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Cerium-Zirconium Oxide Nanocatalysts as Free Radical Scavengers for Biomedical Applications

Permanent Link: http://ufdc.ufl.edu/UFE0022459/00001

Material Information

Title: Cerium-Zirconium Oxide Nanocatalysts as Free Radical Scavengers for Biomedical Applications
Physical Description: 1 online resource (140 p.)
Language: english
Creator: Tsai, Yi-Yang
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: aging, antioxidants, catalyst, ceria, cerium, free, islet, nanoparticles, radicals, zirconia
Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Administering CeO2 nanoparticles into cell cultures was found to improve cell culture?s viability both in vitro and in vivo. In this dissertation, an in vitro study conducted in our laboratory has observed the reduction of endogenous free radical concentration in CeO2 treated cell cultures. Furthermore, it is found that CeO2 nanoparticles scavenge free radicals through catalysis. Based on the findings, a series of CeO2-based nanocrystallites of greater catalytic activities was developed, aiming to achieve the same therapeutic efficacy to cell cultures with lower nanoparticle doses. In this dissertation, zirconium-doped CeO2 (CexZr1-xO2) nanoparticles were synthesized and characterized. Their free radical scavenging activities were tested against harmful endogenous oxygen species, including hydrogen peroxide and superoxide radicals. It is found that the scavenging activity of CexZr1-xO2 nanoparticles was promoted up to four times when scavenging hydrogen peroxide. The scavenging activity of CexZr1-xO2 nanoparticles was promoted up to nine times when scavenging superoxide radicals. Importantly, their free radical scavenging activities to hydrogen peroxide correlate to the reported oxygen vacancy concentrations in the same materials. Their free radical scavenging activities to superoxide radicals correlate to the reported reducibility in the same materials. The results suggest that oxygen vacancies, lattice oxygen, electrons, and holes are involved in free radical scavenging. In addition, it is found that CexZr1-xO2 nanoparticles regulate antioxidant protein?s redox states upon catalysis. This might be a distinct antioxidant defense pathway that reactivates antioxidant protein's function, and to explain the superior protection of CeO2 nanoparticles.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Yi-Yang Tsai.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Sigmund, Wolfgang M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022459:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022459/00001

Material Information

Title: Cerium-Zirconium Oxide Nanocatalysts as Free Radical Scavengers for Biomedical Applications
Physical Description: 1 online resource (140 p.)
Language: english
Creator: Tsai, Yi-Yang
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: aging, antioxidants, catalyst, ceria, cerium, free, islet, nanoparticles, radicals, zirconia
Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Administering CeO2 nanoparticles into cell cultures was found to improve cell culture?s viability both in vitro and in vivo. In this dissertation, an in vitro study conducted in our laboratory has observed the reduction of endogenous free radical concentration in CeO2 treated cell cultures. Furthermore, it is found that CeO2 nanoparticles scavenge free radicals through catalysis. Based on the findings, a series of CeO2-based nanocrystallites of greater catalytic activities was developed, aiming to achieve the same therapeutic efficacy to cell cultures with lower nanoparticle doses. In this dissertation, zirconium-doped CeO2 (CexZr1-xO2) nanoparticles were synthesized and characterized. Their free radical scavenging activities were tested against harmful endogenous oxygen species, including hydrogen peroxide and superoxide radicals. It is found that the scavenging activity of CexZr1-xO2 nanoparticles was promoted up to four times when scavenging hydrogen peroxide. The scavenging activity of CexZr1-xO2 nanoparticles was promoted up to nine times when scavenging superoxide radicals. Importantly, their free radical scavenging activities to hydrogen peroxide correlate to the reported oxygen vacancy concentrations in the same materials. Their free radical scavenging activities to superoxide radicals correlate to the reported reducibility in the same materials. The results suggest that oxygen vacancies, lattice oxygen, electrons, and holes are involved in free radical scavenging. In addition, it is found that CexZr1-xO2 nanoparticles regulate antioxidant protein?s redox states upon catalysis. This might be a distinct antioxidant defense pathway that reactivates antioxidant protein's function, and to explain the superior protection of CeO2 nanoparticles.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Yi-Yang Tsai.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Sigmund, Wolfgang M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022459:00001


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890d43bcb8571e2eec15f6291f2a44ea8934966c







CERIUM-ZIRCONIUM OXIDE NANOCATALYSTS AS FREE RADICAL SCAVENGERS
FOR BIOMEDICAL APPLICATIONS






















By

YI-YANG TSAI


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008



































O 2008 Yi-Yang Tsai

































To my parents, fiancee, and advisors who had been highly encouraging and supportive









ACKNOWLEDGMENTS

There are many teachers and friends to thank for. Without your guidance, inspiration, and

consideration, this work would never be accomplished.

First of all, I would like to thank Dr. Wolfgang Sigmund for his inputs and scientific

advises to this work. On top of the advisory guidance, he also delivered his enthusiasm,

generosity, and philosophy to students. Without his mentoring and broad research interests, this

proj ect would have been plain and lack of innovation. I would also like to thank my committee

members, Drs. Holloway, Moudgil, Batich, and Atkinson for their constructive comments. They

have excellent reputation and are highly acknowledgeable in academia. They are prominent

scholars and are great examples in my future career development. I would like to dedicate this

work to the memory of Dr. Iaonnis Constantinidis, who was an advisor, cheerleader, and sincere

friend of mine.

I would like to thank current and previous colleagues in Sigmund Group. Without them I

would never learn my broad interests and insights in this emerging research proj ect, especially

Drs. Georgios Pyrgiotakis and Amit Daga for setting up great examples for young group

members. I would like to thank those people who have helped me in this project. Many

assistance from them start from my accidental requests, but they had big heart assisting me into

the situation. Jose Oca-Cossio, Kelly Siebein, and Gill Brubaker are especially acknowledged in

this proj ect.

Finally I would like to thank my parents for being supportive through my study in the

United States. I also like to thank my fiancee, Ju-Hsuan Cheng PharmD. She accompanied me

through the tough time, and has created wonderful time as well as memories in my life.











TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ................. ...............8....____......

LIST OF FIGURES .............. ...............9.....


LI ST OF AB BREVIAT IONS ................. ............... 11......... ...

CHAPTER


1 INTRODUCTION ................. ...............15.......... ......

1.1 Motivation............... ...............1
1.2 Obj ectives .................. ............ ...... .......... ........1
1.3 Specific Aims and Expected Outcomes............... ...............19

2 BACKGROUND .............. ...............22....


2.1 Free Radicals in Biology and Medicine............... ...............22
2. 1.1 Free Radical Theory of Aging ................. ...............22........... .
2. 1.2 Modern Free Radical Theory in Biology............... ...............24
2.2 Antioxidant Defense in Biology and Medicine .............. ...............26....
2.2.1 Antioxidant Defense System .............. ...... ..... ... .... ... .............2
2.2.2 Alternative Antioxidant Defense Other Than Direct Free Radical Scavenging.....28
2.3 CeO2 in Catalysis, Biology, and Medicine ................ ...............28.............
2.3.1 CeO2 aS Catalysts .............. ...... ... .. ...............2
2.3.2 CeO2 Nanoparticles in Biomedical Applications .............. ....................2
2.3.3 Free Radicals in Ceo2 ............... .. .. ........... ............3
2.3.4 Hypothesis of CeO2 Nanoparticles in Free Radical Scavenging ................... .........32
2.3.5 Promote Catalytic Activity by Doping Zirconium into CeO2 ................ ...............33
2.3.6 Introduction of CexZrl-xO2 ........._._ ...... .... ...............35

3 TEST HYPOTHESIS WITH CELL CULTURES .............. ...............39....


3.1 M ethodology ................. ...............39................
3.2 Results and Discussion .............. ...............44....
3.3 Summary ................. ...............54.......... .....

4 SYNTHESIS OF CERIUM-ZIRCONIUM OXIDE NANOPARTICLES ...........................55


4.1 Nanoparticle Synthesis .............. ...............56....
4.1.1 Reverse Micelle Synthesis............... ...............5
4. 1.2 Experimental Methods............... ...............57
4.2 Nanoparticle Stabilization .............. ...............59....











4.2. 1 Stabilization Using Buffer .........__........_. ...............59.....
4.3 Particle Size Analysis .............. ... ... ...............60
4.3.1 Dynamic Light Scattering Technique............... ...............6
4.3.2 Agglomerate Size Distribution ................. ...............60........... ...
4.4 Chemical Analysis................ ..... .............6
4.4.1 Inductively Coupled Plasma Spectroscopy .............. ...............62....
4.4.2 Compositions of Final Products .............. ...............63....
4.5 Summary ................. ...............63................

5 STRUCTURAL PROPERTIES AND NONSTOICHIOMETRIC BEHAVIOR OF
CERIUM-ZIRCONIUM Oxide Nanoparticles............... .............6

5.1 Characterization Techniques and Experimental Methods .............. .....................6
5.1.1 Structural Characterization Using TEM ................. ...............65........... .
5.1.2 Structural Characterization Using XRD ................. ...............66..............
5.1.3 Structural Characterization Using Raman ................. ...............67..............
5.2 TEM Results and Discussion............... ...............6
5.3 XRD Results and Discussion............... ...............7
5.4 Raman Results and Discussion............... ...............7
5.5 Summary of TEM, XRD, Raman Results .............. ..................... .......8
5.5.1 Crystalline CexZrl-xO2 Nanoparticles with Homogeneous Particle Size ................82
5.5.2 Phase Transition Detected by XRD and Raman Spectroscopy .............. ................82

6 FREE RADICAL SCAVENGING BY CERIUM-ZIRCONIUM OXIDE
NANOPARTICLES .............. ...............84....


6. 1 Prospective Scavenging Activities in CexZrl-xO2 Nanoparticles ................. ................. 84
6.2 Activities against Hydrogen Peroxide .............. ...............86....
6.2. 1 Experimental Methods ..........._._ ......_..._ ...............86....
6.2.2 Results and Discussion .............. ...............88....
6.3 Activities against Superoxide Radicals .............. ...............93....
6.3.1 Experimental Methods............... ...............94
6.3.2 Results and Discussion ..........._.._._ ...............98......__ ...
6.4 Electron Conduction in Catalysis ..........._.._ ....._.. ........_ ....... .....1
6.4. 1 Experimental Methods. .........._...._ ...............116.._..._ .....
6.4.2 Results and Discussion ..........._.._._ ...............118..._.__ ....
6.5 Summary .........._...._ ......_. ...............118....

7 IMPLICATIONS .............. ...............121....


7. 1 Distinct Antioxidant Defense Pathway ................. ...............121..............
7.2 Implications in Catalysis............... .. .. .. ................12
7.2. 1 Alternative Technique to Inspect ionic Conductivity at Room Temperature.......1 24
7.2.2 Alternative Technique to Inspect Localized Electron Conductivity ....................125

8 CONCLUSIONS .............. ...............127....

APPENDIX













A GLOS SARY ................. ...............129......... .....


LIST OF REFERENCES ................. ...............132................


BIOGRAPHICAL SKETCH ................. ...............140......... ......










LIST OF TABLES


Table page

2-1 Genetic changes and the responded free radical scavengers that affect oxidative
damage in living systems. .............. ...............27....

2-2 Classification of the phases in the CeO2-ZrOz binary system............._._ ........._._.....3 6

3-1 Intracellular amount of CeO2 in PTC-tet cells following 48 hrs of incubation with
media containing CeO2 HanOparticles. .............. ...............49....

4-1 Chemicals and the amounts used to prepare CexZrl-xO2 HanOparticles in reverse
m icelle synthesis. ............. ...............58.....

4-1 Synthesis procedures ................. ...............58........... ....

4-2 Mean agglomeration size (M,,), Dso, and Dgg of CexZrl-xO2 Suspensions obtained
using NanoTrac. .............. ...............61....

4-3 Chemical compositions of final products determined. ................ ......... ................63

5-1 Particle sizes of CexZrl-xO2 HanOparticles averaged from 50 arbitrary selected
particles in TEM micrographs. ............. ...............76.....

5-2 Crystal structures and their sub-phases in CexZrl-xO2, and the classification using
Raman spectroscopy. ............. ...............79.....

6-1 Concentrations for enzyme SOD and CexZrl-xO2 HanOparticles to achieve 50% of
inhibition rate in activity test. ................ ...............110.......... ....

6-2 Reaction rate constants of CexZrl-xO2 HanOparticles against superoxide radicals. ..........11 1










LIST OF FIGURES


Figure page

2-1 Metabolism in a mitochondrion. .............. ...............26....

2-2 Hypothetic mechanism of free radical scavenging in the vicinity of CeOz surface. .........33

2-3 Schematic diagrams showing cubic fluorite (left) and pyrochlore (right) structures
for CeOz/ZrOz and CexZrl-xOz, respectively. .............. ...............34....

2-4 Phase diagram of the CeOz-ZrOz binary system ......___ .... ... .... ......_._.........3

2-5 Phase transition of CeOz-ZrOz binary system .. ..._._._ .... ... .__ ......_. .........3

2-6 X RD and Raman spectra of (1-x) CeO xZrOz. .............. ...............3 8....

3-1 TEM micrographs of CeOz nanoparticles ................. ...............45...............

3-2 XRD pattern of CeOz nanoparticles............... .............4

3-3 Chemical compositions of the tri-sodium citrate buffer. ................ ........................47

3-4 Zeta potentials of CeOz suspensions as a function of pH .................... ...............4

3-5 TEM micrographs of CeOz nanoparticles in PTC-tet cells. ............... ...................5

3-6 Free radical concentration in PTC-tet cells. .............. ...............52....

3-7 The amount of insulin secreted by non-labeled and CeOz labeled PTC-tet cells. .............53

4-2 Optical images of CeOz nanoparticles dispersed in DI water (left), and in sodium
citrate buffer solution solution (right)............... ...............59

4-3 Agglomerate size distribution in CexZrl-xOz suspensions at pH 7.4. .............. .................61

5-1 TEM micrographs of CeOz nanoparticles ................. ...............69...............

5- 2 TEM micrographs of Ceo~sZro.zOz nanoparticles. ............. ...............70.....

5-3 TEM micrographs of Ceo. 7Zro.302 nanoparticles. ................ .............. ......... .....71

5-4 TEM micrographs of Ceo.62r0.402 nanoparticles. ............. ...............72.....

5-5 TEM micrographs of Ce0.4Zro.6O, nanoparticles. ............. ...............73.....

5-6 TEM micrographs of Ceo.,Zro.sOz nanoparticles. ............. ...............74.....

5-7 TEM micrographs of ZrOz nanoparticles. ........... _........._ ....._.. ...........7










5-8 XRD spectra of a series of CexZrl-xO2 HanOparticles ................. ................ ........ .77

5-9 Raman spectra of CexZrl-xO2 HanOparticles. ............. ...............8 1....

6-1 Hypothesized scheme of free radical scavenging by CexZrl-xO2 HanOparticles. ................85

6-2 OSC of CexZrl-xO2 meaSured a pulse chromatographic system at 400 oC. ........................86

6-3 Detection scheme used to determine the peroxide concentration in activity tests.............87

6-4 A) shows the peroxide concentration in the presence of 7 nm commercial CeO2 and
synthesized CexZrl-xO2 HanOparticles over time. B) shows the natural logarithmic
values of the peroxide concentration divided by initial peroxide concentration. ........._....89

6-5 The effective scavenging efficiency K of CexZrl-xO2 HanOparticles vs. OSC and the
amount of superoxide radicals. ............. ...............91.....

6-6 Superoxide radicals produced by hypoxanthine and xanthine oxidase. ............................94

6-7 Principle of WST-1 assay to detect superoxide radicals ................. ................. ......95

6-8 Experimental arrangement of stock solution concentrations, total concentrations, and
experimental procedures. ............. ...............97.....

6-9 The setup of stock solutions and their concentrations in a 96-wells microplate. ..............98

6-10 The results of activity test obtained using UV-Vis ................. ......__. ........._._. 101

6-11 The results of activity tests obtained using UV-Vis. ............. ...... ............... 102

6-12 The results of activity tests measured by microplate reader............_._._ ........_._. .....103

6-13 The results of activity tests measured by microplate reader............_._._ ........_._. .....105

6-14 The results of activity tests measured by microplate reader............_._._ ........_._. .....107

6-15 Inhibition curves of enzyme SOD with different incubation time ................. ...............1 10

6-16 Ce3+ COntents and OSC in CexZrl-xOz nanOparticles. ............... .......... ................1 13

6-17 Principle of the biochemistry based assay to inspect the capability for CexZrl-xO2
nanoparticles to conduct electrons in catalysis. ................ ...............115..............

6-18 Experimental arrangements of stock solution concentrations and experimental
procedures to inspect electron conduction on CexZrl-xO2 HanOparticles. ................... ......117

6-19 Redox states of cytochrome c detected using UV-Vis ................. .........................1 17

7-1 Redox states of cytochrome c in the presence or absence of superoxide radicals. ..........123









LIST OF ABBREVIATIONS


AD

CAT

CeO2

CexZrl-xO2

COX

DLS

DMEM

DNA

DCF

EDS

ELISA

ER

GSH

GPx

HD

HO

HQ

HRP

HX

ICP

IEP

MAIC

MCP

NADH


Alzheimer' s diseases

Catalase

Cerium oxide, cerium dioxide, or sometimes listed as ceria

Zirconium-doped CeO2 Solid solutions

Cytochrome c Oxidase

Dynamic light scattering

Dulbecco's Modified Eagle's Medium

Deoxyribonucleic acid

Reactive oxygen species probe, 2',7' -dichloroflurescin diacetate.

Energy dispersive spectrum

Enzyme-Linked Immuno Sorbent Assay

Endoplasmic reticulum

Glutathione

Glutathione peroxidase

Huntington diseases

Heme oxygenase

Hydroquinone

Horse radish peroxidase

Hypoxanthine

Inductively coupled plasma

Isoelectric point

Maj or Analytical Instrumentation Center at University of Florida

Monocyte cheoattractant protein

Nicotinamide adenine dinucleotide










NADP Nicotinamide adenine dinucleotide phosphate

OSC: Oxygen storage capacity

PBS Phosphate buffered saline

PD Parkinson's disease

PERC Particle Engineering Research Center at University of Florida

PHGPx Phospholipid hydroperoxide glutathione peroxidase

ROS Reactive oxygen species

SAD Selective area diffraction

SOD Superoxide dismutase

TEM Transmission electron microscopy

XO Xanthine oxidase

XRD X-ray diffraction









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CERIUM-ZIRCONIUM OXIDE NANOCATALYSTS AS FREE RADICAL SCAVENGERS
FOR BIOMEDICAL APPLICATIONS
By

Yi-Yang Tsai

August 2008

Chair: Wolfgang M. Sigmund
Major: Materials Science and Engineering

Administering CeO2 HanOparticles into cell cultures was found to improve cell culture's

viability both in vitro and in vivo. In this dissertation, an in vitro study conducted in our

laboratory has observed the reduction of endogenous free radical concentration in CeO2 treated

cell cultures. Furthermore, it is found that CeO2 HanOparticles scavenge free radicals through

catalysis. Based on the findings, a series of CeO2-based nanocrystallites of greater catalytic

activities was developed, aiming to achieve the same therapeutic efficacy to cell cultures with

lower nanoparticle doses.

In this dissertation, zirconium-doped CeO2 (exZrl-xO2) HanOparticles were synthesized

and characterized. Their free radical scavenging activities were tested against harmful

endogenous oxygen species, including hydrogen peroxide and superoxide radicals. It is found

that the scavenging activity of CexZrl-xO2 HanOparticles was promoted up to four times when

scavenging hydrogen peroxide. The scavenging activity of CexZrl-xO2 HanOparticles was

promoted up to nine times when scavenging superoxide radicals. Importantly, their free radical

scavenging activities to hydrogen peroxide correlate to the reported oxygen vacancy

concentrations in the same materials. Their free radical scavenging activities to superoxide









radicals correlate to the reported reducibility in the same materials. The results suggest that

oxygen vacancies, lattice oxygen, electrons, and holes are involved in free radical scavenging.

In addition, it is found that CexZrl-xO2 HanOparticles regulate antioxidant protein's redox

states upon catalysis. This might be a distinct antioxidant defense pathway that reactivates

antioxidant protein's function, and to explain the superior protection of CeO2 HanOparticles.









CHAPTER 1
INTTRODUCTION

In the last decade, many disorders and diseases in mammalians were found to correlate

with the damages caused by endogenous oxidative stress [1,2]. The oxidative stress in living

system is a result of accumulating free radicals, where lipids, proteins, DNA, and other

molecules receive or donate mobile electrons from free radicals [1,3]. The long-term

accumulative damages caused by free radicals finally cause mutation of cells or apoptosis

(programmed cell death) [1,4]. The damages further contribute to tissue injury or dysfunction,

and finally progress to human disorders. For example, (1) neurodegenerative diseases including

Parkinson' s disease (PD), Alzheimer's disease (AD), Huntington's disease (Juvenile HD)

[1,5,6,7]; (2) certain cardiovascular diseases including strokes, heart attacks, ischemia, and

atherosclerosis [1,8,9]; (3) genetic and metabolic diseases including Down's syndrome and

diabetes [1,10,11]; (4) cancers including liver, prostate, lung, breast, and many other cancers

[1,12]; (5) symptoms of aging including osteoporosis [1], were found to be the result of

overproduced endogenous free radicals. Meanwhile, the excess amounts of free radicals were

also a consequence of some human disorders, such as inflammatory disorders [1,13], allergies

[1,14], and infectious diseases including pneumonia [1,15] and HIV [1]. Therefore, the research

on the role of free radicals in pathophysiologic processes and the potential therapies to oxidative

stress related disorders are drawing more and more attention over time.

Mammalian cells fight against detrimental free radicals through antioxidant defense

systems by scavenging the redundant oxidative stress. Scientists have demonstrated that

administrating effective means of free radical scavengers protects biological systems from

oxidative damage of lipids, proteins, DNA, and other molecules [1,16, 17]. The protection in cell

cultures can prevent apoptosis, mutation, and enhance the viability of cell cultures [1,18]. In









living animals, the therapeutic efficacy has been demonstrated to inhibit the symptoms of AD,

PD [1,19], diabetes [1], cancers [1,20], other diseases, and even prolong laboratory animal's

lifespan [21,22,23,24].

The antioxidants or free radical scavengers that carry out antioxidant defenses in

mammalians are listed in the following [1].

* Enzymes that catalytically remove free radicals, such as superoxide dismutase (SOD),
glutathione peroxidase (GPx), catalase (CAT), and other peroxidase enzymes.

* Enzymes that catalytically synthesize compounds that can remove free radicals, such as
heme oxygenase (HO).

* Compounds that physically quench free radicals, such as carotenoids.

* Compounds that act as sacrificial agents to protect more valuable biomolecules, such as
cytochrome c (cyt c), ascorbic acid (vitamin C), a-tocopherol (vitamin E), glutathione
(GSH).

* Enzymes that catalytically recover the sacrificed compounds back into the original state,
such as cytochrome c oxidase (COX).

* Proteins that protect molecules against damage caused by other mechanisms, such as
chaperones.

* Compounds, proteins, or enzymes that regulate redox states of mitochondria, such as GSH
and protein bcl-2.

The free radical scavenging cooperates with complicated chemical and catalytic reactions.

Usually the scavenging processes carried out through redox reactions of a compound/agent, and

the particular compound/agent is activated by the second or more agents. The sequent reactions

cascade reactive free radicals and are terminated with the formation of stable species, such as

oxygen, water, nitric oxides, and carbon dioxide. However, the scavenging mechanisms in vivo

are even more complicated, and many of them remain mystical even after years of research in

this field.









However, the protection from free radical scavengers is not 100% effective. The

protection from free radical scavengers is restricted by their turnover numbers, the uptake rates,

and their distributions in vivo. In general, high doses, routine administration of antioxidants are

inevitable to achieve effective means to living animals. The demands for effective free radical

scavengers are soaring since more and more human diseases, including aging, were found to

connect to oxidative stress. A broad variety of free radical scavengers have been explored,

synthesized, and tested in order to achieve the therapeutic intervention to living systems. More

effective, lipophilic, and even organelle-targeting antioxidants have been developed in order to

satisfy the needs of life sciences.

Most free radical scavengers used for therapy are organic compounds or chelations with

transition metal ions. However, a new platform has been demonstrated to perform similar

antioxidant protection to biological systems. CeO2 HanOparticles were accidently found to

improve brain cell culture's viability and organism longevity up to six fold [25,26]. Although

how these ceramic nanoparticles improve cell's viability remains controversial, we found that

CeO2 HanOparticles reduce endogenous free radical concentrations in cells and may protect cells

as free radical scavengers [27]. Indeed, recent studies, including works published by our group

[27], have demonstrated that CeO2 HanOparticles are able to scavenge hydroxyl radicals [28],

superoxide radicals [29], and peroxides in the absence of cell cultures.

1.1 Motivation

The motivation of this dissertation was originally to improve the transplanted islet' s

viability, in order to increase the off-insulin time when using transplanted islets to treat patients

with type 1 diabetes. Type 1 diabetes is an autoimmune disorder, in which the body's own

immune system attacks the beta cells in the pancreas, the damage causes islets to shut down

insulin production. This fatal disorder causes blindness, kidney and heart failure, limb










amputation, and other diseases associated complications [30]. Recently, pancreatic islet cell

transplantation has been proposed as an ideal treatment to type 1 diabetes; however, damages

caused by mechanical trauma and anti-rej section drugs dramatically decrease the amounts of

preserved islets [3 1,32,33]. Thus, one of the maj or challenges is to increase the preserved islets

mass during isolation and post transplantation [34,35]. To improve the preservation, using CeO2

nanoparticles to improve transplanted islet's viability was proposed.

The motivations of this dissertation are as follow.

* To improve preservation of transplanted islets using CeO2 HanOparticles.

* To identify the mechanism of beneficial efficacy that carried out by CeO2 HanOparticles.

* To develop a new platform according to the concept in materials science. Aim to achieve
the same therapeutic efficacy to biological systems by lowering the applied nanoparticle
dosages.

1.2 Objectives

CeO2 is an excellent catalyst that carries out non-selective catalytic reactions through

exchange of electrons, holes, lattice oxygen, and oxygen ions. In many discussions related to its

surface chemistry, it has been demonstrated that ROS, such as superoxide, peroxide, singlet

oxygen ions, act as intermediates and are involved in the catalytic reactions [36]. Interestingly,

viability enhancement of cell cultures has been broadly achieved by using ROS scavengers.

Therefore, the key hypothesis in this dissertation relies on the fact that CeO2 is a remarkable

catalyst and it may act as ROS scavengers in the viability enhancement. With the hypothesis

aforementioned, there are several obj ectives in this dissertation.

* Test free radical concentrations in CeO2 HanOparticles treated cells, and the relationship
between free radical concentrations and cellular viability.

* Understand free radical chemistry in between biology/nanoparticles interfaces.

* Identify distinct antioxidant defenses performed by CeO2 HanOparticles beyond current
knowledge.










* Promote the beneficial efficacy of CeO2 HanOparticles.

* Achieve the same benefits to biological systems with lower dosages of CeO2-based
nanoparticles.

1.3 Specific Aims and Expected Outcomes

To achieve the obj ectives, it is necessary to develop a series of nanoparticles that exhibits

greater ROS scavenging activities. According to the aforementioned hypothesis, ROS

scavenging properties of CeO2 HanOparticles are a consequence of catalysis and the properties

could be promoted by increasing oxygen vacancies in the materials. Here, it is proposed to dope

zirconium into CeO2 HanOparticles in order to promoting CeO2 HanOparticle's ROS scavenging

activities. There are two reasons choosing zirconium as dopants. First, the oxidation form of

zirconium, zirconia (ZrO2), iS a very biocompatible material, and it has been broadly used as

biomaterials, such as dental materials. Second, zirconium doped CeO2 (exZrl-xO2) materials

have been used to improve the catalytic activity carried out by CeO2. With zirconium ions

substituting cerium ions in the lattice, it is found that CexZrl-xOz has four times more oxygen

vacancies than CeO2. The catalytic activities of CexZrl-xO2 COrrelated with the amount of oxygen

vacancies, so it can be expected to improve up to four times compared to CeO2.

The activities of these CexZrl-xO2 HanOparticles will be tested in response to crucial free radicals

in biological systems, and their activities will be compared with the enzymes that specifically

scavenge these popular free radicals. The free radical scavenging activity will be evaluated

using biochemical assays that have long been used to test enzyme activities.

There are several specific aims in this dissertation.

* Synthesize CeOz nanOparticles, and test their beneficial efficacy to PTC-tet cells, including
free radical concentrations in the treated cultures.

* Synthesize CexZrl-xO2 HanOparticles with monodispersed particle diameters, where x = 0-
1.0.










* Characterize Cexhi-xO2 HanOparticles' structures, and demonstrate the zirconium dopants
incorporated in the CeO2 lattice, and form a solid solution.

* Evaluate the scavenging activities of the synthesized nanoparticles in response to free
radicals that are influential in living systems, i.e. superoxide radicals( (O ) and peroxide
(O0, ), and compare their activities in respect to particular enzymes that scavenge
superoxide radicals and peroxide, i.e. enzyme SOD and CAT.

To attain the objectives of this dissertation, Cex 1-xO2 HanOparticles will be prepared using

reverse micelle synthesis. The structures of the synthesized nanoparticles will be investigated

using transmission electron microscopy (TEM), x-ray diffraction (XRD), and Raman

spectroscopy. The scavenging activities of Cex 1-xO2 HanOparticles will be evaluated with

commercial assays based on biochemical reactions. The following results are expected in this

dissertation.

* Free radical concentrations inhibited in the CeO2 HanOparticle treated cultures.

* Cexhi-xO2 HanOparticles with narrow particle size distribution and with diameters of 3-7
nm, whereas x = 0, 0.2, 0.4, 0.6, 0.7, 0.8, 1.0. The synthesized Cexhi-xO2 HanOparticles
are solid solutions with defined structures.

* Cexhi-xO2 HanOparticles exhibit greater free radical scavenging activity in response to
superoxide radicals and peroxide. Their scavenging activities vary with different amounts
of zirconium doping.

* The free radical scavenging activities of Cex 1-xO2 HanOparticles correlate to the
concentration of oxygen vacancy in their lattice. Free radical scavenging activities of
Cexhi-xO2 HanOparticles could be improved up to four times in in Cex 1-xO2 HanOparticles
with 20-40 % zirconium dopants.

In general, this dissertation covers the synthesis, characterization of nanoparticles, and

investigations to the free radical chemistry in between bio/nano interfaces. The focus of this

dissertation is especially on the investigations of free radical chemistry in between bio/nano

interfaces, which is accomplished by testing the free radical scavenging activity of vacancy

engineered nanoparticles. After all, the contribution in this dissertation gives a comprehensive

understanding to, first, the therapeutic efficacy of CeO2 HanOparticles in biological systems;









second, the mechanism of free radical scavenging by CeO2-based nanoparticles. Based on the

advancement in knowledge, this work may help preparing more effective free radical scavenging

nanoparticles for biomedical applications. In summary, we are aiming to achieve the same

therapeutic efficacy to biological systems by treating lower nanoparticle dosages.









CHAPTER 2
BACKGROUND

In this chapter, the background of free radicals in biology and medicine will be introduced.

After it, the antioxidant defense systems and alternative defense pathways that are used to protect

biological systems from free radical's attack will be introduced. Finally, current research based

on CeO2 HanOparticle's therapeutic efficacy will be listed and described in detail. Most

importantly, the hypothetic mechanism to describe the benefits that CeO2 HanOparticles brought

to biological systems as well as a model that can be used to improve CeO2 HanOparticle's activity

are also covered in this chapter.

2.1 Free Radicals in Biology and Medicine

2.1.1 Free Radical Theory of Aging

Free radicals are species capable of independent existence that contain one or more

unpaired electrons. Because of the unpaired electrons, free radicals tend to be reactive and easily

undergo chemistry with other molecules. There are a broad variety of free radical species in

living systems. This occurs because electrons generated in the metabolic chain react with other

molecules (such as oxygen, proteins, lipids, and DNA, RNA), forming free radicals. The

metabolic chain in mammalian cells for energy generation mostly occurs in mitochondria. By

consuming glucose and oxygen, mitochondria transfer nutrition into energy and yield as

byproducts oxygen radicals (shown in Figure 2-1). In mitochondria, electrons leaked out as

failures occur in the electron transportation chain (shown in Figure 2-1 (a)). The leaked

electrons then react with oxygen or other species, forming oxygen radicals and other free

radicals. Therefore, more than 90% of free radicals are produced by mitochondria, and the

oxidative damage usually initiated from mitochondria.









In biochemistry, the free radicals of interest are often referred to as reactive oxygen species

(ROS), i.e. superoxide radicals (O~ ), peroxides (O, ), and hydroxyl radicals (OH' ), because

the most biologically significant free radicals are oxygen-centered. ROS are involved in the cell

growth, differentiation, progression, and death. Low concentrations of ROS may be beneficial or

even indispensable in processes such as intracellular signaling and defense against micro-

organisms. However, high amounts of reactive ROS participate in cell cycles resulting in

cumulative damages to lipids and cell DNA. The expression of damages results in inflammation,

cancers, age related diseases, and aging to mammalians. In the last decades, Alzheimer's,

Parkinson's diseases, diabetes, cardiovascular diseases, eye disease, cancers, obesity, aging, and

many other diseases are found to relate to oxidative stresses caused by ROS [5,37,38]. However,

not all free radicals are ROS and not all ROS are free radicals. For example, the free radicals

superoxide and hydroxyl radical are ROS, but the ROS hydrogen peroxide (H202) is not a free

radical species, however the term free radicals usually refer to these compounds in biology.

The free-radical theory of aging is that organisms age because cells accumulate free radical

damage with the passage of time. The theory has some immediately attractive features, and is

rational to explain human disorders and aging.

* Free radicals are produced during metabolism, sometimes these free radicals are
detrimental and sometimes are for useful purposes. Once antioxidant defenses do not
scavenge them completely, the ongoing oxidative damage to DNA, lipids, and proteins
causes programmed cell death, cell mutation, or dysfunction of the cell.

* Production of free radicals can be envisaged as the consequence of genes selected because
they confer benefits in early life. For example, facilitating signal transduction in early
stage, diminishes infectious agents.

* The theory can explain the relation between metabolic rate, oxygen consumption and
lifespan. The more oxygen consumed forms higher level of free radicals, thus cause more
oxidative damages or reduce lifespan in mammals. However, higher metabolic rate does
not cause shorter lifespan directly, since the activities of enzymes or proteins maybe
greater so to reduce the damages by free radicals.










* Mitochondria are the energy plant in cells. The electron transport chain involved in
metabolism, however the leakage of electrons in the transportation generate free radicals.
The leakage of electrons is related to aging of mitochondria or insufficient enzymes and
proteins in the chain reaction.

* The accumulation of free radicals causes lipid peroxidation and reduced proteins in
mitochondria. It results in the release of cytochrome c and further lead to cell programmed
death.

* Caloric restriction in mammals often decreases levels of oxidative damage to DNA, lipids,
proteins, and attenuates age-related declines in repair systems.

* Long lived species usually have better antioxidant protection in regulation to rates of
oxygen uptake than shorter-lived species.

In conclusion, free radicals cause oxidative damage to important molecules, further

promote apoptosis or senescence that impair tissue renewal. Free radicals also generate

inappropriate cellular signaling, and contribute to age-related diseases.

2.1.2 Modern Free Radical Theory in Biology

After the free radical theory in biology was proposed, the influences of ROS have been

controversial since some studies found partially promote ROS level may be beneficial to lifespan

of mammals [1]. On one hand, ROS are detrimental to cells and tissues. On the other hand,

higher ROS level may stimulate enzyme activities and increase animal's lifespan [1]. Above all,

it was found the redox states of mitochondrial protein, cytochrome c, are also crucial in

apoptosis. It is because the reduced cytochrome c exhibits lower affinity to inner mitochondrial

membrane. Once the inner mitochondrial membranes are peroxidized, the reduced cytochrome c

can be released to inner mitochondrial membrane space. After all, the accumulated cytochrome

c is released to cytoplasm and reacts with hydrogen peroxide, forming caspase proteins

(precursors of apoptosis signaling). As cytochrome c is released from the inner mitochondria

membrane, electron transportation in metabolic chain becomes short, so more electrons can be









released from the transport chain. This results in dramatic increases of free radical generation,

and finally accelerates the progression of apoptosis (shown in Figure 2-1 (b)).

Free radicals play extremely important roles in the cytochrome c releasing processes. Free

radicals cause lipid peroxidation in inner mitochondrial membranes; superoxide radicals reduce

cytochrome c and peroxide oxidize cytochrome c before they were released to inner

mitochondrial membrane space; peroxide oxidized the reduced cytochrome c in cytoplasm,

forming caspase proteins. Although the redox states of cytochrome c are crucial in apoptosis, it

is not proper to discuss whether the reduced state or oxidized state of cytochrome c is beneficial

to cells. In conclusion, the oxidized cytochrome c is preferable in inner mitochondrial

membrane, while the reduced cytochrome c can't form caspase proteins when cytochrome c is

released [40,41,42,43].

In the modern free radical theory in biology, free radicals are no longer always detrimental

to cells and tissues, but often adequate oxidative stress maybe beneficial to mammals. First of

all, proper oxidative stress stimulates the generation of antioxidant enzymes, proteins, and assists

to defend oxidative stress of detrimental levels. Second, proper oxidative stress adjusts the redox

states of cytochrome c in mitochondria, forming oxidized cytochrome c, which has higher

affinity to bond to mitochondrial membrane. Third, proper oxidative stress reduces cytochrome

c when released, avoiding them from binding with other proteins to form caspase proteins.

Instead of the cumulative damages caused by free radicals, the redox states of cytochrome c that

altered by free radicals play a more influential role in modern free radical theory.












Outer
m to hondrial (1




DNA (I
Inner mitochondria c (1 a
space cause liid lower bonding
perox ationaffinity to mito-
A "'membrane

Inner I IIIIc vt c II

Mit~ho~l~ldlFADH2 FAD2 il T


NADHNAD2H +1/202 H20


Glucosneer



Oxygen radicals

Figure 2-1. Metabolism in a mitochondrion. A) electron transport chain. B) modern free radical
theory causing apoptosis. Figures made according to [1,2,40,41,42,43].


2.2 Antioxidant Defense in Biology and Medicine

2.2.1 Antioxidant Defense System

It has been demonstrated that administering free radical scavengers into biological systems

allows improving cell's viability, cell's survival rates, and mammal's lifespan. Some free radical

scavengers benefit cultures through removing free radicals catalytically, some through quenching

free radicals, some through regulating mitochondrial redox potential, and the others protect

cultures through stimulating the generation of antioxidants. They can be artificial or natural

chemical compounds, proteins, or enzymes that execute electrochemical reactions with free

radicals or regulate the redox states of mitochondria.











There have been broad categories of free radical scavengers reported. In this section,

several maj or free radical scavengers and their functions are listed in Table 2-1.



Table 2-1. Genetic changes and the responded free radical scavengers that affect oxidative
damage in living systems [1].


Antioxidant defense engies

Low molecular mass untiOxidatnt


Repair systems



Availability of transition metal ions "catalytic" for free
radiedl reactions


Targels orf oxidative damage


Uptake or processing of dietary antioxidants

Republe reneiive' specie's productions

Rate at which oxidatively damaged cells die


SOD. ('AT, 110 ,peroxiredoxins

Enzymes synthesizing and catabolizing GSH,
ascorbate, rate, carnosine, bilirubin, biliverdin, etc.
ProfIessome. I.on prorlemase other pmrotease. D)NA
repair. PHO~Px. enzymes that meltabolize cytolexicr
aidc~hydes chaperones
Transferrin, ferritin, caeruloplasmin, metallothionein,
haemopexin. haptoglobin, iron ion transporters, c~oppe-r
ion transporters, lactoferrin, haem oxygenases
Milcrations in confo~rmation of proteins DNA\
chromatin, membranes lipoplroteins etc
Vitamin C, E, carotenoids in guts, 11avonoid

Cytochrom e P'450. NO.X oxcridases pro~duc~ing
superox~ide and pe-ratide. clther peroxlidases
P53, bel-2, bax, genes affecting cell cycles and
apoptosis/necrosis


Many studies have shown that these disorders or diseases caused by oxidative stress could

be soothed or inhibited by administering adequate amounts of free radical scavengers, such as

the most discussed enzymes heme oxygenase-1 (HO-1), enzyme SOD and CAT, into laboratory

animals. Specifically, by exposing pancreatic tissues to SOD mimic compound, cell survival

rates can be improved to three or four times comparing to untreated pancreas during isolation


[44]. Such technical approach has been adopted in islet transplantation nowadays, in order to

increase the preservation mass of transplanted islets. In addition, exposing pancreatic tissues to

HO-1 can improve islet' s function in vivo after transplantation [45]. Specifically, exposing SOD

and CAT-mimic compounds to wild type worms, flies, fungi, can increase their mean lifespan by










44%, 30%, and 600%, respectively [22,23]. Specifically, transgenic mice with over-expressed

CAT in mitochondria can live 20% longer than wild type mice [24]. Overall, introducing

sufficient amounts of free radical scavengers into biological systems allows to elevate cell

culture's viability and survival rates under stress in vitro. Furthermore, in vivo studies show that

the administration of free radical scavengers attenuates oxidative stress and inhibits the

progression of disorders in animal models [13,19].

2.2.2 Alternative Antioxidant Defense Other Than Direct Free Radical Scavenging

Most antioxidant defense pathways are to scavenge free radicals endogenously. However,

there are particular enzymes that actually are not scavengers, but to oxidize the reduced

cytochrome c back into their functionalities in mitochondria. Peroxidases, which are also

antioxidant defense enzymes, use peroxides to oxidize another substrate. This particular

antioxidant defense pathway can be used to elucidate why the adequate amounts of hydrogen

peroxide can benefit a biological system [1,46]. Furthermore, this antioxidant defense pathway

also inhibits cytochrome c released through regulating its redox states, in which the peroxidase

oxidize cytochrome c and so to create its bonding strength with inner mitochondrial membranes.

Amongst the enzyme peroxidases, cytochrome c peroxidase and NADH oxidase are the

most important enzymes in the mitochondrial inner membrane space [47]. In the mitochondrial

electron transportation chain cytochrome c peroxidase plays as complex IV, and it takes

electrons away from the reduced cytochrome c (see Figure 2-1(a)). Lack of cytochrome c

peroxidase, cytochrome c would be reduced by electrons and be released from mitochondria to

trigger apoptosis [41,43]. The same protection is also true by enzyme NADH oxidase [37].

2.3 CeO2 in Catalysis, Biology, and Medicine

CeOz and other CeOz-related materials are excellent catalysts that carry out catalytic

reactions through the exchanging the electrons, holes, lattice oxygen, and oxygen ions with










ligands. Recently, CeOz was found to protect cultures in the stressed circumstances, and has

been proven to improve cell culture's viability.

In this section, the background of CeOz will be introduced based on its catalytic properties.

The hypothesis of free radical scavenging as well as the specific scenario related to catalysis of

CeOz will be discussed. Following the discussion, the studies that used CeOz nanoparticles to

benefit biological systems will be introduced in details. Finally the free radical scavenging

mechanism that carried out by CeOz will be discussed according to the knowledge learned in

references and the understanding of free radical theory in biology.

2.3.1 CeO2 RS Catalysts

Based on its extraordinary catalytic properties, CeOz and its related materials have been

widely applied for the use in various catalytic systems, such as three-way catalysts (catalytic

converters), solid electrolytes in solid oxide fuel cells, gas sensors, catalysts in water-gas shift

reaction, and as oxygen storage materials [48,49,50,51,52,53]. The versatility of CeOz

nanoparticles relies upon its behavior of nonstoichiometric lattice oxygen. In other words, the

catalysis carried out by the concentration of mobile intrinsic defects, undoped CeOz, i.e. oxygen

vacancies, dominates the designated chemical reactions occur via the exchange of oxygen

species in the vicinity of CeOz surfaces [36].

2.3.2 CeO2 Nanoparticles in Biomedical Applications

The therapeutic efficacy of CeOz nanoparticles was first discovered by Rzigalinski et al.

