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Synthesis and Surface Functionalization of
Gold Nanoparticles for Localized Tissue
Heating
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
by
Jiawey Yong
Faculty of Science, Engineering and Technology,
Swinburne University of Technology
August 2014
I
Declaration
I hereby declare that this thesis is my original work and, to the best of my knowledge, this
thesis contains no material previously published or written by another person, except where
due reference is made in the text. None of this work has been submitted for the award of
any other degree at any university. This thesis includes text and figures from 2 of my own
original papers published in the journals. Wherever contributions of others were involved
every effort has been made to acknowledge the contributions of the respective workers or
authors.
Jiawey Yong
August, 2014
II
Abstract
When formed into nanoparticles, some metals such as gold have shown intrinsic surface
plasmon resonance properties in which the surface plasmon resonance (SPR) wavelength in
the visible to near-infrared (NIR) spectrum is tunable by varying the size and shape of the
particles. Amongst the nanoparticles, gold nanorods (GNRs) are efficient photon-to-heat
converters and they strongly absorb NIR light at a wavelength overlapping its longitudinal
SPR and generate localised heating to the surrounding environment. Coupled with optical
sources emitting light at wavelengths within the NIR regime, the absorbing GNRs are
valuable in many biological applications particularly in photothermal therapy. While an
enormous amount of work has been reported on the photothermal ablation of tumors and
pathogenic microorganisms, and photothermally controlled release of biomolecules and
drugs in biological cells, the use of the photothermal capabilities of GNRs in assisting
neural stimulation is a relatively new concept. Conventional neural stimulation relies on
electrical currents to stimulate nerves and the technique has achieved significant
importance in restoring hearing and vision in neural prosthetics over the past decades.
However, electrical currents tend to spread out in tissue, which limits the accuracy with
which different nerve fibres can be stimulated. Alternatively, in conventional infrared
neural stimulation, the absorption of infrared light (typically at 1850 nm) in water and the
delivery of rapid pulses of heat to the nerve cells have proven effective in a wide range of
nerve types and systems. However, the process is relatively inefficient, requiring high
power levels, which places high demands on the laser technology. In addition, infrared light
has a relatively weak penetration into tissue, due to the absorption by water.
In order to address these limitations, this thesis demonstrates the feasibility of applying
stable GNRs in NIR stimulation of primary auditory neurons in vitro. To achieve this goal,
GNRs were first synthesised by means of seed-mediated growth during which their
longitudinal SPR wavelengths were tuned appropriately by either adjusting the
concentration of silver nitrate or gold seeds. Subsequent surface modifications via polymers
and silica encapsulation ensured that the nanorod longitudinal SPR wavelength was shifted
to final position at 780nm, matching the NIR laser wavelength used in this study.
III
Prior to investigating the neural stimulation, spiral ganglion neurons (SGNs), a model of
auditory neurons, were cultured with the silica-coated GNRs and the presence of nanorods
in the vicinity of neurons was investigated by dark-field light scattering and
microspectroscopy. The dark-field microscopy showed light scattering of GNRs from
within the SGNs and the microspectroscopic analysis of the associated SGNs revealed
typical spectral characteristic of GNRs. These results suggested that the silica-coated GNRs
were relatively stable in the cellular environments. Additionally, the scattering from the
silica-coated GNRs exhibited a more preserved scattering spectral profile when in the cell
cultures compared to GNRs with other surface coatings (bare GNRs, and polymer-coated
GNRs), as demonstrated using the NG108-15 cell line.
For NIR stimulation, whole-cell patch-clamp electrophysiology was used to monitor any
electrical response from the neurons upon laser irradiation. The results showed that spiral
ganglion neurons (SGNs) cultured with stable silica-coated GNRs were able to respond to
the 780 nm pulsed NIR laser irradiation by exhibiting enhanced cell electrical activity.
Variable millisecond laser pulse lengths were used, and elevating the laser pulse length
significantly increased the magnitude of cell electrical activity significantly. In particular,
SGNs fired action potentials when exposed to longer laser pulses. On the contrary, when
SGNs were cultured with silica-coated gold nanospheres that absorbed at 530 nm, the 780
nm laser pulses had no significant stimulatory effect on the neurons. Similarly, the 780 nm
laser pulses had no significant effect on the control SGNs without GNRs.
The enhanced cell electrical activity was attributed to the localised heating caused by
resonant absorption in the GNRs. In order to understand the photothermal heating of silica-
coated GNRs associated with the SGNs, indicative temperature changes near the surface of
the neurons were measured by an open patch micropipette. The results revealed
temperature rises between 0.5 ºC and 6.0 ºC depending on the laser pulse length used,
which also formed a correlation with the enhanced electrical activity of the neurons on
exposure to the laser pulses.
This work demonstrates that it is possible to stimulate nerve cells with a wavelength that
has a larger penetration depth than longer wavelength infrared sources, provided that a
strongly absorbing material such as gold nanorods is associated with the target nerves. The
IV
results of this study suggest the potential to use NIR light to improve the effectiveness of
infrared nerve stimulation.
V
VI
Acknowledgement
First and foremost, I have to express my sincere gratitude to Dr. John Fecondo and Dr.
François Malherbe for the opportunity that I have been given to undertake this PhD
research. Special thanks to François who took on the role of coordinating supervisor for the
project towards the end of third year of my PhD. He has given invaluable thoughts,
patience and tireless effort throughout the project. Many thanks go to Dr. Aimin Yu for
taking on a project, which was not part of her central research focus. Her technical skills
and sound thinking in the areas of particle functionalization, project management, and the
review of this thesis and other research publications have been much appreciated. I would
also like to extend my gratitude to Prof. Paul Stoddart for his generous guidance and
support at all stages of the project and the financial assistance during my PhD. My sincere
thanks also go to Dr. Daniel Eldridge and Prof. Sally McArthur for the helpful discussions.
My lab work would not have been completed easily without their help, so I would like to
gratefully acknowledge all technical staff in the chemistry and biochemistry labs at
Swinburne University of Technology for their technical support throughout the course of
my PhD. Big thanks to Savi who assisted in so many ways. My heartfelt appreciation also
goes to all co-workers in Yu group and Stoddart group for their unreserved support and
guidance in a way or another. Thanks all my fellow colleagues who shared my office and
lab, especially Nelson, Li, and Tas for all the laughs during the stressful times.
To collaborators, my sincere acknowledgement goes to Dr. Karina Needham, who
contributed her time on SGN culture preparation and patch-clamp study. Also, I would like
to thank Dr. Sharath Sriram and RMMF staff for allowing me to use their TEM and
assisting me in the use of the instrument. Thanks also to MCN staff and Ricardas Buividas
for their assistance with the use of microspectrometer and Dr. Lorenzo Rosa for the FDTD
simulation.
Finally, a special thanks to my beloved family and Jun for their continuous love and
support.
VII
VIII
Table of Contents
Declaration ............................................................................................................... I
Abstract ................................................................................................................... II
Acknowledgements ............................................................................................... VI
Table of Contents ............................................................................................... VIII
List of Figures .................................................................................................... XIII
List of Tables ................................................................................................... XVIII
Acronyms............................................................................................................ XIX
1. Introduction ........................................................................................................ 1
1.1 Introduction .................................................................................................. 1
1.2 Thesis Overview……………………………………………………………..3
2. Literature Review ............................................................................................... 6
2.1 Gold Nanoparticles ........................................................................................ 6
2.1.1 Synthesis, Shape and Size Control ..................................................... 6
2.1.1.1 Wet-chemical Synthesis of Gold Nanospheres .................................... 6
2.1.1.2 Electrochemical.................................................................................... 9
2.1.1.3 Laser Ablation, UV and Microwave Irradiation ................................ 10
2.1.1.4 Green Synthesis ................................................................................. 10
2.1.2 Fabrication of Gold Nanorods .......................................................... 11
2.1.2.1 Synthesis of Gold Nanorods (Seed-mediated Growth Method) ........ 11
2.1.2.2 Growth Mechanism and the Role of Silver Ions ................................ 14
2.1.2.3 Binary Surfactant System .................................................................. 15
2.1.2.4 High Aspect Ratio Gold Nanorods .................................................... 16
2.1.2.5 Seedless Growth Method ................................................................... 17
2.1.2.6 Templated Synthesis .......................................................................... 17
2.1.2.7 Electrochemical Method .................................................................... 18
2.1.2.8 Photochemical Method ...................................................................... 18
2.1.2.9 Size and Shape Tuning of Pre-Grown Gold Nanorods ...................... 19
IX
2.2 Optical Properties of Gold Nanoparticles ....................................................... 20
2.2.1. Dark-field Light Scattering ............................................................. 23
2.2.2 Two-photon Luminescence Imaging ................................................ 24
2.2.3 Photoacoustic Imaging ..................................................................... 25
2.3 Surface Modification and Functionalization ................................................... 25
2.3.1 Layer-by-layer (LbL) Polyelectrolyte Coatings ............................... 27
2.3.2 Thiol Ligands ................................................................................... 28
2.3.3 Silica Coating ................................................................................... 32
2.3.4 Other Functionalization Strategies ................................................... 34
2.4 Biocompatibility and Biodistribution of Gold Nanoparticles ........................ 36
2.4.1 In vitro .............................................................................................. 36
2.4.2 In vivo and Biodistribution ............................................................... 38
2.5 Photothermal Therapy ..................................................................................... 39
2.5.1 Light and Biological Tissue Interactions .......................................... 39
2.5.2 Photothermal Heating of Biological Tissues .................................... 39
2.5.3 Nanomaterial-based Photothermal Heating ..................................... 40
2.6 Neural Stimulation .......................................................................................... 45
2.6.1 Electrical Stimulation ....................................................................... 47
2.6.2 Photostimulation............................................................................... 47
2.6.2.1 Extrinsic Photoabsorbers for Photothermal Stimulation.................... 50
2.6.2.2 Gold Nanorods for Neural Stimulation .............................................. 51
2.6.2.3 Neuro-targeting and Blood Brain Barrier .......................................... 52
2.6.3 Photovoltaics Interface ..................................................................... 53
3. Synthesis, Surface Modification, and Functionalization of Gold
Nanoparticles .................................................................................................... 57
3.1 Introduction ................................................................................................. 57
3.2 Materials and Methods ................................................................................ 59
3.2.1 Materials ........................................................................................... 59
3.2.2 Preparation of Gold Nanospheres .................................................... 60
3.2.3 Preparation of Gold Nanorods.......................................................... 60
3.2.4 Surface Modification ........................................................................ 61
X
3.2.4.1 Polyelectrolyte (PE) Coating ............................................................. 61
3.2.4.2 PVP Coating ....................................................................................... 61
3.2.4.3 mPEGylation ...................................................................................... 61
3.2.4.4 Silica Coating ..................................................................................... 61
3.2.4.5 FDTD simulation ............................................................................... 62
3.2.4.6 Functionalisation of Silica-coated Gold Nanoparticles ..................... 62
3.2.4.6.1 Amine Silanization and Fluorescent Quantification ............................ 62
3.2.4.5.2 Polydopamine ...................................................................................... 63
3.2.5 Characterisation ................................................................................ 63
3.3 Results ......................................................................................................... 64
3.3.1 Preparation of Gold nanoparticles .................................................... 64
3.3.1.1 Shape Control..................................................................................... 64
3.3.1.2 Longitudinal SPR Band Tuning ......................................................... 66
3.3.1.2.1 Ascorbic Acid....................................................................................... 67
3.3.1.2.2 Silver Nitrate ........................................................................................ 68
3.3.1.2.3 Gold Seeds ........................................................................................... 70
3.3.2 Layer-by-Layer (LbL) Polyelectrolytes Coating.............................. 73
3.3.3 mPEGylation .................................................................................... 75
3.3.4 Silica Coating ................................................................................... 78
3.3.5 FDTD Simulation ............................................................................. 86
3.3.6 Functionalisation of Silica-coated Gold Nanoparticles .................... 87
3.3.6.1 Amines ............................................................................................... 87
3.3.6.2 Polydopamine .................................................................................... 91
3.4 Discussion .................................................................................................... 93
3.5 Conclusion ................................................................................................... 96
4. Dark-field Analysis of Gold Nanoparticles in Neuronal Cells ...................... 99
4.1 Declaration for Chapter 4 ............................................................................ 99
4.2 Introduction ................................................................................................. 99
4.3 Materials and Methods. ............................................................................. 102
4.3.1 Preparation of Immobilized Nanoparticles on PDA-glass
Surface ........................................................................................... 102
XI
4.3.2 Preparation of Neural Cultures ....................................................... 102
4.3.2.1 Primary Cells (Spiral Ganglion Neurons) ........................................ 102
4.3.2.2 NG108-15 Cell Line ........................................................................ 103
4.3.3 Dark-field Light Scattering and Microspectroscopy ........................... 103
4.4 Results ....................................................................................................... 104
4.4.1 Dark-field Light Scattering of Nanoparicles on Glass Slides ........ 106
4.4.2 Dark-field Light Scattering (Primary Cultures of SGNs) .............. 108
4.4.3 Dark-field Light Scattering (NG108-15 Cell Line)........................ 114
4.5 Discussion .................................................................................................. 120
4.6 Conclusion ................................................................................................. 123
5. Photothermal Stimulation of Spiral Ganglion Neurons .............................. 125
5.1 Declaration for Chapter 5 .......................................................................... 125
5.2 Introduction ............................................................................................... 125
5.3 Materials and Methods. ............................................................................. 128
5.3.1 NIR Laser - 780 nm. ....................................................................... 128
5.3.2 Laser Heating of Bulk Nanorod Solutions. .................................... 129
5.3.3 Culture Methods. ............................................................................ 129
5.3.4 Laser Stimulation and in vitro Electrophysiology. ......................... 130
5.3.5 In vitro Local Temperature Measurements. .................................... 131
5.4 Results.......................................................................................................... 132
5.4.1 Laser Heating of Water and Aqueous Gold Nanorods. .................. 132
5.4.2 Laser Stimulation and Whole-cell Patch-clamp
Electrophysiology........................................................................ 135
5.4.2.1 Voltage-clamp. ................................................................................. 138
5.4.2.2 Current-clamp. ................................................................................. 141
5.4.3 Local Temperature Measurements. ................................................ 144
5.5 Discussion .................................................................................................. 150
5.6 Conclusion ................................................................................................. 154
6. Summary and Future Directions ................................................................... 157
6.1 Summary of Findings ................................................................................ 157
6.2 Future Directions ....................................................................................... 159
XII
Bibliography ......................................................................................................... 164
Appendix .............................................................................................................. 205
List of Publications .............................................................................................. 206
XIII
List of Figures
Figure Page
2.1 Schematic representation of the formation of spherical gold nanoparticles
by the citrate reduction method...................................................................... 8
2.2 Schematic illustration of stepwise synthesis of gold nanorods by the seed-mediated silver-assisted growth method ............................................... 13
2.3 Schematic illustration of the formation of gold nanorods in the absence
of silver via surfactant preferential binding or zipping mechanism .............. 15
2.4 Schematic illustration of localized surface plasmon resonance (SPR)
of gold nanoparticles ...................................................................................... 21
2.5 Physical effects arising from the longitudinal plasmon resonance of
gold nanorods induced by NIR laser .............................................................. 24
2.6 Schematic showing general methods employed for surface the
modification and functionalization of gold nanorods .................................... 27
2.7 Absorption spectra of hemoglobins and water in the wavelength range between 400 and 1000 nm ............................................................................. 40
2.8 Schematic depicting neural stimulation by different means ............................. 45
3.1 Synthesis of GNSs ............................................................................................. 65
3.2 Synthesis of GNRs ............................................................................................ 66
3.3 Synthesis of GNRs using variable ascorbic acid concentrations ...................... 68
3.4 Synthesis of GNRs using variable Ag+ concentrations ..................................... 69
3.5 The longitudinal SPR band positions with respect to varying
Ag+ concentrations in the reaction ................................................................. 70
3.6 The UV-vis spectrum of Au seeds used for the growth of GNRs ...................... 71
3.7 Synthesis of GNRs using variable Au seed concentrations .............................. 72
XIV
Figure Page
3.8 The longitudinal SPR band positions with respect to varying
Au seed concentrations in the reaction .......................................................... 72
3.9 PSS coating of GNRs ........................................................................................ 73
3.10 Zeta-potential of GNRs measured after each deposition of PE ....................... 74
3.11 UV-vis absorption spectra of GNRs with PEs ................................................ 75
3.12 Time-dependence of mPEGylation of GNRs, revealed by charges in
zeta potential .................................................................................................. 77
3.13 UV-vis absorption spectra of GNRs before and after mPEGylation ............... 77
3.14 Raman spectra of raw GNRs (red dashed-line) and PEGylated GNRs ........... 78
3.15 A representative TEM image of silica-coated GNRs prepared by using PVP as the surface primer .............................................................................. 79
3.16 UV-vis spectral shift (arrow) as a result of surface coating with silica .......... 80
3.17 UV-vis absorption spectra of silica-coated GNRs in solvents of different
RI................................................................................................................... 80
3.18 TEM images showing GNRs coated with different silica shell thicknesses ... 81
3.19 UV-vis absorption spectra corresponding to the silica-coated GNRs
as shown in Figure 3.18(a), (b) and (c) .......................................................... 81
3.20 Changes in surface potential of GNRs after surface modifications
with mPEG and silica..................................................................................... 82
3.21 UV-vis spectra of GNRs showing the redshift and broadening of
the longitudinal SPR bands after mPEGylation and silica coating ............... 83
3.22 Silica coating of mPEGylated GNRs .............................................................. 84
3.23 UV-vis spectrum of silica-coated PVP/GNSs shows a red-shift (arrow)
of the SPR band after silica coating ............................................................... 85
3.24 TEM image of silica-coated PVP/GNSs ......................................................... 85
XV
Figure Page
3.25 FDTD calculated extinction, absorption, and scattering cross-section spectra
of uncoated (solid curve) and silica (15 nm)-coated (dashed curve) GNRs .. 87
3.26 FTIR spectra of silica-coated GNSs, and amine-grafted silica-coated GNSs . 89
3.27 Fluorescence excitation and emission spectra of primary amine
bound fluorescamine ...................................................................................... 89
3.28 Fluorescamine calibration curves of monoamine standards ............................ 90
3.29 Polydopamine overcoating of silica-coated GNRs ......................................... 92
3.30 ATR-FTIR spectra of polydopamine (top) and
polydopamine/silica-GNRs (bottom) ............................................................. 93
4.1 Dark-field light scattering and microspectroscopy analysis .............................. 105
4.2 Typical spectral profile of the halogen lamp source used in the experiment .... 106
4.3 Dark-field scattering images of GNSs (left) and GNRs (right) on
glass slides ..................................................................................................... 107
4.4 Typical scattering spectra of GNSs (50 nm) and GNRs (48 × 13 nm) on the
PDA-modified glass surface .......................................................................... 108
4.5 Dark-field images showing the SGNs (arrows) surrounded by
other explanted cells ...................................................................................... 109
4.6 Dark-field microspectroscopic analysis of NR-SGNs ...................................... 111
4.7 Scattering spectra acquired from different targets ............................................ 112
4.8 Dark-field microspectroscopic analysis of NS-SGNs ....................................... 113
4.9 Typical bright-field (a) and dark-field (b) images of NG108-15 cells
incubated with PDA/SiO2-GNRs .................................................................. 116
4.10 Representative dark-field image showing internalization of
PDA/SiO2-GNRs in NG108-15 cell nuclei .................................................... 116
4.11 The dark-field image of NG108-15 cells showing scattering from
the SiO2-GNRs ............................................................................................... 117
XVI
Figure Page
4.12 Dark-field images of NG108-15 cells containing (a) PSS-coated GNRs
and (b) bare GNRs ......................................................................................... 117
4.13 Dark-field images of NG108-15 cells (a) without and (b) with
mPEG/GNRs .................................................................................................. 118
4.14 Average scattering acquired from NG108-15 cells for GNRs with
different surface coatings ............................................................................... 119
5.1 Scheme of the experimental setup for bulk heating of nanorod solution .......... 133
5.2 Comparison of laser-induced heating of water with different nanorod
contents .......................................................................................................... 134
5.3 Schematic of experimental setup for simultaneous laser stimulation
and whole-cell patch clamp recordings of a neuron ...................................... 136
5.4 Phase contrast micrograph showing a patched SGN (red arrow) with
microelectrode to the right and the optical fibre to the left of the image ....... 136
5.5 Whole-cell patch-clamp recording of a healthy neuron, showing a typical
response .......................................................................................................... 137
5.6 Averaged voltage-clamp data for a typical neuron in response to laser pulses of different duration ............................................................................ 139
5.7 Averaged voltage-clamp data for NS-SGNs and control SGNs ........................ 139
5.8 Comparison of typical transmembrane currents elicited by 25 ms laser pulses
(red traces) ....................................................................................................... 140
5.9 Dependence of the laser-induced charge on laser pulse duration for the analysed neurons ............................................................................................ 141
5.10 Current-clamped recording of an NR-AN showing subthreshold membrane
potentials (black and blue traces) and an action potential (red trace) ............ 142
5.11 Raw data of current-clamp recording showing action potentials fired
in a SGN in response to 25 ms laser pulses .................................................. 143
5.12 Multiple firing evoked under continuous laser pulse ...................................... 143
5.13 Pipette temperature calibration ........................................................................ 146
XVII
Figure Page
5.14 Data processing of the recorded signals .......................................................... 147
5.15 Typical data processing of the recorded signals .............................................. 148
5.16 Temperature changes as detected by the open-pipette method. ...................... 149
XVIII
List of Tables
Table Page
2.1 Summary of thiol ligand exchange for functionalizing gold nanoparticles ...... 30
2.2 Examples of photoabsorbers used in photothermal therapy .............................. 43
5.1 Variable laser pulse lengths and the equivalent energy per pulse used in the study .................................................................................................... 137
XIX
Acronyms
λmax Wavelength with maximum absorbance
ζ Zeta
ρ Density
AA Ascorbic acid
AAS Atomic absorption spectroscopy
Ag+ Silver ions
APTMS 3-aminopropyltrimethoxysilane AR Aspect ratio
ATR Attenuated total reflection
Au Gold
a.u. Arbitrary absorbance units
BBB Blood brain barrier
CCD Charge-coupled device
CTAB Cetyltrimethylammonium bromide
CW Continuous wave
DMEM Dulbecco’s modified Eagle medium
DMSO Dimethylsulphoxide
DNA Deoxyribonucleic acid
FDTD Finite-difference time-domain
FTIR Fourier transform infrared spectroscopy
GNR Gold nanorod
GNS Gold nanosphere INS Infrared neural stimulation
IR Infrared
ITO Indium tin oxide
LbL Layer-by-layer
mPEG Methoxy-poly(ethylene)glycol
Mw Molecular weight
NH2 Amine
NIR Near-infrared
OD Optical density
PAH Poly(allylamine hydrochloride)
PBS Phosphate buffered saline
PDA Polydopamine
PE Polyelectrolyte
PEG Poly(ethylene)glycol
XX
PSS Poly(styrene sulphonate)
PVP Poly(vinylpyrrolidone)
RI Refractive index
SGN Spiral ganglion neuron SH Thiol
SiO2 Silica
SPR Surface plasmon resonance
TEM Transmission electron microscope
TEOS Tetraethyl orthosilicate
TRPV Transient receptor potential vanilloid
UPD Underpotential deposition
UV Ultraviolet
Vis Visible
1
Chapter 1: Introduction
1.1 Introduction
anosized materials are generally regarded as materials having a structure with at
least one dimension in the nanometer scale which is 1 100 nm. Through
manipulation of size, researchers can potentially modify the properties of a
material, discover new science and develop new devices and technologies. For instance,
research into the use of noble metal nanomaterials has experienced a vast growth when
it became recognised that the bulk properties of the materials change drastically as their
sizes decrease from bulk to small clusters of atoms.1 Two principal factors are
responsible for the properties of nanosized materials differing significantly from their
bulk condition; the increase in relative surface area-to-volume ratio and size-dependent
properties that begin to dominate when matter is reduced to the nanoscale. As the size
decreases, the most notable changes are the optical and electronic properties of the
materials, giving rise to fascinating effects not observed in bulk materials. This has
prompted the use of nanomaterials in diverse fields including biomedical, electronics,
and environmental research. Recent years have witnessed the growing field of
biophotonics, which has a strong focus on the interaction between biological materials
and light. There has been significant and major advancement in biophotonics during the
past decade, for example, laser light is routinely used by ophthalmologists for reshaping
the cornea to improve its focus.2 In recent years, the field of biophotonics has also been
extended by progress in nanotechnology, with the implementation of nanomaterials in
biological materials. This multidisciplinary technology has widened the potential for
innovation in biomedical applications.
For decades, electrical stimulation has been the gold standard for neural stimulation
in which the electrical pulses conducted across neural tissues are applied through a
stimulating electrode, leading to cell depolarisation and generation of action potentials.
Alternatively, neural stimulation by means of laser irradiation using mid-infrared
wavelengths (typically ca. 1450 to 2200 nm) has seen a surge of interest in recent years
and has become widely known as infrared neural stimulation (INS).3, 4
The technique
N
2
can provide several advantages over electrical stimulation: high spatial resolution, no
direct contact required between the stimulation source and target neurons, and no
stimulation artefact when performing stimulation and direct recording simultaneously.5-7
In addition, unlike other means of neural stimulation such as optogenetics and caged
neurotransmitters,8 INS does not require genetic or other alterations prior to stimulating
the target neurons as the technique only relies on the absorption of mid-infrared light by
water surrounding the target tissue. The thermal transient mediated by water absorption
of infrared light is known to be a critical factor contributing to INS.9, 10
Due to the
overtone absorption by water in the mid-infrared region, the infrared light is absorbed
and the photon energy is converted into thermal energy. The subsequent heat dissipation
may lead to processes that are responsible for neural stimulation: (i) transient
temperature-induced reversible changes in cell membrane capacitance,10
and (ii)
activation of temperature-sensitive ion channels.11
INS is indeed a promising
stimulation strategy and has great potential to underpin the next generation of neural
prostheses. However, in many cases, particularly in vivo, the presence of intervening
absorbing tissue layers has limited the efficiency of mid-infrared light in reaching the
target neural tissues. In this context, higher power lasers are often required for
stimulation purposes in order to compensate for the lack of penetration depth.5
Alternatively, careful selection of laser wavelengths used for neural stimulatiom
could provide a solution, such as near-infrared (NIR) wavelengths (typically ca. 650 to
900 nm), where biological tissue is most transparent.12
In this respect, there is a need for
photo-absorbing chromophores in the NIR wavelength range and the ideal
chromophores should possess features including biocompatibility, large absorption
cross section, highly efficient photon-to-heat conversion and preferably imaging
capabilities. Biocompatible gold nanorods are appealing candidates given their strong
extinction cross-sections (absorption and scattering cross-sections) at their plasmon
resonances in the NIR. The extinction coefficient of gold nanorods is several orders of
magnitude larger than those of gold nanospheres and organic chromophores.13
In
addition, gold nanorods are excellent candidates as a multifunctional biological platform
for simultaneous photothermal conversion and imaging.14
In this thesis, a novel method for pulsed NIR laser stimulation of neurons assisted
with highly photo-absorbing gold nanorods is described. The longitudinal plasmon
3
absorption wavelength of the gold nanorods was tuned to match closely with the
incident NIR laser wavelength at 780 nm in order to ensure efficient excitation of
plasmon resonance for maximal photothermal conversion. Besides, gold nanorods were
made biocompatible by coating with polymers and a silica dielectric shell, which has
been effective in reducing the toxicity effects of remnant CTAB present on the nanorod
surface. Furthermore, due to the large extinction cross-sections, not only have gold
nanorods provided efficient photothermal conversion for neural stimulation using the
NIR laser, their light scattering properties have also enabled them to be visualised
directly in neurons by dark-field imaging and detected by spectroscopic analysis.
Depending strictly on the laser pulse lengths or energies, upon laser irradiation, the
heating from the silica-coated gold nanorods has successfully stimulated primary
auditory neurons in vitro as indicated by whole-cell patch-clamp electrophysiology. The
heating effects associated with the nanorods around the neurons were measured by the
open-pipette method, confirming a temperature increase during the laser irradiation.
1.2 Thesis Overview
Here, the thesis contents in the following chapters are summarised.
Because of the wide range of topics relevant to the experimental work conducted in this
thesis (chemistry, materials science, plasmonics, optics, and electrophysiology),
Chapter 2 gives a substantial literature overview on the synthesis, surface modification
and functionalization, optical properties, and relevant biological uses and issues dealing
with noble metal nanoparticles, with particular focus on gold nanoparticles. Given that
gold nanorods were used as photoabsorbers for neural stimulation as described in this
thesis, this overview also emphasizes the capabilities of gold nanorods in bioimaging
and photothermy. Furthermore, the theory and background of neural stimulation using
different methods are discussed.
Chapter 3 describes the preparation and characterisation of nanomaterials used
throughout the thesis. In particular, the wet-chemical synthesis of gold nanoparticles
(nanospheres and nanorods) and the experimental procedures taken to effectively tune
the longitudinal plasmon wavelengths of gold nanorods to the desired wavelength range
are presented. In addition, surface modification and functionalization of gold
4
nanoparticles using polymer and silica coatings are also reported. Gold nanoparticles,
before and after their surface modifications/functionalization, are characterised by a
broad range of techniques including electron microscopy, UV-Vis-NIR
spectrophotometry, and Raman and Fourier transform infrared spectrophotometry
(FTIR). The results are compared morphologically and/or with regards to their colloidal
stability and are discussed throughout the chapter.
In Chapter 4, interactions between the gold nanoparticles and neuronal cells in vitro are
described by referring to dark-field light scattering analyses. Silica-coated gold
nanoparticles associated with the spiral ganglion neurons – a model of primary auditory
neurons of early post-natal rats, were visualised by dark-field microscopy. The stability
information pertinent to the nanoparticles in the vicinity of the neurons was obtained
through the analysis of the scattering spectra acquired using a microspectrometer. The
results are compared with the typical scattering spectra of gold nanoparticles
immobilised stably on a polydopamine-modified substrate. Furthermore, the
interactions between NG108-15 cells – a model neuroblastoma cell line, and gold
nanorods modified/functionalised with polymer and silica coatings were investigated. In
particular, the uptake efficiency of gold nanorods with different surface compositions
was compared qualitatively via dark-field imaging.
Chapter 5 is devoted to demonstrating the feasibility of gold nanorods in assisting the
photothermal stimulation of nerves upon 780 nm laser irradiation. Experiments were
carried out to show that gold nanorods have dominant absorption compared to water at
780 nm. The effect of laser-induced bulk heating was compared between distilled water
and an aqueous solution of gold nanorods. Meanwhile, groups of spiral ganglion
neurons containing silica-coated gold nanorods and, silica-coated gold nanospheres, and
a control with no gold nanoparticles were assessed for any enhanced electrical activity
upon 780 nm laser irradiation and stimulation. Whole-cell patch-clamp
electrophysiology was utilised to monitor the stimulation process and the results are
compared and discussed, taking into account the measured temperature rises during the
laser irradiation.
Chapter 6 concludes this thesis with a summary and a discussion of future perspectives.
5
6
Chapter 2: Literature Review
2.1 Gold Nanoparticles
2.1.1 Synthesis, Shape and Size Control
Historically, Michael Faraday published the earliest method for the formation of
colloidal gold by reduction of chloroaurate (AuCl4–) using phosphorus in the presence
of carbon disulphide as a stabilizing agent. Faraday’s discovery in 1857 led to the
subsequent development of methods for synthesizing or fabricating colloidal gold. Over
the years, there have been many studies on bottom-up and top-down techniques to
synthesize colloidal gold by wet-chemical methods,15, 16
laser ablation,17-19
photochemical,20, 21
and electrochemical methods.22
Wet-chemical synthesis is a rather
popular method compared to all other methods, due to the benefits of a facile procedure,
reasonably low cost, high yields, and environmental friendliness.23
In the wet-chemical
methods, manipulating experimental parameters such as reducing agents, reaction time
and temperature, and stabilizing agents, gold nanoparticles with different shapes such as
rods,15, 16
nanocubes,24
nanoprisms,25, 26
nanostars,27
nanocrosses,28
nanoboxes, and
hollow nanoshells29
and nanocages,30
can be synthesized in a range of sizes. These
approaches are useful in fine-tuning the properties of gold nanoparticles, particularly the
optical properties that are greatly dependent on particle size and shape.31
This section
reviews the synthesizing methods for nanospheric and nanorod gold and their optical
properties.
2.1.1.1 Wet-chemical Synthesis of Gold Nanospheres
The first reproducible method reported for the synthesis of colloidal gold was from
Turkevich and co-workers in 1951, in which the reduction of AuCl4–
was accomplished
by using trisodium citrate at the boiling point of water.32
This citrate reduction method
is also known as the Turkevich method, and the size of spherical nanoparticles produced
is ca. 20 nm. In 1973, Frens refined the Turkevich method and achieved the formation
of spherical gold nanoparticle in a size range between 16 and 150 nm.33
The method
7
tunes the particle size by varying the molar ratio between the trisodium citrate and
AuCl4–. The Turkevich-Frens method is very often used even now for producing
monodisperse gold colloids.
