University of Washington Department of BioengineeringSummer 2010

Design of Silica Coated Gold Nanorods for Photothermal Therapy

Xiaohai Zhang0521394

xiaohai@u.washington.eduFaculty Mentor: Xiaohu Gao

1

Table of ContentsAbstract:........................................................................................................................................................3

A. INTRODUCTION:..............................................................................................................................4

1. Concise Definition of the Project:...................................................................................................4

2. Medical Significance:.......................................................................................................................5

3. Social, Ethical and Economic Considerations:..............................................................................6

a. Economic Issues:..........................................................................................................................6

b. Ethical Issues:...............................................................................................................................7

4. Technical Background:...................................................................................................................7

a. Theory:..........................................................................................................................................7

b. Review of Literature:.................................................................................................................11

c. Previous relevant work:............................................................................................................19

d. Technical issues at the outset of the project:...........................................................................22

B. DESIGN OF TOOLS, DEVICES, AND EXPERIMENTS:...........................................................23

1. Overview of design and research plan:........................................................................................23

2. Materials and Methods:................................................................................................................25

3. Costs:...............................................................................................................................................27

4. Details of Design Process and Statistical Basis for Design of Experiments:.............................27

C. RESULTS:..........................................................................................................................................29

1. Data and Discussion:.....................................................................................................................29

2. Conclusions:...................................................................................................................................37

3. Suggestions for Future Work:......................................................................................................37

D. ACKNOWLEDGEMENTS:.............................................................................................................38

E. REFERENCES:.................................................................................................................................38

2

Abstract:

Gold nanorods (GNR) have been hailed as a multifunctional imaging and therapeutic agent for

treatment of cancer through its unique photoacoustic and photothermal characteristics. GNRs are

able to be excited at specific wavelengths in the near infrared region and produce localized

heating to ablate cancer cells. This type of therapy is minimally invasive and highly selective,

two essential characteristics of an efficacious cancer treatment. However, due to the fact that

GNR excitation wavelength is highly dependent on its anisotropic morphology, its ability to

produce the photothermal effect thus depends on maintaining its physical structure. It has been

shown that laser irradiation often melt GNR structure and decreases its ability to respond to

further excitation. In this study, the sol-gel method of silica coating was applied to gold nanorods

in order to enhance its photothermal stability. GNRs were encapsulated in silica shells of various

thicknesses in attempt to physically encage the GNR during laser excitation and aid in holding

it’s longitudinal to transverse aspect ratio. The silica-coated gold nanorod is a promising way to

increase the efficacy of gold nanorods used in photothermal therapy by giving it enhanced

photothermal stability.

3

A. INTRODUCTION:

1. Concise Definition of the Project:

Photothermal therapy is an experimental method that utilizes heat energy to destroy tissues and

cells as a way to treat diseases such as cancer. With recent developments in nanotechnology,

gold nanoparticles, with their unique ability to dissipate absorbed light energy into heat is being

studied as a tool for cancer treatment. Spherical gold nanoparticles were first studied to be used

for phototherapy; however, their absorption spectrum in the 500nm range severely limited its

ability to become excited in the 650nm to 900nm infrared transmission that penetrates skin and

tissues. An improvement upon the spherical gold nanoparticles is the use of gold nanorods,

which can be tuned to absorb within the optimal transmission range depending on its length-

width ratio. However, energy transfer from laser have shown to cause thermal melting, an effect

that changes the aspect ratio of the nanorods and alters the absorption peak. The goal of this

project is to prevent thermal melting of gold nanorods by encasing the nanorods within a silica

coated shell. To increase the photostability of the nanorods, the silica shell would effectively

serve as a mold that would hold the gold nanorods within its original shape. There are three

important design considerations that need to be considered in the addition of a silica shell on

gold nanorods: First, it is necessary that the silica shell does not prevent the nanorods from being

able to absorb of infrared light. Second, the shell must not interfere with the dissipation of heat

that would eliminate the therapeutic effect. Third, the silica shell thickness must be able to retain

the gold nanorods within its original aspect ratio. In addition to increasing photostability, the use

of silica to coat the gold nanorods also provides a good surface for bioconjugation.

4

2. Medical Significance:

In the United States, cancer accounts for nearly one in every four death. It is the second most

common cause of death only second to hearth disease. The National Cancer Institute estimates

that in 2010, nearly half a million Americans are expected to die of cancer and 1.5 million new

cases are expected to be diagnosed and that around 1.5 million new cases of cancer are expected

to be diagnosed in 2010[1]{, 2010 #1104}.

With an increased federal budget in health research under the new Obama administration,

scientists and physicians are looking harder than ever to eradicate this destructive disease. In

practice, the diagnosis of cancer, and the outcome of treatment is heavily dependent on when the

malignancy is discovered. For this reason, early screening and detection is extremely

advantageous. Studies show that the survival rate for women who were diagnosed with breast

cancer before it metastasize is nearly 98%, but if the tumor metastasized to nearby organs, the 5-

year survival rate is only 27%[2]. Therefore, it is critical that cancer is detected in its early

stages.

With the lack of sufficient sensitivity in early cancer detection, treatments of cancer are usually

very complicated. Current treatments such as chemotherapy or radiation therapy have the

disadvantage of lacking specificity to target tumor tissues and thereby causing debilitating side

effects. For example, less than one percent of administered chemotherapy drugs do not reach the

tumor; therefore, it is essential that a targeted delivery system, in the form of large molecule

drugs or nano-engineered devices be developed to resolve this issue.

5

Gold nanoparticles are an example of a nano-engineered material that has the potential to both

diagnose and treat tumors. Gold is an inert material that has minimal cytotoxic effects. Gold rods

on the nanometer scale have a unique property of becoming excited by infrared light of a specific

wavelength and converting the electromagnetic energy of into heat energy. This property could

be taken advantage of in cancer therapy by localizing the rods onto tumor cells and ablating the

malignant cells via laser excitation. This localized treatment would have minimal side effects due

to its high specificity.

3. Social, Ethical and Economic Considerations:

a. Economic Issues:

Although it may still take years before nanoparticles become a significant clinical tool for cancer

therapy, it is necessary to consider the economic, ethical and social issues that may arise with the

advent of this technology. Economically, the development of nanotechnology drugs initially

requires heavy financial investments in order to conduct enough studies to prove its effectiveness

and safety. Obtaining approval from the Food and Drug Administration will also take substantial

efforts. Although manufacturing the drugs would not be expensive once the process has been

established, it is still likely that nanotechnology drugs would be initially marketed as a type of

high technology treatment with an expensive price tag. Clinically, the gold nanorod ablation

treatment of cancer cells would require special equipment such as a pulse laser machine. The use

of additional machines and technicians would most likely raise the price of the treatment. On a

larger scale, the use of photothermal technology may initially become a treatment in first world

countries performed at specialized institutions.

6

b. Ethical Issues:

There are several ethical issues to consider with the use of nanotechnology in a medical setting.

