Magnetite/Silica
Core-Shell Nanoparticles
for HER-2 Targeted
Magnetic Resonance
Imaging of Breast
Tumours
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy (Applied Chemistry)
Jos Laurie Campbell
School of Applied Sciences
RMIT University
April 2013
i
Declaration
I certify that except where due acknowledgement has been made,
the work is that of the author alone; the work has not been submitted
previously, in whole or in part, to qualify for any other academic award;
the content of the thesis is the result of work which has been carried out
since the official commencement date of the approved research program;
and any editorial work, paid or unpaid, carried out by a third party is
acknowledged; and all ethics, procedures and guidelines have been
followed.
………………………………
Jos Laurie Campbell
…./…./….
ii
Acknowledgements
I must of course first thank my supervisors Professor Suresh K
Bhargava, Dr Vipul Bansal, Dr. Samuel Ippolito and Dr. Kourosh
Kalantar-Zadeh for their support during my research. You gave me
inspiration and guidance throughout my project and made sure I was
mostly pointed in the right direction. From you I learnt to question myself
and see through a challenge rather than to look upon its face. You also
gave me the opportunity to conduct my research with a position in your
labs and for that I am exceptionally grateful.
In particular I must thank Dr Jyoti Arora for her contributions to
my work. We worked in close collaboration throughout the project and
without your input much of the work could not have been completed.
Without you my research would probably have ended up going in some
other direction entirely.
I would also like to thank Prof. Ning Gu and Dr Ma Ming for their
support and hosting me for 6 months at Southeast University Nanjing PR
China during the course of my ENDEAVOUR fellowship in 2011. It was a
wonderful experience and I learnt a great deal from the time in your labs.
Similarly, Prof. C.N.R Rao at ICMS Bangalore India provided me with
support and guidance during 6 weeks spent in your labs. I was warmly
welcomed by your students and thoroughly enjoyed learning from your
group.
There are several staff members of RMIT University that have put up
with me for quite some time now and from whom I have learnt many
important skills. Firstly, Mr Phillip Francis and Mr Peter Rummel who put
in many hours training me in the use of the microscopy facilities, also
their entourage of other technical staff and duty microscopists that keep
the Microscopy and Microanalysis facilities operational. This also includes
the technical staff of the School of Applied Science, Mrs Zahra Homan,
Mrs Nadia Zakhartchouk, Mrs Ruth Cepriano-Hall, Mr Howard Anderson,
iii
Mr Karl Lang and Mrs Diane Mileo for your support, assistance and
occasionally looking the other way.
Throughout my PhD I was supported by a network of family and
friends, I ate your food, drank your beer and slept on your couches. I am
forever grateful for the support you gave me during this period. My
parents unwavering backing and that of my brother and extended family
meant a great deal and I appreciate everything that was done to help me
through this time. My circle of friends who looked after me and bought me
drinks and meals when I couldn’t, made a big difference in the course of
my studies. I hope to be able to pay you all back in some form or another
in the years to come.
Lastly I must thank each and every one of the students I had the
pleasure of working alongside. You gave me a wonderful, if a little
distracting at times, working environment. You sat and talked thoughts
through and helped me focus on the right approach while keeping my
mind open to new and better ideas. Some of you have been by my side
since undergraduate and it is from you that all my fondest memories of
my time at RMIT are formed.
iv
This thesis is dedicated to the memory of my grandfather,
Peter Campbell.
And to the enduring light of my grandmother,
Leni Den Boer.
You taught me that a real answer is one that raises more questions and that it’s best not
to live a life with horse blinkers obstructing your view.
v
Table of Contents
Declaration _____________________________________________________ ii
Acknowledgements ________________________________________________ iii
List of figures _____________________________________________________ 6
List of abbreviations ______________________________________________ 10
Abstract _________________________________________________________ 11
1.1.0 Introduction ________________________________________________ 13
1.1.1 Nanotechnology ____________________________________________ 14
1.1.2 Scale matters and matter is scalable ______________________ 20
1.2.1 Nanotechnology and medicine_______________________________ 24
1.2.2 Properties of materials ____________________________________ 29
1.3.1 MRI contrast _______________________________________________ 29
1.3.2 Magnetism on the nanoscale ______________________________ 30
1.4.1 Functioning of MRI _________________________________________ 34
1.5.1 Weighted images ___________________________________________ 39
1.5.2 Contrast__________________________________________________ 39
1.6.1 Types of magnetism ________________________________________ 43
1.7.1 Production methods of magnetite ___________________________ 45
1.7.2 Aqueous co-precipitation __________________________________ 45
1.7.3 Reactions in constrained environments ____________________ 48
1.7.4 Sol-gel ___________________________________________________ 50
1.7.5 Thermal decomposition ___________________________________ 51
1.8.1 Desired characteristics for MR applications __________________ 55
1
1.8.2 Magnetic properties _______________________________________ 55
1.8.3 Bio-distribution___________________________________________ 56
1.9.1 Surface modification of magnetite ___________________________ 59
1.9.2 Dextran __________________________________________________ 60
1.9.3 Carboxylates _____________________________________________ 61
1.9.4 Silica _____________________________________________________ 61
1.10.1 Stöber method of silica growth _____________________________ 64
1.11.1 Cellular targeted nanoparticles ____________________________ 66
1.12.1 Rationale behind this thesis _______________________________ 69
1.13.1 Chapter summary _________________________________________ 70
1.14.1 References ________________________________________________ 72
2.1.0 Instrumental apparatus ____________________________________ 87
2.1.1 Electron Microscopy ______________________________________ 88
2.1.2 Transmission Electron Microscopy ________________________ 88
2.1.3 X-Ray Diffraction _________________________________________ 89
2.1.4 Atomic Absorption Spectroscopy (AAS)_____________________ 91
2.1.5 Magnetic Resonance Imaging ______________________________ 92
2.1.6 Confocal microscopy ______________________________________ 94
2.1.7 Superconducting Quantum Interference Device (SQUID) ___ 96
2.2.1 References _________________________________________________ 98
3.1.0 Silica coated quasi cubic magnetite as a T2 contrast agent ___ 99
3.1.1 Introduction _______________________________________________ 100
2
3.2.1 Materials and methods ____________________________________ 101
3.2.2 Synthesis of iron oxide nanoparticles _____________________ 101
3.2.3 Silica shell formation process ____________________________ 102
3.3.1 Characterisation___________________________________________ 103
3.3.2 T2 relaxation ____________________________________________ 103
3.3.3 Cell uptake ______________________________________________ 104
3.3.4 MRI study _______________________________________________ 105
3.4.1 Results and discussion ____________________________________ 107
3.4.2 XRD_____________________________________________________ 109
3.4.3 SQUID __________________________________________________ 111
3.4.4 Cell uptake ______________________________________________ 112
3.4.5 Cytotoxicity______________________________________________ 114
3.4.6 Relaxivity ________________________________________________ 115
3.4.7 Tissue imaging __________________________________________ 119
3.5.1 Conclusion ________________________________________________ 121
3.6.1 References ________________________________________________ 123
4.1.0 Targeted MR contrast materials ____________________________ 125
4.1.1 Introduction _______________________________________________ 126
4.2.1 Materials and methods ____________________________________ 128
4.2.2 Reagents, chemicals and assay kits ______________________ 128
4.2.3 Synthesis of magnetite particles __________________________ 128
4.2.4 Synthesis of Fe@SiO nanoparticles _______________________ 129
3
4.2.5 Conjugation of Fe@SiO to Herceptin® ____________________ 131
4.2.6 Assessment of Fe@SiO-HER stability _____________________ 131
4.2.6.1 Conjugation of FITC to Fe@SiO@HER _________________ 131
4.2.7 In vitro studies ___________________________________________ 132
4.2.7.1 Cell culture __________________________________________ 132
4.2.7.2 Cytotoxicity studies __________________________________ 133
4.2.8 Cellular uptake __________________________________________ 133
4.2.8.1 Confocal microscopy _________________________________ 133
4.2.8.2 Flow cytometry ______________________________________ 134
4.2.9 Quantification of HER-2 expression ______________________ 135
4.2.10 MR imaging ____________________________________________ 135
4.2.10.1 MR Imaging in vitro _________________________________ 135
4.2.10.2 Mouse Experiments _________________________________ 136
4.2.10.3 MR imaging in vivo __________________________________ 136
4.3.1 Results and discussion ____________________________________ 137
4.3.2 TEM analysis ____________________________________________ 138
4.3.3 XRD analysis ____________________________________________ 140
4.3.4 Herceptin® conjugation __________________________________ 141
4.4.1 In vitro and in vivo studies _________________________________ 144
4.4.2 Cytotoxicity______________________________________________ 144
4.4.3 Uptake specificity ________________________________________ 146
4.4.4 Cell specific uptake ______________________________________ 148
4.4.5 Phantom cell studies _____________________________________ 152
4
4.4.6 In vivo mouse imaging ___________________________________ 154
4.5.1 Conclusions _______________________________________________ 157
4.6.1 References ________________________________________________ 158
5.1.0 Further investigations _____________________________________ 160
5.1.1 Radio frequency induced heating ___________________________ 161
5.1.2 Introduction _____________________________________________ 161
5.1.3 Experimental and Discussion ____________________________ 163
5.1.4 Conclusion ______________________________________________ 167
5.2.1 DMSA coatings ____________________________________________ 167
5.2.2 Surface coating __________________________________________ 168
5.2.3 Experimental and discussion_____________________________ 169
5.2.4 Conclusion ______________________________________________ 173
5.3.1 Chapter Summary _________________________________________ 174
5.4.1 References ________________________________________________ 176
6.1.0 Conclusions and future work ______________________________ 177
6.1.1 Conclusions _______________________________________________ 178
6.1.2 Future work _____________________________________________ 180
7.1.0 Appendix __________________________________________________ 182
7.1.1 List of publications ______________________________________ 182
7.1.2 Peer reviewed journal articles __________________________ 182
7.1.3 Peer reviewed conference proceedings___________________ 183
7.2.1 List of awards ___________________________________________ 184
5
List of figures
Chapter 1
Figure 1: Scale diagram representing the relative sizes of man-made and
biological nanostructures. ____________________________________________________ 16
Figure 2: Comparison of surface area increase with reduction in unit size
increasing surface area. ______________________________________________________ 18
Figure 3: How the physical properties of gold nanoparticles affect the
observed colour with only change in particle size and shape. ___________________ 23
Figure 4: Reduction of energy via domain wall counter alignment. ______________ 32
Figure 5: Neel wall formation between two counter aligned domains. ___________ 33
Figure 6 : Depiction of a spinning charged particle which generates its own
magnetic field perpendicular to the direction of spin. __________________________ 36
Figure 7: The four stages of signal production: (1) nuclei at rest randomly
aligned, (2) alignment by the external magnetic field B 0, (3) excitation by the
RF pulse and (4) relaxation and signal emission. ______________________________ 38
Figure 8: Comparison of T1 (A) and T2 (B) weighted imaging of the same axial
slice of the brain demonstrating the contrast comparison between tissues
using either weighting regime. ________________________________________________ 41
Figure 9: MR image taken with and without contrast agent. The right hand
image shows a haemorrhage as injected contrast agent has leaked from a
broken blood vessel this aids diagnosis of conditions that would otherwise be
unnoticed in the non-contrast enhanced image. _______________________________ 43
Chapter 2
Figure 1: Magnetic excitation cycle of water protons to produce an MR
contrast image. ______________________________________________________________ 93
Figure 2: Confocal microscope arrangement, __________________________________ 95
6
Figure 3: Schematic of the superconducting loop and Josephson Junctions in
a DC SQUID magnetometer. __________________________________________________ 97
Chapter 3
Figure 1: TEM images of the synthesised Mag particles (A) and (B) Mag@SiO 2
particles demonstrating their size and distribution are shown. ________________ 109
Figure 2 XRD patterns of the as synthesised magnetite (Mag) and silica
coated magnetite (Mag@SiO2) particles _______________________________________ 110
Figure 3: SQUID measurement of the as synthesised magnetite particles. The
value shown is the saturation magnetisation of the Mag@SiO 2 particles in
emu/g at 4 Kelvin. __________________________________________________________ 112
Figure 4: Optical microscopy images of PC3 human prostate cancer cells
(control) grown for 24 h. (A) in the absence of nanoparticles (control), and in
the presence of (B) Mag and (C) Mag@SiO 2 nanoparticles followed by three
washings with PBS __________________________________________________________ 114
Figure 5: Biocompatibility of Mag@SiO2 nanoparticles assessed using MTS
assay after their exposure to PC3 cancer cells for 24 h with respect to
different Fe concentration in Mag@SiO2 nanoparticles. ________________________ 115
Figure 6: Evaluation of Mag@SiO2 nanoparticles as a T2 MR contrast agent is
shown in the form of % signal enhancement with increasing concentration of
Fe. A) demonstrates performance in a phantom studies compared to a pure
saline control; while B) presents results obtained after uptake by PC3 cells. ___ 117
Figure 7: T2-weighted MR images of nude mice with breast tumor obtained
(A) before and (B) after injection of MR contrast agent, obtained using a 3
Tesla MR scanner. __________________________________________________________ 120
Chapter 4
Figure 1: TEM image of as synthesized magnetite and FeO@SiO particles. _____ 139
Figure 2: Histogram outlining the particle size distribution of the as
synthesized magnetite particles. _____________________________________________ 139
7
Figure 3: XRD pattern for the as synthesised 17 nm magnetite particles. ______ 141
Figure 4: % of Herceptin lost from the particles after incubation for up to 24
h in varying pH ranging from 5-10 ___________________________________________ 143
Figure 5: % Herceptin lost from particles after incubation for up to 24 h in
human serum concentrations ranging from 25-100% _________________________ 143
Figure 6: % viability of SKBR3 cells after incubation with increasing
concentrations of Fe@SiO@HER particles over a 24 and 48 hour period. _______ 145
Figure 7: Relative surface expression of HER2 receptor between SKBR3,
BT474 and MCF7 as measured by flow cytometery. ___________________________ 147
Figure 8: Confocal microscopy images of SKBR3 cells after incubation with
FITC- labelled Fe@SiO@HER for 3 hours. _____________________________________ 148
Figure 9: Dose response data for SKBR3, BT474 and MCF7 particle uptake
proportional to increasing particle concentration after 4 hours. _______________ 149
Figure 10: Particle adsorption time course for SKBR3, BT474 and MCF7 cells
up to 24h incubation. _______________________________________________________ 152
Figure 11: Bar graph and corresponding MR images showing contrast for
each cell line after incubation with 50 µg/ml Fe@SiO@HER particles. _________ 153
Figure 12: Histological analysis of BALB/c nude mouse tumour, stained with
anti-HER2 antibody, scale bar 1000 µm. _____________________________________ 155
Figure 13: MRI comparison of SKBR3 tumours in mice 4 hours post injection. _ 156
Figure 14: Signal enhancement values calculated via MR readout for the
tumour sites between the three variables. ____________________________________ 156
Chapter 5
Figure 1: TEM images of the prepared particles. A) as prepared TD Mag, B)
TD Mag with 5 nm SiO2 coating, C) as prepared AQ Mag, D) AQ Mag with 5
nm SiO coating. Scale bar 50 nm. ____________________________________________ 164
8
Figure 2: Induction furnace used for heating experiments at Southeast
University Nanjing. __________________________________________________________ 165
Figure 3: Heating rate comparison between the magnetite produced through
aqueous co precipitation (AC Mag) and thermal decomposition (TD Mag)
before and after a 5 nm layer of SiO2 is added. _______________________________ 166
Figure 4: TEM image of the DMSA coated Mag particles. ______________________ 170
Figure 5: Biocompatibility of DMSA coated magnetite particles at increasing
concentrations over 24 and 48 hours. ________________________________________ 171
Figure 6: SQUID data showing magnetisation saturation of DMSA coated Mag
particles.____________________________________________________________________ 172
Figure 7: Relative signal enhancement of DMSA coated magnetite particles
compared to control under phantom imaging parameters. ____________________ 173
9
List of abbreviations
Atomic adsorption spectroscopy
(AAS)
Aqueous co-precipitation magnetite
(AC Mag)
Bicinchoninic acid
(BCA)
Carbon nanotubes
(CNT)
Computed tomography
(CT)
Dimercaptosuccinic acid
(DMSA)
Echo time
(TE)
Epidermal growth factor receptor
(EGFR)
External magnetic field
(B0)
Fetal bovine serum
(FBS)
Hydrofluoric acid
(HF)
Magnetic resonance
(MR)
Magnetic resonance imaging
(MRI)
Mean fluorescence intensity
(MFI)
Net Magnetisation Vector
(NMV)
Nuclear magnetic resonance
(NMR)
Phosphate buffered saline
(PBS)
Poly (D,L-lactic-co-glycolic acid)
(PLGA)
Positron emission tomography
(PET)
Radio frequency
(RF)
Repetition time
(TR)
Reticuloendothelial system
(RES)
Single echo sequence
(SE)
Specific loss power
(SLP)
Superconducting quantum interference device
(SQUID)
Superparamagnetic iron oxide nanoparticles
(SPIONS)
Teteraethyl orthosilicate
(TEOS)
Thermal decomposition magnetite
(TD Mag)
Transmission electron microscopy
(TEM)
Ultrasound
(US)
X-ray Diffraction
(XRD)
10
Abstract
Contrast agents allow for greater detail of specific internal
structures to be obtained under clinical MR imaging. These images are
used to aid in the diagnosis of conditions or damage including tumours
and other cancerous tissues. Gadolinium has for quite a while been used
as a primary material for this purpose but increasing concerns regarding
toxicity are adding pressure to seek alternate materials. Magnetite offers
many properties that would make it an excellent contrast material and
several products have come to market but are restricted in their
application primarily due to particle size.
This thesis aims to approach the problems associated with
magnetite nanoparticles and their use in intravenous systems. This
requires reducing the overall particle size and ensuring biocompatibility.
This was accomplished by modifying the core particle size by altering the
thermal decomposition parameters and surface coating the material with
a layer of silica. A preliminary investigation was made using quasi cubic
magnetite particles to investigate the biocompatibility and performance as
an MR contrast agent. Based on these findings a second core shell
nanoparticle system was formed using modified techniques to reduce the
overall particle size by shrinking the core magnetite and the silica shell
thickness.
These particles were subsequently surface modified with Herceptin®
to facilitate the selective uptake of the particles into specific cancerous
11
cells. The particles were examined for their biocompatibility, structural
stability and selective uptake into SKBR3 cells in vitro and in vivo in live
breast tumour baring mice. All the studies were undertaken in MRI
scanners in use by clinicians at the Peter MacCallum Cancer Research
Centre. This demonstrated that in a clinical setting, these core shell
particles can be made to selectively target specific tumours in significant
number to cause notable localised contrast at the target site.
12
Chapter 1
1.1.0 Introduction
This chapter will serve to introduce the concepts of nanotechnology
and its various applications with a focus on medical imaging techniques.
The literature presented in this chapter should serve as a basis to justify
the research conducted in the subsequent chapters and highlight the
areas of the field that are lacking and require attention.
13
1.1.1 Nanotechnology
Over time, humans as a species have developed ways to manipulate
our environment to suit our needs. This has been possible due to certain
physiological adaptations as well as a seemingly uncontrollable curiosity
that has driven us to develop technologies to enhance our lives. The
development of the ―pointed stick‖ would likely have been among the first
technological developments sought by our ancestors, but even this
simplistic concept requires understanding of the principles and materials
involved. Our ancestors more than likely had experiences in life where
they learned that pointy things were more unpleasant to be poked with
than blunt things (experience). They would have realised that having a
good pointy stick around was beneficial to their survival and set about
collecting pointy sticks for this purpose (requirement). Arguably the best
pointy sticks were long, straight and rigid; sticks such as this would have
been hard to find (value in attributes). However with a little understanding
of the materials and the desired application, a premium pointed stick
could be crafted from a non-premium material (creative solution).
This
process
of
fulfilling
a
need
by
first
increasing
our
understanding of the world, then applying this new knowledge in creative
ways, is the core of the technological development cycle. It is also the only
reason these clawless, unarmoured and quite tasty humans have survived
to this day. This cycle has allowed us to reach a point in our history when
we have the opportunity to develop tools on a scale that may one day
allow us to control the very atomic structures of materials. As we enter
14
this era of ―Nanotechnology‖ we seek to further develop the tools and
understanding of this world below us, in order to create new and
important tools to solve the problems we face as a species.
Nanotechnology refers broadly to all fields of science that seek to
control at least one dimension of a material structure on the nanoscale, in
order to alter its properties or application. This includes physics,
chemistry, electronics, biology and materials engineering, making it a
truly multidisciplinary field. The term ―nanotechnology‖ comes from the
coupling of the Greek prefix ―Nano‖, meaning ―Dwarf‖ and of course the
word ―technology‖; this size prefix denotes a fraction of one billionth
(1/1,000,000,000th). In the case of the unit of length, one ―nanometre‖ is
equal to a billionth of a metre (0.000000001 m). However the term
nanotechnology is generally used to refer to any material that has one
aspect of its structure controlled on the scale of less than 100 nm. It is
often difficult to put this size range into perspective as it is far below the
common units that humans interact with in everyday life. Figure 1 below
is a simplified diagram used to describe the relationship this size range
has to our everyday world, from the diagram we can already see manmade
developments at and below this scale. Yet we still lack the understanding
required to equal or even come close to the complexities of the biological
world, which for the time being remain the only true nanomachines.
15
Figure 1: Scale diagram representing the relative sizes of man-made and biological
nanostructures, image courtesy of Josh Wolfe's report on nanotechnology:
[http://www.forbeswolfe.com]
Richard Feynman worked tirelessly in the field of quantum
mechanics and to this day, his efforts remain as the cornerstones of
modern physics. Yet it was a semi off the cuff after dinner talk given by
him in 1959 entitled ―There’s plenty of room at the bottom‖ that began
courses of inquiry and serious idle conversation into the realms of
―Nanotechnology‖. In the talk he discussed reaching the limits and
physical boundaries of atomic scale fabrication and proposed shrinking
computer components down until internal connects were made of wires
―10-100 atoms in diameter‖.
When Feynman gave his talk, computers still took up whole rooms;
however now we are beginning to reach his milestones with interconnects
16
now being made at less than 30 nm which is equivalent to Feynman’s 100
atom upper limit. What Feynman envisioned was the fabrication of devices
on an ever decreasing scale, until we are able to directly manipulate each
atom of a material so we can arrange them any way we want. This
approach, making ever smaller devices and components from larger
materials, is referred to as the ―top down‖ method and is used extensively
in the lithographic processes to make computer chips. However, since he
gave his talk there have been discoveries in biology of machines that
already operate at this scale. Biology has become recognised as a field full
of nanotechnology, where incredibly complex systems and machines are
fabricated by molecular sized pumps and engines that researchers are
beginning to understand and reprogram [1-4]. The method used by
biology, by which it forms larger much more complex structures using
small building blocks, is termed the ―bottom up‖ approach.
The simplest advantage nanomaterials have is that of surface area,
a solid 1 cm3 block has a surface area of 6 cm2, if this block is subdivided
into 1 mm cubes; the same volume now has a surface area of 60 cm2. If
you divide further into the nanoscale and make that same 1 cm3 block out
of 1 nm cubes, the available surface area of that volume climbs to
60,000,000 cm2 (Figure 2). This dramatic change in surface area to
volume ratio can affect many aspects of a material properties including
chemical reactivity [5], electrical conductivity [6-8], magnetic properties
[9],
strength,
ductility
and
melting
temperature
[10-12].
Certain
phenomena also begin to manifest in nanomaterials that are not observed
17
in the bulk such as quantum confinement and surface plasmon
resonance [5, 9]. These features make investigating nanomaterials a high
priority for just about every major industry and scientific field. The fruits
of these early investigations have already begun to be born and more than
just hints of interesting materials and their properties have been
uncovered.
Figure 2: Comparison of surface area increase with reduction in unit size increasing
surface area. Image courtesy of the United States National Nanotechnology Initiative [13]
Carbon nanotubes (CNT’s) for example have many useful properties
such as high tensile strength and flexibility [14, 15], electrical properties
[16-19] and hydrophobicity [20-22] which can be applied to many fields.
For example, the semiconductor industry has been steadily improving the
number of transistors that can be fabricated onto a single photo
lithographically printed chip; approximately adhering to Moore’s law of a
doubling of computer power every 2 years. However, it has been accepted
18
that the current methods of fabrication will hit limitations in minimum
feature size once 22 nm feature sizes are obtained. This is not due to
simple fabrication restrictions, which although technically difficult, sub
22 nm features can be obtained; but due to quantum tunnelling, short
channel effects and passive power dissipation occurring as a consequence
of how the materials begin to behave on this scale [23, 24]. The
exceptional
electrical
properties
of
CNT’s
have
been
extensively
investigated in attempts to overcome this problem. Bundles of, or single
CNT’s can be used to replace the channel material used in a transistor,
because CNT’s have strong covalent C-C bonding in the sp2 region, they
remain chemically inert yet capable of conducting large amounts of
electrical charge ballisticly down their quasi 1D structure [25-27]. Their
properties can be tuned by altering the chirality of the tubes [28, 29],
surface or terminal coatings [30, 31], for use in solar cells [32-34],
conductive films [35, 36], textiles [37, 38], oscillators [39, 40] and even
massive scale applications such as creating a space elevator using CNT’s
to construct the cables [41]. ―The space elevator will be made about 50
years after everyone stops laughing‖ Arthur C. Clark. As such, CNT’s are
demonstrating wide reaching applications with very little change in their
structure demonstrating the multifunctionality of a single tool produced
by nanoscience.
For many years, biologists have been working with nanotechnology;
all primary biological systems operate on the nanoscale to some degree.
But it is only in the last few decades the functionality of the pumps and
19
motors that operate at the molecular scale have begun to be understood.
These molecular machines are still out of our power to produce yet every
year more and more is understood about their functioning. However
researchers have been able to begin exploiting bio mimicry and
reappropriation of naturally occurring nanomachines for other purposes,
such as protein kinesin being used as a ―linear walker‖ after extraction
and adhesion to a track surface with a supply of ATP to drive the required
conformational changes [42, 43].
Motors that spin the flagella on bacteria, proton pumps that
selectively pump ions across membranes, the functioning of ribosomes as
they transcribe RNA into proteins, are all demonstrations of true
nanomachines. They demonstrate exquisite precision in their function and
operate with extreme efficiency. Only recently have researchers been able
to begin to fabricate structures on the same scale and although we are
beginning to realise some of the benefits of nano engineered materials in
biology, we are still a long way from designing our own motors, pumps
and protein folders. However we have begun to develop understanding of
how material properties change once particular attributes such as size
and shape are constricted on the nano scale.
1.1.2 Scale matters and matter is scalable
Although much technological advancement has been made by
reducing the size of components, computers being a prime example, mere
20
size reduction is only half the story. In order to fully appreciate the
possibilities and potential of materials engineered on the nanoscale, an
understanding of quantum effects is required. Quantum theory was
developed as a means of describing the behaviour of matter as it departs
from classical mechanics on the atomic and subatomic scale. Although
defining quantum theory to a subset of behaviours, probabilities and
properties of subatomic systems is well outside the realm of this thesis,
one aspect ―Spin‖ will be discussed later in sections 1.3.2 & 1.4.1.
However, what does need to be understood is that once a material is
reduced in size to below certain limits, these effect can begin to manifest
as useful properties that aren’t exhibited in the ―bulk‖ state. This change
is due to the size range being comparable to the De Broglie wavelength of
the charge carriers within the material. Various physical phenomena such
as, conductance, melting point, fluorescence and magnetic permeability
are directly linked to their charge carrier wavelength [44-48]. As such,
once the particle size is reduced to less than that of the charge carrier
wavelength, the wavelength begins to exist in a constricted state and
whatever property that charge imparts can shift. This means that at this
scale morphology becomes increasingly important and that by changing
the particle size, shape, spacing and dielectric conditions, we can actively
manipulate the properties of the material for our needs.
One
clear
example
of
this
change
in
properties
in
metal
nanoparticles is the visible colour shift when the particle size confinement
begins to affect the surface plasmon properties of the surface. Similar to
21
the ―particle in a box‖ principle, the oscillations in the coherent charge
density at the interface between the metal and dielectric layer begin to
blue-shift with decreasing size once below a critical size of about a few
nanometers and is clearly observable with gold nanoparticles. This
property of gold has been known for an exceptionally long time and lead to
the use of gold particles in creating stained glass windows back as far as
the 4th century AD [49, 50]. However it has only been recently that this
property of gold has become understood and that the colour shift is
caused by the surface plasmon of the material. Within metallic materials,
the free electrons often depicted as a ―sea of free electrons‖, have a natural
standing wave frequency of oscillation. However the electrons on the
boundary between the edge of the material and either another material or
vacuum, oscillate with a separate standing wave that runs parallel to the
interface. When light interacts with the surface of a bulk metal, it typically
undergoes elastic interaction with the electrons forming the metallic
bonds and is reflected with only a very small portion being absorbed. This
is the reason most metals in their pure form have a white lustre, as all
frequencies are reflected from the surface.
However if the relative permittivity of the material is such that the
surface plasmon frequency is below that of the interacting light; the
dielectric function of the free electrons transitions from negative
(reflecting) to positive (transmitting). This results in there being an upper
limit for the frequency of reflected light, in the case of gold and copper,
this occurs within the visible light spectrum. The upper frequencies of the
22
incident light are transmitted and the reflected light spectrum is reduced,
resulting in the gold and red colours of those metals. In bulk systems, the
surface plasmon resonance is fixed, based on the atomic properties of the
material. However in nanoparticle systems, the surface plasmon frequency
is confined to the outer layer of the individual particle. Therefore, by
reducing the particle diameter, the surface plasmon resonance can be
―shifted‖ and different transmission spectra can be observed. In this way,
the visible colour of a material can be ―tuned‖ simply by changing the
particle size as demonstrated in Figure 3 below [51].
Figure 3: How the physical properties of gold nanoparticles affect the observed colour
with only change in particle size and shape. Sánchez-Iglesias, A., I. Pastoriza-Santos, et
al. [51]
Tuning material properties by nanoscale manipulation has allowed
for nanomaterials to be applicable to a plethora of fields and applications.
Such as semiconductors [52-55], medical diagnostics [52, 56-59],
23
biosensors [52, 58, 60, 61], information storage [62], single electron
transistors [52, 63-65], solar cells [52, 66, 67], cell tracking [52, 68-71],
energy storage [72-75], environmental protection [76] and smart surfaces
[77-79], to name only a handful. It is difficult to appreciate how a single
advancement can affect so many aspects of our lives but it is becoming
much clearer to both the research and lay community that a great change
in what we understand about the world has given rise to some very useful
tools.
"Every industry that involves manufactured items will be impacted
by nanotechnology research. Everything can be made in some way
better—stronger, lighter, cheaper, easier to recycle—if it’s engineered and
manufactured at the nanometre scale." -- Stan Williams, Director of
Quantum Science Research, HP Labs.
