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Chapter 5. Synthesis of Gold Nanoparticles in Liquid Phase Daeha Seo and Hyunjoon Song

Chapter 5. Synthesis of Gold Nanoparticles in Liquid Phase Daeha Seo and Hyunjoon Song

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November 15, 2012



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Gold Nanoparticles for Physics, Biology and Chemistry



R. Quidant



Fig. 10.3. Transmittance (a, c, and e) and reflectance (b and d) images of engineered tissue constructs

labeled with anti-EGFR/gold conjugates. The tissue constructs consist of densely packed, multiple

layers of cervical cancer (SiHa) cells. The contrast agents were added on top of the tissue phantoms

in 10% PVP solution in PBS (a and b) or in pure PBS (c and d). After incubation for 30 min at

room temperature, the phantoms were transversely sectioned with a Krumdieck tissue slicer, and the

sections were imaged using the Zeiss Leica inverted laser scanning confocal microscope with X10 (a–d)

objective. A small spot on a tissue construct was imaged using X40 oil immersion objective to show

high density of the epithelial cells in the phantom (e). Reflectance images were obtained with 647 nm

excitation. Arrows show the surfaces exposed to the contrast agents. The scale bars are (a–d) 200 µm

and (e) 20 µm (From Ref. 24).



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10.3.1.2 Dark-field microscopy

Beyond the high reflectivity of AuNPs, one can exploit their extraordinary

optical properties associated to their LSPR resonances. A simple way to

use AuNPs as a contrast agent is based on their ability to efficiently scatter

light with frequencies within their plasmon band. El Sayed and coworkers

first suggested fast-screening cancer cells targeted with AuNPs using darkfield microscopy.26 Unlike conventional (bright) transmission microscopy,

in a dark-field microscope, the numerical aperture (NA) of the illumination

condenser does not overlap with the NA of the collection objective lens.

In other words, in absence of any scattering centers, the incident light rays

are not collected and lead to a black (dark) background. This technique

thus enables us to image tiny objects with a large signal over noise. For

instance, a dark field was used to perform the first scattering spectroscopy

of individual plasmonic nanoparticles.27

The data of El Sayed et al. on epithelial cancer cell lines targeted with

gold nanospheres conjugated to anti-EGFR show that this non-scanning, fast

and simple imaging technique enables us to discriminate between cancer

cells and healthy cells after incubation within a solution of the conjugated

AuNPs. Additionally, when combined with linear scattering spectroscopy,

the level of agglomeration of the AuNPs can be assessed. However, the

significant direct scattering from the cell can be a major drawback when

dealing with low concentrations of markers, as it strongly limits the minimum number of AuNPs that can be detected.



10.3.1.3 Enhanced-fluorescence microscopy

Another approach relies on exploiting the ability of AuNPs to enhance

the fluorescence yield of fluorophores located at their vicinity. Through

the adjustment of the particle resonance and the fluorophore-metal distance, one can substantially affect the excitation and decay channels of the

molecule.28−30 For very short distances, typically shorter than 10 nm, the

fluorescence gets quenched as the nonradiative decay channels prevail over

the radiative ones. For larger distances the balance is inverted and fluorescence is enhanced as the result of a combination of processes including

enhanced light absorption, enhanced radiative decay and enhanced reemission of fluorescence to the far field (antenna effect).

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While near-infrared fluorophore-like ICG (Indocyanine Green) has been

extensively used for molecular imaging, it usually suffers from low quantum yields in aqueous media (typically of about 1%) that limit the imaging

sensitivity. Recently, it was shown that the emission of weak NIR fluorophores, such as ICG, could be dramatically enhanced to about 80% by

gold nanoshells consisting of spherical dielectric core coated with a thin gold

layer.31,32 ICG molecules were positioned at an optimum distance of about

10 nm by growing a silica layer around the nanoparticle surface. Interestingly, the spacer layer can also incorporate some Fe3 O4 magnetic nanoparticles that enables using the same complex for both fluorescence imaging and

Magnetic Resonance Imaging (MRI). Despite its lower spatial resolution,

MRI is complementary to fluorescence since it penetrates tissues to depth of

several centimeters. Bardhan and coworkers first demonstrated the suitability of their fluorescent–magnetic contrast agent conjugated to anti-HER2

(Human Epidermal growth factor 2) for in vitro imaging breast cancer cells

that over-express HER2.33 Very recently, the same authors extended their

approach to a mice model34 to track the circulation of the complex through

the mice body over several days. In vivo fluorescent imaging was used to

monitor the concentration of complexes after their injection in the blood

flow (Fig. 10.4). Their data nicely show that due to their bioconjugation,

the complexes remain within the tumor region for longer than in healthy

tissues. There is thus a time window of several hours during which the fluorescent map enables detection and/or treatment (see Section 10.2) of cancer

tissues. It was also observed that the complexes were fully eliminated from

the mice organism after 72 hours.