One necessary question to consider is: what are the needs nano-biotechnology assumes to meet?

Research in new nanotechnology drugs is a forefront in biomedicine and countless of research

capital is being spent to develop new and improved drugs. However, one may argue whether or

not the expense of research is better spent on more basic solutions to public health. This question

is relevant to high end medicine and it is necessary for us to consider whose values do nano-

medicine serves to express and who are the ultimate benefactor from the research. For example,

it is important to consider whether the development of photothermal treatment would ultimately

serve as a tool for the elite portion of the population who are able to afford it, or become a tool

that will benefit the general public.

4. Technical Background:

a. Theory:

Gold Nanorods:

Materials on the nano-scale are small enough to place restrictions on the motions of its electrons,

a property that has led to unique physical and chemical properties as the result of their size. In

nanoparticles made from semi-conductors, motion of electrons is confined by the particle’s size

and shape. This phenomenon, known as quantum confinement, gives rise to the unique

fluorescence and absorption properties of semi-conductor particles [3, 4]. On the other hand,

nanoparticles made from noble metals such as gold are able to use confined resonant photons to

induce oscillations of conduction band electrons in a phenomenon known as Surface Plasmon

Resonance. This property enhances the irradiative properties of metal nanoparticles at the

resonant frequency which makes these nanoparticles an excellent tool for optical detection [5].

7

Another property of plasmonic nanoparticles is that when they are excited by light in resonance

with their surface plasma oscillation, they are able to convert the electromagnetic energy of the

light into heat energy. Heat energy release of the particle is dependent on the laser pulse width

and energy. This is property of the nanoparticles gives it the ability to heat its surrounding

environment by absorbing an outside radiation source, making it an effective agent for

photothermal therapy.

Due to the effect of size on plasmon confinement, the optical features of gold nanorods are

dependent on the shape and size of the particle. Gold nanorods are thus different than gold

nanospheres in that it has two absorption bands in the near-infrared region that corresponds to

the oscillation of electrons on its two axes. The bands are known as transverse and longitudinal,

and each absorb at around 500 nm to 800 nm respectively. The size and morphology dependent

properties of gold nanoparticles allow it to be specifically tuned for various applications. For

example, gold nanorods that absorb light in the infrared region (650-900nm) are useful for

medical applications because of the high penetrance of infrared light through skin.

Surface Plasmon Resonance:

Surface Plasmon Resonance (SPR) describes a phenomenon when electrons of a metal undergo

oscillation induced by a resonant frequency of coherent light. Oscillations involving electrons

moving back and forth on the particle results in a strong absorption of light. The absorption band

of SPR particles is dependent on its morphology and shape. In the case of gold nanorods,

excitation of the long axis results in the longitudinal band and the short axis results in the

8

transverse band. While the transverse band is relatively insensitive to increasing aspect ratio of

the nanorod, the transverse band absorption wavelength was resolved to change proportionally

with the particle’s aspect ratio in the relationship: λ(max) = 95R + 420 [6].

Seed-Mediated Growth Method:

A effective method to synthesize colloidal gold nanorods is through the seed-mediated growth

method that was designed by Wiesner and Wokaun in 1989. Since then, the method has been

modified by Jana et al and subsequently by Nikoobakht and El-Sayed in 2003 into the modern

protocol used today [7, 8]. In the seed mediated growth method, gold seeds are first made by

reducing auric acid in the prescence of cetyltrimethylammonium (CTAB) with the use of sodium

borohydride. Subsequently, the seeds are added to a bulk solution of gold ions dissolved in

bromide (CTAB) solution and reduced by ascorbic acid. Silver ions obtained from silver nitrate

are used to control the aspect ratio. This method allows for creating gold nanorods with aspect

ratios from 1.5 to 4.5 [9]. The mechanism of this procedure has been proposed by Murphy et al.

to be the result of preferential binding CTA+ head groups of CTAB onto the {100} face of face-

centered gold nanoparticles[10]. This asymmetric binding forces gold ions to reduce only on the

{110} side of the gold nanoparticles, resulting in the elongation of the particle during growth.

Sol-Gel Method of Silica Coating:

Silica shell is formed on the gold nanoparticles via the Stober method, a sol-gel technique that

chemically deposits silica on to the surface of gold. More specifically, the gold nanorod is first

solubilized in an aqueous solution via replacing the CTAB groups with thiolated polyethyl glycol

9

(mPEG-SH). Subsequently, tetraethyl orthosilicate (TEOS) particles deposited onto the

PEGylated surface.

In theory, the sol gel method works as TEOS reacts with water in a hydrolysis reaction in a way

that exchanges the OR group of TEOS with OH group. Subsequently, a condensation reaction

occurs that links the hydrolyzed molecules to form siloxane bonds, or Si-O-Si. The deposition,

hydrolysis and condensation reaction occurs to gradually form a thickened layer of silica before

the cessation.

Photothermal Therapy of Cancer:

Photothermal therapy of cancer consists of localizing the gold nanorods onto cancer cells

specifically and stimulates the rods at their resonant frequency with laser to induce heat

dissipation and subsequent destruction of cells. There are two theories to how the cells are

destroyed. In the study conducted by Cortie et al, it was determined that heat generation on

murine macrophage cells apoptosized due to heat stress as the effective cell temperature were

increased to up to 10 degrees by the laser irradiation [11]. On the other hand, Cheng and Wei et

al showed in their studies that more effective absorption of laser was seen when nanoparticles

were located on the membrane. This means that heat was mostly concentrated on the membrane

surface causing perforation of the membrane and subsequent apoptosis of cell [12].

10

b. Review of Literature:

“Targeted Photothermal Lysis of the Pathogenic Bacteria, Pseudomonas aeruginosa, with

Gold Nanorods”[13]

This paper showed an application of targeted photothermal treatment on the bacterium

Psedudomonas aeruginosa. The studies show that gold nanorods, covalently linked with

antibodies, were able to selectively bind to the barium and cause its destruction via near-infared

radiation. This is an important study with regards to this project because it shows the ability of

gold nanorods to be functionalized with biomolecules such as igGs to allow specific targeting.

This study also showed that the destruction of selected cells is significant against those that are

not targeted with the nanorods and that the gold nanorods themselves have minimal cytotoxicity.

“Cancer Cell Imaging and Photothermal Therapy in the Near-Infrared Region by Using Gold

Nanorods”[14]

Gold nanorods are used in this study to act as a dual imaging and therapy agent. An active

targeting scheme of seeking overexpressed EGFR on malignant cells was used. Imaging of HSC,

HOC, and HaCat cells were conducted via surface plasmon resonant absorption spectroscopy

and light scattering imaging. It was shown that the three cells could be distinguished based on

the imaging results. This studied also showed that malignant cells had increased uptake of

nanorods and could be destroyed selectively via laser ablation. A variety of laser power energies

were also tested to determine their effect on the cells. A laser power of 160 mW (20W/cm2) was

determined to cause obvious injuries to normal cells while (10W/cm2) was able to damage

malignant cells.