Although there are many aspects to consider in the field of
nanomaterials; this thesis will focus one specific area, magnetic resonance
imaging (MRI) contrast agents. The following sections will introduce the
topic of MRI and how nanotechnology can play a part in its advancement.
1.2.1 Nanotechnology and medicine
Given the wide variety of nanoparticles and nanomaterial systems
being researched, it is no surprise that much attention has been placed
on
medical
applications
given
their
broad
material
properties.
Nanomaterials can positively benefit medical applications both passively
and actively. Passive interaction is caused by a nanomaterials basic
24
properties
being
beneficial,
such
as
the
antimicrobial
and
anti-
inflammatory properties of silver nanoparticles [80-82]. As such, silver
nanoparticles have found direct application areas such as treatment of
minor cuts and grazes; burns and ulcers, and general wound healing that
would otherwise require topical antiseptic treatment [83-85].
Active nanoparticle applications take advantage of the size and
surface modification of the materials to create specific structures that
actively enhance the materials properties and application, such as hollow
capsules for delivering drugs or other payloads [86]. Nanoparticles or
capsules are typically 5-400 nm in diameter, which makes them small
enough to behave as delivery systems for the much larger (≈10 µm) human
cells. The ability to tailor materials on such an extreme scale allows us to
begin to mimic the ways in which the human body has been working for
hundreds of thousands of years.
Our immune system uses a highly specific set of protein markers to
identify healthy, infected, old or young cells. Now that researchers can
manipulate materials on this scale, we can use the same system to target
and transport therapeutics, toxins or signal molecules directly to specific
cells or tissues increasing efficacy and decreasing side effects and toxicity
[87]. Polymeric nanocapsules have found many applications in this area
given their biocompatibility, controllable synthesis and ease of surface
modification [88-91]. Poly (D,L-lactic-co-glycolic acid) (PLGA) particles
have been used to deliver drugs such as antipsychotics, insulin, anti-
25
inflammatories, chemotherapeutic and anti-cancer drugs to target tissues
[92, 93].
Typically chemotherapy drug doses are more effective as they
increase, however the highly toxic nature of chemotherapy drugs causes
extensive systemic toxicity and side effects. Therefore dosage must be
balanced between damage to tumour cells vs damage caused to healthy
cells via the treatment. This often extends the period patients have to
undergo therapy as the doses must be kept low, which increases
likelihood of tumour cells becoming resistant to the chemotherapy drugs.
However
polymer
effectiveness
in
capsules
made
transporting
from
PLGA
chemotherapy
have
agents
demonstrated
such
as
9-
nitrocamptotecin, cisplatin and paclitaxel directly to tumour sites which
reduces the systemic toxicity of the drugs allowing for higher doses with
reduced side effects, increasing efficacy [92, 93].
Vaccine delivery is another area where polymer nanoparticles have
found application. The same systems for drug delivery can be utilised for
vaccines, by carrying the target surface proteins and stimulating the
immune system to elicit the desired response. Polymer nanoparticles have
demonstrated the ability to trigger both specific and non-specific immune
responses to a desired target [94]. By stripping molecular markers from
target cells and binding them to polymeric nanoparticles; researchers have
been able to initiate an immune system response to cells that would
otherwise escape the detection process [95].
26
The requirements of vaccine delivery are such that larger particles
(sometimes greater than 1 µm) are required because they are readily taken
into macrophages and dendritic cells where they can transmit their
markers to the immune system for cataloguing [95, 96]. Smaller particles
have also demonstrated effectiveness in providing immunogenicity [97,
98]. Reddy et al. demonstrated that immunogenicity was increased when
the particles were able to freely circulate in the lymphatic system for an
extended period due to the decreased particles size. Once internalised into
dendritic cells and macrophages, the target molecules can be transferred
while the carrier particle is easily degraded [99-101]. The versatility of
polymer nanoparticles makes them excellent vectors for vaccine delivery
due to their controllable size, ease of surface coating and biodegradability.
Vaccine and drug delivery systems take advantage of the molecular
properties of nanomaterials to evade the reticuloendothelial system (RES),
target specific cell types and deliver drugs. However these nano vectors
can transport molecular probes to tissues that can aid in the formation of
images for positron emission tomography (PET), computed tomography
(CT) and magnetic resonance (MR) imaging by exploiting the physical
properties of nanomaterials. This was originally suggested by Weissleder
in 1999 when they deemed that the expanding understanding of
molecular mechanisms of disease and their relevant biomarkers could be
combined with advances in medical imaging [102]. Their group was even
able to demonstrate this principle in the pre-clinical setting [103, 104].
27
By combining molecular markers with nanoparticle probes, the
spatial resolution was able to be increased for imaging modalities such as
MR, CT and ultrasound (US). These techniques have a high spatial
resolution and are typically used to diagnose disease prior to imaging with
techniques such as PET or optical imaging, which although highly
sensitive, possess low spatial resolution. It would be considered more
effective if the more highly sensitive nuclear imaging techniques could be
used routinely, however they fall short on several aspects. Exposure to the
ionising radiation used to create the PET image causes damage to both
patient cells and to the sensitive molecular probes being used. This limits
the maximum exposure time and duration of biological processes that can
be imaged. This greatly reduces usefulness for targeted molecular imaging
as quite often longer scanning times may be required to deliver the probe
to the target.
For
these
reasons
combined
imaging
modalities
have
been
developed such as PET/CT and PET/MRI which have greatly enhanced
performance compared to either technique individually. MRI demonstrates
a higher spatial resolution than CT, hence PET/MRI produces the better
combination and is under clinical development. For all these modalities,
molecular markers and probes are required to produce high quality
images. The continual development and implementation of nanomaterials
for these applications remains an integral part of further improving
imaging diagnostics.
28
1.2.2 Properties of materials
Much like our stick wielding cousins, we can see how our experience
that magnetic materials can offer advantages in the application of MR
contrast; has driven our growing interest in the fundamentals of their
design. Due to our advancements in imaging modalities, we now have a
requirement to develop related technologies to improve that which we have
already developed. From our collective research, we have ascertained
many of the behaviours of magnetic particles at this scale. While
attempting to apply this knowledge to MR contrast, we see specific value
in several selected features. This allows us to produce a creative solution
using the knowledge we have amassed about biology and the physical
world, to fulfil the needs of the application. In this way we are still
sharpening sticks, just to a much finer scale and the cycle of technological
development rolls on unchanged.
1.3.1 MRI contrast
Nanofabrication advancements have led to the reliable production of
nanoparticles in a size range that can interact with cells by being
adsorbed, binding to the surface, or carrying signal molecules to target
regions [105-108]. Vast amounts of resources have been allocated to the
further development of nanoparticle systems with biological applications
due to the immense number of medical and research opportunities for
29
targeted delivery, activation or tracking systems [109, 110]. MR imaging
offers a large amount of detail regarding the internal tissues of the body
however is somewhat limited in its ability to differentiate tissues under
some circumstances without the aid of an external contrast agent. These
contrast agents modify the way in which the MR signal is produced from
the tissues in which it is present and due to the magnetic requirements,
are often made from or include metals in their composition.
The magnetic properties of the materials remain the most critical
aspect of an MR contrast agent; however a nanoparticle behaves quite
differently to the way in which a chelated Gd contrast agent exploits its
electron pairs to generate a magnetic dipole. This is due to its generally
greater size than a conventional contrast agent; however this opens up a
small window to exploit the way in which the magnetic properties of
materials change on the nanoscale.
1.3.2 Magnetism on the nanoscale
A material’s magnetic properties stem from the spin of electrons
within the atoms that make up the material. The spin of the electron, both
around the nucleus (angular component) and the rotational spin
(quantum component) coupled with the –ve charge results in a magnetic
dipole moment which forms a small magnetic field. In materials that have
conducive crystal structures, these dipoles can line up and form much
larger dipoles whose field is equal to the sum of its parts [111].
30
Magnetic domains are present in many metals, only they exhibit
little or no net magnetic field due to the equal counter alignment of their
many domain polarities. This can be altered by firmly striking a metal rod
causing an imbalance in electrons and inducing a temporary dipole, or
passing the metal through a static magnetic field to force alignment of the
domains (rubbing a nail on a magnet). Similarly, striking or heating a
permanent magnet contributes energy to the crystal and allows for
domains to form and counter align reducing the net magnetic field of the
material [112].
The domain wall is the physical solution to reducing the
magnetostatic energy of aligned dipoles [113]; although forming the wall
requires energy referred to as ―exchange energy‖, the energy reduction it
grants is greater than the energy consumed in its formation [114]. In some
materials the domains do not counter align enough to completely reduce
the net magnetic field. This is because in highly crystalline materials there
is often an ―easy direction‖ of magnetisation that runs parallel to one of
the crystal planes, this results in the familiar ferromagnetic properties of
fridge and bar magnets. Domain walls require both energy and space to
form; this is one aspect that can be exploited by the nano-engineering of
magnetic materials.
Magnetic domains exist as a result of energy minimisation of
primarily the exchange and anisotropy energies between individual dipoles
[112-114]. A single domain material has a very high magnetostatic energy
associated with it, however if the uniform alignment of the dipoles is
31
broken up into localised domains that allow for flux closure between
poles, the magnetostatic energy of a system can be reduced (Figure 4).
These domains are bordered with zones of gradually rotating dipole
alignments (Figure 5) [111]. Figure 5 illustrates the gradual change in
dipole direction upon formation of a Neel wall between domains. This
leads to a defined width of the domain wall, which is governed by
competition between the exchange and anisotropic energies.
Figure 4: Reduction of energy via domain wall counter alignment.
The exchange energy causes the wall thickness to increase, as it
decreases with less angular difference between neighbouring dipoles [114].
While the anisotropic energy increases as the dipole is rotated further
away from the easy axis, this causes reduction of domain wall thickness.
32
The competition between these two forces balance within a material to
form domain walls of defined thickness based on both the crystallographic
and magnetostatic energy of the material [114]. This results in a critical
particle size of approximately 15-30 nm where a highly crystalline Fe3O4
nanoparticle can simply not have enough diameter to allow a domain wall
to form [115, 116]. This stops the particle from reducing its magnetostatic
energy
by
forming
multiple
domains
and
maintains
a
higher
electromagnetic unit per gram (emu/g) value than multi domain magnetic
materials.
Domain wall
Zones of
opposing
polarity
Figure 5: Neel wall formation between two counter aligned domains.
Instead, at this size range the dipole can ―flip‖ randomly while under
no external field influence [117]. In this way the net field of adjacent
33
particles sums to zero as their dipoles cancel out, minimising their energy.
This is termed superparamagnetisim, whereby a material exhibits no
magnetic behaviour until an external field applied and all the dipoles
within the particle set align and produce a high net field [117].
Superparamagnetic iron oxide nanoparticles or (SPIONS) demonstrate very
little coercivity and return to approximately 0 emu/g once an external
field has been removed. This is highly advantageous for biological and
industrial applications as the particles do not aggregate due to intrinsic
ferromagnetic behaviour, hindering dispersion or cell uptake. Therefore by
manipulating the crystal structure and particle size within the nanoscale,
the magnetic properties of a common material can be tuned for advanced
applications.
1.4.1 Functioning of MRI
MRI imaging has become a very powerful and widely used imaging
technique for visualising internal organs and structures within the body.
The instrument exploits the quantum nature of certain atoms within
tissues in our bodies. Specifically hydrogen nuclei, both due to their
abundance and that their solitary, positive, spinning proton gives them a
relatively large net magnetisation vector (NMV) [115, 116].
With regard to quantum mechanics, spin is one of two types of
angular momentum a particle can exhibit. Orbital angular momentum
relates the particle to another point of reference and is often generalised
34
similarly to angular momentum in classical mechanics (L=r p), while spin
has no analogue in the classical sense and can be generalised as an
individual particle rotating on its own axis. This generalisation was first
proposed
by
Wolfgang
Pauli
but
was
not
properly
named
and
conceptualised until 1925 by Ralph Kronig, George Uhlenbeck and
Samuel Goudsmit [118]. Pauli then revisited the idea in 1927 and set
about establishing the appropriate mathematical framework regarding
spin which was then used by Paul Dirac in 1928 to derive his relativistic
quantum mechanics [119, 120].
Spin is a fundamental characteristic of elementary and composite
particles as well as atomic nuclei. The idea that it represents a value of a
particle rotating on its own axis is correct in so far as the spins do obey
the laws outlined by the principles of quantized angular momenta,
however there were other properties of spin that set it apart from this
description. Firstly, the direction of a spin can be changed but it can
never increase or decrease its speed. Secondly, the spin of a charged
particle is associated with a magnetic dipole moment with a G-factor that
relates the magnetic moment back to the angular momentum and
quantum number of the particle. It is this magnetic dipole moment that is
taken advantage of in the processes required to create an image using an
MRI scanner. Any particle that has a net charge and is in motion will
produce a magnetic field; this effect can be attributed to any atomic
nucleus with an odd mass number (Figure 6). As these nuclei are both
charged and in motion, they exhibit a net magnetic field as stated by the
35
laws
of
electromagnetism
[116].
Therefore
these
nuclei
can
be
manipulated by application of a static external magnetic field (B0) and an
RF pulse to produce MR images of tissues within the body [115].
Figure 6: Depiction of a spinning charged particle which generates its own magnetic field
perpendicular to the direction of spin.
When B0 is applied, the magnetic moments of the hydrogen nuclei
align parallel and anti-parallel with the applied field (Figure 7 panel 2)
[115, 116]. This ―alignment‖ and ―anti-alignment‖ condition is determined
by several factors. Given that the quantum physical properties of the
nuclei are considered in terms of electromagnetic radiation (packets of
energy as opposed to waves) there are only two energy states the nuclei
can obtain ―Low‖ and ―High‖; the proportions of the nuclei in these states
depends on the thermal energy levels of the nuclei [116].
36
Nuclei in the Low state will align parallel with the external field,
while the High energy nuclei will align in the anti-parallel direction until
their energy is overcome by the strength of the externally applied field. As
snap freezing patients prior to imaging tends to be looked down upon by
sticklers in the health industry, clinical scanners emphasise higher field
strengths to overcome the majority of the thermal energy contribution
from the patient [116, 121]. However due to thermal equilibrium, there are
always more nuclei in the low energy state prior to alignment. As the
excess of aligned nuclei overcome the NMV of the anti-parallel nuclei in
the high state, a net moment in alignment with the field is produced
proportional to the difference between the two populations. This becomes
the NMV of the patient and is used to generate the MR signal.
With the net moments of the hydrogen nuclei aligned along the
same axis, an external RF pulse of precise frequency can be applied to the
nuclei to cause the excitation and relaxation required to produce the
signal measured by the scanner. If the RF pulse applied to the nuclei is
equal to the Larmor frequency of the particle; the pulse is in resonance
with the nuclei and an energy transfer can occur (Figure 7 panel 3) [115].
It is this transfer of energy that causes the magnetic moments to shift
away from the lower energy state of alignment with the external field to
the excited state. Once the pulse has been switched off, the nuclei quickly
begin to return to their ground state of alignment with the external field.
During this process, the energy adsorbed from the RF pulse is reemitted and detected (Figure 7 panel 4), while the magnetic moments of
37
the nuclei return to alignment due to de-phasing [116]. This process
produces what is termed ―T1 recovery‖ as the nuclei give up their energy
to their immediate environment during spin lattice relaxation. The nuclei
recovery rate is exponential and referred to as the ―T2 relaxation time‖. T2
relaxation or ―spin-spin‖ relaxation results from nuclei exchanging energy
with adjacent nuclei [115]. This process is outlined in the diagram below.
Figure 7: The four stages of signal production: (1) nuclei at rest randomly aligned, (2)
alignment by the external magnetic field B0, (3) excitation by the RF pulse and (4)
relaxation and signal emission.
With the B0 and tissue temperature being static, the RF pulse power
and duration becomes the critical factor in the imaging process [116]. The
38
repetition time (TR) controls the gap between pulses and available time for
the nuclei to relax before the application of the next pulse for individual
slices of the scan, which is measured in milliseconds (ms); TR determines
T1 relaxation [116]. The echo time (TE) represents the required time from
the pulse input to the peak of output signal which determines the amount
of transverse magnetisation decay that occurs between pulses, hence the
amount of T2 relaxation which is also measured in ms [116].
1.5.1 Weighted images
1.5.2 Contrast
Contrast within an image is defined as having both saturated bright
and dark areas of no signal. Between these extremes, intermediate shades
occur and make up the bulk of the image. Within each slice of the image,
the NMV can be segregated into differing vectors from various tissues in
the patient, discerning the difference between fat, muscle, cerebrospinal
fluid and water. High signal is produced by the tissue having a large
transverse magnetism component which results in a bright signal.
Conversely, low signal is generated from tissues that have a small
transverse component; typically the two extremes of this principle are fat
and water [116].
Water is hydrogen directly linked to oxygen which strongly attracts
electrons away from the hydrogen atom making it more susceptible to the
39
effects of B0, while fat is made up of hydrogen linked to carbon in large
lipid molecules. In the case of fat, the carbon doesn’t attract the electrons
away from the hydrogen as strongly, rendering the hydrogen more
protected from the effects of B0 by its electron cloud [116]. Subsequently
hydrogen in fat recovers more quickly than hydrogen in water and both
differ in brightness in MR images. There are three mechanisms used to
produce contrast within MR imaging, T1 recovery, T2 decay and
proton/spin density (total number of protons per unit volume).
MR images can be ―weighted‖ differently; this means that the
parameters can be changed so that different tissues appear bright or dark.
A T1 weighted image relies on the difference between the T1 times of fat
and water [116]. T1 imaging employs a shorter TR between pulses and
yields more signal from fat, creating bright spots for fatty tissues. In T2
weighted imaging, the TE is the defining signal that determines contrast;
the TE is longer in water than it is in fat and as such produces more
signal and bright areas high in water content. Hence either a T1 (fat bias)
or T2 (water bias) image can be produced by changing the imaging
parameters [116] (Figure 8).
40
Figure 8: Comparison of T1 (A) and T2 (B) weighted imaging of the same
axial slice of the brain demonstrating the contrast comparison between
tissues using either weighting regime.
T1 and T2 imaging modes can be useful when detecting certain
pathologies within a tissue. Tumour sites tend to have higher water
concentration than surrounding tissues and as such appear bright under
T2 weighted imaging, allowing the site to be identified from other
structures in the tissue. There are however many pathologies that are
difficult to identify without the aid of an external contrast enhancement
agent, that affects either the T1 or T2 characteristics of the tissue.
Contrast agents function based on dipole-dipole interactions and
magnetic susceptibility [116]. In general, contrast agents have large
magnetic moments that affect the local magnetic fields of protons [115].
Magnetic susceptibility is a fundamental property of matter and reflects
the extent to which the nucleus can be magnetised by the application of
41
an external magnetic field. ―Enhancement‖ of a T1 weighted image is
achieved by reducing the T1 relaxation time of nearby protons,
brightening the nuclei in the presence of the agent. While as T2 contrast is
based on shortening the T2 decay time, this can be achieved by a material
that has a highly positive magnetic susceptibility [115].
Contrast in this case, is increased by reducing the signal from
protons under influence of B0, darkening the area [115]. An example of
these agents is the paramagnetic gadolinium based agents for T1 and
ferromagnetic substances such as Ferridex™ for T2 weighted contrast.
These materials are used to further enhance the contrast produced during
imaging of tissues or pathologies that would otherwise be difficult to
distinguish, such as tumours, infection, infarction, inflammation or post
traumatic lesions (Figure 9) [115, 116]. The magnetic properties of
common contrast agents can fall under several different classifications,
paramagnetic, superparamagnetic and ferromagnetic.
42
Figure 9: MR image taken with and without contrast agent. The right hand image shows
a haemorrhage as injected contrast agent has leaked from a broken blood vessel this aids
diagnosis of conditions that would otherwise be unnoticed in the non-contrast enhanced
image. Image courtesy of http://www.modernfaqs.com [4]
1.6.1 Types of magnetism
Paramagnetic materials result from unpaired electrons within an
atom, these unpaired electrons produce a very small magnetic moment
around the atom; however these moments orient randomly and cancel
each other out. While in the presence of B0, the moments align and
produce a bulk NMV and express a positive magnetic susceptibility,
enhancing the strength of the local field. Gadolinium (Gd) is an ideal
paramagnetic contrast agent as it has seven unpaired electrons that cause
a decrease in T1 and T2 relaxation times in surrounding atoms [123].
Unfortunately Gd in its elemental form is highly toxic and requires
chelating compounds to reduce its potentially toxic effects. Although Gd
chelates are commercially available and commonly used, there have been
43
concerns regarding chelate disassociation post injection causing toxicity.
Links between Gd contrast agents and the deaths of patients due to
nephrogenic systemic fibrosis has been reported in patients who received
Gd chelates while suffering kidney damage [124].
Superparamagnetism is similar to that of ferromagnetism /
ferrimagnetism but only occurs in sufficiently small nanoparticles. If a
ferromagnetic material such as iron is confined to a small enough size, its
magnetisation direction can flip randomly due to the influence of
temperature. While these random flips are occurring, no net field is being
produced. If an external field B0 is applied, these magnetic moments can
align and produce a much stronger magnetic susceptibility than
paramagnetic materials. Several types of superparamagnetic agents are
being researched or are available for clinical use, such as magnetite,
Fe3O4; maghemite, γ-Fe2O3 ; and haematite α-Fe2O3; as Feridex, Sinerem
and Magnevist respectively, however none of these are available in
Australia currently.
Fe is a common element found in our bodies and is able to be
broken down through an excess iron biological excretion pathway [125].
However, due to technological limitations the size of these iron oxide
particles has limited their usage to gastrointestinal lumen, liver and
spleen imaging. This is due to the particles being too large to remain in
blood circulation long enough as they are rapidly removed by first pass
metabolic processes [126, 127]. Recently these technical limitations are
being overcome and new iron based magnetic particles are being produced
44
using method capable of controlling size and surface chemistry [128-131].
Tailoring the surface chemistry is an important step in increasing both
biocompatibility and allowing possible molecular targeting for cell specific
applications. This can be greatly beneficial as MRI already has
exceptionally high spatial resolution, but the ability to target certain
pathologies with contrast agent, makes this diagnostic tool far more
attractive [102].
1.7.1 Production methods of magnetite
1.7.2 Aqueous co-precipitation
The co-precipitation method for forming Fe based nanoparticles is
the simplest and generally most effective chemical route. Originally
developed by Massart in 1981 [132], his process yielded very poor
monodispersity but was performed without the use of stabilisation
molecules. Now refined and using stabilising molecules, this method can
be
used
to
fabricate
either
Fe3O4 or
γFe2O3 particles by
aging
stoichiometric ratios of ferrous and ferric salts in an aqueous medium at
relatively low temperatures (60-120°C).
Fe2+ + 2Fe3+ +8OH- → Fe3O4 + 4H2O
(1)
Equation 1 demonstrates the basic principle by which this process
yields product, given the thermodynamics of this reaction, precipitation
45
should occur within the pH range of 8-14 with a molar ratio of Fe3+/Fe2+
being 2:1 in a non-oxidising environment [133]. The Fe3O4 produced from
this reaction remains unstable and can be readily oxidised into
maghemite in the presence of oxygen as shown in equation 2.
Fe3O4 + 2H+ → γFe2O3 + Fe2+ + H2O
(2)
As such, the process must be carried out in an oxygen free
environment to avoid hematite or maghemite formation [134]. Maghemite
forms as a result of oxidation and has been found to have reduced
magnetic properties. Therefore oxidation should be avoided while
pursuing magnetic product with a high emu/g value [134]. Magnetic
properties of metals depend largely on crystal structure and as magnetite
and maghemite differ on their spinel structure their magnetic properties
vary. Magnetite fully occupies the octa and tetrahedral sites, while
maghemite possesses cationic vacancies in the octahedral site lowering
symmetry within the structure [135].
This difference in symmetry gives rise to the different magnetic
properties of the two materials. The two oxide states are difficult to
identify using techniques such as XRD given their exceptionally similar
patterns therefore sample purity is difficult to determine [136]. As
maghemite is expected to form to some extent during any process to
produce magnetite, reported values for magnetisation saturation between
the two materials tends to vary but generally measurements of bulk
materials yield values of ≈ 90 emu/g-1 for magnetite and ≈ 65 emu/g-1 for
maghemite [136].
46
Fe3O4 particles are crystalline structures and in general, grow in
accordance to the rules and behaviours of crystal formation systems.
These rules suggest that for homogenous particles to be produced, two
distinct phases of particle formation must be separated, nucleation and
growth. Usually the nucleation phase begins as particle seeds precipitate
out of a supersaturated solution, the solution concentration drops as a
result and the precipitation ceases, leaving a set amount of nanoparticle
seeds. The growth phase occurs as solutes diffuse from the solution to the
surface of the seeds, keeping these two phases separate and controlled
generally leads to higher monodispersity within the particles produced
[137].
The
co-precipitation
method
can
yield
large
quantities
of
nanoparticles fairly efficiently (approx. 50% yield). This is advantageous
for imaging applications as large quantities are required for human
administration; however as kinetic factors are the only controls, the
particle size distribution is difficult to tailor and reproduce. For this
reason, when highly monodisperse particles are required this method is
superseded by others. Due to the production process, nanoparticles
fabricated via the co-precipitation method incorporate polymers, lipids or
dendrimers onto their surfaces which impart particle stability and
reduced aggregation. These surface coatings also present various
attachment sites for later modification for targeting applications.
47
1.7.3 Reactions in constrained environments
Constrained environment reactions are similar in principle to coprecipitation processes, except that these processes involve using either
synthetic or biological nanoreactors to add additional control to the
process via direct physical confinement. These methods were developed in
an attempt to improve the monodispersity of the particle product. This
process has been demonstrated using amphoteric surfactants in nonpolar solvents to create water swollen reverse micelles [138-144] and
phospholipid vesicles using FexOy particles as supports [145, 146]. An
added advantage of this process is that the molecules used to restrict and
control the growth of the particles, may also act later on as a surface
coating; providing stability or desirable surface characteristics.
Controlling the morphology of the fabricated particles using this
method can be achieved primarily by modifying the reactor shell.
Amphiphillic molecules such as lipids have opposing polar charges on the
molecule (hydrophilic/polar head – hydrophobic/non-polar tail). This
causes
spontaneous
formation
of
micelle
or
liposome
structures
depending on the solvent [147]. The final spherical structures arise as a
stacking issue between the broad head and narrow tail, resulting in a
spherical formation. Given that the curvature of the structure is
proportional to the head width vs. tail length ratio, modifying these
characteristics of the lipids gives control of overall reactor size.
These reaction encapsulation principles have also been investigated
using dendrimers and cyclodextrins [148, 149]. Similar to amphiphilic
48
molecules, dendrimers have head and tail groups, but also possess a
branching side chain. In most examples, dendrimers are added during the
magnetic particle synthesis to form a surface coating that infers stability
in water. Magnetic materials produced in this way are referred to as
―magnetodendrimers‖ and have found applications in several medical
fields such as MR imaging and cell tracking [129]. The polymer coating in
this case has a high affinity for cellular membranes due to its surface
charge, resulting in a high rate of non-specific particle uptake [150-152].
Lipid molecules can be used to form a bi-layer based liposome to
encapsulate the nanoparticle after formation. Lipids usually have a polar
head group and two fatty acid chains forming the hydrophobic tail, iron
oxide
particles
encapsulated
in
this
manner
are
referred
to
as
magnetoliposomes [152]. Lipid bilayers are highly functional given their
ability to simply add specific molecules to the outer membrane for either
cellular targeting, reducing RES clearance or drug loading directly onto
the lipid surface [135].
Various other materials such as PEG have been used to reduce the
overall diameter of the magnetic particles and to prevent further oxidation
[153].
PEG
is
known
to
maintain
particle
stability
by
reducing
intermicellar interactions by minimising the interfacial free energy of the
core, preventing aggregation [154]. The increased circulation time of
particles using polymeric molecules is attributed to the large hydrophillic
corona formed around the core which prevents opsonin adsorption,
49
reducing the visibility of the particles to phagocytes, decreasing clearance
by the liver and spleen [155].
The primary advantage of reverse micelle or nanoreactor fabrication
techniques is that a wide variety of shapes and surface features can be
formed by variations in surfactant/co-surfactant or reaction conditions
[156]. While these materials have demonstrated excellent capabilities, they
remain relegated to pre-clinical testing. It is most likely due to the inability
to
produce
the
large
amounts
of
material
required
for
human
administration due to difficult synthesis methods.
1.7.4 Sol-gel
Given
the
success
of
constrained
environment
fabrication
techniques, it is no surprise that the sol-gel process has also been applied
to iron oxide nanoparticle formation. Similarly, the sol-gel method uses
two distinct phases (in this case liquid-solid) to control the reaction
kinetics of the growing particles [157, 158]. Due to the nature of the
reaction process, the nanometric particles (sol) are formed within a
restrictive matrix of the supportive ―gel‖, as the reactions are commonly
performed at room temperature; later heat treatments are required to
refine the crystalline state of the material.
The kinetics of the sol gel process are primarily governed by solvent
temperature, pH and concentration of salt precursors [159-161]. γFe2O3
particles with a size range between 6-15 nm have been fabricated via this
route [162]. This process does offer some advantages, such as the
50
products structure being highly controllable and determined by reaction
conditions. Sol- gel techniques also yield monodisperse materials with
good control over particle diameter, with the flexibility to embed the
product into an inert matrix such as silica during the fabrication process
[163, 164]. However the multi stage fabrication steps, environmental
dependencies, including the high temperature heat treatment (up to
400°C), make it a cumbersome method to quickly establish a reproducible
protocol for good yield.
1.7.5 Thermal decomposition
Given
the
requirement
for
product
monodispersity,
high
temperature thermal decomposition methods have been investigated [135,
165-167].
The
principle
employed
is
the
decomposition
of
an
organometallic compound in a high boiling point solvent in the presence of
a stabilising agent. The high temperatures required to begin to decompose
the metal salts allow for precise control of the nucleation and growth
phases of particle formation [135]. While the final morphology of the
particles can be modified by altering ratios of the starting reagents and
duration of high temperature aging process [168]. This process was
inspired by the use of thermal decomposition to produce high quality
semiconductor crystals in in organic media [169, 170].
A wide range of metal precursors are able to be utilised such as
acetylacetonates, metal cupferronates, carbonyls [171] as well as simple
51
non-toxic metal chlorides [172]. This method can also be utilised to
produce metallic nanoparticles which are of interest in areas such as data
storage due to their higher net magnetisation compared to their oxide
counterparts [173]. Butter et al. produced polymer stabilised metallic Fe
particles ranging from 2-10 nm by the thermal decomposition of [Fe(CO)5]
in the presence of polyisobutene under a nitrogen atmosphere [173]. The
particles remained exceptionally stable in water, however were still
susceptible to oxidation given the change in susceptibility measurements
along with the increase in particle size [168].
