in wastewater treatment plants.

Bio-inspired or biomimetic chemistry deals with the replication of the nature’s fundamental processes, which can help in understanding the functioning of biological systems and develop novel applications. Although a large number of researchers worked towards the replication of natural synthetic pathways through biogenetic syntheses, enzyme mimicry by the small organic molecules and inorganic complexes emerged in leaps and bounds over the years. The development of biomimetic chemistry then continued in designing the molecules that can function like enzymes. And now, with the advent of nanotechnology, nanostructured materials have been shown to exhibit enzyme-like activities (nanozymes). Interestingly, the two distinct fields, biology and materials science, have been integrated to form an entirely new area of research that has captured a great attention. Along with the pronounced application of nanomaterials as drug delivery vehicles, anticancer agents, antimicrobials, etc., research is also focused on designing nanomaterials for the biomimetic applications. The thesis consists of five chapters. The first chapter provides a general overview of the recently discovered nanozymes that mimic heme-peroxidase, oxidase, superoxide dismutase, catalase, haloperoxidase and phosphatase. This chapter also deals with the nanozymes’ application in sensing and immunoassay, and as antioxidants, neuroprotective agents. The factors affecting the nanozymes’ activity and the challenges associated with them is also covered in this chapter. Chapter 2 is divided into two parts and it deals with the biomimetic properties of graphene-based materials. In part A, the remarkable peroxynitrite (PN) reductase and isomerase activities of hemin-functionalized reduced graphene oxide (rGO) is discussed. In part B, the activity of graphene oxide (GO) as peroxide substrate for the glutathione peroxidase (GPx) enzyme is discussed. In chapter 3, the oxidant material, V2O5, is shown to exhibit significant GPx-like antioxidant activity in its nano-form. Chapter 4 deals with the oxidase-like activity of MnFe2O4 nanooctahedrons for the antibody-free detection of major oxidative stress biomarker, carbonylated proteins. In chapter 5, the phosphotriesterase mimetic role of vacancy engineered nanoceria is discussed. instead of H2O2 for glutathione peroxidase (GPx) enzyme. As partial reduction of GO was observed when treated with GPx enzyme due to the fact that large sheet-like structures cannot be accessible to the active site, we studied the reaction with some GPx mimetics (Fig. 2). Varying the concentration of cofactor glutathione (GSH) required for the reaction, GPx mimic, ditelluride, could accomplish the reduction of GO following Michaelis-Menten kinetics. As the structure of GO is elusive and under active investigation, our study highlights the presence of peroxide linkages as integral part of GO other than hydroxyl, epoxy and carboxylic groups. This study also highlights an important fact that the modification of GO by biologically relevant compounds such as redox proteins must be taken into account when using GO for biomedical applications because such modifications can alter the fundamental properties of GO. Figure 2. The GO reductase and decarboxylase activities of GPx mimetic ditelluride compound, suggesting the presence of peroxide linkages on GO. In chapter 3, we have discussed about the novel antioxidant nanozyme that combats oxidative stress. During our attempts in the investigation of antioxidant nanozymes, we surprisingly noticed that the oxidant material, V2O5, shows significant GPx-like antioxidant activity in its nano-form. The Vn readily internalize in the cells and exhibit remarkable protective effects when challenged against reactive oxygen species (ROS). Although Vn has been shown to protect cells from ROS-induced damage, cells treated with bulk V2O5 and few vanadium complexes resulted in generation of ROS and severe toxicity. Detailed investigation on the mechanism of this interesting phenomenon Chapter 4 deals with the development of novel methodology for detection of biomarkers. Inspired by the use of antibodies and enzymes for detection of a specific antigen, we have shown for the first time that the nanozymes can entirely replace antibodies and enzymes in Enzyme-linked Immunosorbent Assays (ELISA). As a specific example, we focused on the antibody-free detection of chief oxidative stress biomarker, carbonylated proteins, as our target. To achieve this, we designed MnFe2O4 nanooctahedrons that can function as oxidase enzyme and form signaling point of detection. We functionalized MnFe2O4 nanooctahedrons with hydrazide terminating groups so that carbonylated proteins can be linked to nanozymes by hydrazone linkage (Fig. 4a). Treatment of various carbonylated proteins (hemoglobin (Hb), Myoglobin (Mb), Cytochrome c (Cyt c), RNase and BSA) coated in well plate with hydrazide-terminated MnFe2O4 nanooctahedrons and then with 3,3’,5,5’-tetramethylbenzidine substrate, resulted in instantaneous detection by well plate reader (Fig. 4b). Considering the challenges and difficulties associated with the conventional methods used to detect such modified proteins, this methodology opens up a new avenue for the simple, cost-effective, instantaneous and entirely antibody-free ELISA-type detection of carbonylated proteins. Our results provide a cumulative application of nanozymes’ technology in oxidative stress associated areas and pave a new way for direct early detection of post translational modification (PTM) related diseases. Figure 4. a) Nanozyme linked to the carbonylated protein coated on a plate through hydrazone linkage. b) General bar diagram showing detection of oxidized (carbonylated) proteins by nanozymes. Synopsis Figure 5. a) A cartoon view of surface of ceria showing vacancy. b) Zoomed portion of high resolution transmission electron microscopic image showing few vacancies on the surface of nanoceria. c) Catalytic mechanism of detoxification of paraoxon at the defect site. In the final chapter, chapter 5, we have discussed about the nanomaterial that can function as phosphotriesterase enzyme. Phosphotriesterase enzyme is a bacterial enzyme that is involved in the rapid hydrolysis of sarin gas-related deadly nerve agents such as paraoxon, parathion and malathion. When encountered with these orgnaophospatetriesters, living beings tend to undergo nerve shock to cause paralysis by inhibiting an extremely important enzyme called acetylcholine esterase. They are also known to cause severe oxidative stress problems and are associated with neurodegenerative disorders. Therefore, curbing the toxic effects and detoxification of these nerve agents is a world-wide concern and many research teams have focused their attention to address this important problem. Working on the development of nanozymes for important problems, we found that nanoceria, especially the vacancy engineered one (Fig. 5a,b), can serve as active mimic of phosphotriesterase enzyme in the presence of N-methylmorpholine (acting as a distal base histidine). Vacancy engineered nanoceria has been shown to catalyze the hydrolysis of high amounts of paraoxon quiet efficiently and within few minutes with very low activation energy and high kcat. Detailed mechanistic investigation revealed that the presence of both Ce(III) and Ce(IV) is very essential for detoxification activity (Fig. 5b). The vacancies on the surface of nanoceria, were the buried Ce(III) ions are directly exposed to the reaction environment, behave as hotspots or enzyme active sites for detoxification reaction (Fig. 5b).