10.3.2 Nonlinear imaging techniques

Another attractive aspect of plasmonic AuNPs is their ability to dramatically

enhance, through their intense local fields, weak nonlinear optical processes

occurring either in the particle itself or in its vicinity.



10.3.2.1 Multiphoton imaging

Among the nonlinear imaging modalities that can benefit from enhanced

plasmonic fields, let’s first mention Second harmonic generation (SHG)

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Fig. 10.4. In vivo Fluorescence monitoring of gold nanoshell/ICG complexes in mice: (a) NIR images

of mice with HER2 low expressing MDAMB231 xenografts (Top) and HER2 overexpressing BT474AZ

xenografts (Bottom) at 0.3, 2, 4, 24, 48, and 72 h after injection of nanocomplexes. (b) Fluorescence

(FL) intensity of tumor-to-body ratio at different time points of mice with BT474AZ xenografts (n)

6) and MDAMB231 xenografts (n) 3) and showing maximum fluorescence at 4 h. (c) Fluorescence

intensity comparison of tumors only between BT474AZ (n) 6) and MDAMB231 (n) 3) showing 71.5%

increase in signal at 4 h in BT474AZ tumors compared to MDAMB231 tumors, p) 0.003 across tumor

types. (From Ref. 34).



microscopy. SHG (also known as frequency doubling) is a nonlinear optical process, in which photons from a pulsed laser source interacting with

a nonlinear material are effectively “combined” to form new photons with

twice the energy, and therefore half the wavelength of the incident photons.35

A unique feature of SHG is that it requires the presence of an asymmetric distribution of the second harmonic sources (non-centrosymmetry). Thus cell

membranes, their proteins and their crucial contribution to cellular physiology are ideally suited to interrogation with such technique. However,

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SHG is a process with poor efficiency that, in practice, requires advanced

detection schemes. It was shown that complexes formed by nonlinear dyes

attached to AuNPs could be used to strongly enhance SHG at the membrane

of cells.36

Similarly to SHG, Third harmonic generation (THG) microscopy, in

which incident photons at frequency ω lead to photons at 3ω, can be boosted

by plasmon nanooptics. The absence of asymmetry requirements, combined

with the high third-order susceptibility χ(3) of gold,37 makes AuNPs excellent examples of efficient sources of THG for cell imaging, without any

need for additional nonlinear molecules. In the experiment by Yelin and

coworkers, AuNPs were grown into the cells from tiny gold seeds to a size

at which they were resonant with the near infrared light from a femto-second

Titanium-Sapphire laser source.38



10.3.2.2 SERS imaging

Another imaging modality that can strongly benefit from AuNPs is Raman

imaging. Raman spectroscopy is a spectroscopic technique used to study

vibrational, rotational, and other low-frequency modes in a system. It relies

on inelastic scattering, or Raman scattering, of monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range. The

laser light interacts with molecular vibrations, phonons or other excitations

in the system, resulting in the energy of the laser photons being shifted

up or down. Consequently, the shift in energy gives information about the

phonon modes in the system and hence is very powerful at identifying the

presence of given specie via its structural fingerprint. However Raman signal is very weak (typically 1 out of 107 photons) and the integration times

needed render imaging very tedious in practice. Nevertheless, it was shown

that metallic nanostructures could dramatically enhance the Raman cross

section by more than ten orders of magnitude39,40 to reach levels of signal comparable to fluorescence and enable single molecule measurements.

The exact mechanism of the so-called Surface Enhanced Raman Scattering (SERS) has, however, been a matter of debate for many years between

two main theories, based on electromagnetic and chemical mechanisms,

respectively. In the electromagnetic theory, the local field enhancement

near resonant AuNPs augments both the excitation field experienced by

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the molecules and their emitted Raman signal. While the electromagnetic

theory of enhancement can be applied regardless of the molecule being studied, it does not fully explain the magnitude of the enhancement observed in

many systems. The chemical mechanism involves charge transfer between

the chemisorbed species and the metal surface. It only applies to specific

cases though, where the molecules have formed a chemical bond with the

surface. After many years of debate, nowadays it is pretty well accepted that

in many SERS experiments, both mechanisms may coexist.40

While SERS was discovered in the 1970s, it has only recently been

exploited for bioimaging.41 The approach that has been considered by several groups is based on preparing a complex consisting of a gold nanoparticle

combined to an efficient Raman active molecule. After a proper bioconjugation, the complex can be used to target cancer cells to map the distribution

of cancer markers.42−44 Lately, the technique has been extended to in vivo

imaging on a mouse model.45 The main advantage of SERS imaging over

other approaches is the possibility to simultaneously detect multiplex analytes by exploiting the sharper bandwidth of Raman peaks as compared to

fluorescence peaks.



