1. Investigating the potential use of cerium oxide nanoparticles as catalytic antioxidants in biomedical areas.
Xuanhao Wu Che Tan
2016/4/29

2. Chemical mechanical polishing and planarization Industrial catalysis
Cerium Oxide (CeO2) nanoparticles (NPs)
Chemical mechanical polishing and planarization
Industrial catalysis
Catalytic converters
Fuel additives
Fuel cells
CeO2 is one of the most interesting oxides industrially nowadays due to their oxygen storage capabilities and their ability to participate in Ce4+/Ce3+ redox process.
This equation implies a reversible reaction. Indeed, solid particle ceria can be thought of as an “oxygen buffer” that either provides or removes oxygen to/from the surrounding environment by responding to a lack or excess of oxygen in that environment. The ability to reversibly extract oxygen atoms from the lattice has obvious utility for the catalytic oxidation of various materials such as carbon monoxide, and other partially oxidized exhaust gases
The oxygen vacancy is that of a missing oxygen atom (or atoms for a di or tri vacancy) in one or more of the eight octants in a ceria unit cell. can actually be quantified by a number called the oxygen storage capacity (OSC). This number is expressed as micromoles of oxygen liberated per gram of starting material
[1] Campbell and Peden, Science, 2005.
[2] Sayle et al., Nanoscale, 2013.
[3] Reed et al., Environ. Sci.: Nano, 2014.

3. - Superoxide dismutase (SOD) activity
- Reactive oxygen species (ROS) scavenging
- Reactive nitrogen species (RNS) scavenging
Antioxidant behaviors of CeO2
3C e O 2 ∙− →3C e O 2
C e O 2 ∙− +2 H + →C e H 2 O 2
Ce4+ reduction (1)
Ce3+ oxidation (2)
H 2 O 2 +2C e H + →2C e H 2 O
peroxide catalase (3)
∙NO+ O 2 ∙− +2 H + →ONO O −
Peroxynitrite formation
mitochondria
Xanthine oxidase
Cerium oxide nanoparticles have since been shown to display a number of antioxidant behaviors, including superoxide dismutase (SOD) activity, catalase mimetic activity, reactive oxygen species (ROS) and reactive nitrogen species (RNS) scavenging.
We can further make the reasonable assumption that the disease pathology is somehow related to the excess superoxide that may be generated by hydrogen peroxide, as has been implicated in Parkinson's disease. In reality, the excess of superoxide ion may be due to malfunctioning of the SOD1 and SOD2 enzymes, but it is mathematically more tractable to pose the equations as an excess of hydrogen peroxide:
[4] Korsvik et al., Chemical Communications, 2007

4. Factors - Size effects - pH effects - Surface-coating effects
Peroxynitrite (ONOO−) is stable under basic conditions
pH values influence the zeta potential of nanoceria
Lattice expansion leads to a
decrease in oxygen release
Oleic acid
Poly (acrylic acid) - Octylamine (PAAOA)
Polyethyleneimine (PEI)…
[5] Hailstone et al., J. Phys. Chem. C, 2009
[6] Liu et al., ES&T, 2009
[7] Lee et al., ACS Nano, 2013

5. Knowledge gaps
Reports provide conflicting data about the reaction of CeO2 NPs with ROS in vitro: anti- or pro- oxidant behavior.
- Nanoceria enables radical scavenging in physiological pH conditions
- Toxic to several types of human cancer cells in vitro, including squamous cell carcinoma, alveolar epithelial cancer cells, and pancreatic carcinomas due to the generation of ROS and the induction of oxidative stress
The impact of undetermined cellular and environmental factors on the manifestation of anti- or pro- oxidant behavior in vitro
- pH
- Particle size
- Surface coating…
Lack of understanding for the behavior of CeO2 NPs in vivo
- Interactions with biologically relevant molecules such as proteins, anions and lipids are likely to alter the behavior of CeO2 NPs in vivo
- Conflicting results between In vivo and in vitro

6. Techniques Transmission electron microscopy (TEM):
- Nanoparticle shape and geometric size
- High-resolution TEM (HRTEM) could show the structure of crystalline CeO2 NPs core.
X-ray diffraction (XRD) pattern:
- Crystallite size
- Compare TEM geometric size to XRD crystallite size to obtain an estimate of the number of crystallites per particle.
X-ray photoelectron spectroscopy (XPS):
- Indicate the presence of a mixed valence state in CeO2 NPs and corresponding binding energy peaks for Ce3+ and Ce4+,
Dynamic light scattering (DLS):
- Hydrodynamic particle size (particle size with its attendant adsorbate and solvation sphere)

7. Objective Main Approach
Understand how the properties (specifically size, charge, and surface coating) of cerium oxide nanoparticle can affect their antioxidative properties both in vitro and in vivo
Main Approach
The free radical scavenging activity of cerium oxide nanoparticles will be investigated and compared in both aqueous solution (cell-free) and in cell cultures (in vitro)
The antioxidative effect of cerium oxide nanoparticles will also be elucidate and compared both in vitro and in vivo using cell cultures and Caenorhabditis elegans (C. elegans) as test models, respectively.