11

“Highly Controlled Silica Coating of PEG-Capped Metal Nanoparticles and Preparation of

SERS-Encoded Particles”[15]

Similar to the concept of this project, this study examined controlled silica coating of PEG-

capped metal nanoparticles. Specifically, this paper studied encoded nanoparticles for

multiplexed screening and imaging. The researchers used thiol modified poly ethylene glycol to

cap different sized gold nanoparticles and used the stober method to grow silica on top of the

PEGylated shells. Control of shell thickness of different particle sizes and shapes were studied

and capability of the particles for multiplexing was determined. The study showed that a

significant excess of mPEG-SH has to be used in order to form homogenous silica shells because

the mPEG has to replace most of the CTAB structures.

“Simulation of the Optical Absorption Spectra of Gold Nanorods as a Function of Their

Aspect Ratio and the Effect of the Medium Dielectric Constant”[16]

This studied provided a theoretical background on the relationship between the absorption of

gold nanorods and its longitudinal plasmon resonance. The researchers showed that the dielectric

constant also affects the maximum absorption wavelength. Thermal reshaping was used to

examine the dependence of medium dielectric constant vs the aspect ratio and the maximum

absorption. This research is relevant to the project as it shows the parameters that would alter the

absorption maximum of gold nanorods, such as PEGylation, as well as solvents with various

dielectric constants.

12

“Spectroscopic determination of the melting energy of a gold nanorod” [17]

The melting energy of a single gold nanorod was studied in colloidal solution. The study showed

that gold nanorods have a threshold pulse energy level. Below the threshold level, it would take

significantly higher number of pulses to melt gold nanorods. Above the threshold level, the

nanorods would take pulses to melt. This paper examined the melting characteristics of gold

nanorods and theoretically calculated the energy required to melt a single nanorod. It is

important for the current study because it provides the ranges of pulses and power to initiate

melting experiments.

“Surface Plasmon Resonance Scattering and Absorption of anti-EGFR Antibody Conjugated

Gold Nanoparticles in Cancer Diagnostics: Applications in Oral Cancer” [18]

The surface plasmon resonance of gold nanoparticles was utilized in this study to label oral

epithelial cell lines. The gold nanorods were conjugated with anti-epidermal growth factor

receptor antibodies in order to specifically bind to the malignant cells. The study showed that the

nanorods had over 600% greater affinity to cancer cells than the controls. The study also

supported the use of light scattering gold nanoparticles to label cancer cells and ultimately

diagnose oral cancer.

“Hyperthermic effects of gold nanorods on tumor cells”[19]

This research article evaluated the effects of plasmonic gold nanorods for the multifunctional

purpose of image-guided therapy and inducing localized hyperthermia for cancer therapy. The

study showed that an increase of 5 degrees Celsius can induce denaturation of proteins and

organized biomolecular assemblies in cancer cells. Human KB epithelium cells were used and

13

cultured for two photon excitation at 1mW. The study showed that nanorods do not need to be

internalized by the cells inorder to cause hyperthermic damage. In fact, the membrane of cells is

the most susceptible to thermal ablation.

“Single-Step Coating of Mesoporous Silica on Cetyltrimethyl Ammonium Bromide-Capped

Nanoparticles” [20]

Mesoporous silica is coated on CTAB capped nanoparticles with different thicknesses. This

study shows that silica can be directly coated on CTAB nanoparticles without an intermediate

step. The authors outline a simple procedure to coat CTAB nanoparticles with silica via direct

addition of TEOS in a basic solution with constant stirring. The silica shells were found to be

uniform and the thickness increased with reaction time. This study also showed that increasing

layers of silica formed on gold nanorods create a spherical shape.

“Gold Nanorods: From Synthesis and Properties to Biological and Biomedical Applications”

[5]

This review paper is an excellent source of primary information regarding the characteristics of

gold nanorods and the basic theory of its plasmonic and photothermal properties. In addition, the

review also examines several vital articles on the study of gold nanorods as well as synthesis and

testing procedures. A discussion on the end use of gold nanorods in biomedical applications

such as gene delivery, photothermal therapy, molecular imaging and biosensing is also provided.

This review provides the general background information required to conduct the current study.

“Silica-Coating and Hydrophobation of CTAB-Stabilized Gold Nanorods”[21]

14

This article examines silica coating of gold nanorods covered with layer by layer wrapping of

polyelectronic polymers such as the negatively charged polystyrene sulfonate (PSS) and the

positively charged poly allylamine hydrochloride (PAH). The final polymer layer consists of

PVP, a negatively charged polymer that enables redispersion of the particles in propanol. The

Stober method was used to create silica shells of different thicknesses. The study also shows that

the longitudinal surface plasmon band of the gold nanorods red shifts after coating because the

silica alters the local refractive index.

“Enhanced thermal stability of silica-coated gold nanorods for photoacoustic imaging and

image-guided therapy”[22]

Thermal stability of silica coated gold nanorods was examined in this study by means of testing

for the particles’ photoacoustic response and optical absorption. Nanosecond laser pulses with

different fluences were used to irradiate the particles and UV-Vis and transmission electron

microscopy (TEM) were used to record the particle responses. It was shown that gold nanorod

core length shrunk during laser-pulse exposure but the silica retained its shape, suggesting that

the gold nanorods may be losing atoms. This is a useful comparison study between silica coated

gold nanorods and uncoated nanorods under photothermal excitation conditions.

“Colloidal Dispersions of Gold Rods: Synthesis and Optical Properties”[23]

This theoretical paper shows the absorption characteristics of gold nanorods of different lengths

and aspect ratios. A mathematical model is provided to calculate the absorption wavelength of

different shaped nanorods based on their length and diameter ratio. The authors showed that

increasing anisotropy of nanorods would red shift the maximum longitudinal wavelengths. This

15

is an important study in that it shows the relationship between nanorod aspect ratio and

absorbance wavelength. The study also provides a mathematical basis to calculate the

absorbance of randomly oriented nanorods in solution.

“Gold nanorods: Synthesis, characterization and applications”[5]

This paper provides a mathematical framework to examine the various effects of gold nanorods

including changes in aspect ratio, solvent polarization, silica shell thickness, etc. Calculated

spectra for gold nanorods of various silica shell thickness is made. Not only such, this review

also explains the reactive effects of silica coating as well as laser-induced structural and

morphological transitions. The review covers behaviors of gold nanorods under laser excitation

of various fluences, pulse widths, frequencies, etc. The theoretical information provides

important background information for the current study.

“Synthesis, Characterization, and Self –Assembly of Size Tunable Gold Nanorods” [24]

This dissertation conducts a thorough characterization, synthesis of gold nanorods. It not only

provides the theoretical background but includes experimental procedures for multiple synthesis

methods. The author conducts an in-depth analysis of the seed method of growing gold nanorods

and explains the role of CTAB on the formation of gold nanorods in great detail. This

dissertation is useful for the current project in that it also delves into various separation methods

and detailed experiment protocols.