The favoured Turkevich-Frens method produces high yield and near-monodisperse
gold nanoparticles with high yield and stability in aqueous solution. This method uses
the citrate to reduce a gold salt in the form of AuCl4– and also to stabilize the
nanoparticles. The advantage of this method is that by altering the amount of citrate, the
size of the particles can be controlled.33
The reduction of AuCl4–
and the formation of
nanospheric gold occurs in three successive steps34
: nucleation, growth and coagulation,
as illustrated in Figure 2.1. Citrate is a weak reducing agent, and as such it is unable to
reduce gold ions at room temperature unless heating is applied. When heated, the citrate
is oxidized and produces acetone decarboxylate which reduces Au (III) to Au (I). The
nucleation step occurs at the same time, during which gold nuclei are created as a
multimolecular complex of Au (I) and acetone decarboxylate. The nuclei decompose
irreversibly to form Au (0) and the number of nuclei that are formed or decomposed
depends greatly on the amount of citrate. During the nucleation step, once a sufficient
number of nuclei are formed, the nucleation process slows down and the growth step
begins.Now excess Au (I) is reduced on the surface of the existing nuclei until all of the
ions are consumed. In the final coagulation step, the formation of larger gold particles is
achieved by several nuclei fusing together. The overall chemical reaction35
in the
synthesis of gold nanospheres by the citrate reduction method is given by:
6������
+ ������ + 5��� → 6���
+ 24���
+ 18��
+ 6���
The successful formation of citrate-capped gold nanospheres is typically indicated by
the characteristic ruby red colour of the suspension. The citrate prevents particle
aggregation by acting as a stabilizing agent and providing the nanoparticle surface with
a negative charge, which confers electrostatically repulsive forces to keep nanoparticles
with similar layers apart from each other, so that the nanoparticles can remain in
solution as a stable colloidal suspension. The size of individual colloidal gold particles
in the solution is determined by the number of gold nuclei formed at the beginning and
thus increasing or decreasing the molar ratio of citrate to AuCl4– can predetermine the
final particle size.33, 34
The citrate reduction method has been extended and modified
over the years. For instance, one-pot synthesis of gold nanospheres using citrate
8
reduction can also produce 3-mercaptopropionate (MPA)-stabilized nanoparticles when
citrate and MPA are simultaneously added and refluxed during the citrate reduction
process.36
Particle enlargement has also been reported in which the aqueous solution
consisting of citrate-capped gold nanospheres and Au(CN)2–
is irradiated with a gamma
source.37
The organic radicals released by the gamma irradiation can cause the reduction
of Au(CN)2–
at the gold nanospheres, and therefore increases the diameter of the
nanoparticles.
Murphy and co-workers have reported a synthesis method for variable sizes (5 nm to
40 nm) of gold nanospheres using seeded growth.38
In this method, the small citrate-
capped gold seeds are first prepared by the reduction of AuCl4–
with sodium
borohydride. The ~3.5 nm gold seeds are added to the growth solution consisting of
cetyltrimethylammonium bromide (CTAB) and ascorbic acid. The size of the
nanospheres can be manipulated by varying the ratio of seed to AuCl4- and the gold
nanospheres produced in this way are separated from impurities such as rod-shaped
gold nanoparticles by centrifugation. Interestingly, this seeded growth method has been
refined and employed by the same group to produce gold nanorods.15
Figure 2.1 Schematic representation of the formation of spherical gold nanoparticles by
the citrate reduction method.
9
Production of smaller colloidal gold (1 to 5 nm) was developed by Brust and co-
workers in 1994.39
This method is referred to as the Brust--Schiffrin method and uses a
two-phase system and alkanethiol stabilization for synthesizing spherical gold
nanoparticles. In this method, AuCl4– is transferred to toluene using
tetraoctylammonium bromide as the phase-transfer reagent and is subsequently reduced
by sodium borohydride in the presence of dodecanthiol. During the synthesis, the
orange colour of AuCl4- in the organic phase turns to deep brown upon addition of
borohydride, indicating the formation of tiny dodecanethiol-capped gold particles that
are soluble in non-polar organic solvents and are very stable due to the strength of the
gold-thiol bond.39
The particle size can be tuned from 1.5 to 5 nm under various
experimental conditions, such as gold to thiol ratio, temperature, and reduction rate. The
Brust-Schiffrin one phase system was later developed and used to produce water soluble
thiolate-protected gold particles in which bifunctional p-mercaptophenol is used as the
ligand.40
The formation of gold-thiolates by Brust and co-workers in the 90’s has
sparked significant research interests in the subsequent uses of the gold-thiolate
complex that is also referred to as monolayer protected clusters (MPCs).41
MPCs were
used several decades ago as remedies for diseases.42
Apart from their therapeutic
potential, gold MPCs have been reported as catalysts,43
wherein controlling the core
size and the number of gold atoms in the formation of gold-thiolate nanoclusters is of
paramount importance.41
The chemical formulae of the gold-thiolate complexes were
later confirmed by electrospray ionisation mass spectroscopy (ESI-MS) analysis.44, 45
This allowed the preparation of MPCs with ‘magic-number’ atomic core mass. For
instance, Qian and Jin used a modified method of Brust to prepare monodisperse
Au144(SCH2CH2Ph)60 nanoparticles.46
Other examples include clusters of: Au24, Au25,
Au38, Au102, Au130, and Au225.
2.1.1.2 Electrochemical
Ma and co-workers demonstrated the preparation of gold nanoparticles via the
electrochemical reduction of AuCl4– in the presence of poly(vinylpyrrolidaone) (PVP)
wherein the size can be controlled by adjusting the ratio of PVP:AuCl4–.47, 48
A double
pulse electrochemical technique is also employed in the preparation of gold
10
nanoparticles for better control of the particle size.49
Gold nanoparticles have also been
synthesized in situ by electrochemical methods.50
2.1.1.3 Laser Ablation, UV and Microwave Irradiation
Laser ablation of metal is a top down method for nanoparticle fabrication and it is
performed in a controllable and contamination-free environment without the use of
reducing agent. Synthesis of gold nanospheres by means of laser ablation was first
demonstrated in 2001 by Mafuné et al.17
In this method, a gold metal plate is immersed
in an aqueous solution of sodium dodecyl sulphate (SDS) and subsequently the gold
plate is subjected to laser irradiation. The particle size range produced in this way is
dependent on the SDS concentration; smaller size particles are produced with higher
SDS concentration. Pulsed laser ablation in liquids has recently emerged as a novel
“green” tool for synthesis of colloidal nanomaterials, including gold. In this method,
laser radiation is used to ablate a solid target immersed in liquid, yielding nanoclusters
which are then released into the liquid, forming a colloidal nanoparticle solution. The
effective fluorescence quenching as well as sensitive SERS detection of rhodamine 6G
was recently demonstrated using laser-ablated and -fragmented gold nanospheres.19
Microwave irradiation can rapidly provide a uniform heating source to the reaction
and produce homogeneous nucleation sites, hence shortening the crystallization time.
This approach is often carried out in the AuCl4–
and a stabilizing agent such as PVP and
surfactants. Particle size can be controlled by the ratio between AuCl4–
and the
stabilizing agents. There have been many examples of microwave-assisted synthesis.51-
54 While using the microwave irradiation approach is faster in producing gold
nanoparticles compared to the citrate reduction method, the particle size and shape
uniformity is often compromised.
2.1.1.4 Green Synthesis
Greener biosynthesis has recently emerged to offer an alternative to chemical
synthesis methods. Despite the fact that metal nanoparticles show toxicity to some
microorganisms,55
bio-production of nanoparticles in living microorganisms has been
reported over the past years, for instance using E.coli,56
Lactobacillus,57
fungus
Verticillium sp.,58
and potentially viruses.59
Ecofriendly plant-based synthesis of gold
nanoparticles has also been widely reported over the past years.60-62
Leaf extracts,63-66
11
mushroom,67
and fruits,62
can, in general, produce nanoparticles with a size range of 5
to 200 nm using the plant materials. A comprehensive review with specific focus on the
plant-based synthesis of metallic nanoparticles has been published elsewhere.68
2.1.2 Fabrication of Gold Nanorods
Being able to synthesise nanorods with different sizes is important; for example, it
has been determined that larger particles are more efficient for both absorption and
scattering, both of which play a dominant role in simultaneous photothermal heating
and bioimaging contrast enhancement.69
Therefore, gold nanorods are synthesised in a
range of different aspect ratios, primarily via wet chemistry methods, which provide a
facile means for controlling nanorod size. For gold nanorod synthesis, bottom-up and
top-down growth methods have been adopted. In the former, gold nanorods are formed
through nucleation in an aqueous solution in the presence of a gold precursor and a
reducing agent. The later approach produces gold nanorods through a combination of
different physical lithography processes and gold deposition. The bottom-up methods
for synthesizing gold nanorods include wet-chemical, electrochemical, sonochemical,
solvothermal, microwave-assisted and photochemical reduction techniques.
2.1.2.1 Synthesis of Gold Nanorods (Seed-mediated Growth Method)
The seed-mediated growth (seeded growth) method is the most common synthesizing
method and is superior to other methods because of the simplicity of the procedure, high
synthesis yield and ease of particle size control, and importantly the method does not
require a high processing temperature. The seeded growth method was first published
by Murphy’s group in 2001.15
In a typical synthesis, citrate-capped small gold seeds
(~3.5 nm) are prepared by reduction of chloroauric ions with sodium borohydride. The
citrate-capped gold seeds are penta-twinned nanocrystals26
that are added to the growth
solution containing Au (I) ions obtained by the reduction of chloroauric ions with
ascorbic acid in the presence of cetyltrimethylammonium bromide (CTAB) surfactant.
The addition of citrate-capped seeds catalyses the further reduction of Au (I) ions to Au
(0) on their surface, leading to rod formation. This earlier method produces low nanorod
12
yield (5%) but the aspect ratios is tunable from 6 to 20.15
In 2003, the method was
refined by Nikoobakht and El-Sayed, who replaced sodium citrate with CTAB in their
seed formation process, making single crystalline CTAB-capped gold seeds.16
The
synthesis protocol is illustrated in Figure 2.2 in which two steps are associated with the
protocol: (i) small CTAB-capped gold seeds (~1.5 nm) are prepared by the reduction of
chloroauric ions in the presence of CTAB with sodium borohydride and (ii) the addition
of the CTAB-capped seed solution to the growth solution in the presence of CTAB
prepared by the reduction of Au (III) complex ions to Au (I) complex ions with ascorbic
acid. The added seeds catalyze the further reduction of Au (I) to form Au (0). Addition
of silver nitrate to the growth solution before seed addition ensures rod formation and
greatly improves the control of the nanorod aspect ratio. This method produces a high
yield of gold nanorods (99%) with tunable aspect ratios from 1.5 to 4.5.16
Using the classical seed-mediated growth method,16
the conversion of ionic gold to
metallic gold is only ~15% efficient as reported by Orendorff and Murphy.70
The
majority of gold ions remain in the growth solution after the nanorod growth is halted.
Recently, Vigderman and Zubarev used excess hydroquinone to replace ascorbic acid as
a reducing agent and produced high purity gold nanorods with significantly improved
ionic-to-metallic gold conversion.71
The synthesized gold nanorods are stabilized by a bilayer of CTAB surfactant, which
provides ammonium CTA+ headgroup cationic surface charge that prevents particle
aggregation in aqueous solutions.72
The first monolayer of CTAB formed on the
nanorods is initiated by an electrostatic interaction between the positively charged
CTA+ headgroup and the negatively charged gold crystal surface. The hydrophobic
alkyl tails in the aqueous solution are not energetically favoured and thus another layer
of CTAB molecules is formed, with the hydrophobic tails interacting with the inner
layer and the CTA+ headgroup pointing outwards, leading to the assembly of a bilayer
of CTAB as the rods elongated.73
13
Figure 2.2 Schematic illustration of stepwise synthesis of gold nanorods by the seed-
mediated silver-assisted growth method.16
CTAB is also a shape-directing surfactant and is used in the formation of anisotropic
nanorods. The concentration of CTAB determines the shape of nanoparticles. For
instance, anisotropically shaped gold nanorods develop as the major product only above
the critical micelle concentration (CMC) of CTAB.15
Below the CMC, the reduction of
AuCl4- primarily results in nanospheres.
38 Therefore, the formation of nanorods during
the seed-mediated growth is also known to be dependent on the purity of CTAB. Garg
and co-workers determined that the presence of bromide is critically important in the
formation of nanorods.74
In their study, gold nanorods were not formed when CTAC or
a 1:2 ratio of CTAC:CTAB is used instead of pure CTAB. Smith et al. investigated the
nanorod synthesis using CTAB obtained from different manufacturers and found that
14
the iodide impurities present in some of the CTAB stocks can influence the growth step
thereby preventing nanorod formation.75, 76
2.1.2.2 Growth Mechanism and the Role of Silver Ions
Although the growth of gold nanorods is the most mature protocol amongst the
synthesis of anisotropic nanoparticles, the exact growth mechanism is not fully
elucidated yet. Various mechanisms that may drive the nanorod growth have been
previously proposed.77-79
In this context, crystallography of metal nanorods has played a
significant role in helping to elucidate the growth mechanism.26, 78
Murphy and co-
workers proposed that the intrinsic multiply-twinned structure of citrate-capped gold
seeds would stretch, causing symmetry breaking to form anisotropic nanoparticles.78
Thereafter, preferential surfactant binding to the side facets of seed nanoparticles can
inhibit the crystal growth on the side, while gold is deposited to the end facet leading to
nanorod elongation (Figure 2.3). This is the earliest mechanism (also known as the
zipping mechanism) initially proposed by making the assumption that rod-shaped
particles pre-existed for preferential binding of surfactant.78
On the other hand, Mulvaney and co-workers proposed an electric field model for the
growth process.79
In this mechanism, chloroauric ions bound to the CTAB micelles are
reduced to Au (I) by ascorbic acid forming CTAB–[AuCl2] metallomicelles. These
micelles then bind to the CTAB-capped gold seed particles through collisions and are
further reduced to Au (0). The seed particle-micelle collisions take place as a result of
electrostatic interaction between the positively charged seed and negatively charged
AuCl2- on the CTAB micelles. Due to the electric field effect, the collisions occur at a
much faster rate at the high curvature tips than the sides of the seeds and thus lead to
nanorod growth. While this mechanism can explain the nanorod formation, it did not
address how the initial tips of the seed nanoparticles are formed.
Although the detailed role of silver ions in controlling the nanorod aspect ratio still
remain unclear, it has been previously proposed that adsorption of the Ag (I) ions in the
form of AgBr at the different higher-energy facets of gold nanoparticles inhibits particle
growth on these crystal facets while allowing growth on less inhibited facets.80
In a
previous study by Liu and Guyot-Sionnest, the crystalline structures of both citrate-
15
capped seeds and CTAB-capped seeds were analysed and compared.26
It was found that
single-crystalline CTAB-capped seeds produce single crystalline nanorods, while the
multiply twinned crystalline citrate-capped seeds produce penta-twinned gold
bipyramids. Based on these crystallographic findings, they proposed that underpotential
deposition (UPD) of silver preferentially occurs on specific gold facets, resulting in the
formation of a silver monolayer. The silver monolayer on any gold facet may take a
longer time to be oxidized and replaced by gold, while other facets without a silver
monolayer may grow upon gold deposition.26, 81
Taking into consideration the
mechanism proposed by Mulvaney and co-workers,79
UPD of silver on certain gold
facets,26
and surfactant preferential binding,78
Orendorff and Murphy have come out
with a proposed growth mechanism70
wherein CTAB–[AuCl2] metallomicelles
collide/bind to the CTAB-capped gold seed particles via electric field interactions,
leading to the breaking of spherical symmetry into different crystal facets. Silver UPD
occurs quickly on the side facets but much slower on the end facets. Hence the particle
elongates as more CTAB–[AuCl2] and/or CTAB surfactant bind preferentially onto the
side facets, and continues to elongate until the end facets are also deposited with
silver.70
Figure 2.3 Schematic illustration of the formation of gold nanorods in the absence of
silver via surfactant preferential binding or zipping mechanism. Adapted from Ref. 82.
2.1.2.3 Binary Surfactant System
A binary surfactant system in seed-mediated growth was first demonstrated by
Nikoobakht and El-sayed.16
A more hydrophobic surfactant, benzyldimethylammonium
chloride (BDAC), is mixed with CTAB for making surfactant mixtures with different
ratios. Increasing the BDAC/CTAB ratio appeared to increase the width of the
nanorods.16
The nanorods synthesized in this way are uniform in size and shape,
16
however the role of BDAC is not well understood. Recently, Ye et al. demonstrated the
synthesis of highly monodisperse gold nanorods with improved overall nanorod
dimensions using a binary surfactant system composed of CTAB and sodium oleate.83
Khlebtsov et al. recently reported the use of a similar binary surfactant mixture to
achieve flexible overgrowth of gold nanorods for fine tuning of LSPR.84
2.1.2.4 High Aspect Ratio Gold Nanorods
In the classical seed-mediated growth approach, CTAB-capped seed and an
appropriate amount of silver nitrate routinely allows one to synthesize gold nanorods
with aspect ratios up to 4.5 and LSPR peaks close to 850 nm.16
Gold nanorods with
higher aspect ratios can be synthesized using a binary surfactant mixture, for example,
CTAB and BDAC in the growth solution allow gold nanorods to grow up to an aspect
ratio of 10 and LSPR up to 1300 nm,16, 85
whereas gold nanorods have been elongated to
aspect ratios of up to 20 in a binary surfactant system consisting of CTAB and Pluronic
F-127 in the absence of silver nitrate.86
On the other hand, a silver-free seeded growth
method with nitric acid as an additive has also been reported to produce high aspect
ratio (~19) gold nanorods.87
However, the formation of a large quantity of impurities
such as spherical nanoparticles and triangular nanoplates is often associated with these
methods, thus the methods require further purification steps to recover the gold
nanorods. Recently, Zhu et al. reported the addition of HCl in the presence of silver
nitrate to facilitate the high yield formation of gold nanorods with aspect ratios of up to
8 and LSPR up to 1100 nm.88
Ye et al. showed that by adding HCl and aromatic salt
additives such as salicylate compounds to the growth solution, high aspect ratio gold
nanorods (~7) could be synthesized with negligible shape impurities.89
The additives
effectively allow a lower amount of CTAB to be used and also improve the size and
shape uniformity of the gold nanorods. Zubarev and co-workers recently demonstrated a
significantly improved and large yield synthesis of high aspect ratio gold nanorods by
using a large excess of hydroquinone as a reducing agent instead of ascorbic acid in the
seeded growth synthesis.71
High purity gold nanorods with LSPR up to 1230 nm can be
synthesized through aging of the growth solution over the course of 6 hours. Zhang et
al. reported a similar approach by implementing a one-pot synthesis in which sodium
17
borohydride is added directly to the nanorod growth solution to initiate seed formation
and particle growth.90
2.1.2.5 Seedless Growth Method
The preparation of seed solution has poor reproducibility because small gold seeds
with a narrow size distribution are difficult to make, which often leads to inconsistency
in the subsequent nanorod growth. Besides, the prepared seed solution is temporally
labile as gold seeds can suffer from Oswald ripening after a certain amount of time. Jana
et al. reported a seedless growth method,91
in which seeds are formed in situ, as opposed
to seeded growth where seeds are formed ex situ. In this method sodium borohydride is
added directly to the nanorod growth solution containing silver ions, ascorbic acid, and
CTAB to initiate particle nucleation and growth.91
Gold nanorods with controllable
aspect ratios can be synthesized by changing the amount of sodium borohydride. Zijlstra
et al. reported a similar seedless growth method; however, the rod length is controlled
by changing the temperature instead of sodium borohydride.92
Recently, seedless
growth is also referred to as one-pot synthesis given that sodium borohydride forms
gold seeds directly in the nanorod growth solution.90, 93
2.1.2.6 Templated Synthesis
Rod-shaped templates can provide a mould for gold nanorod growth. The
prerequisite for this method is that the template material, such as a polycarbonate or
alumina membrane has to possess nanometer sized cylindrical pores so that the
reduction of AuCl4- can occur inside the pores.
94-96 The template can then be dissolved
away leaving the fully grown nanorods with diameter identical to the diameter of the
pores.97
Gold nanorods produced in this way can possess a small diameter ranging from
11 to 16 nm,97-99
which is nearly the diameter of nanorods synthesized by the seed-
mediated growth method.16
However, the length of the rods is relatively variable across
the nanorod array due to uneven deposition of gold.98
A similar approach was recently
reported wherein the mesoporous silica SBA-15 is used as a template to allow gold
seeds to grow anisotropically inside the pores into thin rods with small diameter (6-7
nm) and adjustable aspect ratios under conditions similar to the classical seed-mediated
growth.100
The mesoporous silica template is subsequently etched by HF in ethanol in
18
the presence of 1-dodecanethiol, releasing the gold nanorods which are capped
instantaneously by dodecanethiol ligands. Gao et al. recently adopted silica nanotubes
as the template for gold nanorod growth.101
The inner cavity of the nanotubes is pre-
functionalized with amino groups which later attracted AuCl4- by electrostatic
interactions onto their surface. The selective deposition of gold inside the tubes
following seed-mediated growth allowed the formation of nanorods with fairly uniform
size and aspect ratios of up to 21. The nanorods are released from the silica template by
etching with NaOH in an aqueous solution containing thiol-PEG.101
2.1.2.7 Electrochemical Method
Wang and co-workers published the earliest reports on gold nanorod growth through
electrochemical reduction in the presence of cationic surfactants and co-surfactants.22,
102 In this approach, a thin gold plate anode is oxidized and produced gold nanorods at
the cathode. The electrochemical reactions took place in a mixed surfactant system
consisting of CTAB and tetradodecylammonium bromide (TDTAB). It was later
reported that the presence of silver ions in the system is able to increase rod yield and
length.22
The nanorods synthesized on the surface of the electrode are “harvested” into
the solution by sonication and in general their aspect ratios range from 1 to 7 and
longitudinal plasmon resonance wavelength up to 1050 nm. The exact mechanism of
nanorod growth is not fully understood, but the shape control is thought to be achieved
by the cylindrical micelle formed by the surfactants. The fabricated nanorods are single-
crystalline in nature as determined by crystallography.103
2.1.2.8 Photochemical Method
The photochemical growth approach was first reported by Kim et al. in
which 254 nm ultra violet (UV) light irradiation was able to reduce Au (III) to Au (0)
in a growth solution consisting of a mixed CTAB-TDTAB surfactant system, silver
nitrate, acetone, and cyclohexane.20
This approach produces rather uniform gold
nanorods with aspect ratios up to ~5. A similar work was also carried out by Niidome et
al who demonstrated a synthesis protocol combining chemical and photochemical
reactions.104
In their report, Au (III) is first chemically reduced to Au (I) by ascorbic
acid. Further reduction of Au (I) to Au (0) is driven by the ketyl radicals generated by
19
UV irradiation of acetone present in the growth solution, which also contained a
micellar solution of CTAB, and silver nitrate.104, 105
2.1.2.9 Size and Shape Tuning of Pre-Grown Gold Nanorods
Over the past years, wet chemical synthesis methods have become well established
and tremendous progress has been made on the shape and size tuning of gold nanorods.
The shape and size of gold nanorods can not only be tuned during the seeded growth,16,
106 but also can be tuned by a process known as overgrowth.
107-110 In a typical
overgrowth process, a classical seeded growth approach is coupled with some chemical
modifications, which can lead to changes in morphology of the as-grown gold nanorod,
such as at the two ends of the nanorods. For instance, the as-grown gold nanorods
synthesized by the classical seeded growth usually exhibit spherical-shaped ends.
Excess gold ions left in the growth solution containing the as-grown gold nanorods can
be further reduced by additional ascorbic acid at varying concentrations.107
Due to the
preferential surfactant binding,78
CTAB molecules are known to bind to the sides of the
nanorods, restricting further growth on the sides. Provided that more ascorbic acid is
added to reduce the residual gold ions, the preferential CTAB binding on the pre-grown
gold nanorods causes more gold atoms to be deposited at the nanorod ends, leading to
the formation of dogbone-like nanorods.107
Alternatively, overgrowth can be achieved
by adding a lower pH ‘overgrowth’ solution to the initial growth solution containing the
as-grown gold nanorods.108
In this approach, the authors demonstrated that adjusting the
CTAB concentration and pH of the ‘overgrowth’ solution can alter the morphology of
the as-grown nanorods. The process depends entirely on the overgrowth pathways; for
example, the three reported pathways lead to tip-overgrowth, isotropic overgrowth, and
isotropic overgrowth. The selective overgrowth of gold nanorods was also demonstrated
to be able to alter the morphology of as-grown gold nanorods.111
The overgrowth
process uses small thiol molecules including glutathione and cycteine to block the two
ends of the gold nanorods where the packing density of CTAB molecules is loose,112
leading to transverse overgrowth instead of longitudinal growth upon addition of
‘overgrowth’ solution.111
The gold nanorods produced in this way have a larger
diameter in the middle section but the length of the rods remains unchanged.111
20
In contrast to tranverse overgrowth, the diameter of the nanorods can be shortened by
anisotropic oxidation while leaving the length of the rods unaffected.113
In this method,
the two ends of the nanorods are protected by a Ag2O layer, while etching occurs
preferentially at the side surface of the nanorods. On the other hand, the length of the
nanorods can be selectively shortened while leaving the diameter unchanged by
anisotropic oxidation process.114-116
The approach again takes advantage of the loosely
packed CTAB molecules at the two ends of the nanorods where oxidation takes place
relatively easily compared to the sides of the nanorods. In this way, the aspect ratios of
the nanorods can be tailored over a broad range while keeping the diameter nearly
unaffected.
2.2 Optical Properties of Gold Nanoparticles
Aqueous solutions of gold nanoparticles often exhibit colours that differ from the
colour of bulk gold. For instance, a dispersion of gold nanospheres often appears as
ruby red. This has been attributed to a unique confined optical phenomenon known as
the localized surface plasmon resonance (SPR). This phenomenon occurs when the
conduction electrons in the nano-scale gold are excited into a collective oscillation upon
interacting with the incident light.117
When the oscillation reaches a resonant frequency
matching the frequency of the incident light, gold nanoparticles strongly absorb and
scatter incident light at this SPR frequency and exhibit large electric field enhancements
around the particles. Figure 2.4 illustrates the collective oscillation of delocalized
electrons confined in gold nanospheres and gold nanorods in response to an external
electric field.
21
Figure 2.4 Schematic illustration of localized surface plasmon resonance (SPR) of gold
nanoparticles. (a) Localized SPR in gold nanospheres; (b) and (c) longitudinal and
transverse SPR in gold nanorods, respectively.
The SPR frequency (wavelength) band in the electromagnetic spectrum varies
depending on the nanoparticle size and shape, interparticle spacing (plasmon coupling),
and the dielectric properties of the nanoparticles as well as the refractive index of the
surrounding medium.118, 119
The size and shape of nanoparticles can influence the SPR
because the electric field density on the surface varies with particle geometry.13, 31, 120
Whereas the dielectric properties of nanoparticles are not only due to the electronic
structure of gold material, they are also influenced by the refractive index of the
22
surrounding medium, including any surface-adsorbed molecules which influence the
dipole plasmon resonance.121, 122
In addition, the plasmonic coupling that occurs when
particles are sufficiently close together, such as in aggregates, can also influence the
SPR, resulting in a redshift of the SPR wavelength band.123
The SPR properties of gold nanoparticles have been very useful in many applications
such as biological sensing and imaging,124-128
medical diagnostic and therapeutic
applications.129-131
In addition, the effect of plasmon coupling has been applied in
colorimetric detection of a wide range of analytes,132-135
in which the molecules of
interest can bring two or more nanoparticles close together, resulting in a colour change.
Gold nanoparticles especially those with tips, such as nanorods and nanostars, are also
known to enhance spectroscopic signals, for example, the Raman scattering signal of
Raman-active molecules adsorbed onto the nanoparticles can be enhanced by up to
several orders of magnitude.136
The strong electric field enhancements at the ends of
gold nanorods due to the high curvature have been demonstrated both theoretically and
experimentally.137
Typical Raman signals are relatively weak. Therefore in order to
maximize the signal a sufficient analyte concentration is needed. Gold nanoparticles
adsorbed or in close proximity to the molecules in the analytes can enhance the signal.
This effect is called surface-enhanced Raman scattering (SERS)138
and is due to the
huge enhancement in the absorption and scattering cross sections resulting from SPR of
nanoparticles.139
When two or more nanoparticles are sufficiently close to each other
and forming aggregates, the gap spaces between the nanoparticles create large electric
field enhancements for SERS active molecules. These ‘hot spots’ greatly amplify SERS
signals for molecules in the small gap (<5 nm) and have provided useful tools for
ultrasensitive detection in various applications.140
Mie theory accounts for the localized SPR of spherical and spheroidal metal particles
smaller than the wavelength of light. 141
For gold nanospheres, the SPR absorption band
of the localized SPR spectrum is around 520 nm in the visible region, which results in a
strong absorption in this region. The intense ruby red colour of the particle solution is
due to the red light being transmitted because the gold nanospheres absorb in the green
region. Gans extended Mie’s theory to account for the localized SPR properties of
ellipsoidal-shaped gold nanorods.142, 143
The conduction band electrons in gold nanorods
oscillate along the two axes of the rod: longitudinal and transverse axes. This is due to
23
the different possible orientations of the rod with respect to the electric field of incident
light. The electron oscillation along the transverse direction induces a weak absorption
band in the visible region similar to the absorption band of the gold nanospheres,
whereas the electron oscillation along the longitudinal direction induces a strong
absorption band in the longer wavelength region or near-IR (NIR) (typically 600-1200
nm). Therefore the two plasmon resonance bands (one weak and one strong) have
become the characteristic spectral feature of gold nanorods.
The longitudinal SPR band of gold nanorods is sensitively affected by the rod length-
over-width (aspect ratio, AR) due to the higher polarizability,143
for instance, changing
the aspect ratio of the gold nanorods will shift the longitudinal SPR band across a broad
spectral range, covering the visible and NIR regions. The aspect ratio of gold nanorods
is tunable and is most efficiently controlled by wet-chemical synthesis.143
The typically
range of aspect ratio is between 2.5 and 5 for most bioapplications. The strong
interaction of NIR light with the longitudinal axis of gold nanorods can lead to
absorption and scattering due to the longitudinal plasmon resonance, and during which
important photophysical processes occur, generating electron-hole pairs, luminescence,
and heat (Figure 2.5).144
The absorption, scattering, and/ luminescence properties have
great significance in biological applications, including bioimaging and photothermal
therapy.
2.2.1. Dark-field Light Scattering
Gold nanoparticles exhibit enhanced scattering cross sections at their plasmon
resonance. The scattering cross sections can even be several times greater than that of
the emission from conventional fluorescent dye molecules,145
making them ideal
candidates as imaging contrast agents for biological samples. Importantly, gold
nanoparticles do not suffer from photobleaching or any other forms of optical fatigue
under continuous illumination. As predicted by Mie theory,141
the scattering of gold
nanoparticles increases with particle size. Therefore, larger gold nanoparticles are
typically used for better imaging in cells. The scattering from the particles is observed
against a dark background, and hence it is often called dark-field scattering. Dark-field
scattering microscopy has been widely adopted in visualizing the gold nanoparticles
24
associated with the cells, for example, light scattering from anti-EGFR antibody
conjugated gold nanorods can be observed from the cancer cells as bright orange to red
light.129
Given that the dark-field scattering imaging technique is relatively simple to
perform and does not involve an expensive setup, the technique has become popular
over the years for efficient tracking of gold nanoparticle in cells,146
as well as
monitoring the dynamics of cellular uptake of gold nanoparticles.147
Figure 2.5 Physical effects arising from the longitudinal plasmon resonance of gold
nanorods induced by NIR laser. Adapted from Ref. 144.
2.2.2 Two-photon Luminescence Imaging
Two photon luminescence (TPL) has emerged as a popular choice of bioimaging
technique over the past 10 years.148-150
Gold nanorods are particularly suitable for TPL
due to their large two-photon absorption cross sections, and also the increased light
penetration depth in tissues in the NIR wavelength range. Compared to confocal
fluorescence imaging, the major advantage of TPL is that the fluorescence background
is significantly reduced, and the spatial resolution can be greatly enhanced especially
when femtosecond NIR laser is used for plasmon excitation.148
Besides, TPL imaging
25
with gold nanorods also offers high 3D spatial resolution and a penetration depth
sufficient for deep tissue,151
and in vivo imaging.152
.
2.2.3 Photoacoustic Imaging
Acoustic waves were first realized more than a century ago by Alexander Graham
Bell who demonstrated that the thermal expansion of the metal disk upon light
interaction can generate sound detectable with a stethoscope.153
This phenomenon is
known as the optoacoustic or photoacoustic effect. Over the past decades, photoacoustic
imaging has emerged as biomedical imaging technology that utilizes the basis of
thermoelastic expansion with the assistance of laser light. In a typical photoacoustic
imaging experiment, water content in the body absorbs the infrared (IR) light and
converts the absorbed light energy to thermal energy. The thermal expansion creates
pressure waves that propagate in the medium during which a transducer can be used to
detect the acoustic signals. The depth resolution of photoacoustic imaging can reach
several centimeters in biological tissues, making the imaging technique better than
ultrasound imaging.144
Photoacoustic imaging is often used for imaging tumours in the
body, provided that tumour tissues have increased haemoglobin and water content that
can absorb more radiation and respond much quicker than the normal tissues.154
Gold
nanorods have been widely used to greatly enhance imaging contrast in vitro155
,in
vivo156-158
and in tissue phantoms159
for photoacoustic imaging, relying on the strong
absorption of pulsed NIR laser. Enhanced acoustic waves can be generated when the
incident pulsed laser is absorbed by the nanorods at the targets, leading to the generation
of localized heating which expands the medium and thereby increases photoacoustic
signal.