[25,26]. In the incidence, the lifespan of CeOz nanoparticles treated to neuron primary cells was

found to be improved up to six fold. Since then, the benefits of introducing CeOz nanoparticles

into biological systems were confirmed in other cell lines and tissues both in vitro [27,54,55,56]

and in vivo [57,58]. Among all the studies including this dissertation, it was found that the

intracellular free radical concentration of those treated cultures remained at ordinary level, even










though the cultures were under serious stress. Thus, it is now convincing that CeOz

nanoparticles are free radical scavengers, and protect cultures from apoptosis through antioxidant

defense.

The benefits of introducing CeOz nanoparticles into biological systems were confirmed in

different cultures and tissues both in vitro and in vivo *. Specifically, the introduction of CeOz

nanoparticles with size smaller than 20 nm into primary cultures [25,26], HT22 rodent neuronal

cells [55], or into CRL8798 breast epithelial cells [56], led to improved cellular survival with

survival rates approaching three to four times higher than control cells, when exposed to stressed

environments. The efficacy of CeOz nanoparticles to scavenge reactive oxygen species has also

been demonstrated in vivo. Specifically, in studies involving peroxide induced retinal

degradation, delivery of CeOz nanoparticles directly into the vitreous of the mouse eye prevented

against vision loss by protecting the retina from intracellular peroxides [58]. Another study

investigating spinal cord repair demonstrated that the administration of a single dose of CeOz

nanoparticles provided significant neuron-protection to adult rat spinal cord neurons [54].

Finally, intravenous administration of CeOz nanoparticles protected MCP-mice (monocyte

chemoattractant protein (MCP)-1 transgenic mice) against the progression of cardiac dysfunction

by attenuating myocardial oxidative stress, endoplasmic reticulum (ER) stress, and inflammatory

processes [57].

In these studies and the results that obtained in our works, the benefits of administrating

CeOz nanoparticles are consistent with the reduced oxidative stress in cell cultures. The

reduction of oxidative stress supports the hypothesis that CeOz nanoparticles protect cultures by

SThe dosages of CeOz nanoparticles used in the references are different from the molar concentrations that used in
the following experiments. The concentrations used in the references are based on the assumption of 1,500 cerium
atoms in a single CeOz nanoparticle, so the molecular weight of the particular CeOz nanoparticle is estimated as
172.12 X 1,500 = 258,180 g/mole. The molar concentration used in our studies are based on ionic concentration of
CeOz, where the molecular weight is 172.12 g/mole.









scavenging excess amounts of intracellular free radicals. Although the researches that studying

free radical scavenging properties of CeOz is absent, it has long been demonstrated that oxygen

radicals act as intermediates in the catalysis carried out by CeOz.

2.3.3 Free Radicals in CeO2

The mechanisms of free radical scavenging afforded by CeOz nanoparticles have been

investigated in a variety of non-biological systems including fuel cells [59], catalytic converters

[36,60], and gas sensors [6]. The applications of CeOz nanoparticles rely on the properties to

exchange their lattice oxygen ions, electrons, holes, with reagent molecules. In the exchange

processes, CeOz provides electrons to the reagent molecules, forming bonds in its vicinity, and

finally react with second reagent molecules or lattice oxygen to generate the designated products.

For example, in the oxygen storage process, oxygen molecules first adsorb on CeOz surface,

transform into superoxide radicals (O~ ), peroxide (O, ), then oxygen ions (O O2 ), and

finally migrate into oxygen vacancies (Vj') in the lattice [60,62,63]. The oxygen ions captured

in CeOz lattice or the lattice oxygen in CeOz can be released, forming gaseous oxygen or

becoming involved in other chemical reactions. The widely accepted mechanism to explain the

surface oxygen exchange on CeOz nanoparticles is shown in Equation (2-1). Based on these and

other efforts it is now believed that oxygen exchange on CeOz surface is triggered by the

adsorption of oxygen molecules, followed by a transformation into superoxide radicals and

peroxides, dissociation into oxygen ions, and finally migration into oxygen vacancies in the

lattice. Such reaction has been observed at room temperature [64]. Using spectroscopic

techniques, superoxide and peroxide radicals have been identified as intermediates of surface

oxygen exchange on ceria surface, and the exchange of environmental oxygen and lattice oxygen

has been observed at room temperature [62,64]. In addition, using atomic force microscopy and









scanning tunneling microscopy, both Namai et al. [65] and Esch et al. [63] have observed the

migration of oxygen species into CeO2 S surface oxygen vacancies at room temperature, with the

mobility of oxygen species promoted as temperature increases. The widely accepted formula of

oxygen exchange on CeO2 Surface is as follows:

02(g) 4 2(ads) 4 2(aas) 2 Oads)0 4 20,, a 20,2,1,, (2-1)

Free radicals are involved in chemical reactions catalyzed by CeO2 and oxygen deficient

materials. For example, superoxide radicals on CeO2 behave as active oxygen species in CO

oxidation; N20, NO2, and NO- radicals are detected on CeO2 in NO reduction; hydroxyl radicals

OH on CeO2 are found to participate in the water-gas shift reaction; and peroxide species on

CeO2 are found to oxidize hydrocarbon species CH4, 2zH4, C3H6 even at room temperature. In

general, it has been demonstrated that active free radicals adsorbed on CeO2 Surface behave as

triggers in various chemical reactions, and as a measure of active free radicals determined by the

oxygen vacancy concentration in the lattice [66,67].

2.3.4 Hypothesis of CeO2 Nanoparticles in Free Radical Scavenging

Although the exact process by which CeO2 HanOparticles scavenge free radicals in

biological systems is not known, one could hypothesize that it resembles the mechanism

observed in non-biological systems. The ROS generated in biological systems are reactive.

When CeO2 HanOparticles are close to ROS, the electrons of free radicals may form bonds with

mobile electronic carriers that provided by oxygen vacancies. These oxygen species then

dissociated into oxygen ions and finally diffuse into the lattice. The total reaction becomes the

exchange of environmental oxygen species and lattice oxygen. Thus, we hypothesize that

intracellular free radicals, such as superoxide radicals and peroxides would act as intermediates

in surface oxygen exchange. These oxygen species finally migrate into oxygen vacancies in









CeOz nanoparticles, and lattice oxygen may be emitted to form oxygen molecules in biological

systems. The hypothesis that describes oxygen radical scavenging by CeOz nanoparticles is

illustrated in Figure 2-2.



02- 022-
O 2(g)

02--- 22






Figure 2-2. Hypothetic mechanism of free radical scavenging in the vicinity of CeOz surface.
Oxygen vacancies (V ") provide sites for adsorption of superoxide radicals (O ') and
peroxides (Oa -). These metastable vacancies also thermally activate the transition of
radicals into oxygen ions and migration into CeOz lattice.


2.3.5 Promote Catalytic Activity by Doping Zirconium into CeO2

The catalytic properties of CeOz nanoparticles can be improved according to three

principles. It can be improved intrinsically through exciting the electrons and holes through

band gap. It can be improved extrinsically through adding impurities in the lattice. Or, it can be

improved stoichiometrically by increasing the oxygen vacancy concentration in the lattice. In

general, the catalytic properties of CeOz can be improved by doping a broad variety of elements.

In this dissertation, doping zirconium into CeOz is selected to achieve the improvement [36].

CeOz has a face-centered cubic unit cell with space group Fm3m. In this structure, each

cerium cation coordinates eight equivalent nearest-neighbor oxygen anions at each corner of the

cube, and each anion tetrahedrally coordinates four cations [36]. Both ZrOz and CeOz have

cubic fluorite structures. Mixing the two materials in solid solutions will form a pyrochlore









structure (shown in Figure 2-3). The defect-rich pyrochlore structure is the result of misfit of

zirconium and cerium ionic radii (0.084 nm and 0.097 nm, respectively) in the structure. In

addition, the relatively stable quadrievalent zirconium cations sit on trivalent/quadrivalent

cerium sites leading to the formation of extra oxygen vacancies in the solid solution, thus

decreasing coordination numbers, and expanding their lattice parameters. Several studies have

found that the concentration of trivalent cerium ions in CexZrl-xO2 Solid solutions is promoted as

more quadrievalent zirconium doping in the lattice [68,69,70]. As the results, the improved

catalytic properties in zirconium doped CeO2 are COntributed nonstoichiometrically (i.e. via

increasing oxygen vacancies) and extrinsically (i.e. via other defects). Utilizing this concept, the

concentration of charge carriers in CexZrl-xO2 Solid solutions is promoted and so their catalytic

activities are improved.




















CeO2 /rO2 Ce~zr,. 02 o

Figure 2-3. Schematic diagrams showing cubic fluorite (left) and pyrochlore (right) structures for
CeO2/ZrO2 and CexZrl-xO2, TOSpectively. Figures made according to [36].










The catalytic activities of Cexh;-xO2 HanOparticles are usually evaluated by measuring

their oxygen storage capacity (OSC). OSC represents a measure of the oxygen vacancy

concentration in metal oxides, and directly correlates to a catalyst's performance, such as in

catalytic converters, catalytic combustions, water-gas shift reactions, and oxygen storage

materials. It has been demonstrated that Cex 1-xO2 HanOparticles exhibit up to four times higher

OSC when 20-40% of cerium ions in the solid solutions were substituted by zirconium ions at

low (400 0) and high temperatures (1000 0) [71,72]. As aforementioned, it is hypothesized that

oxygen vacancies mediates free radical scavenging by Cex 1-xO2 HanOparticles. Therefore,

preparing Cex 1-xO2 HanOparticles of variant amounts of lattice oxygen vacancy and test their

activities against free radicals may provide clues to examine the hypothesis that free radical

scavenging is mediated by lattice oxygen vacancies.

2.3.6 Introduction of CexZrz-xO2

Cex 1-xO2 undergoes three maj or phase transformations at ambient temperature with

elevated zirconium concentrations (shown in Figure 2-4) [73,74]. At ambient temperature,

Cexhi-xO2 Sustained in cubic fluorite structure (c) when 0 15% of zirconium containing in the

crystal structure. The cubic fluorite phase undergoes to two metastable phases, t" and t', and then

to tetragonal structure (t) in the intermediate zirconium containing range (15 90 %). The t'

phase is a phase through a diffusionless transition from t phase, and the t" phase is a intermediate

phase between t' and c phases. The t" phase shows no tetragonality of the sublattice and it

exhibits an oxygen displacement from ideal cubic fluorite sites, so t" phase is usually referred to

as a cubic phase. Finally, at higher than 90 % of zirconium containing, Cex 1-xO2 undergoes to

monoclinic or the mixture of tetragonal and monoclinic (m) structures at ambient

temperature. Table 2-2 shows the classification of phases in Cex 1-xO2 with elevated zirconium

concentration in the lattice.








3073


0 2 4 60 80 10
ZrO ctn mol

Fiur 24.Phsedigrm f heCe ,-Z 'rO, biar sytm7]
Table~~~~ 2-.Casfcaino h pae nteCe,7bnrysse 3]

M mm


Mo nocli n ic (m )
Tetragonal (t)
Tetragonal (t ':)
Tetragonal (t ")
Cubic: ('c')


0-10
10-30
30-65
65-80
80)-1 00


N/A
>1
>1
1


P2ile
P42/11111
P4 ,/rlnc
P42/111TI
EmTn3m


The phase transformation of CexZrl-xOz, however, could be dependent on different

synthesis methods, particle size, or tempering procedures. For instance, the phase transformation
was found to postpone to higher zirconium containing range while the particle size was

engineered as small as 10 nm (shown in Figure 2-5) [75]. Specifically, the targeted particle size









in this study is smaller than 10 nm, therefore the phase transformation could be expected in the

nanoparticles containing higher zirconium, when prepared in reverse micelle synthesis.

The phase transformation of CexZrl-xOz can be distinguished by using XRD and Raman

spectroscopy. Using XRD, the transition from cubic to tetragonal phase can be identified by the

tetragonal features next to XRD peak (220). On the other hand, the transition of sub-structures,

i.e. c, t", and t' phases, can be identified using Raman spectroscopy due to the phonon excitation

which is induced by the symmetry of lattice structures. The methodology to distinguish the

crystal structures as well as phase transition is shown in Figure 2-6.


1000










180


0 10Q 20 30 40 50 60 70
ZrO: Centent (mol%~)


Figure 2-5. Phase transition of CeOz-ZrOz binary system. The transitions are particle size
dependent [75]. The phase transitions were distinguished by XRD associated with
Raman spectroscopy. Figure reproduced from [75].


80 90 100











A (B /'x .
/\ ~~~=~ T' Ir g na f at r x= x= 0.4

I:I \\x=0.3





15~x=. 20 2 0 3 0 4 0 0 0 0 0 0 0 0
Raa s=Pft (c -
Fiue26 R n aa pcr f(-)COxr )XDo 1xC3xrso
th haetaniin rm ui furtet ttaonlpas.B)Rmn hf o 1
x)erxrshwte hsetasiin of u-srctrs.Fgrerpodcdfm
[751. ;









CHAPTER 3
TEST HYPOTHESIS WITH CELL CULTURES

The therapeutic efficacy of CeOz nanoparticles has been demonstrated in various cell

cultures. The successful outcomes of viability improvements in the treated cultures have been

demonstrated to be true both in vitro and in vivo. In this chapter, the experiments are designed to

elucidate the mechanism of viability enhancement that contributed by CeOz nanoparticles. For

the experimental setups, CeOz nanoparticles were administered into PTC-tet cells, and the

endogenous oxidative stress in cell cultures was evaluated. The results suggest that CeOz

nanoparticles serve as free radical scavengers in biological systems, and their scavenging

properties most likely are through catalysis. The evidence in this chapter supports the concept

that enhancing the catalytic activities of CeOz nanoparticles may allow decreasing the dosages in

biological systems for the same therapeutic efficacy. This chapter also serves as a demonstration

of previous chapters, where the results implied the alternative platforms providing a novel

therapy to human disorders. The contents in this chapter are based on the paper that the author

published in Nanomedicine [27].

3.1 Methodology

To test the hypothesis and further establish the fundamental knowledge of this technique

for islet transplantation in vivo, we tested PTC-tet cell lines that treated with CeOz nanoparticles

in response to raised oxidative stress. The PTC-tet cell lines are murine insulinoma cells. They

are modified from P cells in islet of Langerhans in the pancreas. The PTC-tet cell lines' function

is similar to primary P cells, so they are optimum cell lines for preliminary studies in

biochemistry .

The PTC-tet cells are treated with the CeOz nanoparticles synthesized in our lab. It is

required to note that the preparation of CeOz nanoparticles in the sequent sections of this chapter









is different to the CeO2 HanOparticles in the other chapters. Essentially, a different surfactant,

lecithin was used to synthesize CeO2 HanOparticles in this chapter, while CexZrl-xO2

nanoparticles in the following chapters were prepared in a reverse micelle system formed by

surfactant, bis(2-ethylhexyl) sulphosuccinate (AOT).

Delineating the in vitro and in vivo scavenging mechanism of CeO2 HanOparticles is

important in furthering our understanding of these particles and extending their potential

biological and medical applications. To do so, it is necessary that CeO2 HanOparticles are

colloidally stable in solution for extended periods of time because biological systems may

require prolonged incubations to achieve optimum efficacy. In the previous works, CeO2

nanoparticles were synthesized and then stabilized in a solution of multivalent dispersants. A

solution of tri-sodium citrate was applied to stabilize Ceo2 HanOparticles by shifting CeO2

nanoparticles' isoelectric point (IEP).

The intracellular concentration of CeO2 HanOparticles in murine insulinoma PTC-tet cells,

a well accepted surrogate for in vitro studies of islet cell viability, was quantified and their ability

to scavenge free radicals in vitro was assessed by exposure to hydroquinone.

Synthesis of CeO2 nanoparticles

Phosphatidylcholine (laboratory grade), toluene (laboratory grade), cerium (III) nitrate

hexahydrate (99.5%, M.W. = 434.22 g/mole), and ammonium hydroxide (NH3 COntent 28~30%)

were purchased from Fisher Scientific and used without further purification. 2.285 gram of

phosphatidylcholine was dissolved in 100 ml of toluene to form reverse micelles. Five mini-liter

of 0. 1 M cerium nitrate aqueous solution was pipetted into the colloidal micelle system, and the

mixture was strongly stirred for 30 min until the system appeared homogeneous. Ten mini-liter

of 1.5 M ammonium hydroxide solution was titrated into the system to initiate electrochemical









reaction. After 45 min of stirring, CeO2 HanOparticles gradually formed in the reverse micelles.

The nanoparticles were collected by centrifugation with a force field of 18 G and then

sequentially rinsed with 50 ml of methanol, 50 ml of ethanol, and 50 ml of water in order to

remove the redundant surfactants. Between each rinse the nanoparticles were collected by

centrifugation at 18 G.

Colloidal stabilization of CeO2 nanoparticles

The CeO2 HanOparticles were dispersed in 100 ml of 0.05 M (13.17g/1) saline/sodium

citrate buffer (Cat. # 821840, MP Biomedical) and ultrasonicated until the appearance of the

suspension changed from turbid to transparent. The pH value of suspension was adjusted to 7.4

using 0.1N citric acid and sodium hydroxide solutions. The suspension was sterilized by

filtration with a 0.2 Cpm filter. The average yields from such preparation were approximately 50

mg of CeO2 HanOparticles stabilized in 100 ml of sodium citrate solution.

Concentration analysis

The yields and concentration of CeO2 Suspensions were determined by Perkin-Elmer

Plasma 3200 inductively coupled plasma spectrometer (ICP). The ICP sample was prepared by

dissolving 1 ml of the suspension in 1 ml of 95% sulfuric acid (Acros Organics). After heating

to 90oC for 24 hrs, the sample was diluted to 5ml with deionized water for ICP measurement.

Structural characterization

The crystal structure and crystallite size of CeO2 HanOparticles were characterized by X-ray

diffraction (XRD) using CuKu radiation (XRD Philips APD 3720). For the preparation of the

XRD sample, CeO2 HanOparticles were extracted before stabilization and dried in air for 24 hrs.

Micrographs and electron diffraction pattern were determined using a JEOL 2010F

transmission electron microscope (TEM) equipped with selected area diffraction (SAD). For the









preparation of TEM samples, CeO2 HanOparticles were extracted before stabilization and dried

on carbon formvar-coated grids (Electron Microscopy Sciences).

Stability tests

The stability of CeO2 Suspensions was determined by measuring the zeta potential of the

suspension using a Brookhaven ZetaPlus. One tenth mini molar of citrate-adsorbed CeO2

nanoparticles and citrate-free Ceo2 HanOparticles were prepared, and 0. 1 vol% of potassium

chloride was added to define the background electrolyte. The pH of the sample was adjusted by

titrating a trace amounts of 0. 1 N hydrochloric acid and 0. 1 N sodium hydroxide into the

suspensions.

Cell culture

Murine insulinoma PTC-tet cells were provided by the laboratory of Shimon Efrat (Albert

Einstein College of Medicine, Bronx, NY) and cultivated as monolayers in Dulbecco' s Modified

Eagle's Medium (DMEM) (Mediatech, Herndon, VA). This medium contains 20 mM glucose

and is supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan, UT), antibiotics (100

U/ml penicillin and 100 ng/ml streptomycin), and L-glutamine to a final concentration of 6 mM

(Sigma, St. Louis, MO). Cultures were maintained at 37 oC under humidified (5% CO2/95% air)

conditions, and appropriate media were completely replaced every 2-3 days.

Quantification of intracellular CeO2 nanoparticles

The intracellular amount of CeO2 HanOparticles was determined by ICP. PTC-tet cells

were incubated in media containing 0, 50, 100 and 200 CLM CeO2 for 48 hrs. At the end of this

incubation period, the cells were washed with PBS 2-3 times to remove all extracellular CeO2

nanoparticles. A pellet of 20-40 millions cells was generated by centrifugation and digested in

1ml of either 95% sulfuric acid for 48 hrs at 60 oC. The sample was then diluted with 5ml with









deionized water before proceeding with the ICP measurements. Samples with 0 CLM CeO2 were

actually prepared as "sham" controls, meaning that a volume of the vehicle solution (i.e., citrate)

equal to the volume of the CeO2 HanOparticle solution was added to the cells.

The same cultures used to determine the intracellular cerium concentration were also used

to visualize the intracellular distribution of the nanoparticles with TEM microscopy. TEM

images of CeO2 exposed and sham treated control PTC-tet cells were obtained with a JEOL

2010F microscope to visualize the intracellular compartmentation of the CeO2 HanOparticles.

Chemical analysis was performed by energy dispersive spectrum (EDS) built in the same TEM

microscope.

Quantification of intracellular free radical concentration

The ability of citrate-coated CeO2 HanOparticles to scavenge free radicals in vivo was

assessed by the following protocol. PTC-tet cells were cultured as monolayers in T-75 flasks

and incubated overnight (~18 hrs) with DMEM media that contained 0, 100 or 200 CLM CeO2

nanoparticles. Each flask contained between 30-40 million cells. At the end of the labeling

period, the cells were rinsed 2-3 times with PBS to remove extracellular CeO2 HanOparticles,

trypsinized, centrifuged, and the cell pellet re-suspended in 20 ml of fresh non-CeO2 COntaining

DMEM. Aliquots of 4 ml were placed in separate centrifuge tubes. The freely suspended cells

were exposed for 15 min to media that were supplemented with an aliquot from a stock solution

of hydroquinone (HQ) so that the final hydroquinone concentration in the media was 1 or 2 mM.

Cells exposed to 0 mM HQ were in fact sham treated with a volume of PBS equal to that of the

stock HQ solution that was added to reach 2 mM. The stock HQ solution was prepared by

dissolving 8 mg HQ in 0.5 ml of dioxane and then diluted with 9.5 ml of PBS. At the end of the

15 min HQ treatment the cells were centrifuged, the media discarded and 200 Cll of 2',7'-









dichloroflurescin diacetate (DCF) (Molecular Probes, Eugene OR) were added to suspend the

cells. Each 200 Cll suspension was placed in a single well of a 96-well plate and the plate was

placed in a Synergy HT reader (Bio-Tek, Winooski, VT) and allowed to incubate at 37 OC for 20

mins. Fluorescence was measured using an excitation filter centered at 480 nm and an emission

filter centered at 520 nm. These experiments were repeated 3 times.

Analytical assays

For all experiments described above the viability of PTC-tet cells following exposure to

CeO2 HanOparticles was assessed by a commercially available assay based on the detection of

MTT (3 -(4, 5-dimethylthi azol -2-yl)-2,5 -diphenyl -tetrazolium bromi de) (Mol ecular Prob es,

Eugene, OR), while the amount of insulin secreted in the media was measured by using mouse

insulin Elisa based immunoassay kit (ALPCO, Windham, NH).

3.2 Results and Discussion

To visualize the CeO2 HanOparticles and measure their physical dimensions, TEM

micrographs of the nanoparticles were obtained. Figure 3-1(a) shows the image and diffraction

pattern (up right) of CeO2 HanOparticles under such preparations. Based on this and similar

images from other preparations, we deduce that the CeO2 HanOparticles are equiaxed, of

monodispersed particle size. The SAD pattern indicates that these CeO2 HanOparticles were

highly crystallized. The size of the CeO2 HanOparticles range between 2 nm and 6 nm for all

preparations synthesized in this study. We arbitrary selected 50 nanoparticles in TEM images

and measured their Feret' s diameter, and the size distribution upon such estimation has been

shown in Figure 3-1(b). The average particle size is 3.7 nm, and the error is estimated as 0.5 nm

due to the contrasts of TEM images.










Figure 3-2 shows an XRD spectrum of the synthesized CeO2 HanOparticles, and the

diffraction peaks indicate that these particles are crystalline CeO2 with fluorite crystal structure.

The diffraction peaks (111), (200), and (220) crystal planes are used to calculate these particles'

crystallite size. Using Scherrer' s equation, Dhki = 0. 8932 /fhkl cos9, the crystallite sizes are

calculated as 3.7+0.6 nm, 3.9+0.7 nm, and 3.6+0.6 nm from diffraction peaks (111), (200), (220),

respectively. The average crystallite size is 3.7+0.2 nm after calculation: this value corroborates

the average particle size determined by TEM and implies that each of these CeO2 HanOparticles

is a single crystallite.



A P Ldsd 2s- B












0l2345678910
10am I 1 Foret Diamter (nm)


Figure 3-1. TEM micrographs of CeO2 HanOparticles that were synthesized using surfactant
lecithin. A) TEM image of CeO2 HanOparticles (scale bar 10 nm). Diffraction
pattern associated with TEM shows that the synthetic nanoparticles are highly
crystallized. B) Particle size distribution calculated from 50 arbitrary selected
particles in TEM images. The average Feret's diameter of synthetic nanoparticles is
3.7 nm.


























20 30P 40 50 60O 70 805 90
Diffraction Angle (28]


Figure 3-2. XRD pattern of CeOz nanoparticles. The mean crystallite size of these particles is
calculated as 3.7+0.2 nm using Scherrer's equation.


In order to minimize the influences from agglomerations and concentration gradients that

may occurr in the growth medium, it is necessary for CeOz nanoparticles to be well dispersed in

the medium. Unfortunately, the isoelectric point (IEP) of CeOz nanoparticles is around 6.5 to

7.5, whereas the CeOz nanoparticles tend to agglomerate at the targeted pH value, 7.4. Here, we

used tri-sodium citrate molecules as dispersants, since tri-sodium citrate has been used to

disperse CeOz nanoparticles through shifting the isoelectric point of CeOz. Using tri-sodium

citrate, the IEP of CeOz is shifted to lower pH values, therefore at pH 7.4 CeOz have strong

negative electrostatic charges on their surface, resulting in stabilization effects. The other

advantage of using tri-sodium citrate is due to its pH buffering properties. Tri-sodium citrate and

its salts have been used to make pH buffers as well as anti-coagulation solutions at pH 7.4.

Therefore, tri-sodium citrate buffer was used as dispersing solution as well as pH buffer in this

study. Figure 3-3 shows the major chemical compositions in tri-sodium citrate buffer. Figure 3-

4 presents the zeta potential of a citrate-adsorbed and a citrate-free CeOz suspension at various










pH values. Comparing both zeta potential profiles, we conclude that IEP of CeO2 Shifts from 8

to approximately 2 when the nanoparticles are stabilized in citrate solution. The shift of IEP is a

result of surface charge modification due to the adsorbed citrate on CeO2 HanOparticles. The zeta

potential of citrate treated nanoparticles shifts from +10 mV to -38 mV at pH 7.4, which

indicates the promotion of surface charges on the CeO2 particle surface, and provides CeO2

nanoparticles sufficient electrostatic repulsion to avoid flocculation. The citrate treated

suspension is able to retain transparency without visible deposition of particles for at least 60

days.



O OH O


/s PJ~-0 O- Naf

H+ H-O O-
H OHNa+





Chemical Compositions of
Saline/Tri-sodium Citrate Buffer:
1. Sodium citrate
2. Sodium chloride
3. Hydrochloric acid
4. Sodium hydroxide



Figure 3-3. Chemical compositions of the tri-sodium citrate buffer.











60 ---a- CeO, In water
--+-- Ceoz in sodium citrate





2 4 6j 8 10 1'2
.20. *, PH value





Figure 3-4. Zeta potentials of CeO2 Suspensions as a function of pH.


Stabilization of CeO2 HanOparticles with citrate imposes two key advantages. First, it

extends the shelf-life of the nanoparticle solution, permitting the performance of longitudinal

studies with the same nanoparticle preparation. Second, it minimizes, if not eliminates, the

precipitation of CeO2 HanOparticles from solution. Consequently, the nanoparticle distribution

within the solution is homogeneous; allowing for uniform delivery of the nanoparticles to cells

either in vitro or in vivo. Alternatively, partial precipitation of CeO2 HanOparticles could lead to

a heterogeneous delivery because the local CeO2 COncentration in the vicinity of cells may be

altered, resulting in a variable intracellular uptake of the nanoparticles.

A key parameter in assessing the efficacy of CeO2 HanOparticles to scavenge reactive

oxygen species is the intracellular concentration and compartmentation of the CeO2

nanoparticles. To quantify the intracellular concentration of CeO2 HanOparticles, PTC-tet cells

were incubated in the modified media containing 0, 50, 100 and 200 C1M CeO2 for 48 hrs. At the

end of this incubation period, the cells were digested and the concentration of CeO2

nanoparticles was determined by ICP. Table 3-1 shows the CeO2 COncentrations in PTC-tet cells

after 48 hours of incubated in different extracellular concentrations. The data show that the









accumulation of CeO2 in PTC-tet cells proportion to the extracellular concentration. However,

exposure of PTC-tet cells to large concentrations of citrate-adsorbed CeO2 HanOparticles in the

culture media (>200 pLM) was detrimental to the cells. This is illustrated by the lower number of

cells that were measured when cells were exposed to media containing 200 pLM CeO2. Since all

flasks used in this experiment were prepared identically, this lower cell count may be attributed

to (1) detachment of cells during the incubation period, (2) a decrease in the rate of proliferation

by the cells or (3) combination of (1) and (2). It is important to note that cell viability was

measured on the cells that were used to quantify the intracellular CeO2 COntent and does not

include cells that may have been detached during incubation with the nanoparticles.

Once the intracellular concentration of CeO2 HanOparticles was determined (Table 3-1), we

proceeded to determine the intracellular allocations of CeO2 HanOparticles. CeO2 HanOparticles

are clearly visible in a TEM picture of PTC-tet cells. Figure 3-5 shows the allocations of CeO2

nanoparticles in the treated cells. These data showed that CeO2 HanOparticles were present

throughout the cytoplasm as well as within organelles such as the mitochondrion, either as small

or large aggregates. TEM cell samples were examined by EDS and the presence of cerium was

clearly detected.


Table 3-1. Intracellular amount of CeO2 in PTC-tet cells following 48 hrs of incubation with
media containing CeO2 HanOparticles.


Extracellular
Amount of
CeO2 ICP Reading Cell Numbers
eonentraion pg) x106 CeO2 in Cell Viability
(pg/cell)

0 0.24 49.8 0.01 >90%
50 2.33 42.3 0.06 >90%
100 4.59 42.6 0.11 >90%
200 7.8 22.2 0.35 >90%


















~-

:- ~
f:
1.
~*
r. L" ~
D
.L ~t' :"'
3~: ~ .? ;~'rb l
4; ~, i
r

~Q;~:~mrid.".oti


Figre3-. EMmicogaps f eO~anpatile in (TCte cels. )Cnrlcl ihu
not exouet ennprils )Alag grgt fC aoatce
positione intectplamo T-e elfloin 8h xouet ei










Figure3aggregat mcorp of CeOz nanoparticlestais poiione witthi thel mitchondrion ofl wtheu




same cell in B and C. The EDS chemical analysis is shown on the upper right corner
of the image, indicates the presence of cerium in the pointed spots. Scale bar 500nm
in A-C, and 100 nm in D.



The effectiveness of these citrate-adsorbed CeOz nanoparticles to scavenge intracellular


free radicals was assessed by exposing PTC-tet cells to 1 or 2 mM HQ for 15 min. Figure 3-6









indicates the effect of HQ exposure on the intracellular free radical concentration. The data

show that in the absence of CeOz nanoparticles such an insult results in an increase in the

intracellular free-radical concentration. However, when cells were preloaded with either 50 or

100 C1M CeOz, the intracellular free radical concentration decreased regardless of the

extracellular hydroquinone concentration. These data represented the effectiveness of CeOz

nanoparticles to scavenge free radicals in vitro. It is important to note that labeling PTC-tet cells

with CeOz nanoparticles did not affect either their viability of the cells as depicted in Table 3-1

or their ability to secrete insulin as it is illustrated in Figure 3-7. These data support earlier

reports demonstrating the neuroprotective, ophtalmoprotective, and cardioprotective properties

of CeOz nanoparticles, although the CeOz concentrations in our work were up to 4 orders of

magnitude higher than in the above reports. However, our data is based on quantitative

measurement of the concentration via full chemical analysis using ICP which should be the

standard applied to measure concentrations. The earlier papers based their calculated

concentrations on a qualitative picture analysis.

Another important observation from our experiments was the lack of cytotoxic effects

associated with the use of the CeOz nanoparticles. This observation is in contrast to a recent in

vitro study on human lung cancer cells that demonstrated a significant decrease in cell viability

with exposure to CeOz nanoparticle [76]. The loss of cell viability was attributed to large

quantities of free radicals generated by the nanoparticles resulting in excessive oxidative stress.

Although the doses of CeOz nanoparticles used in that study is similar to that used in the present

study (i.e., Lin et al. used 3.5-23.3 Cpg/ml for 1-3 days, while our present study used 17.2-34.4

Cpg/ml for 2 days), the effect of the nanoparticles is opposite. Whereas Lin et al. reported

generation of free radicals, we report scavenging of free radicals. This difference in function









may be attributed to the reported photocatalyst-nature of CeO2 HanOparticles (Eg = 3.15eV) upon

the illumination of UV lights [77]. However, higher energy is required to stimulate free radical

formation by our CeO2 HanOparticles, since the broader bandgap energy (3.65eV in terms for 3.7

nm CeO2 HanOparticles) was reported for CeO2 HanOparticles smaller than 20 nm [78] (i.e., Lin et

al. used 20 nm particles whereas we used 3.7 nm particles).


Figure 3-6. Free radical concentration in PTC-tet cells. The solid gray bars represent
intracellular free radical concentrations of non-CeO2 loaded PTC-tet cells following
exposure to 1 or 2 mM hydroquinone (HQ). The shadded and white bars represent
intracellular free radical concentrations of CeO2 labeled PTC-tet cells (50 and 100
CIM respectively) following identical hydroquinone exposures. Each bar is the
average of three measurements and the error bars represent the standard deviation of
the mean. Statistical comparisons amongst the various groups were performed using
a t-test analysis and the asterisks indicate p values <0.03. The only comparison that
was not statistically (NS) significant was that between non-labeled and cells labeled
with 50 CIM CeO2 and exposed to 1 mM HQ. These data represent one of three
independent experiments.


O~ldlCe[h
E ~U ~ sorcct~,
60 1 O ~~I~ CI CeOz I

e 50 1 ~s I
E4g I
U
rv~ 30

m
e -I
10 ~k
O
P OI I
~ r~U Ha 2 nPI Ha










1000-


800-

~a 600-


* 400

200-


0
0 pM CeO, 50 pMI CeO, 100 pMI CeO,


Figure 3-7. The amount of insulin secreted by non-labeled and CeO2 labeled PTC-tet cells.
Labeled cells were exposed to media containing either 50 or 100 CIM CeO2
nanoparticles for 24 hours. Each bar represents the average of two measurements and
the error bars represent the standard deviation of the mean. Similar measurements
were also performed with cells exposed to 50 or 100 CIM CeO2 COntaining media for
48 hours with identical results.


There are many potential applications in medicine that can benefit from the free radical

scavenging abilities of Ceo2 HanOparticles. One such application having a potential for dramatic

impact is that of islet transplantation. One of the many challenges that face islet transplantation

is the loss of islet viability shortly after transplantation due to oxidative stresses induced by

mechanical trauma and/or immunosuppressive medication [79]. Although several antioxidants

such as Heme Oxygenase-1, SOD mimetics, vitamin C and/or E have been shown to improve

oxidative stress in transplanted islets, they either degrade over a relatively short time span once

incorporated into the islets, or the islets require genetic manipulation to express the desired

antioxidant protein [1,79]. The proposed CeO2 HanOparticles are a promising alternative to the

existing methods because they can be delivered intracellularly and possibly targeted to specific

organelles, are stable over long periods of time, do not require the genetic manipulation of cells

and do not affect either the viability or insulin secretion of the host islets.










3.3 Summary

In this study, we presented novel CeOz nanoparticles synthesis based on a reverse micelle

formation technique. The nanoparticles were crystalline, had an average particle size of 3.7 nm,

and could be dispersed in medium for long periods of time due to their coating with citrate.

When tested in vitro, the CeOz nanoparticles were deposited in the cytoplasm of insulin secreting

cells, and the intracellular concentration of CeOz nanoparticles reached 0.35 pg/cell. In addition,

our observation of a reduced intracellular free radical concentration was consistent with the

intracellular concentration of CeOz nanoparticles, which is proportional to the extracellular CeOz

concentrations. The ability of these nanoparticles to scavenge free radicals was maintained in

vivo, providing effective protection to PTC-tet cells against an insult by the free radical

generator, hydroquinone.









CHAPTER 4
SYNTHESIS OF CERIUM-ZIRCONIUM OXIDE NANOPARTICLES

This chapter introduces the preparation procedures prior to analyzing the free radical

scavenging activities in Cexhi-xO2 HanOparticles. The preparation procedures include synthesis

of Cexhi-xO2 HanOparticles using reverse micelle system, dispersion of Cex 1-xO2 HanOparticles

using buffers of multi-valent ions, and evaluating their particle size distributions in the

nanosuspensions. In each preparation procedure, the background of the techniques will be

described in order to provide a thorough understanding.

In order to evaluate free radical scavenging activities in Cex 1-xO2 HanOparticles, it is

important to prepare a system with narrow nanoparticle size distribution, and these nanoparticles

in the particular systems should be well stabilized in a solution. The reason for doing so is to

minimize the influences from the surface area that can be diminished by particle size and

agglomerations. Since reverse micelle synthesis provides the system a relatively narrow

nanoparticle size distribution (usually 3-10 nm), the technique was selected to synthesize Cex 1-_

xO2 HanOparticles.

These synthesized Cexhi-xO2 HanOparticles are then dispersed in a saline/tri-sodium citrate

buffer. The capability for this specific buffer to disperse CeO2 HanOparticles has been described

in Chapter 2. In this buffer, tri-sodium citrate not only acts as an optimum dispersant, but the

saline/tri-sodium citrate/citric acid system also stabilizes the suspension's pH value. A stable pH

value is essential for the activity tests to free radical scavenging, because the fluctuating pH

value can strongly influence the stability of free radicals. The particle size distribution was

measured using particle size analyzer. It is to ensure the suspensions have comparable surface

area and to confirm that particle agglomerations are prevented in the preparation.









Finally, the Ce/Zr ratio and the concentration of CexZrl-xO2 HanOparticles were measured

using chemical analysis methods. This procedure is also crucial, because it shows potential

leaching of cerium or zirconium ions in the synthesis; and it provides precise nanoparticle

concentrations for the activity tests.

4.1 Nanoparticle Synthesis

4.1.1 Reverse Micelle Synthesis

Reverse micelle method was invented as a process for preparing nanoparticles with narrow

particle size distribution in the 1980's. Reverse micelles are the reversed aggregates of normal

surfactant micelles. They are stabilized by the dissolution of the hydrophobic groups located

outside of the reversed micelles in an apolar media in order to minimize the interfacial energy.

In the reverse micelle route, each aqueous precursor is surrounded by a surfactant monolayer,

and each reverse micelle works as a single nanoreactor, and has a size around 2 nm to 10 nm.

The formation of nanoparticles is accomplished by diffusing a small amount of reactant, such as

ammonium hydroxide, into reverse micelles. According to the electrochemical stability,

nucleation and growth occur due to the increasing pH value in these nanoreactors. Therefore,

nanoparticles are formed in reverse micelles. This method has been used to synthesize a variety

of materials, including ceramics and polymeric nanoparticles. Several examples are: CaCO3,

BaCO3 [80], ZrO2 [81], CeO2 [82], CdS [83], proteins, and enzymes [84,85].

The shape and size of the products strongly depend on the synthesis conditions. By

adjusting the water/surfactant ratio to desired conditions, nanoparticles with different shapes

including spherical, planar, cylindrical, discoidal, or even vesicular can be formed [86]. Also,

particle sizes in the range from 1 nm to 100 nm with high crystallinity can be synthesized. The

reverse micelle method increases the homogeneity of chemical composition and facilitates the

preparation of nanoparticles that often have monodispersed particle size.









In order to compare the scavenging activity in our proposed system with other competitive

groups, a surfactant that other groups used to synthesize CeO2 HanOparticles was used instead of

surfactant lecithin.

4.1.2 Experimental Methods

Bis(2-ethylhexyl) sulphosuccinate (AOT, laboratory grade), toluene (laboratory grade),

cerium (III) nitrate hexahydrate (99.5%, M.W. = 434.22 g/mol), zirconyl(IV) nitrate hydrate

(99.5%, M.W. = 231.23 g/mol), and ammonium hydroxide (NH3 COntent 28~30%) were

purchased from Fisher Scientific and used without further purification. One and half grams of

AOT was dissolved in 100 mL of toluene to form reverse micelles. Five mini-liters of precursor

solution was prepared by mixing 0.1 M cerium nitrate and 0.1 M zirconyl nitrate aqueous

solutions according to the Ce/Zr ratios. The precursor solution was then pipetted into the

colloidal micelle system, and the mixture was strongly stirred for 30 minutes until the system

appeared homogeneous. Ten mini-liters of 1.5 M ammonium hydroxide solution was titrated

into the system to initiate the precipitation. The chemicals and their amounts used in the

synthesis are listed in Table 4-1.

After 45 min of stirring, CexZrl-xO2 HanOparticles had gradually precipitated in the reverse

micelles, and the appearance of the system became yellowish. The nanoparticles were collected

by centrifugation with a force field of 18 G and then sequentially rinsed with 50 mL of acetone,

Fifty mini-liters of ethanol, and 50 mL of water in order to remove the redundant surfactants.

Between each rinses the nanoparticles were collected by centrifugation at 18 G. All samples

prepared in the following chapters were synthesized or prepared at room temperature without

further heat treatment. The synthesis procedure is illustrated in Figure 4-1.





Table 4-1. Chemicals and the amounts used to prepare CexZrl-xO2 HanOparticles in reverse
micelle synthesis.


N npoa


Surfactant


Prec~ursor Solution


Oxidizing Agent


Nanoparticles


CeriumNitrate
Toluene AOT
(0.1 M)


Zirconyl Nitrate Ammonium Hydroxide
(0.1 M) (1.5 M)


Ceoz
Cezoar.22
Ceo~z~rosz
Cezoar.42
Ce~zo.4rez
Ce~zo.2rsz
ZTroz


100 ml
100 ml
100 ml
100 ml
100 ml
100 ml
100 ml


1.5 g
1.5 g
1.5 g
1.5 g
1.5 g
1.5 g
1.5 g


5 ml
4 ml
3.5 ml
3 ml
2 ml
1 ml
0


0
I ml
1.5 ml
2 ml
3 ml
4 ml
5 ml


10 ml
10 ml
10 ml
10 ml
10 ml
10 ml
10 ml


H -H. H




Toluene


O Na
O SO


Precursor
Solution


Prcursor solutionl
Scaptured in a
reverse micelle


Strong Stir


(a) Mvicelle Formation




(e) Purification

Methanol


!b) Reverse Micelle Formation




(c) Electrochemical Reaction

Ammonium hydroxide
solution


Ethanol

WMater


O


(d) Co election










1. i


Precursor Ammonium
Microdroplets hydroxide


Figure 4-1. Synthesis procedures.









4.2 Nanoparticle Stabilization


4.2.1 Stabilization Using Buffer

Although the isoelectric point (IEP) of CeO2 TangeS between 6.5 to 7.5 [87,88], using

sodium citrate buffer solution allows to disperse CexZrl-xO2 HanOparticles at pH 7.4 and to retain

pH stability at the same time. The optical images of CeO2 HanOparticles stabilized in aqueous

solutions are shown in Figure 4-2.

A 0.05 M saline/sodium citrate buffer (Cat. # 821840, MP Biomedical) was prepared

according to the manufacturer' s instructions. After the CexZrl-xO2 HanOparticles were rinsed and

collected, the products were dispersed in the sodium citrate buffer. The pH value of each sample

was adjusted to 7.4 by titrating 0. 1 N citric acid (97.5%, Sigma-Aldrich) or 0.1 N NaOH (Sigma-

Aldrich). The suspensions were ultrasonicated overnight until the suspension changed from

turbid to transparent. The suspensions were then filtered through a 0.2 Clm syringe filter in order

to remove unexpected agglomerates.





















Figure 4-2. Optical images of CeO2 HanOparticles dispersed in DI water (left), and in sodium
citrate buffer solution solution (right).









4.3 Particle Size Analysis

4.3.1 Dynamic Light Scattering Technique

Dynamic light scattering (DLS) technique is established for measuring particle size over

the size range from a few nanometers to a few microns. When light hits small particles the light

scatters in all directions so long as the particles are small compared to the wavelength. If the

light source is monochromatic and coherent, then a time-dependent fluctuation in the scattering

intensity can be observed. These fluctuations are due to the fact that the small molecules in

solutions are undergoing Brownian motion and so the distance between the scatterings in the

solution is constantly changing with time. When the coherent source of light having a known

frequency is directed at the moving particles, the light is scattered but at a different frequency.