16

“A Golden Bullet? Selective Targeting of Toxoplasma gondii Tachyzoites Using Antibody

Functionalized Gold Nanorods”[11]

This paper shows a practical application of gold nanorod in the photothermal destruction of

parasitic protozoan. The study consists of a targeted application of gold nanorods conjugated

with antibodies that target to the model protozoan Toxoplasma gondii. Results showed that a

significant reduction in the model survivability was achieved via photothermal therapy at a dose

of ~51W/cm^2.

“PNIPAM Gel-Coated Gold Nanorods for Targeted Delivery Responding to a Near-Infrared

Laser”[25]

This is another example of an end use application of gold nanorods for photothermal therapy. In

this case, gold nanorod was encapsulated in a poly (N-isopropylacrylamide) nanogel. A laser

power of 2.1 W/cm^2 was used, and the authors calculated the input energy required to raise the

gold nanorods by 20 degrees. In addition, the authors also used silica-coated nanorods with a

thickness of 16.7 nm. The longitudinal oscillation increased with the silica shell modified

nanorods. The study showed that when systemically injected, the gold nanorods resided mostly

in the blood in after 10 minutes and lungs after 30 minutes.

“Seed-Mediated Synthesis of Gold Nanorods: Role of the Size and Nature of the Seed”[26]

The paper conducts a thorough study on the seed-mediated method of synthesizing gold

nanorods. The authors examined how the sizes of seeds ultimately affect the type or length of

gold nanoparticles that are synthesized. The study showed that the aspect ratio of nanorods

17

increases as the seed size decreases. They also show that and negatively charged seeds have a

larger effect on changes in dimensions of nanorods at different aspect ratios.

“Preparation and Growth Mechanism of Gold Nanorods (NRs) Using Seed-Mediated Growth

Method”[27]

This article discusses an adapted method of synthesizing gold nanorods from Jana’s original

2001 paper. The author uses CTAB instead of the citrate capped stabilizing agent for seed

growth. For higher ratios of nanorods, the paper also discusses the use of

benzyldimethylhexadecylammoniumchlrodie (BDAC) for mixing along with CTAB. The paper

explains the mechanism by which gold nanorods form from seeds, and the contributions that

CTAB makes in coordinating growth on the ends instead of sides.

“Synthesis of Nanosized Gold-Silica Core-Shell Particles”[28]

Research on the coating of gold nanoparticles with silica at controlled shell thicknesses utilizing

the Stober growth method in ethanol. Study showed that it is possible to manipulate shell

thickness via additions tetraethoxysiline (TES). Research showed that concentrations of TES and

the ethanol/water solvent ratio is critical for the formation of homogenous single core silica

coated particles.

“Synthesis, Assembly, and Biofunctionalization of Silica-Coated Gold Nanorods for

Colorimetric Biosensing” [29]

Research showed the synthesis of silica coated gold nanorods with a modified Stober process for

the optical detection of h igG. Films of silica modified gold nanorods with bioconjugated anti-h

18

igG were fixed onto poly (vinylpyridine) PVV modified quartz substrates. It was shown that the

h-igGs were able to be detected colormetrically as well as by the naked-eye. The authors further

show that it is critical to remove as much unbound CTAB as possible before beginning silica

coating and that vortexing during the reaction is more efficient at producing well defined

particles than other mechanical stirring methods.

“Two-Photon Luminescence Imaging of Cancer Cells Using Molecularly Targeted Gold

Nanorods”[30]

This article demonstrates use of gold nanorods for epithelial cancer cell imaging in tissue

phantom at depth of 75 micrometers. The nanorods were coated with polystyrene sulfonate (PSS)

and functionalized with anti-EGFR antibodies. Study showed that gold nanorods can be used as

bright contrast agents for two photon laser imaging in thick tissues. The excitation wavelength of

the laser was tuned to the longitudinal plasmon resonance frequency and the laser power was

increased 26% with each 20 micrometer depth to maintain similar emission intensity.

c. Previous relevant work:

Encapsulation of Single Quantum Dots with Mesoporous Silica:

A method to encapsulate Quantum Dots (QDs) with mesoporous silica has been developed in the

Gao Lab. Unlike the traditional sol-gel or the microemulsion methods, this new technology has a

simpler procedure that can synthesize particles with high monodispersity, luminescence, stability

and tunable shell thickness. The rationale of this new synthesis method is to first coal Quantum

Dots with CTAB in order to transfer the QDs from chloroform into an aqueous solution. This is

accomplished when the alkyl groups of CTAB intercalate with the QD surface ligands through

19

hydrophobic interactions and the amino head group of CTAB becomes positively charged.

Following addition of CTAB, silane compounds would be able to form mesopores on the CTAB

surface. The thickness of the silica shell is manipulated by varying the ratio of QD/ Silane ratios

and silica between 10 to 30 nanometers are able to be coated. The silica coating of Quantum dots

shows a new method of using silica to coat nanoparticles that may be applied to other objects.

Seed Mediated Gold Nanorod Synthesis:

There are two main methods of synthesizing gold Nanorods, top-down method and bottom up

method. Top down methods mainly consists of lithography methods to product gold nanorods.

With this method, electron-beam lithography or focused ion beam lithography can be used to

create nanostructures either a pre-shaped template by either depositing or removing gold ions.

Although lithographic methods can create highly specific particle shapes, they are generally

experimentally taxing and inefficient for mass production. On the other hand, the seed mediated

gold nanorod synthesis is a chemical reduction method that is able to create highly monodisperse

nanorods quickly and with large yields. The seed mediated method has been used in the Gao lab

to coat gold nanoparticles with silica. The rational of this synthesis method is to reduce gold ions

on gold nanoparticle seeds in the presence of a surfactant and silver ions. The seeding solution is

first created using a strong reducing agent to synthesize small gold nanospheres, and

subsequently placed into a solution with a milder reducing agent. In the second reaction, gold

ions are slowly reduced onto the original seeds in an anisotropic fashion.

20

Sol Gel Silica Coating on PEG Capped Nanorods:

Coating of silica on metal nanoparticles has always been difficult because of the low affinity

between the two materials. Capping agents has traditionally been used to enhance the deposition

of silica on the particle surface. The Stober method of coating is a well-known sol-gel method;

however its reaction requires the stabilization of the particles in ethanol. Research in this area has

been in search of a stabilization agent can prime the particle surface. Previously, materials such

as poly(vinylpyrrolidone) (PVP) (page 2 of Highly controlled), or

polyoxyethylene(5)nonylpheny ether has been used to passivate particle surfaces, however the

most recent development in the area for the coating of CTAB stabilized particles has been the

use of thiol-functionalized polyethylene glycol (mPEG-SH).