Notably, in 2004 Park et al. published a concise method to produce
gram quantities of spherical magnetite nanoparticles with an exceptionally
narrow size distribution using thermal decomposition of a metal-oleate
[165]. This process used a two-step synthesis in which the metal oleate
was first produced using a non-toxic precursor such as iron chloride and
sodium oleate instead of using precursors such as pentacarbonyls which
have been found to be potentially toxic [174].
The process involved thermal decomposition of the iron-oleate
complex in the presence of oleic acid in 1-octadecene as a solvent at
320°C. The monodispersity of the product produced was attributed to the
considerable enthalpy gap between the nucleation and growth phases.
These processes occurred separately as a result of the disassociation of
one of the three oleate ligands at 200-240°C by CO2 elimination which
triggered the nucleation phase. The remaining two oleate ligands remained
attached until 300°C, after which the main growth phase initiates. This
52
process produced 5–22 nm spherical particles after 30 min of aging at
320°C, increasing the aging duration caused a shift in morphology to
hexagonal/cubic particles [165]. Unfortunately the particles produced in
this manner remain capped with oleic acid and as such remain
hydrophobic. In order to use these materials in biological systems, post
fabrication techniques are required to infer the appropriate surface
characteristics for solubility in water.
The thermal decomposition method is also able to produce particles
of differing morphology. Dumestre et al. utilised thermal decomposition to
produce cubic nanostructures with controllable size and shape. Their
group achieved this by decomposing Fe(N(Si(CH3)3)2)2 and H2 when
combined with oleic acid or hexadecylammonium chloride in the presence
of hexadecylamine at 150°C [175]. They established that variations in the
ratio between the acid ligand and amine slightly altered the edge length of
the particles from 7 - 8.3 nm. The group also found that the cubic
structures formed crystalline superlattices with aligned axis whose
interparticle spacing varied (1.6 – 2 nm) in relation to the edge length of
the individual particles forming the lattice [175]. Similar particle tuning
abilities were also reported by Puntes et al. in the fabrication of cobalt
nanodisks by thermal decomposition of cobalt carbonyl in the presence of
linear amines [176]. Nickel and cobalt nanorods can also be formed using
this technique as demonstrated by Dumestre et al. They demonstrated
that not only could they produce cobalt nanorod structures by
decomposition of [Co(η 3-C8H13)( η4-C8H12)] but that these particles would
53
self-assemble
into
superlattices
when
formed
in
the presence
of
trioctylphosphane oxide [177, 178]. All the fabrications methods are
summarised below in table 1.
Synthetic
method
Synthesis
Reaction
period
Solvent
Size
distribution
Shape
control
Yield
Coprecipitation
Easy,
ambient
conditions
Complicated,
ambient
conditions
Minutes
Water
Moderately
narrow
Poor
High
scalable
Hours
Organic
compound
Moderately
narrow
good
Low
Hydrothermal
Simple, high
pressure
Hours
Days
Water
Ethanol
Very narrow
Very
good
Moderate
Thermal
decomposition
Complicated,
inert
atmosphere
Hours
Days
Organic
compound
Very narrow
Very
good
High
scalable
Microemulsion
Sol-gel
Table 1: Comparison of advantages/disadvantages of the differing magnetite nanoparticle
fabrication techniques.
Given the requirements for MRI contrast agents to have high net
magnetisation, narrow size distribution, small size, high product yield and
biocompatibility, we felt that the method outlined by Park et al best suited
our needs [165]. A post fabrication step would be required to infer the
appropriate solubility in water however the requirement of this additional
step was outweighed by the overall physical properties and high yield this
method offered.
54
1.8.1 Desired characteristics for MR
applications
1.8.2 Magnetic properties
The MR imaging process exploits the magnetic interaction of nuclei
with an external magnetic field; contrast enhancement is achieved by a
material that alters this interaction and is fundamentally based on the
magnetic properties of that material [116]. Materials that exhibit
superparamagnetisim offer some of the best magnetic properties for this
application, given their effective ―switching‖ between magnetic and nonmagnetic states with the application of an external field [116, 179].
They have also shown the ability to possess the high saturation
magnetisation values required to generate a larger net effect on
surrounding
protons
during
the
imaging
process.
The
saturation
magnetisation is measured in (emu/g), materials with higher emu/g
values cause a reduction in T1 and T2 relaxation rates [131]. Therefore, a
material with higher emu/g value will increase the contrast locally on a T2
weighted image. A wide range of emu/g readings have been reported for
magnetite nano particles, most range from 30-50 emu/g which is lower
than the 90 emu/g recorded for bulk magnetite [168, 180].
Three main factors are involved in contributing to the magnetisation
saturation of magnetite nanoparticles, these are particle size, crystal
55
structure and particle spacing [181]. The particle size and crystal
structure varies between fabrication conditions and process used, how
these factors affect the magnetisation saturation of the particles has been
discussed previously. However particle spacing is dependant almost solely
on post fabrication coatings that act to reduce the interaction between the
individual magnetic domains. By separating the particles, each unit is
able to behave as an individual domain that aligns with the applied field
rather than an aggregate of domains with a net field [180].
Magnetite particles produced by thermal decomposition possess
these attractive properties, however, without a secondary surface material,
are unable to be applied to biological applications due to their nonfavourable behaviour within the biological environment. For magnetite
particles to be applicable to biological applications they must also possess
desirable biological properties such as low toxicity and high particle
stability in biological media [179]. To achieve this, a surface coating is
required to infer these properties to the particles; surface coatings also
offer attachment sites for targeting or labelling molecules [182]. Magnetite
surface coatings are commonly organic compounds that isolate the core
from the external environment [123, 135, 183].
1.8.3 Bio-distribution
Bio-distribution of a material or compound is exceptionally
important for any nanomaterial with medical applications. There are
56
different bio-distribution requirements for various applications, such as
specific site localisation in the case of targeted MR contrast or systemic
distribution for certain pharmacological compounds. Each requirement
has its own challenges presented by the mammalian body.
Intravenously administered MR contrast media must be able to both
localise at a desired site, to provide the relative contrast, while avoiding
uptake and excretion by the RES. This uptake results from the quick
response of phagocytes that rightly identify the injected particles as
foreign bodies and begin consuming the particles via phagocytosis [184,
185]. However, given that the highest amount of phagocytes are found in
the liver (approx. 90%), this behaviour while detrimental to distributing
the particles to other parts of the body can be used to condense large
amounts of contrast agent within the liver to aid in MR imaging of the
problematic organ [186].
Other areas of the body also naturally take up large numbers of
particles due to phagocyte activity and can subsequently enhance their
relative contrast under MR imaging such as the spleen, lymph nodes and
bone marrow [187]. This process of phagocytic removal is proportional to
both the surface properties and size of the particles with larger and more
highly charged particles being removed faster [126].
Particle size has been found to have a considerable effect on biodistribution. In general it has been established that 90% of particles with
a diameter greater than 60 nm are removed from the blood by
phagocytosis occurring in the liver after 30 minutes [125, 188]. Whereas
57
organs such as the lungs, brain and kidneys show minimal or nondetectable amounts of iron oxide nanoparticles [188]. This distribution
behaviour can be altered by reducing the particle size and altering the
surface coating of the materials [126, 188]. If the application requires that
the injected particles localise at another part of the body other than the
liver, spleen or bone marrow; size and surface modifications are required
that enable the particles to evade the RES long enough to complete their
function. An increase in the blood half-life of iron oxide particles in
relation to both size and surface coating has been reported by several
groups [189, 190]. Overcoming the initial RES response allows particles to
begin to localise at different parts of the body and opens the door for
target specific localisation of particles throughout the body. It was found
that increasing the blood half-life of particles caused distribution to move
to areas such as the kidneys, spleen, lungs, heart instead of %90 uptake
within the liver [131, 191].
One key aspect of iron oxide nanoparticles is their biocompatibility,
as iron is an essential element required by the body it can be adsorbed
and broken down by normal metabolic cycles. The particles coalesce in
the liver for approximately 3 days and 4 days in the spleen [125, 131,
191]. The particles are stored in the lysosomes of the phagocytes that
consumed them until they are broken down and are assimilated by the
normal iron metabolism and incorporated into the haemoglobin of
erythrocytes [131].
58
1.9.1 Surface modification of magnetite
Given the nature of the MR process and the properties of magnetic
nanomaterials, an abundance of applications has begun to be investigated
within the field. Contrast enhancement, targeted imaging, dual imaging
and therapeutic delivery have all been investigated using various
nanoparticle systems [89, 91, 95, 127, 186, 192, 193]. Bio-medical
applications require attentive detail regarding their biological interface, for
both colloidal stability within biological systems and toxicity.
Surface properties of nanomaterials can greatly vary their possible
application; as well as imparting excellent colloidal stability. Without a
suitable surface coating, some iron oxides are hydrophobic, therefore in
water based solutions, large clusters and agglomerates form due to the
hydrophobic interactions between particles [194]. This process produces
clusters with a strong magnetic dipole – dipole attraction which begin to
exhibit ferromagnetic properties as the cluster size increases [194]. With
an increase in cluster size, the single domains of the individual particles
are lost and the net magnetisation of the material is reduced [195]. Given
these factors, surface coating of the magnetic particles is imperative in
order to maintain their efficacy for biological applications. Given the wide
range of potential applications for magnetic nanoparticles, many surface
coatings have been investigated for uncoated particles to enhance their
desirable properties. As several surface coatings such as dendrimers and
lipids have already been discussed, only a brief description of three
common surface coating techniques will be covered in this section.
59
1.9.2 Dextran
Dextran is a polysaccharide composed of α-D-glucopyranosyl units
that can be modified in both its length (1000 – 2,000,000 Da) and may
contain branches formed from its α-1,3 linkages. Dextran has been
commonly used to reduce blood clotting in surgery, as a parenteral
nutrition source, lubricant in eye drops and to apply osmotic pressure to
biological molecules. It has been found to be exceptionally biocompatible
while having a long blood circulation time [182]. The dextran layer is
degraded in the body by intracellular dextranases within macrophages; in
general this occurs to the greatest degree within Kupffer cells and is
excreted in the urine [196]. As the iron oxide is incorporated into the
body’s iron store [197], the combination of the iron oxide core and
biocompatible surface coating is very advantageous. Dextran sulphate has
been applied as a surface coating to promote particle uptake by
macrophages, given its interaction with the membrane receptor (SR-A)
[198]. This property can be exploited to aid in imaging atherosclerotic
plaques as the nanoparticle laden macrophages are known to be first
responders to plaque deposits [199, 200]. For these reasons, dextran
coated magnetic particles have been able to pass industry regulation
standards and have translated into products available on the market.
Some
examples
of
these
are
carboxydextran
coated
magnetite,
Ferucarbotran (Resovist® Japan and Europe) and dextran coated
magnetite and ferumoxides (Endorem® – Europe, Feridex® in the USA
and Japan).
60
1.9.3 Carboxylates
Carboxylates have been utilised to stabilise the surface of
nanoparticles due to their solubility in aqueous media. The coating
process described by Sahoo et al. results in one or two of the carboxylate
groups co-ordinating to the magnetite surface dependant of environmental
pH of the reaction. This leaves at least one of the three carboxylate groups
free to interact with the solvent; this group carries the surface charge that
infers the desired hydrophilicity and stability [201]. The availability of the
carboxylate surface charge provides anchorage sites for additional
molecules for labelling or targeting such as antibodies or fluorescent dyes.
Another commonly used carboxylate coating is dimercaptosuccinic
acid (DMSA). Similarly to citric acid the conjugation occurs through
available carboxylic groups, however the overall stabilisation of the
particle is a result of intermolecular disulphide crosslinking between
surface ligands [181, 202]. DMSA coating provides biocompatibility,
stabilisation and both carboxylic acid and thiol groups available on the
surface
for
further
functionalization
with
desired
molecules
or
components [202]. DMSA surface coatings will be discussed further in
chapter 5 section 5.2.1
1.9.4 Silica
Silica or (silicon dioxide) is an oxidised metalloid of silicon, found
abundantly in nature, in its native form it is almost chemically inert.
61
Stöber was the first to produce silica spheres in the micron range [203],
these materials were subsequently used in abrasives and ceramic
additives. Further refinements to the process yielded greater control of
particle diameter and surface morphology. This opened up applications in
biology using the particles as drug carriers and template materials for
fabrication of nano capsules [86]. However silica can also be used to coat
the surface of iron oxide nanoparticles contributing several beneficial
properties [204]. The physical barrier produced by the silica coating
provides protection from the short range dipole interactions produced by
the magnetic moments of surrounding magnetic particles [204].
This is due to the magnetic dipole interaction being proportional to
1/(distance between dipoles)3 [205]. Without the silica coating the
particles would aggregate due to interactions with biological media and
this would lower their exchange energy whilst under an external field. The
silica coating also infers a negative surface charge which allows particle
dispersions up to high particle densities ranges [206]. These properties
have been attributed to strong, short range Stern layer forces, produced
by (OH¯) groups on the silica surface which render the surface of each
particle negatively charged [206].
The negative charges present on the surface reduce particle
interaction due to electrostatic repulsion. These forces have been found to
influence a range of up to 4 nm into the solvent and are strong enough to
reduce the dispersion interaction between particles [207, 208]. These
silanol groups can be functionalised to allow biomarkers to be tethered to
62
the surface, either by using covalently bonded coupling agents or
electrostatic forces to adhere molecules [209].
Silicon dioxide has also been demonstrated to be biocompatible
[210, 211] and as a result, a silica coated iron oxide product has been
developed and brought to market for gastro intestinal imaging under the
name Gastromark® [210, 212]. This biocompatibility has also widened its
biological applications to gene transfection agents [213, 214], drug
delivery systems [215, 216]. SiO2 is also highly chemically stable even in
the presence of strong acids such as nitric and sulphuric acid.
Hydrofluoric acid (HF) is one of the few acids capable of dissolving SiO2
and for this reason it has been used as a protective/sacrificial layer in the
lithographic process of the semiconductor industry. The abilities of
selective removal, chemical and physical stability also make SiO2 an
excellent template material for nanoparticle synthesis.
The main routes of silica surface coating stem from the original
Stöber process [203], in which a silica precursor such as teteraethyl
orthosilicate (TEOS) in water and ethanol undergo a base mediated
hydrolysis to form micrometre sized spherical silica particles. A more
detailed description of this process is given in section 1.8.5.
This process was later extended to particle coating by using the
desired core particles as a template for the hydrolysis product of silica.
Using this technique, silica shell thickness can be tuned so as to maintain
size uniformity within the product. Coating thicknesses varying between
2-100 nm have been reported [183, 217] without additional stabilising
63
molecules or surfactants. Therefore product can be easily centrifuged from
the growth solution without the need for complicated washing steps.
Another common methodology for silica coating iron oxide particles
is using the particles as seeds for a microemulsion based process similar
to the method established by Massart [132]; initially used to grow the iron
oxide particles in constrained environments [144, 218-220]. However this
process is only capable of yielding polydisperse particles and the
surfactants
used
require
extensive
washing
steps
to
ensure
biocompatibility [221, 222]. This makes them more difficult to translate to
the clinical setting due to volume requirements of the application.
1.10.1 Stöber method of silica growth
Given that this thesis will investigate the application of the Stöber
method in the coating of iron oxide nanoparticles, a more detailed
description of the process and its subsequent application to particle
coating will be discussed here.
The Stöber method first described by Werner Stöber in 1968 was
used to produce monodisperse spherical SiO2 particles and has since been
improved to reproducibly fabricate particles ranging from 50 – 2000 nm in
diameter [203]. The process consists of hydrolysing the silyl ether present
on a silicate ester, commonly tetraethyl orthosilicate (TEOS), in a mixture
of alcohol, water and ammonia to produce particles. There are two
64
conflicting models used to describe the growth process of the SiO2
particles, each with seemingly proven experimental grounding. The La Mer
model describes the process as monomer addition, in which nucleation
occurs rapidly during the initial stages, after which the particle diameter
increases as growth becomes the dominant reaction [223-225]. This offers
one model to describe the homogeneity of the particles produced by the
process. An alternate theory is called controlled aggregation and differs in
that it describes the growth of the silica particles as the result of
aggregation of primary particles, and that final diameter is not only
determined by reaction rates, but also by the size and colloidal stability of
the primary particles [226-228].
SiO2 grown in this manner has demonstrated the ability to have its
deposition controlled both in rate and structure, giving rise to the ability
to create defined nanostructures with solid or porous morphologies [86].
The surface of the silica surface also presents an external negative charge
in the form of silanol (Si—OH–) which offer electrostatic attraction and
attachment sites to positively charged molecules. Specific adsorption
allows for certain molecules to be adsorbed onto the surface or into the
pores of a silica structure which can later be released or combined with
additional molecules in a ―layer by layer‖ approach [229]. If the silica
offers no more functionality it can simply be selectively removed by
etching in HF or potassium hydroxide to leave behind the polymers
templated by the silica creating, structures with application in drug
delivery, micro-reactors and lab on chip devices, [230-232].
65
1.11.1 Cellular targeted nanoparticles
Cell specific nanoparticle internalisation is a research area just
coming
out
of
its
infancy
with
a
range
of
groups
publishing
demonstrations of the principle [233-238]. The process hinges on isolating
a surface marker present on the target cells but one not expressed on the
surface of others, or at least expressed to a far lower extent. Conveniently,
some cancerous tissues have a tendency to over express certain surface
proteins, making them viable targets for antigen modified nanoprobes.
Breast tumours are typically classified by histo-pathological grade,
type and stage; these factors are used to determine the prognosis and
viable treatment pathways such as chemo, adjuvant or immunotherapy.
The tumour types are classified based on the epidermal growth factor
receptor (EGFR) family of tyrosine kinase receptors which contains four
distinct groups, human epidermal growth factor 1 (EGFR/HER1), HER2,
HER3, and HER4 [239].
These receptors are expressed in many different tumour types to
different levels. HER2 has been extensively studied given its prevalence on
the surface of particularly aggressive tumours that have poor prognosis
[239]. In the case of many of these aggressive cell types, HER2 is
overexpressed several orders of magnitude greater than in non-tumorous
cells. This overexpression can be used as a marker to identify these cells
from their surroundings, an attribute that has been exploited for
66
treatments such as Herceptin® [240-242]. Herceptin® is a tumour specific
immunotherapy used in adjuvant and neoadjuvant treatments; it has
demonstrated the ability to stimulate the immune system to attack HER2
expressing tumours; however some patients begin to develop resistance to
the treatments.
Herceptin® was approved by the FDA in 1998 for the treatment of
HER2 positive breast cancers. Herceptin® is a monoclonal antibody
developed to target the extracellular domain of the HER2 surface protein
which was later humanised for clinical use [243]. Although the HER2
receptor is expressed by normal cells, it is expressed up to 100 times more
in many cancerous cells [244]. This large expression variation is the basis
of the success of the Herceptin® treatment, however little is known about
the exact method of action the Herceptin® uses to cause cell death in the
cancerous cell. The consensus of the literature indicates that Herceptin®
functions by binding and being subsequently internalised via receptor
mediated endocytosis. After this phase it is thought to cause cell cycle
arrest by down regulating HER2 which leads to a decline in cell
proliferation, suppression of angiogenesis and cell death. However this
mode of action is also an Achilles heel of the treatment, treating the
cancer in this specific way does put additional pressure on the cancerous
cell population to reduce the number of HER2 surface proteins, which is
the action specifically driven by the treatment. This results in an increase
in cancerous cells that express lower levels of HER2 and thus avoid
treatment. For this reason the treatment is usually used in combination
67
with chemotherapy where a synergistic effect has been observed when
used in conjunction [245].
Although Herceptin® performs as a cancer treatment in its own
right, its ability to specifically target the surface of certain cancerous cell
lines can be utilised as a targeting moiety for a nanoprobe. The targeting
ability of the Herceptin® can be harnessed by conjugating it to the surface
of a nanoprobe which is subsequently internalised by the cancerous cells.
These guided nanoprobes can serve several functions ranging from drug
carriers, contrast enhancement and functional particles such as induction
heating active materials [246-252]. However few of the applications have
developed far enough to be applied in the clinical environment. The most
basic of these applications involves coating a magnetic or radioisotope
labelled nanoparticle in conjunction with Herceptin® to enhance the local
contrast of MR or PET at cell specific locations.
Currently several iron oxide based materials have been applied to
targeted MR contrast, however the materials used lack the physical
attributes required to be clinically useful. This is mostly due to the
particle size being too large, thus causing rapid clearance from the blood
by the RES. The current materials also suffer from poor homogeneity and
low magnetisation, hence large dosages are required in order to achieve
contrast [253-255].
Several studies have successfully demonstrated the adhesion of
Herceptin® to iron oxide based nanoparticles for targeted imaging.
However in each case limitations of the materials have held the product
68
back from advancing towards clinical studies such as small production
scale, low magnetisation or reduced contrast enhancement [256]. One
study by Chen et al. [256] showed that the magnetite particles possessed
a low magnetisation saturation value and even though they successfully
targeted HER2 expressing cells, the particles were only able to yield a
meagre MR contrast. A similar finding was published by Hilger et al. [257]
that demonstrated only a 20% contrast enhancement at the tumour site
with a dosage of 20 mg iron particles per kg body weight. The mass
magnetisation value of the materials used seems to be the dominant
problem amongst many studies which prevents adequate signal at
clinically useable dosages. In one study that overcame this problem, Huh
et al. [258] demonstrated production of magnetite particles which were
subsequently surface modified with Herceptin® to enable cell specific
targeting. Although they were able to produce homogenous magnetite
crystals with a mass magnetisation value of 80 emu/g, this required small
scale
synthesis
with
several
size
separation
phases
to
ensure
homogeneity. In this case the fabrication complexity was the limiting
factor in moving the material forward into clinical trials.
1.12.1 Rationale behind this thesis
With a lack of iron oxide based contrast products available for
clinical use, there remains a reliance on Gd based agents for many
contrast applications. As understanding of potential Gd risks develops,
replacement materials are being sought. This thesis aims to demonstrate
69
that iron oxide can be used as a viable contrast agent capable of
specifically targeting certain cell lines by using surface coatings to
overcome the shortcomings of the material. Disadvantages such as
removal from the circulatory system due to particle size and low
magnetisation saturation can be overcome by production and surface
coating techniques, while particle yields can be increased using improved
production techniques.
The fabrication of two differing types of iron oxide based contrast
agents will be investigated with their subsequent performance as an MR
contrast agent in vitro and in vivo under clinical conditions, examined. The
applicability of an improved version of this material for targeted cellular
uptake will also be examined in chapter 4 under the same conditions.
1.13.1 Chapter summary
This thesis contains 7 chapters which are organised in such a way
as to first outline the subject area before beginning to discuss the
experiments and results for the work conducted. Chapter 1 was to serve
as in overarching introduction to the field and some specifics relating to
the use of nanoparticles in MR contrast.
Chapter 2 deals with the underlying principles of the instruments
used during the characterisation and experimentation phases of the
investigation.
This
chapter
primarily
70
serves
to
ensure
proper
understanding of the instrumentation so that the reader is able to clearly
understand the data presented in chapters 3, 4 and 5.
Chapter 3 presents the fabrication, characterisation and application
of quasi cubic silica coated iron oxide nanoparticles as an MRI contrast
agent. this chapter presents this material as an initial investigation into
the performance of such materials for both biocompatibility and clinical
MR contrast performance.
Chapter 4 builds on the materials discussed in chapter 3 and
investigates the performance of a smaller silica coated iron oxide particle
for targeted imaging of SKBR3 breast tumours. Again the material is
characterised and its performance measured under clinical conditions,
this time targeting the tumour site after intravenous injection.
Chapter 5 deals with some extensions of the work presented in
chapters 3 and 4. Induction heating performance of the material
discussed in chapter 4 is investigated for hyperthermia applications after
a brief introduction to the field. A DMSA surface coating replacing the
silica coating of the quasi cubic particles presented in chapter 3 is also
discussed, along with biocompatibility and MR contrast performance data.
Chapter 6 simply presents a summary of conclusions drawn based
on the body of the thesis chapters. While chapter 7 contains an appendix
of the publications produced during the course of my PhD studies.
71
1.14.1 References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Bustamante C, Chemla YR, Forde NR, Izhaky D. Mechanical processes in
biochemistry. Annual Review of Biochemistry 2004,73:705-748.
Tsunoda SP, Aggeler R, Yoshida M, Capaldi RA. Rotation of the c subunit
oligomer in fully functional F1Fo ATP synthase. Proceedings of the National
Academy of Sciences 2001,98:898-902.
Smith DE, Tans SJ, Smith SB, Grimes S, Anderson DL, Bustamante C.
The bacteriophage [phis]29 portal motor can package DNA against a large
internal force. Nature 2001,413:748-752.
Astumian RD. Thermodynamics and Kinetics of a Brownian Motor.
Science 1997,276:917-922.
Kolanski KW. Surface Science. 1 ed: John Wiley & Sons, Ltd; 2012.
Du X, Skachko I, Barker A, Andrei EY. Approaching ballistic transport in
suspended graphene. Nature Nanotechnology 2008,3:491-495.
White CT, Todorov TN. Carbon nanotubes as long ballistic conductors.
Nature 1998,393:240-242.
Yanson AI, Bollinger GR, van den Brom HE, Agrait N, van Ruitenbeek JM.
Formation and manipulation of a metallic wire of single gold atoms.
Nature 1998,395:783-785.
Khanna SN, Castleman AW. Quantum Phenomina in Clusters and
Nanostructures. 1 ed: Springer; 2003.
Lu L, Shen YF, Chen XH, Qian LH, Lu K. Ultrahigh strength and high
electrical conductivity in copper. Science 2004,304:422-426.
Treacy MMJ, Ebbesen TW, Gibson JM. Exceptionally high Young's
modulus observed for individual carbon nanotubes. Nature 1996,381:678680.
Paul DR, Robeson LM. Polymer nanotechnology: Nanocomposites. Polymer
2008,49:3187-3204.
Initiative NN. What's so special about the nanoscale. In; 2012.
Walters DA, Ericson LM, Casavant MJ, Liu J, Colbert DT, Smith KA, et al.
Elastic strain of freely suspended single-wall carbon nanotube ropes.
Applied Physics Letters 1999,74:3803-3805.
Yu M-F, Files BS, Arepalli S, Ruoff RS. Tensile Loading of Ropes of Single
Wall Carbon Nanotubes and their Mechanical Properties. Physical Review
Letters 2000,84:5552-5555.
Liang W, Bockrath M, Bozovic D, Hafner JH, Tinkham M, Park H. Fabry Perot interference in a nanotube electron waveguide. Nature
2001,411:665-669.
Frank S, Poncharal P, Wang ZL, Heer WAd. Carbon Nanotube Quantum
Resistors. Science 1998,280:1744-1746.
An KH, Kim WS, Park YS, Moon JM, Bae DJ, Lim SC, et al.
Electrochemical Properties of High-Power Supercapacitors Using SingleWalled Carbon Nanotube Electrodes. Advanced Functional Materials
2001,11:387-392.
Niu C, Sichel EK, Hoch R, Moy D, Tennent H. High power electrochemical
capacitors based on carbon nanotube electrodes. Applied Physics Letters
1997,70:1480-1482.
Yang J, Zhang Z, Men X, Xu X, Zhu X. Thermo-responsive surface
wettability on a pristine carbon nanotube film. Carbon 2011,49:19-23.
72
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
Lee CH, Johnson N, Drelich J, Yap YK. The performance of
superhydrophobic and superoleophilic carbon nanotube meshes in wateroil filtration. Carbon 2011,49:669-676.
Benito SP, Lefferts L. Wettability of carbon nanofiber layers on nickel foils.
Journal of Colloid and Interface Science 2011,364:530-538.
Koh M, Mizubayashi W, Iwamoto K, Murakami H, Ono T, Tsuno M, et al.
Limit of gate oxide thickness scaling in MOSFETs due to apparent
threshold voltage fluctuation induced by tunnel leakage current. Electron
Devices, IEEE Transactions on 2001,48:259-264.
Vahala KJ. Quantum box fabrication tolerance and size limits in
semiconductors and their effect on optical gain. Quantum Electronics, IEEE
Journal of 1988,24:523-530.
Li HJ, Lu WG, Li JJ, Bai XD, Gu CZ. Multichannel Ballistic Transport in
Multiwall Carbon Nanotubes. Physical Review Letters 2005,95:086601.
Javey A, Qi P, Wang Q, Dai H. Ten- to 50-nm-long quasi-ballistic carbon
nanotube devices obtained without complex lithography. Proceedings of
the National Academy of Sciences of the United States of America
2004,101:13408-13410.
Kreupl F, Graham AP, Duesberg GS, Steinhögl W, Liebau M, Unger E, et
al. Carbon nanotubes in interconnect applications. Microelectronic
Engineering 2002,64:399-408.
Louie S. Electronic Properties, Junctions, and Defects of Carbon
Nanotubes. In: Carbon Nanotubes. Edited by Dresselhaus M, Dresselhaus
G, Avouris P: Springer Berlin Heidelberg; 2001. pp. 113-145.
O'Connell MJ, Eibergen EE, Doorn SK. Chiral selectivity in the chargetransfer bleaching of single-walled carbon-nanotube spectra. Nat Mater
2005,4:412-418.
Li HM, Cheng FO, Duft AM, Adronov A. Functionalization of single-walled
carbon nanotubes with well-defined polystyrene by "click" coupling.
Journal of the American Chemical Society 2005,127:14518-14524.
Pulikkathara MX, Kuznetsov OV, Khabashesku VN. Sidewall covalent
functionalization of single wall carbon nanotubes through reactions of
fluoronanotubes with urea, guanidine, and thiourea. Chemistry of
Materials 2008,20:2685-2695.
Kamat PV. Meeting the clean energy demand: Nanostructure architectures
for solar energy conversion. Journal of Physical Chemistry C
2007,111:2834-2860.
Wang X, Zhi L, Muellen K. Transparent, conductive graphene electrodes
for dye-sensitized solar cells. Nano Letters 2008,8:323-327.
Zhu Y, Murali S, Cai W, Li X, Suk JW, Potts JR, et al. Graphene and
Graphene Oxide: Synthesis, Properties, and Applications. Advanced
Materials 2010,22:3906-3924.
Watcharotone S, Dikin DA, Stankovich S, Piner R, Jung I, Dommett GHB,
et al. Graphene-silica composite thin films as transparent conductors.
Nano Letters 2007,7:1888-1892.
Wu ZC, Chen ZH, Du X, Logan JM, Sippel J, Nikolou M, et al.
Transparent, conductive carbon nanotube films. Science 2004,305:12731276.
Dalton AB, Collins S, Munoz E, Razal JM, Ebron VH, Ferraris JP, et al.
Super-tough carbon-nanotube fibres - These extraordinary composite
fibres can be woven into electronic textiles. Nature 2003,423:703-703.