The shift in light frequency is related to the size of the particles causing the shift. Due to their

higher average velocity, smaller particles cause a greater shift in the light frequency than larger

particles. It is this difference in the frequency of the scattered light among particles of different

sizes that is used to determine the sizes of the particles. Here, the size distribution of

agglomeration is used to evaluate the stabilization of suspensions, since the degree of

agglomerations may influence the scavenging activity of nanoparticles. The particle size

distributions of the suspensions were measured by NanoTrac (MicroTrac Inc.) at PERC.

4.3.2 Agglomerate Size Distribution

Figure 4-3 shows the agglomerate size distribution in CexZrl-xOz suspensions measured by

DLS. The mean agglomerate size Mn, Dso and Dgg of all suspensions are listed in Table 4-2. The

DLS results show that the CexZrl-xOz nanoparticles, except for ZrOz, are well dispersed in the

sodium citrate buffer solution at pH 7.4. All the suspensions are able to sustain stabilization for

more than 6 months without observable deposition.










































using NanoTrac.


to~llr '100



Figur 4-3 Aglmrt iedsrbto nCx~-Ossesosa H74(=0 .,04

0.,07 .,10.Teago eaesz adnme ecnaeo glmrto r
mesue byNao~ac

Tabl 4-. M an gglo eraionsiz (M,), soandDos f Cx ~-xO susensonsobtine


CeO2

Ceo sZr0 202
Ceo 7ZrO.302

Ce0.6Zr0402

Ceo.4Zro.O 60
Ce0 2Zro sO2

ZrO2


7.0

6.7


5.8

3.9

7.9
8.2

123.0


9.2

6.0

12.0
16.4

248.6


6.3

4.2

8.5
7.2

138.6


WW5~rr
4.9

4.6









4.4 Chemical Analysis

4.4.1 Inductively Coupled Plasma Spectroscopy

The inductively coupled plasma spectroscopy (ICP) is a sophisticated spectroscopic

technique for chemical analysis. The term inductively coupled plasma is a type of plasma source

in which the energy is supplied by electrical currents which are produced by electromagnetic

induction. The ICP system is equipped with monochromators covering the particular spectral

ranges with a grated ruling, and it is operated based on the principle of atomic emission by atoms

ionized in the argon plasma. Light of specific wavelengths is emitted as electrons return to the

ground state of the ionized elements, quantitatively identifying the species present.

The ICP samples are prepared as ions in a solution, since the samples need to be inj ected

through plasma and form an ionized mist. Therefore, acids and bases are usually involved in

sample preparations. The system is capable of analyzing the trace concentration of materials in

both organic and aqueous matrices with a detection limit range of less than 1 ppm.

The Ce/Zr ratios, nanoparticle concentrations in CexZrl-xO2 Suspensions were confirmed by

Perkin-Elmer Plasma 3200 Inductively Coupled Plasma Spectroscopy at Particle Engineering

Research Center (PERC). Sulfuric acids were used to dissolve Ceo2, Since it is reported that

CeO2 Only dissolved in this particular acid. The solubility of CeO2 WAS tested in several acids,

i.e. sulfuric acid, hydrochloric acid, and nitric acid, prior to the concentration analysis, and it was

confirmed that only sulfuric acid was able to completely dissolve Ceo2 priOr to concentration

measurements.

To prepare samples for ICP, an adequate amount of products was dissolved by 95% of

sulfuric acid (Aldrich-Sigma) at 900C overnight. The samples were diluted using DI water prior

to use. Before measuring the cerium and zirconium concentrations in the samples, 100 ppm, 10

ppm, and 1 ppm of cerium ICP standard and zirconium ICP standard (Ricca Chemical) were










used to prepare as standard samples. The nanoparticle concentrations used in this dissertation

were confirmed using ICP.

4.4.2 Compositions of Final Products

The compositions of final products are shown in Table 4-3. The compositions correspond

to the designed Ce Zr ratios, and the standard deviations of the compositions are between

0.00018 to 0.02 (standard deviations were obtained from 2 different suspension preparations).

The ICP results suggest the fact that reverse micelle synthesis method is a remarkable method to

prepare nanoparticles in laboratory scale, and the compositions of solid solutions can be

controlled in fewer than 2% of deviations.


Table 4-3. Chemical compositions of final products determined.
Std. deviation .Std. deviation
Formula Ce ratio Zr ratio Ce/Zr

CeO2 0.987 0 00236 0 011 0.00825 0 99/O 01
Ceo.8Zro.202 0.819 0 00018 0.181 0.00037 0 82/0 18
Ceo.7Zro~sO2 0.712 0 00284 0.288 0.00569 0 71/0 19
Cea.sZro.402 0.633 0 00319 0.367 0.00638 0 63/0 37
Ceo.4Zro~sO2 0.433 0 01026 0.567 0.02052 0 43/0 57
Ceo.2Zr~o.sO2 0.218 0 00626 0.782 0.01252 0 22/0 78
ZrO2 -0 00199 0 00141 1.002 0.00282 0 0/110



4.5 Summary

A series of CexZrl-xO2 HanOparticles using reverse micelle systems was synthesized, and

the chemical compositions of cerium and zirconium (i.e. x and 1-x) were precisely controlled

between 0.0 to 1.0 (x= 0.0, 0.2, 0.4, 0.6, 0.7, 0.8, 1.0). The products were washed and collected

using centrifugation. The sediments could be re-dispersed in an anticoagulation buffer, tri-

sodium citrate buffer at pH 7.4. In this chapter, measuring particle sizes using NanoTrac also

yields the agglomerate sizes of final products. According to the results in DLS measurements,









we concluded that the nanoparticles were well dispersed in a tri-sodium citrate buffer. The

chemical compositions of the final products were confirmed using ICP, and their chemical

compositions corresponded to the initial design. In this chapter, we have confirmed that CexZrl_

xO2 HanOparticles with a series of Ce/Zr ratios can be prepared in reverse micelle synthesis, and

these nanoparticles can be dispersed in sodium citrate buffer. The chemical compositions of the

final products correspond to the initial design.









CHAPTER 5
STRUCTURAL PROPERTIES AND NONSTOICHIOMETRIC BEHAVIOR OF CERIUM-
ZIRCONIUM OXIDE NANOPARTICLES

This chapter discusses the structural properties and nonstoichiometric behavior of CexZrl_

xO2. Several characterization techniques including TEM, XRD, and Raman spectroscopy are

used to confirm the crystal structures, phases, crystallite size, and crystallinity of these

nanoparticles.

It is worth to note that these nanoparticles were synthesized in a designed reverse micelle

system, and they were formed from hydrated cerium/zirconium salts at room temperature. Heat

treatments to these nanoparticles were prevented in the synthesis. The exemption of heat

treatments may cause the residual stress in the lattice, and cause mixture of several phases in

each single nanoparticles. In general, the residual stress may eliminate the symmetry of

structures and exclude the resolution beyond the limitation of the characterization techniques.

5.1 Characterization Techniques and Experimental Methods

5.1.1 Structural Characterization Using TEM

Transmission electron microscopy (TEM) is applied to determine the particle size, crystal

structures, and partially to characterize the crystallinity of CexZrl-xO2 HanOparticles. TEM is a

technique whereby a beam of electrons is transmitted through a specimen, interacting with the

specimen as it passes through it. A contrast is formed from when the transmitted electrons

interact with electrons and nucleus in the specimen and the signal is magnified and focused by an

obj ective lens and appears on an imaging screen, or is detected by a sensor such as a CCD

camera.

Experimental methods

In this section, the Feret particle diameter will be measured through particle analysis in

TEM images. The crystal structures of CexZrl-xO2 HanOparticles will be evaluated by analyzing









the SAD patterns stimulated by the transmission electron beams. The crystallinity of

nanoparticles will be estimated through analysis of the lattice planes/fringes of nanoparticles in

TEM images.

TEM samples were prepared by re-dispersing adequate amounts of CexZrl-xO2

nanoparticles in methanol, sonicating for 5 minutes, and dropping the suspensions on formvar

carbon film supported copper grids (Electron Microscopy Sciences). A JEOL TEM 2010F TEM

located at Maj or Analytical Instrumentation Center (MAIC, UF) was used to characterize these

samples.

The particle size of each sample was determined by averaging the Feret' s diameter of

nanoparticle images which arbitrarily selected in TEM micrographs. The standard deviation of

particle sizes was estimated around 0.5 nm, owing to the resolution of TEM micrographs.

5.1.2 Structural Characterization Using XRD

X-ray diffraction (XRD) is applied to determine the crystallite size, crystal structures of

CexZrl-xO2 HanOparticles. XRD is an x-ray technique based on the principle of scattering. The

electromagnetic x-ray incidents to a specimen, and interacts with its electrons and nucleus. The

contrast only appears when constructive interference occurs in the condition that the diffraction

angles satisfied Bragg's Law. The diffraction pattern of a specimen reveals the characteristics of

the particular material, including crystal structures, crystallite sizes, lattice parameters, etc.

Experimental methods

In this section, Scherrer' s equation will be used to determine the crystallite size in the

range of submicron sizes. The crystal structures of these nanoparticles will be determined by

comparing their diffraction spectra with that of the corresponded crystalline materials.

All CexZrl-xO2 HanOparticles were synthesized using reverse micelle method, washed with

methanol, collected, and dried overnight. A glass slide was cleaned and covered with a double









face tape, and the dried powders were poured on the tape and evenly applied on the tape. The x-

ray diffraction spectra were obtained using XRD Phillips APD 3720 at MAIC, UF.

The crystallite size of each sample was calculated using Scherrer' s equation.

D= (5-1)
pcos B

where D, is the volume weighted crystallite size, r is Scherrer constant falls in the range

of 0.87 to 1.0, Ai is the wavelength of radiation, and P is the integral breadth of the reflection

located at 2 0. In this study, Scherrer' s constant is 0.89, and wavelength of radiation is of 1.54

angstroms [75].

5.1.3 Structural Characterization Using Raman

Raman spectroscopy is applied to determine the crystal structures, their sub-phases, and

phase transformation in the series of synthesized Cex 1-xO2 HanOparticles. Raman spectroscopy

is a spectroscopic technique used in condensed matter physics and chemistry to study vibrational,

rotational, and other low-frequency modes in a system. The vibration relies on inelastic

scattering, or Raman scattering of monochromatic electromagnetic radiation, induced by laser

light interacting with phonons or other excitations in the system, resulting in the energy of the

laser photons being shifted due to the vibrational transitions in the molecules. In this

dissertation, Raman spectroscopy is used to characterize the crystal structures of Cexhi-xO2

nanoparticles. By detecting the vibrational energy induced by the symmetry of lattices, it is

allowed to distinguish the crystal structures and the sub-phases of materials using Raman

spectroscopy.

Experimental methods

All Cex 1-xO2 HanOparticles were synthesized using reverse micelle methods, washed with

methanol, collected, and dried overnight. Commercial 7 nm CeO2 HanOparticles were purchased










from Nanoscale Materials Inc., USA, and commercial 40 nm CeOz nanoparticles were purchased

from Alfa Aesar. Commercial ZrOz nanoparticles (tetragonal phase) were purchased from Alfa

Aesar. The samples were poured on a cleaned glass slide, and tested using Renishaw Bio Raman

at the Particle Engineering Research Center (PERC, UF). The excitation wavelength of incident

radiation of Renishaw Bio Raman is 514 nm.

5.2 TEM Results and Discussion

Figure 5-1 to Figure 5-7 represent TEM micrographs of CexZrl-xOz nanoparticles (x= 0,

0.2, 0.4, 0.6, 0.7, 0.8, 1.0) and their selective area electron diffraction (SAD) patterns. In these

TEM micrographs, nanoparticles prepared in reverse micelle synthesis have particle size between

2 to 7 nm. The particle size distributions of 50 arbitrary picked particle images are recorded

in Table 5-1, and their mean particle sizes are averaged and shown in the same table. The mean

particle sizes of nanoparticles in our preparation are between 3.2 nm of ZrOz to 3.8 nm of

CeazZra~sOz, and the standard deviation of mean particle sizes is around 0.5 nm according to the

resolution of particle images.

From Figure 5-1 to Figure 5-7, we can conclude that synthesis of CexZrl-xOz nanoparticles

using reverse micelle method is possible. Using this method, 2-7 nm of nanoparticles are

prepared, and these nanoparticles remain highly crystallized even though heat treatment was

exempted during preparation. In TEM images, lattice fringes are observed in the particle images,

implying highly ordered crystallization in nanoparticles. Furthermore, well defined rings are

observed in SAD patterns, which reconfirm the ability for this preparation method to synthesize

highly crystallized CexZrl-xOz nanoparticles in nature.














































Figure 5-1. TEM micrographs of CeO2 HanOparticles. A) Micrograph of CeO2 HanOparticles
(magnification 200 kX, scale bar 10 nm). B) Micrograph of CeO2 HanOparticles
(magnification 300 kX, scale bar 10 nm). C) Micrograph showing the lattice fringes
of CeO2 HanOparticles. The fringes imply the highly ordered crystallinity of
nanoparticles (magnification 1,000 kX, scale bar 5 nm). D) SAD pattern of CeO2
nanoparticles. The bright contrasts in the images are the accumulation of scattered
electrons, therefore the ordered ring patterns indicate the polycrystalline materials
detected in the TEM electron beam.














































Figure 5- 2. TEM micrographs of Ceo.sZr0.202 HanOparticles. A) Micrograph of Ceo.sZr0.202
nanoparticles (magnification 200 kX, scale bar 10 nm). B) Micrograph of
Ceo.sZr0.202 HanOparticles (magnification 200 kX, scale bar 10 nm). C) Micrograph
showing the lattice fringes of Ceo.sZr0.202 HanOparticles. The fringes imply the highly
ordered crystallinity of nanoparticles (magnification 600 kX, scale bar 5 nm). D)
SAD pattern of Ceo.sZr0.202 HanOparticles. The bright contrasts in the images are the
accumulation of scattered electrons, therefore the ordered ring patterns indicate the
polycrystalline materials detected in the TEM electron beam.









70














































Figure 5-3. TEM micrographs of Ceo.7Zr0.302 HanOparticles. A) Micrograph of Ceo.7Zr0.302
nanoparticles (magnification 100 kX, scale bar 20 nm). B) Micrograph of
Ceo. 7Zr0.302 HanOparticles (magnification 200 kX, scale bar 10 nm). C) Micrograph
showing the lattice fringes of Ceo. 7Zr0.302 HanOparticles. The fringes imply the highly
ordered crystallinity of nanoparticles (magnification 500 kX, scale bar 5 nm). D)
SAD pattern of Ceo. 7Zr0.302 HanOparticles. The bright contrasts in the images are the
accumulation of scattered electrons, therefore the ordered ring patterns indicate the
polycrystalline materials detected in the TEM electron beam.














































Figure 5-4. TEM micrographs of Ceo.6Zr0.402 HanOparticles. A) Micrograph of Ceo.sZr0.402
nanoparticles (magnification 100 kX, scale bar 20 nm). B) Micrograph of
Ceo.62r0.402 HanOparticles (magnification 200 kX, scale bar 10 nm). C) Micrograph
showing the lattice fringes of Ceo.62r0.402 HanOparticles. The fringes imply the highly
ordered crystallinity of nanoparticles (magnification 500 kX, scale bar 5 nm). D)
SAD pattern of Ceo.62r0.402 HanOparticles. The bright contrasts in the images are the
accumulation of scattered electrons, therefore the ordered ring patterns indicate the
polycrystalline materials detected in the TEM electron beam.














































Figure 5-5. TEM micrographs of Ce0.4Zro.6O2 HanOparticles. A) Micrograph of Ce0.4Zro.6O2
nanoparticles (magnification 100 kX, scale bar 20 nm). B) Micrograph of
Ce0.4ZroeO02 HanOparticles (magnification 200 kX, scale bar 10 nm). C) Micrograph
showing the lattice fringes of Ce0.4Zro.6O2 HanOparticles. The fringes imply the highly
ordered crystallinity of nanoparticles (magnification 600 kX, scale bar 5 nm). D)
SAD pattern of Ce0.4Zro.6O2 HanOparticles. The bright contrasts in the images are the
accumulation of scattered electrons, therefore the ordered ring patterns indicate the
polycrystalline materials detected in the TEM electron beam.















































Figure 5-6. TEM micrographs of CetzZra~sO2 HanOparticles. A) Micrograph of CetzZra~sO2
nanoparticles (magnification 100 kX, scale bar 20 nm). B) Micrograph of
CetzZra~sO2 HanOparticles (magnification 200 kX, scale bar 10 nm). C) SAD pattern
of CetzZra~sO2 HanOparticles. The bright contrasts in the images are the accumulation
of scattered electrons. The contrast of the ring is not strong due to the residual
surfactants on particle surface.











74













I


(
I~ '
*:~
-*-. ;r
c~
~i3 r ;r ,r,.~5i~~~;;i~J3~c~i:


Figure 5-7. TEM micrographs of ZrO2 HanOparticles. A) Micrograph of ZrO2 HanOparticles
(magnification 50 kX, scale bar 50 nm). B) Micrograph ofZrO2 HanOparticles
(magnification 200 kX, scale bar 10 nm). C) Micrograph showing the lattice fringes
of ZrO2 HanOparticles. The fringes imply the highly ordered crystallinity of
nanoparticles (magnification 600 kX, scale bar 5 nm). D) SAD pattern ofZrO2
nanoparticles. The bright contrasts in the images are the accumulation of scattered
electrons, therefore the ordered ring patterns indicate the polycrystalline materials
detected in the TEM electron beam.










Table 5-1. Particle sizes of CexZrl-xO2 HanOparticles averaged from 50 arbitrary selected
particles in TEM micrographs.





CeO2 0(EM 10 14 22 3 1 0 0 3 7
Cesr~o Ji- O: ( 11 27 10 2 0i 0 0i 3.6
C&D. 7Zro.sO2 1 18 22 9 0 0 0 0 3 3
C eo sLro 4a 3 11 26 10 01 0 0 0i 3.4
C&D.4Zro.6O2 0 18 22 6 3 0 1 0 3 5
Cero :2Zr o0: L 6 2 1 0i 0 0i 3.89
ZrO2 1 23 19 6 1 0 0 0 3 2


sThe mean particle size of Ce0.2Zro.sO2 WAS measured from 10 arbitrary selected particle images
due to the limited resolution in TEM micrographs.


5.3 XRD Results and Discussion

Figure 5-8 shows the XRD spectra of synthesized CexZrl-xO2 HanOparticles (x = 0, 0.2, 0.4,

0.6, 0. 7, 0.8, 1.0O). In Figure 5-8, each individual peak in the XRD spectrum of CeO2

nanoparticles fit to the corresponded XRD spectra of crystalline CeO2. The peaks (1 11) in CeO2,

Ceos8Zr0.202, Ce0.7Zr0.302, and Ceo.62r0.402 Slightly shift to a higher diffraction angle, which is a

result of lattice expansion. The shift of peaks (1 11) corresponds to the reported spectra, and is an

evidence of solid solutions throughout the nanoparticles. The tetragonal feature appears in the

XRD spectrum of CeO2, Ce0.8Zr0.202, e0. 7Zr0.302, and Ceoz62r0.402 HanOparticles, which is a

feature of phase transformation from cubic to tetragonal crystal structure. From XRD spectra,

we conclude that CeO2, Ce0.8Zr0.202, Ce0.7Zr0.302, Ce0.6Zr0.402 HanOparticles have cubic fluorite

structure, and the phase transformation occurred when more than 40% of zirconium ions doped

in the solid solution. The results correspond to crystal structures a well as phase transformation

of CexZrl-xO2 HanOparticles (x = 0.6-1.0) reported by Zhang et al. [75]. However, the XRD









spectra of CexZrl-xOz nanoparticles with more than 60% of zirconium dopants do not correspond

to the results reported in the other literatures [36,75]. In the nanoparticles synthesized in our

laboratory, the broadened XRD spectra of Ce0.4Zro.602, Ceo.2Zros02, and ZrOz nanoparticles

show mixed crystal structures in these nanoparticles. The position of the two major peaks

matches the reported XRD spectra of cubic fluorite, tetragonal and monoclinic crystal structures.

Thus, we assume that Ce0.4Zroe02, Ceo.2Zro.s02, and ZrOz nanoparticles are mixtures of cubic,

tetragonal, and monoclinic structures.


20 30 40 50 60 70 80 90
Diffraction Angle (o)

Figure 5-8. XRD spectra of a series of CexZrl-xOz nanoparticles (x= 0, 0.2, 0.4, 0.6, 0.7, 0.8,
1.0). The XRD spectrum of CeOz crystalline is plugged in the bottom to show the
correspondence of XRD between synthesized CeOz and reference data. The
tetragonal feature (t) appears and increases when more zirconium doped into the solid
solutions, indicating phase transformation in the series of nanoparticles.










The crystallite size of CeOz nanoparticles is determined by XRD spectra associated with

Scherrer' s equation, and the crystallite size is calculated to be 3.710.2 nm by averaging the

values obtained from XRD peaks (111), (200), (220). The crystallite size corresponds to particle

size determined by TEM micrographs, and the results implied that each nanoparticle is of a

single crystallite. Accordingly, the crystallite sizes of Ceo.sZro.zOz and Ceo.7Zro.302 nanoparticles

that obtained from their XRD peaks (111), (200), (220) are 2.010.07 nm and 3.410.2 nm,

respectively. However, the Ceoz62r0.402, Ce0.4Zro.602, Ceo.2Zro.s02, and ZrOz nanoparticles have

broadened XRD spectra, therefore Scherrer's equation is not suitable to calculate the crystallite

sizes of these samples.

5.4 Raman Results and Discussion

To identify the phases in CexZrl-xOz, there are six distinct Raman shifts being used. The

shift of each peak may be unique in the samples that prepared in different synthesis methods,

heat treatment, or particle size, therefore, we adopted the wave numbers in Raman spectra that

reported by Zhang et al. [75] in this dissertation. Figure 2-6(b) is the Raman spectra of CexZrl_

xO, solid solutions adopted from Zhang's work. The numbers in the figure represent the features

for identifying c, t, t ', or t phases. In order to simplify the classification, the indications of each

peak are listed in

Table 5-2, where peak 4 is a strong peak pointing toward the main cubic structure; peaks 1-

3 represent the tetragonal distortion in the lattice; peaks 5, 6 contributed by defects and oxygen

displacements that distort the cubic structure. Briefly, by identifying the intensity of Raman shift

peak 4 and peaks 1-3, it is possible to identify metastable t phase in cubic fluorite structure and

phase transformation from cubic to tetragonal structure in CexZrl-xOz nanoparticles.










Table 5-2. Crystal structures and their sub-phases in CexZrl-xOz, and the classification using
Raman spectroscopy.



c ('ubic Stubc 1 Norne 41 and 41 only

t" Cubic Metastable 1 Existing 1, 2, 3, and 4

I'Tet~ragonal Melastble -1 Ei~csling 1-6 and weak 4I

t Tetragonal Stable >1 Existing 1-6 and weak 4



Figure 5-9 shows Raman spectra of commercial 7 nm, 40 nm CeOz nanoparticles, and a

series of synthesized CexZrl-xOz nanoparticles. Due to instrumental limitation, the Raman shifts

in this work was detected down to 200 cml

The Raman spectra of commercial 7 nm, 40 nm, and synthesized CeOz nanoparticles are

shown in Figure 5-9(a). According to their Raman spectra, commercial CeOz nanoparticles and

synthesized CeOz nanoparticles have a very strong peak 4, so their crystal structures are based on

cubic fluorite structure. In the Raman spectra of synthesized CeOz nanoparticles peaks 3, 5, and

6 are noted, so t" phase also exists in the system. This is the result of lattice distortion, due to

the residual lattice stress formed in the synthesis, in which the exemption of heat treatment

caused the phenomenon.

From Figure 5-9(a) to (d), peak 4 in Raman spectra was observed, although the intensity of

peak 4 decreases with increasing zirconium dopants. The results suggest, first, samples CeOz,

Ceos8ZroazOz, Ceo-Zros302, and Ceo.62r0.402 have cubic structure. Second, the cubic structure in

these samples was distorted by zirconium dopants, while the cubic feature (peak 4) gradually

diminished with increasing zirconium dopants. Meanwhile, peaks 3, 5, and 6 remain constant in

these samples, indicating greater portion of t" phase appearing in the cubic based structure when

more zirconium ions were doped into the solid solution. In general, t phase is a metastable










phase in cubic structure, and the axial ratio of its lattice structure equals to one. In conclusion,

CeOz, Ceo.sZro.zOz, Ceo.7Zro.302, and Ceo.62r0.402 have cubic structures, and the ratio of

metastable cubic phase (t") increases when more and more zirconium doped in the crystal

structure. Comparing to the characterization of the same materials that were conducted by

Zhang et al., it is noted that the metastable t phase appears in the undoped CeOz nanoparticles.

The early appearing t phase conflicts the results reported by Zhang et al. [78]. It is believed

that the metastable t phase in CexZrl-xOz nanoparticles with lower zirconium dopants is a result

of lattice distortion. Due to the lack of heat treatment, the residual stress in CexZrl-xOz

nanoparticles may cause lattice distortion.

In Figure 5-9(e), peak 4 disappears while peaks 3, 5, 6 remain sound in the Raman spectra.

The vanished peak 4 is a sign of phase transition from cubic to tetragonal. At this state, the axial

ratio of crystal structure in samples Ce0.4Zro.6O, becomes greater than one and its crystal lattice

no longer belongs to cubic but rectangular. From Figure 5-9(e) to (g), none or weak peak 4 was

observed in the Raman spectra, and peaks 3, 5, 6 remain constant while more and more

zirconium dopants in the materials. Furthermore, peak 2 arises while more than 80% of

zirconium ions doped in the lattice. Perhaps, the rising peak 2 represents phase transition from

metastable t' to t phase. In conclusion, the results suggest that Ce0.4Zroe02, Ceo.2Zro.s02, and

ZrOz nanoparticles are no longer in cubic structure. Instead, they are tetragonal in structure, and

their axial ratio of lattice is greater than one.





















Ilil



200 3)D 4lo 5031 600 70I RU 200 300 400 500 600 700 I00
Ramar Shift (cn ') Raman Shift (cm ')

4 L Synthes zed i4,,.Z 0' D synlhsshed c~S4 ~ o,





5~45



II



200 390 40]0 5010 600 700 800 200 300 400L 5011 600 700 800
Ranin Sill1:21 )Raman Shift rem`

E SynthesizedTpP F ZI Synthesized ( 0







.Y 3 4 e h 0 00 30 40 S 0 0
Raa Shf e aa hf c

Fiur -9 amn pctaofCxh1-O Hnpatcls.A cmeril n eO,40n
COadsnhsize Ce2Hnprils )Cor 022 07 .0.D
Ceo2.42E)C04r 2.F Ce.ZosO.GsyteidZr2H)cm ria
Zr2Hnprils(hw ntefloigpg) h ubr akdi h
spcr eedeie npeiosprgah


























200 300 400 500 600 700 800 200 300 400 500 600 700 800
Rununllllt b !i m-i)) Rimar. Shutil Im.')

Figure 5-9. Continued.


5.5 Summary of TEM, XRD, Raman Results

5.5.1 Crystalline CexZrz-xO2 Nanoparticles with Homogeneous Particle Size

From the lattice fringes, diffraction patterns in TEM images, and defined peaks in XRD

spectra, it can be concluded that Cex 'l-xOz nanoparticles prepared by reverse micelle synthesis

method are crystalline in nature. In addition, the defined Raman shifts in Cexh'1-xOz

nanoparticles support the conclusion that these Cexh'1-xOz nanoparticles are crystalline in nature.

Although other studies have shown that Cex 'l-xOz nanoparticles precipitated in other reverse

micelle systems were amorphous and heat treatments were preferred, we have been able to

perform an improved, more confined reverse micelles system that allows improving crystallinity

of the final products. Observed in TEM, all samples synthesized in the particular reverse micelle

system are homogeneous in size, and their particle sizes are around 3-7 nm in diameters.

5.5.2 Phase Transition Detected by XRD and Raman Spectroscopy

In this work, the information obtained using XRD and Raman spectroscopy has provided

credible conclusions in terms of crystal structures as well as sub-structures of the lattice. Using










XRD, it is found that CeO2, Ce0.8Zr0.202, Ce0.7Zr0.302, and Ceo0e2r0.402 HanOparticles have cubic

fluorite structure, while Ce0.4Zro.6O2, Ce0.2Zro.sO2, and ZrO2 HanOparticles have complex

structures which mixed with cubic fluorite, tetragonal, and monoclinic structures. Perhaps the

mixed crystal structures in Ce0.4Zro.6O2, Ce0.2Zro.sO2, and ZrO2 HanOparticles are a result of

residual stress in the lattice due to exempted heat treatment. Using Raman spectroscopy, the

phase transformation from cubic to tetragonal was found when more than 60% of cerium ions

were replaced with zirconium dopants. In addition, it is found that the more metastable cubic

phase t were formed when the concentration of zirconium dopants increased in the lattice. The

portion of t" phase finally no longer sustained in cubic structure, so the metastable t" phase

finally transformed to tetragonal structure. The crystal structure as well of its sub-structure of

Cex2rl-xO2 HanOparticles can be identified using XRD associated with Raman spectroscopy. In

addition, the phase transformation, sub-phases in Cex2rl-xO2 HanOparticles can be detected by

comparing the shifted peaks of Raman spectra.









CHAPTER 6
FREE RADICAL SCAVENGING BY CERIUM-ZIRCONIUM OXIDE NANOPARTICLES

It is hypothesized that free radical scavenging in CeO2 HanOparticles is a consequence of

catalysis, and the scavenging activity of CeO2 HanOparticles can be improved structurally by

doping zirconium into lattice. To demonstrate our hypothesis is correct, the scavenging activities

of CexZrl-xO2 HanOparticles shall be tested and the obtained activities will be used to compare the

reported OSC in the same materials. By doing so, it may help to understand the scavenging

mechanism carried out by CexZrl-xO2 HanOparticles. In addition, the scavenging mechanism of

these nanoparticles can be promoted further, according to the obtained knowledge.

In this chapter, two of ROS, hydrogen peroxide and superoxide radicals, will be used to

probe the scavenging activities of CexZrl-xO2 HanOparticles. It is because these two oxygen

species are very influential ROS in biological systems, and do play essential roles in cell

metabolism as well as apoptosis. To successfully test nanoparticle's scavenging activities,

several innovative methodologies are applied in this dissertation. These innovative

methodologies are assays that based on biochemical reactions, and these assays are the first time

used to evaluate a metal oxide catalyst' s activity at room temperature. At the end of this chapter,

the free radical scavenging activities of CexZrl-xO2 HanOparticles as well as the scavenging

mechanism will be discussed based on the structural properties of CexZrl-xO2 HanOparticles.

6.1 Prospective Scavenging Activities in CexZrz-xO2 Nanoparticles

As aforementioned, free radical scavenging mechanism carried out by CexZrl-xO2

nanoparticles is a result of surface oxygen exchange. Based on the hypothetic mechanism

proposed in Figure 2-2, there are two prerequisites in the scavenging. First is that the transition

of oxygen species only occur when mobile electrons available in the lattice. Second is that the

exchange only occur when oxygen vacancies present in the lattice. According to the









understanding, a model that is available to describe free radical scavenging by CexZrl-xO2

nanoparticles is illustrated in Figure 6-1.

Since the available mobile electrons can be provided by oxygen vacancies in the lattice, the

scavenging activities of CexZrl-xO2 HanOparticles likely are limited by the concentration of

oxygen vacancies in the lattice. The concentration of oxygen vacancies in CeO2-based materials

is reported to be proportional to their OSC. Therefore, we can presume that free radical

scavenging activities of CexZrl-xO2 HanOparticles follow the level of OSC in the same materials.

Fortunately, OSC of CexZrl-xO2 have been reported, and Figure 6-2 is reproduced from the paper

published by Descorme et al. [62]. Since the catalytic activity of CeO2 HanOparticles can be

promoted up to four times by incorporating 20-40 % of zirconium dopants, it is prospected to

have four times of greater scavenging activities in CexZr -xO2 HanOparticles than in undoped

CeO2 HanOparticles.










Mitochondrion Eeg


10

Ce,Zr _,O,+IOX t CeZr,_ Oz+2e + V + -O2
~c 2

Figure 6-1. Hypothesized scheme of free radical scavenging by CexZr -xO2 HanOparticles.




















05 15 5 6 0


20a ~ ~ aru cotn (ma-%)--------

Figure~~~~~~~~~~~~~~~~~~~I 6-.OCo e*1xmaue us crmtgahcsse t40o.Fgr
rerdue frm[6]

6.2 Aciite agis yrgnPrxd

Amn teractv oxgnseis yrgnprxd n ueoierdcl r h
mostinfuenial OS n mtaboismas ell s pogrmmedcel deth. othof hem avebee
widely~ ~ dicse an thei cocnrtoshv endrcl esrdi ilgcl sytms[]
The~~~~~~ ~~~ cocnrtoso yrgnprxd nbooia ytm eeee sdt ersn h
lvlof enoeosoiaiesrsoree celclresvaiiy[]Ints capeexr
xO, naoatce'saegn civte gis yrgnprxiea hsooia ee r
prsete. hi evl f ydoenpeoxdei slete t imc heefiiecyofths
nanoartclesin ioloica sysems
6.. Ep rimnt Methods

To tethdoe eoiesaegnhdognprxd ouin eemxdwt
CexrlxO,(x 0,0., .4,0.,07,0., 10)susenion ad he esdua proidecocntato









over time was evaluated. Peroxide concentrations in each sample were determined using an

Amplex@ red hydrogen peroxide/peroxidase assay kit (Cat. # A22188, InvitrogenTM). The

principle to measure hydrogen peroxide is illustrated in Figure 6-3. The Amplex@ reagents were

prepared according to manufacturer' s instructions. To prepare samples for activity tests, 100 Cll

of 200 CIM CexZrl-xOz suspensions were prepared in a 96-wells plate (Corning Inc.). A small

amounts of substrate hydrogen peroxide was titrated into 0.25 M phosphate buffer (pH = 7.4),

forming 100 CIM hydrogen peroxide. To initiate the free radical scavenging tests, 100 Cll of 100

CIM hydrogen peroxide solutions were pipetted into each well at the designated time. The

reactions were finally stopped by introducing enzyme horse radish peroxidase and Amplex@

reagents into each well at the designated reaction time. The Amplex@ reagents were also diluted

using 0.25 M phosphate buffer. Each sample's peroxide concentration was determined by

measuring the optical density of the red product, resorufin, at 570 nm absorbance. Optical

density measurements were made using Synergy HT multi-detection microplate reader (BioTech

Instruments Winooski, VT).


,O, HO
OHOO HO O


N Horse Radish Peroxidase N


CH,
10-acetyl-3,7-d ihyd roxyphe noxazi ne resorufin
(Colorless) (Red, absorption at 571nm )

Figure 6-3. Detection scheme used to determine the peroxide concentration in activity tests.
Colorless Amplex@ Red reagent (10-acetyl-3 ,7-dihydroxyphenoxazine) reacts with
peroxide, forming red resorufin. The reaction is catalyzed by enzyme horse radish
peroxidase.









6.2.2 Results and Discussion

The free radical scavenging efficiency of CexZrl-xOz nanoparticles are determined by

measuring the reduction of 50 CIM hydrogen peroxide solutions in 100 CIM CexZrl-xOz

suspensions over time. Figure 6-4(a) shows the peroxide concentration decreased by a series of

CexZrl-xOz nanoparticles over time. Figure 6-4(b) shows the residual hydrogen peroxide

concentration divided by initial peroxide concentration in natural logarithmic scale. In Figure 6-

4(a), it is obvious that Ceo -ZrosO, and Ceoz62r0.402 nanoparticles exhibited the most efficient

radical scavenging property, while Ce0.4Zro.602, Ceo.s2ro.2O, exhibited moderate efficiency and

CeOz, Ceo.zZro.sOz exhibited the lowest efficiency. There is no significant peroxide

concentration change in the sample of ZrOz nanoparticles. In Figure 6-4(b), the slope of each

profile represents the hydrogen peroxide scavenging activities of each sample. From high to

low, the rankings of peroxide scavenging activities in CexZrl-xOz nanoparticles are Ceo. 7Zro.302,

Cece62r0.402, Ce0.4Zroz602, Ceos82roa20, CeOz, Ceo.zZro.sOz, commercial CeOz, and ZrOz,

respectively.

According to the free radical scavenging mechanism illustrated in Figure 2-2, the

scavenging mechanism of hydrogen peroxide can be summerized in Equation (6-4). It is: the

adsorption/desorption of peroxide, the transition of peroxide to oxygen ions, and finally the

diffusion of oxygen ions into oxygen vacancies in the lattice. The oxygen vacancies are then

restored by emitting lattice oxygen molecules. To evaluate the scavenging activities of CexZrl_

xO, nanoparticles, we then simplify Equation (6-4) to Equation (6-5). In Equation (6-3), rcl, rc_ ,

and re,, refer to the adsorption constant, desorption constant, catalytic rate constant, respectively.

The initial condition in each sample is the same and hydrogen peroxide concentration in each

sample is very diluted, thus we are able to ignore the influences from adsorption/desorption (Ky/Krc

I) in the reactions. Therefore, the scavenging activities (i.e. effective rate constant) K can be








































C- omm. CeOz --0-- Syn, CeOz -L Syn, CeosgZro.Or
--7-- Syn. Ceo.7Zro0.3O2 ---- .- S) n. Ceo.6Zr0.40. --3--8- Syn, Ceo.4Zro.60 z
Syn. C~eorZrosO2 OD Syn. ZrOz


calculated using Equation (6-7). The scavenging activity K of each CexZrl-xO2 Samples is shown


in Figure 6-5 in terms of Ce Zr molar ratios.



A L


~i~~......
`B,




`"---:I.-----.~ -~nr


Time (min.)


Figure 6-4. A) shows the peroxide concentration in the presence of 7 nm commercial CeO2 and
synthesized CexZrl-xO2 HanOparticles over time. B) shows the natural logarithmic
values of the peroxide concentration divided by initial peroxide concentration. The
slopes of profiles in B represent the peroxide radical scavenging efficiency.























h
--" 0.4



r~
O

~i~ o~l
c


Time (min.)


Figure 6-4. Continued.




Intrinslc Intrinslc

Of2 + CexZrT-xO2 +Vj'" + 2e Of2 --CexZrT-xO2 +V y' + 2e ~
-1
Intrinslc Intrinsic

<20 --- CexZrt-xO2 +V y' + 2e 20 2- xZrT-xO2 +V yd > (6-1)
Intrinslc Intrinsic




11
0,2- xZrT-xO2 +O 2 -02erO +0- x0r -x2 +V +2eZO 6
22



1 t


0) 20) 40 6 80 100 12 140 160r 180o 200










d[O, ]
= K [Of-]- [Cex z-xO, ]
dt


(6-3)



(6-4)


[Of ]
In
[ Of ] ,,,,,,,


where K =







3.5-


3.0 -



2.0

1.5 -



1. -


K-[Cex Zrt-xO, ]-t


,
KM + [Of2 ]


r, + r
r,


250


200



150



100


O
50


1.5 a



1.0 a



0.5 Z



0.0


Cerium content (x inl CexZr;-xrO?)

Figure 6-5. The effective scavenging efficiency K of Cex 'l-xOz nanoparticles (bar diagram) vs.
OSC (m) and the amount of superoxide radicals (A) detected on Cex 'l-xOz
nanoparticles. The error bars represent the standard deviations of effective
efficiencies between each data points [62].


According to the activity tests, the hydrogen peroxide scavenging activities in Cex 'l-xOz

nanoparticles is enhanced by doping zirconium ions into CeOz lattice. More importantly, the


0.0 0.2 0.4 0.6 0.8 1.0









scavenging activities of CexZrl-xOz correlates to the magnitudes of OSC measured by pulse

chromatographic system [62]. In Figure 6-5, we show that the scavenging activities increase

with the amounts of zirconium dopants in CeOz lattice. The scavenging activity increases to

maximum in Ceo. 7Zro.302 nanoparticles, and gradually decreases when more than 40% of cerium

ions are substituted. There is no distinguishable peroxide radical scavenging property observed

in the case of pure ZrOz nanoparticles. On the other hand, OSC measured at 400 oC increases to

the highest level when 20% to 40% of cerium ions were substituted; then OSC dropped gradually

when more cerium ions were replaced; finally OSC totally diminished in pure ZrOz

nanoparticles. In addition, the scavenging activity of Ceo. 7Zro.302 nanoparticles is four times

greater compared to the undoped CeOz nanoparticles, corresponding to the same enhancement

that occurred in Ceo.632ro.37O, nanoparticles in respect to OSC [48,62]. According to the

correlation, the nanoparticle concentrations utilized to evaluate the effective free radical

scavenging efficiencies can be replaced by the concentration of active sites. Equation (6-8)

shows that the enhanced free radical scavenging efficiency in CexZrl-xOz is a consequence of

improved lattice oxygen vacancies. The correspondence between the free radical scavenging

efficiency and the magnitude of OSC confirms the idea that free radical scavenging is mediated

by oxygen vacancies. Also, the results deliver a message that the enhanced free radical

scavenging activities are achieved by manipulating oxygen vacancies in the CeOz lattice.


In = -K -[FT]-.t (6-5)


It is worth to note that CexZrl-xOz nanoparticles may also exhibit greater superoxide radical

scavenging properties compared to the undoped CeOz. It has been demonstrated that CeOz

nanoparticles exhibited superoxide dismutase mimetic properties, and their catalytic activity is

comparable to enzyme superoxide dismutase [66]. Yet the greater superoxide radical scavenging










properties in CexZrl-xO2 HanOparticles will be demonstrated later. Prior to this work, we found

that Descorme et al. had detected larger amounts of superoxide radical adsorption on CexZrl-xO2

nanoparticles than on the undoped CeO2, even at room temperature. Importantly, the superoxide

radical adsorption on CexZrl-xO2 Surfaces also corresponds to the magnitude of OSC in CexZrl_

xO2 HanOparticles (shown in Figure 6-5) [62].

In summary, their activities were tested in a 50 CIM hydrogen peroxide solution in order to

investigate their free radical scavenging efficiency in biological systems. The free radical

scavenging activities of these CexZrl-xO2 HanOparticles is enhanced up to four times in

Cea.7Zrt.302 HanOparticles and gradually decreased when tetragonal phase appear in the

structures. The effective free radical scavenging activities of CexZrl-xO2 HanOparticles correlates

to the magnitude of OSC in the same materials, where OSC is used to evaluate oxygen vacancy

concentration in metal oxide catalysts. The correlation confirmed that CexZrl-xO2 HanOparticles

scavenge hydrogen peroxide through the exchange of peroxide ions and lattice oxygen, and the

scavenging activities are mediated by oxygen vacancies in the lattice. As consequence, the

enhanced free radical scavenging properties of CeO2 HanOparticles are achieved by increasing

oxygen vacancies in the lattice through doping zirconium into CeO2 HanOparticles. The

improvement is as great as four times compared to the undoped CeO2.

6.3 Activities against Superoxide Radicals

In this section, the scavenging activities of CexZrl-xO2 HanOparticles are tested against

superoxide radicals. Different to hydrogen peroxides, superoxide radicals are defined free

radicals which have unpaired electrons. Since the electron configurations of superoxide radicals

are unpaired, superoxide radicals are much more reactive than hydrogen peroxide and they have

great affinity to become hydrogen peroxides or oxygen molecules by electron exchange.

Therefore, the half life of superoxide radicals is as short as 0.05 second at high concentrations.









Perhaps, the damage caused by superoxide radicals to biological systems may be more influential

than hydrogen peroxide due to their reactivity [1].

6.3.1 Experimental Methods

Due to the short half life of superoxide radicals, it is important to generate this species

continuously in the activity tests. To generate the designed radicals, we used hypoxanthine and

enzyme xanthine oxidase to produce superoxide radicals, hydrogen peroxide, and uric acids. The

hydrogen peroxide produced in this reaction is then removed by adding large amounts of enzyme

CAT, in order to prevent the effects from hydrogen peroxide. The reaction used to generate

superoxide radicals is illustrated in Figure 6-6.