10.3.2.3 Two-photon induced Luminescence

In addition to frequency generation and Raman scattering, there has also

been a growing interest in exploiting a χ(3) -based phenomenon known as

two-photon absorption. Such process is based on the simultaneous absorption of two photons with the same energy that excites the molecules

into higher energy state. In this context, it was shown that the strong

local field resulting from the agglomeration of AuNPs at the surface

of cells could be used to dramatically enhance the two-photon absorption and thus the subsequent fluorescence of adjacent molecules of the

cell membrane.38 Alternatively, one can directly exploit the two-photon

induced luminescence (TPL) from AuNPs. Such a process, first reported

by Mooradian in the 1960s,46 is slightly different from two-photon absorption in molecules, which requires simultaneous absorption of two coherent

photons. TPL-based imaging has recently received lot of attention in the

plasmon nano-optics community as a powerful technique to probe the nearfield optical response of plasmonic nanostructures.47−50 Recently, Durr and

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Fig. 10.5. Two-photon luminescence (TPL) images of cancer cells placed on a coverslip from a cell

suspension. (a) TPL image of unlabeled cells. (b) TPL image of nanorod-labeled cells. Imaging required

9 mW of excitation power in unlabeled cells to get same signal level obtained with only 140 µW for

nanorod labeled cells, indicating that TPL from nanorods can be more than 4000 times brighter than

TPAF from intrinsic fluorophores. (c) TPL image of nonspecifically labeled cells. (From Ref. 51.)



coworkers used TPL microscopy to perform targeted imaging of cancer cells

in three dimensional tissue phantoms.51 In their experiment, gold nanorods

designed to be resonant at 760 nm were bioconjugated with EGFR to target

skin cancer cells (Fig. 10.5). Their data show the distribution of the nanorods

at the cells membrane. Discrete bright spots within the cytoplasm are indicative of endosomal uptake of EGFR receptors with nanorods inside the cells.

More recently, the same approach has been combined with 3D imaging in

vivo to characterize the intestinal blood vessels of a mouse.52



10.3.3 Photo-acoustic imaging

To close this section on the use of AuNPs as contrast agent in bioimaging, we

review recent advances in photo-acoustic microscopy for in vivo imaging.

Photo-acoustic imaging is based on the photo-acoustic effect: upon illumination with laser pulses at optical frequencies, biological tissues adsorb part

of the delivered energy and convert it into heat, leading to transient thermoelastic expansion and thus wideband (e.g., MHz) ultrasonic emission. The

generated ultrasonic waves are then detected by ultrasonic transducers to

form an image with a spatial resolution down to tens of micrometers. Upon

near infrared illumination, the penetration depth is on the order of centimeters making this modality very well suited for in vivo imaging. The optical

absorption in biological tissues can be due to endogenous molecules such

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Fig. 10.6. Ultrasound (a) and photoacoustic (b–f) images of gelatin implants in mouse tissue ex-vivo

at laser illumination wavelength 532, 680, 740, 800, and 860 nm, respectively. The gelatin implants containing the cells with targeted AuNPs (1), control A431 cells (2), the A431 cells mixed with mPEG-SH

coated AuNPs (3), and NIR dye (4) are indicated on the ultrasound image. Tuning the incident wavelength enables discriminating agglomerated AuNPs targeted to cancer cells from the non-targeted

AuNPs. The images measure 44 mm laterally and 11 mm axially (From Ref. 57).



as hemoglobin or melanin. Since blood usually has much larger absorption levels than surrounding tissues, there is sufficient endogenous contrast

for photo-acoustic imaging to visualize blood vessels. Recent studies have

shown that photo-acoustic imaging can be used in vivo for tumor angiogenesis monitoring, blood oxygenation mapping, functional brain imaging,

and skin melanoma detection. However, when aiming at imaging regions

with low absorption, the use of contrast agents is required to provide sufficient signal over noise. In this context, the enhanced absorption of gold

nanoparticles makes them very attractive candidates to boost the ultrasonic

signal.53−55 As a practical example, Wang and coworkers have recently

demonstrated efficient intravascular imaging of macrophages using agglomeration of gold nanospheres as a novel strategy towards monitoring of cardiovascular diseases.56 Interestingly, multi-wavelength imaging (changing

the wavelength of the optical illumination) is an efficient way to discriminate the signal from the agglomerated AuNPs targeted to cancer cells from

the non-targeted AuNPs.57

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In this section, we have reviewed some of the main bio-imaging modalities in which AuNPs can behave as an efficient contrast agent. Lately there

has been a clear tendency towards multimodal plasmonic imaging in which a

single nanoprobe could be used by multiple imaging methods; for instance,

to confirm the development of a disease.