PEG is a non-toxic material that is traditionally used to stabilize nanoparticles in aqueous

solutions. PEG-modified particles have also shown to be resistant against nonspecific protein

adsoption, and resistance to clearance by the reticuloendothelial system. Recent works by

Fernadez-Lopez et al. has also shown that PEG coating is also proficient at solubilizing particles

in ethanol [15]. This phenomenon is taken advantage of to coating nanoparticles for Stober

reaction. The use of thiolated PEG is to take advantage of the well-established thio-gold

chemistry. Studies have shown that mPEG-SH, with its stronger activity with gold nanoparticles,

is able to replace the CTAB coating on gold nanorods. Furthermore, PEG also has strong affinity

for silica as the ether oxygen of PEG can readily bind to silanol through hydrogen bonding. The

previous research on sol gel synthesis silica coated nanorods is an essential aid to the present

research.

21

d. Technical issues at the outset of the project:

Several outstanding issues at the outset of the project have to do with synthesis of high quality

monodisperse silica coated gold nanorods at controlled shell thicknesses. Among of the

difficulties in the synthesis is the loss of particles during washing, aggregation problems,

controlling proper aspect ratios of nanorods and control of shell thickness.

One of the major problems in nanoparticle research is the loss of particles during synthesis,

conjugation or reactions that require washing steps. These problems could be caused by lack of

careful pipetting, inadequate centrifuging speeds, adhesion of particles on reaction vial walls, etc.

Loss of particles can often cause overuse of chemicals in subsequent reactions or too little of the

chemicals remaining for proper analysis (i.e. out of limit of detection of analysis machines). In

order to minimize these problems, it is necessary that the researcher critically examine the

process to determine where the particles are being loss and how to minimize the losses.

Another technical issue is aggregation problems. Nanoparticles tend to aggregate when

destabilized in solution (i.e. when the attractive forces between the particles become greater than

the repulsive forces). This can be prevented by incorporating steric stabilization or coating with

charged polymers. For example, increasing CTAB concentration or PEG conjugation would both

stabilize gold nanorods by increasing charge density of the solution or particle respectively.

Control of nanorod aspect ratio and shell thickness are both problems that need to be addressed

before tests with laser ablation. The difficulty in both processes is that each reaction may be

22

easily altered due to slight variations in synthesis procedures and experimental conditions.

Therefore, precautions need to be taken to ensure repeatability of test conditions.

CTAB purification can also be problematic because the procedure requires the removal of most

CTAB from solution before proceeding to PEG replacement. However, a careful balance must be

achieved because excess CTAB removal would destabilize the gold nanorods causing

aggregations. The washing steps after gold nanorod synthesis, therefore, must be carefully in

order to achieve a balance between particle stability and reaction efficiency.

Analytical methods:

Another major technical challenge to this project is the requirement of heavy analysis tools such

as the Transmission Electron Microscope (TEM) and pulse laser. TEM is required to verify the

physical structure of the synthesized particles and the pulse laser is used to conduct thermal

heating experiments. Due to the fact that TEM and pulse laser are not a common laboratory

tools, it would be difficult to obtain results quickly and efficiently. In combination, it takes

considerable time and effort to obtain the use of these devices.

B. DESIGN OF TOOLS, DEVICES, AND EXPERIMENTS:

1. Overview of design and research plan:

The silica coated gold nanorod is to solve the problem of thermal reshaping of gold nanorods

during photothermal stimulation. Research has shown that when gold nanorods changes shape or

fragments when it is unable to efficiently disperse the absorbed energy. This reshaping causes

nanorods to go under structural changes to melt into spherical particles or fragments into smaller

particle[31].The melting of gold nanorods into spherical particles necessarily changes the particle

23

aspect ratio and alters its absorption wavelength. The implication of this change is that the same

nanorods cannot be excited furthermore by the excitation wavelength and thus losing its

photothermal activity.

The overall aim of this design is to solve the issue of thermal reshaping of gold nanorods and

prolong the lifetime of nanorods being excited at its native absorption wavelength. Additionally,

the particles need to retain its functionality as a bioconjugation template and have minimal

cytotoxicity effects. More specifically, 1) the silica coated gold nanorod must be soluble in

aqueous solution, 2) the silica shell needs to protect the encapsulated gold nanorod from melting

under pulsed excitation, thereby maintaining the same absorption wavelength of the gold

nanorod 3) the silica shell cannot significantly obstruct absorption of light into the rods 4) the

silica gold nanorod needs to be monodisperse, free from aggregation, and have a hydrodynamic

size of approximately 100 nm.

In order to achieve this design, synthesis of monodisperse gold nanorods with similar aspect

ratios, and thereby similar absorption wavelengths needs to be achieved in order to perform a

significant comparison study. Subsequently, silica shells of different thicknesses will need to be

synthesized. Sets of particles of different thicknesses would be tested under pulsed laser at

various relevant photothermal excitation energies to select the design that provides optimum

protection from nanorod reshaping.

Tuning silica with different thicknesses can be controlled by control of reaction conditions

during the Stober synthesis process. More specifically, parameters such as the TEOS to nanorod

24

ratio, reaction time, amount of reducing agent are all variables that can be controlled to produce

particles of different thicknesses. Alternate design considerations include synthesizing gold

nanorods of specific aspect ratio with fixed excitation wavelength. This can be controlled by

varying the amount of seeding solutions added to the growth solution in the seed mediated

bottom-up nanorod growth.

Materials and Methods:

Preparation of Gold Nanorods:

Seed-Solution:

The preparation of the seed solution started with a mixture of 7.5 mL of 0.1 M CTAB in water

and 0.10 mL of 25 mM HAuCl4. 0.6 mL of ice cold 0.01 M NaBH4 was added under vigorous

stirring. The solution turned from yellow to brown in color and was then stirred for a few

minutes. This solution was then put into a 27 °C water bath.

Growth-Solution:

The growth solution was prepared by mixing, CTAB (0.1 M, 55 mL) with 1.2 mL HAuCl4 (25

mM) and 0.4 mL AgNO3 (10 mM). After gentle mixing of the solution, 0.6 mL ascorbic acid

(0.1 M) was added as a mild reducing agent. The color of the growth solution changed rapidly

from bright yellow to colorless.

Seed-mediated Growth:

Finally, an amount of 60 μL of gold seed solution was added, and the mixture was swirled, and

then allowed to sit overnight, resulting in a brown-colored suspension of gold nanorods.

25

Silica Coating of Gold Nanorods:

Passivation in Ethanol:

The as-prepared GNRs solution was purified by centrifugation at 9000 rpm for 15 min, the

supernatant was discarded and the precipitate was re-dispersed in 50 mL water. The process was

repeated two more times to remove excess CTAB. 10 mg of mPEG-SH was dissolved in 1 mL

water, sonicated for 10 min, and then 50 μL of 0.1 M NaBH4 solution was added, the mixture

was sonicated for another 15 min. 10 mL of the GNR solution (0.7 nM) was mixed with the

mPEG-SH solution by gentle vortex for 3 min, and the mixture was stirred for 5 h. mPEG-SH

treated GNRs suspension was purified by centrifugation at 8500 rpm for 12 min, dispersed in

water. After washed for 2 times, the GNRs were dispersed into 2.5 mL pure ethanol.