NH~~aXantine t0
N Oxidase



H H H
terminated by
hypoxanthine uric acid catalase


Figure 6-6. Superoxide radicals produced by hypoxanthine and xanthine oxidase.


To detect the presence of superoxide radicals, a water soluble compound, sodium salt of 4-

[3-4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetaoi]1,3-benzene disulfonate (WST-1, Dojindo),

is used as a superoxide probe. Only react with superoxide radicals, WST-1 salts become an

irreversible, water-soluble formazan dye (shown in Figure 6-7). The dye formation can be

observed at around 450 nm with the maximum density at 438 nm spectrophotometrically [89],

and is not affected by the generation of hydrogen peroxide in the reaction. In this study, the

formation of formazan dye is observed using two techniques, UV-Vis (UV/Vis Perkin-Elmer

Lambda 800) and microplate reader. UV-Vis spectroscopy and microplate reader are both










spectrophotometric basis; however, comparing to microplate reader UV-Vis is a technique with

higher resolution and allows measuring the dye formation continuously. However, the

application of UV-Vis is limited by the numbers of samples that can be measured. Therefore, the

technique based on microplate reader is engaged in order to test multiple samples

simultaneously.



xanthiine
O, 202 WST-1 farrnazan



H,02 207-- WST-1
uri~c aIcid
SOD


Og + Hz,,











WST-1


Figure 6-7. Principle of WST-1 assay to detect superoxide radicals [90].


For the samples prepared for UV-Vis, 0.5 mM EDTA (Aldrich), 0.5 mM hypoxanthine

(Aldrich), 9.65 mU/ml xanthine oxidase (Invitrogen), 0.5 mM WST-1, and 1 mM CexZrl-xOz

nanoparticle suspensions (x = 0, 0.4, 0.6, 0.7, 0.8, 1.0) were prepared in 100 mM phosphate

buffer (pH = 7.4, Aldrich) as stock solutions. Two hundreds micro-liters of each stock solution

except hypoxanthine stock solution were titrated into a 1 ml cuvette, i.e. EDTA, xanthine









oxidase, WST-1, and nanoparticle suspension. To initiate the reaction, 200 Cl1 of hypoxanthine

stock solution was titrated into the cuvette in order to generate superoxide radicals. The total

concentrations of CexZrl-xO2 HanOparticle suspensions were then diluted to 200 CLM in the

reactions. The dye formation was detected by UV-Vis at 450 nm. The absorbance was recorded

and shown in Figure 6-11. In order to obtain the reaction rate constants of CexZrl-xO2

nanoparticle against to superoxide radicals, another sample prepared with enzyme SOD (Cat.

#1901 17, MP Biomedical LLC) was repeated instead of nanoparticle suspensions. In the

experiment, various stock solutions with SOD concentrations (0.25, 0.5, 1, 5, 10, 100 U/ml) were

mixed in the cuvettes instead of 1 mM CexZrl-xO2 HanOparticle suspensions. The total

concentrations of enzyme SOD were then diluted to 0.05, 0. 1, 0.2, 1.0, 2.0, 20 unit/ml in the

reactions. The stock solutions, their final concentrations, and experimental procedures are

illustrated in Figure 6-8.



Step 1:
0.5 mM EITTA, 200 pl
9.65 mUflm xanthine oxdse, 200 Crl
0.5mMWST-1, 200 pl~
Total Cocenwtration in 1 ml sample
1 mM CerZn-.Oz orr SOD 200 Crl
0.1 mM EDTA
1.93 mU/ml xanthine oxidase
Step 2:
0.1 mM WST-1
Initiate reacction by titmating0.mMCrn.2oSO
0.5 mM hypoxanthine, 200 Crl
0.1 mIM hypoxanthine


Step 3:
Observe absorbance at 450 nm
per2 seconds





cuvette










Figure 6-8. Experimental arrangement of stock solution concentrations, total concentrations, and
experimental procedures.

For the samples prepared for microplate reader, different nanoparticle concentrations were

tested in the setups with various superoxide production rates. The superoxide radical production

rates were controlled by the amounts of enzyme xanthine oxidase in the reactions. In the

standard radical production rate, the total concentration of 1.93 mU/ml xanthine oxidase was

used. However, the reaction may take too long to complete. To conquer this disadvantage, the

other two tests with five times (5X) and twenty-five times (25X) of xanthine oxidase (i.e. 9.65

mU/ml and 48.25 mU/ml in total) were repeated using the same experimental procedures.

In the test with standard superoxide production rate (i.e. 1.93 mU/ml xanthine oxidase in

total), 0.5 mM EDTA, 0.5 mM hypoxanthine, 9.65 mU/ml xanthine oxidase, and 0.5 mM WST-1

were prepared in 100 mM phosphate buffer (pH = 7.4) as stock solutions. The control samples

were a series of enzyme SOD with different concentrations. They were 0.01, 0.05, 0. 1, 0.5, 1.0,

2.5, 5.0, 10, 20, 50 U/ml SOD prepared in phosphate buffers. The total concentration of SOD in

the reaction therefore was divided by five, which were 0.002, 0.01, 0.02, 0.1, 0.2, 0.5, 1.0, 2.0,

4.0, 10 U/ml. The samples of our interest were CexZrl-xOz nanoparticle suspensions (x = 0, 0.4,

0.6, 0.7, 0.8, 1.0) with three different concentrations. The nanoparticle concentrations were 0.05,

0.25, 1.0 mM in stock solutions, therefore the total concentrations in the test were 0.01, 0.05, 0.2

mM. After the stock solutions were prepared, 50 Cll of tested samples, control samples, and other

stock solutions (except hypoxanthine solution) were titrated in a 96 well microplate (Corning,

NY) accordingly. Finally, 50 Cll of hypoxanthine solutions were titrated into each well quickly in

order to initiate the reaction. The plate was then placed in microplate reader to read the optical

density absorbancee) at 450 nm. Figure 6-9 shows the arrangement of sample preparation in

microplate and the concentration of each stock solution in the plate.






















Step 1:
a) 0.5 mMEITT~A, 50 pl
b) 9.65 mUlml xanthine oxidase, 50 pl
c) 0. 5 mM WST-1, 50 rl
d) CexZrs..Os or SOD ,50 pl


Step 2:
Filled blank 1, 2, 3
Blankc 1:
a+htewater+hrypoxanthine n
Blankc2: a+b~t+-c+dtwte
Blank 3: a+h+- +water+water


Step 3:
Titrated 50 pl1 of 0.5 mM
hypoxatnthine to imitiate
reaction


soD 50 Ulml Bnlan I Blrank 3||
-Blank 2
son 2n wmi son~ 05 osml son cL ur~mi

son to U/ml LaCd muce2 5 pM sO pM

90D 5 Urnt I mM Cee.s oz02 250 pM 50pM

SOD 2.5 Ulm~l I mM rM Y7ro3G2 250 pM 50 pM

SO L Ulml 1 mM Cae70402 250 pM 50pM

SODO.~5 Ulm~l 1 mM Cae2rozaO2 250 pM 50 pM

soDO0.1 Ulml imM r02 250 pM 50pM~


The activity tests underwent in higher superoxide production rates were carried out under

similar protocols. The concentrations of xanthine oxidase that was used to precede different

superoxide production rates were increased from 1.93 mU/ml to 9.65 mU/ml and 48.25 mU/ml

in total.


Figure 6-9. The setup of stock solutions and their concentrations in a 96-wells microplate.


6.3.2 Results and Discussion

Figure 6-10 shows the results of activity tests using UV-Vis. In Figure 6-10, each profile

represents the absorbance of each sample over time. The absorbance readings are results of

formazan dye formation, which are caused by the interaction of WST-1 and superoxide radicals.

Thus, the dropped optical density in the control sample is a result of protection received from

enzyme SOD. The inhibition from dye formation represents the enzyme activity, so the


Step 4:
Measure optical
density at 450 mn
accoardingly









inhibition is used to calculate the rate constants in the reaction. In Figure 6-10, the inhibition to

dye formation is SOD concentration dependent. The samples with 1 U/ml or higher

concentration of enzyme SOD achieved 100% protection, while the different percentage of

protection is distinguishable in the diagram.

Figure 6-11 shows the results of the same experimental setups but CexZrl-xO2 HanOparticles

were used to scavenge superoxide radicals instead of enzyme SOD. In Figure 6-1 1, Ce0.4Zro.6O2

has greater inhibition percentages to dye formation, indicating a greater superoxide scavenging

activity. Also, ZrO2 has no significant superoxide scavenging activity, while CeO2 has the

lowest activity among all the CeO2-based nanoparticles. The rankings of superoxide scavenging

activity in CexZrl-xO2 HanOparticles are listed as follow according to the results in Figure 6-11.

Ce04Zr0602 06eZr0402 07eZr0302 08eZr0202 > CeO2 > ZrO2

The results can be concluded that zirconium dopants in CeO2 HanOparticles prompt the

scavenging activities against superoxide radicals. The scavenging activities in CexZrl-xO2

nanoparticles become zirconium dopants dependent but not oxygen vacancy concentration

dependent.

Figure 6-12 shows the results of activity tests measured by microplate reader. The

superoxide radicals in this test were produced by 1.93 mU/ml xanthine oxidase and 0.1 mM

hypoxanthine. Each data point was the average of three different measurements, and the

standard errors were included in the marks. Figure 6-12(a) shows the inhibition to dye formation

in samples with enzyme SOD. In Figure 6-12(a), it is obvious that the inhibition to dye

formation is SOD concentration dependent. The SOD concentrations greater than 0.5 U/ml

totally inhibit dye formation made by superoxide radicals. In Figure 6-12(b), inhibition to dye

formation also occurred as 200 CIM CexZrl-xO2 HanOparticles were involved in the reaction.










According to the profiles, Ce0.4Zro.6O, nanoparticles exhibit the greatest superoxide radical

scavenging activity, and the activities of CexZrl-xOz nanoparticles decrease as fewer dopants

incorporated in the solid solutions. In contrast to the CeOz-based nanoparticles, ZrOz

nanoparticles exhibited none or indistinguishable scavenging activity. In Figure 6-12(c) and (d),

inhibition to dye formation is reproducible when lower nanoparticle concentrations present in the

reaction. 50 CIM and 10 CIM CexZrl-xOz nanoparticles also performed distinguishable scavenging

activity against superoxide radicals; however, the protections were not as efficient as in the

systems of 200 CIM Ce0.4Zro.6O, nanoparticles. According to the results in Figure 6-12, it can be

concluded that the rankings of superoxide scavenging activity in CexZrl-xOz nanoparticles are,

Cee)4Zre)60, > Ce()6Zre)40, > Ce, zZr,,0, > Ce,,Zr,,0, > CeO, > ZrO,

The scavenging activities of CexZrl-xOz nanoparticles are zirconium dopants dependent.

The results correspond to the observation in experiments carried out by UV-Vis.

The same experiment was reproduced with higher xanthine oxidase concentrations (9.65

and 48.25 mU/ml) in order to shorten the reaction time. The results are shown in Figure 6-13

and Figure 6-14. In Figure 6-13 and Figure 6-14, the results are reproducible compared to that of

1.93 mU/ml xanthine oxidase. The scavenging activities of CeOz and Ceo.sZro.zOz are very

close, while very limited scavenging activity was observed in ZrOz samples. The superoxide

radical scavenging activities of CexZrl-xOz nanoparticles are ranked as follow,

Cee)4Zre)60, > Ce()6Zre)40, > Ce, zZr,,0, > Ce,,Zr,,0, > CeO, > ZrO,











~~ -m- control
-20 U/ml SOD
S1-- 2 U/ml SOD
-7- -1 U/ml SOD
9 ~0.2, U/ml SOD)
S- 0. 1 U/ml SOD
0.05 U/ml SOD





I





0 2 4 6 8 10

Time (minutes)

Figure 6-10. The results of activity test obtained using UV-Vis. A series of enzyme SOD with
different concentrations (total concentration) protected WST-1 salts against
superoxide radicals. The higher the absorbance indicates the more formazan
formation caused by superoxide radicals.










I -m--- control
-e-200 pM CeO,
-- 200 ClM Ce fZr0.20
.t:-7 200 c'M Ce froaO~0
-fj ~200 CtM Ce fZroAO0
4- 00 M Ce frozO
200 pM Zr~O











0 2 4 6 8 10

Time (minutes)

Figure 6-11. The results of activity tests obtained using UV-Vis. A series of 200 CIM CexZrl-xO2
nanoparticles (x = 0, 0.4, 0.6, 0.7, 0.8, 1.0) with different concentrations protected
WST-1 salts against superoxide radicals. The higher the absorbance indicates the
more formazan formation caused by superoxide radicals. The results have shown that
CexZrl-xO2 HanOparticles exhibit extraordinary scavenging properties against
superoxide radicals.













m I 0 Ulml SOD
4,0 Ulml SOD
2.0 U/mi SOD
7 .0Uml SOD
0.5 Ulml SOD ea1
4 0.2 Ulml SOD e 2
0.1 U/lml SOD) *r~
0.02 Ulml SOD g $ ,a
A 0.01 U/miSOD ,)l *~,.
*0.002 Ulml d
control *






0 50 100 150 200 250 3(00
Time (minutes)


a control
*200pM CeO,
200 pM Ce xZr ,O?
'I 200 pM Ce /rO99
200 pM Ce fZr O
4 "~i~MCeaZr O q
200 IM ZrO


a
D


I III


0 50 100 150 200 250 300
Time (minutes)

Figure 6-12. The results of activity tests measured by microplate reader. A) shows the inhibition
to dye formation in samples with enzyme SOD. B) in samples with 200 CIM CexZrl_
xO2 HanOparticles. C) in samples with 50 CIM CexZrl-xO2 HanOparticles. D) in
samples with 10 CIM CexZrl-xO2 HanOparticles. The superoxide radicals in all samples
were produced by 1.93 mU/ml xanthine oxidase and 0.1 mM hypoxanthine. Each
data point was the averaged result of three samples. Standard errors are shown in each
data point; however most of the error bars are smaller than the marks.
