2.3 Surface Modification and Functionalization
Surface modification and functionalization of colloidal particles represents one of the
most important aspects in the preparation of stable functional nanomaterials. The
purpose of surface modification and functionalization varies from one application to
another, but most frequently aims to make nanoparticles stable under different
26
conditions. This section is intended to review primarily the surface modification and
functionalization of gold nanoparticles. There are many other reviews that can provide
an insight into similar requirements for other colloidal nanoparticles.160
Common
surface modification and functionalization of colloidal gold can be achieved by several
means, for examples, polymer coatings,161-163
thiol ligands,164, 165
and silica coating
(Figure 2.6).166, 167
Polymer coatings mainly rely on the electrostatic
adsorption/deposition of oppositely charged polymers either in a single layer or in a
layer-by-layer (LbL) manner.168
Whereas thiol modification and/or functionalization
take advantage of the strong gold-thiol bond chemistry,169
thiol ligands can be tailored
via click chemistry170
which may contain a spacing group in between an anchoring
group for attachment to the nanoparticle surface, and a terminal functional group(s) that
provide different chemical properties to the nanoparticles, such as surface charge or a
reactive site for subsequent functionalization. Silanization offers the well-understood
Stöber method171
for surface modification, with the added advantage of allowing further
functionalization via silane chemistry.
There has been a great deal of interest in functionalizing the nanorod surface
especially for those particles synthesized by seed-mediated growth methods because the
CTAB bilayer can be easily disrupted in several instances:172-174
(i) when the CTAB
concentration is lower than its CMC, (ii) organic solvents are added to the nanorod
solution, (iii) the salt content in the solution is high, (iv) at extreme pH, and (v) strong
mechanical forces are applied to the gold nanorod solution such as repeated
centrifugation. Therefore the surfaces of gold nanorods are usually modified and
functionalized before they are used in many applications.
27
Figure 2.6 Schematic showing general methods employed for the surface modification
and functionalization of gold nanorods. Adapted from Ref. 175.
2.3.1 Layer-by-layer (LbL) Polyelectrolyte Coatings
Polyelectrolytes are charged (anionic or cationic) polymers that are available in
different chain lengths. Uniform layer-by-layer (LbL) assembly of polyelectrolytes
provides a facile route to tailor the surface properties of nanoparticles with a wide
variety of functional groups. The LbL approach is based on the electrostatic driven LbL
self-assembly which is easily controllable.176
Versatile coating of gold nanocrystals with
LbL polyelectrolytes was first studied by Caruso and co-workers.161, 177
By monitoring
the redshift of the peak plasmon absorption and also a reversal of surface charge of the
nanoparticles, successful adsorption of polyelectrolytes onto the nanoparticles can be
confirmed. Murphy’s group has extended this strategy to coat gold nanorods and
assembled gold nanorods onto modified glass slides.178
In their report, several layers of
PSS(-) and PDADMAC(+) polyelectrolytes were sequentially and successfully
deposited around the nanorods as indicated by charge reversal following each polymer
deposition.
LbL polyelectrolyte coating of gold nanorods confers several advantages. Firstly,
polyelectrolytes have successfully reduced the cytotoxicity effect of CTAB present on
the nanorods,179-181
because CTAB molecules are the sole toxicant to cells.182
Secondly,
28
gold nanorods have poor stability in organic solvents; however, after LbL coating with
an appropriate polyelectrolyte, they can be dispersed into polar organic solvents with
much improved stability.162
This has subsequently enabled certain surface modifications
which require the reaction to occur in the organic phase, such as silica coating through
the sol-gel method.183
Furthermore, polyelectrolyte layers have also been reported to be
able to improve the photothermal stability of gold nanorods,184
thereby enabling
applications in photothermal controlled release. Besides, provided that the surface
charge is positive, gold nanorods can be loaded with negatively charge dye molecules
such as rhodamine 6G,185, 186
and polyelectrolyte layers wrapped around the gold
nanorods could ensure that dye leakage is minimized.
2.3.2 Thiol Ligands
Surface ligands with thiol moieties form strong gold-thiol bond with gold
nanoparticles or nanostuctures.169
The strong covalent binding of the functional thiol
ligands onto the gold surface is significant in surface engineering of nanoparticles for a
broad range of applications. For example, the unique DNA-gold nanoparticle assembly
(or spherical nucleic acids187
) pioneered by Mirkin and co-workers provides an
interesting development based on gold-thiol bonding.188
The use of synthetic thiol-
terminated DNA oligomers for conjugating with gold nanoparticles via gold-thiol
binding has helped to the build plasmonic nanostructures into organized nanoparticle
arrays,189
and has also been developed into an ultrasensitive gold nanoparticle-based
platform for biodiagnosis.188, 190-193
Some recent examples showing the versatile use of
thiol ligands for functionalizing gold nanoparticles are summarized in Table 2.1.
In most cases gold nanoparticles are produced by means of wet-chemical methods,
wherein citrate or CTAB is often used as the stabilizer and capping agent. Therefore,
surface ligand exchange offers a solution to functionalize the nanoparticles with thiol
ligands. The ligand place-exchange reaction was first introduced by Murray’s group,41,
194 in which the terminal thiol group(s) of a ligand can be substituted for the existing
protective ligands or capping agents, thus offering chemical functionality onto gold
nanoparticles.195
Ligand exchanged gold nanoparticles have, in general, better stability
in a variety of conditions such as in aqueous solutions, some organic solvents, and
29
serum media.143, 196
An example of good use of this versatile place-exchange reaction
has been demonstrated in the hydrophobation of gold nanorods via a two-step ligand
exchange.197
Initially, the CTAB bilayers on the gold surface are displaced by thiol-
PEG ligands, which are then exchanged with dodecanethiol so that the nanorods can be
dispersed in non-polar organic solvents such as THF for subsequent integration into a
liquid crystalline matrix.197
30
Table 2.1 Summary of thiol ligand exchange for functionalizing gold nanoparticles.
Type of nanoparticles Ligand exchange type Ligands Notes
Gold nanospheres Citrate – thiol Dithiol Raman tag SERS probes
198, 199 for sensitive detection of cadmium
ions198
and traceable intracellular drug release.199
Thiol-Gadolinium-DTPA In vivo contrast agents in MRI.200
Cysteine-terminated CPPs Subcellular lysosomal active targeting of
nanoparticles.201
Cysteine-terminated pentapeptide
sequence
Entrapment of small drug molecules, cargo drug
delivery into mammalian cells.202
CTAB – thiol Thiol-terminated PEG, heterofunctional
(HS-PEG-NH2) PEG
PEGylation and amino functionalization (for
subsequent maleimide functionalization), nanoparticle-
maleimide forms conjugates with thiolated lipid.203
Gold nanorods CTAB – thiol Heterobifunctional (HS-PEG-COOH)
PEG
Promote self-assembly of superparamagnetic Fe2O3
and form hybrid nanoparticles.204
CTAB (partial) – thiol Thiol-terminated PEG Modification of nanorod tips for overgrowth of other
metals (Ag, Pd, and Pt) on the nanorod surface.205
CTAB – thiol Thiol-terminated CTAB analogue Achieve high surface exchange, high particle stability,
and show enhanced cellular uptake.206
31
CTAB – thiol Cysteine-terminated DNA Photothermal gene release.207
CTAB (partial) – thiol Aromatic dithiols Modification of nanorod tips for end-to-end
assembly.208
CTAB – thiol Thiol-terminated PEG, dodecanthiol Two-step ligand exchange for hydrophobation of
nanorods.197
Gold nanoshells Thiol-terminated ssDNA Photothermal control release of ssDNA/siRNA.209
Gold nanostars Citrate – thiol Cysteine-terminated CPPs Enhance cellular uptake and photothermolysis.210
Gold nano-popcorn CTAB – thiol Rh6G-modified RNA aptamer SERS targeted sensing of prostate cancer cells with
conjugated anti-PSMA antibody211
DTPA, diethylenetriamine-pentaacetic acid; CPP, cell-penetrating peptides; DTPA, diethylenetriamine pentaacetic acid; ssDNA, single stranded
DNA; siRNA, small-interfering RNA
32
A large excess of thiol molecules is often used for the surface functionalization of
gold nanorods due to the relatively strong binding of CTAB on the gold surface.
Previous work by Liz-Marzán and co-workers reported that four thiol-PEG molecules
per nm2
of the nanorod surface is sufficient for transferring gold nanorods from aqueous
solution into ethanol without causing particle aggregation.212
Thiol-PEGs can provide
gold nanorods with not only high stability but also improved biocompatibility. Many
studies have established that CTAB molecules present on the gold nanorods contribute
significant toxicity to the cells.180, 182, 213, 214
Therefore surface exchange of CTAB with
thiol-PEGs can significantly reduce the cytotoxicity of gold nanorods.179
Furthermore,
in vivo studies have consistently demonstrated that the administered PEGylated gold
nanorods can circulate in the blood for a prolonged period due to the reduced non-
specific protein adsorption, thus achieving increased circulation half-lives.196, 215
The high curvature at the two ends of gold nanorods can result in less densely packed
CTAB bilayers compared to those on the side facets. Therefore desorption of CTAB is
more likely to occur at the tips and this also facilitates surface exchange with thiol
molecules. A few reports have made use of this strategy, for instance, biotin
disulphide,112
cysteine216
and glutathione217
for tip-to-tip self-assembly of nanorods, and
glutathione for blocking the two ends of gold nanorods in order to promote transverse
overgrowth.111
Importantly, PEGylated gold nanorods have been shown to provide nucleation sites
for the hydrolyzed silica precursor, tetraethylorthosilicate (TEOS), which in turn can be
grown into a silica shell via the sol-gel method.212
The hybrid silica (shell)-gold (core)
nanorods have significant uses in many different applications which will be discussed in
the next section.
2.3.3 Silica Coating
Another strategy for surface functionalization of gold nanoparticles is to form a layer
of silica on the nanoparticles by the well-known Stöber method, which is the classical
method often employed to synthesize silica nanoparticles.171, 218
The typical reaction
involves hydrolysis of silica precursor, TEOS, in a polar organic solvent such as
33
ethanol, and subsequent condensation during which silanol groups join together and
form siloxane bridges.
The protocol for silica coating on gold nanoparticles was first demonstrated by Liz-
Marzán and co-workers.166, 219
Their initial studies were focused on the silica coating of
citrate-capped gold nanospheres using a silane coupling agent, 3-
aminopropyltrimethoxysilane (APTMS), as a surface primer that displaced citrate and
formed a hydrated silica monolayer on the gold surface given the higher affinity of
amines for gold.166
Successive silica depositions were carried out with sodium silicate in
aqueous solution before finally transferring the surface-primed nanoparticles into
ethanol solution for extensive growth of the silica shell with TEOS.
Subsequent methods for silica coating of gold nanoparticles evolved significantly,
including the use of a surface adsorbed amphiphilic polymer, polyvinylpyrrolidone
(PVP), to stabilize the nanoparticles in ethanol and provide pyridyl groups for
interacting with the silica precursor,220
and direct coating under appropriate conditions
(e.g. vigorous shaking etc) without prior particle surface modification.167, 221
Importantly, there has been significant progress in coating CTAB-capped gold
nanoparticles with silica. The major issue associated with CTAB-capped nanoparticles
such as gold nanorods is that the CTAB in alcohol solution is easily disrupted, resulting
in particle aggregation before silica formation. Furthermore, displacement of bilayer
CTAB molecules by silane coupling agents is relatively challenging due to the strong
binding of CTAB bilayers to the gold surface. The direct silica coating method has been
attempted on gold nanorods,222
and using the surface CTAB as templates, a silica shell
with mesostructure can be directly formed on the nanorods.223
However, the poor
reproducibility of the direct coating method has prompted the need for a better solution.
Liz-Marzán’s group reported that layer-by-layer (LbL) deposition of polyelectrolytes
can be used to wrap around the nanorods, masking the CTAB bilayers and allowing
dispersion of gold nanorods in alcohol solutions such as ethanol or 2-propanol for
subsequent silica coating through the Stöber method.183
Alternatively, thiol-PEG can be
used to displace CTAB molecules on the nanorods and subsequently allow dispersion of
PEGylated gold nanorods in ethanol for silica coating.212
In both instances, the group
was able to demonstrate that the thickness of the smooth silica shell can be controlled
by changing the amount of TEOS or the reaction time.183, 212
34
Silica coating confers a few advantages over other stabilizers. Firstly, silica shells
can provide enhanced colloidal stability to the nanoparticles and the stability is
primarily determined by the thickness of the silica shells and so the distance between
metal core particles.224
Besides, silica coating also improves the photothermal stability
of gold nanorods by reducing the chance of photothermal reshaping of nanorods on
exposure to high fluence ultrafast lasers.225
Most importantly, silica shells offer
multifunctionality to the nanoparticles, for example, pure silica on the surface of gold
nanoparticles can be grafted with a variety of functional groups including aldehyde (–
CHO), amino (–NH2), and carboxyl groups (–COOH), via co-condensation with
organosilanes.226
These functional groups present on the silica shell surface of the
nanoparticles can ensure relatively stable covalent conjugation with biomolecules such
as antibodies.227
Heat generated from the gold nanorods as a result of photothermal events is
transported through the nanorod/fluid interface and into the surrounding fluid (e.g.
water, organic fluids).228
In a previous study by Chen et al., it was reported that silica
shells can improve the thermal transfer (heat dissipation) due to a higher interfacial heat
conductance across the silica to water interface (>1000 MW.m-2
.K-1
) compared to the
gold to water interface (170 MW.m-2
.K-1
).229
This in turn has significantly improved the
amplitude of photoacoustic signals generated from silica-coated gold nanorods
compared to nanorods without silica coating,229
and thus the findings will benefit in vivo
photoacoustic imaging using gold nanorods as contrast agents.230, 231
In addition, the
rapid heat dissipation of the silica-coated gold nanorods may assist the development of
absorber-based photothermal stimulation of nerve cells.232, 233
The silica shell can also be used to protect Raman-active molecules234
or capture
fluorescent dye molecules and upon plasmon excitation the electric field enhancement
around the gold nanorods can improve the optical transitions of the adjacent fluorescent
dye molecules, resulting in enhanced fluorescence or SERS.
2.3.4 Other Functionalization Strategies
Magnetic coating of nanoparticles confers the advantage of easy separation from
precursor materials by using a magnet. The hybrid gold-magnetic core-shell
nanoparticles combine optical and magnetic properties, and offer great potential for
applications in biomedicine and catalysis.235
Methods for the preparation of magnetic
35
shell gold nanorods have been reported.236
Polyelectrolytes such as PSS that consists of
negative charge can attract Fe (II) and Fe (III) in the solution, which may be
coprecipitated to iron oxide particles in situ on the surface of gold nanorods upon
addition of NaOH. This method produces a uniform coating of iron oxide on the gold
nanorods but the magnetic power is rather weak which has been related to the thin
magnetic coating and the large mass of the gold core. Alternatively, premade iron oxide
nanoparticles can be assembled onto gold nanorods by the electrostatic interaction
between iron oxide and CTAB.236
The gold-core magnetic-shell complex synthesized
from the latter method has been reported to possess higher magnetic power over the first
method as measured by a superconducting quantum interference device (SQUID)
magnetometer.
Synthetic lipids have also been used to modify gold nanorods, for example,
phosphatidylcholine can be coated on gold nanorods following a place exchange
reaction.237
In this method, phosphatidylcholine modified gold nanorods can be
obtained by chloroform extraction of CTAB molecules on the nanorods in the presence
of phosphatidylcholine. The phosphatidylcholine coating has significantly improved the
nanorod stability and also the biocompatibility.237
A similar lipid modification approach
was also adopted by Orendorff et al,238
in which the excess Zwitterionic phospholipid
liposomes are used to displace CTAB surfactant molecules from the nanrods.238
Lee et
al. demonstrated the place exchange of CTAB coating with a cationic phospholipid
vesicle using a vesicle-to-nanorod fusion approach.239
In their report the CTAB-capped
gold nanorods were first place-exchanged with a non-ionic surfactant BriJ56, and
subsequently the Brij56-coated nanorods were dispersed in excess liposome
formulations wherein the cationic phospholipid bilayer coating replaced BriJ56 on the
nanorod surface. The cationic phospholipid-coated gold nanorods have enabled
electrostatic adsorption of small-interfering RNA (siRNA) which are stably dispersed in
cell media and showed reduced cytotoxicity compared to CTAB-capped nanorods.239, 240
36
2.4 Biocompatibility and Biodistribution of Gold Nanoparticles
2.4.1 In vitro
Biocompatibility remains as one of the critical prerequisites for any nanomaterials to
be of practical use in biological environments. Extensive toxicological studies have
been carried out based on in vitro tests and have generally involved various gold
nanoparticle types and cell models.241
Other than the standard cell viability assays, some
recent works have also looked into cellular response including analyses of reactive
oxygen species (ROS),242
gene expression,213, 243
and bioimpedance.244, 245
Although
most of these studies have concluded that the gold core is benign and biological inert,
there is still much debate as to whether gold nanoparticles are safe for cells and tissues.
For gold nanoparticles, in general, there are several factors that can contribute to the
cytotoxicity including particle size, shape, surface chemistry, and direct toxicants
released from the nanoparticles.
Firstly, gold nanoparticles with smaller diameters (<2 nm) exhibited more significant
toxicity than those with larger diameters (>10 nm).246
This is primarily due to the
catalytic effect of the small nanoparticles that could induce extremely efficient chemical
reactivity (intracellular ROS) in biological settings, leading to cellular oxidative stress
and subsequently to mitochondrial damage.242
Apart from this, it is established that the
cytotoxicity is potentially linked to the cellular uptake of the nanoparticles, which
affects the cells in a dose-dependent manner. The cellular uptake process is strongly
influenced by the particle size, shape, and surface charge.182
In a study by Chan and co-
workers, cellular uptake appeared to be dependent on the diameter of gold
nanospheres,247
in which 50 nm particles showed the highest uptake amongst all other
sizes (14, 30, 74, and 100 nm). In the same report, the authors compared the effect of
nanoparticle shape on cellular uptake.247
Their results indicated that spherical gold
nanoparticles and the lower aspect ratio gold nanorods have a higher chance of entering
cells compared to high aspect ratio gold nanorods. Qiu et al. have demonstrated a
similar cellular uptake pattern, in which lower aspect ratio gold nanorods showed
greater uptake than higher aspect ratio ones.248
Both studies have suggested that cellular
uptake via receptor-mediated endocytosis requires an energy-dependent process249
and
that larger nanostructures (i.e. nanorods) require more energy in order to enter the
cells.247, 248
Along with size and shape, the surface chemistry of gold nanoparticles is
37
also a key factor that can affect cellular uptake. There have been many reports that
studied the link between the surface net charge and cellular uptake.243, 250, 251
Cationic
nanoparticles were found to enter cells more readily than anionic or neutral
nanoparticles with the same size and shape. This has been attributed to the
predominantly negatively charged cell membrane which interacts more strongly with
the cationic nanoparticles. However, this pattern of nanoparticle-cell interactions is
highly debatable for the following reasons: (i) as pointed out previously by Murphy et
al,252
the charge distribution in cell membranes is heterogeneous, containing not only
negatively charged domains, but also positively charged and non-charged domains, and
(ii) the positively charged nanoparticles should first interact with the serum proteins
associated with the culture medium before reaching the cells. This is a phenomenon
often explained as protein-nanoparticle interactions that form protein coronas around the
nanoparticles, thus altering the initial surface charge of the nanoparticles.182, 253-255
Some reports have also suggested that the cationic nanoparticles no longer possessed
positive surface charge upon suspension in biological media containing serum
proteins.182, 256
In this context, a mechanism for cellular uptake of nanoparticles has
been proposed, wherein the serum albumin that is present in the culture medium is first
adsorbed onto the nanoparticles, and may subsequently bind and facilitate
endocytosis.256
Although the exact mechanism of cellular uptake is still unknown, the
interactions of serum proteins with nanoparticles will have significant implications for
efforts to functionalize nanoparticles especially for targeted delivery purposes.257
While the nanoparticle size, shape, and surface charge are known to influence the
cytotoxicity in one way or another, the toxicants released from the nanoparticles can
also affect the cells’ viability. For instance, extensive in vitro studies have established
that the CTAB molecules on the surface and/or desorbed from the surface of gold
nanorods are highly toxic and can disrupt cell membranes.214
Therefore, tremendous
efforts have been made to avoid CTAB molecules on the nanorod surface from reaching
the cells. In this regards, several notions have emerged over the years, including
modifying the nanorods with other biocompatible materials, such as PEGs,179
poly(acrylic acid),182
poly(diallyldimethylammonium chloride),243
poly(allamine
hydrochloride),182
poly(4-styrenesulfonic acid),180, 243
phosphatidylcholine,237, 258
38
lipofectamine phospholipids,239
silica,259
and multilayer polymer coatings,181
which
have all been able to reduce the cytotoxicity of the gold nanorods.
2.4.2 In vivo and Biodistribution
In vivo experiments performed over the years have generally established that
intravenously administered nanoparticles that have a neutral surface can be retained in
the blood circulation (circulation half-life) for a much longer time than charged
nanoparticles. This is due to the likely formation of particle aggregates induced either
by protein-nanoparticle interactions or by the highly ionic biological conditions
following intravenous administration.257
The protein-particle aggregates are quickly
cleared from the blood without any potential use. Additionally, the host immune system
such as the reticuloendothelial system (RES) recognizes the aggregates as foreign
materials via opsonization due to adsorbed antibodies and complement proteins, and
subsequently the opsonized materials are engulfed by the macrophages inside the liver
and spleen.257
Gold nanoparticles with a neutral surface can be prepared by
functionalizing the nanorods with PEG in order to avoid non-specific protein adsorption
and thus rapid clearance from the in vivo system.196, 215, 260
Furthermore, PEG molecules
with different chain lengths have been shown to affect the circulation half-life of
PEGylated gold nanoparticles, wherein longer chain length can significantly prolong the
circulation half-life.260
Owing to the different sizes of interendothelial pores lining the blood vessels, the
biodistribution of the nanoparticles varies and is mainly determined by the size of the
nanoparticles and also the materials of the nanoparticles (biodegradable or non-
biodegradable). Nanoparticles that possess a size of no larger than 6 nm are cleared
from the body through the renal and urinary systems (excretion pathways).261
However,
larger size nanoparticles (and potential particle aggregates) are primarily accumulated in
liver and spleen.262
Researchers are constantly seeking for answers as to whether the
accumulation of particles in those organs can cause toxicity to the animals. A recent
extensive evaluation of the biodistribution of 155 nm PEG-coated Au nanoshell
particles suggested that the particles may remain in the liver and spleen indefinitely but
did not show any signs of toxic effects in the animals.263
39
2.5 Photothermal Therapy
2.5.1 Light and Biological Tissue Interactions
Photothermal therapy emphasizes the need for converting light energy (photons) into
thermal energy in target objects such as biological tissues, that it can be used for
therapeutic treatments. In fact, interactions between light and biological tissues are
essential and commonly happen in daily life. For instance, exposure to sunlight allows
plants to make sugars such as glucose via photosynthesis, whereas humans require
exposure to UV from sunlight in order to acquire vitamin D in the skin. In some cases,
light can also be a source of cancers such as UV damage to the DNA in melanocytes
leading to melanoma - the most serious form of skin cancer in Australia.
Over the past decades, the basis of light-tissue interactions has promoted many
medical applications, such as lasers in ophthalmology, dermatology, otolaryngology,
and oncology, all of which rely on photothermal treatments with lasers.264
Depending
on the wavelength of laser light, when the light-tissue interactions occur, the light can
be strongly or weakly absorbed, reflected or scattered, and further transmitted. For
example, most tissue chromophores such as oxyhemoglobin, deoxyhemoglobin, water,
melanin, fat and yellow pigments,265
have weak absorption coefficients in the NIR
range from 650 to 900 nm, thus the NIR wavelengths allow deep penetration of light in
tissues (Figure 2.7).12
2.5.2 Photothermal Heating of Biological Tissues
Photothermal heating effects are widely used for light-tissue interactions in
photothermal therapy with medical lasers. In this regards, photons absorbed by tissue
chromophores generate thermal effects via molecular vibration and collisional
relaxation, leading to consequences such as tissue coagulation, protein denaturation and
vaporization, depending on the temperature reached.266
Medical applications including
tissue cutting and welding in surgery, vaporization of tumours, and removal of tattoos,
are often used today. Another interesting aspect of photothermal heating in biological
tissues is the acoustic events induced by rapid (femtoseconds to nanoseconds) pulsed
laser irradiation at the target.266
The pressure waves as a consequence of medium
expansion propagate away from the target as acoustic waves which can be detected via
an ultrasound detector and subsequently translated into a photoacoustic image.
40
.
Figure 2.7 Absorption spectra of hemoglobins and water in the wavelength range
between 400 and 1000 nm. The NIR window is situated between 650 and 900 nm.
Adapted from Ref. 12.
2.5.3 Nanomaterial-based Photothermal Heating
Nanomaterials have been designed and fabricated to facilitate photothermal heating
processes for a variety of applications, from targeted drug release to cancer treatments.
The concept is rather similar to photodynamic therapy in cancer treatments wherein
photosensitisers containing drug molecules can be activated by a light source, thereby
achieving release of the drugs in the tumour sites and causing necrosis.267
These
photosensitisers are made to be activated by laser wavelengths in the NIR range,
conveniently allowing deep tissue penetration. For nanomaterials, two major approaches
are blended for photothermal therapy using NIR lasers; heating of the gold-based
nanomaterials, and/or release of therapeutics upon such heating. The former process
alone is sufficient to cause necrosis to cancer cells due to the photothermal conversion
as a result of plasmon resonances in the gold,268, 269
while the latter takes advantages of
400 500 600 700 800 900 1000
10-4
10-3
10-2
10-1
100
101
102
Wavelength (nm)
Ab
sorp
tio
n c
oeff
icie
nt
(cm-1)
NIR regime
H2O
HbO2
Hb
41
the heat generated from the gold and releases drugs in the local vicinity after collapse of
the thermoresponsive encapsulating.270, 271
The mechanism of heating in gold
nanoparticle-based photothermal therapy involves a series of photophysical events: (i)
interaction of laser lights with the conduction electrons in the gold nanoparticles,
resulting in coherent oscillation of excited electrons, (ii) kinetic energy transfer to the
metal lattice through electron-phonon coupling, and (iii) the lattice temperature rises
and then cools off rapidly (within ~100ps) by dissipating the heat to the surrounding
environment via phonon-phonon relaxation.272
This ability to convert light into heat
through the photophysical plasmon excitation has made gold nanoparticles excellent
candidates for photothermal therapy.
The notion of using NIR-absorbing gold nanoparticles for photothermal therapy in
cancer treatments in vitro and in vivo was first demonstrated in 2003 by Halas et al.
using gold nanoshells (silica core encapsulated in gold shell) targeted to breast
carcinoma cells using the conjugated specific antibody and a laser at 820 nm.268
The
irradiated tumours in the nanoshell-treated animals reached the critical temperature
threshold for hyperthermia, leading to irreversible tissue damage. Their subsequent
work further showed that the PEG-coated gold nanoshells coupled with 808 nm laser
irradiation can selectively kill the tumours in mice via photothermal ablation without
impairing normal activity in the mice.273
The development of nanoparticle-based plasmonic photothermal therapy has surged
over the years, for instance, gold nanorods are continuing to receive attention for
photothermal therapy of cancer cells.129, 269, 274, 275
In addition, several other applications
have arisen, including photothermal treatment of pathogenic microorganisms,276, 277
photothermal heating controlled release of entrapped molecules in nanocarriers,197, 278
release of genetic materials such as DNA and RNAi in target cells,207, 270, 279-281
and
tissue welding.282, 283
In order to induce localized heating yet leave the surrounding healthy cells and
tissues unaffected, the nanoparticles have to be specifically delivered to the targets of
interest. Through surface bioconjugations, gold nanoparticles can be targeted to specific
cell types and heated upon irradiation with the light source. Particles can be conjugated
with cell surface recognition molecules which specifically bind to the target receptor on
the cell membrane via lectin-carbohydrate, ligand-receptor, and antibody-antigen
42
interactions.284, 285
Gold nanorods conjugated with a specific antibody have been a
common strategy used to target the cytoplasmic membrane of certain cell types. For
example, monoclonal anti-epidermal growth factor receptor (anti-EGFR) antibodies
have been used to specifically bind to EGFR present predominantly on the surface of
malignant cancer cells128, 129, 286
. Other than antibodies, folate150, 275
and deltorphin
molecules287
conjugated to gold nanorods have also been reported to play a significant
role in targeting and binding to cancer cells for selective localized photothermal
treatment.
Apart from gold nanoshells and gold nanorods, other gold nanoparticles (mostly
different shapes) have been investigated for photothermal therapy. Furthermore, other
materials such as carbon-based materials have also been reported for their
photoabsorbing and photothermal capabilities. Some examples are summarized in Table
2.2, along with information such as the irradiation source and their applications. Most of
the photoabsorbing materials have been used in photothermal ablation of tumour cells
and their absorption wavelengths are typically in the NIR range, where the water and
tissue is most transparent. Compared with other types of photoabsorbers, such as dye
molecules and carbon nanotubes, gold-based photoabsorbers have the added advantage
of simultaneously being able to provide imaging capabilities and to exert therapeutic
effects.288
43
Table 2.2 Examples of photoabsorbers used in photothermal therapy
Photoabsorber Functionalization Application Mode Source
Temperature
measurement Reference
Gold nanoclusters Primary antibody In vitro tumour cells
hyperthermia Thermal - necrosis
ns laser, 420-570 nm,
80 mJ/cm2
[289]
Gold nanorods PEG In vivo tumour
hyperthermia Thermal - necrosis
CW 808-810 nm,
2 W/cm2
70°C, IR thermal
imaging
[14, 196,
290]
Folate In vitro tumour cells
hyperthermia Thermal - necrosis
fs laser, 765 nm,
48.6 W/cm2
[150]
Folic acid and silica In vitro tumour cells
hyperthermia Thermal - necrosis CW 808 nm, 4W/cm
2 [291]
Elastin-like polypeptide,
PEG
Laser tissue welding, repair
rupture tissue Thermal - repair CW 800 nm, 20W/cm
2 [292]
Laser tissue welding Thermal - repair CW 810 nm, 100-140
J/cm2
>55°C, numerical
temperature model [282, 293]
Gold nanoshells PEG In vivo tumour treatments Thermal - necrosis CW 820 nm, 3.5-4
W/cm2
~74 °C, magnetic
resonance
temperature imaging
[268]
ssDNA, siRNA Controlled release Thermal - therapeutics CW 800 nm, 2.5W/cm2 <37 °C [209]
Gold nanocrosses In vitro tumour cells
hyperthermia Thermal - necrosis CW 900 nm, 4.2 W/cm
2 [28]
Gold nanostars TAT peptide hyperthermia Thermal - necrosis CW 850 nm, 0.2 W/cm2 [210]
Gold nanocages PEG and antibody In vitro tumour cells
hyperthermia Thermal - necrosis
fs laser 810 nm,
1.5 W/cm2
[294]
44
Carbon nanotubes Lipid and PEG In vivo tumour treatments Thermal - necrosis CW 808 nm, 0.6 W/cm2
52.9°C thermal
imaging [290]
DNA In vivo tumour treatments Thermal - necrosis CW 1064 nm, 2.5W/cm2 [295]
PEG In vivo tumour treatments Thermal - necrosis 808 nm, 76W/cm2 [296]
Carbon particles In vitro photostimulation Thermal - stimulation 650- 800 nm,
1.5-5 mW/µm2
50-90°C, focal
boiling [297, 298]
Molydium
disulfide DOX, PEG
Hyperthermia and controlled
drug release Thermal - necrosis
CW 808 nm,
0.35 W/cm2
44-45°C, indicated by
IR thermal imaging [299]
Pd nanosheet DOX Hyperthermia and controlled
drug release Thermal - necrosis CW 808 nm, 1 W/cm
2 [300]
Graphene oxide PEG, targeting peptides In vitro tumour cells
hyperthermia Thermal - necrosis
CW 808 nm, 15.3
W/cm2
52°C, thermal
imaging of pelleted
cells
[301]
PEG In vivo tumour treatments Thermal - necrosis CW 808 nm, 2W/cm2
~50°C thermal
imaging [302]
FePt nanoparticles folate In vitro tumour cells
hyperthermia Thermal - necrosis
fs laser 800 nm,
70 mJ/cm2
[303]
Organic dye heparin–folic acid In vivo tumour treatments Thermal - necrosis CW 808 nm, 0.8 W/cm2
54.6 °C, IR thermal
imaging [304]
Tungsten oxide
In vivo tumour treatments Thermal - necrosis CW 980 nm,
0.72 W/cm2
~50°C, thermal
imaging [305]
45
2.6 Neural Stimulation
In neurotransmission, communication or signalling within the neuron and between
neurons relies heavily on the conduction of electrical signals in the form of action
potentials. The conduction begins near the cell body of a neuron, where an action potential
is generated because of a depolarization that flows along its axons. At the gap junction, the
depolarization continues across the membrane to the receiving neuron (postsynaptic
neuron), triggering another action potential and subsequently the whole conduction process
continues through several cellular units in the nervous system. The depolarization that
generates the action potential in a neuron occurs because ions move across the neuronal
membrane (influx and outflux) through ion channels in accord with a concentration
gradient. One can initiate the process by applying a stimulus that can increase the opening
or closing of the ion channels and the process is referred to as neural stimulation. Neural
stimulation can be performed by several means via external stimuli (Figure 2.8). Common
stimuli reported in the literature include electrical,306, 307
caged neurotransmitters,308
ultrasound,309, 310
infrared light,3, 7, 311-313
and microwaves.314
Figure 2.8 Schematic depicting neural stimulation by different means.