Silica coating of Gold Nanorods:

After adding 0.4 mL of H2O, 15 μL NH4OH (30%) and 4 μL TEOS, the mixture solution was

allowed to stir overnight. The resulted GNR@SiO2 solution was purified with ethanol for three

times.

Laser Excitation and TEM:

Synthesized gold nanorods were first examined by TEM to determine its physical properties and

shell thickness. Subsequently, aliquots of control gold nanorods and two silica coated nanorods

of increasing thicknesses would be placed onto a 96-well plate and loaded onto a pulse-excitation

laser with preset fluence, excitation wavelength, and energy. The samples would be

characterized again under UV-Vis and TEM after laser excitation to look for nanorod reshaping.

26

2. Costs:

Chemical or Equipment Company FW Cost Uses Total

CTAB - decyl trimethyl ammonium bromide, for molecular biology 99% Sigma 346 g/mol in lab n/a $0

NaBH4 - Sodium Borohydride Sigma 37.83 g/mol in lab n/a $0

AgNO3 - Silver Nitrate ACS 99%

Alfar Aesar 169.87 g/mol in lab n/a $0

Ascorbic Acid - L(+) Ascorbic Acid powder JT Baker 176.12 g/mol in lab n/a $0

HAuCl4 - Hydrogen tetrachloroaurate (III) Aldrich 99.90% in lab n/a $0

mPEG-SHLaysan Bio Inc MW 5000 in lab n/a  $0

Copper TEM Grids Grid Size 300 in lab n/a  $0Biology TEM $60 12 $720TEM Training $40 1 $40

Total $760.00

3. Details of Design Process and Statistical Basis for Design of Experiments:

Gold Nanorods:

The goal in the gold nanorod design is to select an aspect ratio that would be within the window

of detection for in the UV-Vis absorption detector. In addition, since PEG functionalization red-

shifts the absorption spectrum of nanorods, it is also important that the shift would be

accommodated.  With the constraint of these two parameters, the longitudinal excitation

wavelength of the gold nanorods needs to be between 500 to 700 nm. Gold nanorod aspect ratio

can be controlled via changing the ratio of seed solution introduced into the growth solution. It is

important to determine the parameters for synthesizing gold nanorods with similar aspect ratios

because the rods that would be used in the next steps should be homogeneous in order to control

27

for excitation wavelength. Statistically, this study would be done in triplicates in order to verify

the relationship between seed to growth solution ratio and aspect ratio of synthesized gold

nanorods.

Silica Coating:

The aim in silica coating is to design a shell that will protect gold nanorods from melting during

laser excitation. Since the melting of gold nanorods would change the aspect ratio of the rods

into spherical particles, the excitation wavelength would inadvertently change as well. The

thickness of the silica coating would affect how much energy it would require for the gold

nanorods to change shape. In order to test the effectiveness of the silica coating, silica coated

particles and regular gold nanorods would be subjected to same experimental conditions. The

laser pulse numbers would be kept the same while the fluence numbers vary. Particles from the

two groups would subsequently be analyzed under UV-Vis and TEM to look for misshaped

particles.

To account for statistical significance, an n = 3 would be used to compare samples of gold

nanorods and silica coated gold nanorods under same laser fluences. The percentage of

misshaped nanorods would be accounted for both before and after laser excitation. Subsequently,

the percentage of misshaped particles from the control group and the experimental group would

be compared with using an unpaired t-test to account for statistical significance. A p-value of

0.05 was chosen to be counted as significant.

28

C. RESULTS:

1. Data and Discussion:

Gold Nanorod Synthesis:

According to the design scheme, gold nanorods were initially synthesized via seed mediated

growth. The targeted longitudinal excitation wavelength window is circa 750- nanometers in

order to accommodate for the shift in spectrum in silica coating. Gold Nanorods were analyzed

with UV-Vis and TEM. The initial seed solution appears dark brown in color, whereas the

growth solution is yellow after the addition of HAuCl4. Specifically, the seed solution was made

by mixing 7.5 mL of CTAB (0.1M) and 48 uL of HAuCl4 at (48 mM) and 1 mL of NaBH4

(0.01M). First, CTAB was dissolved in DIH2O in a 200mL beaker with magnetic stirring on a

hot plate set at 60 degrees C. The solution becomes clear after 10 minutes. Subsequently,

HAuCl4 solution was added drop-wise to a stirring CTAB solution and sodium borohydride was

added afterwards in at once. The sodium borohydride was cool to 20 degrees C before added.

The CTAB-HAuCl4 solution was light yellow before addition of NaBH4 and turns brown after

addition. It is critical that the CTAB solution remains above room temperature to avoid

crystallization of the solution.

The growth solution was synthesized using 45mL CTAB (0.1M) placed in a 50mL conical

centrifuge tube. Subsequently, 515uL HAuCl4 (48mM) was added along with 500uL of AgNO3

(10mM), 300uL of Ascorbic acid (0.1M) and 60uL of (2.4M) HCl. Everything was added

quickly and mixed by inverting the tubes. The addition of ascorbic acid turned the light yellow

solution into a clear and transparent solution. Addition of HCl was used to promote the reaction.

29

The solution was incubated for 15 minutes. Varied amount of seed solution was added to the

growth solution after 15 minutes of incubation time and the solution was placed in a water bath

at 27 degrees C for overnight. Again, it was important that the solution remains above room

temperature at all times during the reaction to avoid crystallization of CTAB. Furthermore, it is

also important that Ascorbic Acid (0.1M) is prepared fresh during each reaction. The failure to

do so lead to an incomplete reduction effect that leads to ineffective nanorod growth (the

absorption wavelength tends to increase).

After 24 hour incubation, the synthesized gold nanorods were purified out of the CTAB solution

via washing with DIH2O. At this time, the gold nanorods have an appearance of either purple or

dark red color. More specifically, the solution was centrifuged for 20 minutes at 9000 RPM. The

supernatant was subsequently removed and the solution re-dispersed in 20 mL DIH2O. This

process was repeated three times. However, it is critical to avoid further washing because

lowering CTAB concentration leads to gold nanorod aggregation. The purification of nanorod

via centrifugation allows selection of higher aspect ratio nanorods. A higher ratio of nanorods to

spherical particles can be interpreted from a higher relative absorbance at longitudinal

wavelength than the transverse wavelength (Figure 2). It is necessary that a highly monodisperse

sample of gold nanorods is synthesized before continuing further.

30

Figure 1: Separation of aggregates and spherical particles and purification of gold nanorods.