Hu L, Pasta M, La Mantia F, Cui L, Jeong S, Deshazer HD, et al.
Stretchable, Porous, and Conductive Energy Textiles. Nano Letters
2010,10:708-714.
73
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
Bachtold A, Hadley P, Nakanishi T, Dekker C. Logic circuits with carbon
nanotube transistors. Science 2001,294:1317-1320.
Sazonova V, Yaish Y, Ustunel H, Roundy D, Arias TA, McEuen PL. A
tunable
carbon
nanotube
electromechanical
oscillator.
Nature
2004,431:284-287.
Wang Z, Ciselli P, Peijs T. The extraordinary reinforcing efficiency of
single-walled carbon nanotubes in oriented poly(vinyl alcohol) tapes.
Nanotechnology 2007,18:455709.
Hess H, Bachand GD, Vogel V. Powering Nanodevices with Biomolecular
Motors. Chemistry – A European Journal 2004,10:2110-2116.
Ernst K-H. Molecular motors: A turn in the right direction. Nature
Nanotechnology 2013,8:7-8.
Balandin AA, Ghosh S, Bao WZ, Calizo I, Teweldebrhan D, Miao F, et al.
Superior thermal conductivity of single-layer graphene. Nano Letters
2008,8:902-907.
Reuse FA, Khanna SN. geometry, electronic-structure, and magnetism of
small ni-n (n=2-6, 8, 13) clusters. Chemical Physics Letters 1995,234:7781.
Andriotis AN, Lathiotakis N, Menon M. Magnetic properties of Ni and Fe
clusters: a tight binding molecular dynamics study. Chemical Physics
Letters 1996,260:15-20.
Shu Q, Yang Y, Zhai Y, Sun D, Xiang H, Gong X-g. Size-dependent Melting
Behavior of Iron Nanoparticles by Replica Exchange Molecular Dynamics.
Nanoscale 2012.
Sharma PK, Jilavi MH, Nass R, Schmidt H. Tailoring the particle size from
μm→nm scale by using a surface modifier and their size effect on the
fluorescence properties of europium doped yttria. Journal of Luminescence
1999,82:187-193.
Wagner FE, Haslbeck S, Stievano L, Calogero S, Pankhurst QA, Martinek
P. Before striking gold in gold-ruby glass. Nature 2000,407:691-692.
Turkevich J. Colloidal gold. Part II. Gold Bulletin 1985,18:125-131.
Sánchez-Iglesias A, Pastoriza-Santos I, Pérez-Juste J, Rodríguez-González
B, García de Abajo FJ, Liz-Marzán LM. Synthesis and Optical Properties of
Gold
Nanodecahedra
with
Size
Control.
Advanced
Materials
2006,18:2529-2534.
Sanchez C, Julian B, Belleville P, Popall M. Applications of hybrid organicinorganic nanocomposites. Journal of Materials Chemistry 2005,15:35593592.
Gudiksen MS, Lauhon LJ, Wang J, Smith DC, Lieber CM. Growth of
nanowire superlattice structures for nanoscale photonics and electronics.
Nature 2002,415:617-620.
Hu JT, Ouyang M, Yang PD, Lieber CM. Controlled growth and electrical
properties of heterojunctions of carbon nanotubes and silicon nanowires.
Nature 1999,399:48-51.
Lieber CM, Wang ZL. Functional nanowires. Mrs Bulletin 2007,32:99-108.
Dupuy AM, Lehmann S, Cristol JP. Protein biochip systems for the clinical
laboratory. Clinical Chemistry and Laboratory Medicine 2005,43:12911302.
Fouillet Y, Jary D, Chabrol C, Claustre P, Peponnet C. Digital microfluidic
design and optimization of classic and new fluidic functions for lab on a
chip systems. Microfluidics and Nanofluidics 2008,4:159-165.
Hoa XD, Kirk AG, Tabrizian M. Towards integrated and sensitive surface
plasmon resonance biosensors: A review of recent progress. Biosensors &
Bioelectronics 2007,23:151-160.
74
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
Jokerst JV, Raamanathan A, Christodoulides N, Floriano PN, Pollard AA,
Simmons GW, et al. Nano-bio-chips for high performance multiplexed
protein detection: Determinations of cancer biomarkers in serum and
saliva using quantum dot bioconjugate labels. Biosensors & Bioelectronics
2009,24:3622-3629.
Kasemo B. Biological surface science. Surface Science 2002,500:656-677.
Wanekaya AK, Chen W, Myung NV, Mulchandani A. Nanowire-based
electrochemical biosensors. Electroanalysis 2006,18:533-550.
Vettiger P, Cross G, Despont M, Drechsler U, Durig U, Gotsmann B, et al.
The "millipede" - Nanotechnology entering data storage. Ieee Transactions
on Nanotechnology 2002,1:39-55.
Goldhaber-Gordon D, Shtrikman H, Mahalu D, Abusch-Magder D, Meirav
U, Kastner MA. Kondo effect in a single-electron transistor. Nature
1998,391:156-159.
Nitzan A, Ratner MA. Electron transport in molecular wire junctions.
Science 2003,300:1384-1389.
Park J, Pasupathy AN, Goldsmith JI, Chang C, Yaish Y, Petta JR, et al.
Coulomb blockade and the Kondo effect in single-atom transistors. Nature
2002,417:722-725.
Suzuki K, Yamaguchi M, Kumagai M, Yanagida S. Application of carbon
nanotubes to counter electrodes of dye-sensitized solar cells. Chemistry
Letters 2003,32:28-29.
Gratzel M. Mesoscopic solar cells for electricity and hydrogen production
from sunlight. Chemistry Letters 2005,34:8-13.
Corr SA, Rakovich YP, Gun'ko YK. Multifunctional magnetic-fluorescent
nanocomposites for biomedical applications. Nanoscale Research Letters
2008,3:87-104.
Fu C-C, Lee H-Y, Chen K, Lim T-S, Wu H-Y, Lin P-K, et al.
Characterization and application of single fluorescent nanodiamonds as
cellular biomarkers. Proceedings of the National Academy of Sciences of the
United States of America 2007,104:727-732.
Lasne D, Blab GA, Berciaud S, Heine M, Groc L, Choquet D, et al. Single
nanoparticle photothermal tracking (SNaPT) of 5-nm gold beads in live
cells. Biophysical Journal 2006,91:4598-4604.
Walling MA, Novak JA, Shepard JRE. Quantum Dots for Live Cell and In
Vivo Imaging. International Journal of Molecular Sciences 2009,10:441491.
Liu KC, Anderson MA. Porous nickel oxide/nickel films for electrochemical
capacitors. Journal of the Electrochemical Society 1996,143:124-130.
Liu C, Li F, Ma L-P, Cheng H-M. Advanced Materials for Energy Storage.
Advanced Materials 2010,22:E28-+.
Guo Y-G, Hu J-S, Wan L-J. Nanostructured materials for electrochemical
energy conversion and storage devices. Advanced Materials 2008,20:28782887.
Gomez-Romero P. Hybrid organic-inorganic materials - In search of
synergic activity. Advanced Materials 2001,13:163-174.
Polshettiwar V, Varma RS. Green chemistry by nano-catalysis. Green
Chemistry 2010,12:743-754.
Niklasson GA, Granqvist CG. Electrochromics for smart windows: thin
films of tungsten oxide and nickel oxide, and devices based on these.
Journal of Materials Chemistry 2007,17:127-156.
Nath N, Chilkoti A. Creating "Smart" surfaces using stimuli responsive
polymers. Advanced Materials 2002,14:1243-+.
75
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
Luzinov I, Minko S, Tsukruk VV. Adaptive and responsive surfaces
through controlled reorganization of interfacial polymer layers. Progress in
Polymer Science 2004,29:635-698.
Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramirez JT,
et al. The bactericidal effect of silver nanoparticles. Nanotechnology
2005,16:2346-2353.
De M, Ghosh PS, Rotello VM. Applications of Nanoparticles in Biology.
Advanced Materials 2008,20:4225-4241.
Nadworny PL, Wang J, Tredget EE, Burrell RE. Anti-inflammatory activity
of nanocrystalline silver in a porcine contact dermatitis model.
Nanomedicine: Nanotechnology, Biology and Medicine 2008,4:241-251.
Sibbald RGBSMDFFM, Contreras-Ruiz JMD, Coutts PRNI, Fierheller
MRNB, Rothman AME, Woo KMRNPAGNC. Bacteriology, Inflammation,
and Healing: A Study of Nanocrystalline Silver Dressings in Chronic
Venous Leg Ulcers. Advances in Skin & Wound Care October
2007;20(10):549-558.
Chaloupka K, Malam Y, Seifalian AM. Nanosilver as a new generation of
nanoproduct in biomedical applications. Trends in Biotechnology
2010,28:580-588.
Chen J, Han C-m, Lin X-w, Tang Z-j, Su S-j. Effect of silver nanoparticle
dressing on second degree burn wound. Zhonghua wai ke za zhi [Chinese
journal of surgery] 2006,44:50-52.
Goethals EC, Elbaz A, Lopata AL, Bhargava SK, Bansal V. Decoupling the
Effects of the Size, Wall Thickness, and Porosity of Curcumin-Loaded
Chitosan Nanocapsules on Their Anticancer Efficacy: Size Is the Winner.
Langmuir 2012,29:658-666.
Singh M, Li XM, Wang HY, McGee JP, Zamb T, Koff W, et al.
Immunogenicity and protection in small-animal models with controlledrelease tetanus toxoid microparticles as a single-dose vaccine. Infection
and Immunity 1997,65:1716-1721.
Pitt GG, Gratzl MM, Kimmel GL, Surles J, Sohindler A. Aliphatic
polyesters II. The degradation of poly (DL-lactide), poly (ε-caprolactone),
and their copolymers in vivo. Biomaterials 1981,2:215-220.
Prescott LF, Nimmo WS. Novel Drug Delivery and Its Therapeutic
Application: UMI Books on Demand; 1989.
Hans ML, Lowman AM. Biodegradable nanoparticles for drug delivery and
targeting. Current Opinion in Solid State and Materials Science 2002,6:319327.
Kumari A, Yadav SK, Yadav SC. Biodegradable polymeric nanoparticles
based drug delivery systems. Colloids and Surfaces B: Biointerfaces
2010,75:1-18.
Dadashzadeh S, Derakhshandeh K, Shirazi FH. 9-nitrocamptothecin
polymeric nanoparticles: cytotoxicity and pharmacokinetic studies of
lactone and total forms of drug in rats. Anti-Cancer Drugs 2008,19:805811.
Derakhshandeh K, Erfan M, Dadashzadeh S. Encapsulation of 9nitrocamptothecin, a novel anticancer drug, in biodegradable
nanoparticles: Factorial design, characterization and release kinetics.
European Journal of Pharmaceutics and Biopharmaceutics 2007,66:34-41.
Molino NM, Bilotkach K, Fraser DA, Ren D, Wang S-W. Complement
Activation and Cell Uptake Responses Toward Polymer-Functionalized
Protein Nanocapsules. Biomacromolecules 2012,13:974-981.
Cruz LJ, Tacken PJ, Rueda F, Domingo JC, Albericio F, Figdor CG.
Targeting nanoparticles to dendritic cells for immunotherapy. In:
76
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
Nanomedicine: Infectious Diseases, Immunotherapy, Diagnostics,
Antifibrotics, Toxicology and Gene Medicine. Edited by Duzgunes N. San
Diego: Elsevier Academic Press Inc; 2012. pp. 143-163.
Xiang SD, Scholzen A, Minigo G, David C, Apostolopoulos V, Mottram PL,
et al. Pathogen recognition and development of particulate vaccines: Does
size matter? Methods 2006,40:1-9.
Nicolete R, dos Santos DF, Faccioli LH. The uptake of PLGA micro or
nanoparticles by macrophages provokes distinct in vitro inflammatory
response. International Immunopharmacology 2011,11:1557-1563.
Semete B, Booysen LIJ, Kalombo L, Venter JD, Katata L, Ramalapa B, et
al. In vivo uptake and acute immune response to orally administered
chitosan and PEG coated PLGA nanoparticles. Toxicology and Applied
Pharmacology 2010,249:158-165.
Reddy ST, Berk DA, Jain RK, Swartz MA. A sensitive in vivo model for
quantifying interstitial convective transport of injected macromolecules
and nanoparticles. Journal of Applied Physiology 2006,101:1162-1169.
Reddy ST, Rehor A, Schmoekel HG, Hubbell JA, Swartz MA. In vivo
targeting of dendritic cells in lymph nodes with poly(propylene sulfide)
nanoparticles. Journal of Controlled Release 2006,112:26-34.
Swartz MA, Hubbell JA, Reddy ST. Lymphatic drainage function and its
immunological implications: From dendritic cell homing to vaccine design.
Seminars in Immunology 2008,20:147-156.
Weissleder R. Molecular imaging: Exploring the next frontier. Radiology
1999,212:609-614.
Mahmood U, Tung CH, Weissleder R. Optical imaging of gene expression
using
enzyme-specific
activatable
contrast
agents.
Radiology
1999,213P:102-102.
Simonova M, Weissleder R, Sergeyev N, Vilissova N, Bogdanov A. Targeting
of green fluorescent protein expression to the cell surface. Biochemical and
Biophysical Research Communications 1999,262:638-642.
Nel AE, Maedler L, Velegol D, Xia T, Hoek EMV, Somasundaran P, et al.
Understanding biophysicochemical interactions at the nano-bio interface.
Nature Materials 2009,8:543-557.
Verma A, Uzun O, Hu Y, Hu Y, Han H-S, Watson N, et al. Surfacestructure-regulated cell-membrane penetration by monolayer-protected
nanoparticles. Nature Materials 2008,7:588-595.
Woo KM, Chen VJ, Ma PX. Nano-fibrous scaffolding architecture
selectively enhances protein adsorption contributing to cell attachment.
Journal of Biomedical Materials Research Part A 2003,67A:531-537.
Ma PX. Biomimetic materials for tissue engineering. Advanced Drug
Delivery Reviews 2008,60:184-198.
Bae Y, Jang WD, Nishiyama N, Fukushima S, Kataoka K. Multifunctional
polymeric micelles with folate-mediated cancer cell targeting and pHtriggered drug releasing properties for active intracellular drug delivery.
Molecular Biosystems 2005,1:242-250.
Portney NG, Ozkan M. Nano-oncology: drug delivery, imaging, and
sensing. Analytical and Bioanalytical Chemistry 2006,384:620-630.
Balamurugan B, Skomski R, Sellmyer D. Magnetic Clusters and
Nanoparticles. In: Nanoparticles: Synthesis Characterisation and
Applications. Edited by S CR, V RR. 1 ed: American Scientific Publishers;
2010.
Weinberg S. The Theory of Quantum Fields. In: Press Sydnicate of the
University of Cambridge; 1996.
Jiles D. Introduction to Magnetism and Magnetic Materials. 2nd ed; 1998.
77
114.
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
Carey R, D. IE. Magnetic domains and techniques for their observation:
Academic Press; 1966.
Weishaupt D, Köchli V, Marincek B. How does MRI work?: an introduction
to the physics and function of magnetic resonance imaging. Second ed:
Springer; 2003.
Westbrook C, Kaut C. MRI in practice. Second edition ed: Blackwell
Science; 1998.
Néel L, Kurti N. The Selected Works of Louis Neel: Gordon & Breach
Publishing Group; 1984.
Van-der-Warden BL, R. K, E. UG. Spin history correspondence:. In:
Massachusetts Institute of Technology; 1921.
Dirac PAM. The quantum theory of the electron. Proceedings of the Royal
Society of London Series a-Containing Papers of a Mathematical and
Physical Character 1928,117:610-624.
Dirac PAM. The quantum theory of the electron - Part II. Proceedings of the
Royal Society of London Series a-Containing Papers of a Mathematical and
Physical Character 1928,118:351-361.
Brown M, Semelka R. Production of net magnetization: John Wiley &
Sons; 2005.
Enterprises NW. Modern Faq's. In: LLC dba; 2013.
Na HB, Song IC, Hyeon T. Inorganic Nanoparticles for MRI Contrast
Agents. Advanced Materials 2009,21:2133-2148.
Ergun I, Keven K, Uruc I, Ekmekci Y, Canbakan B, Erden I, et al. The
safety of gadolinium in patients with stage 3 and 4 renal failure.
Nephrology Dialysis Transplantation 2006,21:697-700.
Pouliquen D, Lejeune JJ, Perdrisot R, Ermias A, Jallet P. Iron-oxide
nanoparticles for use as an mri contrast agent - pharmacokinetics and
metabolism. Magnetic Resonance Imaging 1991,9:275-283.
Chouly C, Pouliquen D, Lucet I, Jeune JJ, Jallet P. Development of
superparamagnetic nanoparticles for MRI: Effect of particle size, charge
and surface nature on biodistribution. Journal of Microencapsulation
1996,13:245-255.
Thorek DLJ, Chen A, Czupryna J, Tsourkas A. Superparamagnetic iron
oxide nanoparticle probes for molecular imaging. Annals of Biomedical
Engineering 2006,34:23-38.
Song YJ, Wang RX, Rong R, Ding J, Liu J, Li RS, et al. Synthesis of WellDispersed Aqueous-Phase Magnetite Nanoparticles and Their Metabolism
as an MRI Contrast Agent for the Reticuloendothelial System. European
Journal of Inorganic Chemistry 2011:3303-3313.
Strable E, Bulte JWM, Moskowitz B, Vivekanandan K, Allen M, Douglas T.
Synthesis and characterization of soluble iron oxide-dendrimer
composites. Chemistry of Materials 2001,13:2201-2209.
Wang YXJ, Hussain SM, Krestin GP. Superparamagnetic iron oxide
contrast agents: physicochemical characteristics and applications in MR
imaging. European Radiology 2001,11:2319-2331.
Weissleder R, Elizondo G, Wittenberg J, Rabito CA, Bengele HH,
Josephson L. Ultrasmall superparamagnetic iron-oxide - characterization
of a new class of contrast agents for mr imaging. Radiology 1990,175:489493.
Massart R. Preparation of aqueous magnetic liquids in alkaline and acidic
media. Ieee Transactions on Magnetics 1981,17:1247-1248.
Jolivet JP, Chaneac C, Tronc E. Iron oxide chemistry. From molecular
clusters to extended solid networks. Chemical Communications 2004:481487.
78
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
147.
148.
Kim DK, Zhang Y, Voit W, Rao KV, Muhammed M. Synthesis and
characterization of surfactant-coated superparamagnetic monodispersed
iron oxide nanoparticles. Journal of Magnetism and Magnetic Materials
2001,225:30-36.
Laurent S, Forge D, Port M, Roch A, Robic C, Elst LV, et al. Magnetic iron
oxide
nanoparticles:
Synthesis,
stabilization,
vectorization,
physicochemical characterizations, and biological applications. Chemical
Reviews 2008,108:2064-2110.
Cornell RM, Schwertmann U. Electronic, Electrical and Magnetic
Properties and Colour. In: The Iron Oxides: Wiley-VCH Verlag GmbH & Co.
KGaA; 2004. pp. 111-137.
Tartaj P, Morales MP, Veintemillas-Verdaguer S, Gonzalez-Carreno T,
Serna CJ. ChemInform Abstract: Synthesis, Properties and Biomedical
Applications of Magnetic Nanoparticles. ChemInform 2008,39:no-no.
Lee KM, Sorensen CM, Klabunde KJ, Hadjipanayis GC. Synthesis and
characterization of stable colloidal fe3o4 particles in water-in-oil
microemulsions. Ieee Transactions on Magnetics 1992,28:3180-3182.
O'Connor CJ, Seip C, Sangregorio C, Carpenter E, Li SC, Irvin G, et al.
Nanophase Magnetic Materials: Synthesis and Properties. Molecular
Crystals and Liquid Crystals Science and Technology Section a-Molecular
Crystals and Liquid Crystals 1999,335:1135-1154.
Liz L, Quintela MAL, Mira J, Rivas J. Preparation of colloidal fe3o4
ultrafine particles in microemulsions. Journal of Materials Science
1994,29:3797-3801.
Dresco PA, Zaitsev VS, Gambino RJ, Chu B. Preparation and properties of
magnetite and polymer magnetite nanoparticles. Langmuir 1999,15:19451951.
Gobe M, Kon-No K, Kandori K, Kitahara A. Preparation and
characterization of monodisperse magnetite sols in WO microemulsion.
Journal of Colloid and Interface Science 1983,93:293-295.
Nassar N, Husein M. Preparation of iron oxide nanoparticles from FeCl3
solid powder using microemulsions. physica status solidi (a)
2006,203:1324-1328.
Santra S, Tapec R, Theodoropoulou N, Dobson J, Hebard A, Tan WH.
Synthesis and characterization of silica-coated iron oxide nanoparticles in
microemulsion:
The
effect
of
nonionic
surfactants.
Langmuir
2001,17:2900-2906.
Dickson DPE, Walton SA, Mann S, Wong K. Properties of magnetoferritin:
A novel biomagnetic nanoparticle. Nanostructured Materials 1997,9:595598.
Wong KKW, Douglas T, Gider S, Awschalom DD, Mann S. Biomimetic
synthesis and characterization of magnetic proteins (magnetoferritin).
Chemistry of Materials 1998,10:279-285.
Seidel C. Physics of amphiphiles: Micelles, vesicles and microemulsions.
Proceedings of the International School of Physics ―Enrico Fermi‖, Cours
XC at Varenna on Lake Como, 19–29 July 1983. Hg. von V. DEGIORGIO
und M. CORTI. ISBN 0-444-86940-9. Amsterdam/Oxford/New
York/Tokyo: North Holland Physics Publishing 1985. XXIV, 888 S., Lwd.,
Dfl. 400.00. Available in USA and Canada from Elsevier Science
Publishers, 52 Vanderbilt Ave., New York, NY 10017, USA. Acta
Polymerica 1986,37:474-474.
Bonacchi D, Caneschi A, Dorignac D, Falqui A, Gatteschi D, Rovai D, et al.
Nanosized iron oxide particles entrapped in pseudo-single crystals
gamma-cyclodextrin. Chemistry of Materials 2004,16:2016-2020.
79
149.
150.
151.
152.
153.
154.
155.
156.
157.
158.
159.
160.
161.
162.
163.
164.
Hou Y, Kondoh H, Shimojo M, Sako EO, Ozaki N, Kogure T, et al.
Inorganic nanocrystal self-assembly via the inclusion interaction of sscyclodextrins: Toward 3D spherical magnetite. Journal of Physical
Chemistry B 2005,109:4845-4852.
Walter GA, Cahill KS, Huard J, Feng HS, Douglas T, Sweeney HL, et al.
Noninvasive monitoring of stem cell transfer for muscle disorders.
Magnetic Resonance in Medicine 2004,51:273-277.
Lee IH, Bulte JWM, Schweinhardt P, Douglas T, Trifunovski A, Hofstetter
C, et al. In vivo magnetic resonance tracking of olfactory ensheathing glia
grafted into the rat spinal cord. Experimental Neurology 2004,187:509516.
Bulte JWM, Cuyper MD. Magnetoliposomes as Contrast Agents. In:
Methods in Enzymology. Edited by Nejat D: Academic Press; 2003. pp.
175-198.
Wan SR, Huang JS, Guo M, Zhang HK, Cao YJ, Yan HS, et al.
Biocompatible superparamagnetic iron oxide nanoparticle dispersions
stabilized with poly(ethylene glycol)oligo(aspartic acid) hybrids. Journal of
Biomedical Materials Research Part A 2007,80A:946-954.
Gaucher G, Dufresne M-H, Sant VP, Kang N, Maysinger D, Leroux J-C.
Block copolymer micelles: preparation, characterization and application in
drug delivery. Journal of Controlled Release 2005,109:169-188.
Kwon GS. Polymeric micelles for delivery of poorly water-soluble
compounds. Critical Reviews in Therapeutic Drug Carrier Systems
2003,20:357-403.
Mulder WJM, Strijkers GJ, van Tilborg GAF, Griffioen AW, Nicolay K.
Lipid-based nanoparticles for contrast-enhanced MRI and molecular
imaging. Nmr in Biomedicine 2006,19:142-164.
Dai Z, Meiser F, Möhwald H. Nanoengineering of iron oxide and iron
oxide/silica hollow spheres by sequential layering combined with a sol–gel
process. Journal of Colloid and Interface Science 2005,288:298-300.
Ismail AA. Synthesis and characterization of Y2O3/Fe2O3/TiO2
nanoparticles by sol–gel method. Applied Catalysis B: Environmental
2005,58:115-121.
Brinker CJ, Scherer GW. Sol-gel science: the physics and chemistry of solgel processing: Academic Press; 1990.
Ennas G, Musinu A, Piccaluga G, Zedda D, Gatteschi D, Sangregorio C, et
al. Characterization of iron oxide nanoparticles in an Fe2O3-SiO2
composite prepared by a sol-gel method. Chemistry of Materials
1998,10:495-502.
Raileanu M, Crisan M, Petrache C, Crisan D, Zaharescu M. Fe2O3-SiO2
nanocomposites obtained by different sol-gel routes. Journal of
Optoelectronics and Advanced Materials 2003,5:693-698.
delMonte F, Morales MP, Levy D, Fernandez A, Ocana M, Roig A, et al.
Formation of gamma-Fe2O3 isolated nanoparticles in a silica matrix.
Langmuir 1997,13:3627-3634.
Niznansky D, Rehspringer JL, Drillon M. Preparation of magnetic
manoparticles (gamma-fe2o3) in the silica matrix. Ieee Transactions on
Magnetics 1994,30:821-823.
Bentivegna F, Ferre J, Nyvlt M, Jamet JP, Imhoff D, Canva M, et al.
Magnetically textured gamma-Fe(2)O(3) nanoparticles in a silica gel
matrix: Structural and magnetic properties. Journal of Applied Physics
1998,83:7776-7788.
80
165.
166.
167.
168.
169.
170.
171.
172.
173.
174.
175.
176.
177.
178.
179.
180.
181.
Park J, An KJ, Hwang YS, Park JG, Noh HJ, Kim JY, et al. Ultra-largescale syntheses of monodisperse nanocrystals. Nature Materials
2004,3:891-895.
Sun SH, Zeng H, Robinson DB, Raoux S, Rice PM, Wang SX, et al.
Monodisperse MFe2O4 (M = Fe, Co, Mn) nanoparticles. Journal of the
American Chemical Society 2004,126:273-279.
Redl FX, Black CT, Papaefthymiou GC, Sandstrom RL, Yin M, Zeng H, et
al.
Magnetic,
electronic,
and
structural
characterization
of
nonstoichiometric iron oxides at the nanoscale. Journal of the American
Chemical Society 2004,126:14583-14599.
Lu A-H, Salabas EL, Schueth F. Magnetic nanoparticles: Synthesis,
protection, functionalization, and application. Angewandte ChemieInternational Edition 2007,46:1222-1244.
Murray CB, Norris DJ, Bawendi MG. Synthesis and characterization of
nearly monodisperse cde (e = s, se, te) semiconductor nanocrystallites.
Journal of the American Chemical Society 1993,115:8706-8715.
Peng XG, Wickham J, Alivisatos AP. Kinetics of II-VI and III-V colloidal
semiconductor nanocrystal growth: "Focusing" of size distributions.
Journal of the American Chemical Society 1998,120:5343-5344.
Farrell D, Majetich SA, Wilcoxon JP. Preparation and characterization of
monodisperse Fe nanoparticles. Journal of Physical Chemistry B
2003,107:11022-11030.
Hyeon T, Lee SS, Park J, Chung Y, Bin Na H. Synthesis of highly
crystalline and monodisperse maghemite nanocrystallites without a sizeselection process. Journal of the American Chemical Society
2001,123:12798-12801.
Butter K, Philipse AP, Vroege GJ. Synthesis and properties of iron
ferrofluids. Journal of Magnetism and Magnetic Materials 2002,252:1-3.
Brief RS, Ajemian RS, Confer RG. Iron pentacarbonyl - its toxicity
detection and potential for formation. American Industrial Hygiene
Association Journal 1967,28:21-&.
Dumestre F, Chaudret B, Amiens C, Renaud P, Fejes P. Superlattices of
Iron Nanocubes Synthesized from Fe[N(SiMe3)2]2. Science 2004,303:821823.
Puntes VF, Zanchet D, Erdonmez CK, Alivisatos AP. Synthesis of hcp-Co
nanodisks. Journal of the American Chemical Society 2002,124:1287412880.
Dumestre F, Chaudret B, Amiens C, Fromen MC, Casanove MJ, Renaud
P, et al. Shape control of thermodynamically stable cobalt nanorods
through organometallic chemistry. Angewandte Chemie-International
Edition 2002,41:4286-4289.
Dumestre F, Chaudret B, Amiens C, Respaud M, Fejes P, Renaud P, et al.
Unprecedented crystalline super-lattices of monodisperse cobalt nanorods.
Angewandte Chemie-International Edition 2003,42:5213-5216.
Gupta AK, Gupta M. Synthesis and surface engineering of iron oxide
nanoparticles for biomedical applications. Biomaterials 2005,26:39954021.
Goya GF, Berquo TS, Fonseca FC, Morales MP. Static and dynamic
magnetic properties of spherical magnetite nanoparticles. Journal of
Applied Physics 2003,94:3520-3528.
Jun Y-w, Huh Y-M, Choi J-s, Lee J-H, Song H-T, KimKim, et al. Nanoscale
Size Effect of Magnetic Nanocrystals and Their Utilization for Cancer
Diagnosis via Magnetic Resonance Imaging. Journal of the American
Chemical Society 2005,127:5732-5733.
81
182.
183.
184.
185.
186.
187.
188.
189.
190.
191.
192.
193.
194.
195.
196.
197.
198.
199.
200.
Berry CC, Curtis ASG. Functionalisation of magnetic nanoparticles for
applications in biomedicine. Journal of Physics D-Applied Physics
2003,36:R198-R206.
Lu Y, Yin YD, Mayers BT, Xia YN. Modifying the surface properties of
superparamagnetic iron oxide nanoparticles through a sol-gel approach.
Nano Letters 2002,2:183-186.
Dobrovolskaia MA, Aggarwal P, Hall JB, McNeil SE. Preclinical studies to
understand nanoparticle interaction with the immune system and its
potential effects on nanoparticle biodistribution. Molecular Pharmaceutics
2008,5:487-495.
Lunov O, Syrovets T, Loos C, Beil J, Delecher M, Tron K, et al. Differential
Uptake of Functionalized Polystyrene Nanoparticles by Human
Macrophages and a Monocytic Cell Line. Acs Nano 2011,5:1657-1669.
Kawamura Y, Endo K, Watanabe Y, Saga T, Nakai T, Hikita H, et al. Use of
magnetite particles as a contrast agent for mr imaging of the liver.
Radiology 1990,174:357-360.
Davis
SS.
Microspheres
and
drug
therapy:
pharmaceutical,
immunological, and medical aspects: Elsevier; 1984.