46
The characterization of neural stimulation is typically carried out by patch-clamp
electrophysiology, in which the bioelectrical activity such as ionic currents that flow across
the cell membrane of the neurons can be measured with a glass micropipette. The patch-
clamp technique was developed after several refinements to the existing techniques.
Initially, Hodgkin and Huxley demonstrated the use of 1 mm diameter glass capillaries
filled with saline and inserted into the giant squid axon for measuring the action potential.
Later, Graham et al. reduced the diameter of the capillaries to several micrometers for
better recordings of the small muscle fibres.315
The first voltage clamp technique was then
reported by Marmont who applied the micropipette to measure, intracellularly, the
membrane voltage and current of individual cells.316 In the early stages of development,
noisy recordings were often obtained due to leaky currents through the cell membrane as a
consequence of poor sealing between the pipette and the cell membrane. Subsequently,
Sakmann and Neher developed the patch-clamp technique that used a blunt tip with small
diameters (0.5 – 2 µm) to form a tight seal with the cell membrane (expressed as a MΩ
seal).317, 318 It was also realized that a gentle suction applied to the membrane through the
micropipette allowed GΩ seal (gigaseal) between the pipette and the patched membrane,
leading to significant improvements in the recordings.319
Over the years, the patch-clamp
technique has been used to measure the bioelectrical activity of neurons upon stimulation.10,
320 The technique has also been used recently to assess the physiological conditions of
hippocampal CA1 neurons after being treated with gold nanoparticles.321
Other than the patch-clamp technique, monitoring physiological changes such as
intracellular calcium transients is also feasible for characterizing neural stimulation.322, 323
The intracellular calcium transients associated with the neural stimulation (via activation of
calcium permeable channels) can primarily be detected by fluorescent dyes such as fluo-4
AM, which are added to the cell culture and upon stimulation, the fluorescence intensity
can be measured by confocal imaging. Compared to the patch-clamp technique, the major
advantage of fluorescence calcium imaging is that the technique allows simultaneous
monitoring of the activity of a large population of neurons.324
47
2.6.1 Electrical Stimulation
Electrical stimulation via a stimulating electrode has long been the gold standard for
neural excitation. The method involves the injection of electrical current into neurons and
excitable tissues. The major advantage of electrical stimulation is that the electrical current
can be delivered in a controllable and quantifiable manner.6 However, there are several
fundamental limitations associated with this technique.325 Firstly, the stimulating electrode
requires physical contact with the tissue, which could either lead to potential toxicity
contributed by the electrode material or tissues damage after being impaled with the
electrodes. Next, relatively large electrodes must be used to avoid electrochemical reactions
in the tissue at high current densities. Due to the large size of the electrodes, spatial
precision of stimulation is difficult to achieve, and this is exacerbated by the spread of
electrical current. Furthermore, for electrophysiological recordings of electrically
stimulated neuronal activity, an electrical artefact from the stimulus can co-exist with the
cell electrical response.325
As a result, efforts are often made to post-process the recording
data for better interpretation. The limitations of electrical stimulation have prompted
interest in alternative solutions, including techniques based on light (photostimulation).
2.6.2 Photostimulation
Over the past decades, tremendous interest has emerged in the manipulation or
stimulation of nerve cells by means of laser light. This is an alternative development to the
conventional electrical stimulation because the method does not require the use of
stimulating electrodes, and hence can avoid unnecessary tissue contact, and also the method
provides spatially selective stimulation.6 Laser light has been used for photostimulation as
early as 1971, when Fork focused 488nm UV light on Aplysia neurons and showed that
action potentials can be induced upon irradiation.311 Later in 1983, Farber and Grinvald
demonstrated the use of a fluorescent dye for photostimulation of leech neurons.326
The
fluorescent dye stained the membrane and was excited by the laser, and the subsequently
released oxygen free radicals were thought to have caused a reversible depolarization in the
neurons. Since then, various photostimulation methods have emerged for manipulating
48
neural activity with lasers in the UV range, infrared (IR), and combined laser sources (two-
photon stimulation). There are several approaches to laser stimulation of nerves, including
photomechanical, photochemical, and photothermal.
In photomechanical stimulation, pressure waves (sound waves) induced by ultrasound327
or ultrafast lasers328
applied externally can cause physiological changes in the lipid
membrane,329 leading to the opening of the ion channels.330 In photochemical stimulation,
photosensitive compounds serve to capture the laser light and subsequently can be
transformed into a source of some chemical stimulus.331 For example, neurotransmitter
caging consists of photosensitive compounds that can undergo a chemical change upon
exposure to a light source. The cage encloses neuroactive molecules with photolabile
protecting groups such as nitrobenzyl.332 Exposure to laser light with an appropriate
wavelength, such as in the UV range, can cause photolysis of the protecting groups, leading
to the activation of the excitatory neurotrasmitters.
Caged glutamate is widely used in this form of photochemical stimulation as there are
many neurons in the central nervous system (CNS) that can be stimulated by glutamate.331
Earlier reports have shown that the caged glutamate can be released at unintended sites due
to the scattering of UV light in tissues. This problem has led to the development of two-
photon uncaging wherein two photolabile protecting groups were attached to the caged
molecules and thus, two photons are required for uncaging.308 Pulsed NIR two-photon
uncaging was also developed which uses wavelengths that have a greater penetration depth
in tissues compared to visible light.333
Photochemical stimulation of nerves may also involve the use of genetically expressed
light-sensitive photoreceptors or ion channels in the cells, known as optogenetics.334
Despite offering spatial precision, the major drawback of this approach is primarily the
need for genetic alteration.
The simplest form of photostimulation is perhaps photothermal stimulation via
irradiation of tissue with infrared (IR) laser pulses, which is also referred to as infrared
neural stimulation (INS). INS was first reported by Izzo et al. in 2006, in which auditory
neurons were stimulated with mid-IR laser. The technique has been suggested as an
49
alternative to the electrical stimulation commonly used in cochlear implants.3 In INS, the
laser light can be delivered to the site of interest through an optical fibre, making no direct
contact with the target nerves. Besides, precise spatial selectivity can be achieved and there
is no electrochemical junction between the stimulation source and the tissue, therefore
electrical artefacts can be eliminated.
INS has been extensively studied by several groups over the past years, with particular
efforts made to understand the mechanism of action. Wells et al. have discussed several
mechanisms that could be responsible for INS, including direct electric field,
photomechanical, photochemical, and photothermal processes, but have ruled out the
former three mechanisms in favour for a transient thermally-mediated mechanism.9 This
photothermal stimulation is recently proposed to be mediated by the absorption of NIR to
IR laser light irradiation (980 – 2400 nm) by water in tissue. This IR spectral range is where
maximum absorption of light by water occurs.
Several subsequent reports followed seeking to understand the mechanism in detail.4
Shapiro et al. have recently proposed that the thermal transient mediated by water
absorption of pulsed IR light could cause rapid membrane electrical capacitance changes
and induce currents that lead to neural excitation in different cell types (oocytes, HEK cells,
artificial lipid bilayers).10
Alternatively, membrane ion channels sensitive to temperature
changes such as the transient receptor potential vanilloid (TRPV) channels can also be
activated as demonstrated by Albert et al. in retinal ganglion cells.11
The activation of
TRPV channels for ion channel gating is dependent on the degree of transient temperature
rise.11
For instance, a typical TRPV1 protein is sensitive to a threshold temperature of 43
°C,335
whereas the TRPV3 protein is sensitive to a threshold temperature of 39 °C.336
Previous reports have shown that the temperature sensitivity of the TRPV1 channels is also
dependent on transmembrane voltage,337
in which the voltage dependence is thought to be
regulated by the C terminal region of the channel which is highly thermally sensitive.338
In
addition, photothermal-induced intracellular calcium activity may also play a role in
modulating excitability of various cell types.322, 323
There is also a report suggesting that
thermal volumetric expansion due to the optoacoustic effect may contribute to the
stimulation.339
50
Photothermal stimulation based on water absorption at IR wavelengths offers great
potential for practical use in medical applications such as neural prostheses, and vision and
hearing restoration strategies. However, there could also be issues like low stimulation
efficiency when using this approach for practical applications, where stimulation targets are
deep within the body (e.g. thalamus, visual cortex, etc). In that case the laser light at IR
wavelengths would be highly attenuated because of thick intrinsic absorbing or scattering
layers above the target structure.5 In this context, relatively high power lasers are required
in order to compensate for the lack of penetration depth.
2.6.2.1 Extrinsic Photoabsorbers for Photothermal Stimulation
Intrinsic photoabsorbers such as water content in tissues, and cellular components have
played important roles in photothermal stimulation. Extrinsic photoabsorbers introduced
exogenously may improve and enhance the photophysical mechanisms of stimulation in
terms of generating a more localized transient heating with a relatively low laser power and
without causing cell damage. Importantly, selection of radiation source and laser
parameters such as wavelength, is important in order to maximize penetration depth and
minimize the laser power used in biological tissues, and will significantly influence
photostimulation. There have been several reports on nanoparticles being used as extrinsic
absorbers, responding to optical297, 298
and magnetic irradiation,340
and converting the
irradiation source into heat that is distributed through the tissue for stimulation.
Ideally, photoabsorbers are designed and fabricated to absorb laser light in the NIR
range between 650 and 900 nm because this spectral range is where tissues have maximum
transparency.12
Migliori et al. reported that carbon particles absorbing at 650 nm can cause
thermal heating of leech neurons.298
Action potentials can be activated upon heating with
50 ms, 250 – 700 µJ laser pulses. Interestingly, carbon particle size played a role in
determining the depolarization magnitude, with the authors claiming that the thermal
energy increased with the particle size, leading to larger depolarization.298
Shoham et al.
demonstrated that cells around black microparticles fired action potentials in response to
51
the projection of intense light patterns at 532 and 800 nm generated using a digital
holographic projection system.297
Other than NIR light in the biological transparency window, magnetic fields also interact
relatively weakly with biological molecules and can penetrate deep into the body. Magnetic
nanoparticles can convert radio-frequency (RF) magnetic fields into heat. Huang et al.
demonstrated the activation of temperature sensitive TRPV1 channels in the plasma
membrane of genetically engineered HEK293 cells and hippocampal neurons by applying
RF to superparamagnetic ferrite nanoparticles targeted to the cells.340 Using a calcium
sensor as an indication of opening of TRPV1 channels, the authors found that an influx of
Ca2+
was triggered by the RF, resulting in membrane depolarisation and subsequent action
potentials in the cells. Although the concept of magnetic field heating of nanoparticles was
applied to the activation of genetically engineered cells, the feasibility of RF responsive
absorber for neural stimulation has great potential in the future development of neural
prostheses.
2.6.2.2 Gold Nanorods for Neural Stimulation
As discussed in Section 2.2, gold nanorods possess a highly tunable plasmon resonance,
a resonant phenomenon whereby light induces collective oscillations of conductive metal
electrons in the gold.285
By controlling the aspect ratio and surface coatings, the plasmon
resonance and the resultant optical absorption of nanorods can be tuned across a broad
range of the spectrum from the visible to NIR. NIR region is where optical absorption in
tissue is minimal and penetration is optimal. Surface plasmon resonances of gold nanorods
can be tuned to efficiently absorb the laser light in the NIR range and turn the laser source
into heat. The localized heating from the nanorods is based on the laser wavelength and
intensity. Depending on the intensity, gold nanorods can be used as “nanoheaters” to induce
hyperthermia in cancer cells,129, 150, 269
or to trigger remote release of entrapped materials
from within the nanocarriers.197, 278, 341
Recently, the potential of gold nanorods for thermal-
and absorber-based neural stimulation has been investigated. Paviolo et al. have
demonstrated that exposure to a NIR laser can induce cellular responses in the NG108-15
52
neuronal cell line containing gold nanorods.232
The cells responded to the laser irradiation
by exhibiting enhanced neurite differentiation. The findings may have potential
implications in neural regeneration. Further analysis of the same cell line incubated with
gold nanorods have suggested that intracellular calcium transients can be activated upon
laser exposure.342 The authors attributed these phenomena to localized heating of the
nanorods due to the plasmon resonance which may (i) perturb the membrane capacitance
and/or open some voltage-sensitive ion channels, (ii) activate temperature sensitive
channels, (iii) result in outflux of the intracellular Ca2+
storage in the organelles. Based on
the promising results of Paviolo et al., further investigation of generation of action
potentials with gold nanorods has been performed in this thesis (see Chapter 5). In this
context, the enhanced membrane depolarization of the auditory neurons has been attributed
to the photothermal stimulation, in which the thermal source in the vicinity of the neurons
is generated from the interaction between the incident laser and the wavelength matching
gold nanorods. In a more recent report, neural stimulation relevant to the work described in
Chapter 5 has been published by Eom et al.343 The work showed the activation of rat sciatic
nerves in vivo using the photothermal effect of 980 nm laser-absorbing gold nanorods
irradiated at variable laser irradiance. Similarly, the enhanced depolarization of neurons in
the nerve bundles has been attributed to localized heating generated by the laser irradiated
gold nanorods.343
2.6.2.3 Neuro-targeting and Blood Brain Barrier
Although promising, extrinsic photoabsorber could face several challenges when applied
in vivo. The major challenge is the specificity of the nanoparticles in providing localized
heating to the target neurons. With an appropriate delivery system, the nanoparticles can be
delivered to the site of interest located in the ray path of the laser beam, and the transient
heat is localized within the target upon laser irradiation. In the case of auditory neurons,
targeted delivery to specific inner ear cell populations can be achieved by functionalizing
the nanoparticles with peptides, for instance, using Tet1 peptide for targeting the
trisialoganglioside clostridial toxin receptor on neurons,344
tyrosine kinase receptor B
(TrkB)-binding peptide for targeting the TrkB receptor,345
and nerve growth factor-derived
53
peptide for targeting the tyrosine kinase receptors and p75 neurotrophin receptors.346
Despite the various ligands that could be used for targeting purposes, there is certainly a
need for multifunctional ligands for successful in vivo delivery. The long-time problem
associated with the delivery of therapeutics to the central nervous system is the blood brain
barrier (BBB). The BBB serves to protect the brain from noxious agents and is also
impermeable to most water-soluble drug molecules.347
Several solutions have been
employed,348
including the invasive direct surgical administration of drugs, and also
encapsulating drugs into lipid carriers such as liposomes and lipid nanoparticles which
could cross the BBB by diffusion.349
Other strategies include functionalizing the
nanocarriers with lipoprotein, which can be recognised by the lipoprotein receptors at the
BBB and thus facilitate transport across the BBB,350, 351
and transferrin, which exhibits high
affinity for the transmembrane glycoprotein TfR.352
In the case of gold nanoparticles, there have been several instances where no lipid carrier
or functionalization is required for crossing the BBB. De Jong et al. have previously
examined the distribution of intravenously injected gold nanospheres with various sizes (10
to 250 nm) in mice, but have only found small (10 nm) particles in the brain.353 Similarly,
Sonavane et al. have shown that only gold nanospheres with smaller sizes (15 and 50 nm)
were able to cross the BBB.354 Sousa et al. have recently shown that gold nanoparticles
with fluorescent dye wrapped in polyelectrolytes were able to reach the brain as confirmed
by X-ray tomography and confocal laser scanning microscopy.355
The authors attributed the
BBB crossing mechanisms to the protein coronas formed around the nanoparticles that
facilitates endocytosis356
but the detailed mechanisms are unclear.
2.6.3 Photovoltaics Interface
Photovoltaics (PV) generate electrical power by means of receiving and converting light
into direct electrical currents using semiconductors. Organic semiconductor photovoltaic
materials that can form an interface with the neurons have been reported to modulate neural
activity upon irradiation with laser lights.357-360
For example, Pappas et al. demonstrated the
layer-by-layer (LbL) assembly of conductive composite films containing HgTe
54
nanoparticles and several layers of poly(dimethyldialylammonium chloride) and used the
conducting films for interfacing with NG108-15 cells.357
Upon 532 nm laser irradiation,
cells exhibited enhanced electrical activity as characterized by patch-clamp
electrophysiology. Ghezzi et al showed that an organic polymer-based photovoltaic blend
was able to promote action potentials in primary hippocampal neurons interfaced with the
photovoltaic material when laser pulses (532 nm) were applied.359
These hybrid materials
provide a promising platform for developing a new generation of neural and retinal
prosthetic devices on the basis of light-activated organic semiconductors.
55
56
57
Chapter 3: Synthesis, Surface Modification, and
Functionalization of Gold Nanoparticles
3.1 Introduction
Metal nanoparticles of gold have been in the spotlight because of their interesting optical
and electronic properties.361 Their uses have been realised since late BC, for instance, gold
nanocrystals have been used to make ruby glass and as colouring agents for ceramics, the
most notable being the Lycurgus cup.362
The plasmonic phenomenon can be observed when
the cup appears ruby red in transmitted light, but turns green in reflected light. More than a
century ago researchers began to understand the physical (optical and electronic) properties
e.g. their electronic configurations obey quantum-mechanical rules, which have been
shown to be size-dependent.363
In the subsequent years, a variety of applications of gold
nanoparticles has emerged.42, 188, 362, 364
Given the high demand, there have been many
studies aiming to synthesise gold nanoparticles with different sizes and shapes in order to
suit different applications.365, 366
The major aspect of synthesis of gold nanoparticles has
been discussed in Chapter 2 (Section 2.1).
It is well known that gold nanoparticles provide great potential for biomedical use such
as imaging and photothermal therapy.367
In particular, gold nanorods (GNRs) are useful
because they absorb most efficiently in the near-infrared (NIR) region compared to gold
nanospheres (GNSs).368
Their capability to absorb NIR light has promoted many
photothermal applications.361
The ability to adjust their longitudinal SPR absorption band
in the NIR wavelength region is of great importance. Seeded growth methods have been
widely adopted in synthesising GNRs.16, 106
CTAB is used as a shape-directing surfactant to
prepare anisotropic particles such as nanorods. A number of factors can contribute to the
dimensions of nanorods, including the concentrations of CTAB, gold ions, silver ions,
ascorbic acid, and gold seeds.285, 369
By controlling these seeded growth conditions, GNRs
can exhibit tunable aspect ratios (length/width). The linear dependence285, 370
of the
58
longitudinal SPR band (���� ) of GNRs on the aspect ratio (R) is given in units of
nanometers by:
���� = 95� + 420 (Eq. 3.1)
Given that the longitudinal SPR band can shift as the nanorod aspect ratio changes,
appropriate seeded growth conditions are often optimized in order to prepare GNRs with
the desired SPR wavelength.
In a typical colloid system, surface modification and functionalization are often
necessary to provide the nanoparticles with more flexibility to suit the intended
applications. Silica is a suitable surface modifier, as the coating provides enhanced
colloidal stability to the nanoparticles and the stability is primarily determined by the
thickness of the silica shells and so the distance between the particle cores.224
Besides,
silica-coated GNRs also show less cytotoxicity than bare GNRs with CTAB coating.181, 227,
229, 259 There have been several reports on the silica coating of different core materials, for
example, silver,371
CdSe/ZnS,372
and magnetite,373
all of which have been used in biological
setting. Prior to silica coating, surface modification of gold nanoparticles, such as layer-by-
layer (LbL) deposition of polyelectrolytes or via thiolated methoxy poly(ethylene)glycol
(mPEG-SH) can result in a more successful silica formation on the nanoparticles through
the sol-gel method.169, 183, 212
This chapter first explores the synthesis of GNSs and GNRs by means of wet chemistry.
The citrate reduction method was used to prepare GNSs. Meanwhile, the silver-assisted
seeded-growth method was used to prepare GNRs. These wet chemical synthesis methods
have significant advantages including their low cost, high yields and uniformity, and
environmental friendliness.23 Although gold nanoparticles syntheses have been extensively
researched for decades, it remains necessary that the fine tuning of the size and SPR band
of the nanoparticles is established in any particular laboratory for specific purposes. For
instance, by following a typical synthesis reported in the literature, one may be able to
synthesise nanoparticles of the desired shape, but the SPR band may require further tuning.
This has prompted the need to identify the appropriate experimental conditions and
parameters in the synthesis protocols. The majority of the work described in this thesis
59
relied heavily on the optical properties of GNRs, in particular to promote the laser-nanorod
interactions. Hence special attention was given to matching the longitudinal SPR band of
the surface modified and non-surface modified GNRs with the incident laser wavelength.
For the synthesis of GNRs, by changing the seeded growth conditions, the tunability of
longitudinal SPR band of GNRs was investigated. The factors (i.e. the concentrations of
ascorbic acid, silver ions and Au seeds) that are crucial in determining the morphology and
the longitudinal SPR band of GNRs were studied. The intention was to find the optimal
conditions for tuning the longitudinal SPR band of GNRs in the near-IR region between
760 and 780 nm, because the longitudinal SPR band of GNRs may exhibit spectral shift
after surface modification and/or functionalization. As-synthesised GNSs and GNRs are
either easily aggregated in extreme pHs, or high salt concentrations, or they are not
compatible with organic solvents.374
Hence, GNSs and/or GNRs were modified by surface
coating with charged-polymers (polyelectrolytes) via layer-by-layer (LbL), PVP
passivation, mPEG-SH (mPEGylation), silica, and polydopamine. The purpose of surface
modification is twofold: (i) to prepare nanoparticles with specific surface coatings for
further in vitro experiments (see Chapter 4 and 5) , and (ii) to prepare stable colloidal
nanoparticles for phase transfer, in which the new solvent system may facilitate further
surface modifications e.g. silica coating via Stöber method. In addition, this Chapter also
addresses common characterisations of the nanoparticles used throughout the thesis.
3.2 Materials and Methods
3.2.1 Materials
Cetyltrimethylammonium bromide (CTAB), hydrogen tetrachloroaurate (III) trihydrate
(HAuCl4∙3H2O, 99.9+%), trisodium citrate, sodium borohydride (NaBH4), silver nitrate
(AgNO3), ascorbic acid, tetraethyl orthosilicate (TEOS), poly(styrene sulphonate) (PSS,
Mw 70,000), poly(allylamine hydrochloride) (PAH, Mw 10,000), poly(vinylpyrrolidone)
(PVP, Mw 10,000), methoxy-PEG-SH (Mn = 5000), ammonium hydroxide (NH4OH) (~28
wt% in water), absolute ethanol and 2-propanol/isopropanol (>99.9%), 3-
aminopropyltrimethoxysilane (APTMS), fluorescamine (>98.0%), ethanolamine (>99%),
anhydrous acetonitrile were purchased from Sigma Aldrich and used as received without
60
further purification. Milli-Q water with a resistance of 18.2 MΩ.cm was used throughout
the experiments. Glassware was cleaned by soaking in aqua regia (HNO3:HCl = 1:3) and
finally washing thoroughly with water.
3.2.2 Preparation of Gold Nanospheres
Monodisperse 20 nm gold nanopsheres were prepared by the citrate reduction method.32
Ten millilitres of 5 mM HAuCl4 was added to 180 mL of H2O and the mixture was heated
to boiling. Ten millilitres of freshly prepared 0.5% (w/v) trisodium citrate in water was then
quickly added to the solution under vigorous stirring. The colour of the mixture turned wine
red within a few minutes and the mixture was cooled to room temperature. The aqueous
gold nanospheres were then filtered through a 0.45 µm syringe filter, and the filtrate was
kept in the fridge for later use.
3.2.3 Preparation of Gold Nanorods
Gold nanorods were synthesised according to a seed-mediated method,106 with minor
modifications so as to adjust the aspect ratio of the nanorods as required. In a typical
synthesis process, at 25 ºC, 600 µL of 10 mM ice-cold NaBH4 was added quickly into a
solution containing 250 µl of 10 mM HAuCl4 and 9.5 mL of 100 mM CTAB under
vigorous stirring. The gold seed solution then turned pale brown-yellow, and was left
undisturbed for 2 hrs at room temperature. For preparing a growth solution, 9.5 mL of 100
mM CTAB, 500 µL of 10 mM HAuCl4, 75 µL of 10 mM AgNO3 were mixed. Then 55 µL
of 100 mM ascorbic acid was injected and the growth solution turned from a brown to
colourless. To initiate nanorod growth, 12 µL of seed solution was added to the mixture,
which was then left overnight at 25 - 27 ºC. The as-synthesised gold nanorods were
centrifuged twice for 12 min at 14,000 rpm to remove excess CTAB in the solution and
finally redispersed in 10 mL of water.
To investigate the shift in the longitudinal SPR band position due to varying
experimental conditions (ascorbic acid, silver nitrate and Au seed concentrations), a
minimum of three samples from different batches were analysed.
61
3.2.4 Surface Modification
3.2.4.1 Polyelectrolyte (PE) Coating
A PE layer-by-layer (LbL) technique was used to prepare gold nanorods appropriate for
transferring to an organic solvent for silica coating. Firstly, 10 mL of PSS (2 mg/mL, 6 mM
NaCl) was added to 10 mL of as-prepared gold nanorods under stirring. The mixture was
then stirred for 3 hr at room temperature. The PSS/GNRs were obtained by centrifugation
at 8,000 rpm for 15 min and finally redispersed in 10 mL of water. Next, 10 mL of
PSS/GNRs were added into 10 ml of PAH (2 mg/mL, 6 mM NaCl) and the mixture was
stirred for 3 hrs. PAH/GNRs were centrifuged twice for 15 min at 8,000 rpm and finally
redispersed in 5 mL of water. Then, 5 mL of PAH/GNRs were added into 2.5 mL of PVP
(2 mg/mL) in water and the mixture stirred overnight. PVP/PE/GNRs were centrifuged
twice for 12 min at 8,000 rpm and finally redispersed in 500 µL of water.
3.2.4.2 PVP Coating
This procedure was used to prepare gold nanospheres with a PVP film over the existing
citrate coating prior to silica coating. An aqueous solution of PVP10 (12.8 mg/mL) was
added to the colloidal gold solution. The mixture was stirred overnight at room temperature
(25 – 27 ºC). The PVP/GNRs were collected by centrifugation and redispersed in 10 mL of
H2O.
3.2.4.3 mPEGylation
mPEGylation was performed to displace CTAB molecules on the gold surface with
methoxy PEG-thiol (mPEG-SH). Typically, to 1 mL of freshly prepared gold nanorods, 100
µL of mPEG-SH (10 mg/mL) previously sonicated with 50 µL of NaBH4 was added. The
mixture was vortexed and incubated at room temperature for 6 hr. Subsequently, the
mixture was centrifuged thrice at 10,000 rpm for 10 min and washed with ethanol and
finally redispersed in ethanol under sonication.
3.2.4.4 Silica Coating
From Section 3.2.4.1, PVP/PE/GNRs were subjected to silica coating. A 1 mL volume
of 2-propanol was added dropwise into 500 µL of PVP/GNRs. Then 1.43 mL of NH4OH
62
(3.84% v/v in 2-propanol) and 400 µL of TEOS (0.97% v/v in 2-propanol) were added into
the mixture and vortexed for 2 hr. The resulting silica/GNRs were washed with 2-propanol
and water by centrifugation at 8,500 rpm and finally redispersed in 2 mL of water.
From Section 3.2.4.2, PVP/GNSs were subjected to the silica coating process.
Isopropanol (1.0 mL) was added to 0.5 mL of PVP-modified gold nanospheres.
Subsequently, water (0.4 mL), ammonia solution (3.84 vol% in 2-propanol, 1.43 mL), and
TEOS (0.97 vol% in 2-propanol, 0.3 mL) were added to the mixture. The reaction mixture
was then vortex mixed for 2 hr to allow homogeneous silica coating. The resulting silica-
coated gold nanospheres were centrifuged thrice at 6,000 rpm for 10 min and washed in
between with ethanol.
From Section 3.2.4.3, PEGylated gold nanorods were subjected to silica coating. To
achieve a 10 nm thick silica coating, typically, 0.4 mL of H2O, 15 µL of NH4OH, and 5 µL
of TEOS were sequentially added into 2.5 mL of PEGylated gold nanorods under
sonication. Sonication was continued for 2 hr and the temperature of the bath water was
controlled to within 25 – 30 ºC. Silica-modified gold nanorods was then centrifuged thrice
at 9000 rpm for 10 min and washed with ethanol in between. The silica-coated gold
nanorods were finally stored at 4 ˚C in 1 mL of ethanol for further use.
3.2.4.5 FDTD Simulation
Finite-difference time-domain modelling was performed to simulate the cross sections of
GNRs and silica modified GNRs. Numerical simulations were carried out by the 3D-FDTD
(Lumerical Solution Inc., Canada). The plasmonic resonance was calculated for a rod-like
particle with 48 nm diameter and 13 nm height. Silica shell thickness was 15 nm.
3.2.4.6 Functionalization of Silica-coated Gold Nanoparticles.
3.2.4.6.1 Amine Silanization and Fluorescent Quantification
One microlitre of pure APTMS was added to 2 mL of the washed silica-coated particles
under stirring. The stirring was continued overnight at room temperature. The solution was
then boiled at 90 °C for 1 hr to promote covalent bonding. Excess reactants were removed
by centrifugation and the particles were washed five times with ethanol/water and finally
63
stored in 1 mL of water for freeze drying. For fluorescent quantification of the NH2 groups,
standard amine solutions were prepared by diluting APTMS or ethanolamine in H2O to a
desired concentration range. Then 90 µL of fluorescamine solution (1 mg/mL in
acetonitrile) was added to a mixture containing 190 µL of borate buffer (0.1 M, pH 8.0) and
20 µL of the analyte. For nanoparticles, 0.1 mg of freeze-dried sample was suspended in
H2O under sonication prior to the assay.
3.2.4.6.2 Polydopamine
Suspensions of 1 mL of SiO2-GNRs were pelleted by centrifugation. Dopamine
hydrochloride (2 mg/mL) was dissolved in 50 mM Tris-HCl, pH 8.5, and added
immediately into the SiO2-GNRs pellets. The mixture was stirred for 10 min, during which
the pinkish colour of the SiO2-GNRs turned light brown. Subsequently, the mixture was
centrifuged at 6000 rpm for 10 min and washed thrice with Tris-HCl, pH 8.5. The
PDA/SiO2-GNRs were suspended and stored in PBS, pH7.4.
3.2.5 Characterisation
UV-vis absorption spectra were collected with a Cary 50 or Cary 300 spectrophotometer
(Agilent, Australia) using a quartz cuvette with 10 mm pathlength. Size distribution and
uniformity of the particles were investigated using a JEOL 1010 transmission electron
microscope (TEM) operating at an accelerating voltage of 100 kV. ImageJ software was
used for image analysis. TEM samples were prepared by adding 20 µL of nanoparticle
solution onto 300 mesh carbon film TEM grids and allowed to dry in air. Zeta (ζ) -
potential was measured by using a Brookhaven 90 Plus particle sizer and zeta potential
analyzer (Brookhaven Instruments Corporation, NY, USA). ATR-FTIR spectra were
collected with a Thermo Nicolet iS5 spectrometer (Thermo Scientific, USA). For Raman
spectroscopy, both CTAB- and mPEGylated-GNRs solutions were dried onto glass
substrates and the nanorod films were studied in a Raman spectrometer (InVia Renishaw,
Wotton-under-Edge, UK) at an excitation wavelength of 785 nm and a laser power of 100
mW. Exposure time was 10 s and the results were averaged from 5 accumulations.
Fluorescence spectra were acquired from a Varian Cary Eclipse fluorescence
64
spectrophotometer using a quartz cuvette with 10 mm pathlength. Fluorescence intensity
was obtained from a microplate reader (POLARstar Omega, BMG Labtech, Germany).
3.3 Results
3.3.1 Preparation of Gold Nanoparticles
3.3.1.1 Shape Control
Spherical and rod shapes gold nanoparticles were synthesised by the citrate reduction
and seed-mediated growth methods, respectively. Figure 3.1(a) shows the TEM image of
the GNSs. Using a concentration of 0.5% (w/v) of citrate, the diameter of the synthesised
GNSs was determined to be 16.8 ± 1.8 nm. The synthesised GNSs exhibit a single SPR
band at 520 nm (Figure 3.1(b)). Figure 3.2(a) and (b) show TEM images of GNRs
synthesised using the typical protocols as described in Section 3.2.3. The distribution of
longitudinal and transverse dimensions of the nanorods is shown in Figure 3.2(c). The
GNRs were synthesised to a dimension (length × width) of 48.6 (± 4.3) x 12.6 (± 1.3) nm.
The UV-vis spectrum of the synthesised GNRs exhibit two SPR bands; the longitudinal
SPR band at 783 nm and the transverse SPR band at 515 nm (Figure 3.2(d)), which is a
typical spectral characteristic for GNRs.
65
Figure 3.1 Synthesis of GNSs: (a) TEM image of GNSs (inset: size distribution), and (b)
UV-vis spectrum showing the SPR band at 520 nm.
66
Figure 3.2 Synthesis of GNRs: (a) and (b) TEM images taken from different area of the
carbon film TEM grid, (c) transverse (green) and longitudinal (red) size distribution of
GNRs. (d) UV-vis spectrum showing the two SPR bands of GNRs, which are labelled as T
(transverse) and L (longitudinal).
3.3.1.2 Longitudinal SPR Band Tuning
The longitudinal SPR band of GNRs is dependent on the particle aspect ratio (AR).
Therefore by adjusting the AR, one can achieve wavelength tuning of the longitudinal SPR.