-C~~lf
a~~iIii~I

I PSII ~C3+~
~~~C~~C3
c;~~
didO **+ rll~irlQ1'1
a ~*+++~*I(*(~~~
glira~ il~rdQ


50 100 150 200 250 3


Control
*1 50 pM CeO,
50 JOM Ce ,xro
r 50 pIM Ceu7 asO?
50 pM Ce ~rO?
r 50 pM Ce ~IrO~0
50 pM 2rO


eY
E
r~&ti


L


Time (minutes)


control
*l I0 yM CeO,
I 0 pM Ce fr O
TT I M Ceo r ~O_
I 0 gM Ce firO,
4 I0 9M Ce fr ,O
10 yM ZrO,


a *
a e *;
eLe
an **


r


d
hr
Yf3
Yi.." S
fr
Eg tfq~i(
~,afr


Time (minutes)


Figure 6-12. Continued.


e -r























































;Y I 41 I I t ~ i I (
0 20 40 60 80 100 120 140 60)

Time (minutes)

Figure 6-13. The results of activity tests measured by microplate reader. A) shows the inhibition
to dye formation in samples with enzyme SOD. B) in samples with 200 CIM CexZrl_
xO2 HanOparticles. C) in samples with 50 CIM CexZrl-xO2 HanOparticles. D) in
samples with 10 CIM CexZrl-xO2 HanOparticles. The superoxide radicals in all samples
were produced by 6.95 mU/ml xanthine oxidase and 0.1 mM hypoxanthine. Each
data point was the averaged result of three samples. Standard errors are shown in each
data point; however most of the error bars are smaller than the marks.


10 U/mi SOD)
e 4.0 Ulmt SOD
2.0 Ulml SOD
V 1.0 U/mi SOD
0.5 U/mnl SOD go
4 0. U/mi SUD as *
0.1 U/ml SOD m s w
S0.02 Ullml SOD I **
* 0.01 Ulml SOD
* 0.002 U/mi
e control eg


0 20 40


Bo I "It o l


Time (minutes)


aI control
*200pM CeO,
?~200 pM Ceo rFIrU29
P 200 I1M Ceo?"eO
200 pM Ceo frMO2
I 200 pM Ces/CO 0
200 rM 2rO,


*
ma s







































control
- 10RM CeO,
10pM CeMr,0,O
V~ 10 M CeograsOC" II a =
10pM Ceo gra40. ma go

I01-M Zr O, e*





I I~
0 20 4 60 80 100 12 140
Tim (mnues


.O


m~0 8
8iQ~


m control
*50 clM CeO,
-r 50 pM Ceg~r,
r 50pM Ceo ro l
50 pM Ce, Zr ~
S50 uMl Ceo Zr,
50) cM ZrO


20 40 60 80 100 )20 1400


f=
,n


Times (minutes)


Figure 6-13. Continued.












I10 U/ml SOD3
4.0 Ulml SOU
~ ~2,0 U/ml SOD
r 1,0 U/mi SOD
0.5 Ulml SOD r8f
.- 0.2 U/mi SOD ;
0.1 Ulml SOD -
0.02i U/ml SOD
o .01 U/ml SOD .








m control
*I 200 pM CeO,










0 5 10 15 20 25 30 35 40
Time (minutes)




to dy fomaio i smples ihezm O. )i ape it 0 MCr
xO2Hanpatices 20C) ien smlswih5 MCeu1x2 a0atcls )i
samleswit 1 00 M Cexrl-xO2 Ha~atce Th sueoxd raia l in alape
wee prouce by' 48 U..25 2 mU/m xatieoiaead01Mhpxnhn.Ec
datapoin wa thel avraedZreublt of the ape.Sadr err r hwnec
data20 pont hoee os fte ro as r mllrta temrs












ii control

1,50 pM Cea rO,
; 950tgMCea~jrO """
S50 yM Ce ,rO ~ n
S50 pM Ce, ~rOla
~~ 50 pM ZrO,


4 44
4 4 j~ 4haI
1 11I 1



0 5 10 15 20 25 30 35 40
Tirne (minutes)


D

I control
SI 0 pM CeraO,
I 0 M Ce~,O,rO
Ir 10 IM Ce,~;~O rO
l 10MCe,,Zr 4
10rpMIelZrO69 n



o -
uI I I I ll 1+ f~
09 5 10 1 0 25 3 5 4
Tie( ints




Figureim 6-14.Contnued









To calculate the reaction rate constants of CexZrl-xO2 againSt superoxide radicals, a method

to compare the inhibition percentages of CexZrl-xO2 HanOparticles and enzyme SOD is adopted.

The inhibition rate is perceived using the equation in the follow,




((Ablank1 Ablank3-) -(Asrample- Ablanik2 )
Activity/(inhibitionrate%) = x 100 (6-6)
(Ablank1 Ablank3)



Where A represents the absorbance or optical density of samples or blanks in each well.

The denominator in the equation represents the increasing amounts of dye formation in the

absence of samples but with superoxide radicals in the reactions. The numerator in the equation

represents the inhibition due to tested samples (i.e. enzyme SOD or CexZrl-xO2 HanOparticles) in

the presence of superoxide radicals. Thus, the equation is to show the inhibition percentage of

dye formation when the tested samples involved in superoxide radical production. To obtain the

rate constant, the amounts of enzymes or nanoparticle concentrations that are required to achieve

50% of inhibition are compared. Using the comparative method, the reaction rate constants of

CexZrl-xO2 HanOparticles can be obtained according to the reaction rate constant of SOD, 1.3-

2.8X109 M^ s^ (at pH = 7.2, M.W. = 32,600 g/mole).

The inhibition curve of enzyme SOD is shown in Figure 6-15, and the calculation is based on the

results in Figure 6-12. The concentrations for enzyme SOD and CexZrl-xO2 HanOparticles to

achieve 50% of inhibition rate are calculated according to the results in Figure 6-12 and Figure

6-15, and the values are shown in Table 6-1.































































Since the concentration of 1.0 U/ml SOD approximately equals to 120 nM SOD [29], the

reaction rate constants of CexZrl-xOz nanoparticles can be obtained using simple calculations.

Due to the high molecular weights of SOD, it is necessary to interpret the reaction rate constant


-a- -30 min.
-*- -60 min.
-120 min.
-r- 180 min.
240 min.
-4 ~- 270 min.


O -



0.6


"I
0.01


i "' I ""'I
I I0

Concentration of SOD (U/ml)


0.2
-.


i



Q~--''


Figure 6-15. Inhibition curves of enzyme SOD with different incubation time. The amounts of
SOD that achieved 50% inhibition rate is used to compare the amounts of CexZrl-xOz
nanoparticles that has achieved the same inhibition.


Table 6-1. Concentrations for enzyme SOD and CexZrl-xOz nanoparticles to achieve 50% of
inhibition rate in activity test.

Concentration to achieve 50%/ inhibition rate (mole/L)


Measured at
.0.150
30 nnn.
60 min. 0.137

120 min. 0.129
180 min. 0.115


~sOr ~aesZmtOr L~aa~8.~~t ~w.rZirr.rCh L~aa*a~s.c~


42.1

40.8

46.1
48.6
49.8
50.3


44.3

45.5

51.3
54.6
56.6
57.6


2983.3

6020. 0

16200.0
5645.5
4773.3
3413.6


240 min.
270 min.


0.102









into the format of (g/1)- s-l instead of Mls^l. The rate constants of all samples are shown in

Table 6-2. In Table 6-2, it is shown that rate constants of CexZrl-xO2 HanOparticles are even

higher than enzyme SOD in respect to scavenging superoxide radicals. Enzyme SOD has been

tested to exhibit the fastest reaction rate among all enzymes, and its reaction rate is only limited

by colloid frequency to superoxide radicals.


Table 6-2. Reaction rate constants of CexZrl-xO2 HanOparticles against superoxide radicals. The
kinetic analysis was measured by microplate reader using WST-1 salts in the samples
with 1.93 mU/ml xanthine oxidase.
MV.W. Iate Constant Rate Constant


SOD 32,600 1.3-2.8X109 39.9X(10
Ce01 172.72 0.6-1.2XI06 3.7-7.1X10~
CeaaZrw.20 163.25 03.6-.1XI06 3.4-7.0X10~
CearZrw~sOl 158.24 4.3-8.0XI06 27.0-503.5X109

CeQ~iaa~me 153.96 5.0-8.4X(10 32.8-54.5XI03
CearZrwaeOt 143.67 5.2-8.9XI06 36.0-61.9X109
ZrOl 123.22 2.7-16.9XIP" 0.021-0.14X105



In Table 6-2, it is found that zirconium dopants are able to improve superoxide radical

scavenging activities of CexZrl-xO2 HanOparticles. Instead of correlating to the OSC, the

scavenging activities of CexZrl-xO2 HanOparticles actually correlate to the amounts of zirconium

dopants in the system. The rankings and magnitude of improvements do not correspond to the

hypothesis.

The results suggest that superoxide radical scavenging activities of CexZrl-xO2

nanoparticles are correlated with the amounts of zirconium dopants in the lattice but not oxygen

vacancy concentrations. The results also conflict what was demonstrated in previous chapters

that hydrogen peroxide scavenging activities of CexZrl-xO2 HanOparticles are correlated with









oxygen vacancy concentrations in lattices. The differences are due to the nature of hydrogen

peroxide and superoxide radicals. Comparing to hydrogen peroxide, superoxide radicals are

more active due to their unpaired electron configurations. In the catalytic reactions with catalase

two steps of chemical reactions are involved. First, hydrogen peroxides donate an oxygen ion to

the reduced catalase, forming an intermediate compound. Second, the intermediate compound

donates its extra oxygen ion to another hydrogen peroxide, forming water and one oxygen

molecule. Except the reactions contributed by electron exchange, it is notices that oxygen ions

also involve in catalysis in the case of hydrogen peroxide. Different to the catalysis to hydrogen

peroxide, the catalytic reactions between superoxide radicals and enzyme SOD do not involve

the exchange of oxygen ions. It is, superoxide radicals only donate their unpaired electrons to

form oxygen molecules or receive electrons to form hydrogen peroxide. In the particular

reaction, the scavenging to superoxide radicals are carried out through electron exchange and no

oxygen ions are involved. Thus, it can be concluded that the catalysis of superoxide radicals

only contributed by the electron exchange with enzyme SOD. However, in the case of CexZrl_

xO2 HanOparticles, the electron exchange not only contributed by oxygen vacancies, but also

contributed by other cationic defects in the lattice.

To further explain what species of cationic defects contribute in superoxide radical

scavenging, it is necessary to investigate the Ce3+ C4+ ratios in CexZrl-xO2 HanOparticles. Figure

6-16 shows the Ce3+ COntents and OSC in CexZrl-xO2 HanOparticles reported by Vidal et al.

[68,69]. In Figure 6-16, the Ce3+ COntents were obtained using magnetic susceptibility

measurements through a magnetic balance under flowing H2 (5%)/He gas. The Ce3+ COntents, or

as reduction percentages, were calculated on a [Ce3+ lC3+ C4+] basis. According to the

reports [68,69], zirconium dopants in the solid solutions promote reduction capability of










nanocrystallites. The results are reproducible at low and high temperatures (473-973 K). The

results shown by Vidal et al. may provide the answer for the results that obtained in the

superoxide radical scavenging tests.

A B



-a68'32-HS s 68/32-HS
+srsm ae 0/0 -EC-50/50-HS
~ 0 15/85-HS l'//p I- 15/85-HS



27 7 7 7 107 27 47 63 87 1
Redutio Temeraur (K Reuto eprt K








thereatio. T e electrn Texchangerele on~ th odcto felectrons ien thale atlyis




Therefre, the reac ntionraes i catalysis of superoxide radicals depends on tembl electron xhn ic




carriers that are available in the scavenging. Doping zirconium into CeOz not only promotes the

oxygen vacancy concentration structurally, but also promotes the Ce3 contents and so to

increase the electron holes. In other words, doping zirconium into CeOz improves superoxide

scavenging activity due to the increased extrinsic cationic defects and nonstoichiometric oxygen

vacancies. The promoted mobile electronic carriers increase the reducibility of Cexh'>-xOz

catalysts [68,69]. This perspective is able to explain why more zirconium dopants in

nanoparticles always improve their activity, and the results do not correspond to oxygen vacancy









concentration in CexZrl-xO2 HanOparticles. The "active sites" in superoxide radical scavenging,

i.e. mobile electronic carriers including oxygen vacancy, electron holes, are listed in the

following equation. Equation 6-13 shows that oxygen vacancies are mobile electronic carriers,

which are contributed from nonstoichiometric defects. Equation 6-14 shows that electron holes

are mobile electronic carriers, electron holes, which are contributed from extrinsic defects.

CexZrt-xO2-3 x Zrt-xO2 + Vj' + 2e (6-7)

CexZrt-xO2 CeX r e Cee Zre + 2h' (6-8)

Here, the model to describe superoxide radical scavenging which mediated by oxygen

vacancies in CexZrl-xO2 HanOparticles is listed in the following equations.

CexZrt-xO2 +Vj' + 2e + OF 4CexZrt-xO2 +V + 2e + 02 (6-9)

CexZrt-xO2 + Vj + 2e + 2H' + Of a CexZrt-xO2 + V + 2e + H202 (6-10)

The model to describe superoxide radical scavenging which mediated by electron holes in

CexZrl-xO2 HanOparticles is listed in the following equation.

Cee + Ce, + Cece + Cez, + 2h' + 0~ Cee + Ce, + Cece + Cez, + h' + 02 (6-17)

Cee + Ce + Cece + Cez, + 2h' + 2H' +OJ- R Ce e + Ce + Cece + Cez, + 2h' + H202
(6-18)

In the equation described above, we understand that zirconium dopants in CexZrl-xO2

nanoparticles improve scavenging activities both against hydrogen peroxide and superoxide

radicals. The improvements are dependent on the increased oxygen vacancy concentration in the

case of hydrogen peroxide, and the improvements in the case of superoxide radicals are

dependent on the promoted mobile electronic carriers. The reason to make this difference is due

to the characteristics of hydrogen peroxide and superoxide radicals. Moreover, most

accumulative oxidative stress occurred in biological systems relies on the transition of electrons









between radicals and molecules, so the optimum free radical scavengers in biological systems are

those that have shown greater activities to remove excess electrons from free radicals. In

conclusion, we can hypothesize that Ce0.4ZroeO2 HanOparticles would be the most effective free

radical scavengers in biological systems.

6.4 Electron Conduction in Catalysis

In this section, the capability for Cex2rl-xO2 HanOparticles to conduct electrons in catalysis

will be inspected and discussed, since the particular property is essential for free radical

scavenging. In previous section, the inhibition percentages of dye formation merely indicated

nanoparticles' superoxide radical scavenging activities. The capability for Cex2rl-xO2

nanoparticles to conduct electrons in catalysis, therefore, would need solid evidence other than

the information we acquired. Here, we adopt a classic assay on biochemistry basis to inspect the

electron conduction on Cex2rl-xO2 HanOparticles. The assay includes a bivalent, reversible

conducting molecule, which is colorimetric only in its reduced state. Thus, the rise or decline of

optical density of this molecule would indicate its redox states. Replace WST-1 salts in the

methodology for scavenging activity test, the redox states of this molecule would indicate the

capability for catalysts to conduct electrons in catalysis. The principle of this method is shown

in Figure 6-17. The colorimetric molecule that used to test electron conduction on Cex2rl-xO2

nanoparticles is cytochrome c (II/III).





Cexlrr Oz

02


Figure 6-17. Principle of the biochemistry based assay to inspect the capability for Cex2rl-xO2
nanoparticles to conduct electrons in catalysis.









6.4.1 Experimental Methods

To generate superoxide radicals, we used hypoxanthine and enzyme xanthine oxidase to

produce the final products including superoxide radicals, hydrogen peroxide, and uric acids. The

hydrogen peroxide produced in such reaction is then removed by adding large amounts of

catalase, in order to prevent the affects of hydrogen peroxide.

To detect the electron conducted on CexZrl-xO2 HanOparticles, a reversible dye, cytochrome

c is conducted in the experiment. Reduced by superoxide radicals, cytochrome c forms

cytochrome c (II), and is colorimetric at 550 nm. In this study, the redox state of cytochrome c is

observed using UV-Vis (UV/Vis Perkin-Elmer Lambda 800).

For the samples prepared for UV-Vis, a stock solution including 0.5 mM EDTA, 12.5 CIM

cytochrome c (Aldrich), in addition of 3,000 unit/ml catalase (Aldrich) is prepared as reagent

solution. 0.5 mM hypoxanthine, 0.9 U/ml xanthine oxidase, and 1 mM CexZrl-xO2 HanOparticle

suspensions (x = 0, 0.4, 0.6, 0.7, 0.8, 1.0) were prepared in 100 mM phosphate buffer (pH = 7.4,

Aldrich) as stock solutions. Before initiating the reaction, 780 Cl1 of reagent solution, 80 Cl~ of

sample solution (i.e. 1 mM CexZrl-xO2 HanOparticle suspensions or 1.875 U/ml SOD samples),

and 20 Cl1 of xanthine oxidase stock solution were titrated into a 1 ml cuvette. To initiate the

reaction, 100 Cl1 of hypoxanthine stock solution was titrated into the cuvette in order to generate

superoxide radicals. The total concentrations of CexZrl-xO2 HanOparticle suspensions were then

diluted to 200 CLM after all. The dye formation was finally detected by UV-Vis at 550 nm. The

stock solutions used in this experiment and the experimental procedures are illustrated in Figure

6-18. The absorbance was recorded and shown in Figure 6-19.











step l:
Cytnchrumme c meagent includes
eanees catalass, 780 pl
0.9 Uhal xanthineasidase, 20
1 mM Ca.ZOI ar SOD) 100 pl

Step2i:

0.5 mM hparranthine, 100 prl


Figure 6-18. Experimental arrangements of stock solution concentrations and experimental
procedures to inspect electron conduction on Cexhi-xO2 HanOparticles.



0.10

0.08
SBlank
0.06 -1 *21C CeiO,
~C Zr, O,
ug0.04 vCe0.7Zr030,

S0.02 / __ Ce Z~lrroe0.
~i ~fZrO,
u 0.0. SOD150 mU/ml
a CeO, w/o superoxide


8-0.04

-0.06


'tI' I' 2" 3 567I

Time (minutes)


Figure 6-19. Redox states of cytochrome c detected using UV-Vis. The higher the absorbance
represents more reduced cytochrome c in the reaction. The declined absorbance
suggests that Cexhi-xO2 HanOparticles received electrons from the reduced
cytochrome c in catalysis.


Step 3:
Observe almabance at 550 mn
par2 socnds









6.4.2 Results and Discussion

In Figure 6-19, the vertical axis shows the redox states of cytochrome c. The higher

absorbance in the particular diagram represents more reduced cytochrome c (II) in the reaction.

In the samples with enzyme SOD protection, the concentration of reduced cytochrome c

increases and stays at high level. It is because SOD merely catalyzes superoxide radicals to

hydrogen peroxide, and the products hydrogen peroxide was immediately removed by enzyme

CAT. On the other hand, the samples with CexZrl-xO2 HanOparticles' protection not only inhibit

the formation of reduced cytochrome c, but also reverse the redox state to its initial state. In

these samples enzyme CAT was also involved, so the agents to reverse redox states of

cytochrome c (from II to III) do not appear to be hydrogen peroxide. It is because the catalysis

from superoxide to hydrogen peroxide requires electrons, so CexZrl-xO2 HanOparticles take

electrons from the reduced cytochrome c and react with other superoxide radicals to form

hydrogen peroxide. The reversed absorbance observed in UV-Vis therefore indicates the

capability for CexZrl-xO2 HanOparticles to conduct electrons in catalysis. In Figure 6-19, more

zirconium dopants in CexZrl-xO2 Solid solutions result in more rapid reversion in redox states. It

is the evidence that CexZrl-xO2 HanOparticles with more zirconium dopants exhibit greater

capability to precede electron conduction. The results obtained in this experiment correspond to

the conclusion in previous section, and these CexZrl-xO2 HanOparticles really are catalyzing free

radicals through electron exchange.

6.5 Summary

In this chapter, the free radical scavenging activities of a series of CexZrl-xO2 HanOparticles

were discussed. Using biochemistry assays, the residual concentration of hydrogen peroxide at

physiological levels was measured over time. In the presence of CexZrl-xO2 HanOparticles,

hydrogen peroxide concentrations in each sample decreased over time. Among all the samples,










Cea7Zrt302 HanOparticles have the greatest peroxide scavenging activity. In addition, the

scavenging activities of Cex2rl-xO2 HanOparticles are correlated with the oxygen vacancy

concentrations in the materials. It is because the catalysis of hydrogen peroxide is determined by

the transition of oxygen ions, so the oxygen vacancy concentrations in Cex2rl-xO2 HanOparticles

become dominant in the scavenging processes.

Using the methodologies based on biochemistry reactions, superoxide radicals were

generated. The scavenging activities of Cex2rl-xO2 HanOparticles to superoxide radicals were

tested using an irreversible dye, and their activities were compared with the fastest superoxide

radical scavenger, superoxide dismutase. The results showed that these Cex2rl-xO2 HanOparticles

have even greater scavenging activity than enzyme SOD. Furthermore, the results showed that

the scavenging activity of Cex2rl-xO2 HanOparticles increases with increasing zirconium dopants

in Cex2rl-xO2 HanOparticles. It is because the zirconium dopants prompt Ce3+ COntents in Cex2rl_

xO2 HanOparticles, and resulting in more mobile electronic carriers. These mobile electronic

carriers conduct the unpaired electrons in free radicals, so the scavenging activities to superoxide

radicals are through electron exchange. It is because the catalysis of superoxide radicals is

determined by the exchange of electrons, so the concentration of mobile electronic carriers in

Cex2rl-xO2 HanOparticles becomes dominant in the reaction. The mobile electronic carriers

include oxygen vacancies, cationic defects that are caused by nonstoichiometric defects and

extrinsic defects.

The capability for Cex2rl-xO2 HanOparticles to conduct electrons in catalysis is inspected

using a bivalent, reversible conducting molecule. In the experiment, electrons in the reduced

cytochrome c were taken away, resulting in non-colorimetric cytochrome c (III). Thus, the

reversed absorbance represents electron conduction from cytochrome c, through Cex2rl-xO2










nanoparticles, and finally to form hydrogen peroxide. The electron conduction in Cex2rl-xO2

nanoparticles relies on the mobile electronic carriers in Cex2rl-xO2 HanOparticles; therefore the

materials with most carriers also have the greatest activity to scavenge free radicals. Since the

accumulation of oxidative damage relies on electron exchange between molecules, such as

proteins, lipids, DNA, and RNA, the optimum free radical scavenger therefore relies on mobile

electronic carrier concentration in Cex2rl-xOz nanOparticles. Thus, we conclude that Ce0.4ZroeO2

nanoparticles would be the most effective free radical scavengers in biological systems, based on

the results in the activity tests.

After this chapter, the remarkable results confirm that the development of Cex2rl-xO2

nanoparticles for biomedical applications requires the knowledge in materials science.

Incorporated with zirconium dopants, nanoparticles that have up to nine times of free radical

scavenging activity compared to undoped CeO2 HanOparticles were developed. After the

knowledge learned in this chapter, we have successfully built up a model that describes hydrogen

peroxide scavenging and superoxide radical scavenging. According to the model, the scavenging

activities of these nanocatalysts can be further promoted if more mobile electronic carriers are

increased.









CHAPTER 7
IMPLICATIONS

7.1 Distinct Antioxidant Defense Pathway

CeO2 HanOparticles have been found to inhibit the progression of organism dysfunction

through regulating its endogenous oxidative stress. It was hypothesized that CeO2 HanOparticles

scavenge free radicals and therefore improve cell cultures' viability. However, we found that

CeO2 HanOparticles are eligible to reinstate the redox states of cytochrome c in the presence of

superoxide radicals. It suggests that protection provided by CeO2 HanOparticles may be

contributed by reactivating cytochrome c's redox states as well. The results also explain the

finding in the previous study that CeO2 HanOparticles restored retinal photoreceptor cells'

function even if CeO2 HanOparticles were administered after cell cultures were damaged [54].

Cytochrome c is a heme protein found loosely bounded with inner membrane of

mitochondria. It is an antioxidant protein with two valences, which can oxidize superoxide

radicals and reduce hydrogen peroxide. Cytochrome c is also a very important protein in

metabolism. It conducts electrons in electron transport chain of mitochondria. Lacking of

cytochrome c, the electrons leak out of the transport chain and forming free radicals in the

sequence. In addition, the over reduced cytochrome c would lose its bonding with inner

membrane of mitochondria and finally be released to cytoplasm. When cytochrome c is released

from mitochondria, they tend to bond with other proteins, forming apoptosome. The apoptosome

triggers apoptosis later on. It has been demonstrated that cytochrome c release requires a two-

step process [91]. Cytochrome c is present as loosely and tightly bound pools attached to the

inner mitochondrial membrane by its association with free radicals, and this interaction must first

be disrupted to generate a soluble pool of this protein. Specifically, solubilization of cytochrome

c involves a breaching of the electrostatic or hydrophobic affiliations that this protein usually









maintains in mitochondrial membrane. Once cytochrome c is solubilized, the reduced

cytochrome c loses its electrostatic bonding with mitochondrial membrane. The lowered

electrostatic bonding is sufficient to trigger cytochrome c's release. Since superoxide radicals

have long been found to reduce cytochrome c, the concept of scavenge superoxide radicals prior

their damage to cytochrome c, or preventing cytochrome c released from mitochondria has been

classified in a novel antioxidant defense pathway (shown in Figure 2-1).

CeOz nanoparticles were found to exhibit exceptional scavenging activity to superoxide

radicals and peroxides. The reaction rate constants of CeOz nanoparticles are superior to enzyme

superoxide dismutase [29] and close to catalase [92]. However, the half life of free radicals is

short, the question remains on how the damaged cells were recovered if nanoceria merely

scavenge free radicals in cell cultures? Beyond CeOz nanoparticles' free radical scavenging

properties, we found that CeOz nanoparticles are eligible to regulate the redox states of

cytochrome c, especially in the presence of superoxide radicals. Figure 7-1 shows the redox

states of cytochrome c in the presence of superoxide radicals, Cea, nanoparticles, and SOD.

The data are extracted from Figure 6-19. In Figure 7-1, profile A shows the reduction of

cytochrome c in the presence of superoxide radicals. Profile B shows the restoration of

cytochrome c as CeOz nanoparticles scavenge superoxide radicals. In profile C, CeOz

nanoparticles slightly restore cytochrome c's redox states in the absence of superoxide radicals.

In profile D, SOD only against superoxide's attack, but does not regulate cytochrome c's redox

states. The redox states of cytochrome c are influential in apoptosis [40,93]. Overwhelmed by

the reduced cytochrome c: (1) electrons would leak from mitochondrial electron transportation

chain, forming detrimental ROS [94]; (2) cytochrome c would be released from mitochondria

due to the lower electrostatic bonding in reduced cytochrome c [95]. The accumulation of ROS









as well as cytochrome c release trigger caspase proteins and finally induce apoptosis. By

regulating the redox states of cytochrome c, CeOz nanoparticles may provide a distinct

antioxidant defense pathway by inhibiting the release of cytochrome c from mitochondrial

membrane.

In this chapter, we found that CexZrl-xOz nanoparticles are capable to regulate the redox

states of cytochrome c (shown in Figure 6-19). In reference to Chen et al.'s work, exposing

CeOz nanoparticles to cultures not only scavenges free radicals in cultures, but also regulates the

redox states of cytochrome c and inhibits cytochrome c release. We suggest that CexZrl-xOz

nanoparticles may contribute a distinct antioxidant defense beyond free radical scavenging. In

consequence, the proposed mechanism is able to explain the previous discovery by Chen et al.

that CeOz nanoparticles restored retinal photoreceptor cells' function even CeOz nanoparticles

were administered after cells were damaged [58].



C- 100 uM CeO,
without superoxide

Cyt c (III) Time
0 I 2~ .) 4 5 6 7 (utes)

aB- 100 uM3 CeOC
against superoxide


D- 150 mU/ml SOD
I againsis superoxide

cyt c (II)


Figure 7-1. Redox states of cytochrome c in the presence or absence of superoxide radicals.









7.2 Implications in Catalysis

In heterogeneous catalysis, the electrochemical reactions comprise a series of electron

exchange, oxygen ion exchange in/between ligands and catalysts. The selection of optimum

catalysts relies on inspecting the catalyst's capability to conduct electrons or oxygen ions in

catalysis. According to the results in previous experiments, the biochemistry based assays that

have been developed in this work are ideal techniques to inspect the interfacial electrochemistry

in the process of catalysts.

7.2.1 Alternative Technique to Inspect ionic Conductivity at Room Temperature

Due to the characteristics of hydrogen peroxide, it is found that the exchange of oxygen

ions is involved in the catalysis for hydrogen peroxide. Based on this concept, hydrogen

peroxide molecules can be treated as probes to inspect ionic conductivity of CeOz-based

materials in catalysis.

In biochemistry, enzyme CAT is used to catalyze hydrogen peroxide. The catalysis of

hydrogen peroxide. The catalysis proceeds in two steps [96] by

(1) Oxidation of CAT by a peroxide

H,O, + Fe(III) E 4 H,O + O = Fe(IV) E(.+) (6-1 1)

(2) Oxidation of the substrates.

H,O, + O = Fe(IV) E(.+) 4 H,O + Fe(III) E + O, (6-12)

Overall,

H,O, ca= >H,O +1/ 20,(-3

In the catalysis, the oxidized enzyme possesses two oxidative equivalents above the native

enzyme and contains highly reactive oxygen bound to the iron. The iron is then promoted to its









quadrivalent states. In the catalysis proceeded by CAT, the catalysis of hydrogen peroxide

depends on its characteristics that conducting its oxygen ions with enzyme CAT.

At low hydrogen peroxide concentration, we have demonstrated that the amount of mobile

oxygen vacancies dominates the catalysis. As an excellent ionic conductor, CxZrl-xeO2 actually

mediates ionic conduction by its mobile oxygen vacancies. It is, the scavenging rates to

hydrogen peroxide scavenging not only indicate the oxygen vacancy concentration in catalyst' s

lattice, but also represent ionic conductivity of CexZrl-xO2.

7.2.2 Alternative Technique to Inspect Localized Electron Conductivity

The catalysis of superoxide radicals are preceded by two steps. Superoxide radicals can

donate electrons to a molecule and become oxygen molecule, or they can receive electrons from

others to form peroxides. In biochemistry, enzyme SOD is used to catalyze superoxide radicals

into hydrogen peroxide and oxygen in free radical scavenging. The catalysis carried out by

enzyme SOD is as follow,

Of +M~'"'' SOD R O2 +M~" SOD (6-14)

Of + 2H' +M~" -SOD aHO 202+"'' SOD (6-15)

Overall,

20, + 2H' H202 (6-16)

In the catalysis carried out by enzymes, it is certainly that the exchange of electrons

involved in the catalysis. However, the scavenging occurred in CexZrl-xO2 HanOparticles remains

uncertain, therefore this section is to discuss the mechanism based on the results obtained in

superoxide radical scavenging tests.

The scavenging to superoxide radicals undergoes a different mechanism comparing to the

scavenging to hydrogen peroxide. In the catalysis to superoxide radicals, a superoxide radical










either donates an electron to catalysts forming oxygen molecules, or receives an electron forming

peroxide. In means that the amount of electrons conducted in catalysts dominates the reaction.

Thus, superoxide radicals work as perfect probes to inspect localized mobile electronic carriers

in catalysis. Utilizing a bivalent, reversible, colorimetric protein cytochrome c, it becomes

possible to "see" the electron conduction in catalysis. The reversed optical density in the

reaction represents the electrons taken away by catalysts, where the electrons are taken away to

form peroxide with other superoxide radicals.










CHAPTER 8
CONCLUSIONS

The in vitro studies in Chapter 3 have shown that CeO2 HanOparticles could relieve

oxidative stress in certain cell cultures. The culture's viability was improved accordingly. In

addition to these successful in vitro results, several obj ectives have been achieved in further,

including synthesis and characterization of Cex2rl-xO2 HanOparticles, evaluation of free radical

scavenging activities of these nanoparticles, and a distinct antioxidant defense pathway that may

contribute to the protection to cultures. In this dissertation, several innovative techniques have

been emerged to evaluate the catalytic properties of Cex2rl-xO2 HanOparticles, and the results are

remarkable. The conclusions are summarized as follow:

* Reverse micelle method is used to synthesize Cex2rl-xO2 HanOparticles of 3-7 nm.

* The synthesized Cex2rl-xO2 HanOparticles are crystalline solid solutions, so heat treatments
can be exempted in the synthesis.

* The synthesized Cex2rl-xO2 HanOparticles are dispersed in sodium citrate buffer. The
agglomeration using such preparation can be ignored, since the agglomerate size
distributions are smaller than 10 nm.

* CeO2 HanOparticles can improve cell culture's viability by relieving its endogenous
oxidative stress.

* Doping zirconium into CeO2 HanOparticles promotes their free radical scavenging
activities. The enhancement comes from the promoted oxygen vacancies as well as mobile
electronic carriers in the lattice.

* For Cex2rl-xO2 HanOparticles to scavenge hydrogen peroxide at low concentration, oxygen
ions are involved in the reactions, therefore oxygen vacancies in the lattice dominate the
scavenging. Among all samples, Ceo.7Zr0.302 HanOparticles exhibit the greatest activity.
The scavenging activities of Cex2rl-xO2 HanOparticles correlate to the oxygen storage
capacity reported in the same materials.

* For Cex2rl-xO2 HanOparticles to scavenge superoxide radicals, the scavenging activity
depends on their capability to conduct electrons in catalysis. Zirconium dopants promote
Ce3+ COntents in CeO2 lattice, so the mobile electronic carrier concentration increases due
to the promoted cationic defects in the lattice. The cationic defects include oxygen
vacancies and electron holes in the lattice, which are contributed from nonstoichiometric










defects and extrinsic defects. After activity tests, Ce0.4ZroeO2 HanOparticles exhibit the
greatest activity of all Cex2rl-xO2 HanOparticles.

*Cex2rl-xO2 HanOparticles can reactivate cytochrome c's redox states in the presence of
superoxide radials. The reinstate of redox states may regulate cytochrome c's function in
metabolism, and further achieve a distinct antioxidant defense.









APPENDIX A
GLOSSARY


A form of programmed cell death in multi-cellular organisms. It is one of
the main types of programmed cell death (PCD) and involves a series of
biochemical events leading to a characteristic cell morphology and death

Protein bcl-2 is an antioxidant protein. An important one states that this is
achieved by activation or inactivation of an inner mitochondrial
permeability transition pore, which is involved in the regulation of matrix
Ca2+, pH, and voltage. It is also thought that some Bcl-2 family proteins
can induce (pro-apoptotic members) or inhibit (anti-apoptotic members)
the release of cytochrome c in to the cytosol which, once there, activates
caspase-9 and caspase-3, leading to apoptosis.


Apoptosis


bcl-2


PTC-tet cells


Pancreatic PTC lines derived from murine insulinomas.


CAT


Carotenoids


CeO2.


CexZrlxO2 :


COX


Cytochrome c


Enzyme catalase. Catalase is a common enzyme found in nearly all living
organisms. Its functions include catalyzing the decomposition of hydrogen
peroxide to water and oxygen. Catalase has one of the highest turnover
rates of all enzymes; one molecule of catalase can convert millions of
molecules of hydrogen peroxide to water and oxygen per second.

Carotene is a precursor to vitamin A, a pigment essential for good vision,
and carotenoids can also act as antioxidants.

Cerium oxide, cerium dioxide, or sometimes listed as ceria.

Zirconium-doped CeO2. The x in the chemical formula represents the
stoichiometry of cerium ions, and (1-x) represents the stoichiometry of
zirconium ions sitting on the cationic sites in the cubic fluorite structures.

The enzyme cytochrome c oxidase or Complex IV is a large
transmembrane protein complex found in bacteria and the mitochondrion.
It is the last enzyme in the respiratory electron transport chain of
mitochondria (or bacteria) located in the mitochondrial (or bacterial)
membrane. It receives an electron from each of four cytochrome c
molecules, and transfers them to one oxygen molecule, converting
molecular oxygen to two molecules of water.

Cytochrome c is a small heme protein found loosely associated with the
inner membrane of the mitochondrion. It is a soluble protein, unlike other
cytochromes, and is an essential component of the electron transfer chain,
where it carries one electron. It is capable of undergoing oxidation and
reduction, but does not bind oxygen. It transfers electrons between
Complexes III and IV.










DCF 2',7'-dichloroflurescin diacetate. A fluorescent dye to detect reactive
oxygen species in biological systems.

Electron transport chain

An electron transport chain associates electron carriers and mediating
biochemical reactions that produce adenosine triphosphate (ATP), which
is a maj or energy intermediate in living organisms. Only two sources of
energy are available to biosynthesize organic molecules and maintain
biochemical and kinetic processes in living organisms: redox reactions.
The schematic diagram of electron transportation chain is shown in Figure
2-1.

Endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is caused by the accumulation of
unfolded proteins in the ER lumen, and is associated with vascular and
neurodegenerative diseases.


Feret' s diameter


I n microscopy, Feret's diameter is the measured distance between parallel
lines that are tangent to an obj ect's profile and perpendicular to the ocular
scale. Generally, Feret's diameter is the greatest distance possible between
any two points along the boundary of a region of interest.

Enzyme glutathione peroxidase. Enzyme glutathione oxidase is an
enzyme that catalyzes glutathione and oxygen to form glutathione
disulfide and hydrogen peroxide.

Glutathione is a tripeptide. It contains an unusual peptide linkage between
the amine group of cysteine and the carboxyl group of the glutamate side
chain. Glutathione, an antioxidant, protects cells from toxins such as free
radicals.

Enzyme heme oxygenase. An enzyme that catalyzes the degradation of
heme. This produces biliverdin, iron, and carbon monoxide.

Hydroquinone. Hydroquinone is a compounds used to induce oxidative
stress in this dissertation.

Inflammation is the complex biological response of vascular tissues to
harmful stimuli, such as pathogens, damaged cells, or irritants. It is a
protective attempt by the organism to remove the injurious stimuli as well
as initiate the healing process for the tissue.

Lipid peroxidation refers to the oxidative degradation of lipids. It is the
process whereby free radicals grab electrons from the lipids in cell
membranes, resulting in cell damage. This process proceeds by a free
radical chain reaction mechanism.


GPx


GSH


HO


HQ


Inflammation





Lipid peroxidation









OSC


Oxidative stress


ROS


Superoxide


SOD


Type 1 diabetes


Oxygen storage capacity. The OSC is determined by measuring oxygen
consumed by the catalyst after reduction under isothermal conditions. It is
a quantitative basis the capability of metal oxide catalysts to release
oxygen under reducing condition, and to uptake oxygen under oxidizing
conditions.

Oxidative stress is caused by an imbalance between the production of
reactive oxygen and a biological system's ability to readily detoxify the
reactive intermediates or easily repair the resulting damage.

Reactive oxygen species. Reactive oxygen species are oxygen species that
are generated from metabolism. They are reactive to other molecules, and
usually cause damage to cells. In free radicals in biology superoxide
radicals, hydroxyl radicals, and hydrogen peroxide are usually appointed
as ROS.

Superoxide radicals. Superoxide is the anion Ol It is important as the
product of the one-electron reduction of dioxygen, which occurs widely in
nature. With one unpaired electron, the superoxide ion is a free radical,
and, like dioxygen, it is paramagnetic.

Enzyme superoxide dismutase. Enzyme SOD is an enzyme found in
almost all living systems. It catalyzes the dismutation of superoxide into
oxygen and hydrogen peroxide. As such, it is an important antioxidant
defense in nearly all cells exposed to oxygen.

Also called juvenile diabetes. It is a form of diabetes mellitus. Type 1
diabetes is an autoimmune disease that results in the permanent
destruction of insulin producing beta cells of the pancreas. Type 1 is lethal
unless treatment with exogenous insulin via injections replaces the
missing hormone.










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BIOGRAPHICAL SKETCH

Yi-Yang Tsai was born in Taiwan in 1976. Before pursuing postgraduate education, he

received B.S. in aeronautical engineering from Feng-Chia University, Taiwan in 1998. After

two years of military services and two years of working experience as R&D engineer, he went

oversea and accepted the graduate education in the United States. He first j oined the Materials

Engineering Department at University of Dayton in 2002, and received his M.S. in 2004. He

j oined the Materials Science and Engineering Department at University of Florida in 2004 under

supervisory of Dr. Wolfgang M. Sigmund, and received his Ph.D. degree in summer 2008.

In the graduate training at University of Dayton, Yi-Yang was specialized in the

characterization techniques, atomic force microscope and an ultrasound integrated atomic force

microscope in the University of Dayton Research Institute (UDRI). Due to his excellent works,

he was awarded a visiting research fellowship in 2003, and has allowed him to travel to the

Fraunhofer Institute for Non-Destructive Testing (IZFP) in Germany for a short term research.

In 2004, he was awarded the DAGSI research fellowship (Dayton Area Graduate Student

Institute). His master thesis was a study that using ultrasound integrated atomic force

microscope to detect nanoscale precipitations and cracks in aluminum alloys. In the two years,

he has published two scientific papers and gave two invited presentations in SPIE conference.

In the Ph.D. training in Dr. Sigmund's research group, Yi-Yang has developed many

innovative techniques in the interdisciplinary research proj ect "Administering CeOz

nanoparticles to enhance cell's viability" assigned by his advisor. In the proj ect Yi-Yang and his

advisor have made several remarkable breakthroughs. They have published several scientific

articles and international patents based on the results. Other than the articles that have been

published, he is preparing manuscripts for paper publications from the remarkable results.





PAGE 1

CERIUM-ZIRCONIUM OXIDE NANOCATALYS TS AS FREE RADICAL SCAVENGERS FOR BIOMEDICAL APPLICATIONS By YI-YANG TSAI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Yi-Yang Tsai 2

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To my parents, fiance, and advisors w ho had been highly encouraging and supportive 3

PAGE 4

ACKNOWLEDGMENTS There are many teachers and friends to thank fo r. Without your guidance, inspiration, and consideration, this work would never be accomplished. First of all, I would like to thank Dr. Wo lfgang Sigmund for his inputs and scientific advises to this work. On top of the advisory guidance, he also delivered his enthusiasm, generosity, and philosophy to students. Without hi s mentoring and broad research interests, this project would have been plain a nd lack of innovation. I would al so like to thank my committee members, Drs. Holloway, Moudgil, Batich, and Atki nson for their constructive comments. They have excellent reputation and are highly acknowledgeable in academia. They are prominent scholars and are great examples in my future car eer development. I woul d like to dedicate this work to the memory of Dr. Iaonni s Constantinidis, who was an a dvisor, cheerleader, and sincere friend of mine. I would like to thank current and previous colleagues in Sigmund Group. Without them I would never learn my broad interests and insights in this emerging research project, especially Drs. Georgios Pyrgiotakis and Amit Daga for setting up great examples for young group members. I would like to thank those people wh o have helped me in this project. Many assistances from them start from my accidental re quests, but they had big heart assisting me into the situation. Jose Oca-Cossio, Kelly Siebein, a nd Gill Brubaker are especially acknowledged in this project. Finally I would like to thank my parents for being supportive through my study in the United States. I also like to thank my fiance, Ju-Hsuan Cheng PharmD. She accompanied me through the tough time, and has created wonderful time as well as memories in my life. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 LIST OF ABBREVIATIONS ........................................................................................................1 1 CHAPTER 1 INTRODUCTION ................................................................................................................ ..15 1.1 Motivation .........................................................................................................................17 1.2 Objectives .........................................................................................................................18 1.3 Specific Aims and Expected Outcomes ............................................................................19 2 BACKGROUND ....................................................................................................................22 2.1 Free Radicals in Biology and Medicine ............................................................................22 2.1.1 Free Radical Theory of Aging ................................................................................22 2.1.2 Modern Free Radical Theory in Biology ................................................................24 2.2 Antioxidant Defense in Biology and Medicine ................................................................26 2.2.1 Antioxidant Defense System ..................................................................................26 2.2.2 Alternative Antioxidant Defense Other Th an Direct Free Radical Scavenging .....28 2.3 CeO2 in Catalysis, Biology, and Medicine .......................................................................28 2.3.1 CeO2 as Catalysts ...................................................................................................29 2.3.2 CeO2 Nanoparticles in Biomedical Applications ...................................................29 2.3.3 Free Radicals in CeO2 ............................................................................................31 2.3.4 Hypothesis of CeO2 Nanoparticles in Free Radical Scavenging ............................32 2.3.5 Promote Catalytic Activit y by Doping Zirconium into CeO2 ................................33 2.3.6 Introduction of CexZr1-xO2 ......................................................................................35 3 TEST HYPOTHESIS WITH CELL CULTURES .................................................................39 3.1 Methodology .....................................................................................................................39 3.2 Results and Discussion .................................................................................................... .44 3.3 Summary ...........................................................................................................................54 4 SYNTHESIS OF CERIUM-ZIRCO NIUM OXIDE NANOPARTICLES .............................55 4.1 Nanoparticle Synthesis .....................................................................................................56 4.1.1 Reverse Micelle Synthesis ......................................................................................56 4.1.2 Experimental Methods ............................................................................................57 4.2 Nanoparticle Stabilization ................................................................................................59 5

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4.2.1 Stabilization Using Buffer ......................................................................................59 4.3 Particle Size Analysis .................................................................................................... ...60 4.3.1 Dynamic Light Scattering Technique .....................................................................60 4.3.2 Agglomerate Size Distribution ...............................................................................60 4.4 Chemical Analysis ............................................................................................................62 4.4.1 Inductively Coupled Plasma Spectroscopy ............................................................62 4.4.2 Compositions of Final Products .............................................................................63 4.5 Summary ...........................................................................................................................63 5 STRUCTURAL PROPERTIES AND NONSTOICHIOMETRIC BEHAVIOR OF CERIUM-ZIRCONIUM Oxide Nanoparticles .......................................................................65 5.1 Characterization Techniques and Experimental Methods ................................................65 5.1.1 Structural Characterization Using TEM .................................................................65 5.1.2 Structural Characterization Using XRD .................................................................66 5.1.3 Structural Characterization Using Raman ..............................................................67 5.2 TEM Results and Discussion ............................................................................................68 5.3 XRD Results and Discussion ............................................................................................76 5.4 Raman Results and Discussion .........................................................................................78 5.5 Summary of TEM, XRD, Raman Results ........................................................................82 5.5.1 Crystalline CexZr1-xO2 Nanoparticles with Homogeneous Particle Size ................82 5.5.2 Phase Transition Detected by XRD and Raman Spectroscopy ..............................82 6 FREE RADICAL SCAVENGING BY CERIUM-ZIRCONIUM OXIDE NANOPARTICLES ...............................................................................................................84 6.1 Prospective Scavenging Activities in CexZr1-xO2 Nanoparticles ......................................84 6.2 Activities against Hydrogen Peroxide ..............................................................................86 6.2.1 Experimental Methods ............................................................................................86 6.2.2 Results and Discussion ...........................................................................................88 6.3 Activities against Superoxide Radicals ............................................................................93 6.3.1 Experimental Methods ............................................................................................94 6.3.2 Results and Discussion ...........................................................................................98 6.4 Electron Conduction in Catalysis ...................................................................................115 6.4.1 Experimental Methods ..........................................................................................116 6.4.2 Results and Discussion .........................................................................................118 6.5 Summary .........................................................................................................................118 7 IMPLICATIONS ..................................................................................................................121 7.1 Distinct Antioxidant Defense Pathway ...........................................................................121 7.2 Implications in Catalysis .................................................................................................124 7.2.1 Alternative Technique to Inspect io nic Conductivity at Room Temperature .......124 7.2.2 Alternative Technique to Inspec t Localized Elect ron Conductivity ....................125 8 CONCLUSIONS ................................................................................................................. .127 APPENDIX 6

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A GLOSSARY .................................................................................................................... .....129 LIST OF REFERENCES .............................................................................................................132 BIOGRAPHICAL SKETCH .......................................................................................................140 7

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LIST OF TABLES Table page 2-1 Genetic changes and the responded free ra dical scavengers that affect oxidative damage in living systems. ..................................................................................................27 2-2 Classification of the phases in the CeO2-ZrO2 binary system. ...........................................36 3-1 Intracellular amount of CeO2 in TC-tet cells following 48 hrs of incubation with media containing CeO2 nanoparticles. ...............................................................................49 4-1 Chemicals and the amounts used to prepare CexZr1-xO2 nanoparticles in reverse micelle synthesis. ...............................................................................................................58 4-1 Synthesis procedures. ..................................................................................................... ....58 4-2 Mean agglomeration size ( Mn), D50, and D95 of CexZr1-xO2 suspensions obtained using NanoTrac. .................................................................................................................61 4-3 Chemical compositions of final products determined. ......................................................63 5-1 Particle sizes of CexZr1-xO2 nanoparticles averaged from 50 arbitrary selected particles in TEM micrographs. ..........................................................................................76 5-2 Crystal structures a nd their sub-phases in CexZr1-xO2, and the classification using Raman spectroscopy. .........................................................................................................79 6-1 Concentrations for enzyme SOD and CexZr1-xO2 nanoparticles to achieve 50% of inhibition rate in activity test. ..........................................................................................110 6-2 Reaction rate constants of CexZr1-xO2 nanoparticles against su peroxide radicals. ..........111 8

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LIST OF FIGURES Figure page 2-1 Metabolism in a mitochondrion. ........................................................................................26 2-2 Hypothetic mechanism of free radica l scavenging in the vicinity of CeO2 surface. .........33 2-3 Schematic diagrams showing cubic fluorit e (left) and pyrochlor e (right) structures for CeO2/ ZrO2 and CexZr1-xO2, respectively. .....................................................................34 2-4 Phase diagram of the CeO2-ZrO2 binary system. ...............................................................36 2-5 Phase transition of CeO2-ZrO2 binary system.. .................................................................37 2-6 XRD and Raman spectra of (1-x)CeO2xZrO2. .................................................................38 3-1 TEM micrographs of CeO2 nanoparticles. .........................................................................45 3-2 XRD pattern of CeO2 nanoparticles. ..................................................................................46 3-3 Chemical compositions of the tri-sodium citrate buffer. ...................................................47 3-4 Zeta potentials of CeO2 suspensions as a function of pH. .................................................48 3-5 TEM micrographs of CeO2 nanoparticles in TC-tet cells. ...............................................50 3-6 Free radical concentration in TC-tet cells. .......................................................................52 3-7 The amount of insulin se creted by non-labeled and CeO2 labeled TC-tet cells. .............53 4-2 Optical images of CeO2 nanoparticles dispersed in DI water (left), and in sodium citrate buffer solution solution (right). ...............................................................................59 4-3 Agglomerate size distribution in CexZr1-xO2 suspensions at pH 7.4. .................................61 5-1 TEM micrographs of CeO2 nanoparticles. .........................................................................69 52 TEM micrographs of Ce0.8Zr0.2O2 nanoparticles. ..............................................................70 5-3 TEM micrographs of Ce0.7Zr0.3O2 nanoparticles. ..............................................................71 5-4 TEM micrographs of Ce0.6Zr0.4O2 nanoparticles. ..............................................................72 5-5 TEM micrographs of Ce0.4Zr0.6O2 nanoparticles. ..............................................................73 5-6 TEM micrographs of Ce0.2Zr0.8O2 nanoparticles. ..............................................................74 5-7 TEM micrographs of ZrO2 nanoparticles. .........................................................................75 9

