Single molecule with dual function on nanogold: Biofunctionalized construct for in vivo photoacoustic imaging and SERS biosensing

Article abstract of DOI:10.1002/adfm.201404341

Multimodal imaging provides complimentary information that is advantageous in studying both cellular and molecular mechanisms in vivo, which has tremendous potential in pre-clinical research and clinical translational imaging. It is desirable to design probes for multimodal imaging that can be administered minimally but provides multifaceted information. Herein, we demonstrate the complementary dual functional ability of a nanoconstruct for molecular imaging in both photoacoustic (PA) and surface-enhanced Raman scattering (SERS) biosensing simultaneously in tandem. To realize this, a group of NIR active organic molecules are designed and synthesized that possess both SERS and PA activity. Nanoconstructs realized by anchoring such molecules onto gold nanoparticles are demonstrated for targeting cancer biomarkers in vivo while providing complimentary information about biodistribution and targeting efficiency. In future, such nanoconstructs could play a major role in identifying surgical margins and also for disease monitoring in translational medicine. A group of NIR active organic molecules are designed and synthesized that possess both surface-enhanced Raman scattering (SERS) and photoacoustic (PA) activity simultaneously. The complementary dual functional ability of the nanoconstruct realized by anchoring such molecules onto gold nanoparticles is used for the targeted detection and imaging of cancer biomarker and also for a biodistribution study using PA imaging and SERS biosensing in tandem.

Full text of DOI:10.1002/adfm.201404341

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Single Molecule with Dual Function on Nanogold:
Biofunctionalized Construct for In Vivo Photoacoustic
Imaging and SERS Biosensing
U. S. Dinish, Zhegang Song, Chris Jun Hui Ho, Ghayathri Balasundaram,
Amalina Binte Ebrahim Attia, Xianmao Lu, Ben Zhong Tang,* Bin Liu,*
and Malini Olivo*
techniques such as fluorescence and bio-
Multimodal imaging provides complimentary information that is advanta-
geous in studying both cellular and molecular mechanisms in vivo, which
has tremendous potential in pre-clinical research and clinical translational
imaging. It is desirable to design probes for multimodal imaging that can be
administered minimally but provides multifaceted information. Herein, we
demonstrate the complementary dual functional ability of a nanoconstruct for
molecular imaging in both photoacoustic (PA) and surface-enhanced Raman
scattering (SERS) biosensing simultaneously in tandem. To realize this, a
group of NIR active organic molecules are designed and synthesized that pos-
sess both SERS and PA activity. Nanoconstructs realized by anchoring such
molecules onto gold nanoparticles are demonstrated for targeting cancer
biomarkers in vivo while providing complimentary information about biodis-
tribution and targeting efficiency. In future, such nanoconstructs could play a
major role in identifying surgical margins and also for disease monitoring in
translational medicine.
luminescence provide the best spatial
resolution, low cost, and ease of operation,
but they are limited by the poor penetra-
tion depth (typically few mm). Moreover,
fluorescence imaging for in vivo studies
is limited by several other factors such as
(i) small Stokes shift and broad spectral
profile that limit the multiplex sensing
in a complex medium, (ii) strong auto-
fluorescence from superficial tissue
layers, and (iii) rapid photobleaching of
fluorophores.[3–6]
In this context, we demonstrate a dual
optical modality molecular imaging and
sensing technique by combining pho-
toacoustic imaging (PAI) and surface-
enhanced Raman scattering (SERS).