To synthesise GNRs with variable positions of longitudinal SPR, the standard seed-
mediated method was used, but with varying parameters that may affect the overall
67
morphology and aspect ratio of the nanorods. There are a variety of factors that can
contribute to the differences in particle morphology and aspect ratio.369
Herein, three
parameters were studied in detail; concentration of ascorbic acid (AA), silver ions (Ag+),
and Au seeds.
3.3.1.2.1 Ascorbic Acid
Ascorbic acid (AA) is a mild reducing agent that is used to reduce Au (I) to Au (0). This
reduction can be observed when the brown-yellow colour of the solution turned colourless.
In a typical experiment, AA was added up to a final concentration of 5.4 × 10-4
M. Using
this final concentration, the nanorods had an aspect ratio of 3.73 (Figure 3.3(a)). Given that
all other concentrations of the reactants are constant, it can be observed that the
morphology of the nanorods changes with increasing final concentration of AA (Figure
3.3(a) – (c)). The nanorods show a decrease in length and an increase in width, giving rise
to aspect ratio of 2.9 (Figure 3.3(b)). Further increase in the AA concentration resulted in a
unique morphology i.e. dumbbell-like structures (Figure 3.3(c)). The particles were
polydisperse in size and shape, with less than 15% rod yield (by shape).
The UV-vis absorption spectra of the synthesised nanorods are shown in Figure 3.3(d).
The plasmon band position was found to be in the range of 781 ± 5 nm, when the final
concentration of AA was 5.4 × 10-4
M. When the concentration was increased to 6.4 × 10-4
M, shortening of the nanorods resulted in the plasmon band position in the visible
wavelength range (680 ± 12 nm). Further increase in the concentration (8.4 × 10- 4
M)
resulted in a plasmon band position in the range of 745 ± 7 nm, however the rod-shape was
compromised. In short, the nanorod longitudinal SPR band shifted significantly (~100 nm)
towards the blue as the initial AA concentration was increased by nearly 20%. This finding
is in consistent with a report by Sau and Murphy.106
Further increases in the AA
concentration not only affected the length and width (aspect ratio) of the GNRs, but also
reduced the rod yield significantly. The majority of the particles exhibited a dogbone- or
dumbbell-like structure when the final concentration exceeded 8.4 × 10-4 M. The decrease
in rod yield may be due to the fast reduction rate, as the concentration increased, the
reduction of Au(III) to Au(0) became faster, thereby inducing the growth of seed particles
in all directions and forming more complex particle shapes.106
68
Figure 3.3 Synthesis of GNRs using variable ascorbic acid concentrations. From (a) to (c)
TEM images of GNRs yielded as a result of increasing ascorbic acid concentrations. (d)
Typical UV-vis spectra showing the varying longitudinal SPR bands.
3.3.1.2.2 Silver Nitrate
Unlike the case of AA, while keeping the concentration of all other reactants fixed,
increasing the Ag+
concentration (from 6.4 × 10-5
M to 8.3 × 10-5
M) did not significantly
alter the morphology of the nanorods. However, a spectral shift was apparent as the
concentration increased. Figure 3.4 shows the UV-vis spectra of GNRs synthesised using
different concentrations of Ag+
in the reaction. As the Ag+ concentration increased, the
69
longitudinal SPR band of the GNRs shifted towards 800 nm. Redshifts in the plasmon band
are an indication of an increase in the average aspect ratio. Indeed, the overall aspect ratio
of the nanorods increased from 3.5 to 3.85 as a result of increasing Ag+ concentration. The
band positions corresponding to the concentration of Ag+
used in the reactions are presented
in Figure 3.5. As opposed to AA, changing the initial Ag+ concentration in the reaction by
nearly 20% did not shift the longitudinal SPR band of the GNRs as much as when changing
the concentration of AA to the same extent. The presence of additional Ag+ did not vary the
overall shape of the GNRs to either dogbone- or dumbbell-like structure like in the case of
AA, but rather improves the nanorod formation and aspect ratio of the nanorods.366
This is
consistent with the concept of underpotential deposition (UPD) of Ag+ which occurs
preferentially at the side {110} facets of gold: while the Ag monoloyer stabilises the {110}
facet, other facets (such as nanorod tips) grow faster due to being less covered with Ag,
therefore facilitating the formation of rod shape particles.369
Figure 3.4 Synthesis of GNRs using variable Ag+ concentrations. UV-vis spectra of GNRs
showing a redshift (arrow) in the longitudinal SPR bands as the Ag+ concentration
increased.
70
Figure 3.5 The longitudinal SPR band positions with respect to varying Ag+
concentrations
in the reaction.
3.3.1.2.3 Gold Seeds
CTAB-capped Au seeds are very small in size (~1.5 nm)26, 369
and are used to promote
the growth of GNRs on the {110} and {100} crystal faces of gold.70
Within this size range,
it was very difficult to characterize the seeds by using electron microscopy. Additionally,
Ostwald ripening is likely to occur when the small particles are dried on the TEM grid
therefore the ‘true’ size of those particles may not be known unless thiol molecules are
added as the capping agent prior to the characterisation.26
Figure 3.6 shows UV-vis
absorption spectrum of Au seeds. It can be observed that there is no specific absorption
band across the wavelength range between 450 nm and 900 nm because of the very small
size of the particles.
While keeping the other experimental conditions fixed, increasing the Au seed
concentration (from 2.08 × 10-7
M to 5.81 × 10-7
M) did not significantly alter the
morphology of nanorods. However, a spectral redshift was apparent as the concentration
increased (Figure 3.7), indicating changes to the aspect ratio of the nanorods. Within this
71
concentration range, the spectra shifted between 765 and 805 nm. The aspect ratio of the
nanorods changes from 3.5 to 3.9 as a result of increasing Ag+ concentration. The band
positions corresponding to the concentration of Au seed used in the reactions are shown in
Figure 3.8. As opposed to AA, changing the Au seed concentration in the reaction by more
than 20% did not shift the longitudinal SPR band of the GNRs as much as when changing
the concentration of AA to the same extent.
Figure 3.6 The UV-vis spectrum of Au seeds used for the growth of GNRs.
72
Figure 3.7 Synthesis of GNRs using variable Au seed concentrations. Typical UV-vis
spectra of GNRs showing a redshift (arrow) in the longitudinal SPR bands as the Au seed
concentration increased.
Figure 3.8 The longitudinal SPR band positions with respect to varying Au seed
concentrations in the reaction.
73
3.3.2 Layer-by-layer (LbL) Polyelectrolyte Coating
The surface of as-synthesised GNRs bore a positive charge due to the presence of the
CTAB bilayer. This has provided a convenient means for surface modification via LbL
coating with charged polymers or polyelectrolytes (PE). The deposition of PEs onto the
GNRs was based on electrostatic interaction.161 PSS is negatively charged and therefore
was used as the first layer of PE coating. The UV-vis absorption spectrum of the modified
GNRs after PSS coating is shown in Figure 3.9(a), from which an absorption band at ~225
nm can be attributed to the presence of PSS.375
Figure 3.9(b) shows the TEM image of
PSS-coated GNRs (PSS/GNRs). PAH is a positively PE and PVP is a weakly negative
charged PE. These two PEs were used as the LbL pair to achieve subsequent coatings on
PSS/GNRs. In a typical LbL coating process, it is noteworthy that two phenomena can
change: (i) nanorod surface charge reverses, and (ii) longitudinal SPR band shifts. Figure
3.10 summarises the measured ζ-potential of GNRs with different surface coatings. These
data show the reversal of surface charge after the deposition of each PE layer (in the order
of PSS, PAH, and PVP). These phenomenological changes on the particle surface are in
consistent with the reported literature with the surface charge changing in accordance with
the netcharge of each polymer layer.178, 185
Figure 3.9 PSS coating of GNRs: (a) Gold nanorods coated with PSS, the additional band
at 226 nm (arrow) indicates the presence of PSS, (b) the associated TEM image (note that a
thin layer of PSS is not visible at this magnification)
74
Meanwhile, LbL PE coating caused the longitudinal SPR band of the GNRs to shift.
Figure 3.11 shows the UV-vis spectral shifts that were observed due to the process of LbL
coating. Firstly, coating with PSS resulted in a major ~20 nm blue-shift of the longitudinal
SPR band of the as-synthesised CATB-capped GNRs, which is consistent with the finding
by Guo et al.376 Subsequently, surface coating with PAH and PVP resulted in a further but
minor (~2 nm) blue-shift. These polymer coatings have contributed to spectral blue-shift,
instead of red-shift that is typical of observations for the LbL coating of GNRs due to the
increase in the local dielectric function as a result of polymeric surface adsorption events.178
The reason is not well understood but could be due to the effects of coupling between
GNRs side-by-side as a result of polymer coatings.409 Here, PVP was used to form an outer
layer of polymer on the GNRs for facilitating silica formation (Section 3.3.4). PVP is an
amphiphilic polymer, and upon coating slight changes to the characteristic SPR bands were
observed. In particular, the longitudinal SPR band became less intense as indicated by the
transverse-to-longitudinal SPR band ratio (Figure 3.11). This can be attributed to some loss
of signal due to sample loss during purification by centrifugation. The broadening of the
spectral band towards long wavelengths suggests a small amount of nanorod aggregation
because of the relatively low surface charge (as indicated by the zeta potential) and thus
less repulsion from the nanoparticles.
Figure 3.10 Zeta-potential of GNRs measured after each deposition of PE. The PE layers
were formed in the order of PSS, PAH, and PVP.
75
Figure 3.11 UV-vis absorption spectra of GNRs with PEs. During the coating of PEs, the
longitudinal SPR band shifted to the left (arrow). Vertical dashed lines indicate the peak
positions.
3.3.3 mPEGylation
Surface displacement of CTAB molecules on the nanorods with methoxy PEG-SH was
made possible due to the strong affinity of thiol groups for the gold surface.169 However,
due to the relatively strong Au-Br bond, complete removal of CTAB from the nanorod
surface can present significant challenges. In this context, surface exchange with mPEG-SH
typically requires a significant amount of time.164
Herein, a time-dependent study of surface
displacement/exchange was carried out. Centrifugation was used to promote surface
desorption of CTAB molecules in the presence of mPEG-SH. The high speed centrifugal
force may help in desorption of CTAB and at the same time allow interaction between the
nanorods and mPEG-SH. The mixture of GNRs and mPEG-SH was incubated for varying
lengths of time (0, 30, 120, 240 and 360 min) before the mixture was subjected to
76
centrifugation at 10,000 rpm for 10 min. Figure 3.12 shows a sub-linear (R2 = 0.977)
decrease in the surface potential of GNRs with incubation time. The decreasing trend in ζ-
potential over time suggests that the CTAB molecules present on the nanorods were
displaced by the neutral mPEG-SH and the process is dependent on the incubation time. As
observed from the zeta potential measurement, the displacement of CTAB with amphiphilic
PEG decreases the positive-charge density of Au NRs over time. This may result in some
aggregation, as indicated by the slight broadening of the longitudinal SPR towards long
wavelengths (Figure 3.13). This is mainly attributed to the reduction in electrostatic
repulsion after thiol passivation, but also the lesser degree of steric effect during the thiol
replacement. The longitudinal SPR band redshifted by ~5 nm after mPEGylation, this
observation suggests the occurrence of surface exchange of molecules (CTAB to mPEG).
In order to investigate the disappearance of CTAB and the formation of gold-thiol bonds
on the nanorods, Raman spectroscopic analysis was carried out on the raw Au nanorod
samples and mPEGylated Au nanorod samples. Figure 3.14 compares the Raman spectra of
raw GNRs and mPEGylated GNRs in the “fingerprint region”. The peak at 180 cm-1
is
present in the spectrum of raw GNRs, which is assigned to the Au-Br bond.377 The peak at
261 cm-1
is only present in mPEGylated GNRs, which can be attributed to the Au-S
bond.164 Both of the Raman peaks can be observed in the case of partial displacement of
CTAB.
77
Figure 3.12 Time dependence of mPEGylation of GNRs, revealed by charges in zeta
potential.
Figure 3.13 UV-vis absorption spectra of GNRs before and after mPEGylation.
0
5
10
15
20
25
30
0 50 100 150 200 250 300 350 400
ζ-p
ote
nti
al
(mV
)
Time (min)
78
Figure 3.14 Raman spectra of raw GNRs (red dashed-line) and mPEGylated GNRs (blue
line).
3.3.4 Silica Coating
PVP/PEs/GNRs prepared via LbL (Section 3.2.4.1) were subjected to silica coating
using TEOS as a silica precursor. Figure 3.15 shows a typical TEM image of silica-coated
GNRs prepared in this way. It can be observed that the coating is homogeneous around the
nanorods. Additionally, silica coating of PVP/PEs/GNRs resulted in a ~20 nm redshift in
the longitudinal SPR band (Figure 3.16). The longitudinal SPR bands redshifted
significantly which could be attributed to the higher refractive index of amorphous silica
(1.46) surrounding the nanorods compared to the lower refractive index of isopropanol
(1.38) as a solvent.183
An increase in refractive index around gold nanoparticles produces a
decrease in the restoring force on the electron oscillation associated with the plasmon
modes.183
Due to the inert chemistry of the silica shell, it is possible to suspend the core-shell
particles in a variety of polar organic solvents without forming aggregates. Figure 3.17
shows the UV-vis spectra of silica-coated GNRs in different solvents. Four solvents with
79
different refractive indexes (RI) were investigated: water (1.33), ethanol (1.36), isopropanol
(1.38), and DMSO (1.48). It can be observed that the longitudinal SPR band shifted
towards red when the nanorods were suspended in the solvents of increasing refractive
index. This is because of the change in the local refractive index surrounding the
nanorod.162
Figure 3.15 A representative TEM image of silica-coated GNRs prepared by using PVP as
the surface primer.
The silica shell thickness can be controlled by adjusting the concentration of TEOS
before adding it into the system that contains PVP/PEs/GNRs. To demonstrate the
feasibility of changing the shell thickness, the concentration of TEOS was varied from
0.97% to 1.5% (v/v% in isopropanol). As shown in the TEM images in Figure 3.18, silica-
coated nanorods have different silica shell thicknesses: (a) 32.4 ± 5.8, (b) 57.7 ± 3, and (c)
62.7 ± 4.3, as a result of 0.97%, 1.2%, and 1.5% of TEOS, respectively. The shell thickness
was calculated by halving the transverse diameter of the core-shell nanostructures (see inset
of Figure 3.18(c)). The increase in the silica shell thickness also contributed to a minor
redshift in the longitudinal SPR band of the GNRs (Figure 3.19). However a broadening of
the SPR band is more apparent as the shell thickness increases. This observation is also in
consistent with the findings by Liz-Marzán’s group.167, 183, 212
80
Figure 3.16 UV-vis spectral shift (arrow) as a result of surface coating with silica. Dashed
lines represent the peak position of the longitudinal SPR.
Figure 3.17 UV-vis absorption spectra of silica-coated GNRs in solvents of different RI.
The red shift increases with increasing RI.
81
Figure 3.18 TEM images showing GNRs coated with different silica shell thicknesses. The
average silica thicknesses were measured as (a) 32.4 ± 5.8 nm, (b) 57.7 ± 3 nm, and (c)
62.7 ± 4.3 nm, which is calculated by halving the transverse diameter of the core-shell
nanostructures as illustrated by the inset of (c).
Figure 3.19 UV-vis absorption spectra corresponding to the silica-coated GNRs as shown
in Figure 3.18(a), (b) and (c). Inset: areas under the spectra are filled to highlight the
broadening of the longitudinal SPR bands.
82
Meanwhile, mPEGylated GNRs prepared in Section 3.2.4.3 were also subjected to silica
coating. TEOS was used as a silica precursor and the mixture of TEOS and the
mPEGylated GNRs in ethanol was sonicated. As shown in Figure 3.20, the surface charge
of the as-synthesised GNRs went from positive (~ +34 mV) to nearly neutral (~ +2 mV)
upon surface exchange with mPEG-SH, and subsequently became negatively charged after
coating with silica (~ -26 mV). The initial positive surface charge of the as-synthesised
GNRs was due to the presence of CTAB on the nanorods. Subsequently, the presence of
neutral PEG on the surface of the nanorods reduced the surface charge substantially to
nearly zero. After silica coating, the silica surface is terminated by hydroxyl groups which
resulted in a negative surface charge on the nanoparticles, as indicated by the negative zeta-
potential.
Figure 3.20 Changes in surface potential of GNRs after surface modification with mPEG
and silica.
Silica coating of mPEGylated GNRs resulted in a ~15 nm redshift in the longitudinal
SPR band (Figure 3.21). The general characteristic of the band is preserved, suggesting that
83
the silica coating did not cause any significant aggregation of the nanorods. Additionally,
the broadening of the transverse and longitudinal SPR bands can be observed. This could
be due to the increase in the amount of spherical particles as a result of shape
transformation following continuous sonication during the coating process, which could be
contributed by ultrasonic bulk heating effect.378 TEM images in Figure 3.22(a) and (b)
show the mPEGylated GNRs and the silica-coated nanorods, respectively. It can be
observed that following silica coating, more spherical particles were present. The silica
coating prepared in this way has a relatively thin shell (~13.5 nm) compared to the method
previously described. Figure 3.22(c) presents the silica thickness size distribution.
Figure 3.21 UV-vis spectra of GNRs showing the redshift and broadening of the
longitudinal SPR bands after mPEGylation and silica coating.
84
Figure 3.22 Silica coating of mPEGylated GNRs. TEM images taken (a) before, and (b)
after the silica coating. (Red arrows point out some particles that have potentially
undergone shape transformation). (c) Size distribution of silica shell thickness.
Silica coating of GNSs was carried out by first coating the as-synthesised GNSs with
PVP. The coating was performed with slow overnight stirring because PVP is weakly
charged and the citrate-capped nanoparticle surface is moderately negatively charged. The
PVP/GNSs were then subjected to silica coating by vortexing the mixture of TEOS and
PVP/GNSs in isopropanol. Figure 3.23 shows the UV-vis spectrum of PVP/GNSs before
and after the silica coating. It can be observed that the redshift was ~5 nm following the
coating. Unlike the case of silica coating of PVP/PEs/GNRs, this spectral shift is relatively
small, which may suggest that the smaller the contact surface area (nanospheres), the lesser
85
the redshift in the observed band position.379 Figure 3.24 shows a typical TEM image of
the silica-coated GNSs. From the TEM image, the homogeneous silica shell can be
observed and the shell thickness was found to be 20 ± 5 nm.
Wavelength (nm)
No
rma
lize
d A
bso
rb
an
ce (
a.u
.)
400 500 600 700 800
0.0
0.5
1.0 PVP coating
Silica coating
Figure 3.23 UV-vis spectrum of silica-coated PVP/GNSs shows a red-shift (arrow) of the
SPR band after silica coating.
Figure 3.24 TEM image of silica-coated PVP/GNSs.
86
3.3.5 FDTD Simulation
The photothermal conversion efficiency of GNRs is dependent on their effective
absorption cross section. Therefore it is important that the coating around the nanorods does
not greatly affect the absorption cross section. To investigate the effect of silica coating on
the absorption cross section, finite-difference time-domain (FDTD) simulation was
performed to calculate the extinction cross sections of both of the uncoated and coated
GNRs at the longitudinal SPR wavelength. Figure 3.25 shows the FDTD simulated cross
section spectra of GNRs with a dimension of 48 × 13 nm, before and after the 15 nm silica
coating. The total extinction cross section is the sum of the absorption and scattering cross
sections. In Figure 3.25, it can be observed that the amplitude of the scattering cross section
is approximately seven times lower than the absorption cross section. Upon silica coating,
the total extinction cross section showed a small increase in the amplitude, which is mainly
contributed by the increase in the absorption (7.8%) and scattering cross sections (1.3%).
These findings are consistent with similar studies by Chen et al.229
and Liu et al.380
The
spectral redshift can also be observed when silica layer was included in the calculation,
which agrees with the experimental observation. However the redshift is greater (~40 nm)
than the experimental measurements despite the silica shell thickness is similar (i.e. 15 nm).
Additionally, unlike the experimental case, spectral broadening is not observed in this
simulation after the silica coating. These differences could be due to the geometrical
(polydispersity of the nanorods) and environmental factors (such as particle-particle
interactions) that applied to the real nanorod samples but not to the simulation (in which
only single nanorod is simulated).380 Although FDTD simulation of GNRs with different
silica shell thicknesses is beyond the scope of this study, increasing the silica shell
thickness is expected to shift the SPR band further towards red in the FDTD model as well
as increase the extinction cross sections as demonstrated in the previous study.380
87
Figure 3.25 FDTD calculated extinction, absorption, and scattering cross-section spectra of
uncoated (solid curve) and silica (15 nm)-coated (dashed curve) GNRs.
3.3.6 Functionalisation of Silica-coated Gold Nanoparticles
3.3.6.1 Amines
To examine the grafting efficiency of silanized-amine, the silica-coated GNSs were
chosen as the nanoparticle model. The amine grafting was carried out using 3-
aminopropyltrimethoxysilane (APTMS). FTIR spectra of non-APTMS-treated and
APTMS-treated samples were acquired and analyzed (Figure 3.26). The presence of Si-O-
Si is confirmed by the peaks at 1020-1100 cm-1
while the peaks at 950 and 800 cm-1
are
attributed to the asymmetric bending and stretching vibration of Si-OH, respectively.
Meanwhile, the absorption peaks at 1635 cm-1
and 3000-3600 cm-1
for both spectra are
assigned to O-H of silica lattice vibrations and bending mode of physically absorbed water
molecules.381 For APTMS-treated samples, the asymmetric and symmetric stretching
88
vibrations of C-H bonds present in the propyl group of attached APTMS were observed in
the spectrum by peaks at 2920 and 2850 cm-1
, respectively. The two peaks are not present
in the spectrum for the non-APTMS-treated nanoparticles, and therefore indicating the
presence of APTMS molecules bonded to the silica shell surface. The FTIR analysis cannot
conclusively prove the presence of the amine groups at the nanoparticles. The expected
peak assigned to the N-H bending at around 1650-1580 cm-1
is not visible. This is attributed
to an overlap with the peak at 1635 cm-1
which is already present in the spectrum. Overall,
the intensity of the peaks corresponding to the NH2 was rather weak, which may be
expected considering the small proportion of the silane surface monolayer compared to the
relatively thick silica coating.
Subsequently, fluorescamine was used to quantify the number of amine groups on the
nanoparticle. It is noteworthy that only the primary amine can interact with fluorescamine
to form a fluorescent product. From the fluorescence spectrum as shown in Figure 3.27, the
excitation and emission wavelengths were 390 nm and 488 nm, respectively. The
fluorescamine standard curves were measured at the peak excitation and emission
wavelengths and plotted as a function of the amount of amine present in hydrolyzed
APTMS and ethanolamine (Figure 3.28). Both APTMS and ethanolamine are monoamines.
From the standard curves in Figure 3.28, the slopes of lines A and B are 20 and 19,
respectively. Given that both of the standards have the same molar concentration of NH2,
the resulting fluorescence intensity is rather close and as expected.
89
Figure 3.26 FTIR spectra of silica-coated GNSs, and amine-grafted silica-coated GNSs.
Figure 3.27 Fluorescence excitation and emission spectra of primary amine bound
fluorescamine. Excitation and emission peaks are 390 nm and 488 nm, respectively.
90
Figure 3.28 Fluorescamine calibration curves of monoamine standards: (a) fresh APTMS
and (b) ethanolamine.
To calculate the number of amines per nanoparticle, the density of the core-shell particles
was first worked out using Eq. 3.2-3.4.
Vcs �4
3πRcs
3 (Vc =
4
3πRc
3) (Eq. 3.2)
Vs = Vcs - Vc (Eq. 3.3)
ρcs = (ρcVc + ρsVs) / Vcs (Eq. 3.4)
In these equations, V, R, and ρ are volume, radius, and density, respectively. Subscripts of
cs, c and s correspond to core-shell, core and shell, respectively. The sampling of over 100
particles from the TEM images gives an average Rcs of 33.5 nm and Rc of 7.5 nm. The
density of the core-shell particles is then calculated to be 2.84 g/cm3, which equals to 2.24 x
1015
nanoparticles per gram. The present fluorescent experiments indicated that 0.1 mg of
91
nanoparticles would generate an average fluorescent intensity of 1782 FIU. From the two
standard curves of APTMS and ethanolamine, this corresponds to 8.6 × 10-9
and 9.3 × 10-9
moles of amines, respectively. Given the number of nanoparticles per gram, the average
number of surface grafted amine is then calculated to be 2.40 ± 0.05 × 104 per nanoparticle.
This amount equates to an average amine surface density of~1.6 molecules per nm2, which
is relatively close to a similar study performed by Chen et al. in which ~2.0 amine per nm2
was measured using the fluorescamine method.382
Using conductometric titrations, Kralj et
al. found that ~2.3 amine per nm2 are present on the silica-coated iron oxide nanoparticles
after the grafting process with APTMS.383
Theoretically, there are 4.6 Si-OH groups per
nm2 of silica surface which represents the main source of reactive groups for amine
silanization.384
The reason for the low surface density of amine at the silica shell in this
work is likely due to a low grafting efficiency as it would be influenced by experimental
parameters including temperature and pH.385
3.3.6.2 Polydopamine
Polydopamine (PDA) was used in the present study in order to improve surface amine
present in the outer surface of silica-coated gold nanoparticles. This procedure was carried
out to overcoat the silica-coated GNRs. Firstly, the overcoating resulted in a redshift (~15
nm) and broadening of the longitudinal SPR band (Figure 3.29(a)), suggesting a successful
overcoating step due to the deposition of PDA that increases the surface dielectric constant.
PDA had self-polymerised onto the silica surface, which resulted in a roughening of the
particles’ surfaces as evident from the TEM analysis (Figure 3.29(b)). Additionally, during
the overcoating step, PDA appears to have formed particles by itself as byproducts which
were not successfully removed by centrifugation. The time (10 min) used for the
overcoating step was deemed sufficient as a greater redshift in the SPR band could result if
a longer time was used.386
This finding is also consistent with a similar study by Black et
al., who performed direct coating of PDA onto bare GNRs, and it was reported that the
redshift in the longitudinal SPR began as soon as 10 min after the addition of dopamine
hydrochloride solution, indicating the spontaneous self-polymerisation of dopamine onto
the nanorods.386
Figure 3.30 presents the FTIR spectra of PDA, and PDA overcoated on the
92
silica surface of GNRs. The peaks at 1515 and 1605 cm-1 are assigned to the indole or
indoline structures associated with the polydopamine.387
The presence of Si-O-Si in silica is
also confirmed by the peaks at 1020-1100 cm-1.
Figure 3.29 Polydopamine overcoating of silica-coated GNRs. (a) UV-vis spectrum of
silica-coated GNRs before and after the polydopamine overcoating. (b) TEM image
showing the polydopamine overcoated on the surface of silica-coated GNRs, together with
polydopamine particles. Inset: Initial silica-coated GNRs.
93
Figure 3.30 ATR-FTIR spectra of polydopamine (top) and polydopamine/silica-GNRs
(bottom). The dotted lines at 1515 and 1605 cm-1
indicates the indole or indoline structures
of polydopamine.387
3.4 Discussion
This chapter presented results on the synthesis and surface modification of gold
nanoparticles. There is a need to address the appropriate experimental conditions and
parameters in the synthesis protocols because the SPR band is highly dependent on the
particle size and aspect ratio.16 In this context, the concentrations of AA, Ag+ and Au seeds
in the seeded growth reaction were investigated. Compared to increasing the AA
concentration, the control over the aspect ratio (and so the SPR band) of the nanorods is
greatly improved when the concentrations of Ag+
or Au seeds were increased to the same
extent. For instance, increasing the final concentration of AA in the reaction by more than a
20% appeared to shorten and change the morphology of the GNRs (to dogbone- or
dumbbell-like structure), resulting in a pronounced change to the plasmon band positions.
However, increasing the final concentration of Ag+
or Au seeds in the reaction by more
94
than 20% appeared to change (minor change under the experimental conditions described
here) the aspect ratio and the plasmon band positions of the nanorods. Comparing the three
components in the seed-mediated growth, fine tuning of the longitudinal SPR band of
GNRs in the region between 760 nm and 780 nm is more easily achieved by changing the
concentration of Ag+ and Au seeds than the concentration of AA. The GNRs synthesised in
that wavelength region are ideal for subsequent uses in the current work, including surface
modification (Section 3.2.4) and biological analyses232, 342, 388
carried out as part of this
thesis (Chapter 4 and 5). In particular, it is important to match the longitudinal SPR band of
the surface modified and non-surface modified GNRs with the incident laser wavelength at
780 nm. While synthesising GNRs with higher aspect ratio and longer longitudinal SPR
wavelength (>1000 nm) is beyond the scope of the thesis, that goal has been achieved by
other groups and reported in some recent literature.83, 89
GNRs produced using a higher concentration of AA in the reaction have a unique
morphology: dogbone- or dumbbell-like structure, which have great potential in providing
‘hot spots’ for enhancing SERS signals of the fingerprint molecules.389
Interestingly, such a
unique shape of dogbone- or dumbbell-like Au nanocrystals has been previously
synthesised by means of electrochemical390
and anisotropic oxidation.109
With regard to the
nanoparticle yield, it has been claimed that the GNRs synthesised herein by the standard
synthesis protocol can reach milligram scale per batch.70, 91
Methods to significantly
increase the yield have been investigated, for example, Lohse et al. developed a reactor for
high-throughput synthesis of gold nanoparticles of spherical and rod shapes and the yield is
on the gram scale.391
Therefore the technique can be adapted or improved in the future to
achieve large scale synthesis.
This chapter also reported the results of surface modification of the as-synthesised gold
nanoparticles. Firstly, for LbL coating of GNRs with PE, low molecular weight PEs were
chosen in order to avoid bridging flocculation between nanoparticles.392
Layers of PSS (-),
PAH (+) PEs and PVP were sequentially and successfully deposited around the GNRs on
the basis of electrostatic interaction and the results were indicated by nanorod surface
charge reversal following each polymer deposition. This LbL coating of GNRs has
provided PSS/GNRs and PVP/PEs/GNRs for biological analyses and silica coating (Section
95
3.2.4), respectively. Meanwhile, thiolated mPEG was used to modify the nanorod surface
and produced mPEGylated GNRs. It is well known that thiol molecules form strong bonds
with Au nanoparticles or nanostuctures.169 A time-dependent study was carried out to
examine the efficiency of CTAB displacement by the mPEG-SH. High curvature at the tips
of the GNRs leads to a less dense CTAB bilayer compared to that on the side facets.112
Therefore, a much faster and easier desorption of CTAB is likely to occur at the tips and
this also facilitates surface exchange with thiol molecules. A few reports have made use of
this strategy to build unique GNR nanostructures, for instance, using biotin disulphide,112
cysteine217
, and glutathione217
for tip-to-tip self-assembly of GNRs, in addition, glutathione
was also used for blocking the tips of GNRs in order to promote transverse overgrowth on
GNRs.111
The extent of CTAB displacement was indicated by the decrease in zeta-potential
of the nanorods. It was found that the surface charge reduced slightly until at least 4 hrs of
incubation with the mPEG-SH. This finding was also supported by the Raman spectrum of
mPEGylated GNRs, in which the Au-Br peak diminished upon successful surface
displacement. Therefore, to prepare mPEGylated GNRs through the typical surface
displacement, a standard incubation time of 4 hr was used. For instance, subsequent silica
coating was performed on the batch of mPEGylated GNRs that had been incubated for at
least 4 hrs to ensure complete and homogeneous silica coating.
Other than surface modification with polymers, silica coating was also carried out for
the GNSs and GNRs. The current work makes use of PVP, which carries pyridyl groups, as
well as methoxy PEG, which contains ether oxygens, to interact and form hydrogen
bonding with hydroxyl groups of the hydrolyzed TEOS. On the silica shell surface, the
hydroxyl groups provide reactive sites for further condensation. For example, the silanol
groups (Si-OH) were further condensed to covalent siloxane bonds (Si-O-Si) that lead to
the formation of thicker silica coating layers. Therefore increasing the concentration of
TEOS increased the thickness of the silica shell grown on the nanorods as observed in the
current study. GNRs with silica shells are relatively stable in polar organic solvents.