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5-8 XRD spectra of a series of CexZr1-xO2 nanoparticles. ........................................................77 5-9 Raman spectra of CexZr1-xO2 nanoparticles. ......................................................................81 6-1 Hypothesized scheme of free radical scavenging by CexZr1-xO2 nanoparticles. ................85 6-2 OSC of CexZr1-xO2 measured a pulse chromatographic system at 400 C. ........................86 6-3 Detection scheme used to determine the peroxide concentration in activity tests. ............87 6-4 A) shows the peroxide concentratio n in the presence of 7 nm commercial CeO2 and synthesized CexZr1-xO2 nanoparticles over time. B) shows the natural logarithmic values of the peroxide concentration divi ded by initial peroxide concentration. ..............89 6-5 The effective scavenging efficiency K of CexZr1-xO2 nanoparticles vs. OSC and the amount of superoxide radicals. ..........................................................................................91 6-6 Superoxide radicals produced by hypoxanthine and xa nthine oxidase. ............................94 6-7 Principle of WST-1 assay to detect superoxi de radicals. ...................................................95 6-8 Experimental arrangement of stock solution concentrations, tota l concentrations, and experimental procedures. ...................................................................................................97 6-9 The setup of stock solutions and their concentrations in a 96-wells microplate. ..............98 6-10 The results of activity te st obtained using UV-Vis. .........................................................101 6-11 The results of activity te sts obtained using UV-Vis. .......................................................102 6-12 The results of activity tests measured by microplate reader. ...........................................103 6-13 The results of activity tests measured by microplate reader. ...........................................105 6-14 The results of activity tests measured by microplate reader. ...........................................107 6-15 Inhibition curves of enzyme S OD with different incubation time. ..................................110 6-16 Ce3+ contents and OSC in CexZr1-xO2 nanoparticles. .......................................................113 6-17 Principle of the biochemistry based assay to inspect the capability for CexZr1-xO2 nanoparticles to conduct el ectrons in catalysis. ...............................................................115 6-18 Experimental arrangements of stock so lution concentrations and experimental procedures to inspect electron conduction on CexZr1-xO2 nanoparticles..........................117 6-19 Redox states of cytochrome c detected using UV-Vis. ....................................................117 7-1 Redox states of cytochrome c in the pres ence or absence of s uperoxide radicals. ..........123 10

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LIST OF ABBREVIATIONS AD Alzheimers diseases CAT Catalase CeO2 Cerium oxide, cerium dioxide, or sometimes listed as ceria CexZr1-xO2 Zirconium-doped CeO2 solid solutions COX Cytochrome c Oxidase DLS Dynamic light scattering DMEM Dulbeccos Modified Eagles Medium DNA Deoxyribonucleic acid DCF Reactive oxygen species probe, 2 ,7-dichloroflurescin diacetate. EDS Energy dispersive spectrum ELISA Enzyme-Linked Immuno Sorbent Assay ER Endoplasmic reticulum GSH Glutathione GPx Glutathione peroxidase HD Huntington diseases HO Heme oxygenase HQ Hydroquinone HRP Horse radish peroxidase HX Hypoxanthine ICP Inductively coupled plasma IEP Isoelectric point MAIC Major Analytical Instrumentati on Center at University of Florida MCP Monocyte cheoattractant protein NADH Nicotinamide adenine dinucleotide 11

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NADP Nicotinamide adenine dinucleotide phosphate OSC: Oxygen storage capacity PBS Phosphate buffered saline PD Parkinsons disease PERC Particle Engineering Research Center at University of Florida PHGPx Phospholipid hydroperoxide glutathione peroxidase ROS Reactive oxygen species SAD Selective area diffraction SOD Superoxide dismutase TEM Transmission electron microscopy XO Xanthine oxidase XRD X-ray diffraction 12

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CERIUM-ZIRCONIUM OXIDE NANOCATALYS TS AS FREE RADICAL SCAVENGERS FOR BIOMEDICAL APPLICATIONS By Yi-Yang Tsai August 2008 Chair: Wolfgang M. Sigmund Major: Materials Scie nce and Engineering Administering CeO2 nanoparticles into cell cultures was found to improve cell cultures viability both in vitro and in vivo In this dissertation, an in vitro study conducted in our laboratory has observed the reduction of endogenous free radical concentration in CeO2 treated cell cultures. Furtherm ore, it is found that CeO2 nanoparticles scavenge free radicals through catalysis. Based on the findings, a series of CeO2-based nanocrystallites of greater catalytic activities was developed, aiming to achieve the sa me therapeutic efficacy to cell cultures with lower nanoparticle doses. In this dissertation, zirconium-doped CeO2 ( CexZr1-xO2) nanoparticles were synthesized and characterized. Their free radical scave nging activities were tested against harmful endogenous oxygen species, including hydrogen peroxide and superoxi de radicals. It is found that the scavenging activity of CexZr1-xO2 nanoparticles was promoted up to four times when scavenging hydrogen peroxide. The scavenging activity of CexZr1-xO2 nanoparticles was promoted up to nine times when scavenging super oxide radicals. Importantly, their free radical scavenging activities to hydrogen peroxide correlate to the re ported oxygen vacancy concentrations in the same materials. Their free radical scavenging ac tivities to superoxide 13

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14 radicals correlate to the reported reducibility in the same materials. The results suggest that oxygen vacancies, lattice oxygen, elec trons, and holes are involved in free radical scavenging. In addition, it is found that CexZr1-xO2 nanoparticles regulate anti oxidant proteins redox states upon catalysis. This might be a distinct antioxidant defense pa thway that reactivates antioxidant proteins func tion, and to explain the superior protection of CeO2 nanoparticles.

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CHAPTER 1 INTRODUCTION In the last decade, many disorders and diseases in mammalians were found to correlate with the damages caused by endogenous oxidative st ress [1,2]. The oxidativ e stress in living system is a result of accumulating free radi cals, where lipids, pr oteins, DNA, and other molecules receive or donate mobile electrons from free radicals [1,3]. The long-term accumulative damages caused by free radicals fina lly cause mutation of cells or apoptosis (programmed cell death) [1,4]. The damages further contribute to tissue injury or dysfunction, and finally progress to human disorders. For ex ample, (1) neurodegenera tive diseases including Parkinsons disease (PD), Alzheimers diseas e (AD), Huntingtons disease (Juvenile HD) [1,5,6,7]; (2) certain cardiovascular diseases incl uding strokes, heart attacks, ischemia, and atherosclerosis [1,8,9]; (3) gene tic and metabolic diseases including Downs syndrome and diabetes [1,10,11]; (4) cancers in cluding liver, prostate, lung, breast, and many other cancers [1,12]; (5) symptoms of aging in cluding osteoporosis [1], were found to be the result of overproduced endogenous free radicals. Meanwhile the excess amounts of free radicals were also a consequence of some human disorders, su ch as inflammatory disorders [1,13], allergies [1,14], and infectious diseases in cluding pneumonia [1,15] and HIV [1 ]. Therefore, the research on the role of free radicals in pathophysiologic processes and the potential therapie s to oxidative stress related disorders are drawing more and more attention over time. Mammalian cells fight against detrimental free radicals through antioxidant defense systems by scavenging the redundant oxidative stre ss. Scientists have demonstrated that administrating effective means of free radical scavengers protects biological systems from oxidative damage of lipids, pr oteins, DNA, and other molecules [1,16,17]. The protection in cell cultures can prevent apoptosis, mutation, and enha nce the viability of cell cultures [1,18]. In 15

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living animals, the therapeutic efficacy has been demonstrated to inhibit the symptoms of AD, PD [1,19], diabetes [1], cancers [1,20], other diseases, and even prol ong laboratory animals lifespan [21,22,23,24]. The antioxidants or free radical scavengers that carry out antioxidant defenses in mammalians are listed in the following [1]. Enzymes that catalytically remove free radi cals, such as superoxide dismutase (SOD), glutathione peroxida se (GPx), catalase (CAT), and other peroxidase enzymes. Enzymes that catalytically synthesize compounds that can remove fr ee radicals, such as heme oxygenase (HO). Compounds that physically quench free radicals, such as carotenoids. Compounds that act as sacrificia l agents to protect more valuable biomolecules, such as cytochrome c (cyt c), ascorbic acid (vitamin C), -tocopherol (vitamin E), glutathione (GSH). Enzymes that catalytically reco ver the sacrificed compounds back into the original state, such as cytochrome c oxidase (COX). Proteins that protect molecules against da mage caused by other mechanisms, such as chaperones. Compounds, proteins, or enzymes that regulate redox states of mitochondria, such as GSH and protein bcl-2 The free radical scavenging cooperates with comp licated chemical and catalytic reactions. Usually the scavenging processes carried out through redox reactions of a compound/agent, and the particular compound/agent is activated by the second or more agents. The sequent reactions cascade reactive free radicals and are terminated with the formation of stable species, such as oxygen, water, nitric oxides, and carbon dioxi de. However, the scavenging mechanisms in vivo are even more complicated, and many of them rema in mystical even after years of research in this field. 16

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However, the protection from free radical s cavengers is not 100% effective. The protection from free radical scavenge rs is restricted by their tur nover numbers, the uptake rates, and their distributions in vivo In general, high doses, routin e administration of antioxidants are inevitable to achieve effective means to living an imals. The demands for effective free radical scavengers are soaring since more and more human diseases, includi ng aging, were found to connect to oxidative stress. A broad variety of free radical scavengers have been explored, synthesized, and tested in order to achieve the th erapeutic intervention to living systems. More effective, lipophilic, and even or ganelle-targeting antioxidants have been developed in order to satisfy the needs of life sciences. Most free radical scavengers used for therapy are organic compounds or chelations with transition metal ions. However, a new platform has been demonstrated to perform similar antioxidant protection to biological systems. CeO2 nanoparticles were accidently found to improve brain cell cultures viab ility and organism longevity up to six fold [25,26]. Although how these ceramic nanoparticles im prove cells viability remains controversial, we found that CeO2 nanoparticles reduce endogenous free radical con centrations in cells and may protect cells as free radical scavengers [27]. Indeed, recen t studies, including works published by our group [27], have demonstrated that CeO2 nanoparticles are able to scav enge hydroxyl radicals [28], superoxide radicals [29], and peroxi des in the absence of cell cultures. 1.1 Motivation The motivation of this dissertation was originally to improve the transplanted islets viability, in order to increase the off-insulin time when using transplanted islets to treat patients with type 1 diabetes. Type 1 diabetes is an autoimmune disorder, in which the body's own immune system attacks the beta cells in the pa ncreas, the damage cause s islets to shut down insulin production. This fatal disorder cau ses blindness, kidney and heart failure, limb 17

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amputation, and other diseases associated compli cations [30]. Recentl y, pancreatic islet cell transplantation has been proposed as an ideal treatment to t ype 1 diabetes; however, damages caused by mechanical trauma and anti-rejection drugs dramatically decrease the amounts of preserved islets [31,32,33]. Thus, one of the major challenges is to increase the preserved islets mass during isolation and post tr ansplantation [34,35]. To im prove the preservation, using CeO2 nanoparticles to improve transplanted islets viability was proposed. The motivations of this di ssertation are as follow. To improve preservation of transplanted islets using CeO2 nanoparticles. To identify the mechanism of benefi cial efficacy that carried out by CeO2 nanoparticles. To develop a new platform according to the con cept in materials science. Aim to achieve the same therapeutic efficacy to biological systems by lowering the applied nanoparticle dosages. 1.2 Objectives CeO2 is an excellent catalyst that carries out non-selectiv e catalytic re actions through exchange of electrons, holes, lattice oxygen, and oxygen ions. In many discussions related to its surface chemistry, it has been demonstrated that ROS, such as superoxide, peroxide, singlet oxygen ions, act as intermediates and are involved in the catalytic reactions [36]. Interestingly, viability enhancement of cell cultures has been broadly achieved by using ROS scavengers. Therefore, the key hypothesis in this dissertation relies on the fact that CeO2 is a remarkable catalyst and it may act as ROS scavengers in th e viability enhancement. With the hypothesis aforementioned, there are several objectives in this dissertation. Test free radical concentrations in CeO2 nanoparticles treated cel ls, and the relationship between free radical concentrations and cellular viability. Understand free radical chemistry in between biology/nanoparticles interfaces. Identify distinct antioxida nt defenses performed by CeO2 nanoparticles beyond current knowledge. 18

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Promote the beneficial efficacy of CeO2 nanoparticles. Achieve the same benefits to biolog ical systems with lower dosages of CeO2-based nanoparticles. 1.3 Specific Aims and Expected Outcomes To achieve the objectives, it is necessary to de velop a series of nanoparticles that exhibits greater ROS scavenging activities. Accord ing to the aforementioned hypothesis, ROS scavenging properties of CeO2 nanoparticles are a consequence of catalysis and the properties could be promoted by increasing oxygen vacancies in the materials. Here, it is proposed to dope zirconium into CeO2 nanoparticles in order to promoting CeO2 nanoparticles ROS scavenging activities. There are two reasons choosing zircon ium as dopants. First, the oxidation form of zirconium, zirconia ( ZrO2), is a very biocompatible material and it has been broadly used as biomaterials, such as dental materials. Second, zirconium doped CeO2 ( CexZr1-xO2) materials have been used to improve the catalytic activity carried out by CeO2. With zirconium ions substituting cerium ions in the lattice, it is found that CexZr1-xO2 has four times more oxygen vacancies than CeO2. The catalytic activities of CexZr1-xO2 correlated with the amount of oxygen vacancies, so it can be expected to improve up to four times compared to CeO2. The activities of these CexZr1-xO2 nanoparticles will be te sted in response to crucial free radicals in biological systems, and their activities will be compared with the enzymes that specifically scavenge these popular free radicals. The free radical scavenging activity will be evaluated using biochemical assays that have long been used to test enzyme activities. There are several specific ai ms in this dissertation. Synthesize CeO2 nanoparticles, and test thei r beneficial efficacy to TC-tet cells, including free radical concentrations in the treated cultures. Synthesize CexZr1-xO2 nanoparticles with monodispersed particle diameters, where x = 01.0. 19

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Characterize CexZr1-xO2 nanoparticles structures, and demonstrate the zirconium dopants incorporated in the CeO2 lattice, and form a solid solution. Evaluate the scavenging activities of the s ynthesized nanoparticles in response to free radicals that are influential in living systems, i.e. superoxide radicals ( 2O ) and peroxide ( 2O ), and compare their activities in respect to particular enzymes that scavenge superoxide radicals and peroxi de, i.e. enzyme SOD and CAT. 2To attain the objectives of this dissertation, CexZr1-xO2 nanoparticles will be prepared using reverse micelle synthesis. The structures of the synthesized nanoparticles will be investigated using transmission electron microscopy (TEM ), x-ray diffraction (XRD), and Raman spectroscopy. The scav enging activities of CexZr1-xO2 nanoparticles will be evaluated with commercial assays based on bioche mical reactions. The following re sults are expected in this dissertation. Free radical concentratio ns inhibited in the CeO2 nanoparticle treated cultures. CexZr1-xO2 nanoparticles with narrow particle size distribution and with diameters of 3-7 nm, whereas x = 0, 0.2, 0.4, 0.6, 0.7, 0.8, 1.0. The synthesized CexZr1-xO2 nanoparticles are solid solutions with defined structures. CexZr1-xO2 nanoparticles exhibit greater free radica l scavenging activity in response to superoxide radicals and peroxi de. Their scavenging activitie s vary with different amounts of zirconium doping. The free radical scavenging activities of CexZr1-xO2 nanoparticles correlate to the concentration of oxygen vacancy in their lat tice. Free radical scavenging activities of CexZr1-xO2 nanoparticles could be improved up to four times in in CexZr1-xO2 nanoparticles with 20-40 % zirconium dopants. In general, this dissertation covers the synthesis, characterization of nanoparticles, and investigations to the free radical chemistry in between bio/nano interfaces. The focus of this dissertation is especially on th e investigations of free radica l chemistry in between bio/nano interfaces, which is accomplished by testing th e free radical scavenging activity of vacancy engineered nanoparticles. After all, the contribution in this dissertation gives a comprehensive understanding to, first, the therapeutic efficacy of CeO2 nanoparticles in biological systems; 20

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21 second, the mechanism of free radical scavenging by CeO2-based nanoparticles. Based on the advancement in knowledge, this work may help preparing more effective free radical scavenging nanoparticles for biomedical appl ications. In summary, we ar e aiming to achieve the same therapeutic efficacy to biological system s by treating lower nanoparticle dosages.

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CHAPTER 2 BACKGROUND In this chapter, the background of free radicals in biology and medicine will be introduced. After it, the antioxidant defense systems and altern ative defense pathways that are used to protect biological systems from free radicals attack will be introduced. Finally, current research based on CeO2 nanoparticles therapeutic efficacy will be listed and described in detail. Most importantly, the hypothetic mechanism to describe the benefits that CeO2 nanoparticles brought to biological systems as well as a model that can be used to improve CeO2 nanoparticles activity are also covered in this chapter. 2.1 Free Radicals in Biology and Medicine 2.1.1 Free Radical Theory of Aging Free radicals are species capable of indepe ndent existence that contain one or more unpaired electrons. Because of th e unpaired electrons, free radical s tend to be reactive and easily undergo chemistry with other molecules. There are a broad variety of free radical species in living systems. This occurs because electrons ge nerated in the metabolic chain react with other molecules (such as oxygen, proteins, lipids, and DNA, RNA), forming free radicals. The metabolic chain in mammalian cells for energy ge neration mostly occurs in mitochondria. By consuming glucose and oxygen, mitochondria transfer nutrition into energy and yield as byproducts oxygen radicals (shown in Figure 2-1 ). In mitochondria, electrons leaked out as failures occur in the electron transportation chain (shown in Figure 2-1 (a)). The leaked electrons then react with oxyge n or other species, forming oxygen radicals and other free radicals. Therefore, more than 90% of free radicals are produced by mitochondria, and the oxidative damage usually initiated from mitochondria. 22

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In biochemistry, the free radicals of interest are often referred to as reactive oxygen species (ROS), i.e. superoxide radicals ( ), peroxides ( ), and hydroxyl radicals ( ), because the most biologically significant free radicals are oxygen-centered. ROS are involved in the cell growth, differentiation, progr ession, and death. Low concentrati ons of ROS may be beneficial or even indispensable in processes such as in tracellular signaling and defense against microorganisms. However, high amounts of reactive ROS participate in cell cycles resulting in cumulative damages to lipids and cell DNA. The expression of damages results in inflammation, cancers, age related diseases, and aging to ma mmalians. In the last decades, Alzheimers, Parkinsons diseases, diabetes, ca rdiovascular diseases, eye diseas e, cancers, obesity, aging, and many other diseases are found to relate to oxidative stresses caused by ROS [5,37,38]. However, not all free radicals are ROS and not all ROS are free radicals. For example, the free radicals superoxide and hydroxyl ra dical are ROS, but the ROS hydrogen peroxide (H2O2) is not a free radical species, however the term free radicals usually refer to these compounds in biology. 2O 2 2OOHThe free-radical theory of aging is that organisms age because cells accumulate free radical damage with the passage of time. The theory has some immediately attractive features, and is rational to explain human disorders and aging. Free radicals are produced during metabolism, sometimes these free radicals are detrimental and sometimes are for useful purposes. Once antioxidant defenses do not scavenge them completely, the ongoing oxidati ve damage to DNA, lipids, and proteins causes programmed cell death, cell muta tion, or dysfunction of the cell. Production of free radicals can be envisaged as the consequence of genes selected because they confer benefits in earl y life. For example, facilita ting signal transduction in early stage, diminishes infectious agents. The theory can explain the relation between metabolic rate, oxygen consumption and lifespan. The more oxygen consumed forms higher level of free radicals, thus cause more oxidative damages or reduce lifespan in mamma ls. However, higher metabolic rate does not cause shorter lifespan dire ctly, since the activities of enzymes or proteins maybe greater so to reduce the damages by free radicals. 23

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Mitochondria are the energy pl ant in cells. The electron transport chain involved in metabolism, however the leakage of electrons in the transportation generate free radicals. The leakage of electrons is related to agi ng of mitochondria or insufficient enzymes and proteins in the chain reaction. The accumulation of free radicals causes lipid peroxidation and reduced proteins in mitochondria. It results in th e release of cytochrome c and further lead to cell programmed death. Caloric restriction in mammals often decrease s levels of oxidative damage to DNA, lipids, proteins, and attenuates age-relate d declines in repair systems. Long lived species usually have better antioxi dant protection in re gulation to rates of oxygen uptake than shorter-lived species. In conclusion, free radicals cause oxidative damage to important molecules, further promote apoptosis or senescence that impair tissue renewal. Free radicals also generate inappropriate cellular signaling, and contribute to age-related diseases. 2.1.2 Modern Free Radical Theory in Biology After the free radical theory in biology wa s proposed, the influences of ROS have been controversial since some studies found partially promote ROS level may be beneficial to lifespan of mammals [1]. On one hand, ROS are detrimen tal to cells and tissues. On the other hand, higher ROS level may stimulate enzyme activities a nd increase animals lifespan [1]. Above all, it was found the redox states of mitochondrial pr otein, cytochrome c, are also crucial in apoptosis. It is because the reduced cytochrome c exhibits lower affinity to inner mitochondrial membrane. Once the inner mitochondrial membrane s are peroxidized, the reduced cytochrome c can be released to inner mitochondrial membrane space. After all, the accumulated cytochrome c is released to cytoplasm and reacts with hydrogen peroxide, forming caspase proteins (precursors of apoptosis signaling) As cytochrome c is released from the inner mitochondria membrane, electron transportation in metabolic chain becomes short, so more electrons can be 24

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released from the transport chain. This results in dramatic increases of free radical generation, and finally accelerates the progression of apoptosis (shown in Figure 2-1 (b)). Free radicals play extremely important roles in the cytochrome c releasing processes. Free radicals cause lipid peroxidation in inner mitoc hondrial membranes; super oxide radicals reduce cytochrome c and peroxide oxidize cytochrome c before they were released to inner mitochondrial membrane space; peroxide oxidize d the reduced cytochrome c in cytoplasm, forming caspase proteins. Although the redox states of cytochrome c are crucial in apoptosis, it is not proper to discuss whether the reduced state or oxidized state of cytochrome c is beneficial to cells. In conclusion, the oxidized cytochrome c is preferable in inner mitochondrial membrane, while the reduced cytochrome c cant form caspase proteins when cytochrome c is released [40,41,42,43]. In the modern free radical theory in biology, free radicals are no longer always detrimental to cells and tissues, but often adequate oxidative stress maybe beneficial to mammals. First of all, proper oxidative stress stimulates the generati on of antioxidant enzymes, proteins, and assists to defend oxidative stress of detrimental levels Second, proper oxidative stress adjusts the redox states of cytochrome c in mitochondria, fo rming oxidized cytochrome c, which has higher affinity to bond to mitochondrial membrane. Th ird, proper oxidative stress reduces cytochrome c when released, avoiding them from binding with other proteins to form caspase proteins. Instead of the cumulative damages caused by free ra dicals, the redox states of cytochrome c that altered by free radicals play a more influen tial role in modern free radical theory. 25

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Figure 2-1. Metabolism in a mitochondrion. A) elect ron transport chain. B) modern free radical theory causing apoptosis. Figures made according to [1,2,40,41,42,43]. 2.2 Antioxidant Defense in Biology and Medicine 2.2.1 Antioxidant Defense System It has been demonstrated that administering free radical scavengers into biological systems allows improving cells viability, cells survival rates, and mamma ls lifespan. Some free radical scavengers benefit cultures th rough removing free radicals cataly tically, some through quenching free radicals, some through regul ating mitochondrial redox potential, and the others protect cultures through stimulati ng the generation of antioxidants. They can be artificial or natural chemical compounds, proteins, or enzymes that execute electrochemical reactions with free radicals or regulate the re dox states of mitochondria. 26

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There have been broad categories of free radi cal scavengers reported. In this section, several major free radical scavengers and their functions are listed in Table 2-1 Table 2-1. Genetic changes and the responded free radical scavengers th at affect oxidative damage in living systems [1]. Many studies have shown that these disorders or diseases caused by oxidative stress could be soothed or inhibited by administering adequa te amounts of free radical scavengers, such as the most discussed enzymes heme oxygenase-1 (HO-1), enzyme SOD and CAT, into laboratory animals. Specifically, by exposing pancreatic tissues to SOD mimic compound, cell survival rates can be improved to three or four times co mparing to untreated pa ncreas during isolation [44]. Such technical approach has been adopted in islet transplantation nowadays, in order to increase the preservation mass of transplanted islets. In additi on, exposing pancreatic tissues to HO-1 can improve islets function in vivo after transplantation [ 45]. Specifically, exposing SOD and CAT-mimic compounds to wild type worms, flies, fungi, can increase their mean lifespan by 27

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44%, 30%, and 600%, respectively [22,23]. Specif ically, transgenic mice with over-expressed CAT in mitochondria can live 20 % longer than wild type mice [24]. Overall, introducing sufficient amounts of free radical scavengers into biological sy stems allows to elevate cell cultures viability and su rvival rates under stress in vitro. Furthermore, in vivo studies show that the administration of free radical scavengers attenuates oxidative stress and inhibits the progression of disorders in animal models [13,19]. 2.2.2 Alternative Antioxidant Defense Oth er Than Direct Free Radical Scavenging Most antioxidant defense pathways are to s cavenge free radicals endogenously. However, there are particular enzymes that actually ar e not scavengers, but to oxidize the reduced cytochrome c back into their functionalities in mitochondria. Peroxidases, which are also antioxidant defense enzymes, us e peroxides to oxidize another s ubstrate. This particular antioxidant defense pathway can be used to elucidate why the adequa te amounts of hydrogen peroxide can benefit a biological system [1,46]. Furthermore, th is antioxidant defense pathway also inhibits cytochrome c released through regul ating its redox states, in which the peroxidase oxidize cytochrome c and so to create its bondin g strength with inner mitochondrial membranes. Amongst the enzyme peroxidases, cytochro me c peroxidase a nd NADH oxidase are the most important enzymes in the mitochondrial inne r membrane space [47]. In the mitochondrial electron transportation chain cytochrome c pe roxidase plays as complex IV, and it takes electrons away from the reduced cytochrome c (see Figure 2-1 (a)). Lack of cytochrome c peroxidase, cytochrome c would be reduced by el ectrons and be released from mitochondria to trigger apoptosis [41,43]. The sa me protection is also true by enzyme NADH oxidase [37]. 2.3 CeO2 in Catalysis, Biology, and Medicine CeO2 and other CeO2-related materials are excellent catalysts that carry out catalytic reactions through the exchanging the electrons, holes, lattice oxygen, and oxygen ions with 28

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ligands. Recently, CeO2 was found to protect cultures in the stressed circumstances, and has been proven to improve cell cultures viability. In this section, the background of CeO2 will be introduced based on its catalytic properties. The hypothesis of free radical scavenging as well as the specific s cenario related to catalysis of CeO2 will be discussed. Following the discussion, the studies that used CeO2 nanoparticles to benefit biological systems will be introduced in details. Fina lly the free radical scavenging mechanism that carried out by CeO2 will be discussed according to the knowledge learned in references and the understanding of free radical theory in biology. 2.3.1 CeO2 as Catalysts Based on its extraordinary catalytic properties, CeO2 and its related materials have been widely applied for the use in various catalytic sy stems, such as three-way catalysts (catalytic converters), solid electrolytes in solid oxide fuel cells, gas sensor s, catalysts in water-gas shift reaction, and as oxygen storage materi als [48,49,50,51,52,53]. The versatility of CeO2 nanoparticles relies upon its behavior of nonstoic hiometric lattice oxygen. In other words, the catalysis carried out by the concentration of mobile intrin sic defects, undoped CeO2, i.e. oxygen vacancies, dominates the designated chemical reactions occur via the exchange of oxygen species in the vicinity of CeO2 surfaces [36]. 2.3.2 CeO2 Nanoparticles in Biomedical Applications The therapeutic efficacy of CeO2 nanoparticles was first discovered by Rzigalinski et al. [25,26]. In the incidence, the lifespan of CeO2 nanoparticles treated to neuron primary cells was found to be improved up to six fold. Si nce then, the benefits of introducing CeO2 nanoparticles into biological systems were confirmed in other cell lines and tissues both in vitro [27,54,55,56] and in vivo [57,58]. Among all the studies including this dissertation, it was found that the intracellular free radical concentrat ion of those treated cultures remained at ordinary level, even 29

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though the cultures were under serious stress. Thus, it is now convincing that CeO2 nanoparticles are free radical scav engers, and protect cu ltures from apoptosis through antioxidant defense. The benefits of introducing CeO2 nanoparticles into biological systems were confirmed in different cultures and tissues both in vitro and in vivo*. Specifically, the introduction of CeO2 nanoparticles with size smaller than 20 nm in to primary cultures [25,26], HT22 rodent neuronal cells [55], or into CRL8798 breast epithelial cell s [56], led to improved cellular survival with survival rates approachin g three to four times higher than co ntrol cells, when exposed to stressed environments. The efficacy of CeO2 nanoparticles to scavenge reactive oxygen species has also been demonstrated in vivo. Specifically, in studies invol ving peroxide induced retinal degradation, delivery of CeO2 nanoparticles directly into the vitreous of the mouse eye prevented against vision loss by protecting the retina from intracellular peroxide s [58]. Another study investigating spinal cord repair demonstrated that the administrati on of a single dose of CeO2 nanoparticles provided significant neuron-protection to adult rat spinal cord neurons [54]. Finally, intravenous administration of CeO2 nanoparticles protected MCP-mice (monocyte chemoattractant protein (MCP)-1 transgenic mice) against the progression of cardiac dysfunction by attenuating myocardial oxidativ e stress, endoplasmic reticulum (ER) stress, and inflammatory processes [57]. In these studies and the results that obtained in our works, the benefits of administrating CeO2 nanoparticles are consistent with the redu ced oxidative stress in cell cultures. The reduction of oxidative stress supports the hypothesis that CeO2 nanoparticles protect cultures by The dosages of CeO2 nanoparticles used in the references are differe nt from the molar concentrations that used in the following experiments. The concentrations used in the references are based on the assumption of 1,500 cerium atoms in a single CeO2 nanoparticle, so the molecular weight of the particular CeO2 nanoparticle is estimated as 172.12 X 1,500 = 258,180 g/mole. The molar concentration used in our studies are based on ionic concentration of CeO2, where the molecular weight is 172.12 g/mole. 30

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scavenging excess amounts of intrac ellular free radicals. Although the researches that studying free radical scavenging properties of CeO2 is absent, it has long been demonstrated that oxygen radicals act as intermediates in the catalysis carried out by CeO2. 2.3.3 Free Radicals in CeO2 The mechanisms of free radical scavenging afforded by CeO2 nanoparticles have been investigated in a variety of nonbiological systems including fuel cells [59], catalytic converters [36,60], and gas sensors [6]. The applications of CeO2 nanoparticles rely on the properties to exchange their lattice ox ygen ions, electrons, holes, with reag ent molecules. In the exchange processes, CeO2 provides electrons to the reagent molecules, forming bonds in its vicinity, and finally react with second reagent molecules or la ttice oxygen to generate the designated products. For example, in the oxygen storage process, oxygen molecules first adsorb on CeO2 surface, transform into superoxide radicals ( ), peroxide ( ), then oxygen ions ( ), and finally migrate into oxygen vacancies ( ) in the lattice [60,62,63]. The oxygen ions captured in CeO2 lattice or the lattice oxygen in CeO2 can be released, forming gaseous oxygen or becoming involved in other chemical reactions. The widely accepted mechanism to explain the surface oxygen exchange on CeO2 nanoparticles is shown in Equati on (2-1). Based on these and other efforts it is now belie ved that oxygen exchange on CeO2 surface is triggered by the adsorption of oxygen molecules, followed by a tr ansformation into superoxide radicals and peroxides, dissociation into oxygen ions, and fi nally migration into oxygen vacancies in the lattice. Such reaction has been observed at room temperature [64]. Using spectroscopic techniques, superoxide and peroxi de radicals have been identified as intermediates of surface oxygen exchange on ceria surface, and the exchan ge of environmental oxygen and lattice oxygen has been observed at room temperature [62,64]. In addition, using atomic force microscopy and 2OV2 2OO 2O O 31

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scanning tunneling microscopy, both Namai et al. [65] and Esch et al. [63] have observed the migration of oxygen species into CeO2s surface oxygen vacancies at room temperature, with the mobility of oxygen species promoted as temperature increases. The widely accepted formula of oxygen exchange on CeO2 surface is as follows: 2 2 )(2)(2)(2)(222lattice ads ads ads ads gOO OOOO (2-1) Free radicals are involved in chemical reactions catalyzed by CeO2 and oxygen deficient materials. For example, superoxide radicals on CeO2 behave as active oxygen species in CO oxidation; N2O, NO2, and NOradicals are detected on CeO2 in NO reduction; hydroxyl radicals on CeO2 are found to participate in the water-gas shift reaction; and peroxide species on CeO2 are found to oxidize hydrocarbon species CH4, C2H4, C3H6 even at room temperature. In general, it has been demonstrated th at active free radicals adsorbed on CeO2 surface behave as triggers in various chemical reactions, and as a measure of active free radicals determined by the oxygen vacancy concentration in the lat tice [66,67]. OH2.3.4 Hypothesis of CeO2 Nanoparticles in Free Radical Scavenging Although the exact process by which CeO2 nanoparticles scavenge free radicals in biological systems is not known, one could hypothesize that it resembles the mechanism observed in non-biological system s. The ROS generated in biol ogical systems are reactive. When CeO2 nanoparticles are close to ROS, the electr ons of free radicals may form bonds with mobile electronic carriers that provided by oxygen vacancies. These oxygen species then dissociated into oxygen ions and finally diffuse in to the lattice. The to tal reaction becomes the exchange of environmental oxygen species and lattice oxygen. Thus, we hypothesize that intracellular free radicals, such as superoxide radicals and peroxides would act as intermediates in surface oxygen exchange. These oxygen specie s finally migrate into oxygen vacancies in 32

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CeO2 nanoparticles, and lattice oxygen may be em itted to form oxygen molecules in biological systems. The hypothesis that de scribes oxygen radical scavenging by CeO2 nanoparticles is illustrated in Figure 2-2 Figure 2-2. Hypothetic mechanism of free ra dical scavenging in the vicinity of CeO2 surface. Oxygen vacancies (Vo ..) provide sites for adsorption of superoxide radicals (O2 .-) and peroxides (O2 2-). These metastable vacancies also thermally activate the transition of radicals into oxygen ions and migration into CeO2 lattice. 2.3.5 Promote Catalytic Activity by Doping Zirconium into CeO2 The catalytic properties of CeO2 nanoparticles can be improved according to three principles. It can be improve d intrinsically throug h exciting the electr ons and holes through band gap. It can be improved extr insically through adding impurities in the lattice. Or, it can be improved stoichiometrically by increasing the oxyge n vacancy concentration in the lattice. In general, the catalytic properties of CeO2 can be improved by doping a broad variety of elements. In this dissertation, doping zirconium into CeO2 is selected to achieve the improvement [36]. CeO2 has a face-centered cubic unit cell with sp ace group Fm3m. In this structure, each cerium cation coordinates eight equi valent nearest-neighbor oxygen an ions at each corner of the cube, and each anion tetrahedrally co ordinates four cations [36]. Both ZrO2 and CeO2 have cubic fluorite structures. Mixi ng the two materials in solid so lutions will form a pyrochlore 33

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structure (shown in Figure 2-3 ). The defect-rich pyrochlore stru cture is the result of misfit of zirconium and cerium ionic radii (0.084 nm and 0.097 nm, respectively) in the structure. In addition, the relatively stable quadricvalent zirconium cations sit on trivalent/quadrivalent cerium sites leading to the formation of extra oxygen vacancies in the solid solution, thus decreasing coordination numbers, and expanding their la ttice parameters. Several studies have found that the concentration of trivalent cerium ions in CexZr1-xO2 solid solutions is promoted as more quadricvalent zirconium doping in the lattice [68,69,70]. As the results, the improved catalytic properties in zirconium doped CeO2 are contributed nonstoic hiometrically (i.e. via increasing oxygen vacancies) and extrinsically (i.e. via other defects). Utilizing this concept, the concentration of charge carriers in CexZr1-xO2 solid solutions is promoted and so their catalytic activities are improved. Figure 2-3. Schematic diagrams show ing cubic fluorite (left) and pyr ochlore (right) structures for CeO2/ZrO2 and CexZr1-xO2, respectively. Figures made according to [36]. 34

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The catalytic activities of CexZr1-xO2 nanoparticles are usually evaluated by measuring their oxygen storage capacity (O SC). OSC represents a measure of the oxygen vacancy concentration in metal oxides, and directly correl ates to a catalysts performance, such as in catalytic converters, catalytic combustions, water-gas shift reactions, and oxygen storage materials. It has been demonstrated that CexZr1-xO2 nanoparticles exhibit up to four times higher OSC when 20-40% of cerium ions in the solid so lutions were substituted by zirconium ions at low (400) and high temperatures (1000) [71,72]. As aforementioned, it is hypothesized that oxygen vacancies mediates free radical scavenging by CexZr1-xO2 nanoparticles. Therefore, preparing CexZr1-xO2 nanoparticles of varian t amounts of lattice oxygen v acancy and test their activities against free radicals may provide clues to examin e the hypothesis that free radical scavenging is mediated by lattice oxygen vacancies. 2.3.6 Introduction of CexZr1-xO2 CexZr1-xO2 undergoes three major phase transfor mations at ambient temperature with elevated zirconium conc entrations (shown in Figure 2-4 ) [73,74]. At ambient temperature, CexZr1-xO2 sustained in cubic fluorite structure (c) when 0 15% of zirconium containing in the crystal structure. The cubic fluorite pha se undergoes to two metastable phases, t" and t', and then to tetragonal structure (t) in the intermediate zirconium containing range (15 90 %). The t' phase is a phase through a diffusionless transition from t phase, and the t" phase is a intermediate phase between t' and c phases. The t" phase shows no tetragonality of the sublattice and it exhibits an oxygen displacement from ideal cubic fluorite sites, so t" phase is usually referred to as a cubic phase. Finally, at highe r than 90 % of zirconium containing, CexZr1-xO2 undergoes to monoclinic or the mixture of tetragonal and monoclinic (m) structures at ambient temperature. Table 2-2 shows the classification of phases in CexZr1-xO2 with elevated zirconium concentration in the lattice. 35

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Figure 2-4. Phase diagram of the CeO2-ZrO2 binary system [73]. Table 2-2. Classificatio n of the phases in the CeO2-ZrO2 binary system [36]. The phase transformation of CexZr1-xO2, however, could be dependent on different synthesis methods, particle size, or tempering procedures. For in stance, the phase transformation was found to postpone to higher zirconium c ontaining range while the particle size was engineered as small as 10 nm (shown in Figure 2-5 ) [75]. Specifically, the targeted particle size 36

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in this study is smaller than 10 nm, therefore th e phase transformation could be expected in the nanoparticles containing higher zirconium, when prepared in reverse micelle synthesis. The phase transformation of CexZr1-xO2 can be distinguished by using XRD and Raman spectroscopy. Using XRD, the transition from cubi c to tetragonal phase can be identified by the tetragonal features next to XRD peak (220). On the other ha nd, the transition of sub-structures, i.e. c, t", and t' phases, can be identified using Ra man spectroscopy due to the phonon excitation which is induced by the symmetry of lattice st ructures. The methodology to distinguish the crystal structures as well as phase transition is shown in Figure 2-6 Figure 2-5. Phase transition of CeO2-ZrO2 binary system. The transitions are particle size dependent [75]. The phase transitions were distinguished by XRD associated with Raman spectroscopy. Figure reproduced from [75]. 37

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38 Figure 2-6. XRD and Raman spectra of (1-x)CeO2xZrO2. A) XRD of (1-x)CeO2xZrO2 show the phase transition from cubic fluorite to tetragonal phase. B) Raman shift of (1x)CeO2xZrO2 show the phase transition of sub-st ructures. Figures reproduced from [75].

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CHAPTER 3 TEST HYPOTHESIS WITH CELL CULTURES The therapeutic efficacy of CeO2 nanoparticles has been dem onstrated in various cell cultures. The successful outcomes of viability improvements in the treated cultures have been demonstrated to be true both in vitro and in vivo. In this chapter, the experiments are designed to elucidate the mechanism of viability enhancement that contributed by CeO2 nanoparticles. For the experimental setups, CeO2 nanoparticles were administered into TC-tet cells, and the endogenous oxidative stress in cell cultures was evaluated. The re sults suggest that CeO2 nanoparticles serve as free radical scavengers in biological systems, and their scavenging properties most likely are through catalysis. The evidence in this chapter supports the concept that enhancing the catalytic activities of CeO2 nanoparticles may allow decreasing the dosages in biological systems for the same therapeutic efficacy. This chapter also serves as a demonstration of previous chapters, where the results imp lied the alternative plat forms providing a novel therapy to human disorders. The contents in this chapter are ba sed on the paper that the author published in Nanomedicine [27]. 3.1 Methodology To test the hypothesis and fu rther establish the fundamental knowledge of this technique for islet transplantation in vivo, we tested TC-tet cell lines that treated with CeO2 nanoparticles in response to raised oxidative stress. The TC-tet cell lines are murine insulinoma cells. They are modified from cells in islet of Langerh ans in the pancreas. The TC-tet cell lines function is similar to primary cells, so they are optimum cell lines for preliminary studies in biochemistry. The TC-tet cells are treated with the CeO2 nanoparticles synthesized in our lab. It is required to note that the preparation of CeO2 nanoparticles in the sequent sections of this chapter 39

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is different to the CeO2 nanoparticles in the other chapters. Essentially, a different surfactant, lecithin was used to synthesize CeO2 nanoparticles in this chapter, while CexZr1-xO2 nanoparticles in the following chapters were pr epared in a reverse micelle system formed by surfactant, bis(2-ethylhexyl) sulphosuccinate (AOT). Delineating the in vitro and in vivo scavenging mechanism of CeO2 nanoparticles is important in furthering our understanding of these particles and ex tending their potential biological and medical applications. To do so, it is necessary that CeO2 nanoparticles are colloidally stable in solution for extended pe riods of time because biological systems may require prolonged incubations to achieve opt imum efficacy. In the previous works, CeO2 nanoparticles were synthesized and then stabilize d in a solution of multivalent dispersants. A solution of tri-sodium citrate was applied to stabilize CeO2 nanoparticles by shifting CeO2 nanoparticles isoele ctric point (IEP). The intracellular concentration of CeO2 nanoparticles in murine insulinoma TC-tet cells, a well accepted surrogate for in vitro studies of islet cell viability, was quantified and their ability to scavenge free radicals in vitro was assessed by exposure to hydroquinone. Synthesis of CeO2 nanoparticles Phosphatidylcholine (laboratory grade), toluen e (laboratory grade), cerium (III) nitrate hexahydrate (99.5%, M.W. = 434.22 g/mo le), and ammonium hydroxide (NH3 content 28~30%) were purchased from Fisher Scientific and us ed without further purif ication. 2.285 gram of phosphatidylcholine was dissolved in 100 ml of toluen e to form reverse micel les. Five mini-liter of 0.1 M cerium nitrate aqueous solution was pipette d into the colloidal micelle system, and the mixture was strongly stirred for 30 min until th e system appeared homogeneous. Ten mini-liter of 1.5 M ammonium hydroxide solution was titrated into the system to initiate electrochemical 40

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reaction. After 45 min of stirring, CeO2 nanoparticles gradually formed in the reverse micelles. The nanoparticles were collected by centrifuga tion with a force field of 18 G and then sequentially rinsed with 50 ml of methanol, 50 ml of ethanol, and 50 ml of water in order to remove the redundant surfactants. Between each rinse the nanoparticles were collected by centrifugation at 18 G. Colloidal stabilization of CeO2 nanoparticles The CeO2 nanoparticles were dispersed in 100 ml of 0.05 M (13.17g/l) saline/sodium citrate buffer (Cat. # 821840, MP Biomedical) a nd ultrasonicated until th e appearance of the suspension changed from turbid to transparent. The pH value of suspension was adjusted to 7.4 using 0.1N citric acid and sodium hydroxide solutions. The suspension was sterilized by filtration with a 0.2 m filter. The average yields from su ch preparation were approximately 50 mg of CeO2 nanoparticles stabilized in 100 ml of sodium citrate solution. Concentration analysis The yields and concentration of CeO2 suspensions were determined by Perkin-Elmer Plasma 3200 inductively coupled plasma spectrometer (ICP). The ICP sample was prepared by dissolving 1 ml of the suspension in 1 ml of 95% sulfuric acid (Acros Organics). After heating to 90 C for 24 hrs, the sample was diluted to 5ml with deionized water for ICP measurement. Structural characterization The crystal structure a nd crystallite size of CeO2 nanoparticles were characterized by X-ray diffraction (XRD) using CuK radiation (XRD Philips APD 3720). For the preparation of the XRD sample, CeO2 nanoparticles were extracte d before stabilization and dried in air for 24 hrs. Micrographs and electron diffraction patte rn were determined using a JEOL 2010F transmission electron microscope (TEM) equipped with selected area diffraction (SAD). For the 41

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preparation of TEM samples, CeO2 nanoparticles were extracted before stabilization and dried on carbon formvar-coated grids (Ele ctron Microscopy Sciences). Stability tests The stability of CeO2 suspensions was determined by meas uring the zeta potential of the suspension using a Brookhaven ZetaPlus. On e tenth mini molar of citrate-adsorbed CeO2 nanoparticles and citrate-free CeO2 nanoparticles were prepared, and 0.1 vol% of potassium chloride was added to define the background electr olyte. The pH of the sample was adjusted by titrating a trace amounts of 0.1 N hydrochloric acid and 0.1 N sodium hydroxide into the suspensions. Cell culture Murine insulinoma TC-tet cells were provided by the la boratory of Shimon Efrat (Albert Einstein College of Medicine, Bronx, NY) and cu ltivated as monolayers in Dulbeccos Modified Eagles Medium (DMEM) (Mediatech, Herndon, VA). This medium contains 20 mM glucose and is supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan, UT), antibiotics (100 U/ml penicillin and 100 ng/ml streptomycin), and L-glutamine to a final concentration of 6 mM (Sigma, St. Louis, MO). Cultures were maintained at 37 C under humidified (5% CO2/95% air) conditions, and appropriate media were completely replaced every 2-3 days. Quantification of intracellular CeO2 nanoparticles The intracellular amount of CeO2 nanoparticles was determined by ICP. TC-tet cells were incubated in media containing 0, 50, 100 and 200 M CeO2 for 48 hrs. At the end of this incubation period, the cells were washed with PBS 2-3 times to remove all extracellular CeO2 nanoparticles. A pellet of 20-40 millions cells was generated by centrifugation and digested in 1ml of either 95% sulfuric acid for 48 hrs at 60 C. The sample was then diluted with 5ml with 42

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deionized water before proceeding with the ICP measurements. Samples with 0 M CeO2 were actually prepared as sham controls, meaning that a volume of the vehicle solution (i.e., citrate) equal to the volume of the CeO2 nanoparticle solution was added to the cells. The same cultures used to determine the intrac ellular cerium concentration were also used to visualize the intracellular distribution of the nanoparticle s with TEM microscopy. TEM images of CeO2 exposed and sham treated control TC-tet cells were obtained with a JEOL 2010F microscope to visualize the in tracellular compartmentation of the CeO2 nanoparticles. Chemical analysis was performed by energy disp ersive spectrum (EDS) built in the same TEM microscope. Quantification of intracellular free radical concentration The ability of citrate-coated CeO2 nanoparticles to scavenge free radicals in vivo was assessed by the following protocol. TC-tet cells were cultured as monolayers in T-75 flasks and incubated overnight (~18 hrs) with DMEM media that contained 0, 100 or 200 M CeO2 nanoparticles. Each flask contained between 30-40 million cells. At th e end of the labeling period, the cells were rinsed 2-3 time s with PBS to remove extracellular CeO2 nanoparticles, trypsinized, centrifuged, and the cell pellet re-s uspended in 20 ml of fresh non-CeO2 containing DMEM. Aliquots of 4 ml were placed in separa te centrifuge tubes. The freely suspended cells were exposed for 15 min to media that were supp lemented with an aliquot from a stock solution of hydroquinone (HQ) so that the final hydroquinone concentration in the media was 1 or 2 mM. Cells exposed to 0 mM HQ were in fact sham treat ed with a volume of PBS equal to that of the stock HQ solution that was added to reach 2 mM. The stock HQ solution was prepared by dissolving 8 mg HQ in 0.5 ml of di oxane and then diluted with 9.5 ml of PBS. At the end of the 15 min HQ treatment the cells were cent rifuged, the media discarded and 200 l of 2,743

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dichloroflurescin diacetate (DCF) (Molecular Pr obes, Eugene OR) were added to suspend the cells. Each 200 l suspension was placed in a single well of a 96-well plate and the plate was placed in a Synergy HT reader (Bio-Tek, Winooski, VT) and allowed to incubate at 37 C for 20 mins. Fluorescence was measured using an excitation filter centered at 480 nm and an emission filter centered at 520 nm. These e xperiments were repeated 3 times. Analytical assays For all experiments describe d above the viability of TC-tet cells following exposure to CeO2 nanoparticles was assessed by a commercially available assay based on the detection of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny l-tetrazolium bromide) (Molecular Probes, Eugene, OR), while the amount of insulin secreted in the media was measured by using mouse insulin Elisa based immunoassa y kit (ALPCO, Windham, NH). 3.2 Results and Discussion To visualize the CeO2 nanoparticles and measure their physical dimensions, TEM micrographs of the nanoparticles were obtained. Figure 3-1 (a) shows the image and diffraction pattern (up right) of CeO2 nanoparticles under such preparations. Based on this and similar images from other preparations, we deduce that the CeO2 nanoparticles are equiaxed, of monodispersed particle size. The SAD pattern indicates that these CeO2 nanoparticles were highly crystallized. The size of the CeO2 nanoparticles range between 2 nm and 6 nm for all preparations synthesized in this study. We arbitrary selected 50 nanoparticles in TEM images and measured their Ferets diameter, and the size distribution upon such estimation has been shown in Figure 3-1 (b). The average particle size is 3.7 nm, and the error is estimated as 0.5 nm due to the contrasts of TEM images. 44