Weissleder R, Stark DD, Engelstad BL, Bacon BR, Compton CC, White DL,
et al. Superparamagnetic iron-oxide - pharmacokinetics and toxicity.
American Journal of Roentgenology 1989,152:167-173.
Moore A, Marecos E, Bogdanov A, Weissleder R. Tumoral distribution of
long-circulating dextran-coated iron oxide nanoparticles in a rodent
model. Radiology 2000,214:568-574.
Islam T, Wolf G. The pharmacokinetics of the lymphotropic nanoparticle
MRI contrast agent ferumoxtran-10. Cancer Biomarkers 2009,5:69-73.
Jain TK, Reddy MK, Morales MA, Leslie-Pelecky DL, Labhasetwar V.
Biodistribution, clearance, and biocompatibility of iron oxide magnetic
nanoparticles in rats. Molecular Pharmaceutics 2008,5:316-327.
Cheon J, Lee J-H. Synergistically Integrated Nanoparticles as Multimodal
Probes for Nanobiotechnology. Accounts of Chemical Research
2008,41:1630-1640.
Jennings LE, Long NJ. 'Two is better than one'-probes for dual-modality
molecular imaging. Chemical Communications 2009:3511-3524.
Hamley IW. Nanotechnology with soft materials. Angewandte ChemieInternational Edition 2003,42:1692-1712.
Mendenhall GD, Geng YP, Hwang J. Optimization of long-term stability of
magnetic fluids from magnetite and synthetic polyelectrolytes. Journal of
Colloid and Interface Science 1996,184:519-526.
Schulze E, Ferrucci JT, Poss K, Lapointe L, Bogdanova A, Weissleder R.
Cellular uptake and trafficking of a prototypical magnetic iron-oxide label
in-vitro. Investigative Radiology 1995,30:604-610.
Briley-Saebo K, Bjornerud A, Grant D, Ahlstrom H, Berg T, Kindberg GM.
Hepatic cellular distribution and degradation of iron oxide nanoparticles
following single intravenous injection in rats: implications for magnetic
resonance imaging. Cell and Tissue Research 2004,316:315-323.
Platt N, Gordon S. Is the class A macrophage scavenger receptor (SR-A)
multifunctional? — The mouse’s tale. The Journal of Clinical Investigation
2001,108:649-654.
de Winther MPJ, van Dijk KW, Havekes LM, Hofker MH. Macrophage
scavenger receptor class A - A multifunctional receptor in atherosclerosis.
Arteriosclerosis Thrombosis and Vascular Biology 2000,20:290-297.
Libby P, Aikawa M. Stabilization of atherosclerotic plaques: New
mechanisms and clinical targets. Nature Medicine 2002,8:1257-1262.
82
201.
202.
203.
204.
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
215.
216.
Sahoo Y, Goodarzi A, Swihart MT, Ohulchanskyy TY, Kaur N, Furlani EP,
et al. Aqueous Ferrofluid of Magnetite Nanoparticles: Fluorescence
Labeling and Magnetophoretic Control. The Journal of Physical Chemistry
B 2005,109:3879-3885.
Fauconnier N, Pons JN, Roger J, Bee A. Thiolation of maghemite
nanoparticles by dimercaptosuccinic acid. Journal of Colloid and Interface
Science 1997,194:427-433.
Stober W, Fink A, Bohn E. Controlled growth of monodisperse silica
spheres in micron size range. Journal of Colloid and Interface Science
1968,26:62-&.
Sun YK, Duan L, Guo ZR, Yun DM, Ma M, Xu L, et al. An improved way to
prepare superparamagnetic magnetite-silica core-shell nanoparticles for
possible biological application. Journal of Magnetism and Magnetic
Materials 2005,285:65-70.
Rocchicciolideltcheff C, Franck R, Cabuil V, Massart R. Surfacted
ferrofluids - interactions at the surfactant-magnetic iron-oxide interface.
Journal of Chemical Research-S 1987:126-126.
Mulvaney P, Liz-Marzan LM, Giersig M, Ung T. Silica encapsulation of
quantum dots and metal clusters. Journal of Materials Chemistry
2000,10:1259-1270.
Israelachvili J. Intermolecular and Surface Forces, Second Edition: With
Applications to Colloidal and Biological Systems (Colloid Science):
Academic Press; 1992.
Hartley PG, Larson I, Scales PJ. Electrokinetic and direct force
measurements between silica and mica surfaces in dilute electrolyte,
solutions. Langmuir 1997,13:2207-2214.
Ulman A. Formation and structure of self-assembled monolayers.
Chemical Reviews 1996,96:1533-1554.
Hahn PF, Stark DD, Lewis JM, Saini S, Elizondo G, Weissleder R, et al. 1st
clinical-trial of a new superparamagnetic iron-oxide for use as an oral
gastrointestinal contrast agent in mr imaging. Radiology 1990,175:695700.
Chen HY, Cui SS, Zhu HY, Tu ZZ, Shan LL, Xue JP, et al. In vivo
monitoring of Organ-Selective distribution of CdHgTe/SiO(2) Nanoparticles
in mouse model. In: Reporters, Markers, Dyes, Nanoparticles, and
Molecular Probes for Biomedical Applications Iii. Edited by Achilefu S,
Raghavachari R. Bellingham: Spie-Int Soc Optical Engineering; 2011.
Trewyn BG, Nieweg JA, Zhao Y, Lin VSY. Biocompatible mesoporous silica
nanoparticles with different morphologies for animal cell membrane,
penetration. Chemical Engineering Journal 2008,137:23-29.
Shi M, Liu Y, Xu M, Yang H, Wu C, Miyoshi H. Core/Shell Fe3O4@SiO2
Nanoparticles Modified with PAH as a Vector for EGFP Plasmid DNA
Delivery into HeLa Cells. Macromolecular Bioscience 2011,11:1563-1569.
Bardi G, Malvindi MA, Gherardini L, Costa M, Pompa PP, Cingolani R, et
al. The biocompatibility of amino functionalized CdSe/ZnS quantum-dotDoped SiO(2) nanoparticles with primary neural cells and their gene
carrying performance. Biomaterials 2010,31:6555-6566.
Rosenholm JM, Sahlgren C, Linden M. Multifunctional Mesoporous Silica
Nanoparticles for Combined Therapeutic, Diagnostic and Targeted Action
in Cancer Treatment. Current Drug Targets 2011,12:1166-1186.
Wang T-T, Chai F, Wang C-G, Li L, Liu H-Y, Zhang L-Y, et al. Fluorescent
hollow/rattle-type mesoporous Au@SiO2 nanocapsules for drug delivery
and fluorescence imaging of cancer cells. Journal of Colloid and Interface
Science 2011,358:109-115.
83
217.
218.
219.
220.
221.
222.
223.
224.
225.
226.
227.
228.
229.
230.
231.
232.
233.
Fang CL, Qian K, Zhu JH, Wang SB, Lv XX, Yu SH. Monodisperse alphaFe(2)O(3)@SiO(2)@Au core/shell nanocomposite spheres: synthesis,
characterization and properties. Nanotechnology 2008,19.
Runowski M, Grzyb T, Lis S. Bifunctional luminescent and magnetic
core/shell type nanostructures Fe3O4@CeF3:Tb3+/SiO2. Journal of Rare
Earths 2011,29:1117-1122.
Atarashi T, Kim YS, Fujita T, Nakatsuka K. Synthesis of ethylene–glycolbased magnetic fluid using silica-coated iron particle. Journal of
Magnetism and Magnetic Materials 1999,201:7-10.
Chen F, Bu W, Chen Y, Fan Y, He Q, Zhu M, et al. A Sub-50-nm
Monosized Superparamagnetic Fe3O4@SiO2T2-Weighted MRI Contrast
Agent: Highly Reproducible Synthesis of Uniform Single-Loaded Core–
Shell Nanostructures. Chemistry – An Asian Journal 2009,4:1809-1816.
Philipse AP, Vanbruggen MPB, Pathmamanoharan C. Magnetic silica
dispersions - preparation and stability of surface-modified silica particles
with a magnetic core. Langmuir 1994,10:92-99.
Tartaj P, Serna CJ. Synthesis of Monodisperse Superparamagnetic
Fe/Silica Nanospherical Composites. Journal of the American Chemical
Society 2003,125:15754-15755.
Matsoukas T, Gulari E. Dynamics of growth of silica particles from
ammonia-catalyzed hydrolysis of tetra-ethyl-orthosilicate. Journal of
Colloid and Interface Science 1988,124:252-261.
Matsoukas T, Gulari E. Monomer-addition growth with a slow initiation
step: A growth model for silica particles from alkoxides. Journal of Colloid
and Interface Science 1989,132:13-21.
Matsoukas
T,
Gulari
E.
Self-sharpening
distributions
revisitedâ€‖polydispersity in growth by monomer addition. Journal of
Colloid and Interface Science 1991,145:557-562.
Bogush GH, Zukoski Iv CF. Studies of the kinetics of the precipitation of
uniform silica particles through the hydrolysis and condensation of silicon
alkoxides. Journal of Colloid and Interface Science 1991,142:1-18.
Bogush GH, Zukoski Iv CF. Uniform silica particle precipitation: An
aggregative growth model. Journal of Colloid and Interface Science
1991,142:19-34.
Lee K, Sathyagal AN, McCormick AV. A closer look at an aggregation
model of the Stöber process. Colloids and Surfaces A: Physicochemical
and Engineering Aspects 1998,144:115-125.
Ariga K, Hill JP, Ji Q. Layer-by-layer assembly as a versatile bottom-up
nanofabrication technique for exploratory research and realistic
application. Physical Chemistry Chemical Physics 2007,9:2319-2340.
Ariga K, Nakanishi T, Michinobu T. Immobilization of biomaterials to
nano-assembled films (self-assembled monolayers, Langmuir-Blodgett
films, and layer-by-layer assemblies) and their related functions. Journal
of Nanoscience and Nanotechnology 2006,6:2278-2301.
Pillai V, Kumar P, Hou MJ, Ayyub P, Shah DO. Preparation of
nanoparticles of silver-halides, superconductors and magnetic-materials
using water-in-oil microemulsions as nano-reactors. Advances in Colloid
and Interface Science 1995,55:241-269.
Ai H, Jones SA, Lvov YM. Biomedical applications of electrostatic layer-bylayer nano-assembly of polymers, enzymes, and nanoparticles. Cell
Biochemistry and Biophysics 2003,39:23-43.
Lee H, Ahn C-H, Park TG. Poly lactic-co-(glycolic acid) -Grafted Hyaluronic
Acid Copolymer Micelle Nanoparticles for Target-Specific Delivery of
Doxorubicin. Macromolecular Bioscience 2009,9:336-342.
84
234.
235.
236.
237.
238.
239.
240.
241.
242.
243.
244.
245.
246.
247.
248.
249.
Nurunnabi M, Cho KJ, Choi JS, Huh KM, Lee Y-h. Targeted near-IR QDsloaded micelles for cancer therapy and imaging. Biomaterials
2010,31:5436-5444.
Fan CX, Gao WH, Chen ZX, Fan HY, Lie MY, Deng FJ, et al. Tumor
selectivity of stealth multi-functionalized superparamagnetic iron oxide
nanoparticles. International Journal of Pharmaceutics 2011,404:180-190.
Cruz LJ, Tacken PJ, Rueda F, Domingo JC, Albericio F, Figdor CG.
Targeting nanoparticles to dendritic cells for immunotherapy, in
Nanomedicine: Infectious Diseases, Immunotherapy, Diagnostics,
Antifibrotics, Toxicology and Gene Medicine, N. Duzgunes, Editor. 2012,
Elsevier Academic Press Inc: San Diego. p. 143-163.
Xiao Z, Levy-Nissenbaum, E Alexis F, Luptak A, Teply BA, Chan JM, Shi J,
Digga E, Cheng J, Langer R, Farokhzad OC. Engineering of Targeted
Nanoparticles for Cancer Therapy Using Internalizing Aptamers Isolated
by Cell-Uptake Selection. Acs Nano, 2012. 6(1): p. 696-704.
Llevot, A. and D. Astruc, Applications of vectorized gold nanoparticles to
the diagnosis and therapy of cancer. Chemical Society Reviews, 2012.
41(1): p. 242-257.
Nicholson RI, Gee JMW, Harper ME. EGFR and cancer prognosis.
European Journal of Cancer 2001,37:S9-S15.
Izumi Y, Xu L, di Tomaso E, Fukumura D, Jain RK. Tumor biology Herceptin acts as an anti-angiogenic cocktail. Nature 2002,416:279-280.
Molina MA, Codony-Servat J, Albanell J, Rojo F, Arribas J, Baselga J.
Trastuzumab (Herceptin), a humanized anti-HER2 receptor monoclonal
antibody, inhibits basal and activated HER2 ectodomain cleavage in
breast cancer cells. Cancer Research 2001,61:4744-4749.
Normanno N, Campiglio M, De Luca A, Somenzi G, Maiello M, Ciardiello F,
et al. Cooperative inhibitory effect of ZD1839 (Iressa) in combination with
trastuzumab (Herceptin) on human breast cancer cell growth. Annals of
Oncology 2002,13:65-72.
Carter P, Presta L, Gorman CM, Ridgway JBB, Henner D, Wong WLT, et al.
Humanization of an anti-p185her2 antibody for human cancer-therapy.
Proceedings of the National Academy of Sciences of the United States of
America 1992,89:4285-4289.
Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell
2000,103:211-225.
Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et
al. Use of Chemotherapy plus a Monoclonal Antibody against HER2 for
Metastatic Breast Cancer That Overexpresses HER2. New England Journal
of Medicine 2001,344:783-792.
Diagaradjane P, Orenstein-Cardona JM, Colon-Casasnovas NE,
Deorukhkar A, Shentu S, Kuno N, et al. Imaging epidermal growth factor
receptor expression in vivo: Pharmacokinetic and biodistribution
characterization of a bioconjugated quantum dot nanoprobe. Clinical
Cancer Research 2008,14:731-741.
Park H, Lee S, Chen L, Lee EK, Shin SY, Lee YH, et al. SERS imaging of
HER2-overexpressed MCF7 cells using antibody-conjugated gold
nanorods. Physical Chemistry Chemical Physics 2009,11:7444-7449.
Vo-Dinh T, Alarie JP, Cullum BM, Griffin GD. Antibody-based nanoprobe
for measurement of a fluorescent analyte in a single cell. Nature
Biotechnology 2000,18:764-767.
Yong K-T, Ding H, Roy I, Law W-C, Bergey EJ, Maitra A, et al. Imaging
Pancreatic Cancer Using Bioconjugated InP Quantum Dots. Acs Nano
2009,3:502-510.
85
250.
251.
252.
253.
254.
255.
256.
257.
258.
Mi Y, Liu X, Zhao J, Ding J, Feng, S. Multimodality treatment of cancer
with herceptin conjugated, thermomagnetic iron oxides and docetaxel
loaded nanoparticles of biodegradable polymers. Biomaterials, 2012.
33(30): p. 7519-7529.
Aydin, R.S.T., Herceptin-decorated salinomycin-loaded nanoparticles for
breast tumor targeting. Journal of biomedical materials research. Part A,
2013. 101(5): p. 1405-15.
Zhao, J., Y. Mi, and S.-S. Feng, Targeted co-delivery of docetaxel and
siPlk1 by herceptin-conjugated vitamin E TPGS based immunomicelles.
Biomaterials, 2013. 34(13): p. 3411-3421.
Corot C, Robert P, Idée J-M, Port M. Recent advances in iron oxide
nanocrystal technology for medical imaging. Advanced Drug Delivery
Reviews 2006,58:1471-1504.
Budde MD, Frank JA. Magnetic Tagging of Therapeutic Cells for MRI.
Journal of Nuclear Medicine 2009,50:171-174.
Long CM, Bulte JWM. In vivo tracking of cellular therapeutics using
magnetic resonance imaging. Expert Opinion on Biological Therapy
2009,9:293-306.
Chen TJ, Cheng TH, Chen CY, Hsu SCN, Cheng TL, Liu GC, et al. Targeted
Herceptin-dextran iron oxide nanoparticles for noninvasive imaging of
HER2/neu receptors using MRI. Journal of Biological Inorganic Chemistry
2009,14:253-260.
Hilger I, Trost R, Reichenbach JR, Linss W, Lisy MR, Berndt A, et al. MR
imaging of Her-2/neu protein using magnetic
nanoparticles.
Nanotechnology 2007,18.
Huh YM, Jun YW, Song HT, Kim S, Choi JS, Lee JH, et al. In vivo
magnetic resonance detection of cancer by using multifunctional magnetic
nanocrystals. Journal of the American Chemical Society 2005,127:1238712391.
86
Chapter 2
2.1.0 Instrumental apparatus
Precise fabrication of nanomaterials requires a great deal of
materials characterisation and analysis. In order to properly understand
the structure and elemental make-up of the particles, many different
instruments were used to identify certain aspects of the materials as they
were being fabricated and applied. The following chapter is designed to
provide a basic understanding of these instruments and their operating
principles including, transmission electron microscopy (TEM), confocal
microscopy, X-ray Diffraction (XRD), atomic adsorption spectroscopy
(AAS),
superconducting
quantum
interference
magnetic resonance imaging (MRI).
87
device
(SQUID)
and
2.1.1 Electron Microscopy
Electron microscopy must be employed to visualise surface features
less than 200 nm in diameter, this being the absolute lower resolution
limit to light microscopy. Electron microscopes make use of a high voltage
beam of electrons either passing through or scattering from the sample
instead of light. The maximum resolution of any microscope is given by
the equation below, where λ represents the wavelength of the beam (either
visible light or a high voltage stream of electrons) and (alpha) signifies onehalf of the angular aperture. As a high voltage electron beam has a
wavelength in the range of a few Angstroms, an electron microscope can
in theory resolve images down to the atomic level, however in common
practice 1-0.5 nm is considered easily achievable.
2.1.2 Transmission Electron Microscopy
TEM is an electron microscopy technique that allows for thin
samples to be observed on a 2D plane by passing an accelerated electron
beam through a sample supported on a carbon film on a copper mesh. At
lower resolution the image produced simply provides contrast between
densities in the sample material as electron adsorption increases with
increasing density, this yields contrast on a phosphorescent plate at the
88
end of the microscope column. The contrast is a direct result of the beam
interacting with the different elements within the sample as their
scattering potentials vary. The scattering potentials for different elements
can be inferred from the Rutherford formula shown below, where (K) is the
deflection potential, (r) is the distance between the electron and the
nucleus, (z) the positive charge and (e) the electron charge. This equation
demonstrates the relationship between scattering potential and atomic
number, as one increases so does the other, leading to the clear contrast
between materials under TEM.
Several types of TEM were used to capture images presented in this
thesis, a JEOL 2010 high resolution transmission electron (HRTEM)
microscope operated at an accelerating voltage of 200 kV and JEOL 1010
TEM microscope with an operating voltage of 100 kV.
2.1.3 X-Ray Diffraction
In this thesis XRD was used to determine the crystalline type and
nature of various metals and metal oxides. In most cases the samples
were in powder form, which consisted of many randomly oriented grains of
sample. The instrument uses the elastic scattering of X-rays with a
wavelength
of
0.1
Angstroms
to
produce
diffraction
patterns
representative of the atomic configuration within the sample. The X-ray
89
beam primarily interacts with the electrons within the target, this
interaction, when occurring at the correct angle, causes X-ray photons to
be elastically deflected. These X-rays are picked up by a detector and the
required angle and number of detected photons is recorded. In crystalline
materials, the atoms are arranged in periodic patterns and will produce
regular scattering patterns in the form of concentric rings. The spacing
and intensity of these rings can be mapped out to form a fingerprint of a
particular crystal configuration of the element.
These diffraction patterns reveal the distribution of the atoms within
the sample and their arrangement relative to each other. When the atoms
are arranged regularly, as they are in crystalline materials, the scattered
photons can constructively interfere, producing sharp interference
maxima. The Bragg equation (shown below) is generally used to relate the
aspects of X-ray diffraction, where n represents the order of diffraction, λ
is the wavelength of the X-rays, d the interplanar spacing and θ denotes
the scattering angle.
All the XRD patterns displayed in this thesis were obtained using a
Bruker D8 ADVANCE X-Ray diffractometer, fitted with a graphitemonochromated copper tube source (Cu Kα radiation, λ = 1.5406 Å) and a
scintillation counter detector.
90
2.1.4 Atomic Absorption Spectroscopy (AAS)
AAS is generally used as a quantitative technique for measuring an
analyte concentration within a sample. The basic principle of the
technique relies on the sample being atomised, typically by an airacetylene flame and then subjected to a well-defined quantity of element
specific radiation. The wavelength of the radiation is chosen specifically
for the element under scrutiny due to the required electron transition
energies of each element; this is what gives this technique its selectivity.
The measured absorbance of radiation for the sample must be compared
to a series of samples of known concentration to create a calibration curve
and as such relates to the Beer-Lambert law shown below, where A is the
measured absorbance, a(λ) is the absorptivity coefficient, b is the path
length and c is the analyte concentration.
The Beer-Lambert law cannot completely describe the experimental
results given that nonuniformity within the sample, scattering of light
from particles, shifts in chemical equilibria and refraction index as a
function of concentration adversely affect the outcomes. This is why a
calibration curve is required for direct comparison of absorbance readings
of the unknown analyte compared to known concentrations. In this thesis
AAS is used to determine the concentration of Fe present in samples and
to allow adjustment of particle concentration to known quantities prior to
experiments. The AAS instrument used was a Varian AA280FS Fast
Sequential Atomic Absorption Spectrometer.
91
2.1.5 Magnetic Resonance Imaging
MRI is regarded as a powerful imaging tool because of its high
spatial resolution capability, non-invasive nature and its capability to
avoid ionizing radiation. This technique is able to exploit nuclear magnetic
resonance (NMR) principles to produce cross sectional images of a living
organism, the basic process is outlined in the below diagram. In general,
MRI scanners are tuned to effect water protons within the tissues of the
subject, prior to being placed in the strong uniform magnetic field of the
scanner, the magnetic moments of the water protons are randomly
oriented with respect to each other (Figure 1 panel 1). Once placed inside
the magnetic field, these magnetic moments align with the applied field
axis in one of two orientations (Figure 1 panel 2). A very finely tuned
polarised radio frequency (RF) pulse is then applied to the protons, this
pulse is precisely tuned to resonate with only the water protons, this
pulse contributes enough energy to the protons to force a spin transition
away from the alignment of the applied magnetic field (Figure 1 panel 3).
The water protons then decay from this excited state and re-align their
magnetic moments with the applied external field, this process re-emits
RF radiation which is detected by the scanner and interpreted into an
image based on the time taken for the protons to relax to the ground state
(Figure 1 panel 4).
92
Figure 1: Magnetic excitation cycle of water protons to produce an MR contrast image.
In order to form a coherent image, the relative position of each
emitted signal must be deciphered. This is accomplished by applying an
additional gradient field across the sample which makes the magnetic field
strength vary based on position within the target. This translates into a
predictable
change
in
emitted
radio
frequency
from
the
protons
comparative to their position and their relative location can be obtained
using inverse Fourier transformation. MRI experiments undertaken within
this thesis were completed on a on a 3.0 Tesla clinical Siemens Trio MRI
scanner using a 12-channel head coil and the following parameters: T2weighted imaging, spin echo sequence, transverse orientation, echo time
(TE) = 76 ms, repetition time (TR) = 2000 ms, matrix 320x320, slice
93
thickness= 1.70mm. Specific scanning protocols will be mentioned as the
relevant experiments are discussed.
2.1.6 Confocal microscopy
Confocal microscopy takes its name from the fact that there are two
focal points, one on each of the illumination and detection light paths.
Typically mixes of krypton/argon and helium/neon gas lasers are used to
produce a range of very specific wavelengths to be sent down the
excitation light path. After passing through the first pinhole to remove out
of focus light the light is reflected off a beam splitter and sent to the
objective lens. In fluorescence microscopy this beam splitter is replaced by
a dichromic filter which reflects only certain wavelengths. On the return
path the emitted light passes through the beam splitter or dichromic filter,
and passes through the final pinhole before the detector to remove
unfocused light.
94
Figure 2: Confocal microscope arrangement, image courtesy of Dirk Butcher from The
Marder Lab [1]
This pinhole setup allows for high resolution imaging of a very thin
focal plane within the sample and much sharper imaging of fluorescence
within samples due to the removal of out of focus light. In this thesis
confocal microscopy is used to image the interaction of human cells with
nanomaterials and the instrument used was a Nikon A1 Confocal
microscope.
95
2.1.7 Superconducting Quantum Interference
Device (SQUID)
SQUID is a highly sensitive technique used to measure magnetic
fields and can quantify the magnetic properties of a material. It operates
by measuring the difference in the voltage across a superconducting loop
broken by either 2 (DC) or 1 (RF) Josephson junctions. These junctions
are formed by either a thin insulating layer or constriction between two
identical
superconductors.
Essentially,
each
superconductor
has
associated with it an arbitrary phase θ, while two weakly connected
superconductors the phase is equal to δ = θ1 – θ2. This results in a phase
difference between the junctions given the bias current that is passing
through each side of the loop. This electrical behaviour is exceptionally
stable as the flux loop can only maintain flux in multiples of the flux
quantum and if an external flux is applied, an observable voltage change
between the junctions occurs. In this way, if a material with magnetic
properties is placed inside the ring, and a known external field is applied,
the magnetic flux of the sample can be inferred by the difference to the
expected voltage between the junctions at known magnetic flux input.
96
I
Josephson Junctions
i
V
i
I
Figure 3: Schematic of the superconducting loop and Josephson Junctions in a DC
SQUID magnetometer.
SQUID is an exceptionally sensitive technique, so sensitive in fact it
is able to measure the magnetic fields produced by biological systems.
However the superconducting property of the loops requires that they
operate at exceptionally low temperatures, some as low as 2 K, and
require a liquid helium bath to operate. In this thesis SQUID is used to
quantify the magnetic properties of magnetite nanoparticles, the model
used was a Quantum Design MPMS-XL5 operating at 4K.
97
2.2.1 References
1.
Butcher, D. Guide to microscopy. 2012 [cited 2012 02/06/2012]; Available from:
http://www.bio.brandeis.edu/marderlab/microscopyB.html.
98
Chapter 3
3.1.0 Silica coated quasi cubic magnetite as a
T2 contrast agent
This
chapter
presents
and
discusses
the
fabrication,
characterisation and application of quasi cubic particles produced by
thermal decomposition. The particles were characterised by TEM, XRD
and SQUID before having their biocompatibility and cell uptake compared.
The quasi cubic particles were then applied as a contrast agent for MR
imaging under clinical conditions.
99
3.1.1 Introduction
The
following
chapter
documents
the
fabrication
and
characterisation of silica coated magnetite nanoparticle for use as T2weighted MRI contrast agents. The production methodologies and
materials characterisation regarding
structure, cytotoxicity, particle
stability and relaxivity will be discussed, as will in vivo, in vitro
applications.
The initial goal was to produce a magnetite@silica (@ denotes a
―magnetite core-silica shell‖ structure) with a total diameter of less than
100 nm, along with good particle stability, high production yield, high
magnetisation saturation and low cyto-toxicity. As previously discussed in
section 1.6.5, it was determined that the thermal decomposition method
proposed by Park et al. [1] was the most promising magnetite fabrication
technique; based on the high yield, magnetisation and size monodispersity
parameters.
The main drawback of this method is the hydrophobicity of the
resulting particles, this was to be overcome by the addition of a silica
coating based on the Stöber process [2], similar to that presented by Fang
et al. [3]. This silica coating process was chosen for its ease of large batch
production, particle stability and availability of attachment sites for later
modification with targeting molecules. This chapter discusses both silica
coated and uncoated particles and will denote the coated particles as
―Mag@SiO2‖ and the uncoated simply as ―Mag‖.
100
3.2.1 Materials and methods
All chemicals were purchased from Sigma-Aldrich and used as
received without further modification. The prostate cancer cells (PC3 cell
line) were purchased from American Type Culture Collection (ATCC).
CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) kit
was purchased from Promega Corporation.
3.2.2 Synthesis of iron oxide nanoparticles
The process used to fabricate the magnetic nanoparticles for use in
this study was thermal decomposition of organic iron precursors as
reported by Park et al. [1] with significant modification. The iron-oleate
compound was synthesised by dissolving 5.4 g iron chloride and 18.25 g
sodium oleate in a solution of 70 ml hexane, 40 ml ethanol and 30 ml
distilled water. The solution was homogenised, then refluxed at 70 °C for 4
hours, then the organic layer containing the iron-oleate complex was
separated and washed with 30 ml distilled water in a separating funnel.
All remaining solvent was removed from the compound by heating in an
oven overnight at 100 °C, leaving the waxy iron-oleate residue behind. 9 g
(1.42 Mol) of the iron-oleate was then dissolved in 63.3 ml 1-octadecene
and refluxed under nitrogen at 320 °C in the presence of 1.425 g oleic acid
as a capping agent. After aging at 320 °C for 30 min, the solution was
allowed to cool to room temperature. To precipitate the magnetite
particles, 250 ml ethanol was added to the black mixture and sonicated
101
for 30 minutes. The particles were centrifuged out of solution and were
collected and washed 3 times in ether and ethanol. All solvent was then
removed and the particles were allowed to dry under a flow of nitrogen to
yield the magnetite as a dry powder.
3.2.3 Silica shell formation process
Silica-coated iron oxide nanoparticles (Mag@SiO2) were prepared
using a method significantly modified from Fang et al and Morel et al [3, 4]
wherein controlled hydrolysis of silica precursor in the presence of
magnetite nanoparticles was performed. In the present approach, preformed magnetic particles were used as nucleating sites for subsequent
hydrolysis of silica precursor around them. In our process, the particles
were first stabilised using a layer of citric acid by refluxing the particles in
ethanol and 2 ml solution of 5 mg/ml trisodium citrate for 6 hours. The
resulting particles were then washed twice in water and then redispersed
in 15 ml ethanol. To this solution 2 mL deionized water (MilliQ) and 1 mL
of ammonia (25% solution) was added while immersed in a sonicator
programmed to switch on for 1 in every 10 min. Further, an overhead
stirrer was used to mix the solution while 4 mL of 1:60 (TEOS : ethanol)
was added at the rate of 0.4 ml/h using a syringe pump. The coating
process was allowed to continue for 12 hours at room temperature. The
silica coated iron oxide nanoparticles were centrifuged, washed three
times with ethanol and redispersed in MilliQ-water.
102
3.3.1 Characterisation
The relevant XRD, TEM and SQUID instrumentation information
has been previously discussed in chapter 2. The following pertains to
certain instruments or procedures that may have a large variation in their
conditions of use which need to be specified for appropriate comparison.