Spectral shifts were observed and as expected in different solvents, because of the change
in the local dielectric constant.162
The ability to detect small molecules based on spectral
shifts has been demonstrated on the basis of changing local refractive index.393, 394
96
In the previous literature, it was demonstrated that surface charge can affect the uptake
of nanoparticles into cells.213
To examine the cellular uptake (Chapter 4), the silica surface
of GNSs or GNRs was modified with surface amines. Surface functionalization with
amines was first tested with a silanized amine, APTMS, as amine source to achieve post-
grafting onto the silica surface of GNSs. The amines present were found to be relatively
low on the surface of silica-coated GNSs, which is possibly due to a low grafting
efficiency. Therefore a polymer with more amines was used in the subsequent surface
functionalization. The silica-coated GNRs were overcoated with polydopamine (PDA) in a
process of oxidation under alkaline conditions. The biocompatible PDA contains abundant
functional groups especially indole; therefore it was used to modify the silica surface. The
formation of PDA in aqueous dopamine is driven by the oxidation of dopamine catechols to
quinones at alkaline pH, forming dihydroxyindole, which later cross-linked to form
melanin.395 There are significant differences between the APTMS and PDA in terms of
bond formation on the silica surface; the former strictly relies on the formation of covalent
linkages via silanization, while the latter self-polymerises onto the silica surface via charge-
transfer, hydrogen bonding, and π-π stacking interactions.387
3.5 Conclusion
In summary, samples of GNSs and GNRs were synthesised using citrate reduction and
silver-assisted seeded growth methods, respectively. For the synthesis of GNRs, fine
tuning of the nanorod longitudinal absorption band in the wavelength region between 760
and 800 nm was found to be more easily and consistently achievable by manipulating the
concentration of Ag+ or gold seeds instead of AA. The work is significant in that GNRs can
be synthesised with a desired longitudinal SPR band so as to compensate for known
spectral band shifts following the necessary surface modification and/or functionalization
as a result of changes in the local dielectric function. GNRs coated with PSS,
PSS/PAH/PVP (LbL PEs), methoxy PEG, silica shell, and silica/polydopamine were
successfully prepared and characterised. Each of the surface coatings contributed to
longitudinal band shifts of varying degree. In addition, GNSs coated with PVP, and silica
shell were also successfully prepared and characterised. The nanospheres coatings also
97
resulted in shifts in the transverse SPR band, but to a much lesser extent than the
longitudinal band shifts in GNRs. GNRs with varying silica shell thickness were prepared
by adjusting the concentration of TEOS in the system. Changes in the shell thickness also
altered the spectral profile e.g. broadening of the longitudinal absorption band. However
the absorption bands were able to be tuned to ~780 nm, which was suitable for the
requirements of the laser study in Chapter 5. Surface functionalization of silica-coated
GNSs with amines via aminosilane grafting was attempted, but the lengthy protocols did
not yield a satisfactory outcome in terms of the number of surface amines. On the other
hand, polydopamine was successfully functionalized onto the silica-coated GNRs by
spontaneous self-polymerisation under oxidative and alkaline conditions and the abundant
functional groups including indole could help in the subsequent work in Chapter 4. The
results and the materials prepared herein supported the further work reported in this thesis.
98
99
Chapter 4: Dark-field Analysis of Gold Nanoparticles in
Neuronal Cells
4.1 Declaration for Chapter 4
Some of the research presented in this chapter have been published as:
• J. Yong, W.G.A. Brown, K. Needham, B.A. Nayagam, A. Yu, S.L. McArthur and P.R.
Stoddart. “Dark-field microspectroscopic analysis of gold nanorods in spiral ganglion
neurons”, Proc. SPIE 8923, 2013.
4.2 Introduction
Gold nanoparticles have shown great promise as imaging contrast agents128, 146, 291
and
photothermal therapeutic agents269, 396 in vitro and in vivo. Both of these applications are
based on the absorption and scattering which are two striking intrinsic optical properties of
gold nanoparticles. The optical behavior is based on the resonance between collective
oscillations of electrons in the conduction band and the incident light field, also known as
the localized surface plasmon resonance (LSPR). The optical cross-section (absorption and
scattering cross-sections) as a result of LSPR are dependent on particle size and shape due
to the volumetric radiative capacity,397
for example, given the larger volume, 80 nm gold
nanospheres (GNSs) have a larger optical cross-section than smaller gold nanospheres (<20
nm),13
while gold nanorods (GNRs) have optical cross-sections an order of magnitude
higher than GNSs.397
Both light absorption and scattering properties of gold nanoparticles
have been well known for various applications in biological imaging, photothermal therapy,
sensor and electronic devices.398
The single absorption and scattering peak of the spherical
nanoparticles typically falls in the wavelength range from 520 to 560 nm, depending on the
size and surface dielectric properties of the nanospheres. It was later found that GNRs can
be synthesized with a greater range of absorption and scattering peak wavelengths by
changing the length-to-width ratio of the nanorods.16, 399 As shown in Chapter 3, GNRs
100
exhibit two absorption peaks, corresponding to the transverse and longitudinal axes of the
nanorods with respect to the polarization of the incident light. Through wet chemical
synthesis, the longitudinal peak can be tailored from the visible to near-infrared wavelength
(NIR, 650 – 900 nm) region, where biological tissue is most transparent.12
This has
benefited bioapplications such as hyperthermia therapy129, 150, 269, 275 and DNA/drug
delivery207, 270, 280, 400, 401
, details of which are discussed in Chapter 2 (Section 2.5.3).
Apart from absorption, the light scattering properties of gold nanospheres and nanorods
have been used primarily in cellular imaging by dark-field microscopy.146, 402, 403
The
nanorods scatter light more intensely with a wavelength dependence corresponding to the
longitudinal SPR peak.361 Although the particles themselves are much smaller than the
diffraction-limitted resolution of an optical microscope, this scattered light allows direct
visualization of the nanorod distribution.
Analysis of the distribution of the gold nanoparticles in contact with or inside cells can
contribute to an improved understanding of biologically relevant processes such as
intracellular delivery. For instance, it is often required to verify the presence of GNRs
inside cells in order to establish their stability for photothermal therapies. Undoubtedly,
monitoring cell death in the case of hyperthermia of tumor cells or the expression of
fluorescent proteins inside cells in the case of gene delivery can both serve to indirectly
indicate the presence of GNRs. However, in the case where GNRs are to be used for other
purposes such as modulating the normal cellular responses,232, 342 validating the presence of
GNRs in the vicinity of the cells may not be straightforward. These studies either require
fluorescent molecules bound to the nanoparticles or the use of electron microscopy. The
former can complicate the investigation, given that fluorescence dyes can desorb from the
nanoparticles due to changing pH in the extracellular and intracellular environments and
can suffer from photobleaching, while the later can be lengthy in terms of sample
preparation. Furthermore, tracking the nanoparticles in heterogenous samples such as
primary cultures can pose significant challenges, particularly where a specific cell is of
particular interest and may only be present as a small fraction of the population. Moreover,
in primary neuronal cultures, different cell types can exhibit significant differences in their
capacity to take up the nanoparticles.404 Therefore it is necessary to select and analyze each
individual cell in order to acquire information pertinent to the nanoparticles.
101
Spectroscopic analysis provides spectral information which can be correlated to the
presence of the nanoparticles in the cells and their stability information. Previous
nanoparticle and single cell imaging analysis has been based on spectral acquisition,
including Raman microspectroscopy and dark-field microspectroscopy.405
While these have
largely been investigated using cell lines, the literature regarding spectroscopic analysis of
heterogenous populations of primary neuronal cultures and nanoparticles is rather limited.
This chapter explores the use of dark-field light scattering and microspectroscopy to
analyse gold nanoparticles that are associated with neuronal cells. Neuronal cells are
chosen as the experimental model because of its research value in the subsequent
investigations of gold nanoparticle (absorber)-based photothermal neural stimulation. For
dark-field light scattering analysis, we first prepared stable GNS and GNR films on
polydopamine (PDA)-modified glass surfaces by immersion. The spectral library of the
immobilized nanoparticles could then be compared with spectra acquired from the cells,
providing a useful means for particle identification. PDA can undergo oxidative self-
polymerisation in aqueous dopamine hydrochloride and form an adhesion layer on virtually
all kind of material surfaces.395
Previous works have also demonstrated the feasibility of
adsorbing metal ions onto the PDA surface.406-408
For in vitro analysis, two models of neuronal cultures were used; primary rat cultures of
spiral ganglion neurons (SGNs) isolated from the early post-natal rats, and NG108-15
neuroblastoma cell lines. Silica-coated GNRs and GNSs (SiO2-GNRs and SiO2-GNSs)
were used as stable imaging contrast agents to probe spiral ganglion neurons (SGNs) in
primary rat cultures. The associations of both types of gold nanoparticles with the SGNs
were confirmed via dark-field microspectroscopy. Given the complex nature of the isolated
primary cultures, neurons were visually distinguished from other cells by their round
appearance and the associated scattering spectra of the gold nanoparticles were collected
from individual neurons.
Subsequently, the effects of GNR surface coatings on the particle internalization in
NG108-15 cell lines were investigated qualitatively by dark-field light scattering and
microspectroscopy. GNRs with five different coatings were prepared as described in
Chapter 3 and used for the cell study; polydopamine/silica (PDA/SiO2-GNRs), silica (SiO2-
GNRs), PSS (PSS/GNRs), CTAB (bare GNRs), and PEG (mPEG/GNRs). Each of the
102
coatings differs in their surface charges, but all of the nanorods have an initial longitudinal
SPR wavelength at 780 nm before incubation with the cultures. Therefore the extent to
which the SPR peak shifts can be observed and correlated with particle stability in the
cultures.
4.3 Materials and Methods
4.3.1 Preparation of Immobilized Nanoparticles on PDA-glass Surface
Glass slides (Delta Technology, USA) were cleaned with 2-propanol under sonication
and rinsed with milli-Q water and air dried by nitrogen. Dopamine hydrochloride (Sigma,
USA) was dissolved in 10 mM Tris HCl, pH 8.5 at 2g/L. The cleaned glass slides were
immersed into the dissolved dopamine hydrochloride and left overnight (~12-15 hr).
Subsequently, the slides were rinse with milli-Q water and air dried by nitrogen. The PDA-
glass slides were immersed into nanoparticle solutions of 50 nm GNSs (BBI International,
USA) or GNRs (AR ~3.78) and left overnight, and then rinsed with milli-Q water and air
dried by nitrogen.
4.3.2 Preparation of Neural Cultures
4.3.2.1 Primary Cells (Spiral Ganglion Neurons)
Cultures of dissociated early postnatal rat spiral ganglion neurons (SGNs) were prepared
as described in detail in Section 5.3.3. Briefly, dissociated cells were plated on the poly-
ornithine/laminin-coated glass coverslips. Cultures of dissociated neurons were incubated at
37ºC, 10% CO2 for up to 48 h. The culture medium was replenished daily. Subsequently,
concentrated nanorod and nanosphere samples were diluted to a particle optical density of
~ 0.18 at their respective maximum wavelength (λmax) with culture medium and added to
the neuronal cultures for overnight incubation (~15 to 17 hr) at 37 ºC under CO2
atmosphere. Neuronal cultures were rinsed with PBS to remove excess nanorods and fixed
in 4% paraformaldehyde (Sigma, USA) for 10 min. Cells were then rinsed with PBS again
to remove the 4% paraformaldehyde and finally the coverslips were mounted on the glass
slides with Aquatex mounting agent with an RI of 1.5 (Merck, Australia).
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4.3.2.2 NG108-15 Cell Line
NG108-15 mouse neuroblastoma x rat glioma hybrid cells were obtained from the
European Collection of Cell Cultures (ECACC; Health Protection Agency Culture
Collections, Porton Down, UK) and grown in Dulbecco’s Modified Eagle Medium
(DMEM) containing 10% (w/v) foetal calf serum (FCS), 1% (w/v) L-glutamine, 1% (w/v)
penicillin/streptomycin (pen/strep) in a humidified atmosphere with 5% CO2 at 37 °C.
DMEM, FCS, L-glutamine, and pen/strep were purchased from Invitrogen, Australia. Cells
used for experiments were 70% - 80% confluent in culture and harvested mechanically. For
experiments, 2 × 104 cells/cm
2 cells were seeded onto Millicell EZ slides (Merck Millipore,
Australia) and allowed to attach to the surface (or reach 75% confluence). The cell
monolayer was rinsed twice with PBS, pH 7.5 (Gibco; Invitrogen, Australia).
For incubation, GNR samples in the culture medium were added to the cells at a particle
optical density range of ~ 0.18. After exposure to GNRs for 24 hr, the cells were washed
twice by PBS buffer to remove unbound GNRs. Cells were fixed with 4%
paraformaldehyde for 10 min to halt cellular processes. After washing for 3 times with
PBS, the cells were mounted with Aquatex mounting agent with a RI of ~1.5 (Merck,
Australia) and visualized under the microscope.
4.3.3 Dark-field Light Scattering and Microspectroscopy
Bright-field and dark-field images of the neurons were taken with an inverted
microscope (Eclipse Ti-U, Nikon, Japan) using a brightfield and an oil immersion type
darkfield condenser (NA 0.9-1.45) and a 60×/1.25 oil immersion iris objective (PlanFluor).
The colour images were taken with the camera (Digital Sight DS-Vi1 or DS-Ri1, Nikon,
Japan). The shutter was controlled by the computer software (NIS-Elements, Nikon, Japan)
which also performs auto white balance. The scattering spectra were acquired using an
Isoplane SCT-320 imaging spectrometer coupled with a ProEM CCD camera (Princeton
Instruments). No filter or polarization optics was inserted during the spectral acquisitions.
The acquisitions were synchronized to the computer via the LightField software version
4.0. Acquisition time was 100 ms and each spectrum was obtained from a ~5 µm slit width
using the 60× oil immersion objective. The wavelength range from 400 to 1000 nm was
acquired using a 150 g/mm diffraction grating. Typically, the raw signals were corrected by
104
subtracting from the dark currents that were measured by keeping the camera shutter
closed. The raw data were normalized using the following equation:
���� = �������� ��������
��������� �������� (Eq. 4.1)
where ���� is the calculated scattering intensity for each wavelength, ������� is the signal
intensity of a given pixel, and ������ and ���� ���� are the signal intensity of the dark
current and the halogen lamp, respectively. For nanoparticle-SGN analysis (�′���), the
calculated signals were further corrected by subtracting the average normalized control cell
background spectrum, ��������:
���� = ���� − �������� (Eq. 4.2)
For nanoparticle-NG108-15 cell analysis (����), the following equation is used:
���� = �������� ��������
��� �� ���� �������� (Eq. 4.3)
where ����� ����� is the average signal intensity of the halogen lamp and control NG108-15
cells (typically n = 5 to 10 cells in the culture without nanoparticle treatment).
4.4 Results
Figure 4.1 illustrates the experimental setup that was used for dark-field imaging and
microspectroscopic analysis of the nanoparticles associated with the cells throughout the
Chapter. The light is collected into the dark-field condenser where a hollow cone of light is
formed which focuses a narrow beam of light onto the sample at an angle greater than the
numerical aperture of the objective (Figure 4.1(a)). The scattered light is collected by the
105
objective lens and delivered to the spectrometer via a microslit. The light that enters the
microslit is then dispersed by a grating into different colours (wavelengths) which are
picked up by the high sensitivity CCD (Figure 4.1(b)). The halogen lamp was used as the
light source and it has a broad spectral profile ranging from 400 to 900 nm as analyzed by
the spectrometer (Figure 4.2).
Figure 4.1. Dark-field light scattering and microspectroscopy analysis; (a) the combined
microscopy and spectroscopy setup that was used to acquire scattered light images and
spectra, (b) spectrometer that disperses the scattered light into corresponding wavelengths.
106
Wavelength (nm)
Inte
nsi
ty (
Cou
nts
)
400 500 600 700 800 900 1000
0
2000
4000
6000
8000
Figure 4.2 Typical spectral profile of the halogen lamp source used in the experiment.
4.4.1 Dark-field Light Scattering of Nanoparticles on Glass slides
Depending on the nanoparticle shape, the colour of the scattered light can be different.
The scattered light should match the SPR wavelength of the nanoparticles. For example,
GNSs scatter green light provided the SPR wavelength is in the green region. Meanwhile,
GNRs scatter orange to red light because the longitudinal SPR is dominant and is in the
orange-red region of the visible range. Figure 4.3 presents dark-field scattering images of
GNSs and GNRs. The nanoparticles were adsorbed onto the PDA film on the glass surface
following overnight immersion. From the dark-field images, it can be observed that GNSs
scatter green light, while dark-field image of GNRs shows prominent orange to red colour
of the scattered light. The 50 nm bare GNSs were chosen over the smaller nanospheres for
this experiement because the larger nanospheres have higher scattering efficiency.13
Meanwhile, the bare GNRs used in this section have been synthesised to a dimension of
~48 × 13 nm by a seeded growth method (Section 3.2.3). It should be noted that both of the
nanoparticle films were observed under an oil-immersion dark-field objective, and the
index-matching oil (~1.5) was used to enhance the clarity. The white spots and wrong
coloured spots that appear in the figures can be attributed to larger contaminant particles
107
(such as dust particles), aggregation and variations in nanoparticle size/shape and
orientation on the surface.
Calculated using Eq. 4.1, dark-field microspectroscopic analysis of the PDA films
containing GNSs or GNRs revealed scattering spectra with major peaks at 563 nm and 800
nm, respectively (Figure 4.4). Both of the spectra exhibit the expected spectral profiles and
the major SPR peaks correspond to their associated particle shape, i.e. a single SPR peak
for GNSs and two SPR peaks for GNRs. For GNRs immobilized on the surface, while the
longitudinal SPR peak is extremely intense, the scattering spectrum only showed a minor
transverse SPR peak. This could be due to the orientation of the nanorods on the surface
with respect to the light.409
Figure 4.3 Dark-field scattering images of GNSs (left) and GNRs (right) on glass slides.
Scale bars are 10 µm.
108
Figure 4.4 Typical scattering spectra of GNSs (50 nm) and GNRs (48 × 13 nm) on the
PDA-modified glass surface. The SPR peaks correspond to the particle shapes.
4.4.2 Dark-field Light Scattering (Primary Cultures of SGNs)
The present study served to provide information relating to the availability and stability
of nanoparticles in spiral ganglion neurons (SGNs), which were present at levels of only
about 10 to 20% in these isolated primary cultures. This information assisted in interpreting
the work on neural stimulation with GNRs and short-wavelength near-infrared lasers using
the same neurons as the experimental model (see Chapter 5). This chapter examines the
particle scattering from the SGNs in primary cultures using dark-field imaging and
microspectroscopy. Firstly, as seen in the dark-field images in Figure 4.5, the SGNs appear
mophologically as round, sometimes exhibitting a lemon-like shape, and with a typical size
of at least 10 µm. Given the heterogeneous nature of the primary neuronal cultures, the
unique shape of the SGNs helps in their identification amongst the other cells that were
present, including dissociated modiolar cultures (MC) such as the Schwann cells or
fibroblasts. The present study involved the analysis of SGNs that have been treated with
silica-coated GNRs (SiO2-GNRs) and silica-coated GNSs (SiO2-GNSs) using the combined
109
dark-field imaging and scattering microspectroscopy. Scattering spectra of SiO2-GNRs and
SiO2-GNSs associated with the neurons were acquired by microspectroscopy.
Figure 4.5 Dark-field images showing the SGNs (arrows) surrounded by other explanted
cells. Scale bars are 10 µm.
In a typical experiment, the nanorods within the SGNs (NR-SGNs) can be observed by
the colourful scattering that they exhibit. Figure 4.6(a) shows the dark-field image of a
SGN containing SiO2-GNRs. The same neuron was also captured by the CCD camera of
the spectrometer in imaging mode, which contains no true colour information (Figure
4.6(b)). Pseudo colour has been used to depict the associated nanorods which showed
greater intensity than the cell surface. Subsequently, the line of spectral acqusition was
positioned along the cross-section of interest, where the scattered light is collected into the
spectrometer via the microslit. The scattered light from each spot in the region of interest is
projected onto the slit and then dispersed along a row of pixels on the CCD, providing the
associated scattering spectra for further processing. The signals were normalized by the
average spectrum of the halogen white light and then subtrated from the normalized
spectrum of the SGN background (Eq. 4.1 and 4.2). Figure 4.6(c) shows the processed
scattering spectra acquired from the SGN along the acqusition line. The spectra exhibit
typical features of the scattering of GNRs, particularly the transverse and longitudinal SPR
peaks. The scattering peak at ~550 nm may be attributed to the transverse plasmon
110
resonance of the nanorods, together with the spherical gold nanoparticles that remain as a
by-product of the nanorod synthesis. Meanwhile, the broad maximum in the 780 to 850 nm
wavelength region is attributed to the longitudinal plasmon peak of the GNRs. Compared
with the typical longitudinal SPR peak of GNRs present on the glass surface (Figure 4.4), it
is noticeable that the peaks have broadened significantly, indicating some particle
agglomeration in the cultures. Particle agglomeration is likely to occur in the cells given the
pH-changing intracellular environment.462
Besides, particle agglomeration in the cells can
also occur when triggered by intracellular molecules such as glutathione.252
Other than
broadening, the SPR peaks also exhibit varying degrees of spectral shift, suggesting
changes in the surrounding environment that are reflected in the associated refractive index.
Subsequently, a series of scattering spectra of the nanorods was acquired from different
targets present in the primary cultures including SGNs randomly selected from the
coverslips. All of the spectra were normalized by the average halogen lamp source
spectrum using Eq. 4.1. Apart from the control SGNs that were not treated with the SiO2-
GNRs, it can be seen that all of the spectra exhibit transverse and longitudinal plasmon
peaks, regardless of the acquisition target, e.g., NR-SGNs, dissociated modiolar cultures
(MC) such as the Schwann cells or fibroblasts, and surface of the coverslip (Figure 4.7). In
general, it can be observed that the longitudinal peaks of the spectra fall in the wavelength
range from 775 to 790 nm, suggesting minor and variable spectral shifts from the initial
position of 780 nm. These observations suggest that the SiO2-GNRs remain reasonably
stable following incubation with the cultures.
111
Figure 4.6 Dark-field microspectroscopic analysis of NR-SGNs. (a) A dark-field image
and (b) the corresponding falsecolour image taken from the spectrometer CCD reveal the
presence of nanorods in the vicinity of the neuron. The dashed-line represents the cross-
section at which the spectra were aquired. (c) typical scattering spectra acquired from the
grating-dispersed light passing through the slit (inset) selected from three different points
along the cross-section of the cell (dashed-line in image (a) and (b)). The dotted line
indicates the initial 780 nm peak position of the nanorods.
112
Figure 4.7 Scattering spectra acquired from different targets; the dotted line indicates the
initial 780 nm longitudinal SPR peak position of the nanorods.
Meanwhile, Figure 4.8(a) presents the dark-field light scattering image of a SGN in the
culture that was treated with the SiO2-NSs (NS-SGNs). Although the scattered light of
GNSs in the vicinity of the SGNs was difficult to visualize directly from the dark-field
images, the microspectrometer was sensitive enough to detect the scattering signals from
the associated neurons. Indeed, the spectra obtained at various points along the acquisition
line from the NS-SGNs also exhibit a broad SPR band with the maxima appearing in the
wavelength range between 550 and 650 nm (Figure 4.8(b)). Although the general shape of
the spectra still resembles the typical SPR of GNSs, some broadening of the SPR peaks is
apparent. Similar to the GNRs, particle agglomeration may have occurred when the SiO2-
NSs were added to the cultures. It is worth noting that the higher noise level in the 750 to
1000 nm wavelength region can be attributed to interference from the cellular components,
as well as the influence of reduced CCD sensitivity in this range. From the dark-field light
113
scattering images of SGNs (Figure 4.6(a) and Figure 4.8(a)), GNRs associated with the
neurons scattered light more intensely than GNSs. This could mainly be attributed to the
higher scattering cross-section of the rods compared to the spheres due to the volumetric
radiative capacity.397
Figure 4.8 Dark-field microspectroscopic analysis of NS-SGNs. (a) Dark-field image of an
NS-SGN, the dashed-line represents the cross-section at which the spectra were aquired. (b)
Scattering spectra acquired from the slit pattern (inset) and selected from three points along
the cross-section of the cell (dashed-line in image (a)). The dotted line indicates the initial
550 nm peak position of the nanospheres.
114
4.4.3 Dark-field Light Scattering (NG108-15 Cell Line)
In vitro dark-field imaging takes advantage of the colourful light scattering from the
gold nanoparticles, which are highly distinct in the cells. Using dark-field microscopy, this
section examines the qualitative effects of gold nanorod surface coatings on the interactions
with the NG108-15 neuronal cells. Note that the quantitative cytotoxic effects of GNRs,
PSS/GNRs, SiO2-GNRs on NG108-15s have previously been reported, based on a range of
cell counting assays.232
While that study established that there were no significant long term
(up to 4 days) toxic effects from any of the particles, but it did not look at the detailed
distribution and uptake of GNRs in the cells. Nanorods with surface coatings including
polydopamine/silica (PDA/SiO2-GNRs), silica (SiO2-GNRs), PSS (PSS/GNRs), CTAB
(bare nanorods), and PEG (mPEG/GNRs) were used in this study. The preparation and
characterization of these materials have been discussed in detail in Chapter 3.
Firstly, PDA contains abundant functional groups and is self-polymerised onto the silica
surface of the nanorods. As shown in Section 3.3.5, the presence of PDA confers positive
charge to the nanorods following PDA overcoating on the silica surface. Figures 4.9(a) and
(b) show the typical bright-field and dark-field images of the NG108-15 cells containing
PDA/SiO2-GNRs, respectively. From the dark-field image, it can be observed that the
scattering of the particles has the same focal plane as the cells, indicating that the majority
of the NG108-15 cells have taken up the nanorods during the incubation. This is confirmed
by the colourful bright scattering from the particles within the cells, rather than confined to
the cell surface, in which case the scattering of the particles could form a ring around the
cell (same focal plane) or could be out of focus (top of cell membrane).410
It is noticeable
that the nanorods are mainly localised in the cytoplasm and even nucleus of the cells. As
can be seen from the dark-field image in Figure 4.10, some of the cells accumulated a large
number of nanoparticles inside the nucleus. The PDA layer contains abundant functional
groups, including indoles,411
which confer a positive surface potential. There could also be
some minor functional groups such as the hydroxyls that may offer a negative charge to the
nanorods. This combination of positive and negative charges on the same nanoparticle may
have helped in the cellular and organelle uptake, given the increased interaction
opportunities with the charged cell membranes.412
115
On the other hand, without PDA, SiO2-GNRs did not seem to accumulate in the nucleus
of the cells after the same incubation time (Figure 4.11). However, it can be observed that
the nanorods were also internalized by the cells and are localised mainly in the cell
cytoplasm. However, from the dark-field images the overall difference is not apparent with
regards to nanorods internalization. Indeed, with regard to cytoplasmic internalization of
the nanorods, from the dark-field images there is no apparent difference between the
PDA/SiO2-GNRs and the silica-shell particles without PDA. As opposed to PDA coating,
SiO2-GNRs possess a negative surface charge as reported in Section 3.3.4.
The present study also used dark-field imaging to examine NG108-15 cells treated with
negatively-charged PSS/GNRs and the positively-charged bare nanorods. From the dark-
field scattering images, the results were compared with regards to nanorod internalization.
Figures 4.12(a) and (b) present dark-field images of cells containing PSS/GNRs and bare
GNRs, respectively. The scattering of the particles has the same focal plane as the cells,
suggesting the near proximity of the nanorods to the cells. The pattern of association
between the cells and PSS/GNRs and bare GNRs is rather similar i.e., the nanorods are
internalized and evenly dispersed in the cytoplasm, but display only a minor internalization
in the nucleus. Additionally, the light scattering from the nanorods within the cells is less
bright compared to the cases when either PDA/SiO2-GNRs or SiO2-GNRs were used. This
may suggest i) particle agglomeration in the cultures which resulted in less bioavailability
due to particles lost during the washing step, and/or ii) the presence of silica coating on the
nanorods which slightly enhanced their scattering efficiency. As can be seen from the
FDTD calculations (Section 3.3.5), the scattering cross-section of the nanorods increased
slightly after the silica coating.
116
Figure 4.9 Typical bright-field (a) and dark-field (b) images of NG108-15 cells incubated
with PDA/SiO2-GNRs.
Figure 4.10 Representative dark-field image showing internalization of PDA/SiO2-GNRs
in NG108-15 cell nuclei.
117
Figure 4.11 The dark-field image of NG108-15 cells showing scattering from the SiO2-
GNRs.
Figure 4.12 Dark-field images of NG108-15 cells containing (a) PSS-coated GNRs and (b)
bare GNRs.
Subsequently, nanorods with near neutral surface charge were also examined. The PEG
coating on the surface confers very little surface charge to the nanorods, with a zeta-
potential of only ~1.2 (see Section 3.3.3). Figure 4.13(a) and (b) shows the dark-field
images of untreated NG108-15 cells and cells treated with the mPEG/GNRs, respectively.
118
It can be observed that the nanorods formed particle aggregates which appear as bright
yellowish clumps. However, from the dark-field image, no apparent particle light scattering
can be observed from within the cells cultured with mPEG/GNRs (Figure 4.13(b)). This
could be due to reduced non-specific binding to cellular membranes as a result of the near
neutral surface charge of the nanorods shielded by the PEG chains. This observation is
consistent with other similar studies.213, 413
Figure 4.13 Dark-field images of NG108-15 cells (a) without (b) with mPEG/GNRs. The
particle clumps are pointed out by the arrows.
Although the presence of nanorods can be tracked by analysing the dark-field image of
the cells, one cannot conclusively derive information about the stability of the particles.
Once again the spectral content of the scattering signals of the nanoparticles within the cells
can be useful in this regard. Figure 4.14 presents the scattering spectra acquired from the
NG108-15 cells treated with nanorods containing the various different surface coatings:
PDA/SiO2-GNRs, SiO2-GNRs, PSS/GNRs, mPEG/GNRs, and bare nanorods. All of the
spectra were normalized by the average spectrum (halogen lamp source and control cell
background) (Eq. 4.3). Each spectrum represents the average signals acquired from at least
3 cells. In general, the average scattering spectra acquired from the cells resemble the shape
of the typical nanorod spectral profiles, i.e. containing transverse and longitudinal SPR
peaks. The scattering peak at ~550 nm may be attributed to the transverse plasmon
119
resonance of the nanorods, together with the spherical gold nanoparticles that remain as a
by-product of the nanorods synthesis. The maximum in the 780 to 800 nm wavelength
region is attributed to the longitudinal plasmon peak of the GNRs. All of the nanorod
samples added into the NG108-15 cells have an initial longitudinal SPR at ~780 nm. As can
be seen from the scattering spectra, varying degrees of peak red-shifts and broadening have
occurred as a result of the nanorod interaction with the cells. The most notable changes can
be observed for the PSS/GNRs and bare GNRs. There is a 15 nm redshift in the major
peak; however the typical spectral characteristics of the GNRs have remained, suggesting
changes to the local environment of the nanorods within the cells due to the different
refractive indexes.414 No apparent redshift can be detected for spectra of PDA/SiO2-GNRs
and SiO2-GNRs acquired from the NG108-15 cells, possibly due to the presence of silica
shell and the sensitivity to the changing refractive indexes had reduced.
Figure 4.14 Average scattering acquired from NG108-15 cells for GNRs with different
surface coatings. The dotted line indicates the initial 780 nm peak position of the nanorods
120
4.5 Discussion
For the purpose of dark-field light scattering analysis, the immobilisation of GNSs and
nanorods onto the PDA-modified surface by substrate immersion was intended to minimize
agglomeration of the nanoparticles. This may help in the formation of more isolated
nanoparticles on the surface compared to the drop-cast method in which particles are
packed closely after solvent evaporation.415
When particle-particle distance is maximized,
the scattering peaks of the surface immobilized gold nanoparticles appear narrow, which
may be used to provide a spectral reference library. The PDA underwent spontaneous
oxidative polymerization in a dopamine solution at alkaline pH following a simple
immersion and formed a surface-adherent PDA film on the glass surface,395
hence retaining
the nanoparticles efficiently by adhesion. Despite subsequent immersion in the index-
matching oil environment, most of the nanoparticles were retained on the PDA surfaces,
allowing further analyses by dark-field light scattering.
Many studies involving analysis of cells and gold nanoparticles have made use of dark-
field imaging,128, 129, 146, 259, 291, 416
in which the comparisons are often made between the
cells with and without nanoparticles or time-dependent cellular uptake studies are
performed. Dark-field light scattering is a relatively simple procedure and does not require
the fluorescent dyes or further staining processes. Besides, it is known that the scattering
light of gold nanoparticles is much brighter than the fluorescent dyes.120
However, care
must be taken to select appropriate controls for background subtraction and normalization
against the source spectrum.
SGNs have contributed significant research results in several studies including
conventional electrical stimulation,417
long-wavelength infrared stimulation,4 and
pharmacophysiology.418
In Chapter 5, SGNs are used throughout the investigation of
photothermal stimulation assisted by silica-coated GNRs and GNSs. Hence, it was
advantageous to analyse the interaction and association of the nanoparticles with the SGNs
in these heterogeneous populations of primary neuronal cultures using dark-field light
scattering and microspectroscopy.
From the spectral analysis, all of the SGNs examined have shown positive spectral
characteristics of GNRs or GNSs, suggesting that these neurons have received
121
nanoparticles in a relatively stable and functional form. The stability of the gold core has
been enhanced by silica coating, which offers a barrier against particle aggregation.183, 212
This can be observed from the SPR peaks that have not diminished after incubation with
the cultures. The change in the spectral properties due to agglomeration is typically
manifested as broadening and varying degrees of spectral shift and is most apparent when
two or more bare GNRs are in close proximity to each other (< 2 nm).409, 419
Since this does
not necessarily apply here given the silica coating, the spectral shifts and broadening
observed in this work have been attributed to the change in the local environment,414
primarily due to the interaction of the nanoparticles with cellular components that have
different refractive indexes, for instance, membrane (1.37), cytoplasm and organelles (1.38
- 1.41).414
This is not surprising, given that silica-coated gold nanorods have previously
been used to detect molecules with high sensitivity based on changes in refractive index.394
The present microspectroscopic study is significant in that it has confirmed the presence
of functional GNRs and nanospheres within the SGNs. This is significant in the context of
Chapter 5, which is concerned with the photothermal effects of 780 nm laser illumination
on the NR-SGNs. While the presence of SiO2-NSs in the vicinity of SGNs is also validated
herein, the nanoparticles are off-resonance at the laser wavelength at 780 nm and therefore
they provide a useful control case to test whether any effects are due to the SPR or simply
due to the presence of the nanoparticles.