PAI is a noninvasive sensitive imaging
modality that taps on the benefit of optical
techniques along with high-frequency
ultra sound in such a way that its key features are benefited
and superior to each of the individual imaging techniques.[7–11]
PAI is realized through the absorption of a pulsed light by a
contrast agent present in the tissue, which causes a rapid
and transient temperature rise (in the order of mK), leading
to a localized thermo-elastic expansion.[12] When a NIR nano-
second pulsed laser beam is scanned through the target to be
imaged, the resultant ultrasonic wave profile can be detected
using special transducers. These data can be used to recon-
struct 2D or 3D optical absorption maps with high spatial res-
olution (<100 µm) and with a penetration depth up to a few
1. Introduction
In vivo molecular imaging and sensing provide the means to
study cellular and molecular processes that have tremendous
potential in biomedical research and clinical applications.[1,2]
Among the various small-animal imaging modalities, even
though each technique has its merits and demerits, no single
technique is capable of achieving the most important require-
ments such as high sensitivity, high spatial and temporal reso-
lution, low cost, fast response time, high-throughput, and high
multiplexing capability.[3] In small-animal imaging, optical
Dr. U. S. Dinish, Dr. C. J. H. Ho, Dr. G. Balasundaram, Dr. A. B. E. Attia,
Prof. M. Olivo
Bio-Optical Imaging Group
Singapore Bioimaging Consortium (SBIC)
Agency for Science, Technology and Research, Singapore
11 Biopolis Way, #01–02, Helios, Singapore 138667
E-mail: malini_olivo@sbic.a-star.edu.sg
Prof. X. Lu, Prof. B. Liu
Department of Chemical and Biomolecular Engineering
4 Engineering Drive 4, National University of Singapore
Singapore 117585
E-mail: cheliub@nus.edu.sg
Prof. B. Liu
Institute of Materials Research and Engineering
Agency for Science, Technology and Research (A*STAR), Singapore
3 Research Link, Singapore 117585
Z. Song, Prof. B. Z. Tang
Department of Chemistry, Division of Biomedical Engineering
The Hong Kong University of Science and Technology
Clear Water Bay, Kowloon, Hong Kong
E-mail: tangbenz@ust.hk
Prof. M. Olivo
School of Physics
National University of Ireland
Galway, Ireland
DOI: 10.1002/adfm.201404341
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centimeters. Furthermore, laser excitation at multiple wave-
lengths (multispectral opto-acoustic tomography (MSOT))
will help in generating multispectral spectroscopic imaging of
intrinsic and extrinsic molecular species in one go.[13] In PAI,
endogenous contrast agents like hemoglobin, melanin, lipids,
or a variety of exogenous molecular imaging agents such as
carbon nanotubes, small molecules, and metallic nanoparticles
can be employed.[14–19] Endogenous contrast agents offer better
tissue access and also help in following transient changes,
while exogenous agents significantly improve the sensitivity
and allow the targeted detection of biochemical species.[20]
On the other hand, SERS is recently being explored as an
effective biosensing optical modality for various pre-clinical
applications due to its inherent ability to generate enhanced
Raman signal from analytes when they are in close proximity
to nanoroughened noble metal surfaces like silver (Ag) or gold
(Au).[21–24] SERS provides the most promising advantages like
multiparameter molecular analysis and multiplexing poten-
tial, which is due to the narrow “fingerprint” Raman spectrum
unique to the chemical species. The in vivo SERS is realized
by SERS-active nanoparticles (NPs) known as SERS nano-
tags, which are constructed by attaching strong Raman-active
reporter molecules (RMs) onto AuNPs[25–28] and encapsulating
them in a polyethylene glycol (PEG)/silica/bovine serum
albumin shell.[3,29–33] The encapsulation provides the physical
robustness, stable signal, protection from the biochemical envi-
ronment, and means for bioconjugation. These nanotags can
be easily functionalized with various receptor moieties for spe-
cific and active in vivo targeting of biomarkers.
The most challenging aspect in combining SERS with PAI
for in vivo molecular imaging is the realization of chromo-
phore/nanoconstruct, which is simultaneously active at both
modalities, i.e., the RMs used to construct SERS nanotag
should be PA-active at the desired excitation wavelength. In one
of the such pioneering works, Kircher et al. used conventional
commercial Raman active molecules such as trans-1,2-bis
(4-pyridyl)-ethylene (BPE) attached to AuNPs. In this nanocon-
struct, BPE provides the SERS spectrum while the plasmon
absorption peak of AuNPs (≈530 nm) is used to generate PA
signal. It is obvious that two separate nanomaterials indepen-
dently act as “excitation probes” for SERS and PAI.[34] Moreover,
since the AuNPs absorb light in the visible region, it has lim-
ited tissue penetration depth. To overcome this, in the second
study, the same research group used Au nanorods (AuNRs),
which have longitudinal surface plasmon resonance (LSPR) in
the NIR region to construct the nanoprobe and used the LSPR
to tap on the PA signal.[20] However, AuNRs have been proved
to have poor photostability upon laser illumination[35] and for in
vivo SERS application, AuNPs are generally preferred.[36]
(4) strong NIR absorption and low fluorescence quantum yield
for deep penetration and strong PA signal; and (5) good solu-
bility in polar solvents for bioconjugation. Specifically, we have
developed three dye molecules based on the Cy7 core. Changing
the heterocycle rings attached to the central core or directly con-
jugating aromatic rings to the central core is expected to shift
the absorption maxima and the characteristic Raman peaks.