0.5 1 1.5 2 2.5 3 3.5 4 4.50

0.5

1

1.5

2

2.5

3

3.5

Purification of Gold Nanorods

1 - GNR, 2- GNR Wash (1) , 3-GNR Wash (2), 4-GNR Wash (3)

Lon

gitu

dina

l to

Tra

nsfe

rsve

Int

ensi

ty

Rat

io

Figure 2: Separation of aggregates and spherical particles. Increase of longitudinal absorption to transverse absorption ratio following multiple washes.

UV-Vis absorption data of synthesized gold nanorods was used to examine the aspect ratio

equation proposed by Yan et al. More specifically, a specific sample of gold nanorods was

31

400 450 500 550 600 650 700 750 800 850 900 950 1000 10500

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3

3.3

3.6GNR - Wash 1GNR - Wash 2GNR - Wash 3GNR

Wavelength [nm]

Ab

sorb

an

ce

Wavelength (nm)

Absorption

Purification of Gold Nanorods

shown to have an excitation wavelength of 705nm using UV-Vis. According to the proposed

equation, the aspect ratio of rods having such a wavelength should be 3 (equation 1). The

equation was shown to be relevant as particles that were examined under TEM were found to

have very similar aspect ratio to the predicted value (figure 4).

Figure 3: Gold nanorod excitation wavelength: 705nm longitudinal and 510nm transverse.

1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 402468

101214

Gold Nanorod Aspect Ratio

Aspect Ratio

Freq

uenc

y

Figure 4: Histogram of aspect ratios found in a sample of gold nanorods. The most frequent is 3:1 longitudinal to transverse.

32

Wavelength (nm)

Absorbance

Gold Nanorod Excitation Peak

mPEG-SH Addition and Silica Coating:

mPEG-SH is conjugated on to the gold nanorods upon the completion of purification. More

specifically, the final gold nanorod solution was dispersed in 25 mL of DI H2O. 10 mg of

5000MW mPEG-SH was thawed and weighed and dissolved in a 1mL H2O solution. The sample

was subsequently sonicated and incubated for 10 minutes. After sonication, this sample, along

with 50uL of (0.1) NaBH4 was added to a 10mL stirring solution of the previously prepared gold

nanorods and incubated for 5 hours. In this procedure, it is important that the mPEG-SH is

thawed before being weighed to avoid under-calculating the actual weight due to frozen water

crystals.

The mPEG-SH gold nanorods were washed in DIH2O two times and transferred to 2.5mL of

ethanol to prepare for sol-gel silica growth. A UV-Vis absorption spectrum was taken of this

sample. A slower spin speed of 8500 RPM was used during the washing step at 25 degrees C and

12 minutes. The washing step was done carefully because particles are easily lost in the material

transfer process. It was generally noticed that the excitation wavelength of mPEG-SH coated

gold nanorods had an excitation wavelength redshift of 10 – 20 nm (Figure 5).

The Stober process started with addition of 0.5 mL of H2O into a stirring ethanol solution of

mPEG-SH to make a 1:5 water to ethanol ratio. Subsequently, 0.5mL of 15uL NH4OH (30%)

was added into the mixing reaction vial. Finally, a varied amount of 99% TEOS between 4uL to

10uL was added into the mPEG-SH gold nanorod solution and incubated overnight. The samples

were taken out of the ethanol solution after incubation, washed two times with ethanol and

imaged with TEM.

33

Results of the silica coating showed that coating of gold nanorods in ethanol resulted in a redshift

in excitation spectrum. This shift is around 30-40nm apart from the original nanorods and 20nm

from the mPEG-SH coated nanorods (Figure 5). The shift can be attributed to the change in local

refractive index of the particles and shows that there has indeed been a surficial coating of silica

particles on the surface of the gold nanorods. Furthermore, TEM images also show a thin layer of

silica coating (5.2 nm +/- 0.4 nm, n = 30) formed uniformly on the outer borders of the gold

nanorods. Initially, the silica coated gold nanorods were subjected to heavy aggregation and

fusion of multiple particles, this problem was solved by shortening the TEOS and NH4OH

reaction from five hours as described in the procedure to two hours. Additional sonication and

lowering the centrifugation speed during washing steps were also essential to obtain

monodisperse particles. Furthermore, difficulties were encountered in the synthesis of thick

shelled silica coated nanorods as the silica coating seemed to be independent of the amount of

initial TEOS and NH4OH added. This study showed that that instead of bulk addition of TEOS

and the reducing agent, it was necessary that separate additions after brief incubation time was

necessary to achieve coating of thicker silica shells (Figure 8).

34

Figure 5: Red shift in excitation wavelength with subsequent coating of gold nanorod with PEG and silica.

0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-5.5 5.5-6.5 6.5-7.5 7.5-8.50

5

10

15

20

25

0 0 02

22

5

0 0

Silica Thickness

Shell Thickness (nm)

Fre

quen

cy

Figure 6: Histogram of silica shell thickness found in a sample of silica coated gold nanorods. Most shell thickness is between 3.5 to 5.5 nm on all sides.

35

400 450 500 550 600 650 700 750 800 850 900 950 1000 10500.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8GNRGNR+ mPEGSHGNR+ mPEGSH + Si

Wavelength [nm]

Abs

orba

nce

Absorbance

Wavelength (nm)

Silica Coating Induced Red Shift

Figure 7: Transmission Electron Microscopy image of 5nm thick silica coated gold nanorods synthesized from single addition of TEOS and NH4OH in the sol-gel growth method.

Figure 8: Transmission Electron Microscopy image of 30nm thick silica coated gold nanorods. This was synthesized using multiple additions of TEOS along with the ammonium hydroxide at subsequent time intervals of two hours.

36

50 nm50 nm

2. Conclusions:

In this study, gold nanorods were produced with high consistency with seed mediated growth.

The theoretical excitation wavelength in relation with actual aspect ratio was predicted by

existing formula and confirmed with UV-Vis and TEM. The process of seed mediated growth

was used to create gold nanorods with highly uniform aspect ratios. The growth procedure was

designed to synthesize gold nanorods within a 600-800nm excitation window in order to

accommodate excitation via infrared laser within that wavelength. Subsequently, the gold

nanorods were primed with mPEG –SH to replace the CTAB stabilizers and made soluble in

ethanol. The sol-gel method of depositing silica on the gold nanorods was used to uniformly

coat gold nanorods with a silica shell. Morphology of the particle was characterized by TEM and

the excitation wavelength by UV-Vis. A visible redshift of the excitation wavelength after silica

coating was apparent due to the change in refractive index of the surface. Finally, the sol-gel

procedure was modified to create gold nanorods with thicker silica shells of over 15 nanometers.

The structure was further confirmed by TEM. This study showed that silica shells may be used to

encapsulate gold nanorods as a cage to protect its morphological integrity during electromagnetic

excitation during photohermal treatments.