3.3.2 T2 relaxation
The T2 relaxation was investigated by first preparing solutions of
the magnetic particles with increasing concentration, from 10 to 100
µg/mL of particles in MilliQ-water. The solutions (1mL) were then scanned
in triplicate, using Milli-Q water as a control. Scanning was undertaken
on a clinical 3.0 T Siemens Trio MRI scanner, fitted with a 12 channel
head coil. T2 relaxation was determined using a single echo sequence (SE)
and constant repetition times (TR) of 200 ms, and multiple echo times (TE)
ranging between 0.99 – 100 ms. The signal was fitted to the established
mono-exponential expression by plotting as a function of TE. The T2 rate
was calculated by plotting the reciprocal of the relaxation rate at a TE
value of 10.86 ms. This was a function of the relative molar iron
concentration as measured by AAS, the T2 value was calculated by linear
regression of the slope of this line. The T2 values used are averages
calculated from experiments in triplicate with error calculated as standard
error of the mean.
103
3.3.3 Cell uptake
Human prostate cancer cells (PC3 cell line) were routinely cultured
at 37°C in a humidified atmosphere with 5% CO2 using RPMI 1640
medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin,
1% streptomycin/penicillin and 1 mM L-glutamine. For sub-culturing,
PC3 prostate cancer cells were detached by washing with phosphate
buffered saline (PBS) and incubating with trypsin-EDTA solution (0.25%
trypsin, 1 mM EDTA) for 5 min at 37°C, followed by washing and
incubation with supplemented RPMI 1641 medium. For cell uptake, the
cells were first seeded in 24-well polystyrene dishes for 24 h, followed by
incubation with nanoparticles for 24 h at 37°C in complete cell media, and
subsequent three times washing of cells with PBS, before imaging under
an inverted microscope. For cytotoxicity assays, the viability of PC3
prostate cancer cells exposed to Mag@SiO2 nanoparticles in the absence of
cell growth medium was determined. A CellTiter 96 AQueous One Solution
Cell
Proliferation
Assay
(Promega)
kit
containing
the
tetrazolium
compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS), was used to monitor cell viability
according to the manufacturer's protocols. MTS colour change was
monitored using a plate reader at 490 nm, and cell viability data was
plotted by considering the viability for the untreated cells as 100%.
Experiments were performed in triplicates, and error bars represent
standard experimental errors.
104
3.3.4 MRI study
MRI studies were performed for nanoparticle solutions stored in
phantoms, in PC3 prostate cancer cells after nanoparticle uptake, and in
a mouse model with breast cancer. For phantom MRI studies, phantoms
were prepared in Eppendorf tubes with Mag@SiO2 nanoparticles at three
different Fe concentrations (0.18 mM, 0.9 mM, 1.79 mM) and a saline
solution without any nanoparticles was used as a control. For in vitro MRI
studies, PC3 cancer cells were cultured using the above protocol in 24
well polystyrene plates, and incubated for 24 h with Mag and Mag@SiO2
nanoparticles at three different concentrations (0.18 mM, 0.9 mM, 1.79
mM) and a control with cells but no nanoparticles. MRI measurements for
phantoms and PC3 cells were performed with a clinical 3.0 Tesla Clinical
Siemens Trio MRI scanner using a 12-channel head coil and the following
parameters: T2-weighted imaging, gradient echo sequence, multiple echo
time (TE) ranging from 0.99–100 ms, repetition time (TR) = 200 ms, matrix
128×128, slice thickness of 3 mm.
Relaxation rates (R2) were determined by using a single echo
sequence (SE) with a constant TR of 200 ms and multiple TE ranging from
0.99–100 ms. The signal was plotted as a function of echo time and fitted
to obtain the R2 values. The R2 values of the Mag@SiO2 in phantoms and
PC3 cells were determined by plotting the relaxivity at a TE of 10.86 ms,
as a function of molar iron concentration in respective samples, and
extracting the T2 value from the slope by linear regression of data points
obtained at lower Fe concentration values.
105
Only lower Fe concentrations were used to determine the T2 values,
because with increasing Fe concentrations above a particular threshold,
the MR signals tend to lose their linearity. For the in vitro MRI
measurements in phantoms and PC3 cells, enhancement of the R2 signal
within the PC3 cells was calculated by:
Where R2 is the average of triplicate scans read directly from the scanner.
For in vivo MRI experiments, breast tumour bearing mice were
developed in-house, anaesthetised with ketamine (80 mg/kg body weight)
and xylazine (5 mg/kg body weight), and placed within the 12-channel
head coil. Images were acquired before and after injection of 100 µL of
Mag@SiO2 particles suspension of 100 µg/mL concentration in saline
locally at the tumour site. A T2-weighted spin echo sequence was acquired
with TE/TR of 60/200 ms, a slice thickness of 3 mm and a 128x128
matrix. Data analysis was performed manually by placing ROIs in tumour
and tissue areas on the images. It is important to note that all MR
measurements were undertaken in a clinical scanner under guidance and
supervision of technicians from the Peter MacCallum Cancer Research
Centre. As such this data represents clinically obtained data as all
imaging parameters were constrained to those of clinical facilities.
106
3.4.1 Results and discussion
TEM
analysis
of
the
pristine
and
silica
coated
magnetic
nanoparticles is shown in Figure 1. The overall morphology of the particles
produced was polydisperse and quasi-cubic; however the synthesis
method did yield gram quantities of particles ranging from 40-50 nm in
diameter in a single batch. From the TEM images it is possible to
determine internal material boundaries within the individual quasi-cubes,
this leads us to the possibility that the quasi-cubes formed as a result of
aggregation of several smaller spherical particles late in the growth
process. Due to the novel morphology of the particles produced it was
decided to continue characterisation of the material for the MR contrast
application despite the polydispersity of the product.
The shelf life of commercially available MRI contrast agents is in fact
one of the major limitations associated with clinical applicability of such
materials. Previously, silica shell coatings have demonstrated increased
biocompatibility and particle stability, as well as a facile surface for
further bio-functionalisation in different nanomaterials [5-7]. Therefore, to
provide chemical stability to these quasi cubic magnetic nanoparticles, a
silica shell was grown around quasi-cubic magnetite particles (within 3
days of their synthesis), thereby producing core-shell, magnetite-silica,
nanoparticles Figure 1b.
The controlled silica coating of Mag nanoparticles led to formation of
Mag@SiO2 core-shell structures with an approximately 20 nm silica shell
107
around a 45 nm quasi-cubic magnetite nanoparticle (Figure 1 and inset).
Low magnification TEM analysis of Mag@SiO2 core-shell structures
indicated that most of the magnetite nanoparticles retained their quasicubic morphology after silica coating, and more than 75% of particles in
the sample were found to be individually coated with a silica shell.
However, less than 25% of structures consisted of either two or three or
no Mag particles within the silica shell. Notably, this type of particle
distribution is typical of a chemical synthesis route, which is not
necessarily always explicitly acknowledged in the prevailing literature.
Additionally, we observed that after coating Mag nanoparticles with silica,
the Mag@SiO2 particles remained stable in phosphate buffer saline (PBS)
solution up to 1 mg/mL concentration, as well as in the readilydispersible powder form for at least up to 6 months. The TEM image
shown in Figure 1 was acquired after 6 months of storage of Mag@SiO2
nanoparticles at room temperature and was similar to those imaged
immediately after synthesis. This suggests that a silica coating over Mag
nanoparticles can significantly improve their stability for long-term
storage conditions, thus retaining their medical properties by improving
their shelf life. This is one of the crucial parameters for developing MRIbased contrast agents for clinical and commercial applications.
108
Figure 1: TEM images of the synthesised Mag particles (A) and (B) Mag@SiO2 particles
demonstrating their size and distribution are shown.
3.4.2 XRD
The particles produced by the thermal decomposition method were
examined under XRD and the resulting patterns are displayed in Figure 2.
The pattern produced, fitted well with the JCPDS card file for Fe3O4
magnetite (card file number 75-0449) and is typical of spinel phases of
iron oxide. Although it is difficult to rule out the presence of maghemite
from XRD analysis, given the similarity of the diffraction patterns, there
was no dominance of 221, 203 or 116 peaks in the sample that would
indicate the presence of a large amount of maghemite within the product.
Although it is expected that some maghemite and hematite would have
been present simply due to post fabrication oxidation, however the
functional mass of the product was determined to be magnetite from the
XRD analysis.
109
*
(422)
(511)
(400)
(220)
(440)
Counts (a.u)
(311)
Mag
Mag@SiO2
20
30
40
2°
50
60
70
Figure 2 XRD patterns of the as synthesised magnetite (Mag) and silica coated magnetite
(Mag@SiO2) particles
Figure 2 also demonstrates the XRD pattern obtained from the
sample after silica coating. Due to the surface of the magnetite being
coated in approximately 20 nm of silica, less of the magnetite signal was
obtained, however the (311) and (440) major peaks were still visible. An
additional peak was observed at 29.1° 2θ in the sample coated with silica,
this peak may be attributed to a β-magnetite silica mixed phase (220) that
formed at the boundary between the core and shell structures (JSPDS file
110
no 73-0963). The silica coating only adds a few low angle amorphous
humps to the spectra and an overall dampening of signal intensity from
the magnetite.
3.4.3 SQUID
To fulfil the requirements of an MR contrast agent it was essential
for the material to exhibit a high net magnetisation value. To determine
the magnetic susceptibility of the Mag@SiO2, the material was analysed
using a SQUID magnetometer. The curve in Figure 3 illustrates the
magnetic behaviour exhibited by the Mag@SiO2, which reaches a
maximum value of 74.4 emu g-1, that is comparable to the commercially
available Resovist [8]. The curve also shows low hysteresis and
remanence, suggesting a predominantly superparamagnetic behaviour.
With the high emu value and superparamagnetic nature, it was expected
that the material would produce good contrast during the MR imaging
process.
111
emu/g
80
60
40
20
-22.50k
-15.00k
-7.50k
Field (Oe)
0
0.00
7.50k
15.00k
22.50k
20
-20
10
-40
-400.0
0
0.0
400.0
-60
-10
-80
-20
Figure 3: SQUID measurement of the as synthesised magnetite particles. The value
shown is the saturation magnetisation of the Mag@SiO 2 particles in emu/g at 4 Kelvin.
The insert displays a magnification of the central axis.
3.4.4 Cell uptake
A cell uptake study was undertaken using human prostate cancer
PC3 cells to determine the comparative ability of the uncoated Mag
particles and the Mag@SiO2 to be internalised by human cells (Figure 4).
Both the Mag@SiO2 and Mag particles were incubated with PC3 cells for
24 hours at a concentration of 50 µg/ml. When PC3 cells were exposed to
Mag nanoparticles, it was observed that bare Mag nanoparticles tended to
form large aggregates (of dimensions similar to cell size) over a 24 h
exposure period; which restricted their ability to be internalised by PC3
cells (Figure 4B). As can be inferred from Figure 4B, these large clusters of
bare Mag nanoparticles predominantly attach to the exterior of the cells,
112
and are difficult to be internalized by PC3 prostate cancer cells.
Conversely, after SiO2 coating, Mag@SiO2 nanoparticles remain welldispersed in the solution even after 24 h, which facilitates their more
efficient uptake by PC3 cells, as can be seen from a higher density of
Mag@SiO2 nanoparticles inside PC3 prostate cancer cells (Figure 4C).
Our
group
and
others
have
previously
demonstrated
that
nanoparticle size and aggregation in biological media can play a crucial
role in cellular uptake processes, as non-specific uptake of sub-100 nm
nanoparticles is generally observed via endocytosis mechanism of the cells
[9-14]. Aggregation of Mag nanoparticles in biological media, and
subsequent aggregation reduction after silica coating, clearly suggests the
advantage
of
Mag@SiO2
core-shell
nanoparticles
over
bare
Mag
nanoparticles for biological applications. Based on results from cell uptake
studies, bare Mag nanoparticles were found to be unsuitable for biological
applications due to aggregation. Therefore only Mag@SiO2 nanoparticles
were chosen for further studies regarding their suitability for MRI
applications.
113
Figure 4: Optical microscopy images of PC3 human prostate cancer cells (control) grown
for 24 h. (A) in the absence of nanoparticles (control), and in the presence of (B) Mag and
(C) Mag@SiO2 nanoparticles followed by three washings with PBS. Scale bar 50 µm.
3.4.5 Cytotoxicity
Before exploring any further MR applications of the Mag@SiO2
nanoparticles,
biocompatibility
profiling
of
these
particles
was
accomplished using an in vitro MTS-based cytotoxicity assay (Figure 5),
which is a common procedure for biocompatibility analysis [15]. Particle
uptake was then correlated to the relative cytotoxicity to PC3 cells across
increasing particle concentrations ranging from 1-100 µg/ml. As an
internal control, the MTS assay was run with mirrored concentrations of
nanoparticles and MTS dye without cells. The reduction of the dye caused
by nanoparticles without cell media was subtracted from data to ensure
the viability measurements were not false positives and set a baseline for
each individual concentration. Previous studies indicate that iron oxide
nanoparticles show negligible toxicity at lower concentrations but can be
mildly toxic at higher concentrations. [16-19] It is evident from Figure
5 that Mag@SiO2 nanoparticles did not significantly affect PC3 cell
114
viability up to concentrations of 50 µg/mL, where greater than 80% cell
viability was still maintained. However, further increases in particle
concentration equivalent to 100 µg/ml Fe resulted in a cell viability loss of
up to 30%. This suggests the Mag@SiO2 nanoparticles may be suitable for
MRI applications if the administered dosage is below that which induces
toxicity, yet is still able to produce significant contrast during imaging.
However, this aspect may require further detailed investigation, wherein
the effect of Mag@SiO2 nanoparticles on cytokine production profile of
cells will need to be investigated.
Figure 5: Biocompatibility of Mag@SiO2 nanoparticles assessed using MTS assay after
their exposure to PC3 cancer cells for 24 h with respect to different Fe concentration in
Mag@SiO2 nanoparticles.
3.4.6 Relaxivity
Relaxivity is a measure of the efficiency of a MR contrast agent to
enhance the proton relaxation rate and increase the efficiency to which
115
image contrast is produced during MRI [20]. The relaxivity of the
Mag@SiO2 was assessed on a 3 Tesla clinical MRI scanner to measure its
performance as a T2 MR contrast agent. The relaxivity measurements
were performed both on nanoparticles as suspension in phantoms, as well
as after being internalised by PC3 prostate cancer cells. Mag@SiO2
nanoparticles were found to have a high relaxivity value of 263.23
L/mmol/s in cell free suspensions, and 230.90 L/mmol/s after uptake by
PC3 cells. Having a high relaxivity value along with high mass
magnetisation values are important considerations when developing T2
contrast agents. This is due to the spin-spin relaxation process of protons
in water molecules surrounding the nanoparticles, being facilitated by the
large magnitude of magnetic spins from within the nanoparticles [21, 22].
Mag@SiO2 nanoparticles with high mass magnetization and high relaxivity
values may therefore result in strong T2-weighted MR signal intensity
decreases as measured by MRI [23]. This is critical in allowing nanomolar
activity of contrast agents, which will reduce the overall required contrast
agent dose to the patients.
116
Figure 6: Evaluation of Mag@SiO2 nanoparticles as a T2 MR contrast agent is shown in
the form of % signal enhancement with increasing concentration of Fe. A) demonstrates
performance in a phantom studies compared to a pure saline control; while B) presents
results obtained after uptake by PC3 cells.
The relaxivity data in Figure 6 also suggests a reduction in the
relaxivity value of Mag@SiO2 nanoparticles in PC3 cells after cellular
uptake compared with that in suspension. This finding corroborates well
with previous studies, which showed that the relaxivities of native iron
oxide nanoparticles were higher compared to those after accumulation in
the cells [24, 25]. The mechanisms responsible for this effect have not yet
been fully understood, however it may possibly be attributed to the
confinement of nanoparticles within endosomes of the target cells. This
might cause a build-up of magnetic field inhomogeneity after sub-cellular
compartmentalization, which would conversely be absent in uniformly
distributed nanoparticles in suspensions [26].
Additionally, the different geometrical arrangement of nanoparticles
in suspensions vs. that which occurs in cells may alter antiferromagnetic
117
coupling as a result of clustering within the sub-cellular compartments
and may play some further role in reducing relaxivity values after cellular
uptake [6, 26]. There is also some expected loss of particles as not all the
Mag@SiO2 is taken into the cells prior to being washed, resulting in a
lower signal.
In comparison to the relaxivity values of 230–269 L/mmol/s
observed for Mag@SiO2 nanoparticles in this study, the commercial
Resovist nanoparticle contrast agent has been reported to have a lower
value of 151 L/mmol/s [8]. The observed relaxivity value of Mag@SiO2
nanoparticles prepared in this study is also higher than those reported for
undoped magnetite particles (218 L/mmol/s) in recent detailed studies
[27]. For doped magnetic particles, it has been reported that high
relaxivities of up to 358 L/mmol/s can be achieved by doping magnetite
with Mn (MnFe2O4) [28]. However, potential leaching of Mn during
administration of these MR contrast agents in the body might pose
cytotoxicity issues [19] and to the best of my knowledge, the author was
the first to report undoped Mag@SiO2 nanoparticles with such high
relaxivity values in literature [29].
In addition, the relaxivity studies performed as a function of
different concentrations of Fe in Mag@SiO2 nanoparticles, both as a
suspension in phantoms Figure 6 (A), and after 24 h of nanoparticle
uptake by PC3 prostate cancer cells Figure 6 (B); revealed that the
Mag@SiO2 nanoparticles act as excellent T2-weighted contrast agents.
This contrast value is visualised by an image darkening effect,
118
demonstrated by drop in R2 (
) signal intensity with increasing Fe
concentrations. This is clearly visible in Figure 6 when comparing the 100
µg/mL Fe concentration to the 50 µg/ml. Under these conditions the
Mag@SiO2 nanoparticles provide a signal enhancement of ∼90% in
comparison to more than 70% signal enhancement during imaging of PC3
prostate cancer cells. This is considered significant signal enhancement in
comparison to most of the previously reported materials, in which
generally only 15–20% signal enhancement has been observed [6]. Such
strong MR signal enhancement is expected from Mag@SiO2 nanoparticles
because of their relatively high relaxivity and saturation magnetization
values.
3.4.7 Tissue imaging
The Mag@SiO2 particles had demonstrated biocompatibility and
excellent T2 contrast production in both phantom and cell MR studies,
however performance under these conditions is rarely comparable to that
which occurs in vivo. To investigate the performance of the Mag@SiO2
under such conditions, 10 µg of Mag@SiO2 particles were injected directly
into a breast tumour site of a nude mouse and imaged on the same
clinical 3 Tesla scanner. The images in Figure 7 illustrate the contrast
achieved within tissue directly at the site of injection compared to the
surrounding tissue. This final experiment demonstrates the ability for
119
Mag@SiO2 nanoparticles to effectively operate as a T2 weighted MR
contrast agent within tissue.
Figure 7: T2-weighted MR images of nude mice with breast tumour obtained (A) before
and (B) after injection of MR contrast agent, obtained using a 3 Tesla MR scanner.
120
3.5.1 Conclusion
There are many important factors that must be considered when
developing an MR contrast agent and the Mag@SiO2 nanoparticles
fabricated in this initial study have successfully addressed a majority of
them.
The
core
magnetite
nanoparticles
possess
both
high
net
magnetisation values along with superparamagnetic behaviour; and are
capable of being fabricated in batches large enough to be viable for clinical
applications.
The addition of the silica coating rendered the particle stable in
water, reduced aggregation, and cause little cytotoxicity within the clinical
dosage range required to elicit a contrast response. In this study, a facile,
large-scale
synthesis
of
quasi-cubic
magnetite
and
Mag@SiO2
nanoparticles of sub-100 nm size has been demonstrated. The Mag@SiO2
nanoparticles reported here have a shelf life of more than 6 months, and
are efficiently internalised by human cells without causing significant
aggregation or cellular toxicity.
The
biological
half-life
of
smaller
silica-coated
iron
oxide
nanoparticles (>60 nm) is expected to be further increased due to their
reduced interaction with the body fluids. This study therefore clearly
underlines the importance of SiO2 coating towards improving the uptake
of Mag@SiO2 nanoparticles by PC3 prostate cancer cells and improving
the shelf life of MR contrast agents. The Mag@SiO2 nanoparticles act as
121
promising T2 contrast agents offering a potentially viable option as a
commercial MR contrast agent.
This
is
attributable
to
their
small
size,
high
MR
signal
enhancement, relative biocompatibility, longer shelf life, and highly
modifiable silica surface chemistry which will allow the adhesion of
multiple molecular markers for targeted MRI in further studies discussed
in chapter 4. These characteristics of a T2 contrast agent are highly
desirable for magnetic resonance imaging applications at the pre-clinical
level and for later use clinically. Given the success of this material
towards its application, further modifications to both the structure and
surface coating have been investigated for application towards targeted
MR contrast using cell specific targeting molecules. This application and
the experimental results are discussed in the following chapter.
The work described in this chapter was reviewed and subsequently
published in PLOS 1 under the title ―Quasi-cubic magnetite/silica coreshell nanoparticles as enhanced MRI contrast agents for cancer imaging‖
[29]
122
3.6.1 References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Park J, An KJ, Hwang YS, Park JG, Noh HJ, Kim JY, et al. Ultra-large-scale
syntheses of monodisperse nanocrystals. Nature Materials 2004,3:891-895.
Stober W, Fink A, Bohn E. Controlled growth of monodisperse silica spheres in
micron size range. Journal of Colloid and Interface Science 1968,26:62-&.
Fang CL, Qian K, Zhu JH, Wang SB, Lv XX, Yu SH. Monodisperse alphaFe(2)O(3)@SiO(2)@Au
core/shell
nanocomposite
spheres:
synthesis,
characterization and properties. Nanotechnology 2008,19.
Morel AL, Nikitenko SI, Gionnet K, Wattiaux A, Lai-Kee-Him J, Labrugere C, et al.
Sonochemical approach to the synthesis of Fe3O4@SiO2 core-shell nanoparticles
with tunable properties. Acs Nano 2008,2:847-856.
Bardhan R, Chen WX, Perez-Torres C, Bartels M, Huschka RM, Zhao LL, et al.
Nanoshells with Targeted Simultaneous Enhancement of Magnetic and Optical
Imaging and Photothermal Therapeutic Response. Advanced Functional Materials
2009,19:3901-3909.
Hilger I, Trost R, Reichenbach JR, Linss W, Lisy MR, Berndt A, et al. MR imaging
of Her-2/neu protein using magnetic nanoparticles. Nanotechnology 2007,18.
Goloverda G, Jackson B, Kidd C, Kolesnichenko V. Synthesis of ultrasmall
magnetic iron oxide nanoparticles and study of their colloid and surface
chemistry. Journal of Magnetism and Magnetic Materials 2009,321:1372-1376.
Vogl TJ, Schwarz W, Blume S, Pietsch M, Shamsi K, Franz M, et al. Preoperative
evaluation of malignant liver tumors: comparison of unenhanced and SPIO
(Resovist)-enhanced MR imaging with biphasic CTAP and intraoperative US.
European Radiology 2003,13:262-272.
Ohmori M, Matijevic E. Preparation and properties of uniform coated inorganic
colloidal particles .8. silica on iron. Journal of Colloid and Interface Science
1993,160:288-292.
Ohmori M, Matijevic E. Preparation and properties of uniform coated colloidal
particles .7. silica on hematite. Journal of Colloid and Interface Science
1992,150:594-598.
Murakami Y, Kenjo A, Sadoh T, Yoshitake T, Miyao M. Solid-phase crystallization
of beta-FeSi2 thin film in Fe/Si structure. Thin Solid Films 2004,461:68-71.
Walkey CD, Olsen JB, Guo H, Emili A, Chan WCW. Nanoparticle Size and Surface
Chemistry Determine Serum Protein Adsorption and Macrophage Uptake. Journal
of the American Chemical Society, 2012. 134(4): p. 2139-2147.
Florez, Herrmann C, Cramer JM, Hauser CP, Koynov K, Landfester K, et al., How
Shape Influences Uptake: Interactions of Anisotropic Polymer Nanoparticles and
Human Mesenchymal Stem Cells. Small, 2012. 8(14): p. 2222-2230.
Albanese A, Tang PS, Chan WCW. The Effect of Nanoparticle Size, Shape, and
Surface Chemistry on Biological Systems, in Annual Review of Biomedical
Engineering, Vol 14, M.L. Yarmush, Editor. 2012. p. 1-16.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application
to proliferation and cytotoxicity assays. Journal of Immunological Methods
1983,65:55-63.
Alberola AP, Radler JO. The defined presentation of nanoparticles to cells and
their surface controlled uptake. Biomaterials 2009,30:3766-3770.
Nel A, Xia T, Madler L, Li N. Toxic potential of materials at the nanolevel. Science
2006,311:622-627.
Prina-Mello A, Crosbie-Staunton K, Salas G, Puerto MM, Volkov Y.
Multiparametric Toxicity Evaluation of SPIONs by High Content Screening
Technique: Identification of Biocompatible Multifunctional Nanoparticles for
Nanomedicine. Ieee Transactions on Magnetics, 2013. 49(1): p. 377-382.
Chang Y, Lee GH, Kim TJ, Chae KS. Toxicity of Magnetic Resonance Imaging
Agents: Small Molecule and Nanoparticle. Current Topics in Medicinal Chemistry,
2013. 13(4): p. 434-445.
123
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Weissleder R, Stark DD, Engelstad BL, Bacon BR, Compton CC, White DL, et al.
Superparamagnetic iron-oxide - pharmacokinetics and toxicity. American Journal
of Roentgenology 1989,152:167-173.
Jun Y-w, Huh Y-M, Choi J-s, Lee J-H, Song H-T, KimKim, et al. Nanoscale Size
Effect of Magnetic Nanocrystals and Their Utilization for Cancer Diagnosis via
Magnetic Resonance Imaging. Journal of the American Chemical Society
2005,127:5732-5733.
Koenig SH, Kellar KE. Theory of 1/t-1 and 1/t-2 nmrd profiles of solutions of
magnetic nanoparticles. Magnetic Resonance in Medicine 1995,34:227-233.
Wang YXJ, Hussain SM, Krestin GP. Superparamagnetic iron oxide contrast
agents: physicochemical characteristics and applications in MR imaging.
European Radiology 2001,11:2319-2331.
Muller K, Skepper JN, Posfai M, Trivedi R, Howarth S, Corot C, et al. Effect of
ultrasmall superparamagnetic iron oxide nanoparticles (Ferumoxtran-10) on
human monocyie-macrophages in vitro. Biomaterials 2007,28:1629-1642.
Wuang SC, Neoh KG, Kang E-T, Pack DW, Leckband DE. HER-2-mediated
endocytosis of magnetic nanospheres and the implications in cell targeting and
particle magnetization. Biomaterials 2008,29:2270-2279.
Mansoor A. Nanotechnology for Cancer Therapy. 1 ed: CRC Press; 2007.
Sun SH, Zeng H, Robinson DB, Raoux S, Rice PM, Wang SX, et al. Monodisperse
MFe2O4 (M = Fe, Co, Mn) nanoparticles. Journal of the American Chemical Society
2004,126:273-279.
Lee J-H, Huh Y-M, Jun Y-w, Seo J-w, Jang J-t, Song H-T, et al. Artificially
engineered magnetic nanoparticles for ultra-sensitive molecular imaging. Nature
Medicine 2007,13:95-99.
Campbell JL, Arora J, Cowell SF, Garg A, Eu P, Bhargava SK, et al. Quasi-cubic
magnetite/silica core-shell nanoparticles as enhanced mri contrast agents for
cancer imaging. PLoS ONE 2011,6.
124
Chapter 4
4.1.0 Targeted MR contrast materials
The previous chapter focused on the fabrication and application of
silica coated quasi cubic magnetic nanoparticles and their performance as
an MR contrast agent. In this chapter we will investigate the application of
such a material for the purpose of cell specific MR enhancement using
further particle and surface modification.
125
4.1.1 Introduction
The use of iron oxide based nanoprobes for targeted MR imaging
has gained increasing attention, however very few of the materials
demonstrated all the required attributes that the application requires.
Colloidal stability, biocompatibility, small size distribution, low toxicity,
high magnetisation values and simplistic fabrication of clinically relevant
quantities; are all required of the material before it can be considered
applicable in a broad sense. This chapter aims to present and discuss the
fabrication of a magnetite based Herceptin modified nanoprobe based on
the Mag@SiO2 material presented in the previous chapter.
The Mag@SiO2 presented previously possessed many of the
attributes required to satisfy the requirements for a magnetite based
targeted MR nanoprobe, however it fell short on several aspects. The
overall particle size needs to be reduced to ensure a long blood circulation
time and the surface needs further modification to accommodate the
attachment of a targeting antibody. Due to the difference to the previous
material and addition of subsequent components, this chapter will denote
the Mag@SiO2 particles as ―Fe@SiO‖ to avoid confusion and highlight the
difference to the material previously produced.
Imaging modalities can offer a way to monitor the response of
patients to immune therapies and identify those that have stopped
responding to the treatments. This is possible if an MR contrast agent is
able to specifically target cancerous cells that overexpress certain surface
126
proteins such as HER2, the following chapter will discuss the role of
magnetic nanoparticles for targeted imaging and the development of one
such material based on the previously discussed Mag@SiO2.
Currently, targeted imaging using iron oxide nanoparticles remains
in the preclinical setting due the particles used being rapidly cleared from
the body due to their large particle size. This was the first aspect of the
previous Mag@SiO2 material that required improvement. Through finer
control
of
the
fabrication
process
and
reduction
of
oleic
acid
concentration, smaller and more homogenous particles were able to be
fabricated. Biocompatibility of these new particles had to be re-confirmed
given the stark contrast in possible properties given size reduction. The
silica surface coating process also needed greater control to produce a
thinner surface that allowed further functionalisation. This was achieved
by decreasing the rate of TEOS hydrolysis on particles pre functionalised
with a layer of citric acid.
The surface of the particles then had to allow for the strong
adhesion of targeting molecules such as the antibody Herceptin®. This
was accomplished by simple amine functionalisation to create a strong
electrostatic bond. The toxicity of these magnetite-core\silica shell,
Herceptin® coated particles (Fe@SiO@HER) was examined, as was the
stability of the Herceptin® adhesion under physiological conditions. The
methods and results of these modifications to the material will be
discussed in the following sections along with the performance of this
material as a targeted MRI contrast agent both in vitro and in vivo.
127
4.2.1 Materials and methods
4.2.2 Reagents, chemicals and assay kits
All chemicals were purchased from Sigma Aldrich Pty LTD and used
as received without further modification. Cell lines (SKBR3, BT474 and
MCF7) were purchased from American Type Culture Association (ATCC).
CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) kit
was purchased from Promega Corporation. BCA protein assay was
purchased from Invitrogen. Herceptin® was a generous gift from the
Pharmacy Department at The Peter MacCallum Cancer Centre.