As a proof-of-concept, we developed a bimodal imaging nano-
construct by anchoring the synthesized molecules onto AuNPs
followed by encapsulation and bioconjugation to target the
cancer biomarkers in vivo using a murine xenograft model.
Such nanoconstructs demonstrated molecular specific “finger-
print” SERS sensing in vivo, which is complemented with the
imaging capability of PA to understand the localization and
biodistribution of the particles. Antibody conjugated nanocon-
structs (targeted probe) showed maximum tumor localization
by ≈6 h after injection while it was ≈24 h in the case of antibody
free nanoconstruct (nontargeted probe). Moreover, targeted
probe showed longer retention at tumor compared to non-
targeted probe, while both probes showed clearance from the
mouse body by ≈72 h.
Combination of the superb sensing capability of SERS
with deep tissue penetration (≈up to 4–5 cm) of PA imaging
at micrometer resolution can be used to sense and visualize
small quantities of biomarker expression at various stages of
progression or remission of cancer. In future, the complimen-
tary dual functional imaging and sensing capabilities offered by
PA and SERS could help in the identification of the tumor tis-
sues residing under the surface of normal tissues to completely
remove the microscopic tumor deposits and also in determina-
tion of surgical margins in translational research.
2. Results and Discussion
2.1. Synthesis of Cyanine Molecules and their Optical
Characterization
All heptamethine cyanine derivatives were synthesized through
condensation reaction between bisaldehyde (1) and the cor-
responding indolenine or benzothiazole salts in toluene/n-
butanol mixtures (v/v = 3:7).[37] The reactions proceeded at
120 °C for 12 h to yield green to dark green solids, with consid-
erable yields above 80%. Cy7-Cl and Cy7(S)-Cl were synthesized
by 1 and 2 under the condition for the condensation reaction
mentioned above. Cy7-OTPE was prepared by nucleophilic sub-
stitution of the vinyl chlorine on the cyclohexane bridgehead
of Cy-Cl with 3 in a strong basic condition[38] (Scheme 1). All
compounds have been purified and characterized by standard
spectroscopic techniques including NMR and high resolution
mass spectroscopy (HRMS). The UV–vis absorption and pho-
toluminescence (PL) spectra of all the Cy7 derivatives in dime-
thyl sulfoxide (DMSO) are provided in Figure S1 and Figure S2,
Supporting Information. The absorption maxima of Cy7-Cl,
Cy7(S)-Cl, and Cy7-OTPE are at 786, 808, and 778 nm, and their
PL spectra show emission maxima at 820, 835, and 805 nm,
respectively. This difference clearly demonstrates that the slight
modification in molecular structure has obvious impact on
their optical properties.
So far, there is no report on a single molecule that could be
used for simultaneous read outs in both PAI and SERS detec-
tion. In this context, we synthesize a group of NIR-active mol-
ecules that can act as both SERS and PAI read-out probe simul-
taneously. The molecular design takes the following factors into
consideration: (1) inherently high Raman cross section of cya-
nine structure; (2) matching the light absorption of the dye and
laser excitation to favor surface enhanced resonance Raman
scattering (SERRS)[26]; (3) highly symmetric dye structures
to minimize Raman bands for potential spectral multiplexing;
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Scheme 1. Synthetic route to Cy7-Cl, Cy7(S)-Cl, and Cy7-OTPE.