Following this trend, in the following section we will discuss how the

same complexes used for imaging can lead to promising novel cancer therapy based on local photo-heating.



10.4 Photothermal Properties of Gold Nanoparticles

and their Application to Photothermal

Cancer Therapy

In vivo local delivery of heat has raised a growing interest in particular for

local tissue ablation. Conventional methods include laser-induced therapy,

Microwave and Radio Frequency ablation, and magnetic and focused ultrasound ablation. However, all of these approaches suffer from a common

limitation, which arises from the fact that heating is nonspecific, hence it

leads to the damage of healthy tissues. Alternatively, the use of magnetic

fields to heat magnetic particles targeted to tissues was suggested. Similarly,

in 2003 Hirsch and coworkers proposed to use AuNPs as local heat sources

controllable by an external laser illumination.58 As well as ablation, it has

also been suggested to exploit a more moderate temperature increase in

gold complexes for local drug delivery. Interestingly, photothermal therapy

is fully compatible with molecular targeting used for diagnosis and the same

gold complex could thus be used to detect the disease and treat it. In this

last section we first discuss the optimization of heat generation in AuNPs

before reviewing the latest advances in photothermal cancer therapy and

thermal-induced drug delivery.



10.4.1 Optimizing heat generation in gold nanoparticles

When a metal nanoparticle is illuminated, part of the intercepted light is

scattered in the surroundings while the other part gets absorbed and ultimately dissipated into heat.59 The efficiency of each of these processes can

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be characterized by σsca and σabs , the elastic scattering and the absorption

cross-sections, respectively. The sum of these two processes leads to light

attenuation characterized by the extinction cross-section σext :

σext = σsca + σabs



(3)



Depending on the size and the shape of the nanoparticle, the balance between

scattering and absorption can vary substantially.59−62 For instance, while

small gold spheres (<10 nm in diameter) mainly act as invisible nanosources of heat,63,64 scattering processes dominate for diameters larger than

∼50 nm.59 Here, we focus on the absorption processes and the subsequent

heat generation. The general expression of the absorption cross section for

a nanoparticle illuminated by a plane wave is (in mks units):

σabs =



k

ε0 |E0 |2



J(εω )|E(r)|2 dr



(4)



V



where k = 2πn/λ0 = nω/c is the wave vector, n is the refractive index of

the surrounding medium, εω is the permittivity of the nanoparticle material

at frequency ω, E0 the electric field amplitude of the incoming light considered as a plane wave and E(r) the total electric field amplitude. J(εω )

denotes the imaginary part of the dielectric function. The integral is calculated over the nanoparticle volume V. The power of heat generation Q inside

the nanostructure is directly proportional to σabs :

Q = σI = σcε0 |E|2



(5)



where I = ncε0 |E0 |2 is the intensity of the incoming light and using

Equation 10.4 we obtain:

Q=



n2 ω

J(εω )

2



|E(r)|2 dr =

V



q(r) dr



(6)



V



where q(r) = (n2 ω/2)J(εω )|E(r)|2 is the volumetric power density of heat

generation. This expression shows that the quantity of generated heat is

governed by the electric field intensity within the metal. Consequently, the

drastic influence of the particle geometry on the plasmon mode distribution offer some degree of control for designing efficient nano heat sources,

remotely controllable by laser illumination.

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Fig. 10.7. Heat generation in gold nanoparticles: (Left) Evolution of the heat power spectrum with the

particle aspect ratio (at constant gold volume) (Right) 3D mapping of the heat power density computed

for the four nanoparticle shapes at their respective plasmon resonances.



Recently the Green Dyadic Method (GDM)12,13 has been used to quantify the influence of the geometry of a gold AuNPs on its heating efficiency.

The GDM makes it possible to map the spatial distribution of the heat

power density inside the nanoparticles, providing further insight into the

influence of the particle shape and the illumination conditions on the origin

of heat. Figure 10.7 displays calculations of heat power spectra Q(ω) for

different geometries of gold nanoparticles surrounded by water and illuminated by a plane wave. We fix the intensity of the incoming light at

1 mW · µm−2 = 105 W · cm−2 .

To illustrate the influence of the particle geometry, the heat generation

of a sphere progressively elongating into a rod-like structure at a constant

volume (4πreff /3, where reff = 25 nm) is shown in Fig. 10.7 (left panel).

The successive nanorods aspect ratios are 1:1 (sphere), 1.4:1, 2:1 and 3:1.

Two major features arise from the calculations. First the LSP resonance

markedly depends on the nanoparticle shape. A redshift is indeed expected

for nanorods compared with spheres.

Beyond the resonance redshift, a substantial increase of the heating efficiency is observed, by a factor 5 from the sphere to the 3:1 nanorod. The

GDM can be efficiently employed to understand this feature. Figure 10.7

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