4.2.3 Synthesis of magnetite particles
The core magnetite particles were again synthesised using the high
temperature thermal decomposition method previously used in chapter 3,
modified from the work presented by Park et al. (2004) [1]. However
different aging duration and capping agent quantities were used in order
to produce a material with more desirable properties for the targeting
application.
An iron-oleate complex was first formed by dissolving 5.4 g of iron
chloride and 18.25 g of sodium oleate in a solution comprised of 40 ml
ethanol, 30 ml distilled water and 70 ml hexane. Once homogenised, the
solution was refluxed at 70°C for 4 hours, followed by separation of the
upper organic layer using a separatory funnel, washing and evaporating
128
off hexane, thereby leaving a waxy iron oleate complex. The iron oxide
nanocrystals were formed by dissolving 9.0 g of the iron oleate complex in
1.425 g of oleic acid and 63.3 mL of 1-octadecene, followed by reflux
under nitrogen until it reached 320 °C, at which point the temperature
was held for 20 minutes and the solution was then allowed to cool to room
temperature.
In order to gain greater control of the reaction an additional
measure to ensure all water removal from the iron oleate complex was
used. During the high temperature reflux step, the solution was first
taken to 110 °C and all water vapour allowed to escape prior to continuing
the temperature ramp to 320 °C. After cooling the solution to room
temperature, 250 mL of ethanol was added to the solution and the
magnetite particles were separated via centrifugation, followed by three
wash cycles with ethanol. A yield of up to 9 g of magnetic nanoparticles
per reaction was achieved under these laboratory conditions.
4.2.4 Synthesis of Fe@SiO nanoparticles
The as synthesised magnetite was then washed with 20 mmol nitric
acid solution to remove any surface contamination prior to silica coating.
The coating process was carried out much like it was before, using a
modification of the Stöber method [2] described by Fang et al. [3], however
a thinner layer of silica was to be deposited in this case.
129
2 mg of magnetite particles were first made hydrophilic by surface
coating with citric acid. This was accomplished by refluxing the particles
in 5 ml ethanol and 1 ml trisodium citrate solution (5 mg/ml) overnight.
The resulting particles were washed from solution via centrifugation and
washed 3 times in ethanol and MilliQ-water.
These magnetite particles were then added to 15 ml of ethanol and
3.75 ml Milli-Q water (4:1) and sonicated for 15 minutes. The water in the
sonicator bath was maintained at 28°C to ensure a constant reaction rate.
During the coating process the sonicator was switched on for 2 out of
every 5 minutes. The magnetite particles were continuously stirred via an
overhead stirrer fitted with a glass screw style stirring rod while the 1:160
/ TEOS : ethanol solution was injected into the solution via a syringe
pump at 0.1 ml/h.
Immediately prior to beginning the TEOS injection, 0.5 ml ammonia
(28%) was added to the ethanol, water and magnetite solution. This
process was allowed to continue for up to 3 hours, the thickness of the
silica shell could be altered by increasing or reducing the reaction time.
The silica coated particles were then centrifuged out of solution and
washed 3 times in ethanol and twice in water prior to redispersion in
MilliQ-water.
130
4.2.5 Conjugation of Fe@SiO to Herceptin®
The as prepared silica coated magnetite particles were surface
treated with cysteamine to provide binding sites for the addition of the
Herceptin® antibody. The silica coated particles were incubated in the
presence of excess cysteamine (5 mg/ml) at room temperature on a rotary
shaker for 2 hours (9 ml MilliQ-water, 1 ml cysteamine solution). The
particles were then washed 3 times with MilliQ-water to remove excess
cysteamine.
2 ml Herceptin® solution (1 mg/ml) was added to cysteamine coated
particles in 3 ml MilliQ-water and incubated with the cysteamine
functionalised particles at room temperature on a rotary shaker for 2
hours. The amount of Herceptin® conjugated on the surface of the silica
coated magnetite was quantified using a BCA protein assay kit according
to the manufacturer’s instructions.
4.2.6 Assessment of Fe@SiO-HER stability
4.2.6.1 Conjugation of FITC to Fe@SiO@HER
In order to establish the stability of the silica-cysteamineHerceptin® linkage, firstly Herceptin® had to be conjugated with a FITC
marker molecule, this was accomplished using a FluoroTag FITC
conjugation kit (Sigma, USA). The Herceptin® was first purified using a
Sephadex G-25M column (Pharmacia, Sweden) and then added to the
131
FITC
using
the
manufacturer’s
small
protocol.
scale
The
conjugation
procedure
FITC-Herceptin®
following
complex
was
the
then
conjugated to the surface of the Fe@SiO via the cysteamine linker after 2
hours incubation time at room temperature. The FITC-Herceptin®cysteamine-silica particles were then washed 3 times in MilliQ-water via
centrifugation.
The now FITC conjugated Herceptin® functionalised (Fe@SiO@HERFITC) particles were dispersed in water and exposed to pH variances of 5,
7.14, and 10 as well as human serum concentrations of 25%, 50% and
100%. Release of the Herceptin® from the surface of the Fe@SiO was
determined by the increase in fluorescence within the supernatant caused
by loss of Herceptin® after 24 hours, compared to standards prepared
using known concentrations of Herceptin®-FITC molecules.
4.2.7 In vitro studies
4.2.7.1 Cell culture
Breast cancer cell lines, SKBR-3, BT474 and MCF-7 cells were
routinely cultured at 37˚C in a humidified atmosphere with 5% CO2 using
RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1%
penicillin, 1% streptomycin/penicillin and 1mM L-glutamine (Sigma
Aldrich). For sub-culturing, cells were detached by washing with
phosphate-buffered saline (PBS) and incubating with trypsin-EDTA
132
solution (0.25% trypsin, 1 mM EDTA) for 4 mins at 37 ˚C, followed by
washing and incubation with supplemented RPMI 1641 medium.
4.2.7.2 Cytotoxicity studies
To
assess
cytotoxicity
of
the
iron
oxide-silica
Herceptin®
nanoparticles on breast cancer cells, the viability of SKBR-3 cells
incubated with the Herceptin® labeled particles was assessed after 24 and
48 hours in vitro. SKBR-3 cells were seeded into 24-well plates for 24
hours, after which medium without serum was added with iron oxidesilica nanoparticles at concentrations ranging between 1 to 100 µg/ml. A
CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega,
USA) containing the tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS)
was
used to monitor cell viability according to the manufacturer’s instructions
and the MTS colour change was measured at 490 nm using a microplate
reader and cell viability was plotted against 100% viability for untreated
cells.
4.2.8 Cellular uptake
4.2.8.1 Confocal microscopy
SKBR3, BT474 and MCF-7 cell lines were cultured and seeded onto
a 6-well slide glass chamber. All cell lines were incubated with 1 ml of 50
133
µg/ml Fe@SiO@HER-FITC nanoparticles for 4 hours at 37oC and cellular
uptake was assessed by confocal microscopy. Thirty minutes prior to
imaging, a Hoechst nuclear stain was added to the cells and then washed
several times with PBS to aid in the identification of live cells within the
culture.
4.2.8.2 Flow cytometry
To quantify the
uptake of the iron oxide-silica
Herceptin®
nanoparticles by SKBR3, BT474 and MCF7 cells, dose response and time
course studies were performed using flow cytometry. Cells were grown to
90% confluence in 24-well plates and for the dose-response study; 10
µg/ml, 50 µg/ml and 100 µg/ml of iron oxide-silica Herceptin®
nanoparticles were added to cells and incubated for 24 hours. For timecourse studies, Fe@SiO@HER nanoparticles conjugated to FITC were
incubated at varying time points of 0.5, 1, 3, 5 and 24 hours, at a
constant concentration of 50 µg/ml. After incubation for both doseresponse and time-course studies, the cells were detached from the plates
with trypsin-EDTA and washed twice in PBS and resuspended in FACS-fix
buffer for analysis by flow cytometry and a minimum of 8,000-10,000
viable cells were assayed (FACSCanto, BD Biosciences, RMIT Flow
Cytometry Facility, Bundoora, Australia).
134
4.2.9 Quantification of HER-2 expression
Quantification of HER-2 expression levels between SKBR-3, BT474
and MCF-7 cells was assessed by flow cytometry. Cells were cultured
using the previously mentioned method, seeded onto 24-well plates and
grown to 90% confluency. Herceptin® was added to the cells at a
concentration of 10 µg/ml for 30 min at 4oC followed by two washes and
then incubation with FITC-anti-human IgG secondary antibody for 30 min
at 4oC. The removal of the primary antibody from these treatments was
used as a measurement of non-specific binding of the secondary antibody.
The cells were washed and then analysed by flow cytometer as previously
described. The mean fluorescence intensity (MFI %) was determined by
subtracting the mean channel number for the background from the mean
channel number for the primary antibody treated cells.
4.2.10 MR imaging
4.2.10.1 MR Imaging in vitro
Iron
oxide
nanoparticles
were
prepared
and
conjugated
to
Herceptin® using the described protocols. SKBR-3, BT474 and MCF-7
cells were cultured, detached using trypsin-EDTA solution, washed and
seeded onto 6-well plates and incubated with supplemented RPMI 1641
medium without serum. Each cell line was treated with 1 ml of 50 µg/ml
of Fe@SiO@HER and incubated for 5 hours at 37˚C. Before imaging on
MRI, the cells were washed 3 times with PBS and then fixed with
135
glutaraldehyde. A T2-weighted gradient echo sequence was used with the
same parameters as the phantom studies. Signal enhacement of each of
the cell lines was calculated relative to the control using:
∆R2/R2control
100.
4.2.10.2 Mouse Experiments
We obtained ~6 week old female BALB/c nude mice (ARC) in
accordance with the guidelines and under approval of the Animal Ethics
Committee of RMIT University (RMIT AEEC approval number: 0906). The
nude mice were injected subcutaneously with equal volume of SKBR3
cells (2.5 x 106) and Matrigel (BD, USA) into the right flank. A full tumour
growth analysis was conducted to ensure growth of the tumours as well as
the average time to reach a maximum diameter of approximately 1 cm.
4.2.10.3 MR imaging in vivo
Nude mice (n = 15) bearing SBKR3 tumours were studied by MRI
when the subcutaneous tumours reached a diameter of approximately 1
cm. A solution of Fe@SiO@HER particles (400 µg Fe) was infused via the
tail vein of a group of mice and a solution of Fe@SiO particles (400 µg) was
infused via the tail vein of another group of mice. The MRI of ketaminexyalazine-anesthetised mice was performed at 4 and 24 hours using a 3.0
T magnetic resonance scanner and a heel coil. All animals were measured
using a T2-weighted sequence.
136
4.3.1 Results and discussion
The previous study demonstrated many of the advantages of using a
material such as this for MR contrast enhancement, however the particles
investigated in chapter 3 had several physical characteristics that could
be improved upon for use in targeted MR contrast enhancement. Firstly
the size and morphology of the material required greater control; this was
achieved through modifying the amount of oleic acid present in last phase
of the fabrication. This change was based on work by Song et al. [4]
whereby the size and shape of the particles produced by the high
temperature decomposition method was modified by altering the amount
of oleic acid present in the reaction. By reducing the amount of oleic acid
and decreasing the aging duration of the process, spherical magnetite
particles with an average diameter of 17 nm could be obtained. The
thickness of the silica coating on the particles was also decreased in order
to form a core shell system with an overall diameter of less than 60 nm.
This was required to increase the blood half-life of the particles so
that they could localise at the target site without being removed by the
RES prior to imaging. Previously in chapter 3 we presented in vivo imaging
data produced by directly injecting the particles to the target site. However
in this chapter we will be delivering the particles via intravenous injection
and allowing the particles to self-direct to the tumour site with the aid of
targeting antibodies.
137
Given the fundamental changes made to the contrast material, all
relevant characterisations had to be made using the new material. The
following sections discuss the results obtained while examining the
particle size, solubility, biocompatibility, targeting ability and image
contrast capabilities, of the newly synthesised particles.
4.3.2 TEM analysis
Figure 1 shows the TEM images of both the as synthesised and
silica coated iron oxide nanoparticles. The micrograph of the as
synthesised particles (Figure 1(A)) shows that they have a mean particle
diameter of 17 nm and are both spherical and have a homogenous size
distribution. Figure 1 (B) shows the particles after silica coating in which a
silica shell of ~10 nm has been coated onto the surface of the particles to
infer stability in water, biocompatibility and provide anchorage sites for
the addition of the Herceptin® antibody. The image shows that the silica
coating has formed in a smooth continuous layer around the outside of
the magnetite particle, the majority of the particles contain only a single
core and very little aggregation has occurred during the coating process.
138
Figure 1: TEM image of as synthesized magnetite and Fe@SiO particles, Scale bar is
equal to 50 nm.
14
16
18
20
22
Particle diameter (nm)
Figure 2: Histogram outlining the particle size distribution of the as synthesized
magnetite particles.
For successful MRI applications in vivo, the iron oxide nanoparticles
need to have an overall size smaller than 60 nm in diameter [5]. This
consideration arises because studies have reported that particles larger
than this size limit cell targeting capability and reduce uptake efficiency
[5]. Another problem that occurs due to larger particle size is non-specific
uptake in the RES as well as biological instability [6]. In our previous
investigation we reported iron oxide silica nanoparticles with a diameter of
greater than 60 nm, making these particles unsuitable for in vivo targeted
139
MRI. Therefore the method of synthesis was modified to reduce the size of
the particles making them suitable to in vivo. The TEM images illustrates
that the total particle diameter after silica coating is approx. 50 nm, which
should reduce the RES uptake and should infer a longer blood circulation
time and greater uptake at the target site.
4.3.3 XRD analysis
The XRD analysis in Figure 3 shows little difference to the quasi
cubic particles discussed in the previous chapter. The main feature that
sets them apart is the broadening of the peaks in this sample. This is due
to the smaller overall particle size of this material; however all of the
characteristic peaks remain, albeit to slightly different ratios. The sample
presented an acceptable match with the same magnetite card file as before
(75-0449) however in this case the dominance of the (311) peak is not
absolute. The (440) peak has increased in intensity significantly which
may be due to either the size of the particle being restricted or a more
controlled thermal deposition process. Again we expect there to be
hematite and maghemite present in the material and it is not possible for
XRD to adequately distinguish between the two oxidation states, however
given the colour and magnetic performance of the particles, we determine
that the majority of the material present in the sample remains as
magnetite.
140
(440)
(311)
(220)
(511)
(400)
Counts (a.u)
20
40
2θ °
60
80

Figure 3: XRD pattern for the as synthesised 17 nm magnetite particles.
4.3.4 Herceptin® conjugation
Herceptin® was then conjugated to the surface of the silica coated
magnetite particles using cysteamine as a crosslinker. The cysteamine
molecule binds to the surface of the silica via attachment to the silanol
groups present on the surface, this leaves free amine groups on the
surface being presented from the tail end of the cysteamine molecule.
These amine groups provide an anchorage site for the carboxylic groups
present on the tail end of the FC region of the Herceptin® antibody.
The
chemistry
of
the
nanoparticles-cysteamine-Herceptin®
conjugation indicates the particles are bound electrostatically. The
formation
of
the
Fe@SiO@HER
nanoparticles
141
was
confirmed
and
quantitated by a bicinchoninic acid (BCA) protein assay kit. After
conjugation, the particles were washed of any free unbound Herceptin®
and the remaining material had its protein concentration quantified. This
demonstrated that the Herceptin® had both bound to the surface of the
silica and that it did so at a concentration of 0.66 µg of Herceptin® per 1
µg of iron oxide silica particles.
For in vitro and in vivo studies, stability of the Herceptin®
conjugation within the application environment is critically important. In
order to determine that the Herceptin® was sufficiently bound to the
surface, the iron oxide silica Herceptin® nanoparticles were incubated
over a 24 hour period across a range of pH and human serum
concentrations. The Herceptin® was first conjugated with FITC markers
and then attached to the silica surface. The particles were then incubated
under the varying conditions for 24 hours and centrifuged at 4000 rpm to
pellet out the particles and the Herceptin® that remained bound. The
supernatant was analysed and the proportion of free Herceptin® was
calculated using a standard curve produced by a known concentration of
FITC-Herceptin® conjugates.
142
4
% Herceptin lost
pH 5
pH 7.4
pH 10
3
2
1
0
1h
3h
5h
8h
24 h
Figure 4: % of Herceptin® lost from the particles after incubation for up to 24 h in
varying pH ranging from 5-10
7
25% Serum
50% Serum
100% Serum
% Herceptin lost
6
5
4
3
2
1
0
1h
3h
5h
8h
24 h
Figure 5: % Herceptin® lost from particles after incubation for up to 24 h in human
serum concentrations ranging from 25-100%
The results in Figures 4 and 5 show that across this range of pH
and human serum, the Herceptin® conjugation holds up exceptionally
well with only up to 3.7 % being lost from the surface. It can therefore be
concluded that under these conditions very little Herceptin® is lost due to
the effective linkage via the cysteamine. These stability results are similar
143
to other studies that have reported stable particle conjugations in various
pHs indicating that competitive displacement in vivo is unlikely [7].
4.4.1 In vitro and in vivo studies
The in vivo and in vitro studies presented demonstrate that the
Fe@SiO@HER particles were capable of producing localised MR contrast at
the site of cells expressing a specific surface marker. The particles were
also
capable
of
localising
at
the
target
site
after
intravenous
administration in suitable concentration to provide substantial contrast
enhancement. This data suggests that the Herceptin® functionalised
particles were able to specifically target cells that over expressed the
HER2 surface receptor present in many breast tumours.
4.4.2 Cytotoxicity
It is well known that iron oxide particles can produce cell toxicity in
high concentrations [8-10] and that silica coating has previously
demonstrated to provide an increase in biocompatibility as well as particle
stability [11, 12]. It is also plainly understandable that Herceptin® is
cytotoxic to tumour cells as it undergoes internalisation via receptormediated endocytosis at the HER2 receptor site and then causes down
regulation of HER2 production eventually causing cell death. [13-14]
144
To investigate the cytotoxic potential of the iron oxide silica
Herceptin® nanoparticles, MTS based assays were conducted on SKBR3
cells (Figure 6). SKBR-3 cells were incubated with iron oxide silica
Herceptin® nanoparticles for 24 and 48 hours at varying concentrations
ranging from 1-100 µg/ml. Figure 6 shows that lower concentrations (1-30
µg/ml) have no significant toxicity on SKBR-3 cells however the toxic
effect increases between 50-100 µg/ml.
24 hours
48 hours
100
% Viability
80
60
40
20
0
0
1
3
5
10
30
50
100
Particle concentration g/ml
Figure 6: % viability of SKBR3 cells after incubation with increasing concentrations of
Fe@SiO@HER particles over a 24 and 48 hour period.
These results are consistent with the low concentration of
Herceptin® which is far lower than the therapeutic dosage when used as
an independent treatment, having little toxic effect on the cells. The cell
145
death trends that are observable are quite similar to the previous study
examining the cell toxicity of Mag@SiO2 particles in chapter 3 undergoing
non-specific uptake. This means that in this case, the Herceptin® is
behaving purely as a targeting moiety and is causing no additional
cytotoxicity. Similar results have been reported for larger iron oxide
particles that have been conjugated with Herceptin® and compared their
cytotoxicity to SKBR3 cells. [15-19]
4.4.3 Uptake specificity
We then examined the in vitro binding specificity and efficiency by
dose response and time course of Fe@SiO@HER nanoparticles by breast
cancer cell lines that express different amounts of HER2 and analysed by
flow cytometry. Initially, expression of HER2 was assessed in SKBR3,
BT474 and MCF-7 cell lines. Analysis by flow cytometry showed that
SKBR3 showed the highest HER2 expression followed by BT474 and then
MCF-7 (Figure 7).
146
Relative expression of HER2/neu
Surface expression MFI%
1000
800
600
400
200
0
SKBR3
BT474
MCF7
Figure 7: Relative surface expression of HER2 receptor between SKBR3, BT474 and
MCF7 as measured by flow cytometry.
The results of this study are consistent with the literature which
indicates that amongst SKBR3, BT474 and MCF-7, SKBR3 cells have the
highest HER2 expression [20, 21]. Although the expression levels of these
cell lines are well known and widely reported, it was important to confirm
expression levels as factors such as passage number, freezing, thawing
and cell culture techniques can decrease expression levels within
individual samples. The results are shown in Figure 7 where it can be
clearly seen that SKBR-3 cells significantly overexpress the HER2 surface
protein in comparison to the BT474 and MCF7 cell lines.
147
4.4.4 Cell specific uptake
In order to achieve targeted imaging, the Fe@SiO@HER particle
must be able to specifically bind to target cells. In this case Herceptin® is
being used to target cells that overexpress the surface protein HER2,
however as the HER2 protein is expressed in many cell types to a far
lesser extent; quantification of particle uptake vs. surface expression must
be compared. To first demonstrate that highly expressing SKBR-3 cells
would internalise the particles, FITC-labeled Fe@SiO@HER nanoparticles
were analysed by confocal microscopy. SKBR-3 cells were incubated with
50 µg of FITC-labeled Fe@SiO@HER nanoparticles for 3 hours at 37oC,
after this time the cells demonstrated significant uptake (Figure 8).
Figure 8: Confocal microscopy images of SKBR3 cells after incubation with FITC- labelled
Fe@SiO@HER for 3 hours. Scale bar 30 µm.
The images in this study are similar to that of previous studies that have
looked at the uptake of Herceptin® conjugated nanoparticles by SKBR-3
cells [15, 22]. It is well known that HER2 is involved in receptor mediated
148
endocytosis, therefore resulting in the Herceptin®-tagged nanoparticles
being accumulated within the cells.
The
mechanism
of
cellular
uptake
by
the
Fe@SiO@HER
nanoparticles should be mediated by, and dependant on, Herceptin®.
Therefore cells that express higher numbers of HER2 surface proteins
should internalise more particles. To confirm this, a dose response and
time course response of Fe@SiO@HER nanoparticles by SKRBR3, BT474
and
MCF7
was
conducted.
Fe@SiO@HER-FITC
For
nanoparticles
the
were
dose
response
incubated
study,
at
the
different
concentrations (5, 10, 50, 100 µg/ml) for 24 hours with the SKBR3,
BT474 and MCF7 and then analysed by flow cytometry.
1600
1400
MFI%
1200
SKBR3
BT474
MCF7
1000
800
600
400
200
0
0
5
10
50
100
Concentration in g/ml
Figure 9: Dose response data for SKBR3, BT474 and MCF7 particle uptake proportional
to increasing particle concentration after 4 hours.
149
The results indicate that level of FITC fluorescence increases as the
concentration of the Fe@SiO@HER nanoparticles increases in all cell lines
suggesting, that more nanoparticles are being internalised with increasing
exposure duration (Figure 10). Furthermore, the level of iron oxide silica
Herceptin® FITC nanoparticle uptake is consistent with the amount of
HER2 expression shown in Figure 7 for each of the cells lines, indicating
specific HER2 mediated uptake.
The results from this study are in accordance with cellular uptake
studies of human serum albumin nanoparticles conjugated to Herceptin®
which
also
demonstrate
increasing
uptake
of
nanoparticles
as
concentrations climb [23, 24]. It is interesting to note that most of the
studies that use nanoparticles conjugated to Herceptin® do not show dose
response uptake studies and instead only show the differences in uptake
via MRI.
For example the study by Chen et al [15] investigated the use of
Herceptin® conjugated dextran-coated iron oxide nanoparticles in vitro
and vivo in 4 human breast cancer cells lines. This method is essential in
understanding
phantoms
and
the
MRI
contrast
enhancement
in
vitro
systems.
However
this
behaviour
is
not
between
useful
in
understanding the behaviour of the tagged nanoparticles in in vitro
biological systems.
In addition to dose response, the time required for uptake of
nanoparticles is of critical importance when considering further clinical
applications of such a material. Time-dependent cellular uptake of
150
Fe@SiO@HER
nanoparticles
were
carried
out
using
a
constant
concentration of 50 µg Fe and were incubated with SKBR3, BT474 and
MCF7 for 30 mins, 1 hr, 3 hr, 5 hr and 24 hr (Figure 10). Similarly, the
results show that as the time increases, nanoparticle uptake also
increases and the rate of uptake appears to be consistent with the surface
expression of the cell lines as shown in Figure 7. These findings are
concurrent with previous studies suggesting uptake to be associated with
HER2 receptors [21, 25].
As with the dose response studies, there is limited data available on
time course response studies of nanoparticles conjugated to Herceptin®
except for one study showing uptake of HSA conjugated to Herceptin® in
SBKR3, BT474 and MCF-7 cells over a 24 hour time period [21]. The
results showed time dependant uptake of the nanoparticles and the
influence of incubation times greater than 5 hours to be associated with
non-specific uptake.
151
Particle uptake MFI %
2000
1750
1500
1250
SKBR3
BT474
MCF7
1000
750
500
250
0
0
0.5
1
3
Time (Hrs)
5
24
Figure 10: Particle adsorption time course for SKBR3, BT474 and MCF7 cells up to 24h
incubation.
4.4.5 Phantom cell studies
The ability to provide contrast in phantoms and then in vitro using
MRI is one fundamental aspect in targeted imaging. Another important
aspect is the ability for the targeted nanoparticles to demonstrate selective
binding at the target cells. In this case, the Fe@SiO@HER particles show
selective binding as indicated by the dose and time course response
studies, (Figure 9 and Figure 10). This selective binding is also effective
enough to accumulate enough particles at the target site to generate
significant contrast in a clinical MR scanner. This can be visualised in
Figure 11 where SKBR3 cells appear darker relative to BT474 and MCF-7
cell lines which is also confirmed by a plot of the drop in T2 values against
the cell lines (Figure 11). The signal enhancement relative to the control of
SKBR-3, BT474 and MCF-7 cells were calculated to be 52.3%, 34.7% and
152
12.5% respectively, compared to their control. With a maximum T2 value
of 252.7 L/mmol/s in cell free suspensions, this value is only slightly less
than that produced by the larger quasi cubic particles presented in
chapter 3 which had a maximum value of 263.23 L/mmol/s.
Figure 11: Bar graph and corresponding MR images showing contrast for each cell line
after incubation with 50 µg/ml Fe@SiO@HER particles.
These results are consistent with the surface expression data from
Figure 7 suggesting that as the uptake of Fe@SiO@HER particles is
proportional to the surface expression of HER2 within the cell line. This is
confirmed by the higher T2 contrast observed in SKBR-3 cells compared
to either the BT474 or MCF-7 when washed after incubation with equal
amounts of Herceptin® conjugated iron oxide particles.
On the basis of our successful verification that selective targeting
can be achieved in vitro, we wanted to further examine their performance
in an in vivo environment. For this study SKBR-3 tumours were to be
153
grown in BALB/c nude mice up to a diameter of 8mm2 over 12 days. The
tumours were to be dissected and immunohistochemical analyses run to
ensure expression of HER2 within the tumours grown in vivo. The tumour
carrying mice were then be intravenously injected with Fe@SiO@HER
particles and then MR images taken of the tumour sites to determine if
significant uptake occurred. This experiment was approved by the RMIT
AEEC, approval number 0906 and all animal treatment procedures were
adhered to stringently.
4.4.6 In vivo mouse imaging
Nude mice (n=15) were subcutaneously injected with SKBR3 cell
lines which overexpress HER2 receptors and the tumours were allowed to
grow to 8mm2 prior to beginning the experiment. In order to ensure that
the SKBR-3 tumours grown in vivo expressed HER2 surface proteins,
immunohistochemical
analysis
of
SKBR3
xenograft
tumours
was
performed. Following the growth of xenograft SKBR3 tumours in BALB/c
nude mice, the mice were sacrificed; their tumours were dissected and
processed for histological analysis. Figure 12 shows the staining pattern
of HER2 in SKBR3 xenograft tumours grown in BALB/c nude mice. The
inset shows clear membranous (M) and cytoplasmic (C) staining when
stained with an anti-HER2 antibody. This pattern of staining was
consistent with tumours that are expressing the HER2 surface protein.
154
Figure 12: Histological analysis of BALB/c nude mouse tumour, stained with anti-HER2
antibody, scale bar 1000 µm.
MR imaging of the mice was performed at 4 hours after intravenous
tail injection. The study consisted of two control groups (saline and
Fe@SiO) and one experimental group, Fe@SiO@HER. The first set of
control subjects received only saline and the second set received 400 µg of
Fe@SiO nanoparticles with no Herceptin® bound to its surface. The
experimental group received 400 µg Fe@SiO@HER nanoparticles. Figure
13 shows the MR images at 4 hours post injection of the three groups. The
results
indicate
that
by
4
hours
post
injection,
uptake
of
the
Fe@SiO@HER particles can be seen as indicated by the contrast
enhancement relative to the control.
155
Signal enhancement %
Figure 13: MRI comparison of SKBR3 tumours in mice 4 hours post injection, tumour
regions highlighted.
60
50
40
30
20
10
0
Control
Fe@SiO
Fe@SiO@HER
Figure 14: Signal enhancement values calculated via MR readout for the tumour sites
between the three variables.
To further confirm the specific binding, the signal enhancement was
also calculated against the second control group which received Fe@SiO
nanoparticles (Figure 14). By directly comparing these results we can
confirm that it is the addition of the Herceptin® targeting moiety to the
surface of the particles that allows the material to be specifically targeted
towards HER2 expressing cells. We can also conclude that not only do the
156
particles specifically target cells that over-express the HER2 protein; but
that they do so in such number that significant contrast can be added to
an MR image by the intravenous injection of the particles under clinical
conditions.
4.5.1 Conclusions
We have been able to demonstrate the applicability of silica coated
magnetite particles using a targeting moiety, for cell specific contrast
enhancement. Under clinical conditions using clinical scanning protocols,
we were able to produce significant contrast at a target site in breast
tumour bearing mice 4 hours post injection. This clearly demonstrates
that the particle circulation time is great enough to overcome the RES
uptake and excretion issues that have rendered other nanomaterials
ineffective
for
this
application.
The
Fe@SiO@HER
material
has
demonstrated low cytotoxicity at clinical dosages, the ability to produce
significant contrast and good functional stability under a range of pH and
serum concentrations. The particles themselves can be made in large
enough amounts that they can be considered for human clinical
application and the method of production provides good size and shape
homogeneity.
157
4.6.1 References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Park J, An KJ, Hwang YS, Park JG, Noh HJ, Kim JY, et al. Ultra-large-scale
syntheses of monodisperse nanocrystals. Nature Materials 2004,3:891-895.
Stober W, Fink A, Bohn E. Controlled growth of monodisperse silica spheres in
micron size range. Journal of Colloid and Interface Science 1968,26:62-&.
Fang CL, Qian K, Zhu JH, Wang SB, Lv XX, Yu SH. Monodisperse alphaFe(2)O(3)@SiO(2)@Au core/shell nanocomposite spheres: synthesis, characterization
and properties. Nanotechnology 2008,19.