It is noteworthy that the nanoparticles used in this study are non-targeted, or non-cell-
specific, as they are lacking conjugation with cell-specific ligands. Hence, there could be
two possible mechanisms to account for the non-specific cellular uptake; particle-cell
interactions, and/or cell migration. The former can be explained by protein adsorption onto
the nanoparticles, which facilitates interaction with the cells via receptor-mediated
processes.249 The nanoparticles may form nanoparticle-protein coronas around the cells due
to the proteins present in the cellular culture media such as the serum proteins.253, 420, 421
In
the case of the SGNs, it would appear that the protein-coated nanoparticles (protein coronas
around the nanoparticles) are taken up by the cells. The cell migration process, in which
sedimented nanoparticles are “vacuumed” into the cells,147
is unlikely in the case of SGNs
given the nature of primary rat neuronal cultures422 which slowly proliferate ex vivo and
122
experience limited cell migration. On the other hand, nanoparticle uptake in the case of
immortal cell lines like NG108-15 may involve one or both of these mechanisms.147, 253
The living cell membrane is predominantly negatively charged due to the phospholipids
bilayer structure and the negatively charged head groups.423, 424
Additionally, the cell
membranes contain proteins that are made up of amino acids that are usually negative at
physiological pH. Therefore cellular uptake can be influenced by the surface charge of the
nanoparticles. Previous reports have established that the surface chemistry of nanoparticles
can have a great impact on nanoparticle uptake in cell lines.182, 243, 412
For instance,
positively-charged amine group-carrying nanoparticles have been observed to accumulate
in cells to a much greater degree than non-ionic PEG-coated nanoparticles.425 The NG108-
15 cell study described herein seems to have followed a similar trend, in which the PDA
coating assisted the internalization and accumulation of nanorods in the cytoplasm and
nucleus, while fewer mPEG/GNRs were associated with the cells. The PDA layer contains
abundant functional groups that provide mainly positive charge on the nanoparticle and
there are also examples where these properties of PDA have provided a strong adhering
affinity to some proteins.386, 426-428
On the other hand, near neutral PEG coating resulted in a
less efficient uptake of mPEG/GNRs by the cells. PEG-coated nanoparticles are also well-
known for their long circulation half-life in vivo due to reduced unspecific protein
binding.196, 215, 429
The present dark-field light scattering analysis of SiO2-GNRs, PSS/GNRs and bare
GNRs in the NG108-15 cell line is also significant in that nanorods have been traced inside
the NG108-15 cell cytoplasm. The results were also validated by confocal microscopic
analysis232, 430
in which Rhodamine-B-labeled nanorods were observed inside the cell
cytoplasm. Serum proteins in the culture medium may adsorb onto these nanorods, forming
nanoparticle-protein coronas around the cells and thus helping with the particle
internalization.421
Additionally, nanoparticle-protein interactions can change the size,
shape, and aggregation state of the nanoparticles, depending on the surface coatings, which
may explain the observed redshifts and broadening of the SPR peaks. The small degree of
spectral change does not appear to affect the overall photothermal function of the nanorods,
as these nanorods with different surface chemistries have also been applied to the
123
investigation of neurite outgrowth and intracellular calcium signaling in NG108-15s under
the influence of laser irradiation, with results published in several reports.232, 342, 388
In the present study, although the internalization of the nanorods was confirmed based
on the evidence of imaging the light scattering of the nanoparticles in the same focal plane
as the cells, the nanoparticle distribution may be further clarified in future work by
performing a 3-D reconstruction of multiple focal planes.410, 431
This may assist in
understanding of the overall distribution of the nanoparticles within the cell. The
internalized nanoparticles can also be visualized and tracked by high resolution TEM.416
In
addition, the nanoparticle uptake can also be analyzed quantitatively by inductively coupled
plasma (ICP), in which cells containing gold nanoparticles are digested with aqua regia and
the concentration of gold ions measured.182, 354, 432, 433
4.6 Conclusion
In this chapter, the feasibility of using both dark-field light scattering and
microspectroscopy to monitor gold nanoparticles in contact with primary neuronal cultures
of SGNs and NG108-15 immortal cell lines has been demonstrated. The dark-field light
scattering microscopy provided a straightforward means to identify interactions between
the gold nanoparticles and the cells. Additionally, microspectroscopic analysis of the
scattering has provided information pertinent to the stability state of the nanoparticles in the
cells. The microspectroscopic analysis revealed typical spectral characteristic of GNRs and
nanospheres from NR-SGNs and NS-SGNs in the heterogeneous neuronal cultures,
respectively, and the spectra showed matching profiles with the spectral library collected
from stably immobilized gold nanoparticles on PDA/glass surfaces. From the in vitro dark-
field imaging, the extent to which the nanorod surface chemistry affects average particle
uptake in the NG108-15 cell follows the sequence PDA/SiO2 >SiO2 >PSS ≥CTAB >PEG.
From the microspectroscopic analysis, apart from the mPEG/GNR-treated cells, all other
cells showed typical spectral characteristics of GNRs after treatment with nanorods of
different coatings. Nevertheless, minor spectral shifts were detected on interactions with the
cells. These studies have contributed to an improved understanding of nanoparticle-cell
interactions and will support the future development of this thesis.
124
125
Chapter 5: Photothermal Stimulation of Spiral Ganglion
Neurons
5.1 Declaration for Chapter 5
Part of the results presented in this chapter has been published as:
• J. Yong, K. Needham, W.G.A. Brown, B.A. Nayagam, A. Yu, S.L. McArthur and P.R.
Stoddart. “Gold nanorods-assisted near-infrared stimulation of primary auditory
neurons”, Advanced Healthcare Materials, 2014.
5.2 Introduction
Millions of people worldwide434
suffer from hearing and vision loss which may be
caused by birth defects, aging, trauma, and certain other common degenerative diseases.
Neural prostheses offer artificial means to restore hearing and vision and have come closer
to reality during the past few decades. Implantable neural stimulators such as cochlear
implants (bionic ear) and retinal prostheses have gained significant attention and have
recently been approved by the U.S. Food and Drug Administration (FDA). The implants
rely on electrodes to interface with the inactive nerve cells that normally transmit the
sensory input, and generate electric currents to stimulate action potentials in the cells. An
action potential is important in cell-to-cell communication and is an electrical signal that
originates from the cell body and propagates along the nerve axon to dendrites of another
cell or to an effector cell. Electrical stimulation is currently the gold standard in neural
activation. Research into the electrical nerve stimulation has been going on for decades,
with the earliest clinical experiments being performed in the 1960s.435 However, there are
inherent limitations, such as a lack of spatial precision, electrical stimulation artifact and
the electrodes may create an inflammatory response in the nerve and electrodes.6
As an alternative approach to electrical stimulation, infrared neural stimulation uses
short pulses of infrared (IR) light to stimulate neurons. The light can be delivered to the site
126
of interest through an optical fiber, thus providing localized stimulation without any need
for direct contact with the target neurons. This may pave the way for a future generation of
neural prostheses. The thermal transient mediated by water absorption of the light is known
to be a critical factor contributing to IR neural stimulation.9, 10
The stimulation uses an
optical source in the IR region (typically ca. 1450 to 2200 nm) because of the overtone
absorption by water in this range. Water absorbs the IR light and upon the release of heat,
two secondary processes leading to excitation have recently been proposed: (i) temperature-
induced reversible changes in membrane capacitance due to perturbation in the distribution
of ions adjacent to the cell membrane,10
and (ii) activation of temperature-sensitive ion
channels.11
Although IR neural stimulation offers great potential for practical use in medical
applications, there are also potential limitations for interfacing with deep or three-
dimensional structures, for example in the retina or brain. Such applications may require
the use of light at wavelengths that can pass through unaffected tissue with minimal
intrinsic absorption and scattering. In addition, thick absorbing or scattering layers above
the target structure may reduce the efficiency of neural stimulation.5 Relatively high power
lasers are required in order to compensate for the lack of penetration depth, thus increasing
the risk of thermal damage at the surface. In comparison, the near-IR region between 650
and 900 nm is known as the biological transparency window, where minimal absorption of
light by water content in tissue is taking place.12
Low-power diode lasers with lower energy
consumption are able to penetrate more deeply into cells and tissue.268
Combined with the use of nanoparticles, near-IR laser light can open up new
opportunities for deep tissue treatment. Given the importance of thermal transients in
triggering the neural response, recent work has demonstrated that extrinsic absorbers
responding to both optical297, 298
and magnetic340
sources can be used to excite neurons.
Amongst absorbing materials, gold nanorods (GNRs) can be tailored to strongly absorb
laser light in a relatively narrow NIR range by varying the size, aspect ratio and surface
dielectric properties of the nanoparticles.397
Upon irradiation, GNRs can produce rapid
heating due to photon-to-heat energy conversion. The photothermal transduction efficiency
of GNRs is among the highest compared with other types of gold nanoparticles.69
The
127
photothermal capability of GNRs has been used for several purposes (see Section 2.5.3).
The feasibility of applying the absorbing material in a biological setting is also important.
GNRs have the advantage of widespread use in biology, which has led to a deep body of
knowledge about biocompatibility and for applications in labelling, delivery and sensing.436
In two recent studies, laser-exposed GNRs were known to increase neurite length and
generate calcium transients in the NG108-15 neural cell line.232, 342
The findings strongly
suggested that the cells respond to heat generation associated with the plasmon resonance
as induced by in the GNRs by the NIR laser. However, there were no direct measurements
of cell electrical activity and no direct evidence for the occurrence of action potentials in
that work. Cell electrical activity can be measured in vitro by the whole-cell patch-clamp
recording technique (see Section 2.6).437
The technique has been widely adopted in the
study of infrared neural stimulation,10, 11, 320
in which enhanced electrophysiological
phenomena such as electrical currents carried by ions through transmembrane ion channels,
were described in individual cells. The research reported herein will make use of the patch-
clamp technique to study any increase in the electrical activity of neuronal cells as a result
of photothermal stimulation using a NIR laser both with and without GNRs.
This chapter firstly explores the feasibility of laser-induced heating of bulk aqueous
GNRs and compares it with that of water, using the NIR laser source. Having understood
the photothermal feasibility of GNRs, this chapter further addresses the major hypothesis,
i.e. the photothermal stimulation of rat spiral ganglion neurons (SGNs) cultured with silica-
coated gold nanorods (SiO2-GNRs) and illuminated by a NIR diode laser emitting at 780
nm. As discussed in Chapter 3, the GNRs were tuned to absorb maximally at the incident
laser wavelength via i) optimization of aspect ratio, and ii) silica coating that changes the
surface dielectric properties of the nanorods. Meanwhile, SGNs were cultured directly from
early postnatal rats and were used as the in vitro model. SGNs are auditory neurons whose
cells bodies lie in the spiral ganglion and are strung along the auditory portion of the
cochlea.438
While this model has been extensively studied in vitro,5, 418, 439, 440
the literature
regarding SGNs and nanoparticles is rather limited. Additionally, unlike immortalized
neuronal cell lines, primary neuronal cultures have not been modified by any means, and
therefore more closely mimic the natural state of cells in vivo.441
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In the present study, SiO2-GNRs are used as photoabsorbers and nanoheaters, which
could provide transient heating to the neurons upon exposure to the NIR pulsed laser. In
Chapter 3 and 4, it is learnt that GNRs coated in silica are more stable against particle
agglomeration in different conditions (i.e. in solvents and cell culture). Therefore the choice
of using SiO2-GNRs is appropriate for the current neuronal cells and 780 nm laser study.
Using the same laser source, the photothermal stimulation effects of silica-coated gold
nanospheres (SiO2-GNSs) on SGNs are also examined. In Chapter 4, dark-field
microspectroscopic analysis was used and has successfully identified the presence of SiO2-
GNRs and SiO2-GNSs inside the SGNs. The major difference between the two types of
nanoparticles is the degree to which they absorb 780 nm laser light; nanorods have a higher
laser absorption than nanospheres because of their longitudinal SPR band that match the
incident laser wavelength. In addition, the photothermal stimulation effects of 780 nm
exposure of SGNs without nanoparticle treatment are also examined. While gold
nanospheres (GNSs) are off-resonance at the incident laser wavelength of 780 nm, water
also has a relatively low absorption coefficient at the incident NIR wavelength. Therefore
neither of these absorbers were expected to generate sufficient heat to stimulate the SGNs.
Whole-cell patch-clamp electrophysiology was utilized to conduct direct measurements of
any electrical activity from the individual neuronal cells. Variable laser pulse lengths were
used in order to understand the correlation between the laser pulse energy and the cell
electrical activity. The localised temperature increase associated with the irradiated
nanorods in the vicinity of SGNs was also measured by means of an open patch electrode.
5.3 Materials and Methods
5.3.1 NIR laser – 780 nm
A class IIIB single mode fiber-coupled diode laser (OptoTech, Melbourne, Australia)
with the output λ = 780 ± 5 nm and variable peak power (Max ≥ 100 mW) was used
throughout the experiments. The optical fiber (SMF-28, Corning) has a numerical aperture
(NA) of 0.14, and a fiber diameter of 125 µm (8.2 µm core). Laser power was measured by
a handheld laser power meter integrated with a silicone sensor (LaserCheck, Coherent
129
Scientific, South Australia). Where appropriate, laser pulses were automatically controlled
manually by a function generator (TDS1002, Tektronix, United States)
5.3.2 Laser Heating of Bulk Nanorod Solutions
Suspensions of bare GNRs and SiO2-GNRs used in this section were prepared as
described in Section 3.2. One hundred µL of the bare GNRs or SiO2-GNRs suspension
(optical density at 780 nm ~1.8, equivalent to ~33 ppm and ~41 ppm of gold ions,
respectively, see Appendix I) was added to a narrow-bottom tube containing a K-type
thermocouple. The laser fibre was stably positioned 2.4 mm above the sample for
continuous laser irradiation and care was taken to avoid direct interaction of the laser beam
with the thermocouple. The laser beam area was calculated using the following formula:
� = tan������ �� � (Eq. 5.1)
where � is the beam radius and � is the distance between the fiber tip and the sample.
During the laser irradiation, temperature changes were logged into a computer with the
software PicoLog v5 (Pico Technology Ltd.).
5.3.3 Culture Methods
This protocol involved animals and was approved by the Animal Research and Ethics
Committee of the Royal Victorian Eye and Ear Hospital, Victoria, Australia and was
carried out by Dr. Karina Needham. SGN cultures were prepared from post-natal day four
to seven Wistar rat pups as described previously.418, 442 Briefly, animals were anaesthetized
and following a craniotomy, the bulla was dissected from the temporal bone under sterile
conditions and placed into chilled Neurobasal media (NBM, Invitrogen) containing: N2 and
B27 supplements (Invitrogen), l-glutamine (Invitrogen), Penicillin/Streptomycin and 4.5
g/L d-glucose (Invitrogen). The cochleae were then gently isolated from the bulla and the
organ of Corti carefully removed from the modiolus. All modioli were digested in a sterile
solution of Ca2+
and Mg2+
-free Hank’s balanced salt solution (Gibco), containing 0.025%
trypsin (Calbiochem) and 0.001% DNase I (Roche) and incubated at 37ºC for 10 min.
Subsequently, enzymatic digestion was terminated by addition of 1 mL fetal calf serum
(FCS) (ThermoTrace). Modioli were centrifuged at 2000 rpm for 10 min and after which,
130
the supernatant was discarded and the digested tissue was resuspended in MEM
comprising: penicillin/streptomycin, non-essential amino acids, 1% FCS and DNase I.
Digested cochleae were gently triturated using a series of sequentially smaller gauge
needles (18Ge - 23G), then centrifuged at 2000 rpm for 10 min. The supernatant was
discarded and the cell pellet was resuspended in NBM. Subsequently, the cell suspension
was plated onto glass coverslips pre-coated with poly-ornithine (500 mg/mL; Sigma) and
mouse laminin (0.01 mg/mL; Invitrogen). BDNF and NT3 (Millipore) were used at a final
concentration of either 10 ng/mL or 50 ng/mL each in NT cultures. Dissociated neuronal
cultures were incubated at 37ºC supplied with 10% CO2 for up to 3 days. The medium was
replenished daily.
5.3.4 Laser Stimulation and in vitro Electrophysiology
Whole-cell patch-clamp recordings were made from one to three days in vitro cultures.
For nanoparticle studies, the optical density (OD) of the nanoparticle samples (SiO2-GNRs
and SiO2-GNSs) was adjusted to be equivalent at their respective absorption maxima (λmax):
780 nm for SiO2-GNRs and 525 nm for SiO2-GNSs. Briefly, concentrated nanoparticle
samples (for sample preparation, see Section 3.2) were adjusted to an optical density of
~0.18 with culture medium and added to the neuronal cultures for overnight incubation
(~15 to 17 hr). Given the optical density (OD525 and OD780) of ~0.18, the concentration of
gold ions as measured by atomic absorption spectroscopy (AAS) were ~13 ppm and ~7
ppm for SiO2-GNRs and SiO2-GNSs, respectively (see Appendix I). Cultures incubated
with SiO2-GNRs and SiO2-GNSs are designated as NR-SGNs, NS-SGNs, respectively. On
the day of recordings, glass coverslips with cultured neurons were transferred to the
recording chamber of a microscope (AxioExaminer D1, CarlZeiss Pty Ltd, Germany) fitted
with a 40× water-immersion objective lens.
During the laser stimulation and patch-clamp recordings, cultures were superfused with
an external solution containing the following composition: 137 mM NaCl, 5 mM KCl, 10
mM HEPES, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose (pH 7.4; 300-305 mOsmol/kg).
Superfusion of the cultures was administered via a gravity-fed system. Recording
131
microelectrodes (borosilicate, 1.0 mm outer diameter, 0.58 mm inner diameter, 75 mm
length, 2-6 MΩ) were filled with an internal solution containing: 115 mM K-gluconate, 10
mM HEPES, 7 mM KCl, 0.05 mM EGTA, 2 mM Na2ATP, 2 mM MgATP, 0.5 mM
Na2GTP (pH 7.3; 290-295 mOsmol/kg). All chemicals were purchased from Sigma-Aldrich
(Sydney, Australia) unless otherwise indicated.
Laser light from the laser diode was delivered via a 125 µm (8.2 µm core) diameter
optical fiber (SMF-28, Corning) which was aligned with the target cell body using a
micromanipulator. Peak laser power was kept constant at 90 mW, with pulse lengths
controlled by a function generator and triggered by the patch-clamp data acquisition system
(Digidata 1440A, Molecular Devices). Whole-cell patch-clamp recordings were made at
room temperature (~21-25 °C) and signals were recorded with a Multiclamp 700B
amplifier (Molecular Devices, Sunnyvale, CA, USA) and synchronised with AxoGraph X
analysis software (AxoGraph Scientific, Sydney, Australia).
Neurons were visually identified by a phase-bright, round soma (diameter of ~10-20
µm) and prominent nucleus. Records were digitized at 50 kHz and filtered at 10 kHz. Series
resistance was routinely compensated online (up to 70%), and in current clamp, pipette
capacitance neutralization and bridge balance were utilized to compensate errors due to
series resistance. Corrections for liquid junction potential (12.8 mV) were made offline
using JPCalcW (Prof P. H. Barry, Sydney).
5.3.5 In vitro Local Temperature Measurements
The temperature increase under laser exposure in the vicinity of neurons containing gold
nanorods was measured by recording the resistance of a calibrated borosilicate pipette.
Ohm’s law is applied:
� =�
� (Eq. 5.2)
where I is the current, V is the voltage, and R is the resistance. The micropipette was
positioned ~2 µm apart from the neurons and should provide an indication of heat
distribution from the surface of the neurons to the surrounding bath solution. Pipette
132
resistance was first calibrated following the method described by Yao et al.443
The pipette
was immersed in a bath solution similar in composition with the external solution used for
patch-clamp recordings. The bath solution was preheated to ~40°C using a TC-324B
temperature controller (Warner Instruments, MA, USA) and allowed to cool to room
temperature while the pipette resistance and the temperature were simultaneously recorded.
Pipette resistance was measured by applying a 5 mV pulse, while the bath temperature was
monitored with a K-type thermocouple. A linear calibration relationship was fitted to the
natural logarithm of pipette current versus the inverse of absolute temperature. This
calibrated relationship was used to obtain an estimate of the activation energy (Ea) from the
slope of the plot. Given Ea and the initial pipette current (I0) at ambient temperature (T0),
the temperature rise in the vicinity of the neurons was calculated from the relationship:443
� = � �
��−
�
���� � �
����
��
(Eq. 5.3)
Due to the relatively low signal-to-noise (S/N) ratio in open pipette recording, the raw
signals associated with the temperature information were affected by periodic noise from
the environment. Subsequently, the periodic noise was analysed using curve fitting analysis
software, TableCurve 2D v5.0 (Systat Software, CA, USA). The signals were then
subtracted from a sine wave generated from the sine function:
� = � + �� �����
+ �� (Eq. 5.4)
where a is the vertical shift, b is the amplitude, c is the horizontal shift , ��
is the period of
the function.
5.4 Results
5.4.1 Laser Heating of Water and Aqueous Gold Nanorods
To demonstrate the feasibility of laser-induced heating of bulk aqueous GNRs and
compares it with that of water, the experiment as depicted in Figure 5.1 was prepared. The
temperature effects of the nanorod suspensions and distilled water under continuous
133
exposure to the 780 nm laser can be observed and compared in this study. First, the distance
between the fiber tip and the sample surface was ~2.4 mm, therefore the laser spot size was
~0.36 mm2. Next, using the laser power of 90 mW, the irradiance used was calculated to be
25 W/cm2, although it must be noted that this is more representative of the peak irradiance,
rather than the average laser irradiance over the entire sample.
Figure 5.1. Scheme of the experimental setup for bulk heating of nanorod solution
It was first established that the continuous laser exposure contributed a relatively small
(ΔT ~1.6 °C) increase in the temperature when water was irradiated for 20 minutes (Figure
5.2(a)). Subsequently, it was demonstrated that the laser exposure was able to induce
heating in both of the nanorod solutions. The temperature increased significantly in bare
GNR and SiO2-GNR solutions during the first five minute and reached ~7 °C and 10.7 °C,
respectively (Figure 5.2(a)). After 20 minutes of laser exposure, the temperature increase
that was detected in bare GNR and SiO2-GNR solutions was 9 °C and 13.6 °C,
respectively.
Throughout the laser irradiation, it appears that two phases are associated with the
heating process: initial rapid heating, followed by a gradual heating until the laser is turned
off. The first phase of heating occurs during the first minute of irradiation, after which the
heating rate drops with time. This is because of heat conduction, when heat loss to the
surrounding environment by conduction is proportional to the temperature difference until a
balance between the rate of laser heating and heat transfer to the surrounding is reached.
Figure 5.2(b) shows the UV-vis spectra of SiO2-GNRs and bare GNRs used in this
134
experiment, where the peak maxima are shown to be close to the incident wavelength at
780 nm.
Comparing the bulk heating of bare GNRs and water, the laser exposure resulted in
about 7 times greater temperature increase in aqueous GNRs than in water. Meanwhile, the
laser resulted in about 5 °C more temperature increase in aqueous SiO2-GNRs than in bare
GNRs. The significant temperature difference between the nanorods and water is due to the
water being a weak absorber whereas GNRs have a large absorption coefficient at the
incident wavelength. On the other hand, the temperature difference between SiO2-GNRs
and bare GNRs could be explained by i) the SPR peak of GNRs which is slightly lower
than the SiO2-GNRs at 780 nm, and/or ii) the increase in the absorption cross section of
SiO2-GNRs due to the presence of silica shell. Finite difference time domain (FDTD)
calculations (Section 3.3.5) have shown that the amplitude of the extinction cross section
increases after the silica coating (15 nm silica shell). Besides, there is also a possibility that
the presence of silica shell may improve the thermal conductivity (from gold to surrounding
medium), resulting in a more efficient heat transfer through the silica layer.229
Figure 5.2 Comparison of laser-induced heating of water with different nanorod contents.
(a) The temperature elevation profiles of the nanorod solutions and water under laser
exposure are plotted as a function of irradiation time. (b) UV-vis absorption spectra of the
nanorod solutions used; the dashed-line indicates the laser wavelength at 780 nm.
135
5.4.2 Laser Stimulation and Whole-cell Patch-clamp Electrophysiology
The experimental setup for simultaneous laser irradiation and whole-cell patch-clamp
studies is depicted in Figure 5.3. The phase contrast micrograph in Figure 5.4 shows a
micropipette electrode patched neuron and the optical fibre that has been moved into
position for laser delivery. From the phase contrast microscope, SGNs in the heterogeneous
neural populations exhibited features such as a round cell body, phase-bright, round soma
(diameter of ~10-20 µm) and prominent nucleus. Using a micromanipulator, the patch
clamp micropipette was moved to the neuron of interest and pressed against the membrane.
Subsequently, suction was applied to assist in the formation of high resistance seal
(typically in the gigaohm scale). The high resistance seal was required in order to
electronically isolate the transmembrane currents with little competing noise, and also to
provide enhanced mechanical stability to the recording.437
Cultures of SGNs treated with the SiO2-GNRs or SiO2-GNSs are designated as NR-
SGNs and NS-SGNs, respectively, while the SGNs without nanoparticles are designated as
control SGNs. All of the neurons included in the patch-clamp study were first checked for
the ability to fire action potentials in response to a brief intracellular current test pulse.
Figure 5.5(a) presents an episode of the recordings showing a selected action potential fired
in a NR-SGN in response to depolarizing current injection. The mean action potential
amplitudes were 108 ± 3.2 mV (n = 23) across the recorded population. Additionally, the
underlying fast sodium current and sustained outward potassium currents were also
observed in voltage-clamp as shown in Figure 5.5(b). These physiological features are
typical of observations in functional SGNs.444
Although gold nanoparticles are generally
known to be non-toxic to cells, and silica coating can significantly improve the
biocompatibility,181 in some cases where a significantly higher particle dose (optical density
~ 0.5) was added to the SGN cultures, the SGNs did not exhibit the normal physiological
features. Additionally, the patch-clamp microelectrode was not able to establish a good seal
with the membrane in those cases. Therefore patch-clamp recording was not able to be
made from these neurons. These are indications of apoptotic cultures known to this study
and hence a more appropriate particle dose was chosen for study.
136
Figure 5.3 Schematic of the experimental setup for simultaneous laser stimulation and
whole-cell patch clamp recordings of a neuron.
Figure 5.4 Phase contrast micrograph showing a patched SGN (red arrow) with
microelectrode to the right and the optical fibre to the left of the image.
137
Figure 5.5 Whole-cell patch-clamp recording of a healthy neuron, showing a typical
response for: (a) a single action potential fired in response to a depolarizing current
injection, and (b) fast sodium currents during membrane depolarization in voltage-clamp
(arrow). The asterisk indicates the electrode artefact.
For laser irradiation, the peak power of the laser diode was kept constant at 90 mW. The
laser pulse lengths and the repetition rate were controlled with an external function
generator. The laser pulse lengths and the equivalent energy per pulse used in the current
photothermal stimulation study are summarised in Table 5.1.
Table 5.1 Variable laser pulse lengths and the equivalent energy per pulse used in the study
Pulse length Energy/pulse Pulse length Energy/pulse
25 µs 2.25 µJ 2.5 ms 0.225 mJ
50 µs 4.5 µJ 5 ms 0.45 mJ
100 µs 9 µJ 10 ms 0.9 mJ
250 µs 22.5 µJ 25 ms 2.25 mJ
500 µs 45 µJ 50 ms 4.5 mJ
1 ms 90 µJ
138
5.4.2.1 Voltage-clamp
Voltage-clamp measures the transmembrane current by holding the membrane
potential constant. In voltage-clamp recordings, NR-SGNs exhibited repeatable current
waveforms in response to laser illumination. Inward transmembrane currents at a
holding potential of –73 mV were consistently evoked on exposure to laser pulses.
Representative data in Figure 5.6 show changes in transmembrane current flow in
response to laser pulse lengths of 25 µs to 1 ms (corresponding to pulse energies of ~2
µJ to 90 µJ, respectively). The shape of the laser-induced current response can be seen
to vary as the pulse length was changed. The inward current commenced immediately at
the onset of the laser pulse and returned to the initial value with a small overshoot
(outward current) when the illumination was turned off. At the same holding potential,
the NS-SGNs and control SGNs were also exposed to the similar short laser pulses: 25
µs, 250 µs, and 1 ms. As shown in Figure 5.7, the laser did not evoke any significant
transmembrane current response in these SGNs. However, when longer laser pulses
were used, all of the neuron groups exhibit some level of current response. The extent to
which the laser pulses evoke transmembrane current varies; for example, in comparing
the peak response amplitude of the two types of nanoparticles, the 25 ms laser pulse
elicited ~1 pA in the NS-SGNs, while the same pulse length evoked >50 pA in the NR-
SGNs (Figure 5.8). Similarly, the laser irradiation evoked very little observable current
response in the control SGNs. It should be noted that in Figure 5.8c, slowly inactivating
outward current is not observed as opposed to Figure 5.6. Considering the nature of
primary cultures, this could be due to cell-to-cell variations (e.g. ion channels).
The enhanced cell electrical activity upon laser exposure can be reflected by the
increase in transmembrane current, whereby the NR-SGNs showed the dominant effect.
Figure 5.9 presents the total laser-induced charge generated by the NR-SGNs (n = 12),
NS-SGNs (n = 6), and control SGNs (n = 5) in response to multiple laser pulses of
various durations. The total charge was calculated from the average area under the
current response curves of the voltage clamp recordings. Higher total charge
corresponds to a larger peak current response from the neurons and/or a longer duration
current flow. To a first approximation, the laser-induced charge for the NR-SGNs
appears to increase linearly with pulse duration (R2 = 0.988). The figure confirms that
139
the NR-SGNs produce a much larger transmembrane current response than either the
NS-SGNs or the control SGNs.
Cu
rren
t (p
A)
1 ms
500 µs
250 µs
100 µs
50 µs
25 µs
100 µs
50
0
-50
-100
-150
Figure 5.6 Averaged voltage-clamp data for a typical neuron in response to laser pulses
of different duration. Dashed-line indicates the onset of the laser. All neurons were held
at –73 mV.
Figure 5.7 Averaged voltage-clamp data for NS-SGNs and control SGNs. (a) a NS-
SGN in response to laser pulses of different pulse duration (from top to bottom: 25 µs,
140
250 µs, 1 ms), and (b) a control SGN under similar conditions to (a). Red bars indicate
the timing and duration of laser pulses. All neurons were held at –73 mV.
Figure 5.8 Comparison of typical transmembrane currents elicited by 25 ms laser pulses
(red traces). Raw data of voltage-clamp recordings from (a) control SGN, (b) NS-SGN,
and (c) NR-SGN. All neurons were held at –73 mV. Note that the pulse repetition rate
was not observed to have any effect on the current response, at the repetition rates
shown here.
141
Figure 5.9 Dependence of the laser-induced charge on laser pulse duration for the
analysed neurons.
5.4.2.2 Current-clamp
Current-clamp applies a known constant or time-varying current and measures the
change in membrane potential caused by the applied current. In current-clamp
configuration, voltage responses could be clearly evoked in NR-SGNs subjected to laser
illumination, whereas no significant change was observed in the NS-SGNs and control
neurons. Subthreshold depolarization of the membrane potential was observed in the
NR-SGNs when short laser pulses (< 25 ms) were applied. Additionally, in the NR-
SGNs analyzed, laser pulse lengths of 25 ms successfully evoked action potentials.
Representative data in Figure 5.10 show an example of subthreshold potentials of a NR-
SGN induced by 1 ms and 10 ms laser pulses, together with an action potential elicited
by a single laser pulse of 25 ms. It was observed that the membrane depolarization
commenced with the onset of the laser illumination. In addition, increasing pulse
duration consistently increased the depolarized membrane potential. Pulse lengths of 25
ms may allow the local depolarization to exceed the threshold potential and thereby
142
generate an action potential. Figure 5.11 presents a typical instance where action
potentials in the NR-SGNs were fired in response to pulsed laser exposure. It can be
observed that the action potentials are synchronized closely with the laser pulses at a
repetition rate of ~6 Hz. On several occasions, an extended laser pulse duration was
also investigated to examine the effect of longer laser exposures on the firing rate. A
laser pulse lasting ~1 s was applied to NR-SGNs and the neurons were observed to elicit
sustained action-potential trains. Figure 5.12 shows an example of multiple spikes that
were fired in a NR-SGN at random intervals throughout the pulse. In this data, the
minimum time spacing between multiple spikes is ~50 ms, suggesting ~20 Hz
maximum firing rate under current laser conditions. For the system to work practically
in neural stimulation, higher peak powers could potentially improve maximum firing
rates, for example, cochlear implants typically require firing rates of hundreds of Hz.445
Figure 5.10 Current-clamped recordings of an NR-SGN showing subthreshold
membrane potentials (black and blue traces) and an action potential (red trace), evoked
in response to 1, 10, and 25 ms laser pulses, respectively. Colour bars indicate the onset
and duration of laser pulses.
143
Figure 5.11 Raw data of current-clamp recording showing action potentials fired in a
SGN in response to 25 ms laser pulses. Red trace and grey shadings indicate the timing
and duration of laser pulses. Laser pulses were preceded by a brief intracellular current
test pulse to evoke an action potential (asterisk).
Figure 5.12 Multiple firing evoked under continuous laser pulse. Red trace indicates the
timing and duration of laser pulse. Laser pulses were preceded by a brief intracellular
current test pulse to evoke an action potential (asterisk).