2.2. SERS Activity of the Molecules
it crosses the phantom–water interface. This study shows that
all three cyanine molecules possess both SERS and PA activity
simultaneously. However, due to the physisorption of these
molecules on to AuNPs, SERS signals were not stable, which
should be addressed when developing the nanoconstruct for in
vivo applications. To circumvent this limitation, we chose the
Initially, we tested the SERS activity of these three molecules
(Cy7-Cl, Cy7(S)-Cl, and Cy7-OTPE) after mixing each of them
with 60 nm AuNPs and incubating for 15 min. Strong SERS
signals were observed even at a low laser power of 50 µW and
10 s integration time as shown in Figure 1A. Among these mol-
ecules, Cy7-Cl and Cy7-OTPE produced stronger SERS signals
while Cy7(S)-Cl produced the weakest signal. Since these mol-
ecules possess absorbance in NIR wavelength, 785 nm laser
excitation resulted in a strong signal due to SERRS. However,
there was fluctuation of the signal intensity. This could be
attributed to the nonstable and weaker physisorption of the
RMs onto AuNPs, which may lead to desorption from the sur-
face of AuNPs. To avoid this, covalent anchoring of the RMs
onto AuNPs is necessary when developing SERS-active NPs for
biosensing applications.[26,33]
2.3. PA Activity in Phantom
After demonstrating strong SERS activity of these cyanine mol-
ecules, we tested their PA activity in phantom at a concentra-
tion of 5 × 10−6 M. As shown in Figure 1B–D, the strong bright
spot in the circular well on the left side indicates the PA signal
while the right side channel is filled with control solution. It
was found that Cy7-Cl (Figure 1B) produced the highest PA
signal (6.2×) followed by Cy7-OTPE (Figure 1D, 5.5×), while
Cy7(S)-Cl produced the lowest signal (Figure 1C, 3×), which is
similar to the trend that was observed in the SERS experiment
as well. The strong PA signals on the phantom boundaries
was due to the artifacts caused by the mismatches in optical
properties between the phantom (made up of polyurethane)
and the surrounding water medium, as light is refracted when
Figure 1. A) SERS spectra of the three cyanine molecules under 785 nm
laser excitation. Structures of the molecules are provided. PA signal from
B) Cy7-Cl, C) Cy7(S)-Cl, and D) Cy7-OTPE in phantom. In (B–D), the
bright spot on the left circle represents the PA signal while the right side
circle represents the control.
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Scheme 2. Synthetic route to Cy7-lip.
best molecule, Cy7-Cl that possesses the highest PA and SERS
activity, which is modified to incorporate the lipoic acid (LA)
functional group so that it can covalently attach onto AuNPs to
generate stable signal.
Cy7-lip molecules is compared to other standard RMs such as
malachite green isothiocyanate (MGITC), Cy5, and Rhodamine
6G (Rh6G) under the same experimental conditions (Figure S9,
Supporting Information). It demonstrates that Cy7-lip possesses
≈1 order stronger SERS intensity than the next best molecule,
which is Cy5. We also monitored the stability of the SERS signal
of the Cy7-lip nanoconstruct by measuring the intensity of its
most prominent peak (523 cm−1) over a period of 3 weeks. SERS
signal intensity was very stable with a reduction of only ≈5%
over this period. (Figure S10, Supporting Information).
Any nanoconstruct that possesses absorbance in the NIR
region can be used for in vivo PA imaging and our nanocon-
struct showed absorbance in the NIR region, as shown in
Figure 2B. For comparison, the spectrum of the pure AuNPs is
also provided. Subsequently, we measured the PA activity of the
nanoconstruct in phantom using the pure AuNPs solution as
the control. As shown in Figure 2C, the signal on the left side
of the channel confirms the PA activity of the nanoconstruct
with ≈5-fold stronger signal as compared to the control solution.
Figure 2D shows the transmission electron microscope (TEM)
image of the PEG encapsulated nanoconstruct that demonstrates
mono-dispersity. The PEG encapsulation will help in mini-
mizing the aggregation along with providing a stable encapsula-
tion layer for the RMs. It was also confirmed that after the encap-
sulation, the morphology of the particles remained intact with
an average diameter of ≈60 nm. The hydrodynamic diameter of
the PEG encapsulated nanoconstruct was measured by dynamic
light scattering (DLS) to be ≈87 nm. Furthermore, photostability
of the nanoconstruct under prolonged continuous laser irradia-
tion (in both PA and SERS) for 5 min is shown in Figure S11,
Supporting Information. The obtained stable PA and SERS sig-
nals indicate the excellent photostability of the nanoconstruct.
2.4. Synthesis of Cy7-lip Molecule
Separately, Cy7–2OH was synthesized from 1 and 4, which was
further modified with LA via esterification reaction to furnish
Cy7-lip (Scheme 2). The product was characterized by standard
spectroscopic techniques including NMR and HRMS, which
revealed the right structure with high purity (Figures S3–S8,
Supporting Information). The final product of Cy7-lip shows
similar UV and PL spectra as that of Cy7-Cl indicating that side
chain modification does not obviously affect the optical prop-
erties of the molecule (Figure S1 and Figure S2, Supporting
Information).