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Figure 3-2 shows an XRD spectrum of the synthesized CeO2 nanoparticles, and the diffraction peaks indicate that th ese particles ar e crystalline CeO2 with fluorite crystal structure. The diffraction peaks (111), (200), and (220) crystal planes are used to calc ulate these particles crystallite size. Usi ng Scherrers equation, Dhkl = 0.89 / hkl cos, the crystallite sizes are calculated as 3.7 0.6 nm, 3.9 0.7 nm, and 3.6 0.6 nm from diffraction p eaks (111), (200), (220), respectively. The average crysta llite size is 3.7.2 nm after calculation: this value corroborates the average particle size determined by TEM and implies that each of these CeO2 nanoparticles is a single crystallite. Figure 3-1. TEM micrographs of CeO2 nanoparticles that were s ynthesized using surfactant lecithin. A) TEM image of CeO2 nanoparticles (scale ba r 10 nm). Diffraction pattern associated with TEM shows that the synthe tic nanoparticles are highly crystallized. B) Particle size distribution calculated from 50 arbitrary selected particles in TEM images. The average Feret s diameter of synthetic nanoparticles is 3.7 nm. 45

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Figure 3-2. XRD pattern of CeO2 nanoparticles. The mean crysta llite size of thes e particles is calculated as 3.7.2 nm using Scherrers equation. In order to minimize the influences from aggl omerations and concentration gradients that may occurr in the growth me dium, it is necessary for CeO2 nanoparticles to be well dispersed in the medium. Unfortunately, th e isoelectric point (IEP) of CeO2 nanoparticles is around 6.5 to 7.5, whereas the CeO2 nanoparticles tend to agglomerate at th e targeted pH value, 7.4. Here, we used tri-sodium citrate molecules as dispersants, since tri-sodium citrate has been used to disperse CeO2 nanoparticles through shifti ng the isoelectric point of CeO2. Using tri-sodium citrate, the IEP of CeO2 is shifted to lower pH values, therefore at pH 7.4 CeO2 have strong negative electrostatic charges on their surface, resulting in stabilization effects. The other advantage of using tri-sodium citrate is due to its pH buffering prope rties. Tri-sodium citrate and its salts have been used to make pH buffers as well as anti-coagulati on solutions at pH 7.4. Therefore, tri-sodium citrate buffer was used as dispersing solution as well as pH buffer in this study. Figure 3-3 shows the major chemical compositions in tri-sodium citrate buffer. Figure 34 presents the zeta potential of a citrate-adsorbed and a citrate-free CeO2 suspension at various 46

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pH values. Comparing both zeta potentia l profiles, we conclude that IEP of CeO2 shifts from 8 to approximately 2 when the nanopar ticles are stabilized in citrate so lution. The shift of IEP is a result of surface charge modificatio n due to the adsorbed citrate on CeO2 nanoparticles. The zeta potential of citrate treated na noparticles shifts from +10 mV to -38 mV at pH 7.4, which indicates the promotion of surface charges on the CeO2 particle surface, and provides CeOnanoparticles sufficient electrostatic repulsion to avoid flocculation. The citrate treated suspension is able to retain tr ansparency without visible depos ition of particles for at leas days. 2 t 60 Figure 3-3. Chemical compositions of the tri-sodium citrate buffer. 47

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Figure 3-4. Zeta potentials of CeO2 suspensions as a function of pH. Stabilization of CeO2 nanoparticles with citrate imposes two key advantages. First, it extends the shelf-life of the nanoparticle solution, permitting th e performance of longitudinal studies with the same nanoparticle preparation. Second, it minimizes, if not eliminates, the precipitation of CeO2 nanoparticles from solution. Conseque ntly, the nanoparticle distribution within the solution is homogene ous; allowing for uniform delivery of the nanoparticles to cells either in vitro or in vivo. Alternatively, part ial precipitation of CeO2 nanoparticles could lead to a heterogeneous delivery because the local CeO2 concentration in the vi cinity of cells may be altered, resulting in a vari able intracellular uptake of the nanoparticles. A key parameter in assessing the efficacy of CeO2 nanoparticles to scavenge reactive oxygen species is the intrace llular concentration and compartmentation of the CeO2 nanoparticles. To quantify the intracellular co ncentration of CeO2 nanoparticles, TC-tet cells were incubated in the modified media containing 0, 50, 100 and 200 M CeO2 for 48 hrs. At the end of this incubation period, the cells we re digested and th e concentration of CeO2 nanoparticles was determined by ICP. Table 3-1 shows the CeO2 concentrations in TC-tet cells after 48 hours of incubated in di fferent extracellular concentrati ons. The data show that the 48

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accumulation of CeO2 in TC-tet cells proportion to the extr acellular concentration. However, exposure of TC-tet cells to large concentrations of citrate-adsorbed CeO2 nanoparticles in the culture media (>200 M) was detrimental to the cells. This is illustrated by the lower number of cells that were measured when cells were exposed to media containing 200 M CeO2. Since all flasks used in this experiment were prepared id entically, this lower cell count may be attributed to (1) detachment of cells during the incubation period, (2) a decrease in the ra te of proliferation by the cells or (3) combination of (1) and (2). It is important to note that cell viability was measured on the cells that were used to quantify the intracellular CeO2 content and does not include cells that may have been detached during incubation with the nanoparticles. Once the intracellular concentration of CeO2 nanoparticles was determined ( Table 3-1 ), we proceeded to determine the in tracellular allocations of CeO2 nanoparticles. CeO2 nanoparticles are clearly visible in a TEM picture of TC-tet cells. Figure 3-5 shows the allocations of CeO2 nanoparticles in the treated cells These data showed that CeO2 nanoparticles were present throughout the cytoplasm as well as within organelles such as th e mitochondrion, either as small or large aggregates. TEM cell samples were ex amined by EDS and the presence of cerium was clearly detected. Table 3-1. Intracellular amount of CeO2 in TC-tet cells following 48 hrs of incubation with media containing CeO2 nanoparticles. Extracellular CeO2 concentration ( M) ICP Reading (g) Cell Numbers (x106) Amount of CeO2 in Cell (pg/cell) Viability 0 0.24 49.8 0.01 >90% 50 2.33 42.3 0.06 >90% 100 4.59 42.6 0.11 >90% 200 7.8 22.2 0.35 >90% 49

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Figure 3-5. TEM micrographs of CeO2 nanoparticles in TC-tet cells. A) Control cell without not exposure to CeO2 nanoparticles. B) A large aggregate of CeO2 nanoparticles positioned in the cytoplasm of a TC-tet cell following a 48 hr exposure to media containing 100 M CeO2 nanoparticles. C) Small aggregate of CeO2 nanoparticles that are distributed throughout the cytoplasm of the same cell in B. D) A small aggregate of CeO2 nanoparticles that is positioned within the mitochondrion of the same cell in B and C. The EDS chemical analysis is shown on the upper right corner of the image, indicates the presence of cerium in the point ed spots. Scale bar 500nm in A-C, and 100 nm in D. The effectiveness of these citrate-adsorbed CeO2 nanoparticles to scavenge intracellular free radicals was assessed by exposing TC-tet cells to 1 or 2 mM HQ for 15 min. Figure 3-6 50

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indicates the effect of HQ e xposure on the intracellu lar free radical concentration. The data show that in the absence of CeO2 nanoparticles such an insult re sults in an increase in the intracellular free-radical concentrat ion. However, when cells were preloaded with either 50 or 100 M CeO2, the intracellular free radical concentration decreased regardless of the extracellular hydroquinone concentration. These data represented th e effectiveness of CeO2 nanoparticles to scavenge free radicals in vitro. It is important to note that labeling TC-tet cells with CeO2 nanoparticles did not affect either their viability of the cells as depicted in Table 3-1 or their ability to secrete insu lin as it is illustrated in Figure 3-7 These data support earlier reports demonstrating the neuroprotective, oph talmoprotective, and car dioprotective properties of CeO2 nanoparticles, although the CeO2 concentrations in our work were up to 4 orders of magnitude higher than in the above reports. Ho wever, our data is based on quantitative measurement of the concentration via full chem ical analysis using ICP which should be the standard applied to measure concentrations The earlier papers based their calculated concentrations on a qualitative picture analysis. Another important observation from our experi ments was the lack of cytotoxic effects associated with the use of the CeO2 nanoparticles. This observati on is in contrast to a recent in vitro study on human lung cancer cells that demonstr ated a significant decr ease in cell viability with exposure to CeO2 nanoparticle [76]. The loss of cell viability was attributed to large quantities of free radicals generate d by the nanoparticles resulting in excessive oxidative stress. Although the doses of CeO2 nanoparticles used in that study is si milar to that used in the present study (i.e., Lin et al. used 3.5-23.3 g/ml for 1-3 days, while our present study used 17.2-34.4 g/ml for 2 days), the effect of the nanopartic les is opposite. Whereas Lin et al. reported generation of free radicals, we report scavenging of free radicals. This difference in function 51

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may be attributed to the re ported photocatalyst-nature of CeO2 nanoparticles (Eg = 3.15eV) upon the illumination of UV lights [77]. However, high er energy is required to stimulate free radical formation by our CeO2 nanoparticles, since the broader ba ndgap energy (3.65eV in terms for 3.7 nm CeO2 nanoparticles) was reported for CeO2 nanoparticles smaller than 20 nm [78] (i.e., Lin et al. used 20 nm particles whereas we used 3.7 nm particles). Figure 3-6. Free radical concentration in TC-tet cells. The solid gray bars represent intracellular free radical concentrations of non-CeO2 loaded TC-tet cells following exposure to 1 or 2 mM hydr oquinone (HQ). The shadded and white bars represent intracellular free radical concentrations of CeO2 labeled TC-tet cells (50 and 100 M respectively) following identical hydroqui none exposures. Each bar is the average of three measurements and the error bars represent the st andard deviation of the mean. Statistical comparisons amongst the various groups were performed using a t-test analysis and the asterisks indicat e p values <0.03. The only comparison that was not statistically (NS) significant was that between non-labeled and cells labeled with 50 M CeO2 and exposed to 1 mM HQ. Thes e data represent one of three independent experiments. 52

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Figure 3-7. The amount of insulin secreted by non-labeled and CeO2 labeled TC-tet cells. Labeled cells were exposed to media containing either 50 or 100 M CeO2 nanoparticles for 24 hours. Each bar represen ts the average of two measurements and the error bars represent the standard devi ation of the mean. Similar measurements were also performed with cells exposed to 50 or 100 M CeO2 containing media for 48 hours with identical results. There are many potential applications in medici ne that can benefit from the free radical scavenging abilities of CeO2 nanoparticles. One such application having a potential for dramatic impact is that of islet transpla ntation. One of the many challenge s that face islet transplantation is the loss of islet viability shortly after tran splantation due to oxida tive stresses induced by mechanical trauma and/or immunosuppressive medication [79]. Although several antioxidants such as Heme Oxygenase-1, SOD mimetics, vita min C and/or E have been shown to improve oxidative stress in transplanted islets, they eith er degrade over a relatively short time span once incorporated into the islets, or the islets require genetic manipulation to express the desired antioxidant protein [1,79]. The proposed CeO2 nanoparticles are a promis ing alternative to the existing methods because they can be delivered in tracellularly and possibly targeted to specific organelles, are stable over long periods of time, do not require the genetic manipulation of cells and do not affect either th e viability or insulin secr etion of the host islets. 53

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54 3.3 Summary In this study, we presented novel CeO2 nanoparticles synthesis based on a reverse micelle formation technique. The nanopartic les were crystalline, had an average particle size of 3.7 nm, and could be dispersed in medium for long periods of time due to their coating with citrate. When tested in vitro, the CeO2 nanoparticles were deposited in th e cytoplasm of insulin secreting cells, and the intracellular concentration of CeO2 nanoparticles reached 0.35 pg/cell. In addition, our observation of a reduced intr acellular free radical concentrat ion was consistent with the intracellular concentration of CeO2 nanoparticles, which is proportional to the extracellular CeO2 concentrations. The ability of these nanoparticles to scavenge free radicals was maintained in vivo, providing effec tive protection to TC-tet cells against an insult by the free radical generator, hydroquinone.

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CHAPTER 4 SYNTHESIS OF CERIUM-ZIRCO NIUM OXIDE NANOPARTICLES This chapter introduces the preparation pro cedures prior to analyzing the free radical scavenging activities in CexZr1-xO2 nanoparticles. The preparati on procedures include synthesis of CexZr1-xO2 nanoparticles using reverse mi celle system, dispersion of CexZr1-xO2 nanoparticles using buffers of multi-valent ions, and evalua ting their particle size distributions in the nanosuspensions. In each preparation procedur e, the background of the techniques will be described in order to prov ide a thorough understanding. In order to evaluate free ra dical scavenging activities in CexZr1-xO2 nanoparticles, it is important to prepare a system with narrow nanopa rticle size distribution, and these nanoparticles in the particular systems should be well stabiliz ed in a solution. The r eason for doing so is to minimize the influences from the surface area th at can be diminished by particle size and agglomerations. Since reverse micelle synthe sis provides the system a relatively narrow nanoparticle size distribution (u sually 3-10 nm), the technique was selected to synthesize CexZr1xO2 nanoparticles. These synthesized CexZr1-xO2 nanoparticles are then dispersed in a saline/tri-sodium citrate buffer. The capability for this specific buffer to disperse CeO2 nanoparticles has been described in Chapter 2. In this buffer, tri-sodium citr ate not only acts as an op timum dispersant, but the saline/tri-sodium citrate/citric acid system also stabilizes the suspensions pH value. A stable pH value is essential for the activity tests to free radical scavenging, because the fluctuating pH value can strongly influence the st ability of free radicals. The particle size distribution was measured using particle size analyzer. It is to ensure the suspensions have comparable surface area and to confirm that part icle agglomerations are prev ented in the preparation. 55

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Finally, the Ce/Zr ratio and the concentration of CexZr1-xO2 nanoparticles were measured using chemical analysis methods. This proced ure is also crucial, because it shows potential leaching of cerium or zirconium ions in the sy nthesis; and it provides precise nanoparticle concentrations for the activity tests. 4.1 Nanoparticle Synthesis 4.1.1 Reverse Micelle Synthesis Reverse micelle method was invented as a pr ocess for preparing nanoparticles with narrow particle size distribution in the 1980s. Reverse micelles are the reversed aggregates of normal surfactant micelles. They are stabilized by the dissolution of the hydrophobic groups located outside of the reversed micelles in an apolar media in order to minimize the interfacial energy. In the reverse micelle route, each aqueous precursor is surrounded by a surfactant monolayer, and each reverse micelle works as a single nanore actor, and has a size around 2 nm to 10 nm. The formation of nanoparticles is accomplished by diffusing a small amount of reactant, such as ammonium hydroxide, into reverse micelles. According to the electrochemical stability, nucleation and growth occur due to the increasing pH value in these nanoreactors. Therefore, nanoparticles are formed in reverse micelles. Th is method has been used to synthesize a variety of materials, including ceramics and polymer ic nanoparticles. Several examples are: CaCO3, BaCO3 [80], ZrO2 [81], CeO2 [82], CdS [83], proteins, and enzymes [84,85]. The shape and size of the products strongl y depend on the synthesis conditions. By adjusting the water/surfactant ra tio to desired conditions, nanopart icles with different shapes including spherical, planar, cylindr ical, discoidal, or even vesicu lar can be formed [86]. Also, particle sizes in the ra nge from 1 nm to 100 nm with high cr ystallinity can be synthesized. The reverse micelle method increases the homogeneit y of chemical composition and facilitates the preparation of nanoparticles that ofte n have monodisperse d particle size. 56

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In order to compare the scavenging activity in our proposed system with other competitive groups, a surfactant that othe r groups used to synthesize CeO2 nanoparticles was used instead of surfactant lecithin. 4.1.2 Experimental Methods Bis(2-ethylhexyl) sulphosuccinate (AOT, labor atory grade), toluene (laboratory grade), cerium (III) nitrate hexahydrate (99.5%, M.W. = 434.22 g/mol), zirconyl(IV) nitrate hydrate (99.5%, M.W. = 231.23 g/mol), and ammonium hydroxide (NH3 content 28~30%) were purchased from Fisher Scientific and used withou t further purification. One and half grams of AOT was dissolved in 100 mL of toluene to form reverse micelles. Five mini-liters of precursor solution was prepared by mixing 0.1 M cerium nitrate and 0.1 M zirc onyl nitrate aqueous solutions according to the Ce/Zr ratios. The precursor solutio n was then pipetted into the colloidal micelle system, and the mixture was strongly stirred for 30 mi nutes until the system appeared homogeneous. Ten mini-liters of 1.5 M ammonium hydroxide solution was titrated into the system to initiate the precipitation. The chemicals and their amounts used in the synthesis are listed in Table 4-1 After 45 min of stirring, CexZr1-xO2 nanoparticles had gradually precipitated in the reverse micelles, and the appearance of the system becam e yellowish. The nanoparticles were collected by centrifugation with a force field of 18 G and th en sequentially rinsed with 50 mL of acetone, Fifty mini-liters of ethanol, a nd 50 mL of water in order to re move the redundant surfactants. Between each rinses the nanopart icles were collected by centrif ugation at 18 G. All samples prepared in the following chapters were synthe sized or prepared at room temperature without further heat treatment. The synt hesis procedure is illustrated in Figure 4-1 57

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Table 4-1. Chemicals and the amounts used to prepare CexZr1-xO2 nanoparticles in reverse micelle synthesis. Figure 4-1. Synthe sis procedures. 58

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4.2 Nanoparticle Stabilization 4.2.1 Stabilization Using Buffer Although the isoelectri c point (IEP) of CeO2 ranges between 6.5 to 7.5 [87,88], using sodium citrate buffer solution allows to disperse CexZr1-xO2 nanoparticles at pH 7.4 and to retain pH stability at the same tim e. The optical images of CeO2 nanoparticles stab ilized in aqueous solutions are shown in Figure 4-2 A 0.05 M saline/sodium citrate buffer (Cat. # 821840, MP Biomedical) was prepared according to the manufacturers instructions. After the CexZr1-xO2 nanoparticles were rinsed and collected, the products were dispersed in the sodium citrate buffer. The pH value of each sample was adjusted to 7.4 by titrating 0.1 N citric ac id (97.5%, Sigma-Aldrich) or 0.1 N NaOH (SigmaAldrich). The suspensions were ultrasonicate d overnight until the su spension changed from turbid to transparent. The suspensions were th en filtered through a 0.2 m syringe filter in order to remove unexpected agglomerates. Figure 4-2. Optical images of CeO2 nanoparticles dispersed in DI water (left), and in sodium citrate buffer solution solution (right). 59

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4.3 Particle Size Analysis 4.3.1 Dynamic Light Scattering Technique Dynamic light scattering (DLS) technique is established for measuring particle size over the size range from a few nanometers to a few micr ons. When light hits sm all particles the light scatters in all directions so l ong as the particles are small comp ared to the wavelength. If the light source is monochromatic and coherent, then a time-dependent fluctu ation in the scattering intensity can be observed. These fluctuations ar e due to the fact that the small molecules in solutions are undergoing Brownian motion and so the distance between the scatterings in the solution is constantly changing with time. When the coherent source of light having a known frequency is directed at the moving particles, th e light is scattered but at a different frequency. The shift in light frequency is related to the size of the particles causing the shift. Due to their higher average velocity, smaller particles cause a gr eater shift in the light frequency than larger particles. It is this difference in the frequency of the scattered light among particles of different sizes that is used to determine the sizes of the particles. Here, the size distribution of agglomeration is used to evaluate the stab ilization of suspensions since the degree of agglomerations may influence the scavenging activity of nanoparticles. The particle size distributions of the suspensions were meas ured by NanoTrac (MicroTrac Inc.) at PERC. 4.3.2 Agglomerate Size Distribution Figure 4-3 shows the agglomerate size distribution in CexZr1-xO2 suspensions measured by DLS. The mean agglomerate size Mn, D50 and D95 of all suspensions are listed in Table 4-2 The DLS results show that the CexZr1-xO2 nanoparticles, except for ZrO2, are well dispersed in the sodium citrate buffer solution at pH 7.4. All the suspensions are able to sustain stabilization for more than 6 months w ithout observable deposition. 60

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Figure 4-3. Agglomerate size distribution in CexZr1-xO2 suspensions at pH 7.4 (x= 0, 0.2, 0.4, 0.6, 0.7, 0.8, 1.0). The agglomerate size and numb er percentage of agglomeration are measured by NanoTrac. Table 4-2. Mean a gglomeration size (Mn), D50, and D95 of CexZr1-xO2 suspensions obtained using NanoTrac. 61

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4.4 Chemical Analysis 4.4.1 Inductively Coupled Plasma Spectroscopy The inductively coupled plasma spectroscopy (ICP) is a sophisticated spectroscopic technique for chemical analysis. The term inductively coupled plasma is a type of plasma source in which the energy is supplied by electrical currents which are produced by electromagnetic induction. The ICP system is equipped with mo nochromators covering the particular spectral ranges with a grated ruling, and it is operated ba sed on the principle of atomic emission by atoms ionized in the argon plasma. Light of specific wa velengths is emitted as electrons return to the ground state of the ionized elements, quantit atively identifying the species present. The ICP samples are prepared as ions in a so lution, since the samples need to be injected through plasma and form an ionized mist. Theref ore, acids and bases are usually involved in sample preparations. The system is capable of analyzing the trace concentration of materials in both organic and aqueous matrices with a detection limit range of less than 1 ppm. The Ce/Zr ratios, nanoparticle concentrations in CexZr1-xO2 suspensions were confirmed by Perkin-Elmer Plasma 3200 Inductively Coupled Plasma Spectroscopy at Particle Engineering Research Center (PERC). Sulfuric acids were used to dissolve CeO2, since it is reported that CeO2 only dissolved in this partic ular acid. The solubility of CeO2 was tested in several acids, i.e. sulfuric acid, hydrochloric aci d, and nitric acid, prior to the c oncentration analysis, and it was confirmed that only sulfuric acid was able to completely dissolve CeO2 prior to concentration measurements. To prepare samples for ICP, an adequate amount of products was dissolved by 95% of sulfuric acid (Aldrich-Sigma) at 90 C overnight. The samples were diluted using DI water prior to use. Before measuring the cerium and zirc onium concentrations in the samples, 100 ppm, 10 ppm, and 1 ppm of cerium ICP standard and zi rconium ICP standard (Ricca Chemical) were 62

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used to prepare as standard samples. The nanopa rticle concentrations us ed in this dissertation were confirmed using ICP. 4.4.2 Compositions of Final Products The compositions of final products are shown in Table 4-3 The compositions correspond to the designed Ce/Zr ratios, and the standard deviations of the compositions are between 0.00018 to 0.02 (standard deviations were obtained from 2 different suspension preparations). The ICP results suggest the fact that reverse micelle synthesis me thod is a remarkable method to prepare nanoparticles in laboratory scale, and the compositions of solid solutions can be controlled in fewer than 2% of deviations. Table 4-3. Chemical compositions of final products determined. 4.5 Summary A series of CexZr1-xO2 nanoparticles using reverse mice lle systems was synthesized, and the chemical compositions of cerium and zirconium (i.e. x and 1-x) were precisely controlled between 0.0 to 1.0 (x= 0.0, 0.2, 0.4, 0.6, 0.7, 0.8, 1.0). The products were washed and collected using centrifugation. The sediments could be re-dispersed in an anticoagulation buffer, trisodium citrate buffer at pH 7.4. In this chapte r, measuring particle si zes using NanoTrac also yields the agglomerate sizes of final products. According to the results in DLS measurements, 63

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64 we concluded that the nanoparticles were well dispersed in a tr i-sodium citrate buffer. The chemical compositions of the final products were confirmed using ICP, and their chemical compositions corresponded to the initial design. In this chapter, we have confirmed that CexZr1xO2 nanoparticles with a series of Ce/Zr ratios can be prepared in re verse micelle synthesis, and these nanoparticles can be dispersed in sodium c itrate buffer. The chemical compositions of the final products correspond to the initial design.

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CHAPTER 5 STRUCTURAL PROPERTIES AND NONSTOICHIOMETRIC BEHAVIOR OF CERIUMZIRCONIUM OXIDE NANOPARTICLES This chapter discusses the structural pr operties and nonstoichiom etric behavior of CexZr1xO2. Several characterization techniques in cluding TEM, XRD, and Raman spectroscopy are used to confirm the crystal structures, phases, crystallite size, and crystallinity of these nanoparticles. It is worth to note that these nanoparticles we re synthesized in a de signed reverse micelle system, and they were formed from hydrated cerium/zirconium salts at room temperature. Heat treatments to these nanoparticles were prevente d in the synthesis. The exemption of heat treatments may cause the residual stress in the lattice, and cause mixture of several phases in each single nanoparticles. In general, the residual stress may eliminate the symmetry of structures and exclude the reso lution beyond the limitation of th e characterization techniques. 5.1 Characterization Technique s and Experimental Methods 5.1.1 Structural Characterization Using TEM Transmission electron microscopy (TEM) is applie d to determine the particle size, crystal structures, and partially to characterize the crystallinity of CexZr1-xO2 nanoparticles. TEM is a technique whereby a beam of electrons is tr ansmitted through a specimen, interacting with the specimen as it passes through it. A contrast is formed from when the transmitted electrons interact with electrons and nucleus in the specim en and the signal is magnified and focused by an objective lens and appears on an imaging screen, or is detected by a sensor such as a CCD camera. Experimental methods In this section, the Feret part icle diameter will be measured through particle analysis in TEM images. The crystal structures of CexZr1-xO2 nanoparticles will be evaluated by analyzing 65

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the SAD patterns stimulated by the transmissi on electron beams. Th e crystallinity of nanoparticles will be estimated thr ough analysis of the lattice plan es/fringes of nanoparticles in TEM images. TEM samples were prepared by re-dispersing adequate amounts of CexZr1-xO2 nanoparticles in methanol, sonicating for 5 mi nutes, and dropping the suspensions on formvar carbon film supported copper grids (Electron Micr oscopy Sciences). A JEOL TEM 2010F TEM located at Major Analytical Instrumentation Cent er (MAIC, UF) was used to characterize these samples. The particle size of each sample was determ ined by averaging the Ferets diameter of nanoparticle images which arbitrarily selected in TEM micrographs. The standard deviation of particle sizes was estimated around 0.5 nm, owing to the resolution of TEM micrographs. 5.1.2 Structural Characterization Using XRD X-ray diffraction (XRD) is applied to determine the crystallite size, crystal structures of CexZr1-xO2 nanoparticles. XRD is an x-ray technique based on the principle of scattering. The electromagnetic x-ray inci dents to a specimen, and interacts w ith its electrons and nucleus. The contrast only appears when constr uctive interference occu rs in the condition that the diffraction angles satisfied Braggs Law. The diffraction pattern of a specimen reveals the characteristics of the particular material, including crystal structures, crystallite sizes, lattice parameters, etc. Experimental methods In this section, Scherre rs equation will be used to determ ine the crystallite size in the range of submicron sizes. The crystal structures of these nanoparticles will be determined by comparing their diffraction spectra with that of the corresponded crystalline materials. All CexZr1-xO2 nanoparticles were synthesized using reverse micelle method, washed with methanol, collected, and dried overnight. A gl ass slide was cleaned and covered with a double 66

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face tape, and the dried powders were poured on th e tape and evenly applied on the tape. The xray diffraction spectra were obtained us ing XRD Phillips APD 3720 at MAIC, UF. The crystallite size of each sample was calculated using Scherrers equation. cos vD (5-1) where is the volume weighted crystallite size, vD is Scherrer constant falls in the range of 0.87 to 1.0, is the wavelength of radiation, and is the integral breadth of the reflection located at 2 In this study, Scherrers constant is 0.89, and wavelength of radiation is of 1.54 angstroms [75]. 5.1.3 Structural Characterization Using Raman Raman spectroscopy is applied to determine th e crystal structures, their sub-phases, and phase transformation in the series of synthesized CexZr1-xO2 nanoparticles. Raman spectroscopy is a spectroscopic technique used in condensed matter physics and chemistry to study vibrational, rotational, and other low-freque ncy modes in a system. The vibration relies on inelastic scattering, or Raman scattering of monochromatic electromagnetic radiation, induced by laser light interacting with phonons or other excitations in the system, resulting in the energy of the laser photons being shifted due to the vibrati onal transitions in the molecules. In this dissertation, Raman spectroscopy is used to characterize the crystal structures of CexZr1-xO2 nanoparticles. By detecting the vibrational ener gy induced by the symmetry of lattices, it is allowed to distinguish the crystal structures and the sub-phases of materials using Raman spectroscopy. Experimental methods All CexZr1-xO2 nanoparticles were synthesized using reverse micelle methods, washed with methanol, collected, and dried overnight. Commercial 7 nm CeO2 nanoparticles were purchased 67

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from Nanoscale Materials Inc., USA, and commercial 40 nm CeO2 nanoparticles were purchased from Alfa Aesar. Commercial ZrO2 nanoparticles (tetragonal phase ) were purchased from Alfa Aesar. The samples were poured on a cleaned gla ss slide, and tested using Renishaw Bio Raman at the Particle Engineering Research Center (PER C, UF). The excitation wavelength of incident radiation of Renishaw Bio Raman is 514 nm. 5.2 TEM Results and Discussion Figure 5-1 to Figure 5-7 represent TEM micrographs of CexZr1-xO2 nanoparticles (x= 0, 0.2, 0.4, 0.6, 0.7, 0.8, 1.0) and their selective area elec tron diffraction (SAD) patterns. In these TEM micrographs, nanoparticles prepared in revers e micelle synthesis have particle size between 2 to 7 nm. The particle size di stributions of 50 arbitrary picked particle images are recorded in Table 5-1 and their mean particle sizes are averaged and shown in the same table. The mea particle sizes of nanoparticles in ou r preparation are between 3.2 nm of ZrO2 to 3.8 nm of Ce0.2Zr0.8O2, and the standard deviation of mean part icle sizes is around 0.5 nm according to the resolution of particle images. n From Figure 5-1 to Figure 5-7 we can conclude that synthesis of CexZr1-xO2 nanoparticles using reverse micelle method is possible. Us ing this method, 2-7 nm of nanoparticles are prepared, and these nanoparticle s remain highly crystallized ev en though heat treatment was exempted during preparation. In TEM images, latt ice fringes are observed in the particle images, implying highly ordered crystallization in nanopar ticles. Furthermore, well defined rings are observed in SAD patterns, which reconfirm the ab ility for this preparat ion method to synthesize highly crystallized CexZr1-xO2 nanoparticles in nature. 68

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Figure 5-1. TEM micrographs of CeO2 nanoparticles. A) Micrograph of CeO2 nanoparticles (magnification 200 kX, scale bar 10 nm). B) Micrograph of CeO2 nanoparticles (magnification 300 kX, scale bar 10 nm). C) Micrograph showing the lattice fringes of CeO2 nanoparticles. The fringes imply the highly ordered crystallinity of nanoparticles (magnification 1,000 kX, scal e bar 5 nm). D) SAD pattern of CeO2 nanoparticles. The bright contrasts in th e images are the accumulation of scattered electrons, therefore the orde red ring patterns indicate the polycrystalline materials detected in the TEM electron beam. 69

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Figure 52. TEM micrographs of Ce0.8Zr0.2O2 nanoparticles. A) Micrograph of Ce0.8Zr0.2O2 nanoparticles (magnification 200 kX, scale bar 10 nm). B) Micrograph of Ce0.8Zr0.2O2 nanoparticles (magnification 200 kX, scale bar 10 nm). C) Micrograph showing the lattice fringes of Ce0.8Zr0.2O2 nanoparticles. The fringes imply the highly ordered crystallinity of nanoparticles (ma gnification 600 kX, scale bar 5 nm). D) SAD pattern of Ce0.8Zr0.2O2 nanoparticles. The bright co ntrasts in the images are the accumulation of scattered elec trons, therefore the ordered ring patterns indicate the polycrystalline materials detect ed in the TEM electron beam. 70

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Figure 5-3. TEM micrographs of Ce0.7Zr0.3O2 nanoparticles. A) Micrograph of Ce0.7Zr0.3O2 nanoparticles (magnification 100 kX, scale bar 20 nm). B) Micrograph of Ce0.7Zr0.3O2 nanoparticles (magnification 200 kX, scale bar 10 nm). C) Micrograph showing the lattice fringes of Ce0.7Zr0.3O2 nanoparticles. The fringes imply the highly ordered crystallinity of nanoparticles (ma gnification 500 kX, scale bar 5 nm). D) SAD pattern of Ce0.7Zr0.3O2 nanoparticles. The bright co ntrasts in the images are the accumulation of scattered elec trons, therefore the ordered ring patterns indicate the polycrystalline materials detect ed in the TEM electron beam. 71

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Figure 5-4. TEM micrographs of Ce0.6Zr0.4O2 nanoparticles. A) Micrograph of Ce0.6Zr0.4O2 nanoparticles (magnification 100 kX, scale bar 20 nm). B) Micrograph of Ce0.6Zr0.4O2 nanoparticles (magnification 200 kX, scale bar 10 nm). C) Micrograph showing the lattice fringes of Ce0.6Zr0.4O2 nanoparticles. The fringes imply the highly ordered crystallinity of nanoparticles (ma gnification 500 kX, scale bar 5 nm). D) SAD pattern of Ce0.6Zr0.4O2 nanoparticles. The bright co ntrasts in the images are the accumulation of scattered elec trons, therefore the ordered ring patterns indicate the polycrystalline materials detect ed in the TEM electron beam. 72

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Figure 5-5. TEM micrographs of Ce0.4Zr0.6O2 nanoparticles. A) Micrograph of Ce0.4Zr0.6O2 nanoparticles (magnification 100 kX, scale bar 20 nm). B) Micrograph of Ce0.4Zr0.6O2 nanoparticles (magnification 200 kX, scale bar 10 nm). C) Micrograph showing the lattice fringes of Ce0.4Zr0.6O2 nanoparticles. The fringes imply the highly ordered crystallinity of nanoparticles (ma gnification 600 kX, scale bar 5 nm). D) SAD pattern of Ce0.4Zr0.6O2 nanoparticles. The bright co ntrasts in the images are the accumulation of scattered elec trons, therefore the ordered ring patterns indicate the polycrystalline materials detect ed in the TEM electron beam. 73

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Figure 5-6. TEM micrographs of Ce0.2Zr0.8O2 nanoparticles. A) Micrograph of Ce0.2Zr0.8O2 nanoparticles (magnification 100 kX, scale bar 20 nm). B) Micrograph of Ce0.2Zr0.8O2 nanoparticles (magnification 200 kX, scale bar 10 nm). C) SAD pattern of Ce0.2Zr0.8O2 nanoparticles. The bright contrast s in the images are the accumulation of scattered electrons. The contrast of the ring is not strong due to the residual surfactants on particle surface. 74

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Figure 5-7. TEM micrographs of ZrO2 nanoparticles. A) Micrograph of ZrO2 nanoparticles (magnification 50 kX, scale bar 50 nm). B) Micrograph of ZrO2 nanoparticles (magnification 200 kX, scale bar 10 nm). C) Micrograph showing the lattice fringes of ZrO2 nanoparticles. The fringes imply th e highly ordered crystallinity of nanoparticles (magnification 600 kX, scal e bar 5 nm). D) SAD pattern of ZrO2 nanoparticles. The bright contrasts in th e images are the accumulation of scattered electrons, therefore the orde red ring patterns indicate the polycrystalline materials detected in the TEM electron beam. 75

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Table 5-1. Particle sizes of CexZr1-xO2 nanoparticles averaged from 50 arbitrary selected particles in TEM micrographs. The mean particle size of Ce0.2Zr0.8O2 was measured from 10 arbitrary selected particle images due to the limited resolu tion in TEM micrographs. 5.3 XRD Results and Discussion Figure 5-8 shows the XRD spectra of synthesized CexZr1-xO2 nanoparticles (x = 0, 0.2, 0.4, 0.6, 0.7, 0.8, 1.0). In Figure 5-8 each individual peak in the XRD spectrum of CeO2 nanoparticles fit to the corresponde d XRD spectra of crystalline CeO2. The peaks (111) in CeO2, Ce0.8Zr0.2O2, Ce0.7Zr0.3O2, and Ce0.6Zr0.4O2 slightly shift to a higher diffraction angle, which is a result of lattice expansion. The sh ift of peaks (111) corr esponds to the reported spectra, and is an evidence of solid solutions thr oughout the nanoparticles. The te tragonal feature appears in the XRD spectrum of CeO2, Ce0.8Zr0.2O2, Ce0.7Zr0.3O2, and Ce0.6Zr0.4O2 nanoparticles, which is a feature of phase transformation fr om cubic to tetragonal crystal structure. From XRD spectra, we conclude that CeO2, Ce0.8Zr0.2O2, Ce0.7Zr0.3O2, Ce0.6Zr0.4O2 nanoparticles have cubic fluorite structure, and the phase transformation occurred when more than 40% of zirconium ions doped in the solid solution. The results correspond to crystal structures a well as phase transformation of CexZr1-xO2 nanoparticles (x = 0.6-1.0) reported by Zh ang et al. [75]. However, the XRD 76

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spectra of CexZr1-xO2 nanoparticles with more than 60% of zirconium dopants do not correspond to the results reported in the ot her literatures [36,75]. In the nanoparticles synthesized in our laboratory, the broadened XRD spectra of Ce0.4Zr0.6O2, Ce0.2Zr0.8O2, and ZrO2 nanoparticles show mixed crystal structures in these nanoparticles. The position of the two major peaks matches the reported XRD spectra of cubic fluorite, tetragonal and monoclinic crystal structures. Thus, we assume that Ce0.4Zr0.6O2, Ce0.2Zr0.8O2, and ZrO2 nanoparticles are mixtures of cubic, tetragonal, and monoclinic structures. Figure 5-8. XRD spectra of a series of CexZr1-xO2 nanoparticles (x= 0, 0.2, 0.4, 0.6, 0.7, 0.8, 1.0). The XRD spectrum of CeO2 crystalline is plugged in the bottom to show the correspondence of XRD between synthesized CeO2 and reference data. The tetragonal feature (t) appears and increases when more zirconium doped into the solid solutions, indicating phase transformation in the se ries of nanoparticles. 77

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The crystallite size of CeO2 nanoparticles is determined by XRD spectra associated with Scherrers equation, and the crys tallite size is calculated to be 3.7.2 nm by averaging the values obtained from XRD peaks (111), (200), (220). The crystallite size co rresponds to particle size determined by TEM micrographs, and the resu lts implied that each nanoparticle is of a single crystallite. Accordingl y, the crystallite sizes of Ce0.8Zr0.2O2 and Ce0.7Zr0.3O2 nanoparticles that obtained from their XRD peaks (111) (200), (220) are 2. 0.07 nm and 3.4.2 nm, respectively. However, the Ce0.6Zr0.4O2, Ce0.4Zr0.6O2, Ce0.2Zr0.8O2, and ZrO2 nanoparticles have broadened XRD spectra, therefore Sc herrers equation is not suitable to calculate the crystallite sizes of these samples. 5.4 Raman Results and Discussion To identify the phases in CexZr1-xO2, there are six distinct Rama n shifts being used. The shift of each peak may be unique in the samples that prepared in different synthesis methods, heat treatment, or particle size, therefore, we adopted the wave numbers in Raman spectra that reported by Zhang et al. [75] in this dissertation. Figure 2-6 (b) is the Raman spectra of CexZr1xO2 solid solutions adopted from Zhangs work. The numbers in the figure represent the features for identifying c, t, t, or t phases. In order to simplify the classification, the in dications of each peak are listed in Table 5-2 where peak 4 is a strong peak pointing to ward the main cubic structure; peaks 13 represent the tetragonal distortion in the latt ice; peaks 5, 6 contributed by defects and oxygen displacements that distort the cubic structure. Briefly, by identifying the intensity of Raman shift peak 4 and peaks 1-3, it is po ssible to identify metastable t phase in cubic fl uorite structure and phase transformation from cubic to tetragonal structure in CexZr1-xO2 nanoparticles. 78

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Table 5-2. Crystal structures and their sub-phases in CexZr1-xO2, and the classification using Raman spectroscopy. Figure 5-9 shows Raman spectra of commercial 7 nm, 40 nm CeO2 nanoparticles, and a series of synthesized CexZr1-xO2 nanoparticles. Due to instrume ntal limitation, the Raman shifts in this work was detected down to 200 cm-1. The Raman spectra of commercial 7 nm, 40 nm, and synthesized CeO2 nanoparticles are shown in Figure 5-9 (a). According to their Raman spectra, commercial CeO2 nanoparticles and synthesized CeO2 nanoparticles have a very strong peak 4, so their crystal structures are based on cubic fluorite structure. In the Raman spectra of synthesized CeO2 nanoparticles peaks 3, 5, and 6 are noted, so t phase also exists in the system. This is the result of lattice distortion, due to the residual lattice stress formed in the synthe sis, in which the exemption of heat treatment caused the phenomenon. From Figure 5-9 (a) to (d), peak 4 in Raman spectra was observed, although the intensity of peak 4 decreases with increas ing zirconium dopants. The results suggest, first, samples CeO2, Ce0.8Zr0.2O2, Ce0.7Zr0.3O2, and Ce0.6Zr0.4O2 have cubic structure. S econd, the cubic structure in these samples was distorted by zirconium dopants, while the cubic feature (peak 4) gradually diminished with increasing zirconium dopants. Meanwhile, peaks 3, 5, and 6 remain constant in these samples, indicat ing greater portion of t phase appearing in the cu bic based structure when more zirconium ions were doped into the solid solution. In general, t phase is a metastable 79

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phase in cubic structure, and the axial ratio of its lattice structure equals to one. In conclusion, CeO2, Ce0.8Zr0.2O2, Ce0.7Zr0.3O2, and Ce0.6Zr0.4O2 have cubic structur es, and the ratio of metastable cubic phase (t) increases when more and more zirconium doped in the crystal structure. Comparing to the characterization of the same materials that were conducted by Zhang et al., it is note d that the metastable t phase appears in the undoped CeO2 nanoparticles. The early appearing t phase conflicts the results reported by Zhang et al. [78]. It is believed that the metastable t phase in CexZr1-xO2 nanoparticles with lower zirconium dopants is a result of lattice distortion. Due to the lack of heat treatment, the residual stress in CexZr1-xO2 nanoparticles may cause lattice distortion. In Figure 5-9 (e), peak 4 disappears while peaks 3, 5, 6 remain sound in the Raman spectra. The vanished peak 4 is a sign of phase transition from cubic to tetragonal. At this state, the axial ratio of crystal structure in samples Ce0.4Zr0.6O2 becomes greater than one and its crystal lattice no longer belongs to cubic but rectangular. From Figure 5-9 (e) to (g), none or weak peak 4 was observed in the Raman spectra, and peaks 3, 5, 6 remain constant while more and more zirconium dopants in the materi als. Furthermore, peak 2 ar ises while more than 80% of zirconium ions doped in the latti ce. Perhaps, the rising peak 2 represents phase transition from metastable t to t phase. In conclusion, the results suggest that Ce0.4Zr0.6O2, Ce0.2Zr0.8O2, and ZrO2 nanoparticles are no longer in c ubic structure. Instead, they ar e tetragonal in structure, and their axial ratio of lattice is greater than one. 80

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Figure 5-9. Raman spectra of CexZr1-xO2 nanoparticles. A) commercial 7 nm CeO2, 40 nm CeO2, and synthesized CeO2 nanoparticles. B) Ce0.8Zr0.2O2. C) Ce0.7Zr0.3O2. D) Ce0.6Zr0.4O2. E) Ce0.4Zr0.6O2. F) Ce0.2Zr0.8O2. G) synthesized ZrO2. H) commercial ZrO2 nanoparticles (shown in the following page). The numbers marked in the spectra were defined in previous paragraph. 81

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Figure 5-9. Continued. 5.5 Summary of TEM, XRD, Raman Results 5.5.1 Crystalline CexZr1-xO2 Nanoparticles with Homogeneous Particle Size From the lattice fringes, diffraction patterns in TEM images, and defined peaks in XRD spectra, it can be concluded that CexZr1-xO2 nanoparticles prepared by reverse micelle synthesis method are crystalline in nature. In addition, the defined Raman shifts in CexZr1-xO2 nanoparticles support the conclusion that these CexZr1-xO2 nanoparticles are crys talline in nature. Although other studies have shown that CexZr1-xO2 nanoparticles precipitated in other reverse micelle systems were amorphous and heat treatmen ts were preferred, we have been able to perform an improved, more confined reverse mice lles system that allows improving crystallinity of the final products. Observed in TEM, all samples synthesized in the particular reverse micelle system are homogeneous in size, and their pa rticle sizes are around 37 nm in diameters. 5.5.2 Phase Transition Detected by XRD and Raman Spectroscopy In this work, the information obtained us ing XRD and Raman spectroscopy has provided credible conclusions in terms of crystal structures as well as sub-structures of the lattice. Using 82

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83 XRD, it is found that CeO2, Ce0.8Zr0.2O2, Ce0.7Zr0.3O2, and Ce0.6Zr0.4O2 nanoparticles have cubic fluorite structure, while Ce0.4Zr0.6O2, Ce0.2Zr0.8O2, and ZrO2 nanoparticles have complex structures which mixed with cubic fluorite, tetragonal, and monoclinic st ructures. Perhaps the mixed crystal structures in Ce0.4Zr0.6O2, Ce0.2Zr0.8O2, and ZrO2 nanoparticles are a result of residual stress in the la ttice due to exempted heat treatmen t. Using Raman spectroscopy, the phase transformation from cubic to tetragonal was found when more than 60% of cerium ions were replaced with zirconium dopants. In additio n, it is found that the more metastable cubic phase t were formed when the concentration of zirc onium dopants increased in the lattice. The portion of t phase finally no longer sustained in cubic structure, so the metastable t phase finally transformed to tetragonal st ructure. The crystal structure as well of its sub-structure of CexZr1-xO2 nanoparticles can be identified using XR D associated with Raman spectroscopy. In addition, the phase transformation, sub-phases in CexZr1-xO2 nanoparticles can be detected by comparing the shifted peaks of Raman spectra.

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CHAPTER 6 FREE RADICAL SCAVENGING BY CERIUM -ZIRCONIUM OXIDE NANOPARTICLES It is hypothesized that free radical scavenging in CeO2 nanoparticles is a consequence of catalysis, and the scavenging activity of CeO2 nanoparticles can be improved structurally by doping zirconium into lattice. To demonstrate our hypothesis is correct, the scavenging activities of CexZr1-xO2 nanoparticles shall be tested and the obtained activities wi ll be used to compare the reported OSC in the same materials. By doi ng so, it may help to understand the scavenging mechanism carried out by CexZr1-xO2 nanoparticles. In addition, the scavenging mechanism of these nanoparticles can be promoted furt her, according to the obtained knowledge. In this chapter, two of ROS, hydrogen peroxide and superoxide radicals, will be used to probe the scavenging activities of CexZr1-xO2 nanoparticles. It is because these two oxygen species are very influential ROS in biological systems, and do play essential roles in cell metabolism as well as apoptosis. To successfu lly test nanoparticles scavenging activities, several innovative methodologies are applie d in this dissertation. These innovative methodologies are assays that based on biochemical reactions, and these assa ys are the first time used to evaluate a metal oxide cata lysts activity at room temperatur e. At the end of this chapter, the free radical scav enging activities of CexZr1-xO2 nanoparticles as we ll as the scavenging mechanism will be discussed based on the structural properties of CexZr1-xO2 nanoparticles. 6.1 Prospective Scavenging Activities in CexZr1-xO2 Nanoparticles As aforementioned, free radical scavenging mechanism carried out by CexZr1-xO2 nanoparticles is a result of surface oxygen ex change. Based on the hypothetic mechanism proposed in Figure 2-2 there are two prerequisites in the s cavenging. First is that the transition of oxygen species only occur when mobile electrons available in the lattice. Second is that the exchange only occur when oxygen vacancies pr esent in the lattice. According to the 84

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understanding, a model that is available to describe free radical scavenging by CexZr1-xO2 nanoparticles is illustrated in Figure 6-1 Since the available mobile electrons can be pr ovided by oxygen vacancies in the lattice, the scavenging activities of CexZr1-xO2 nanoparticles likely are lim ited by the concentration of oxygen vacancies in the lattice. The concentration of oxygen vacancies in CeO2-based materials is reported to be proportional to their OSC. Therefore, we can presume that free radical scavenging activities of CexZr1-xO2 nanoparticles follow the level of OSC in the same materials. Fortunately, OSC of CexZr1-xO2 have been reported, and Figure 6-2 is reproduced from the paper published by Descorme et al. [62]. Since the catalyt ic activity of CeO2 nanoparticles can be promoted up to four times by incorporating 20-40 % of zirconium dopants, it is prospected to have four times of greater scavenging activities in CexZr1-xO2 nanoparticles than in undoped CeO2 nanoparticles. Figure 6-1. Hypothesized scheme of free radical scavenging by CexZr1-xO2 nanoparticles. 85

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Figure 6-2. OSC of CexZr1-xO2 measured a pulse chromatogra phic system at 400 C. Figure reproduced from [62]. 6.2 Activities against Hydrogen Peroxide Among the reactive oxygen species, hydrogen pe roxide and superoxide radicals are the most influential ROS in metabolism as well as pr ogrammed cell death. Both of them have been widely discussed and their concentrations have been directly measured in biological systems [1]. The concentrations of hydrogen peroxide in biological systems were even used to represent the levels of endogenous oxidative stress or even cell cultures viability [1]. In this chapter, CexZr1xO2 nanoparticles scavenging activities against hydrogen peroxide at physiological level are presented. This level of hydroge n peroxide is selected to mimic the efficiency of these nanoparticles in biological systems. 6.2.1 Experimental Methods To test hydrogen peroxide scavenging, hydroge n peroxide solutions were mixed with CexZr1-xO2 (x= 0, 0.2, 0.4, 0.6, 0.7, 0.8, 1.0) suspensions and the residual peroxide concentration 86

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over time was evaluated. Peroxide concentrations in each sample were determined using an Amplex red hydrogen peroxide/peroxidase as say kit (Cat. # A22188, Invitrogen). The principle to measure hydrogen pe roxide is illustrated in Figure 6-3 The Amplex reagents were prepared according to manufacturers instructions. To prepare sa mples for activity tests, 100 l of 200 M CexZr1-xO2 suspensions were prepared in a 96wells plate (Corning Inc.). A small amounts of substrate hydrogen peroxide was ti trated into 0.25 M phosphate buffer (pH = 7.4), forming 100 M hydrogen peroxide. To initiate th e free radical scavenging tests, 100 l of 100 M hydrogen peroxide solutions were pipetted in to each well at the designated time. The reactions were finally stopped by introducing enzyme horse radish peroxidase and Amplex reagents into each well at the designated reaction time. The Amplex reagents were also diluted using 0.25 M phosphate buffer. Each samples peroxide concentration was determined by measuring the optical density of the red produc t, resorufin, at 570 nm absorbance. Optical density measurements were made using Synergy HT multi-detection microplate reader (BioTech Instruments Winooski, VT). Figure 6-3. Detection scheme used to determine the peroxide concentration in activity tests. Colorless Amplex Red reagent (10-acety l-3,7-dihydroxyphenoxazine) reacts with peroxide, forming red resorufin. The reacti on is catalyzed by en zyme horse radish peroxidase. 87

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6.2.2 Results and Discussion The free radical scavenging efficiency of CexZr1-xO2 nanoparticles are determined by measuring the reduction of 50 M hydrogen peroxide solutions in 100 M CexZr1-xO2 suspensions over time. Figure 6-4 (a) shows the peroxide concentr ation decreased by a series of CexZr1-xO2 nanoparticles over time. Figure 6-4 (b) shows the residua l hydrogen peroxide concentration divided by initial peroxide con centration in natural logarithmic scale. In Figure 64(a), it is obvious that Ce0.7Zr0.3O2 and Ce0.6Zr0.4O2 nanoparticles exhibited the most efficient radical scavenging property, while Ce0.4Zr0.6O2, Ce0.8Zr0.2O2 exhibited moderate efficiency and CeO2, Ce0.2Zr0.8O2 exhibited the lowest efficiency. There is no significant peroxide concentration change in the sample of ZrO2 nanoparticles. In Figure 6-4 (b), the slope of each profile represents the hydrogen peroxide scaven ging activities of each sample. From high to low, the rankings of peroxide scavenging activities in CexZr1-xO2 nanoparticles are Ce0.7Zr0.3O2, Ce0.6Zr0.4O2, Ce0.4Zr0.6O2, Ce0.8Zr0.2O2, CeO2, Ce0.2Zr0.8O2, commercial CeO2, and ZrO2, respectively. According to the free radical scavenging mechanism illustrated in Figure 2-2 the scavenging mechanism of hydrogen pe roxide can be summerized in Equation (6-4). It is: the adsorption/desorption of peroxide, the transition of peroxide to oxygen ions, and finally the diffusion of oxygen ions into oxygen vacancies in the lattice. The oxyge n vacancies are then restored by emitting lattice oxygen molecules. To evaluate the scavenging activities of CexZr1xO2 nanoparticles, we then simplify Equation (6-4 ) to Equation (6-5). In Equation (6-3), 1, -1, and t, refer to the adsorption consta nt, desorption constant, catalytic rate constant, respectively. The initial condition in each sample is the same and hydrogen peroxide concentration in each sample is very diluted, thus we are able to ignore the influences from adsorption/desorption (1/1) in the reactions. Therefore, the scavenging activities (i.e. effective rate constant) K can be 88