8. variety of size and surface coatings
Objective 1: Synthesis and characterization of cerium oxide nanoparticles with
variety of size and surface coatings
Synthesis of cerium oxide nanoparticles: Thermal decomposition
Phase Transfer of cerium oxide nanoparticles to aqueous solution:
N2 gas
Ce(NO3)36H2O with oleylamine in 1-octadecene
Temperature control by thermocouple
Phase transfer into water using probe sonicator:
Ligands: - Oleic acid (-)
- PEI (+)
(Polyethyleneimine)
Purification
1. Ethanol, Acetone (wash)
2. Hexane (wash)
3. Centrifugation
Re-disperse in nonpolar solvent

9. variety of size and surface coatings (continued)
Objective 1: Synthesis and Characterization of cerium oxide nanoparticles with
variety of size and surface coatings (continued)
Expected Outcomes:
Characterization of cerium oxide nanoparticles:
TEM – Morphology and size of the nanoparticles
DLS – Hydrodynamic size and level of aggregation
Zeta-potential – Nanoparticle surface charge
FTIR – Nanoparticle surface composition
XPS – Oxidation state of the nanoparticle
Lee et al. 2013

10. Absorbance at 530 nm was recorded every minute
Objective 2: Investigating the performance of free radical scavenging in a cell-free aqueous solution and in vitro cell culture
Oxygen-radical absorbance capacity (ORAC) assay
Deionized Water:
Phosphate buffered saline (PBS):
Fibroblast cell culture:
W/WO CeO2 nanoparticle +
Radical initiator
(AAPH)
Absorbance at 530 nm was recorded every minute
fluorescent indicator (β-phycoerythrin)

11. Objective 3: Investigating the antioxidant effect of cerium oxide nanoparticles in vivo using Caenorhabditis elegans (c. elegans) as a model organism
Why C. elegans?
Established biology
Short life cycle
It is multicellular eukaryotic organism
just like us
Readily scorable life traits
Transparency of the body
Ease of cultivation
High sensitivity to various types of
stresses (especially oxidative stress)

12. B. Preparation of synchronized worms
Objective 3: Investigating the antioxidant effect of cerium oxide nanoparticles in vivo using Caenorhabditis elegans (c. elegans) as a model organism
C. elegans cultivation
Maintenance
at 20⁰ C
bacteria lawn :
E. coli strain OP50
nematode growth medium (NGM) plate
N2 wild-type strain
B. Preparation of synchronized worms
All of the assays in this study should start with synchronized worms in the L1
stage to avoid the influence of developmental stage on the test.

13. Worm Synchronize
add 0.5 N NaOH & 1% NaOCl
Wash X3
Syncronized worm
the survived eggs hatched and were termed as L1 larvae
8 hrs
kills the nematodes but the eggs( inside and outside) of the body survived
H. Wang 2009

14. Objective 3: Lifespan Assay
synchronized L1 larvae (t = 0)
control
nano CeO2 exposed
until reproduction cease
every 3 days
Transfer every day
until none alive

15. Survival monitored each hour
Objective 3: Thermotolerance & Oxidative Stress Resistance
nano CeO2 exposure
control
synchronized L1 larvae (t = 0)
lives were counted
Survival monitored each hour
Incubated 72h at 20°C
Transfer to 35⁰C
Transfer to NGM plates containing 2 mM H2O2
10 h

16. Scientific Implication
Physical and chemical properties of a nanoparticle determine the interaction of biomolecules with nanoparticles when the nanoparticles are exposed in a biofluid. Thus, fundamental studies on the effect of size and surface coating of nanoparticles, are prerequisites for the rational design of nanoparticles for biomedical applications.
Nanoceria is consider as a reactive oxygen species scavenger, and have potential biomedical application in the treatment of neurodegenerative diseases. However, the functional nanoceria must be first delivered to the brain for such applications. Therefore, it is important to designthe surface of a nanoparticle to selectively adsorbed specific plasma proteins that promote target site localization.
Given the complexity of biological interactions, in order to predict the biological outcome of nanoparticle interactions, it is important to study these effects in intact, living systems. Gaining insight into the nature of these complex interactions is critical for the optimum design of nanoceria for future applications.

17. Reference
[1] Campbell, C. T., & Peden, C. H. (2005). Oxygen vacancies and catalysis on ceria surfaces. Science, 309(5735),
[2] Sayle, T. X., Molinari, M., Das, S., Bhatta, U. M., Möbus, G., Parker, S. C., ... & Sayle, D. C. (2013). Environment-mediated structure, surface redox activity and reactivity of ceria nanoparticles. Nanoscale, 5(13),
[3] Reed, K., Cormack, A., Kulkarni, A., Mayton, M., Sayle, D., Klaessig, F., & Stadler, B. (2014). Exploring the properties and applications of nanoceria: is there still plenty of room at the bottom?. Environmental Science: Nano, 1(5),
[4] Korsvik, C., Patil, S., Seal, S., & Self, W. T. (2007). Superoxide dismutase mimetic properties exhibited by vacancy engineered ceria nanoparticles.Chemical Communications, (10),
[5] Hailstone, R. K., DiFrancesco, A. G., Leong, J. G., Allston, T. D., & Reed, K. J. (2009). A study of lattice expansion in CeO2 nanoparticles by transmission electron microscopy. The Journal of Physical Chemistry C, 113(34),
[6] Liu, X., Ray, J. R., Neil, C. W., Li, Q., & Jun, Y. S. (2015). Enhanced Colloidal Stability of CeO2 Nanoparticles by Ferrous Ions: Adsorption, Redox Reaction, and Surface Precipitation. Environmental science & technology,49(9),
[7] Lee, S. S., Song, W., Cho, M., Puppala, H. L., Nguyen, P., Zhu, H., ... & Colvin, V. L. (2013). Antioxidant properties of cerium oxide nanocrystals as a function of nanocrystal diameter and surface coating. ACS nano, 7(11),