3. Suggestions for Future Work:

Silica coated gold nanorods represent a class of stable photothermal inducing agents that may be

used in optical imaging, drug delivery, or other biomedical systems. The current work shows that

those surfaces of nanoparticles may be engineered in order to preserve or expand its original

function. The process of silica coating may be applicable to many other nanoparticles and have

been tried in quantum dots and gold nanospheres. In the future, silica modified gold

37

nanoparticles may be further modified with targeting antibodies or other bio-molecules to

achieve targeted delivery of the photothermal particles. With targeted delivery, particles do not

have to only rely on the enhanced permeability and retention effect to deliver efficacious doses

of agents to cancer cells, but instead, achieve direct and targeted delivery that can minimize the

amount of particles used and cytotoxicity effects.

D. ACKNOWLEDGEMENTS:

I would like to thank my PI, Dr. Xiaohu Gao; my mentor, Emily Hu, and all the members of the

Gao lab for their help and contributions in this project.

E. REFERENCES:

1. (2010) Cancer Facts & Figures 2010.2. (2009) Cancer Facts & Figures 2009.3. Narayanan, R. and M.A. El-Sayed, Catalysis with transition metal nanoparticles in colloidal

solution: nanoparticle shape dependence and stability. J Phys Chem B, 2005. 109(26): p. 12663-76.

4. Narayanan, R. and M.A. El-Sayed, FTIR study of the mode of binding of the reactants on the Pd nanoparticle surface during the catalysis of the Suzuki reaction. J Phys Chem B, 2005. 109(10): p. 4357-60.

5. Huang, X., S. Neretina, and M.A. El-Sayed, Gold Nanorods: From Synthesis and Properties to Biological and Biomedical Applications. Advanced Materials, 2009. 21(48): p. 4880-4910.

6. Yan, B., Yang, and Y. Wang, Comment on “Simulation of the Optical Absorption Spectra of Gold Nanorods as a Function of Their Aspect Ratio and the Effect of the Medium Dielectric Constant”. The Journal of Physical Chemistry B, 2003. 107(34): p. 9159-9159.

7. Pelton, M., et al., Ultrafast resonant optical scattering from single gold nanorods: Large nonlinearities and plasmon saturation. Physical Review B, 2006. 73(15): p. 155419.

8. Muskens, O.L., N. Del Fatti, and F. Vallee, Femtosecond response of a single metal nanoparticle. Nano Lett, 2006. 6(3): p. 552-6.

9. Zweifel, D.A. and A. Wei, Sulfide-Arrested Growth of Gold Nanorods. Chem Mater, 2005. 17(16): p. 4256-4261.

10. Busbee, B., S. Obare, and C. Murphy, An Improved Synthesis of High-Aspect-Ratio Gold Nanorods. Advanced Materials, 2003. 15(5): p. 414-416.

11. Pissuwan, D., et al., A golden bullet? Selective targeting of Toxoplasma gondii tachyzoites using antibody-functionalized gold nanorods. Nano Lett, 2007. 7(12): p. 3808-12.

12. Tong, L., et al., Gold Nanorods Mediate Tumor Cell Death by Compromising Membrane Integrity. Advanced Materials, 2007. 19(20): p. 3136-3141.

13. Norman, R.S., et al., Targeted photothermal lysis of the pathogenic bacteria, Pseudomonas aeruginosa, with gold nanorods. Nano Lett, 2008. 8(1): p. 302-6.

38

14. Huang, X., et al., Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J Am Chem Soc, 2006. 128(6): p. 2115-20.

15. Fernandez-Lopez, C., et al., Highly controlled silica coating of PEG-capped metal nanoparticles and preparation of SERS-encoded particles. Langmuir, 2009. 25(24): p. 13894-9.

16. Link, S., M.B. Mohamed, and M.A. El-Sayed, Simulation of the Optical Absorption Spectra of Gold Nanorods as a Function of Their Aspect Ratio and the Effect of the Medium Dielectric Constant. The Journal of Physical Chemistry B, 1999. 103(16): p. 3073-3077.

17. Link, S. and M.A. El-Sayed, Spectroscopic determination of the melting energy of a gold nanorod. The Journal of Chemical Physics, 2001. 114(5): p. 2362-2368.

18. El-Sayed, I.H., X. Huang, and M.A. El-Sayed, Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer. Nano Lett, 2005. 5(5): p. 829-34.

19. Huff, T.B., et al., Hyperthermic effects of gold nanorods on tumor cells. Nanomedicine (Lond), 2007. 2(1): p. 125-32.

20. Zhan, Q., et al., A study of mesoporous silica-encapsulated gold nanorods as enhanced light scattering probes for cancer cell imaging. Nanotechnology, 2010. 21(5): p. 055704.

21. Pastoriza-Santos, I., J. Pérez-Juste, and L.M. Liz-Marzán, Silica-Coating and Hydrophobation of CTAB-Stabilized Gold Nanorods. Chemistry of Materials, 2006. 18(10): p. 2465-2467.

22. Chen, Y.S., et al., Enhanced thermal stability of silica-coated gold nanorods for photoacoustic imaging and image-guided therapy. Opt Express, 2010. 18(9): p. 8867-78.

23. van der Zande, B.M.I., et al., Colloidal Dispersions of Gold Rods:  Synthesis and Optical Properties. Langmuir, 1999. 16(2): p. 451-458.

24. Park, K., Synthesis, Characterization, and Self-Assembly of Size Tunable Gold Nanorods. 2006.25. Kawano, T., et al., PNIPAM gel-coated gold nanorods for targeted delivery responding to a near-

infrared laser. Bioconjug Chem, 2009. 20(2): p. 209-12.26. Rayavarapu, R.G., et al., Iodide impurities in hexadecyltrimethylammonium bromide (CTAB)

products: lot-lot variations and influence on gold nanorod synthesis. Langmuir, 2010. 26(7): p. 5050-5.

27. Nikoobakht, B. and M.A. El-Sayed, Preparation and Growth Mechanism of Gold Nanorods (NRs) Using Seed-Mediated Growth Method. Chemistry of Materials, 2003. 15(10): p. 1957-1962.

28. Dorozhkin, S.V., Nanosized and nanocrystalline calcium orthophosphates. Acta Biomater, 2010. 6(3): p. 715-34.

29. Wang, C., et al., Synthesis, Assembly, and Biofunctionalization of Silica-Coated Gold Nanorods for Colorimetric Biosensing. Advanced Functional Materials, 2006. 16(13): p. 1673-1678.

30. Durr, N.J., et al., Two-Photon Luminescence Imaging of Cancer Cells Using Molecularly Targeted Gold Nanorods. Nano Letters, 2007. 7(4): p. 941-945.

31. Link, S., et al., Laser Photothermal Melting and Fragmentation of Gold Nanorods: Energy and Laser Pulse-Width Dependence. The Journal of Physical Chemistry A, 1999. 103(9): p. 1165-1170.

39