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calculated using Equation (6-7). The scavenging activity K of each CexZr1-xO2 samples is shown in Figure 6-5 in terms of Ce/Zr molar ratios. Figure 6-4. A) shows the peroxide concentr ation in the presence of 7 nm commercial CeO2 and synthesized CexZr1-xO2 nanoparticles over time. B) shows the natural logarithmic values of the peroxide concentration divi ded by initial peroxide concentration. The slopes of profiles in B represent the pe roxide radical scavenging efficiency. 89

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Figure 6-4. Continued. Intrinsic O xx Intrinsic X O xx Intrinsic O xx Intrinsic O xx Intrinsic O xx Intrinsic O xxeVOZrCeOO OOZrCeO VOZrCeO eVOZrCeO eVOZrCeOeVOZrCeO 2 2 1 22 2 2 221 2 2 21 2 21 2 21 21 2 2 21 2 25 4 3 2 1 1 (6-1) 21 2 2 21 2 2 1 1 21 2 22 1 OZrCeOO OZrCeOOZrCeOxx t xx xx (6-2) 90

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] [][ ][21 2 2 2 2OZrCeOK dt Odxx (6-3) tOZrCeK O Oxx Initial ] [ ][ ][ ln21 2 2 2 2 (6-4) where 1 1 2 2, ][ t M M tO K Figure 6-5. The effective scavenging efficiency K of CexZr1-xO2 nanoparticles (bar diagram) vs. OSC ( ) and the amount of superoxide radicals ( ) detected on CexZr1-xO2 nanoparticles. The error bars represent the standard deviations of effective efficiencies between each data points [62]. According to the activity tests, the h ydrogen peroxide scavenging activities in CexZr1-xO2 nanoparticles is enhanced by doping zirconium ions into CeO2 lattice. More importantly, the 91

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scavenging activities of CexZr1-xO2 correlates to the magnitudes of OSC measured by pulse chromatographic system [62]. In Figure 6-5 we show that the scavenging activities increase with the amounts of zirconium dopants in CeO2 lattice. The scavenging activity increases to maximum in Ce0.7Zr0.3O2 nanoparticles, and gradually decreases when more than 40% of cerium ions are substituted. There is no distinguishab le peroxide radical scavenging property observed in the case of pure ZrO2 nanoparticles. On the other hand, OSC measured at 400 C increases to the highest level when 20% to 40% of cerium i ons were substituted; then OSC dropped gradually when more cerium ions were replaced; finally OSC totally diminished in pure ZrO2 nanoparticles. In addition, the scavenging activity of Ce0.7Zr0.3O2 nanoparticles is four times greater compared to the undoped CeO2 nanoparticles, corresponding to the same enhancement that occurred in Ce0.63Zr0.37O2 nanoparticles in respect to OSC [48,62]. According to the correlation, the nanoparticle concentrations uti lized to evaluate the effective free radical scavenging efficiencies can be replaced by the concentration of active sites. Equation (6-8) shows that the enhanced free ra dical scavenging efficiency in CexZr1-xO2 is a consequence of improved lattice oxygen vacancies The correspondence between the free radical scavenging efficiency and the magnitude of OSC confirms th e idea that free radical scavenging is mediated by oxygen vacancies. Also, the results deliver a message that the enhanced free radical scavenging activities are achieved by manipulating oxygen vacancies in the CeO2 lattice. tVK O OO Initial ][ ][ ][ ln2 2 2 2 (6-5) It is worth to note that CexZr1-xO2 nanoparticles may also exhib it greater superoxide radical scavenging properties compared to the undoped CeO2. It has been demonstrated that CeO2 nanoparticles exhibited superoxide dismutase mimetic properties, and their catalytic activity is comparable to enzyme superoxide dismutase [66] Yet the greater superoxide radical scavenging 92

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properties in CexZr1-xO2 nanoparticles will be demonstrated la ter. Prior to this work, we found that Descorme et al. had detected larger amounts of superoxide radical adsorption on CexZr1-xO2 nanoparticles than on the undoped CeO2, even at room temperature. Importantly, the superoxide radical adsorption on CexZr1-xO2 surfaces also corresponds to the magnitude of OSC in CexZr1xO2 nanoparticles (shown in Figure 6-5 ) [62]. In summary, their activities were tested in a 50 M hydrogen peroxide solution in order to investigate their free radical scavenging efficien cy in biological systems. The free radical scavenging activities of these CexZr1-xO2 nanoparticles is enhanced up to four times in Ce0.7Zr0.3O2 nanoparticles and gradually decreased when tetragonal phase appear in the structures. The effective free ra dical scavenging activities of CexZr1-xO2 nanoparticles correlates to the magnitude of OSC in the same materials, where OSC is used to evaluate oxygen vacancy concentration in metal oxide catalyst s. The correlation confirmed that CexZr1-xO2 nanoparticles scavenge hydrogen peroxide through the exchange of peroxide ions and lattice oxygen, and the scavenging activities are mediated by oxygen vacancies in the lattice. As consequence, the enhanced free radical sc avenging properties of CeO2 nanoparticles are achieved by increasing oxygen vacancies in the lattice through doping zirconium into CeO2 nanoparticles. The improvement is as great as four times compared to the undoped CeO2. 6.3 Activities against Superoxide Radicals In this section, the scavenging activities of CexZr1-xO2 nanoparticles are tested against superoxide radicals. Different to hydrogen peroxides, supero xide radicals are defined free radicals which have unpaired electrons. Since th e electron configurations of superoxide radicals are unpaired, superoxide radicals are much more reactive than hydrogen peroxide and they have great affinity to become hydrogen peroxides or oxygen mol ecules by electron exchange. Therefore, the half life of super oxide radicals is as short as 0. 05 second at high concentrations. 93

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Perhaps, the damage caused by superoxide radicals to biological systems may be more influential than hydrogen peroxide due to their reactivity [1]. 6.3.1 Experimental Methods Due to the short half life of superoxide radicals, it is important to generate this species continuously in the activity tests. To genera te the designed radicals, we used hypoxanthine and enzyme xanthine oxidase to produce superoxide radicals, hydrogen peroxide, and uric acids. The hydrogen peroxide produced in th is reaction is then removed by adding large amounts of enzyme CAT, in order to prevent the effects from hydrog en peroxide. The reaction used to generate superoxide radicals is illustrated in Figure 6-6 Figure 6-6. Superoxide ra dicals produced by hypoxanthi ne and xanthine oxidase. To detect the presence of supe roxide radicals, a water soluble compound, sodium salt of 4[3-4iodophenyl)-2-(4-nitrophenyl)-2H -5-tetrazolio]-1,3-benzene disulfonate (WST-1, Dojindo), is used as a superoxide probe. Only react with superoxide ra dicals, WST-1 salts become an irreversible, water-soluble formazan dye (shown in Figure 6-7 ). The dye formation can be observed at around 450 nm with the maximum dens ity at 438 nm spectro photometrically [89], and is not affected by the generation of hydrogen pe roxide in the reaction. In this study, the formation of formazan dye is observed using tw o techniques, UV-Vis (UV/Vis Perkin-Elmer Lambda 800) and microplate r eader. UV-Vis spectroscopy and microplate reader are both 94

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spectrophotometric basis; however comparing to microplate reader UV-Vis is a technique with higher resolution and allows measuring the dye formation continuously. However, the application of UV-Vis is limited by the numbers of samples that can be measured. Therefore, the technique based on microplate reader is e ngaged in order to test multiple samples simultaneously. Figure 6-7. Principle of WST-1 assay to detect superoxi de radicals [90]. For the samples prepared for UV-Vis, 0.5 mM EDTA (Aldrich), 0.5 mM hypoxanthine (Aldrich), 9.65 mU/ml xanthi ne oxidase (Invitrogen), 0.5 mM WST-1, and 1 mM CexZr1-xO2 nanoparticle suspensions (x = 0, 0.4, 0.6, 0.7, 0.8, 1.0) were prepared in 100 mM phosphate buffer (pH = 7.4, Aldrich) as stock solutions. Two hundreds micro-liters of each stock solution except hypoxanthine stock solution were titrated into a 1 ml c uvette, i.e. EDTA, xanthine 95

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oxidase, WST-1, and nanoparticle suspen sion. To initiate the reaction, 200 l of hypoxanthine stock solution was titrated into the cuvette in order to generate superoxide radicals. The total concentrations of CexZr1-xO2 nanoparticle suspensions were then diluted to 200 M in the reactions. The dye formation was detected by UV-Vis at 450 nm. The absorbance was recorded and shown in Figure 6-11 In order to obtain the reaction rate constants of CexZr1-xO2 nanoparticle against to superoxi de radicals, another sample prepared with enzyme SOD (Cat. #190117, MP Biomedical LLC) was repeated instead of nanoparticle suspensions. In the experiment, various stock solutions with SOD concen trations (0.25, 0.5, 1, 5, 10, 100 U/ml) were mixed in the cuvettes instead of 1 mM CexZr1-xO2 nanoparticle suspensions. The total concentrations of enzyme SOD were then diluted to 0.05, 0.1, 0.2, 1.0, 2.0, 20 unit/ml in the reactions. The stock solutions, their final concentrations, and experimental procedures are illustrated in Figure 6-8 96

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Figure 6-8. Experimental arrange ment of stock solution concentra tions, total concentrations, and experimental procedures. For the samples prepared for microplate reader, different nanoparticle concentrations were tested in the setups with vari ous superoxide producti on rates. The supero xide radical production rates were controlled by the amounts of enzyme xanthine oxidase in the reactions. In the standard radical production rate, the total concentration of 1. 93 mU/ml xanthine oxidase was used. However, the reaction may take too long to complete. To conquer this disadvantage, the other two tests with five times (5X) and twenty -five times (25X) of xanthine oxidase (i.e. 9.65 mU/ml and 48.25 mU/ml in total) were repeated using the same expe rimental procedures. In the test with standard superoxide producti on rate (i.e. 1.93 mU/ml xanthine oxidase in total), 0.5 mM EDTA, 0.5 mM hypoxanthine, 9.65 mU/ml xanthine oxidase, and 0.5 mM WST-1 were prepared in 100 mM phosphate buffer (pH = 7. 4) as stock solutions. The control samples were a series of enzyme SOD with different concentrations. They were 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10, 20, 50 U/ml SOD prepared in phosph ate buffers. The total co ncentration of SOD in the reaction therefore was divided by five, which were 0.002, 0.01, 0.02, 0.1, 0.2, 0.5, 1.0, 2.0, 4.0, 10 U/ml. The samples of our interest were CexZr1-xO2 nanoparticle suspensions (x = 0, 0.4, 0.6, 0.7, 0.8, 1.0) with three different concentr ations. The nanoparticle concentrations were 0.05, 0.25, 1.0 mM in stock solutions, th erefore the total concentrations in the test were 0.01, 0.05, 0.2 mM. After the stock solutions were prepared, 50 l of tested samples, control samples, and other stock solutions (except hypoxanthine solution) were titrated in a 96 well microplate (Corning, NY) accordingly. Finally, 50 l of hypoxanthine solu tions were titrated into each well quickly in order to initiate the reaction. The plate was then placed in microplate read er to read the optical density (absorbance) at 450 nm. Figure 6-9 shows the arrangement of sample preparation in microplate and the concentration of each stock solution in the plate. 97

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The activity tests underwent in higher super oxide production rates were carried out under similar protocols. The concentrations of xanthi ne oxidase that was used to procede different superoxide production rates were increased from 1.93 mU/ml to 9.65 mU/ml and 48.25 mU/ml in total. Figure 6-9. The setup of stock solutions and their concentrations in a 96-wells microplate. 6.3.2 Results and Discussion Figure 6-10 shows the results of activity tests using UV-Vis. In Figure 6-10 each profile represents the absorbance of each sample over time. The absorbance readings are results of formazan dye formation, which are caused by the interaction of WST-1 and superoxide radicals. Thus, the dropped optical density in the control sample is a resu lt of protection received from enzyme SOD. The inhibition from dye formation represents the enzyme activity, so the 98

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inhibition is used to calculate the ra te constants in the reaction. In Figure 6-10 the inhibition to dye formation is SOD concentration depende nt. The samples with 1 U/ml or higher concentration of enzyme SOD achieved 100% prot ection, while the different percentage of protection is distinguis hable in the diagram. Figure 6-11 shows the results of the same experimental setups but CexZr1-xO2 nanoparticles were used to scavenge superoxide radicals instead of enzyme SOD. In Figure 6-11 Ce0.4Zr0.6O2 has greater inhibition percentage s to dye formation, indicating a greater superoxide scavenging activity. Also, ZrO2 has no significant superoxide scavenging activity, while CeO2 has the lowest activity among all the CeO2-based nanoparticles. The ranki ngs of superoxide scavenging activity in CexZr1-xO2 nanoparticles are listed as follow according to the results in Figure 6-11 2 2 22.08.023.07.024.06.026.04.0ZrO CeOOZrCeOZrCeOZrCeOZrCe The results can be conclude d that zirconium dopants in CeO2 nanoparticles prompt the scavenging activities against superoxide radicals. The scavenging activities in CexZr1-xO2 nanoparticles become zirconium dopants dependent but not oxygen vacancy concentration dependent. Figure 6-12 shows the results of activity tests measured by microplate reader. The superoxide radicals in this test were produced by 1.93 mU/ml xanthine oxidase and 0.1 mM hypoxanthine. Each data point was the averag e of three different measurements, and the standard errors were included in the marks. Figure 6-12 (a) shows the inhibiti on to dye formation in samples with enzyme SOD. In Figure 6-12 (a), it is obvious that the inhibition to dye formation is SOD concentration dependent. Th e SOD concentrations greater than 0.5 U/ml totally inhibit dye formation made by superoxide radicals. In Figure 6-12 (b), inhibition to dye formation also occurred as 200 M CexZr1-xO2 nanoparticles were involved in the reaction. 99

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According to the profiles, Ce0.4Zr0.6O2 nanoparticles exhibit the gr eatest superoxide radical scavenging activity, and the activities of CexZr1-xO2 nanoparticles decrease as fewer dopants incorporated in the solid solutions. In contrast to the CeO2-based nanoparticles, ZrO2 nanoparticles exhibited none or indist inguishable scavenging activity. In Figure 6-12 (c) and (d), inhibition to dye formation is re producible when lower nanoparticle concentrations present in the reaction. 50 M and 10 M CexZr1-xO2 nanoparticles also performe d distinguishable scavenging activity against superoxide radicals; however, the protections were not as efficient as in the systems of 200 M Ce0.4Zr0.6O2 nanoparticles. According to the results in Figure 6-12 it can be concluded that the rankings of superoxide scavenging activity in CexZr1-xO2 nanoparticles are, 2 2 22.08.023.07.024.06.026.04.0ZrO CeOOZrCeOZrCeOZrCeOZrCe The scavenging activities of CexZr1-xO2 nanoparticles are zirconium dopants dependent. The results correspond to the observation in experiments carried out by UV-Vis. The same experiment was reproduced with higher xanthine oxidase concentrations (9.65 and 48.25 mU/ml) in order to shorten the reaction time. The results are shown in Figure 6-13 and Figure 6-14 In Figure 6-13 and Figure 6-14 the results are reproducible compared to that of 1.93 mU/ml xanthine oxidase. The scavenging activities of CeO2 and Ce0.8Zr0.2O2 are very close, while very limited scav enging activity was observed in ZrO2 samples. The superoxide radical scavengi ng activities of CexZr1-xO2 nanoparticles are ranked as follow, 2 2 22.08.023.07.024.06.026.04.0ZrO CeOOZrCeOZrCeOZrCeOZrCe 100

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Figure 6-10. The results of activity test obtaine d using UV-Vis. A series of enzyme SOD with different concentrations (total concen tration) protected WST-1 salts against superoxide radicals. The higher the absorbance indicates the more formazan formation caused by superoxide radicals. 101

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Figure 6-11. The results of activity tests obtained using UV-Vis. A series of 200 M CexZr1-xO2 nanoparticles (x = 0, 0.4, 0.6, 0.7, 0.8, 1.0) with different concentrations protected WST-1 salts against superoxide radicals. The higher the absorbance indicates the more formazan formation caused by superoxide radicals. The results have shown that CexZr1-xO2 nanoparticles exhibit extraordinar y scavenging properties against superoxide radicals. 102

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Figure 6-12. The results of activity tests measured by microplate reader. A) shows the inhibition to dye formation in samples with enzyme SOD. B) in samples with 200 M CexZr1xO2 nanoparticles. C) in samples with 50 M CexZr1-xO2 nanoparticles. D) in samples with 10 M CexZr1-xO2 nanoparticles. The superoxide radicals in all samples were produced by 1.93 mU/ml xanthine oxidase and 0.1 mM hypoxanthine. Each data point was the averaged result of three samples. Standard errors are shown in each data point; however most of the error bars are smaller than the marks. 103

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Figure 6-12. Continued. 104

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Figure 6-13. The results of activity tests measured by microplate reader. A) shows the inhibition to dye formation in samples with enzyme SOD. B) in samples with 200 M CexZr1xO2 nanoparticles. C) in samples with 50 M CexZr1-xO2 nanoparticles. D) in samples with 10 M CexZr1-xO2 nanoparticles. The superoxide radicals in all samples were produced by 6.95 mU/ml xanthine oxidase and 0.1 mM hypoxanthine. Each data point was the averaged result of three samples. Standard errors are shown in each data point; however most of the error bars are smaller than the marks. 105

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Figure 6-13. Continued. 106

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Figure 6-14. The results of activity tests measured by microplate reader. A) shows the inhibition to dye formation in samples with enzyme SOD. B) in samples with 200 M CexZr1xO2 nanoparticles. C) in samples with 50 M CexZr1-xO2 nanoparticles. D) in samples with 10 M CexZr1-xO2 nanoparticles. The superoxide radicals in all samples were produced by 48.25 mU/ml xanthine oxidase and 0.1 mM hypoxanthine. Each data point was the averaged result of three samples. Standard errors are shown in each data point; however most of the error bars are smaller than the marks. 107

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Figure 6-14. Continued. 108

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To calculate the reactio n rate constants of CexZr1-xO2 against superoxide radicals, a method to compare the inhibition percentages of CexZr1-xO2 nanoparticles and enzy me SOD is adopted. The inhibition rate is perceived using the equation in the follow, 100 ) ( ) ( %) (3 1 2 3 1 blank blank blank sample blank blankAA AAAA rate inhibition Activity (6-6) Where A represents the absorbance or optical density of samples or blanks in each well. The denominator in the equation represents th e increasing amounts of dye formation in the absence of samples but with superoxide radicals in the reactions. The numerator in the equation represents the inhibition due to te sted samples (i.e. enzyme SOD or CexZr1-xO2 nanoparticles) in the presence of superoxide radical s. Thus, the equation is to s how the inhibition percentage of dye formation when the tested samples involved in superoxide radical pro duction. To obtain the rate constant, the amounts of enzymes or nanopartic le concentrations that are required to achieve 50% of inhibition are compared. Using the comp arative method, the reaction rate constants of CexZr1-xO2 nanoparticles can be obtained according to th e reaction rate constant of SOD, 1.32.8109 M-1s-1 (at pH = 7.2, M.W. = 32,600 g/mole). The inhibition curve of enzyme SOD is shown in Figure 6-15 and the calculation is based on the results in Figure 6-12 The concentrations for enzyme SOD and CexZr1-xO2 nanoparticles to achieve 50% of inhibition rate are ca lculated according to the results in Figure 6-12 and Figure 6-15, and the values are shown in Table 6-1 109

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Figure 6-15. Inhibition curves of enzyme SOD with different incubation time. The amounts of SOD that achieved 50% inhibition rate is used to compare the amounts of CexZr1-xO2 nanoparticles that has achieved the same inhibition. Table 6-1. Concentrations for enzyme SOD and CexZr1-xO2 nanoparticles to achieve 50% of inhibition rate in activity test. Since the concentration of 1. 0 U/ml SOD approximately equa ls to 120 nM SOD [29], the reaction rate constants of CexZr1-xO2 nanoparticles can be obtained using simple calculations. Due to the high molecular weights of SOD, it is necessary to interpret the reaction rate constant 110

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into the format of (g/l)-1s-1 instead of M-1s-1. The rate constants of all samples are shown in Table 6-2. In Table 6-2, it is shown that rate constants of CexZr1-xO2 nanoparticles are even higher than enzyme SOD in respec t to scavenging superoxide radicals. Enzyme SOD has been tested to exhibit the fastest reaction rate among a ll enzymes, and its reacti on rate is only limited by colloid frequency to superoxide radicals. Table 6-2. Reaction rate constants of CexZr1-xO2 nanoparticles against supe roxide radicals. The kinetic analysis was measured by microplate reader using WST-1 salts in the samples with 1.93 mU/ml xanthine oxidase. In Table 6-2, it is found that zirconium dopant s are able to improve superoxide radical scavenging activities of CexZr1-xO2 nanoparticles. Instead of correlating to the OSC, the scavenging activities of CexZr1-xO2 nanoparticles actually correlate to the am ounts of zirconium dopants in the system. The rankings and magnit ude of improvements do not correspond to the hypothesis. The results suggest that superoxide radical scavenging activities of CexZr1-xO2 nanoparticles are corre lated with the amounts of zirconium dopants in the lattice but not oxygen vacancy concentrations. The resu lts also conflict what was dem onstrated in previous chapters that hydrogen peroxide scavenging activities of CexZr1-xO2 nanoparticles are correlated with 111

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oxygen vacancy concentrations in lattices. The differences are due to the nature of hydrogen peroxide and superoxide radical s. Comparing to hydrogen per oxide, superoxide radicals are more active due to their unpaired electron configurations. In the catalytic reactions with catalase two steps of chemical reactions are involved. First, hydrogen per oxides donate an oxygen ion to the reduced catalase, forming an intermediate compound. Second, the intermediate compound donates its extra oxygen ion to another hydrogen peroxide, forming water and one oxygen molecule. Except the reactions contributed by el ectron exchange, it is not ices that oxygen ions also involve in catalysis in th e case of hydrogen peroxide. Differ ent to the catalysis to hydrogen peroxide, the catalytic reactions between supe roxide radicals and enzy me SOD do not involve the exchange of oxygen ions. It is, superoxide radicals only donate their unpaired electrons to form oxygen molecules or receive electrons to form hydrogen peroxide. In the particular reaction, the scavenging to superoxide radicals are carried out through electron exchange and no oxygen ions are involved. Thus, it can be concluded that the ca talysis of superoxide radicals only contributed by the electron exchange with enzyme SOD. However, in the case of CexZr1xO2 nanoparticles, the electron exchange not onl y contributed by oxygen vacancies, but also contributed by other cationic defects in the lattice. To further explain what species of cationi c defects contribute in superoxide radical scavenging, it is necessary to investigate the Ce3+/Ce4+ ratios in CexZr1-xO2 nanoparticles. Figure 6-16 shows the Ce3+ contents and OSC in CexZr1-xO2 nanoparticles reported by Vidal et al. [68,69]. In Figure 6-16 the Ce3+ contents were obtained us ing magnetic susceptibility measurements through a magne tic balance under flowing H2 (5%)/He gas. The Ce3+ contents, or as reduction percentages, were calculated on a [ Ce3+]/[Ce3++ Ce4+] basis. According to the reports [68,69], zirconium dopants in the soli d solutions promote reduction capability of 112

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nanocrystallites. The results are reproducible at low and high temperat ures (473-973 K). The results shown by Vidal et al. may provide the an swer for the results that obtained in the superoxide radical scavenging tests. Figure 6-16. A) Ce3+ contents and B) OSC in CexZr1-xO2 nanoparticles. CZ100/0-HS ( CeO2); CZ80/20-HS ( Ce0.8Zr0.2O2); CZ68/32-HS ( Ce0.68Zr0.32O2); CZ50/50-HS ( Ce0.5Zr0.5O2); CZ15/85-HS ( Ce0.15Zr0.85O2). Figures reproduced from [69]. As previously mentioned, catalysis of super oxide radicals depends on electron exchange in the reaction. The electron ex change relies on the conduction of electrons in the catalysis. Therefore, the reaction rates in catalysis of superoxide radicals depend on the mobile electronic carriers that are available in the scavenging. Doping zirconium into CeO2 not only promotes the oxygen vacancy concentration struct urally, but also promotes the Ce3+ contents and so to increase the electron holes. In other words, doping zirconium into CeO2 improves superoxide scavenging activity due to the increased extrin sic cationic defects and nonstoichiometric oxygen vacancies. The promoted mobile electronic carriers increase the reducibility of CexZr1-xO2 catalysts [68,69]. This perspective is able to explain why more zirconium dopants in nanoparticles always improve their activity, and the results do not correspond to oxygen vacancy 113

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concentration in CexZr1-xO2 nanoparticles. The active sites in superoxide radical scavenging, i.e. mobile electronic carriers including oxygen vacancy, electron holes, are listed in the following equation. Equation 6-13 shows that oxygen vacancies are mobile electronic carriers, which are contributed from nonstoichiometric defects. Equation 6-14 shows that electron holes are mobile electronic carriers, electron holes, which are contributed from extrinsic defects. eVOZrCeOZrCeO xx xx221 21 (6-7) hCeCeCeCeOZrCeZr Ce X Zr X Ce xx2' 21 (6-8) Here, the model to describe superoxide radical scavenging which mediated by oxygen vacancies in CexZr1-xO2 nanoparticles is listed in the following equations. 2 21 2 212 2 OeVOZrCeOeVOZrCeO xx O xx (6-9) 22 21 2 212 22 OHeVOZrCeOHeVOZrCeO xx O xx (6-10) The model to describe super oxide radical scavenging which mediated by electron holes in CexZr1-xO2 nanoparticles is listed in the following equation. 2 2 '2 OhCeCeCeCeOhCeCeCeCeZr Ce X Zr X Ce Zr Ce X Zr X Ce (6-17) 22 2 '2 22 OHhCeCeCeCeOHhCeCeCeCeZr Ce X Zr X Ce Zr Ce X Zr X Ce (6-18) In the equation described above, we understand that zirconium dopants in CexZr1-xO2 nanoparticles improve scavenging activities bo th against hydrogen peroxide and superoxide radicals. The improvements are dependent on th e increased oxygen vacancy concentration in the case of hydrogen peroxide, and the improvement s in the case of superoxide radicals are dependent on the promoted mobile electronic carrie rs. The reason to make this difference is due to the characteristics of hydrogen peroxide and superoxide radicals. Moreover, most accumulative oxidative stress occurred in biologic al systems relies on the transition of electrons 114

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between radicals and molecules, so the optimum fr ee radical scavengers in biological systems are those that have shown greater activities to remove excess electrons from free radicals. In conclusion, we can hypothesize that Ce0.4Zr0.6O2 nanoparticles would be the most effective free radical scavengers in biological systems. 6.4 Electron Conduction in Catalysis In this section, the capability for CexZr1-xO2 nanoparticles to conduct el ectrons in catalysis will be inspected and discussed, since the particular property is essential for free radical scavenging. In previous section, the inhibition percentages of dye formation merely indicated nanoparticles superoxide radical scavenging activities. The capability for CexZr1-xO2 nanoparticles to conduct electrons in catalysis, therefore, would need solid evidence other than the information we acquired. Here, we adopt a cl assic assay on biochemistry basis to inspect the electron conduction on CexZr1-xO2 nanoparticles. The assay incl udes a bivalent, reversible conducting molecule, which is colorimetric only in its reduced state. Thus, the rise or decline of optical density of this molecule would indica te its redox states. Replace WST-1 salts in the methodology for scavenging activity te st, the redox states of this molecule would indicate the capability for catalysts to conduct electrons in cata lysis. The principle of this method is shown in Figure 6-17 The colorimetric molecule that used to test electron conduction on CexZr1-xO2 nanoparticles is cytochrome c (II/III). Figure 6-17. Principle of the biochemistry based assay to inspect the capability for CexZr1-xO2 nanoparticles to conduct el ectrons in catalysis. 115

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6.4.1 Experimental Methods To generate superoxide radi cals, we used hypoxanthine and enzyme xanthine oxidase to produce the final products including superoxide radicals, hydrogen pe roxide, and uric acids. The hydrogen peroxide produced in such reaction is then removed by adding large amounts of catalase, in order to prevent th e affects of hydrogen peroxide. To detect the electron conducted on CexZr1-xO2 nanoparticles, a revers ible dye, cytochrome c is conducted in the experiment. Reduced by superoxide radicals, cytochrome c forms cytochrome c (II), and is colorimetric at 550 nm. In this study, the redox st ate of cytochrome c is observed using UV-Vis (UV/Vis Perkin-Elmer Lambda 800). For the samples prepared for UV-Vis, a st ock solution including 0.5 mM EDTA, 12.5 M cytochrome c (Aldrich), in addi tion of 3,000 unit/ml catalase (Ald rich) is prepared as reagent solution. 0.5 mM hypoxanthine, 0.9 U/ml xanthine oxidase, and 1 mM CexZr1-xO2 nanoparticle suspensions (x = 0, 0.4, 0.6, 0. 7, 0.8, 1.0) were prepared in 100 mM phosphate buffer (pH = 7.4, Aldrich) as stock solutions. Be fore initiating the reaction, 780 l of reagent solution, 80 l of sample solution (i.e. 1 mM CexZr1-xO2 nanoparticle suspensions or 1.875 U/ml SOD samples), and 20 l of xanthine oxidase stock so lution were titrated into a 1 ml cuvette. To initiate the reaction, 100 l of hypoxanthine stock soluti on was titrated into the cuve tte in order to generate superoxide radicals. The total concentrations of CexZr1-xO2 nanoparticle suspensions were then diluted to 200 M after all. The dye formation was finally detected by UV-Vis at 550 nm. The stock solutions used in this experiment and th e experimental procedur es are illustrated in Figure 6-18. The absorbance was recorded and shown in Figure 6-19 116

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Figure 6-18. Experimental arra ngements of stock so lution concentrations and experimental procedures to inspect electron conduction on CexZr1-xO2 nanoparticles. Figure 6-19. Redox states of cytochrome c det ected using UV-Vis. The higher the absorbance represents more reduced cytochrome c in the reaction. The declined absorbance suggests that CexZr1-xO2 nanoparticles received el ectrons from the reduced cytochrome c in catalysis. 117

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6.4.2 Results and Discussion In Figure 6-19 the vertical axis shows the redox states of cytochrome c. The higher absorbance in the particular diagram represents more reduced cytochrome c (II) in the reaction. In the samples with enzyme SOD protection, the concentration of reduced cytochrome c increases and stays at high level. It is becau se SOD merely catalyzes superoxide radicals to hydrogen peroxide, and the produc ts hydrogen peroxide was imme diately removed by enzyme CAT. On the other hand, the samples with CexZr1-xO2 nanoparticles protec tion not only inhibit the formation of reduced cytochrome c, but also reverse the redox state to its initial state. In these samples enzyme CAT was also involved, so the agents to reverse redox states of cytochrome c (from II to III) do not appear to be hydrogen peroxide. It is because the catalysis from superoxide to hydrogen peroxide requires electrons, so CexZr1-xO2 nanoparticles take electrons from the reduced cytochrome c and r eact with other superoxide radicals to form hydrogen peroxide. The reversed absorbance observed in UV-Vis therefore indicates the capability for CexZr1-xO2 nanoparticles to conduct elec trons in catalysis. In Figure 6-19 more zirconium dopants in CexZr1-xO2 solid solutions result in more rapi d reversion in redox states. It is the evidence that CexZr1-xO2 nanoparticles with more zirconium dopants exhibit greater capability to precede electron conduction. The resu lts obtained in this experiment correspond to the conclusion in previous section, and these CexZr1-xO2 nanoparticles really are catalyzing free radicals through electron exchange. 6.5 Summary In this chapter, the free radical scavenging activities of a series of CexZr1-xO2 nanoparticles were discussed. Using biochemistry assays, the residual concentration of hydrogen peroxide at physiological levels was measured over time. In the presence of CexZr1-xO2 nanoparticles, hydrogen peroxide concentrations in each sample decreased over time. Among all the samples, 118

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Ce0.7Zr0.3O2 nanoparticles have the greatest peroxide scavenging activity. In addition, the scavenging activities of CexZr1-xO2 nanoparticles are correlated with the oxygen vacancy concentrations in the materials. It is because the catalysis of hydrogen peroxide is determined by the transition of oxygen ions, so th e oxygen vacancy concentrations in CexZr1-xO2 nanoparticles become dominant in the scavenging processes. Using the methodologies based on biochemist ry reactions, superoxide radicals were generated. The scavenging activities of CexZr1-xO2 nanoparticles to superoxide radicals were tested using an irreversible dye, and their activit ies were compared with the fastest superoxide radical scavenger, superoxide dismutas e. The results showed that these CexZr1-xO2 nanoparticles have even greater scavenging activity than enzyme SOD. Furthermore, the results showed that the scavenging activity of CexZr1-xO2 nanoparticles increases with increasing zirconium dopants in CexZr1-xO2 nanoparticles. It is becaus e the zirconium dopants prompt Ce3+ contents in CexZr1xO2 nanoparticles, and resulting in more mobile el ectronic carriers. These mobile electronic carriers conduct the unpaired electrons in free radical s, so the scavenging activities to superoxide radicals are through electron exchan ge. It is because the catalysi s of superoxide radicals is determined by the exchange of electrons, so the concentration of mobile electronic carriers in CexZr1-xO2 nanoparticles becomes dominant in the r eaction. The mobile electronic carriers include oxygen vacancies, cationic defects that are caused by nonstoichiometric defects and extrinsic defects. The capability for CexZr1-xO2 nanoparticles to conduct electrons in catalysis is inspected using a bivalent, reversible c onducting molecule. In the experi ment, electrons in the reduced cytochrome c were taken away, resulting in noncolorimetric cytochrome c (III). Thus, the reversed absorbance repres ents electron conduction from cytochrome c, through CexZr1-xO2 119

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120 nanoparticles, and finally to form hydrogen peroxide. The el ectron conduction in CexZr1-xO2 nanoparticles relies on the mob ile electronic carriers in CexZr1-xO2 nanoparticles; therefore the materials with most carriers also have the greates t activity to scavenge free radicals. Since the accumulation of oxidative damage relies on electr on exchange between molecules, such as proteins, lipids, DNA, and RNA, the optimum free radical scavenger therefore relies on mobile electronic carrier concentration in CexZr1-xO2 nanoparticles. Thus, we conclude that Ce0.4Zr0.6O2 nanoparticles would be the most effective free radi cal scavengers in biolog ical systems, based on the results in the activity tests. After this chapter, the remarkable results confirm that the development of CexZr1-xO2 nanoparticles for biomedical a pplications requires the knowle dge in materials science. Incorporated with zirconium dopa nts, nanoparticles that have up to nine times of free radical scavenging activity compared to undoped CeO2 nanoparticles were developed. After the knowledge learned in this chapter, we have su ccessfully built up a mode l that describes hydrogen peroxide scavenging and s uperoxide radical scavenging. According to the model, the scavenging activities of these nanocatalysts can be further promoted if more mobile electronic carriers are increased.

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CHAPTER 7 IMPLICATIONS 7.1 Distinct Antioxidant Defense Pathway CeO2 nanoparticles have been f ound to inhibit the progression of organism dysfunction through regulating its endogenous oxidative stress. It was hypothesized that CeO2 nanoparticles scavenge free radicals and ther efore improve cell cultures viab ility. However, we found that CeO2 nanoparticles are eligib le to reinstate the redox states of cytochrome c in the presence of superoxide radicals. It sugge sts that protec tion provided by CeO2 nanoparticles may be contributed by reactivating cytochrome cs redox st ates as well. The results also explain the finding in the previous study that CeO2 nanoparticles restored retinal photoreceptor cells function even if CeO2 nanoparticles were administered af ter cell cultures we re damaged [54]. Cytochrome c is a heme protein found l oosely bounded with inner membrane of mitochondria. It is an antioxi dant protein with two valences which can oxidize superoxide radicals and reduce hydrogen peroxide. Cytochro me c is also a very important protein in metabolism. It conducts electr ons in electron transport chain of mitochondria. Lacking of cytochrome c, the electrons leak out of the transport chain and forming free radicals in the sequence. In addition, the over reduced cyto chrome c would lose its bonding with inner membrane of mitochondria and finally be released to cytoplasm. When cytochrome c is released from mitochondria, they tend to bond with other proteins, formi ng apoptosome. The apoptosome triggers apoptosis later on. It has been demons trated that cytochrome c release requires a twostep process [91]. Cytochrome c is present as loosely and tig htly bound pools attached to the inner mitochondrial membrane by its association with free radicals, a nd this interaction must first be disrupted to generate a soluble pool of this protein. Specifi cally, solubilization of cytochrome c involves a breaching of the elect rostatic or hydrophobic affiliati ons that this protein usually 121

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maintains in mitochondrial membrane. Once cytochrome c is solubilized, the reduced cytochrome c loses its electro static bonding with mitochondr ial membrane. The lowered electrostatic bonding is sufficient to trigger cytochrome cs release. Since superoxide radicals have long been found to reduce cytochrome c, the concept of scavenge su peroxide radicals prior their damage to cytochrome c, or preventing cytochrome c releas ed from mitochondria has been classified in a novel antioxidant defe nse pathway (shown in Figure 2-1). CeO2 nanoparticles were found to exhibit exceptional scavengi ng activity to superoxide radicals and peroxides. Th e reaction rate constants of CeO2 nanoparticles are superior to enzyme superoxide dismutase [29] and clos e to catalase [92]. However, th e half life of free radicals is short, the question remains on how the damaged cells were re covered if nanoceria merely scavenge free radicals in cell cultures? Beyond CeO2 nanoparticles free radical scavenging properties, we found that CeO2 nanoparticles are e ligible to regulate the redox states of cytochrome c, especially in the presence of superoxide radicals. Figure 7-1 shows the redox states of cytochrome c in the presence of supero xide radicals, CeO2 nanoparticles, and SOD. The data are extracted from Figure 6-19 In Figure 7-1 profile A shows the reduction of cytochrome c in the presence of superoxide ra dicals. Profile B show s the restoration of cytochrome c as CeO2 nanoparticles scavenge superoxide radicals. In profile C, CeO2 nanoparticles slightly rest ore cytochrome cs redox states in th e absence of superoxide radicals. In profile D, SOD only against superoxides at tack, but does not regulate cytochrome cs redox states. The redox states of cytochrome c are in fluential in apoptosis [40,93]. Overwhelmed by the reduced cytochrome c: (1) electrons would leak from mito chondrial electron transportation chain, forming detrimental ROS [94]; (2) cytochrome c would be released from mitochondria due to the lower electrostatic bonding in reduced cytochrome c [95]. The accumulation of ROS 122

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as well as cytochrome c releas e trigger caspase proteins and finally induce apoptosis. By regulating the redox states of cytochrome c, CeO2 nanoparticles may provide a distinct antioxidant defense pathway by inhibiting the release of cyto chrome c from mitochondrial membrane. In this chapter, we found that CexZr1-xO2 nanoparticles are capable to regulate the redox states of cytochrome c (shown in Figure 6-19 ). In reference to Chen et al.s work, exposing CeO2 nanoparticles to cultures not only scavenges free radicals in cultures, but also regulates the redox states of cytochrome c and inhibits cytochrome c release. We suggest that CexZr1-xO2 nanoparticles may contribute a distinct antioxida nt defense beyond free radical scavenging. In consequence, the proposed mechanism is able to explain the previous di scovery by Chen et al. that CeO2 nanoparticles restored retinal photoreceptor cells function even CeO2 nanoparticles were administered after cells were damaged [58]. Figure 7-1. Redox states of cytochrome c in th e presence or absence of superoxide radicals. 123

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7.2 Implications in Catalysis In heterogeneous catalysis, th e electrochemical reactions co mprise a series of electron exchange, oxygen ion exchange in/between liga nds and catalysts. The selection of optimum catalysts relies on inspecting the catalysts capab ility to conduct electrons or oxygen ions in catalysis. According to the result s in previous experiments, the biochemistry based assays that have been developed in this work are ideal tech niques to inspect the interfacial electrochemistry in the process of catalysts. 7.2.1 Alternative Technique to Inspect io nic Conductivity at Room Temperature Due to the characteristics of hydrogen peroxi de, it is found that the exchange of oxygen ions is involved in the catalysis for hydrogen peroxide. Based on this concept, hydrogen peroxide molecules can be treated as probes to inspect i onic conductivity of CeO2-based materials in catalysis. In biochemistry, enzyme CAT is used to catalyze hydrogen peroxide The catalysis of hydrogen peroxide. The catalysis proceeds in two steps [96] by (1) Oxidation of CAT by a peroxide )(.)IV( )III(2 22 EFeOOHEFeOH (6-11) (2) Oxidation of the substrates. 2 2 22)III( )(.)IV( OEFeOHEFeOOH (6-12) Overall, 2 2 222/1 OOH OHcatalase (6-13) In the catalysis, the oxidized enzyme possesses two oxidative equivalents above the native enzyme and contains highly reactive oxygen bound to the iron. The iron is then promoted to its 124

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quadrivalent states. In the catalysis proceed ed by CAT, the catalysis of hydrogen peroxide depends on its characteristics that conduc ting its oxygen ions with enzyme CAT. At low hydrogen peroxide concentration, we ha ve demonstrated that the amount of mobile oxygen vacancies dominates the catalysis. As an excellent ionic conductor, CxZr1-xeO2 actually mediates ionic conduction by its mobile oxygen v acancies. It is, the scavenging rates to hydrogen peroxide scavenging not only indicate the oxygen vacancy concentration in catalysts lattice, but also repres ent ionic conductivity of CexZr1-xO2. 7.2.2 Alternative Technique to Inspect Localized Electron Conductivity The catalysis of superoxide radicals are pr eceded by two steps. Superoxide radicals can donate electrons to a molecule and become oxygen molecule, or they can receive electrons from others to form peroxides. In biochemistry, en zyme SOD is used to ca talyze superoxide radicals into hydrogen peroxide and oxygen in free radica l scavenging. The catalysis carried out by enzyme SOD is as follow, SODMOSOD MOn 2 )1n( 2 (6-14) SOD MOHSODMHOn )1n( 22 22 (6-15) Overall, 22 222 OHHO (6-16) In the catalysis carried out by enzymes, it is certainly that the exchange of electrons involved in the catalys is. However, the scavenging occurred in CexZr1-xO2 nanoparticles remains uncertain, therefore this section is to discu ss the mechanism based on the results obtained in superoxide radical scavenging tests. The scavenging to superoxide radicals under goes a different mechanism comparing to the scavenging to hydrogen peroxide. In the catalysis to superoxide radicals, a superoxide radical 125

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126 either donates an electron to catalysts forming oxyge n molecules, or receives an electron forming peroxide. In means that the amount of electrons conducted in catal ysts dominates the reaction. Thus, superoxide radicals work as perfect probes to inspect localized mobile electronic carriers in catalysis. Utilizing a bivalent, reversible, colorimetric protein cytochrome c, it becomes possible to see the electron conduction in catalysis. The reversed optical density in the reaction represents the electrons taken away by catalysts, where th e electrons are taken away to form peroxide with other superoxide radicals.

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CHAPTER 8 CONCLUSIONS The in vitro studies in Chapter 3 have shown that CeO2 nanoparticles could relieve oxidative stress in certain cell cultures. The cu ltures viability was improved accordingly. In addition to these successful in vitro results, several objectives have been achieved in further, including synthesis and characterization of CexZr1-xO2 nanoparticles, evalua tion of free radical scavenging activities of these nanoparticles, and a distinct antioxidant de fense pathway that may contribute to the protection to cu ltures. In this dissertation, several innovative techniques have been emerged to evaluate the catalytic properties of CexZr1-xO2 nanoparticles, and the results are remarkable. The conclusions are summarized as follow: Reverse micelle method is used to synthesize CexZr1-xO2 nanoparticles of 3-7 nm. The synthesized CexZr1-xO2 nanoparticles are crystalline solid solutions, so heat treatments can be exempted in the synthesis. The synthesized CexZr1-xO2 nanoparticles are dispersed in sodium citrate buffer. The agglomeration using such preparation can be ignored, since the agglomerate size distributions are smaller than 10 nm. CeO2 nanoparticles can improve cell cultures viability by relieving its endogenous oxidative stress. Doping zirconium into CeO2 nanoparticles promotes their free radical scavenging activities. The enhancement comes from the promoted oxygen vacancies as well as mobile electronic carriers in the lattice. For CexZr1-xO2 nanoparticles to scavenge hydrogen per oxide at low concentration, oxygen ions are involved in the reac tions, therefore oxygen vacancies in the lattice dominate the scavenging. Among all samples, Ce0.7Zr0.3O2 nanoparticles e xhibit the greates t activity. The scavenging activities of CexZr1-xO2 nanoparticles correlate to the oxygen storage capacity reported in the same materials. For CexZr1-xO2 nanoparticles to scavenge superoxi de radicals, the scavenging activity depends on their capability to conduct electr ons in catalysis. Zi rconium dopants promote Ce3+ contents in CeO2 lattice, so the mobile electronic carrier concentration increases due to the promoted cationic def ects in the lattice. The cationic defects include oxygen vacancies and electron holes in the lattice, which are contri buted from nonstoichiometric 127

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128 defects and extrinsic defects. After activity tests, Ce0.4Zr0.6O2 nanoparticles exhibit the greatest activity of all CexZr1-xO2 nanoparticles. CexZr1-xO2 nanoparticles can reactivat e cytochrome cs redox states in the presence of superoxide radials. The reinstate of redox states may regulat e cytochrome cs function in metabolism, and further achieve a distinct antioxidant defense.

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APPENDIX A GLOSSARY Apoptosis A form of programmed cell death in multi-cellular organisms. It is one of the main types of programmed cell d eath (PCD) and involves a series of biochemical events leading to a ch aracteristic cell morphology and death bcl -2 Protein bcl -2 is an antioxidant protein. An important one states that this is achieved by activation or inactivat ion of an inner mitochondrial permeability transition pore, which is involved in the regulation of matrix Ca2+, pH, and voltage. It is also thought that some Bcl-2 family proteins can induce (pro-apoptotic members) or inhibit (anti-a poptotic members) the release of cytochrome c in to th e cytosol which, once there, activates caspase-9 and caspase-3, leading to apoptosis. TC-tet cells Pancreatic TC lines derived from murine insulinomas. CAT Enzyme catalase. Catalase is a common enzyme found in nearly all living organisms. Its functions include cat alyzing the decomposition of hydrogen peroxide to water and oxygen. Catala se has one of the highest turnover rates of all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second. Carotenoids Carotene is a pr ecursor to vitamin A, a pigm ent essential for good vision, and carotenoids can also act as antioxidants. CeO2: Cerium oxide, cerium dioxide, or sometimes listed as ceria. CexZr1-xO2: Zirconium-doped CeO2. The x in the chemical formula represents the stoichiometry of cerium ions, and ( 1-x ) represents the stoichiometry of zirconium ions sitting on the cationic sites in the cubic fluorite structures. COX The enzyme cytochrome c oxidase or Complex IV is a large transmembrane protein complex found in bacteria and the mitochondrion. It is the last enzyme in the resp iratory electron transport chain of mitochondria (or bacteria) located in the mitochondrial (or bacterial) membrane. It receives an electron from each of four cytochrome c molecules, and transfers them to one oxygen molecule, converting molecular oxygen to two molecules of water. Cytochrome c Cytochrome c is a small heme protein found loosely associated with the inner membrane of the mitochondrion. It is a soluble prot ein, unlike other cytochromes, and is an essential com ponent of the electr on transfer chain, where it carries one electron. It is capable of undergoing oxidation and reduction, but does not bind oxygen. It transfers electrons between Complexes III and IV. 129

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DCF 2,7-dichloroflurescin diacetate. A fluorescent dye to detect reactive oxygen species in biological systems. Electron transport chain An electron transport chain associ ates electron carriers and mediating biochemical reactions that produce ad enosine triphosphate (ATP), which is a major energy intermediate in living organisms. Only two sources of energy are available to biosynthesize organic molecules and maintain biochemical and kinetic processes in living organisms: redox reactions. The schematic diagram of electron transportation chain is shown in Figure 2-1. Endoplasmic reticulum stress Endoplasmic reticulum (ER) stre ss is caused by the accumulation of unfolded proteins in the ER lumen, a nd is associated with vascular and neurodegenerative diseases. Ferets diameter I n microscopy, Feret's diamet er is the measured distance between parallel lines that are tangent to an object's profile and perpendicular to the ocular scale. Generally, Feret's diameter is the greatest distance possible between any two points along the boundary of a region of interest. GPx Enzyme glutathione peroxidase. Enzyme glutathione oxidase is an enzyme that catalyzes glutathione and oxygen to form glutathione disulfide and hydrogen peroxide. GSH Glutathione is a tripep tide. It contains an unusua l peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side chain. Glutathione, an antioxidant, protects cells fr om toxins such as free radicals. HO Enzyme heme oxygenase. An enzyme that catalyzes the degradation of heme. This produces biliverd in, iron, and carbon monoxide. HQ Hydroquinone. Hydroquinone is a co mpounds used to induce oxidative stress in this dissertation. Inflammation Inflammation is the complex biological response of va scular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. Lipid peroxidation Lipid peroxidati on refers to the oxidative degradation of lipids. It is the process whereby free radicals grab el ectrons from the lipids in cell membranes, resulting in cell damage This process proceeds by a free radical chain reaction mechanism. 130

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OSC Oxygen storage capacity. The OS C is determined by measuring oxygen consumed by the catalyst after reducti on under isothermal conditions. It is a quantitative basis the capability of metal oxide catalysts to release oxygen under reducing condition, a nd to uptake oxygen under oxidizing conditions. Oxidative stress Oxidative st ress is caused by an imbalance between the production of reactive oxygen and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. ROS Reactive oxygen species. Reactive oxygen species are oxygen species that are generated from metabolism. They are reactive to other molecules, and usually cause damage to cells. In free radicals in biology superoxide radicals, hydroxyl radica ls, and hydrogen peroxide are usually appointed as ROS. Superoxide Superoxide radicals Superoxide is the anion O2 It is important as the product of the one-electron reduction of dioxygen, which occurs widely in nature. With one unpaired electron, the superoxide ion is a free radical, and, like dioxygen, it is paramagnetic. SOD Enzyme superoxide dismutase. Enzyme SOD is an enzyme found in almost all living systems. It catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, it is an important antioxidant defense in nearly all cells exposed to oxygen. Type 1 diabetes Also called juvenile diabetes. It is a form of diabetes mellitus. Type 1 diabetes is an autoimmune diseas e that results in the permanent destruction of insulin produc ing beta cells of the pancreas. Type 1 is lethal unless treatment with exogenous insulin via injections replaces the missing hormone. 131

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140 BIOGRAPHICAL SKETCH Yi-Yang Tsai was born in Taiwan in 1976. Before pursuing postgra duate education, he received B.S. in aeronautical engineering from Feng-Chia University, Taiwan in 1998. After two years of military services and two years of working experience as R&D engineer, he went oversea and accepted the graduate education in the United States. He firs t joined the Materials Engineering Department at Univ ersity of Dayton in 2002, and received his M.S. in 2004. He joined the Materials Science and Engineering De partment at University of Florida in 2004 under supervisory of Dr. Wolfgang M. Sigmund, a nd received his Ph.D. degree in summer 2008. In the graduate training at University of Dayton, Yi-Yang was specialized in the characterization techniques, atomic force micros cope and an ultrasound integrated atomic force microscope in the University of Dayton Research Institute (UDRI). Due to his excellent works, he was awarded a visiting research fellowship in 2003, and has allowed him to travel to the Fraunhofer Institute for Non-Destructive Testing (IZFP) in Germany for a short term research. In 2004, he was awarded the DAGSI research fellowship (Dayton Area Graduate Student Institute). His master thesis was a study th at using ultrasound inte grated atomic force microscope to detect nanoscale precipitations and cracks in aluminum alloys. In the two years, he has published two scientific papers and gave two invited presentati ons in SPIE conference. In the Ph.D. training in Dr. Sigmunds re search group, Yi-Yang has developed many innovative techniques in th e interdisciplinar y research project Administering CeO2 nanoparticles to enhance cells viability assigned by his advisor. In the project Yi-Yang and his advisor have made several remarkable breakthro ughs. They have publis hed several scientific articles and international patent s based on the results. Other than the articles that have been published, he is preparing manuscripts for paper publications from the remarkable results.