144
5.4.3 Local Temperature Measurements
From the results gathered during voltage- and current-clamp recording, it is
understood that the significantly enhanced electrical activity of the NR-SGNs is
dependent on the laser pulse length used, whereas a significantly lower electrical
response can be observed in the control SGNs and NS-SGNs under the same laser
conditions. Therefore it is postulated that this stimulation is due to the localized heating
caused by resonant absorption in the nanorods. The observation that the electrical
activity of the NR-SGNs is dependent on the laser pulse length is consistent with this
hypothesis, as longer laser pulses would be expected to generate larger temperature
changes. Such a correlation was subsequently validated by measuring the local
temperature change near the surface of NR-SGNs during laser illumination. The
temperature rise was detected and measured by using an open patch pipette. Resistance
variation as a result of temperature change is reflected by an increase in current flow
through the open pipette and was expected to correlate with any laser-induced
temperature change, in accordance with the calibrated temperature dependence.
The open pipette is the borosilicate micropipette that is also used in the whole-cell
patch-clamp study, however the pipette tip is slightly bigger so as to improve the
sensitivity towards the change in the current signals. Prior to the temperature
measurement, the open pipette was calibrated by first recording the resistance
dependence with respect to bath temperature. The microscope image in Figure 5.13(a)
shows the thermocouple positioned closely to the open pipette in the bath of
extracellular solution. The thermocouple recorded the temperature changes (~41 ºC to
~22 ºC) while the pipette microelectrode simultaneously measured the resistance
variation. Figure 5.13(b) shows the relationship of the bath temperature and current
flow with time. It can be observed that while the temperature decreased with time, the
current flow increased. The resistivity of the borosilicate material decreases with
increasing temperature, therefore the results suggested that Ohm’s law was obeyed
because current and resistance are inversely related if voltage is clamped (Eq. 5.2).
Subsequently, Arrhenius plots were constructed, from which the activation energy can
be derived from the slope of the linear curve. An example of the Arrhenius plot is
shown in Figure 5.13(c), and the linear slope of the plot has an R2 value of 0.9976. The
average activation energy was 3.4 ± 0.2 kcal/mol (n = 3), which is reasonably close to
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the value estimated by Yao et al.443 Given this activation energy, the temperature
change was calculated by the Eq. 5.3.
The temperature change due to laser heating of the nanorods was measured in the
surrounding bath of the NR-SGNs with the calibrated pipette positioned ~2 µm from the
illuminated neuron. The temperature profiles could be constructed based on the
recorded current signals. Further data processing was required because the recorded
signals pertinent to the temperature information were relatively small compared to the
background electrical noise, contributing to a low signal-to-noise (S/N) ratio. A periodic
50 Hz noise could sometimes be observed, which may be due to the intrinsic noise in
the pipette and/or noise of the current-to-voltage converter picked up during the
recording. Therefore, the data processing was accomplished by using the software
TableCurve 2D. The software analysed the periodic noise obtained as a result of the
recordings and then fitted the data with a sine wave matching the frequency, amplitude
and phase of the periodic noise. An example of the data processing is shown in Figure
5.14(a). Part of the data containing the periodic noise was isolated out for the analysis.
As shown in Figure 5.14(b) the sine wave function was fitted to the noise and
subsequently, taking the information of the fitted sine wave curve, Eq. 5.4 was used to
generate an extended sine wave curve, which was subtracted from the full data set.
Figure 5.15(a) gives an example of the signal subtraction. After the subtraction, the
periodic noise was successfully removed and this data processing had generated the
recorded temperature elevation profile for 1 ms laser pulse (Figure 5.15(b)). Note that
attempts to remove this 50 Hz noise in the frequency domain by Fourier analysis were
unsuccessful, as they tended to distort the shape of the temperature response.
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Figure 5.13 Pipette temperature calibration. (a) Microscopic image showing the
thermocouple bead (red arrow) and the open patch pipette (green arrow), (b)
Relationship of temperature and current with time, together with exponential fitting; as
the bath temperature decrease overtime, the current increases, (c) Arrhenius plot of
pipette current used to measure the time course of temperature changes induced by laser
stimulation. Each point is a single measurement.
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Figure 5.14 Data processing of the recorded signals. Screenshots from TableCurve 2D
showing (a) unprocessed raw temperature data calculated from Eq. 5.4, red box indicate
the data sectioned out and subjected to the fitting analysis, and (b) fitting analysis of the
sectioned data using curve-fit waveform function.
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Figure 5.15 Typical data processing of the recorded signals. (a) Raw temperature data
(blue trace) and sine wave (red trace) generated from the curve fitting function. (b)
Processed temperature data; inset: magnified view of the first 100 ms. Grey shading
indicates the timing of the laser pulse.
Figure 5.16(a) shows typical temperature elevation profiles recorded by the open
patch pipette at the surface of a single NR-SGN subjected to 1, 10, and 25 ms laser
pulses. It can be observed that the temperature rises immediately from the
commencement of the laser pulse at 0 ms and then decayed over several milliseconds
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after the illumination had ceased. The thermal decay is related to heat transfer (primarily
through conduction) from the irradiated target neuron to the surrounding environment.
It can be observed that a temperature plateau is approached when the pulse duration was
25 ms, which is indicative of a balance between the rate of laser heating and conductive
heat losses.446
The thermal relaxation time taken to cool to 50% of the peak temperature
for 1, 10, and 25 ms laser pulses is relatively consistent (~3 ms). Figure 5.16(b) presents
the mean peak temperature changes identified from at least three NR-SGNs for various
pulse lengths. The mean peak temperature shows a sublinear increase with pulse length.
Figure 5.16 Temperature changes as detected by the open-pipette method. (a) Temporal
temperature profiles for 1, 10 and 25 ms pulse durations, and (b) mean peak temperature
changes as a function of pulse duration measured from at least three NR-SGNs.
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5.5 Discussion
All plasmonic nanomaterials possess an intrinsic property known as the absorption
cross section which determines their ability to interact with and absorb light at a
particular wavelength. Such information pertinent to the nanomaterials is of particular
significance since photothermal efficiencies depend heavily on the light absorption.
GNRs are amongst the highest absorbing nanomaterials69 and have a larger absorption
cross section than GNSs and even conventional light-absorbing dyes.397 Therefore the
highly efficient light-to-heat conversion can benefit the use of GNRs as absorbers for
photothermal applications. Prior to examining the two modalities (NIR 780 nm light and
nanorods) as a stimulus to trigger electrical activity in neuronal cells, the level of laser-
induced temperature increase was compared in pure water and the GNR solutions. In the
NIR wavelength range, water is a weak chromophore, with an absortion coefficient
~0.005 mm-1.447 The results from the bulk heating suggested that at 780 nm, the water
absorbs relatively weakly and thus exhibits poor photon-to-heat conversion compared to
aqueous nanorods. It is also likely that this particular wavelength in the NIR region can
penetrate deeply in biological tissue because of the relatively low absorption by water as
well as other absorbing chromophores.12, 448 In contrast, the solution with GNRs can be
heated up on continuous exposure to the laser and the heating rate of the GNR solution
was ~5 times higher than that of water in the first minute of laser irradiation. Compared
to bulk heating, the temperature rise is expected to be relatively faster and highly
localised around an individual gold nanorod depending on the laser pulse fluence.378, 449
Provided the transient heating caused by the nanorods can satisfy the expected threshold
(~15 K/s) for heating rate and change of temperature, the photothermal stimulation of
neuronal cells could be achieved.450 At single particle level, GNR can easily fullfill this
threshold, reaching several thousands of K/s with pulsed laser.449
As observed from the control SGNs, patch-clamp recordings revealed no significant
stimulatory effect by the 780 nm laser alone, eliminating the direct electric field of the
laser as the possible stimulus. This finding is consistent with a similar study by Wells et
al.9 For SGNs containing SiO2-GNSs, the patch-clamp results should be treated with
caution. The stability state of the nanoparticles in contact with the neurons could
substantially affect the results. For example, in principle GNSs absorb weakly in the
NIR wavelength range and should generate very little temperature effect due to the poor
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light-to-heat conversion. However, particle agglomeration can significantly shift the
surface plasmon resonance wavelength towards the red, leading to some degree of light
absorption. This could explain the small current response observed in some NS-SGNs
on exposure to longer laser pulses (Figure 5.8(b)). In Chapter 4, the dark-field
microspectroscopic analysis carried out to the NS-SGNs also revealed significant
spectral redshift and broadening after the incubation. While both of the spectroscopic
and patch-clamp results seem to confirm a small amount of SiO2-GNSs agglomeration,
the extent to which it defeats the current hypothesis is minimal. Compared to the control
SGNs and NS-SGNs, the NR-SGNs exhibited significantly enhanced electrical
responses (both current and voltage responses) upon laser irradiation.
For the temperature measurements, the open pipette approach adopted herein
provides an important indication of the temperature changes. It is assumed that the
temperature of the particles associated with the NR-SGNs has equilibrated with that of
the surrounding water on the timescales relevant to this work.449 Note that this approach
differs from other published work in which local temperature changes due to water
absorption were measured by irradiating the tip of an open patch pipette immersed in
the extracellular bath.443, 451 The temperature measurements could also be carried out by
other means, such as using fluorescent molecules, given that the fluorescence intensity
is inversely proportional to the temperature, or nanoparticle-based thermometry such as
nanodiamonds452 and luminescent nanoparticles.453 However these methods require co-
incubation of additional exogenous components, which may confound the current
nanoparticle study. As can be seen from the temperature measurements, the peak
temperature increases with increasing laser pulse lengths, and correlates with the
electrical activity in the NR-SGNs. The temperature and cell electrical activity as
described herein suggests that there is potential to regulate the electrical activity by
adjusting the laser pulse energy.
Interestingly, the laser pulse energies used to achieve a temperature change sufficient
to promote cell electrical activity was relatively low compared to the typical laser
energies used in infrared neural stimulation. For instance, in a previous report by Wells
et al., an average surface temperature increase of 3.66 ºC was measured from the rat
sciatic nerve surface with an infrared camera while applying infrared neural
stimulation.9 This temperature change is comparable to the 3.5 ºC shift measured for a 1
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ms laser pulse in the present study. However, Wells et al. used a laser pulse energy of
1.13 mJ at 2120 nm, whereas 0.09 mJ was applied from the 780 nm laser diode in the
present study. This suggests that the presence of nanorods can enhance the local heating
due to optical absorption.
In conventional infrared neural stimulation, laser wavelengths greater than 1400 nm
are used and water is the primary chromophore that is responsible for light-to-heat
conversion. In comparison, water absorption is about two to three orders of magnitude
lower at shorter wavelengths in the range between 650 and 900 nm.12, 447 The
wavelength range is known as the biological transparency window, where maximum
NIR laser penetration can be reached.12 Additionally, the longitudinal SPR peak of
GNRs is also tunable within the wavelength range, prompting their good use in the
biomedical fields.175, 454 Assuming a molar of GNRs, the absorption coefficient is ~108
mm–1 at 785 nm,70 compared to approximately 0.005 mm-1 for water, so the nanoparticle
absorption is dominant at this wavelength. Moreover, within this NIR wavelength
range, the reduced scattering coefficient is relatively low ~7 mm-1.455 The thermal
transients observed during laser illumination match the temporal profile modelled for
the heating of water during infrared neural stimulation,446 suggesting that the
stimulation of the neurons by NIR laser-induced heating of SiO2-NRs may be following
similar mechanisms to infrared stimulation.
A recent study of infrared stimulation by Shapiro et al. showed that localized
transient heating in water induced a reversible change in the electrical capacitance of the
cell membrane.10 Temperature-induced changes in capacitance were demonstrated to
result in depolarizing currents in Xenopus laevis oocytes and HEK cells,10 and in
principle should be proportional to the temporal rate of change in the temperature. The
current work suggests a similar dependence, given the temperature profiles presented in
Figure 5.16(a) and the transmembrane currents shown in Figure 5.6.
The activation of temperature-sensitive ion channels, including the transient receptor
potential vanilloid (TRPV) channels,337, 456 has also been implicated in infrared neural
stimulation.11 For instance, a typical TRPV1 protein is sensitive to a threshold
temperature of 41°C.335 Previous work by Huang et al. has demonstrated that the
TRPV1 expressed in hippocampal neurons can be activated by radio-frequency heating
of magnetic nanoparticles targeted to the plasma membrane of the neurons.340 The
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spatially-confined temperature rise of 13°C at the particle surface was measured from
the lifetime of fluorescent dyes tethered to the nanoparticles.
Clearly, given the temperature that was measured at a distance relatively close to
the NR-SGNs using the open pipette, direct activation of TRPV1 is unlikely because the
threshold temperature of 41°C was not reached during laser illumination using any of
the laser pulse lengths (energies). While it is possible that the threshold was reached at
the cell surface, it is also conceivable that other temperature- and voltage-sensitive ion
channels are involved. Laser-induced temperature rises could directly activate some
voltage-dependent Na+ and K+ channels, provided there is sufficient change in
capacitance. On the other hand, recent work by Brown et al. with the same neuronal
model has not been able to demonstrate action potentials under illumination at laser
wavelengths of 1550 nm and 1870 nm.320 Considering that the transmembrane current
changed with the rapid temperature rise, the effect may be due to the capacitive
mechanism proposed by Shapiro et al.10 or by subtle structural changes in the cell
membrane of the neurons e.g. local phase changes in the lipid membrane457 as suggested
by Migliori et al.298 Interestingly, a similar effect has been reported when exposing the
cell membrane to ultrasound.329 In that case, the effect of the GNRs may be explained
primarily by changes in the membrane capacitance as a result of temperature-dependent
conductance changes in biological membrane, although via a different mechanism than
previously proposed.10
Although action potentials were not consistently evoked in the NR-SGNs using a
longer laser pulse length, these observations were obtained at the maximum laser power
available in the current laser system and so the present study was not able to provide a
robust stimulation threshold. However, cell-to-cell variability in physiology and cell-
nanoparticle interactions may also account for some of the differences in electrical
response. There has been some recent work that also demonstrated the feasibility of
using extrinsic absorbers for neural stimulation. Very recently, it has been shown that
GNR-assisted IR neural stimulation requires at least 5 mJ laser pulse energy at 980 nm
for stimulating compound nerve action potentials in rat sciatic nerves in vivo.343 This
laser pulse energy is about two times the minimum energy (2.25 mJ) used in the present
study for NR-SGNs to fire action potentials in vitro. The difference could be due to the
use of different stimulation targets (i.e. nerve bundles vs. individual neuron) as well as
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neuron types (i.e. muscle neurons vs. auditory neurons). Meanwhile, Farah et al. used a
digital holography system to project intense light patterns (532 and 800 nm) onto black
microparticles and demonstrated the activation of rat cortical neurons surrounded by the
particles.297 Their work showed that minimum pulse durations of 100 µs and energies in
the range of 1 µJ were able to stimulate neurons as indicated by calcium transients.
Besides, Migliori et al. reported that carbon particles absorbing laser light at 650 nm can
cause thermal heating in Xenopus oocytes and leech neurons in vitro.298 In that work,
action potentials were activated upon laser heating with 50 ms pulse lengths, and 250–
3500 µJ laser pulse energies. The laser pulse duration and energy used in the present
study for NR-SGNs to fire action potentials were slightly shorter and lower compared to
the report of Migliori et al. However, it is highly conceivable that the laser parameters
and the surface properties of GNRs could be optimized to the benefit of lowering the
pulse lengths and energies used to achieve similar observations of IR neural stimulation
in vitro. For example, further improvements in efficiency will be driven by locating the
nanoparticles closer to the cell membrane or specific ion channels that mediate the
process, plus a laser with higher peak power for shorter pulses and less wasted heat
spreading to the environment.
5.6 Conclusion
This chapter demonstrated the photothermal capability of GNRs and the proof-of-
concept for using SiO2-GNRs and a 780 nm laser to achieve absorber-based
photothermal neural stimulation in vitro. Under continuous 780 nm laser exposure, bulk
heating of nanorod suspensions has revealed a significant temperature increase
compared to pure water. Although the temperature elevated relatively slowly over the
10 min period of continuous laser heating in bulk aqueous solution, the heating curve is
expected to rise much faster at the single-particle level. This has benefited heat-based
neural stimulation. The present in vitro study has demonstrated significant enhancement
in current and voltage activities in the SGNs cultured with SiO2-GNRs under the 780
nm pulsed laser exposure. Some of the NR-SGNs were also observed to fire action
potentials in response to the laser irradiation. In contrast, when the SGNs were cultured
with SiO2-GNSs that typically absorbed at 530 nm, the 780 nm light had no significant
effect on the neurons. The effect of laser irradiation is also negligible for control
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neurons without any absorbing nanoparticles. Temperature measurements in the local
environment of the NR-SGNs have revealed indicative temperature rises of between 0.5
ºC and 6.0 ºC depending on the pulse length, which forms a correlation with the
enhanced electrical activity of the neurons on exposure to the laser pulses. The
mechanism appears to be photothermal in origin, but the full details in this nanoparticle-
mediated case may be subtly different from conventional infrared neural stimulation.
Nonetheless, the improved efficiency of the process has therapeutic potential in
photothermally-based neural prosthetic devices.
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157
Chapter 6: Summary and Future Directions
6.1 Summary of Findings
The studies described here have demonstrated for the first time in vitro that laser
exposure of gold nanorods (GNRs) can enhance electrical activity as well as stimulate
action potentials in spiral ganglion neurons (SGNs) of rats. Based on the voltage- and
current-clamp results presented in Chapter 5, the stimulation of SGNs containing silica-
coated gold nanorods (NR-SGNs) appeared to be dependent on the laser pulse energies
(2 µJ to 4.5 mJ). Laser energies as low as 2 µJ were able to elicit inward transmembrane
currents and sub-threshold transmembrane potentials. As the laser pulse energy
increased, the magnitude of the inward currents and sub-threshold potentials was
observed to increase. Lower pulse energies did not generate action potentials in the
SGNs but produced significantly enhanced current and voltage activity compared to the
control neurons. However, action potentials were fired in the neurons when a laser pulse
energy of 2.25 mJ was used. Importantly, the neurons were still viable after firing action
potentials as indicated by the intracellular current test pulse.
The stimulation effect did not appear to be generated by the 780 nm laser in itself,
because the control SGNs did not response in the same way as NR-SGNs upon laser
irradiation under the same conditions. Moreover, the effect appears to be associated
with the longitudinal SPR absorption of the nanorods, as SGNs containing silica-coated
gold nanospheres (NS-SGNs) showed no significant electrical response. Therefore it
was postulated that GNRs in the vicinity of neurons had generated transient heating in
response to laser irradiation and that this heating contributed to the significant
stimulatory effects in the neurons. In this context, two relevant aspects with regard to
the GNRs were addressed: their presence and location in the vicinity of the neurons, and
the temperature effect that they elicited during laser irradiation. First, cultures of NR-
SGNs were examined by dark-field scattering microscopy and the light scattering of the
GNRs was observed directly from the neurons through the microscope (Chapter 4). In
addition, scattering spectra were acquired from the NR-SGNs using a
microspectrometer and the results showed that all of the neurons exhibited positive
spectral characteristics of GNRs, i.e. the presence of both longitudinal and transverse
158
surface plasmon resonance peaks, hence suggesting the presence of nanorods inside the
neurons with minimal aggregation. It was also important to analyse NS-SGNs to ensure
the presence of silica-coated gold nanospheres (SiO2-GNSs) in the cultures. Therefore
in Chapter 4, it was demonstrated that all of the NS-SGNs examined have exhibited
typical spectral characteristic of GNSs.
Next, the open pipette method was adopted to measure the temperature effects
associated with the laser irradiation of NR-SGNs. Laser pulse energies used in the
stimulation were investigated in order to elucidate the temperature change (ΔT) near the
surface of the NR-SGNs. The results showed that the ΔT was in accord with the laser
pulse energies used, in other words, the ΔT increased with increasing laser pulse energy
and was also presented as a sub-linear relationship with pulse energy. The ΔT was
found to be in the range of 0.5 to 6 °C across all the pulse energies studied. The open
pipette method described here may serve only to provide an indication of the true
temperature rise, given that the pipette can only be positioned as close as 2 µm from the
neurons, while the heating effect is known to be highly localised in the vicinity of the
nanoparticles and thus the neurons. However, the sub-linear temperature increase also
appeared to correlate with the enhanced electrical activity of the neurons, implying that
the temperature measurements provide reasonable approximation.
Successful application of GNRs in the stimulation of SGNs as shown in Chapter 5
had also highlighted the importance of appropriate tuning of the longitudinal plasmon
wavelength for maximum laser-particle interactions. For instance, SiO2-GNSs that were
off-resonance at 780 nm showed negligible response to 780 nm laser irradiation, and
thus no enhancement of electrical activity was apparent in the NS-SGNs. In Chapter 3,
it was shown that the fine tuning of the longitudinal plasmon resonance wavelength of
GNRs was much more easily controlled through manipulating the concentrations of
silver nitrate and gold seeds compared to that of ascorbic acid in the seed-mediated
growth method. This has allowed GNRs to be synthesized with a longitudinal plasmon
wavelength in the near-infrared (NIR) range and also for subsequent surface
modification and functionalization. Using this method, GNRs were synthesized with a
longitudinal plasmon wavelength at ~760 nm. Subsequent surface modifications and
functionalization using polymer and silica coatings redshifted the plasmon wavelength
to ~780 nm (Chapter 3). These stable GNRs with different surface compositions were
159
applied in various studies including cellular uptake experiments in Chapter 4, neural
stimulation in Chapter 5, and several other studies for investigating biocompatibility
and neural regeneration in the NG108-15 neuroblastoma cell line,232, 388 as well as
generation of calcium transients in NG108-15s on exposure to 780 nm laser light.342
In general, surface modification and functionalization are essential for improving
biocompatibility and cellular uptake.243, 420 In Chapter 4, GNRs with different surface
coatings were analysed for their capabilities in contributing to cellular uptake in the
NG108-15 cell line. GNRs were visualized in the cells by dark-field microscopy and the
results showed qualitatively that the ease of cellular uptake of GNRs was dependent on
their surface coatings. Microspectroscopic studies carried out to analyse the cells
containing these nanorods have shown that the spectral characteristics of the nanorods
were preserved following incubation with the cells. However minor spectral shifts were
observed for nanorods with CTAB and PSS coatings upon interactions with the cells,
suggesting changes in the local refractive index in the cellular environments which were
sensitively detected by the nanorods due to the relatively thin surface coatings compared
to nanorods with silica coating. Therefore GNRs with silica coating were the preferred
for the biological studies, including in neural stimulation, as described in this thesis.
6.2 Future Directions
In future work, synthesis of gold nanoparticles can be scaled up using a high-
throughput system which may be accomplished by developing a fluidic reactor that can
produce large amount of high quality nanoparticles.391 The high-throughput reactor
should also be equipped with the capability for particle surface modification. The effort
of this future work may significantly shorten the time required for preparing gold
nanoparticles.
It is anticipated that more research will follow on from this work to extend the GNR-
assisted neural stimulation into different primary neurons, tissue slices and in vivo
proof-of-concept. However, some outstanding challenges must be addressed before the
system can become practically viable. First, there is a need for monitoring the long-term
biocompatibility of GNRs in the neural system. Material-wise, the gold core is known to
be benign and biological inert.420 For example, preclinical biocompatibility screenings
160
of gold shell nanoparticles have shown no toxicity in animal models more than a year
after administration.263 However, long-term assessment of nanotoxicity in the neural
system has not been reported. Immune responses in the CNS, such as activation of
astrocytes and brain phagocytes, can provide an indication of neurotoxicity,458 and has
been demonstrated in a short-term study using semiconductor nanoparticles in vitro and
in vivo.459 A similar approach can be adopted for assessing the long-term
biocompatibility of GNRs in neural tissues. In addition to the core material, the surface
composition(s) can potentially also contribute to cytotoxicity. While the amorphous
silica material on the GNRs shows less cytotoxicity compared to bilayer CTAB
molecules,181 their long-term stability with regards to silica dissolution460 in the culture
conditions may require further evaluation. Any materials that are released by the
particles or expose the CTAB layer could contribute to the extent of cytotoxicity in the
cultures and therefore requires more thorough investigation.
Next, for GNRs to work practically in auditory neural stimulation, some level of
targeting of nanoparticles is required for delivering the particles selectively to the nerve
cells/tissue. More localized heating of the cell membrane or specific photothermally-
responsive receptors on the neuronal cells will allow even more efficient stimulation
with less heat wasted in the surrounding tissue. There have been a few recent reports
focusing on the delivery of polymersome nanoparticles to the inner ear/cochlea using
synthetic peptide ligands such as Tet1 peptide for targeting to the trisialoganglioside
clostridial toxin receptors on neurons,344 tyrosine kinase receptor B (TrkB)-binding
peptide for targeting to the TrkB receptors,345 and nerve growth factor-derived peptide
for targeting to the tyrosine kinase receptors and p75 neurotrophin receptors.346 These
targeting strategies can be used in future study for functionalizing the nanoparticles.
However, the functionalization of the GNRs with targeting ligands is further
complicated by the need to avoid compromising particle stability, because the optical
properties such as absorption efficiency are inherently link to colloidal stability. The
lifetime of the particles in the tissue also needs to be assessed, as this has a major
impact on the sorts of applications that can be supported. For example, bionic
applications require reliable long term localization of particles with the neurons,
whereas fundamental studies of the nervous system can be conducted on a much shorter
timeframe.
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Furthermore, there is also a need to address a more consistent stimulation threshold.
The present studies were only able to stimulate action potentials in the neurons with
laser pulse energies of about 2 mJ. Additionally, action potentials were not consistently
evoked in all of the neurons. It is understood that cell-to-cell variability in the uptake of
GNRs may account for some of the differences in electrical response, leading to
significant challenges in identifying a reliable stimulation threshold. Therefore the
future development of the system will require optimization of all of the conditions
including particle dose and seeding density of primary neurons. In addition, it is also
sensible to characterize the electrical activity of the neurons by means of voltage-
sensitive dyes or by monitoring the calcium transients which are also associated with
the stimulation.322, 461 These methods allow direct microscopic observation of electrical
activity in a population of neurons compared to the single-cell measurements from
whole-cell patch-clamp recordings.
The localised increase and decay in temperature in the laser exposed neurons may
require a better measurement. The present open-pipette method has only allowed
temperature measurements at a distance of about 2 µm away from the neurons, which
may not represent the ‘true’ temperature at the localised area. Future studies may
address this outstanding challenge by integrating a much more localized thermometry
system using nanosized thermometers such as nanodiamonds, which have been able to
measure intracellular temperature changes with high spatial resolution and high
sensitivity.452
In addition to addressing the outstanding challenges as mentioned above, the exact
mechanisms of action during photothermal neural stimulation should be further
explored. Particularly, the role of membrane capacitance and TRPV or other channels in
the process requires further elucidation. It is still not clear whether mechanosensitive
ion channels are also responding to the thermal expansion associated with the heating,
or whether there are changes in capacitance occurring due to phase changes in the lipid
structure of the cell membrane. This issue is important for two reasons: firstly, to assist
with more accurate targeting of nanoparticles to obtain the most effective response, and
secondly, to provide a solid fundamental understanding to underpin future clinical
applications.
162
The research described here has developed a novel means for stimulating nerves and
as such the method has great potential for fundamental studies in neurophysiology and
the future development of neural prosthetic devices.
163
164
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Appendix I
Figure A1 Standard curve constructed for the determination of gold ion concentration
The concentrations of gold ions released from the nanoparticles following acid
digestion with aqua regia were measured quantitatively by atomic absorption
spectrometry (AAS, Varian SpectrAA-20 spectrometer, Varian, Inc.), calibrated with
commercially available gold standard stock solutions (Fluka, Australia).
Table A1 Gold ion concentration for different types of nanoparticles measured by AAS.
Nanoparticle Au Conc.
(ppm)
Optical
density (OD)
at λmax
Section
SiO2-GNSs 7.3 ± 1.2 0.18 4.2.2.1 5.2.4
SiO2-GNRs 13.1 ± 0.4 0.18 4.2.2.1 5.2.4
Bare GNRs 33.2 ± 1.5 1.8 5.2.2
SiO2-GNRs 41 ± 1.8 1.8 5.2.2
206
List of Publications
Journal Articles
1. J. Yong, P.R. Stoddart, A. Yu. Laser heating of gold nanorods coated in silica and silica/polydopamine. (Manuscript in preparation).
2. J. Yong, P.R. Stoddart, A. Yu. Thiol-based surface functionalization of inorganic nanoparticles and their recent advances. (Manuscript in preparation).
3. C. Paviolo, A.C. Thompson, J. Yong, W.G.A. Brown, P.R. Stoddart. Nanoparticle-enhanced infrared neural stimulation. Journal of Neural
Engineering. (2014, Accepted). 4. J. Yong, K. Needham, W.G.A. Brown, B.A. Nayagam, S.L. McArthur, A. Yu,
and P.R. Stoddart. (2014) Gold nanorods-assisted near-infrared stimulation of primary auditory neurons. Advanced Healthcare Materials. DOI: 10.1002/adhm.201400027
5. C. Paviolo, J. W. Haycock, J. Yong, A. Yu, P. R. Stoddart and S. L. McArthur. (2013) Laser exposure of gold nanorods can increase neuronal cell outgrowth. Biotechnology and Bioengineering. DOI: 10.1002/bit.24889
Peer-reviewed Conference Proceedings
1. J. Yong, W.G.A. Brown, K. Needham, B.A. Nayagam, A. Yu, S.L. McArthur
and P.R. Stoddart. Dark-field microspectroscopic analysis of gold nanorods in spiral ganglion neurons. Proc. SPIE 8923 Micro+Nano Materials, Devices, and Applications, Melbourne, Australia, 2013. DOI: 10.1117/12.2033767
2. C. Paviolo, J.W. Haycock, J. Yong, A. Yu, S.L. McArthur, P.R. Stoddart. Plasmonic properties of gold nanoparticles can promote neuronal activity. SPIE Photonics West, San Francisco, USA, 2013. DOI:10.1117/12.2002291
Conference with Published Abstracts
1. K. Needham, W.G.A. Brown, J. Yong, B.A. Nayagam, P.R. Stoddart. Infrared and nanoparticle-enhanced stimulation of auditory neurons in vitro. ARO 37th MidWinter Meeting, SanDiego, California, USA, 2014
2. W. G. A. Brown, J. Yong, K. Needham, B.A. Nayagam, P. R. Stoddart. Infrared and nanoparticle-assisted stimulation of auditory neurons in vitro. 3rd International Symposium Frontiers in Neurophotonics, Bordeaux, France, 2013
207
3. J. Yong, K. Needham, W.G.A. Brown, B.A. Nayagam, A. Yu, S.L. McArthur and P.R. Stoddart. Gold Nanorods Can Promote Infrared Neural Stimulation. 3rd International Conference on: Medical Bionics Engineering Solutions for Neural Disorders. Phillip Island, Melbourne, Australia, 2013.
4. C. Paviolo, J.W. Haycock, J. Yong, A. Yu, S.L. McArthur, P.R. Stoddart. Neurite outgrowth in neuronal cells is promoted by laser exposure of gold nanoparticles. AIP conference, NSW, Australia. 2012.
5. C. Paviolo, J.W. Haycock, J. Yong, A. Yu, S.L. McArthur, P.R. Stoddart. Plasmonic properties of gold nanoparticles can induce intracellular calcium transients. AIP conference, NSW, Australia, 2012.
6. J. Yong, Q. Wang, F. Malherbe, A. Yu. Investigation of antimicrobial properties of carbon nanotube/silver nanoparticles hybrid films. Australian Society of Microbiology Annual Meeting, Brisbane, Australia, 2012.
7. J. Yong, P.R. Stoddart, A. Yu, F. Malherbe. Synthesis and characterization of amino-functionalized gold-silica core-shell nanoparticles. International Conference on Nanotechnology, Kuantan, Malaysia, 2012.
Other Publications
1. L. Fu, J. Yong, G. Lai, T. Tamanna, S. Notley, A. Yu. (2014) Nanocomposite coating of multilayered carbon nanotube-titania. Materials and Manufacturing
Processes. DOI: 10.1080/10426914.2014.880465 2. J. Yong, Q. Wang, H. J. Ng, F. Malherbe and A. Yu. (2013) Antibacterial
properties of multi-walled carbon nanotube-silver nanoparticles hybrid thin films.
Nanoscience and Nanotechnology Letters. DOI: 10.1166/nnl.2013.1686 3. S. Xu, J. Yong, G. Lai, H. Zhang, Y. Wu and A. Yu. (2013) Functionalized
mesostructured cellular forms for loading and release of streptomycin. Chemistry
Letters. DOI:10.1246/cl.2013.235 4. G. Lai, H. Zhang, J. Yong, and A. Yu. (2013) In situ deposition of gold
nanoparticles on polydopamine functionalized silica nanosphere for ultrasensitive nonenzymatic electrochemical immunoassay. Biosensors and Bioelectronics. DOI: 10.1016/j.bios.2013.03.029
5. R. Kubiliūte, K. Maximova, A. Lajevardipour, J. Yong, J. S. Hartley, A. S. M. Mohsin, P. Blandin, J. W. M. Chon, M. Sentis, P. R. Stoddart, A. Kabashin, R. Rotomskis, A.H.A Clayton, and S. Juodkazis. (2013) Ultra-pure, water-dispersed Au nanoparticles produced by femtosecond laser ablation and fragmentation. International Journal of Nanomedicine. DOI: 10.2147/IJN.S44163
6. A. Yu, Q. Wang, J. Yong, F. Malherbe, P. J. Mahon, F. Wang, H. Zhang and J. Wang. (2012) Silver nanoparticle-carbon nanotube hybrid films: preparation and electrochemical sensing. Electrochimica Acta. DOI: 10.1016/j.electacta. 2012.04.024