Song M, Zhang Y, Hu S, Song L, Dong J, Chen Z, et al. Influence of morphology and
surface exchange reaction on magnetic properties of monodisperse magnetite
nanoparticles. Colloids and Surfaces A: Physicochemical and Engineering Aspects
2012,408:114-121.
Jun Y-W, Lee J-H, Cheon J. Nanoparticle Contrast Agents for Molecular Magnetic
Resonance Imaging. In: Nanobiotechnology II: Wiley-VCH Verlag GmbH & Co. KGaA;
2007. pp. 321-346.
Jun Y-w, Lee J-H, Cheon J. Nanoparticle Contrast Agents for Molecular Magnetic
Resonance Imaging. In: Nanobiotechnology II: More Concepts and Applications.
Edited by Mirkin CA, M.Niemeyer C. Weinheim: Wiley-VCH; 2007.
Cortez C, Tomaskovic-Crook E, Johnston A, Radt B, Cody S, Scott A, et al. Targeting
and Uptake of Multilayered Particles to Colorectal Cancer Cells. Advanced Materials
2006,18:1998-2003.
Weissleder R, Stark DD, Engelstad BL, Bacon BR, Compton CC, White DL, et al.
Superparamagnetic iron oxide: pharmacokinetics and toxicity. Am. J. Roentgenol.
1989,152:167-173.
Prina-Mello A, Crosbie-Staunton K, Salas G, Puerto MM, Volkov Y. Multiparametric
Toxicity Evaluation of SPIONs by High Content Screening Technique: Identification
of Biocompatible Multifunctional Nanoparticles for Nanomedicine. Ieee Transactions
on Magnetics, 2013. 49(1): p. 377-382.
Chang Y, Lee GH, Kim TJ, Chae KS. Toxicity of Magnetic Resonance Imaging Agents:
Small Molecule and Nanoparticle. Current Topics in Medicinal Chemistry, 2013.
13(4): p. 434-445.
Ohmori M, Matijevic E. Preparation and properties of uniform coated colloidal
particle 7. silica on hematite. Journal of Colloid and Interface Science 1992,150:594598.
Ohmori M, Matijevi E. Preparation and properties of uniform coated inorganic
colloidal particles .8. silica on iron. Journal of Colloid and InterfaceSscience
1993,162:288-292.
Valabrega G, Montemurro F, Aglietta M. Trastuzumab: Mechanism of action,
resistance and future perspectives in HER2-overexpressing breast cancer. Annals of
Oncology 2007,18:977-984.
Ludyga N, Anastasov N, Rosemann M, Seiler J, Lohmann N, Braselmann H, et al.
Effects of Simultaneous Knockdown of HER2 and PTK6 on Malignancy and Tumor
Progression in Human Breast Cancer Cells. Molecular Cancer Research, 2013.
11(4): p. 381-392.
Chen T, Cheng T, Chen C, Hsu S, Cheng T, Liu G, et al. Targeted Herceptin-dextran
iron oxide nanoparticles for noninvasive imaging of HER2/neu receptors using MRI.
Journal of Biological Inorganic Chemistry 2009,14:253-260.
Aydin, R.S.T., Herceptin-decorated salinomycin-loaded nanoparticles for breast
tumor targeting. Journal of biomedical materials research. Part A, 2013. 101(5): p.
1405-15.
Xiao Z, Levy-Nissenbaum E, Alexis F, Luptak A, Teply BA, Chan JM, et al.
Engineering of Targeted Nanoparticles for Cancer Therapy Using Internalizing
Aptamers Isolated by Cell-Uptake Selection. Acs Nano, 2012. 6(1): p. 696-704.
Llevot A, and Astruc D. Applications of vectorized gold nanoparticles to the diagnosis
and therapy of cancer. Chemical Society Reviews, 2012. 41(1): p. 242-257.
158
19.
20.
21.
22.
23.
24.
25.
Kumar A, Ma H, Zhang X, Huang K, Jin S, Liu J, et al. Gold nanoparticles
functionalized with therapeutic and targeted peptides for cancer treatment.
Biomaterials, 2012. 33(4): p. 1180-1189.
Chen X, Yeung TK, Wang Z. Enhanced Drug Resistance in Cells Coexpressing ErbB2
with EGF Receptor or ErbB3. Biochemical and Biophysical Research Communications
2000,277:757-763.
Steinhauser I, Spänkuch B, Strebhardt K, Langer K. Trastuzumab-modified
nanoparticles: Optimisation of preparation and uptake in cancer cells. Biomaterials
2006,27:4975-4983.
Huh Y-M, Jun Y-w, Song H-T, Kim S, Choi J-s, Lee J-H, et al. In Vivo Magnetic
Resonance Detection of Cancer by Using Multifunctional Magnetic Nanocrystals.
Journal of the American Chemical Society 2005,127:12387-12391.
Taheri A, Dinarvand R, Atyabi F, Ghahremani MH, Ostad SN. Trastuzumab
decorated methotrexate-human serum albumin conjugated nanoparticles for
targeted delivery to HER2 positive tumor cells. European Journal of Pharmaceutical
Sciences 2012,47:331-340.
Steinhauser I, Langer K, Strebhardt K, Spänkuch B. Uptake of plasmid-loaded
nanoparticles in breast cancer cells and effect on Plk1 expression. Journal of Drug
Targeting 2009,17:627-637.
Wartlick H, Michaelis K, Balthasar S, Strebhardt K, Kreuter J, Langer K. Highly
Specific HER2-mediated Cellular Uptake of Antibody-modified Nanoparticles in
Tumour Cells. Journal of Drug Targeting 2004,12:461-471.
159
Chapter 5
5.1.0 Further investigations
The previous chapters outlined the development and application of a
silica coated magnetite particle for MR contrast applications. This included
the modification and attachment of Herceptin® biomarkers to allow for
specific targeting on the cellular level of this material. This chapter aims to
investigate some of the further applications this material may yield. A Brief
introduction to induction heating will be presented prior to experimental
and discussion information. However, as much of the reasoning and
pertinent literature has been discussed in chapter 1, only a very brief
overview of DMSA coatings will be covered prior to its discussion. These brief
investigations serve only to highlight the additional application possibilities
based on the materials previously presented and not in themselves full
studies. Although some data is presented and discussed, it is outside the
purview of this thesis to completely investigate these applications.
160
5.1.1 Radio frequency induced heating
5.1.2 Introduction
Among their interesting properties, magnetic nanoparticles also
possess the ability to be used as nanoscopic heating elements. This is due to
their ability to release excess adsorbed RF energy as heat, as their magnetic
field is forced to alternate direction. This is due to the particle having some
magnetic hysteresis that does not spontaneously realign with the applied
field. This delay in alignment causes energy loss in the form of heat, which
is transferred to the environment. This principle has been well understood
for many years and magnetic induction heaters are used in industry and
even in modern kitchens as stovetops. However, the ability for a nanoscopic
probe to emit heat has possible applications in medicine; specifically in
experimental cancer treatments [1-5].
Hyperthermia (raising temperature), treatments have been used in the
fight against cancer for some time and established methods have yielded
several approaches using localised heating to weaken or kill cancerous cells.
Given the delicate mechanisms that run the cells in our bodies, little rise in
temperature is required to upset a cells functioning, ~42+ °C [6-8].
Commonly the methods of heating the affected area are via infra-red,
ultrasound, RF ablation or blood perfusion. Although these methods can be
targeted at specific areas, they are still unable to distinguish healthy from
cancerous tissue, while perfusion is completely systemic. However the
161
properties of targeted nanoprobes capable of cell specific localisation, offer
new opportunities and enhanced performance to these treatments.
Effective hyperthermia treatments are tantalising partly because they
are a physical treatment and as such there is less build-up of toxins like
during chemotherapy. This reduces possible side effect and allows for more
rigorous and repetitive treatments with less ill effects on the patients healthy
tissues. Often hyperthermia treatments are used in conjunction with
conventional treatments. This is due to the hyperthermia treatment making
the cancerous cells more vulnerable to treatments such as chemo and
radiotherapy [2-5, 9].
Though promising, these treatments are still limited by the current
technological state of the heating materials. Targeted nanoprobes capable of
emitting sufficient heat at a target site would make these treatments far
more effective. Furthermore, these materials could be combined with
thermoresponsive polymers to allow the targeted and RF induced release of
drug molecules. This second approach would make best use of the
synergism between the two treatment styles, temperature and chemical; and
may offer a new range of treatment practices for cancerous tissue.
To this end, we investigated the potential RF induced heating
properties of the Fe@SiO particles fabricated and discussed in chapter 4. It
is currently understood that particles that produce the best specific loss
power (SLP) ratios are those that fall between the dimensions over which the
material
is
transitioning
between
its superparamagnetic
and
stable
ferromagnetic states [1]. Given the approximate range of this transition for
162
our materials being 20 nm, it was likely that our material did possess these
properties [1].
5.1.3 Experimental and Discussion
This work was overseen and carried out in the hyperthermia materials
labs at Southeast University in Nanjing PR China under the guidance of
Professor Gu Ning and Dr. Ma Ming during the course of an ENDEAVOUR
fellowship. Heating rate experiments were undertaken using an induction
heating oven (Figure 2) at 13 amps and 400 kHz. Temperature was read
every 30 seconds using a precision alcohol thermometer. Samples
containing approximately 2 mg of Fe were coated in 5 nm of SiO2 using the
methods described earlier in section 4.2.4. The particles were dispersed via
sonication in 5 ml MilliQ-water to yield a particle solution of approximately
10 µM Fe. Two different materials had their resultant heating rates
compared; these were magnetite produced via aqueous co-precipitation (AC
Mag) and thermal decomposition (TD Mag).
The particles were characterised via TEM, images are shown below in
Figure 1. The comparative particle shape polydispersity can be observed
between the two production methods. Thermal decomposition (Figure 1 A)
produces smaller, more homogenous spherical particles which have been
presented previously in chapter 4. Aqueous co-precipitation on the other
hand, produces larger triangular and cubic nanoparticles of a more
polydisperse nature (Figure 1 C). The TEM images show that a uniform
163
coating of SiO2 deposited on the surface of the particles however some
aggregation has occurred possibly due to the coating process.
This aggregation may alter the performance of the particles as heating
elements due to the interaction of individual dipoles affecting the hysteresis
properties of the particles. This may be a source of concern and interest in
further studies, however in this case we are only seeking to reveal the basic
heating properties of TD Mag before and after a silica coating has been
applied. Given that similar aggregation has occurred in both samples we feel
that for the purposes of this basic study, the materials remain comparable
for basic properties.
Figure 1: TEM images of the prepared particles. A) as prepared TD Mag, B) TD Mag
with 5 nm SiO2 coating, C) as prepared AQ Mag, D) AQ Mag with 5 nm SiO coating. Scale
bar 50 nm.
164
Figure 2: Induction furnace used for heating experiments at Southeast University Nanjing,
China.
The materials produced via aqueous co-precipitation were known to
perform well at induction heating yet poorly as MR contrast due to their low
magnetisation values. This material was prepared and included as a positive
control and performance comparison for the materials produced via thermal
decomposition. The graph below demonstrates the increase in temperature
over time for the materials tested.
165
Figure 3: Heating rate comparison between the magnetite produced through aqueous co
precipitation (AC Mag) and thermal decomposition (TD Mag) before and after a 5 nm layer of
SiO2 is added. The dotted line shows the time taken to reach the clinically useful 44°C
mark.
As expected the materials produced by thermal decomposition
increased the temperature of the water more slowly, however they were able
to reach 44 °C after 6-8 minutes. From this preliminary data we can see that
the Fe@SiO particles produced as a base for targeted MR contrast in the
previous chapter (TD Mag) also possess the ability to act as nanoprobes
capable of RF induced heating. Both materials show a decrease in heating
rates with an added SiO2 layer which is to be expected. However the time
taken to reach 44°C remains below 10 min and would still be easily
accommodated in a clinical timeframe.
166
5.1.4 Conclusion
We have previously shown that the Fe@SiO particles can be
conjugated with biomarkers which allow them to specifically target certain
cell lines for uptake (Chapter 4). We have now demonstrated that these
same materials can be used to some extent for RF induced heating
applications. The data presented merely indicate that the materials have
heating capabilities and further testing would be required in order to
determine if these properties are at all significant in a clinical sense.
However these materials may display enough heating capability to be used
in conjunction with thermally induced drug release systems. To these ends
we believe that these materials deserve further investigation along these
lines and may provide an interesting platform for future targeted MR
contrast/hyperthermia/drug delivery systems.
5.2.1 DMSA coatings
As discussed previously in chapter 1 section 1.8.3, DMSA is a wellknown biocompatible surface coating. It offers many properties that may
make it desirable for medical applications, such as available carboxylic and
thiol groups for further functionalization. However the layer created is not
thick enough to fully separate the magnetic dipoles of the particles to the
same extent as the silica coating that was used throughout chapters 3 and
167
4. Also, as there are both thiol and carboxylic groups on the surface, this
may cause additional complication for further functionalisation.
However DMSA is exceptionally biocompatible and behaves as an
excellent particle stabiliser with a relatively easy coating process. To these
ends, the materials produced in chapter 3, the quasi cubic ―Mag‖ particles,
were examined for their comparative performance in some circumstances
similar to those of the Mag@SiO2 particles. The depth of the study was
however, not to the same extent as previously discussed. This was partly
because the MRI scanners we were using for the research were clinically
active machines; this meant that we could not allocate time on the
instrument for less promising materials. However we were interested in the
properties of DMSA as a biocompatible surface coating and how it affected
the magnetic properties of the particles in contrast to the silica.
The particles were examined for their biocompatibility, magnetisation
saturation and subsequent phantom MR performance. These experiments
were done in conjunction with those discussed in chapter 3 and follow the
same methodologies. Given the short nature of this chapter please refer to
chapter 3 for specific experimental imaging information.
5.2.2 Surface coating
5 mg of Mag particles were dispersed in 10 ml dry toluene and 2.5 mg
(14 µmol) DMSA (Sigma). The solution was sonicated for 20 minutes and
then placed on a rotary shaker at room temperature for 24 hours. The
168
particles were centrifuged out of solution and washed 3 times in ether and 3
times in MilliQ-water. The particles were re dispersed in 5 ml MilliQ-water
and adjusted to 100 µg/ml using AAS for loss quantification.
5.2.3 Experimental and discussion
Figure 4 shows the morphology of the Mag particles after coating with
DMSA. Given the low electron density of the organic DMSA layer, it is not
expected that there be much of a visible layer on the surface under TEM
examination. However given the close stacking of the particles to each other
after DMSA coating, it can be deduced that the surface coating remains
relatively thin. The particles also do not seem to be engulfed in an
amorphous glob of DMSA but this was unlikely given the exceptionally low
quantity of DMSA used in the coating process.
169
Figure 4: TEM image of the DMSA coated Mag particles, scale bar 50 nm.
Identically to the process used in section 3.3.1, biocompatibility of the
DMSA coated particles was ascertained via MTS assay. Viability was
assessed across an increasing concentration range and at both 24 and 48
hours. The particles show very little toxicity in Figure 5 at low to mid-range
concentrations, the only significant decline occurs at the maximum 100
µg/ml concentration. However there is significantly larger error variance in
the data for these samples compared to those of the Mag@SiO2 particles
discussed in chapter 3. This may be due to DMSA inducing cell proliferation
or simply decreasing uptake, however DMSA has been readily used in
cellular uptake studies by other groups [10-12]. This data demonstrates the
high level of biocompatibility that DMSA has been known to exhibit and
should serve to encourage its use when such properties are required.
170
120
24 hours
48 hours
% cell viability
100
80
60
40
20
0
1
3
5
10
30
g/ml Fe
50
100
Figure 5: Biocompatibility of DMSA coated magnetite particles at increasing concentrations
over 24 and 48 hours.
The SQUID data presented in Figure 6 demonstrates the lessening of
the magnetisation saturation of the material while its individual dipoles are
allowed into closer proximity than the same material used in chapter 3. This
lessening of the emu/g value was expected and is reinforced by the
lessening of the obtained signal intensity and subsequent contrast reduction
(Figure 7).
171
Figure 6: SQUID data showing magnetisation saturation of DMSA coated Mag particles.
The phantom study echoed the results from the SQUID analysis, lower
emu/g values translated into less T2 contrast versus the water control.
Below in Figure 7 the relative contrast is shown for the DMSA coated Mag
particles at an increasing concentration compared to the control at 0 µg Fe.
Given the results discussed in section 3.4.5 regarding the decrease in
contrast signal after cellular uptake for the Mag@SiO2 particles, we would
expect similar behaviour from the DMSA coated material.
172
Figure 7: Relative signal enhancement of DMSA coated magnetite particles compared to
control under phantom imaging parameters.
5.2.4 Conclusion
The cell viability study demonstrated the excellent biocompatibility of
DMSA as is widely known and used in literature [10-12]. However, likely due
to the exceptionally thin coating produced by the DMSA, there is a greater
dipole interaction between the particles which allows for a lowering of the
magnetic susceptibility. This is observable in the TEM and SQUID data
(Figures 4 & 6) which results in less overall contrast in the phantom images,
(Figure 7).
Given the results from these brief experiments, DMSA may provide an
interesting
avenue
for
particle
stabilisation
and
biocompatibility
improvements for these materials. Far deeper study would be needed to
173
properly ascertain its potential, but it may offer good properties for
applications in cell tracking where specific targeting is not required. It would
be expected that the shelf life of this material would be less than that of the
silica coated Fe but again further testing would be required. In order to
overcome the dipole separation issue, DMSA may be used in conjunction
with a silica layer to infer greater biocompatibility. However the surface may
need pre-treatment prior to the addition of any additional surface
functionalization due to the mix of available surface groups presented by
DMSA.
5.3.1 Chapter Summary
These
brief
investigations
have
demonstrated
some
extended
versatility of the materials developed in this thesis. The successful
demonstration of the ability to target the Fe@SiO particles to specific sites
within an animal model is offered greater significance by the materials
ability to act as a hyperthermia agent. Further study is required in order to
establish if this material is a viable material for hyperthermia applications.
However targeted drug delivery may offer greater promise given the materials
structural properties and lower overall heat transfer rate.
The greatly enhanced biocompatibility of DMSA may be used for nontargeting applications or as a surface layer on a composite material to imbue
biocompatibility. The surface coating process is simple and can yield large
amounts of product which is a good advantage for a material heading into
174
any clinical setting. If used as an upper coating on the surface of
nanoprobes it may require blocking of either the thiol of carboxylic groups to
enable selective attachment depending on the biomarker binding technique
used.
175
5.4.1 References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Hergt R, Dutz S, Mueller R, Zeisberger M. Magnetic particle hyperthermia:
nanoparticle magnetism and materials development for cancer therapy. Journal of
Physics-Condensed Matter 2006,18:S2919-S2934.
Anderson RL, Kapp DS. Hyperthermia in cancer-therapy - current status .12.
Medical Journal of Australia 1990,152:310-315.
Herman TS, Teicher BA, Jochelson M, Clark J, Svensson G, Coleman CN. Rationale
for use of local hyperthermia with radiation-therapy and selected anticancer drugs
in locally advanced human malignancies. International Journal of Hyperthermia
1988,4:143-158.
Hiraoka M, Nagata Y, Mitsumori M, Sakamoto M, Masunaga S. Current status and
perspectives of hyperthermia in cancer therapy. In: Portable Synchrotron Light
Sources and Advanced Applications. Edited by Yamada H, MochizukiOda N, Sasaki
M; 2004. pp. 102-105.
Kim JH. Combined hyperthermia and radiation-therapy in cancer-treatment current status. Cancer Investigation 1984,2:69-80.
Cavalier.R, Ciocatto EC, Giovanel.Bc, Heidelbe.C, Johnson RO, Margotti.M, et al.
Selective heat sensitivity of cancer cells - biochemical and clinical studies. Cancer
1967,20:1351-&.
Robinson JE, Wizenber.Mj, McCready WA. Combined hyperthermia and radiation
suggest an alternative to heavy particle therapy for reduced oxygen enhancement
ratios. Nature 1974,251:521-522.
Dewey WC. Arrhenius relationships from the molecule and cell to the clinic.
International Journal of Hyperthermia 1994,10:457-483.
Overgaard J. The current and potential role of hyperthermia in radiotherapy.
International Journal of Radiation Oncology Biology Physics 1989,16:535-549.
Liu Y, Chen Z, Wang J. Internalization of DMSA-Coated Fe3O4 Magnetic
Nanoparticles into Mouse Macrophage Cells. In: Future Material Research and
Industry Application, Pts 1 and 2. Edited by Thaung KS; 2012. pp. 1221-1227.
Shubayev VI, Pisanic TR, II, Jin S. Magnetic nanoparticles for theragnostics.
Advanced Drug Delivery Reviews 2009,61:467-477.
Wang Y, Wang Y, Wang L, Che Y, Li Z, Kong D. Preparation and Evaluation of
Magnetic Nanoparticles for Cell Labeling. Journal of Nanoscience and
Nanotechnology 2011,11:3749-3756.
176
Chapter 6
6.1.0 Conclusions and future work
This chapter will present an overall summary of the findings
presented throughout the body of this thesis along with a discussion of the
future work that will be carried out.
177
6.1.1 Conclusions
The treatment of cancers remains one of the few infuriatingly unsolved
and poorly understood major health concerns of the Common Era. A host of
treatments have been developed that enable us to stave off the effects and in
some cases heal the afflicted, but a great deal more can be done. New
developments in technology have allowed researchers to manipulate
materials on unprecedented scales and harness the properties of the
quantum realm to our ends. With these developments come new materials
and approaches to old problems including the treatments of cancer.
Current imaging techniques such as MRI use contrast agents to
enhance the imaging process used to diagnose and treat cancerous tissues.
Many of these contrast agents are based on gadolinium which has excellent
properties, yet is not applicable in all cases and may see greater phase out
pressure in the future due to toxicity concerns. Iron oxides offer an
alternative to gadolinium, but have for a long time been limited in its
application due to particle size constraints.
The specific aim of this thesis was to demonstrate that iron oxide
based particles can be produced that not only can operate as functional T2
contrast agents, but can also specifically target tumorous tissues. It was the
underlying aim to develop a targeted contrast agent that could be suitably
applied within clinical settings. This was accomplished using thermal
decomposition techniques to produce the core magnetite materials which
were then surface stabilised using a silica coating. Two primary materials
178
were fabricated during the course of this research and were discussed
separately in chapters 3 and 4.
The quasi cubic particles presented in chapter 3 served as a trial run
to first establish the properties of such a material and secondly to examine
the performance of silica coated magnetite particles as T2 contrast agents.
This ―Mag@SiO2‖ material proved to be biocompatible and behaved as
excellent contrast agent in both in vitro and in vivo studies. Despite its good
performance, it did however lack the small size and particle size distribution
to prove viable for further studies.
What we learned from fabricating the Mag@SiO2 particles we applied
to the production of a smaller more controlled core shell particle which we
termed Fe@SiO. The particles were less than 60 nm in diameter and
demonstrated similar biocompatibility and cell uptake compared to that of
the previous material. The Fe@SiO particles were subsequently modified to
carry a Herceptin® biomarker which enabled them to specifically target
cellular uptake by cancerous cells. This was confirmed by comparing uptake
rates with HER2 surface expression on breast cancer cells which correlated
well. Finally this material was successfully trailed in vivo as a breast tumour
specific contrast agent by targeting SKBR3 breast tumours grown in mice
intravenous tail injection.
The successfully application as a viable targeted T2 contrast agent, of
a silica coated magnetite based nanoparticle has been demonstrated. The
data shows significant darkening at the tumour site 4 and 24 hours post
injection and a high level of stability and biocompatibility was illustrated
179
through MTS assays and confocal microscopy. This material offers a
promising platform for targeted MR contrast using silica coated magnetite
with possible further applications in hyperthermia and drug delivery
systems.
6.1.2 Future work
As has been discussed in chapter 5, there are many further
applications of the materials presented in this thesis. However with
particular interest, we will be pursuing the development of targeted drug
delivery platforms based on the core shell materials presented in chapter 4.
This work will investigate the performance of core/shell magnetite
nanoparticles coated with drug laden thermoresponsive polymer and
Herceptin® as a targeted, RF induced release, drug delivery platform. We
have been fortunate to receive a category 1 research grant in the form of a
Victorian Postdoctoral Research Fellowship in order to pursue this research
in collaboration with Prof. Sanjiv Gambhir from the Molecular Imaging
Program Stanford (MIPS). This research funding will allow two years to be
spent developing these materials within Prof. Gambhirs group and a third
year at RMIT University upon returning Australia.
As many of the nanoparticle factors have already been addressed
during the course of my PhD investigations, specific attention will be
focused on optimising the core magnetite materials so that maximum
180
heating potential is gained with minimal loss of beneficial magnetization
values used for MR contrast.
Fabrication of outer coatings of thermoresponsive polymer layers and
subsequent drug loading and release profiles will be the primary focus after
core optimisation. The targeting capabilities of the particles will be further
developed under the guidance of world leading researchers in that specific
topic at MIPS.
We intend to develop a modular nanoparticle platform that can be
applied to different applications via changing only the drug carried or the
attached targeting moiety. Clinical application will remain in the forefront of
the expectations of the materials, as such all the production methods will be
developed such that clinical quality and quantity can be produced.
181
Chapter 7
7.1.0 Appendix
7.1.1 List of publications
7.1.2 Peer reviewed journal articles
1. Yaacob, M.H., Ahmad, M. Z., Sadek, A. Z., Ou, J. Z., Campbell, J.L.,
Kalantar-Zadeh, K., Wlodarski, W., (2013) Optical response of WO3
nanostructured thin films sputtered on different transparent
substrates towards hydrogen of low concentration. Sensors and
Actuators, B: Chemical,. 177
2. Dumee, L., Campbell, J.L., Sears, K., Schultz, J., Finn, N., Duke, M.,
Gray, S. (2012). The impact of hydrophobic coating on the performance
of carbon nanotube bucky-paper membrane distillation. Desalination,
DOI 10.1016
3. Ou, J. Z., Yaacob, M. H., Campbell, J.L., Breedon, M., Kalantar-zadeh,
K., & Wlodarski, W. (2012). H 2 sensing performance of optical fiber
coated with nano-platelet WO 3 film. Sensors and Actuators, B:
Chemical, 166(167), 1-6.
4. Campbell, J.L., Arora, J., Cowell, S. F., Garg, A., Eu, P., Bhargava, S.
K., et al. (2011). Quasi-cubic magnetite/silica core-shell nanoparticles
as enhanced MRI contrast agents for cancer imaging. PLoS ONE, 6(7),
5. Ramanathan, R., Campbell, J.L., Soni, S. K., Bhargava, S. K., &
Bansal, V. (2011). Cationic amino acids specific biomimetic
silicification in ionic liquid: A quest to understand the formation of 3-D
structures in diatoms. PLoS ONE, 6(3).
6. Yaacob, M. H., Campbell, J.L., Wisitsoraat, A., & Wlodarski, W. (2011).
Gasochromic response of Pd/NiO nanostructured film towards
hydrogen. Sensor Letters, 9(2), 898-901.
182
7. Rahmani, M. B., Breedon, M., Lau, D., Campbell, J.L., Moafi, A.,
McCulloch, D. G., et al. (2011). Gas sensing properties of
interconnected ZnO nanowires. Sensor Letters, 9(2), 929-935.
8. Ou, J. Z., Campbell, J.L., Yao, D., Wlodarski, W., & Kalantar-Zadeh, K.
(2011). In situ Raman spectroscopy of H 2 gas interaction with layered
MoO 3. Journal of Physical Chemistry C, 115(21), 10757-10763.
9. Ou, J. Z., Yaacob, M. H., Breedon, M., Zheng, H. D., Campbell, J.L.,
Latham, K., et al. (2011). In situ Raman spectroscopy of H 2
interaction with WO 3 films. Physical Chemistry Chemical Physics,
13(16), 7330-7339.
10. Kayani, A. A., Zhang, C., Khoshmanesh, K., Campbell, J.L., Mitchell,
A., & Kalantar-zadeh, K. (2010). Novel tuneable optical elements based
on nanoparticle suspensions in microfluidics. Electrophoresis, 31(6),
1071-1079.
11. Khoshmanesh, K., Zhang, C., Campbell, J.L., Kayani, A. A., Nahavandi,
S., Mitchell, A., et al. (2010). Dielectrophoretically assembled particles:
Feasibility for optofluidic systems. Microfluidics and Nanofluidics, 9(45), 755-763.
12. Campbell, J.L., Breedon, M., Latham, K., & Kalantar-Zadeh, K. (2008).
Electrowetting of superhydrophobic ZnO nanorods. Langmuir, 24(9),
5091-5098.
7.1.3 Peer reviewed conference proceedings
1. Campbell, J.L., Arora, J,. Bhargava, S.K,. Bansal, V. Targeted MRI
contrast using Herceptin labeled Fe3O4@SiO2 particles. Proceedings of
ICONN 2012
2. Campbell, J.L, O'Mullane, A., Bansal, V. & Bhargava, S. Templated
synthesis of metallic and bimetallic nanoshells with applications in
electrocatalysis, Proceeding of CHEMECA 2010
3. Ou, J., Yaacob, M.H., Campbell, J.L., Kalantar-Zadeh, K., Wlodarski,
W. H 2 sensing performance of optical fiber coated with nano-platelet
WO 3 film. 24th Eurosensors conference, Procedia Engineering, Vol (5)
1204-1207, 2010.
183
4. Campbell, J.L,. Lodhia, J,. Cowell, S,. Eu, P,. Bhargava, S.K,. Bansal V.
Enhanced MRI contrast using DMSA and silica coated magnetite
nanoparticles. Proceedings of ICONN 2010
5. Campbell, J.L, Breedon, M., Wlodarski, W., Kalantar-zadeh, K.
Superhydrophobic and superhydrophilic surfaces with MoOx
submicron structures. Proceedings of SPIE 2008
7.2.1 List of awards
Year
Award
2013:
Recipient of Victorian Postdoctoral Research Fellowship
Category 1 government grant valued in excess of $250,000.00 to fund 2
years further research at Stanford University and 1 Year after returning
to RMIT.
2012:
RMIT Higher Degree by Research Publication Excellence Award
2012:
C.N.R. Rao Postgraduate Research Excellence Award in
Materials Science.
Spent six weeks as a visiting researcher under the direct supervision
of Professor C.N.R. Rao at ICMS in Bangalore, India.
2011:
Recipient of ENDEAVOUR Cheung Kong Research Fellowship
Spent six months at Southeast University to learn large-scale
fabrication of magnetic particles and test their induction heating
properties.
2011:
Best Poster Award, ICONN Conference
2010:
John A. Brodie Medal for Achievement in Chemical Engineering,
Awarded for best paper submitted to the CHEMECA Conference.
2008:
Grant Ready Award for Excellence,
RMIT Business Plan Competition
2008:
Citipower & Powercore Australia Energy Innovation Award,
RMIT Business Plan Competition
184
fin.
185