2.5. Characterization of Nanoconstruct with Cy7-lip Reporter
Molecule
SERS and PA active nanoconstruct was developed by using Cy7-
lip as the RM and anchoring it onto AuNPs. In order to realize
a stable nanoconstruct, the concentration of the RM anchored
on to the AuNPs must be optimized. To understand this, we
developed the nanoconstruct at various RM concentrations of
0.1 × 10−6, 0.5 × 10−6, 1 × 10−6, 2 × 10−6, 3 × 10−6, 5 × 10−6, and
10 × 10−6 M. At higher concentrations of the RM, NPs showed
aggregation and resulted in lower SERS intensity and also
showed non-repeatable absorption spectra. We found that the
nanoconstruct with optimized 1 × 10−6 M Cy7-lip concentration
resulted in a stable SERS signal. When the concentration of the
Cy7-lip was lower than 1 × 10−6 M, SERS and PA signals from
the nanoconstruct were weak. Due to the covalent binding of the
LA moiety onto AuNPs, the nanoconstruct demonstrated strong
SERS signal as shown in Figure 2A. Strong SERS intensity of the
2.6. Cell Viability of the Nanoconstruct
Before introducing the nanoconstruct into animals, their cyto-
toxicity was assessed on oral squamous carcinoma cell line
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Figure 2. A) SERS spectra of the nanoconstruct with Cy7-lip reporter molecule. B) Absorption spectra of the nanoconstruct. C) PA study of the nano-
construct in phantom – the bright spot on the left represents the PA signal while the right side circle represents the control. D) TEM image of the PEG
encapsulated nanoconstruct.
(OSCC) cells. 100 µL of 1 OD unit of AuNPs, PEG encapsu-
lated nanoconstruct (Probe-PEG), and epidermal growth factor
receptor (EGFR) antibody conjugated nanoconstruct (Probe-w
ab) were incubated with the cells for 3 h and cell viability was
measured after 24 and 48 h. All the particles exhibited close to
100% viability at both time points as shown in Figure 3.
Non-background-corrected maximum intensity projection
(MIP) PA images of transverse slices through the mouse at two
2.7. Multimodal In Vivo Tumor Detection
2.7.1. PA Imaging
Nanoconstruct with Cy7-lip as RM was conjugated with anti-
EGFR antibody for targeted detection of EGFR biomarker in
OSCC xenograft model. This bioconjugated nanoconstruct was
injected (200 µL) into the tail vein of the mouse, which served
as the test group (targeted) animals while animals injected
with nanoconstructs without antibody conjugation served as
the control group (nontargeted) animals. After injection of the
nanoconstruct, both longitudinal PAI and SERS detection were
carried out at the targeted tumor location along with other ana-
tomical organs such as the liver, spleen, and kidney. PA signals
were collected at 6 different excitation wavelengths 680, 700,
750, 800, 850, and 900 nm based on the absorption maxima
and minima of the nanoconstruct, oxy- and deoxy-hemoglobin
at 1, 4, 6, 24, 48, 72, and 96 h post-injection.
Figure 3. Cell viability study using Cell Counting Kit-8 on OSCC cells
exposed to pure AuNPs, PEG encapsulated nanoconstruct (Probe-PEG)
and antibody-conjugated nanoconstruct (Probe-w ab) after 24 and 48 h.
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Figure 4. A) In vivo MIP transverse and coronal images of mouse at t = 6 and 24 h post injection of the nanoconstruct. The white solid arrows and
dotted ellipses show peak probe accumulation at the tumor site for a targeted probe at t = 6 h, and a nontargeted probe at t = 24 h. The low background
MSOT signal (purple) is attributed to intrinsic tissue absorption. Semi-quantitative biodistribution of the probe measured by PA signal intensity at
various time points from B) targeted and C) nontargeted probes.
time points (6 and 24 h post-injection) for targeted and nontar-
geted probe at the tumor site are shown in Figure 4A. It can be
seen that the nanoconstruct undergoing the active targeting at
the tumor location showed maximum signal at 6 h while the
nontargeted nanoconstruct showed maximum tumor accu-
mulation at 24 h. This implies faster localization of the conju-
gated probe, as compared to that of the nontargeted probe at
the tumor. This is mainly due to the weak localization of the
nontargeted probe in the tumor via enhanced permeation and
retention (EPR) effect, where it generally requires 6 to 24 h to
reach the maximum.[39,40] Complete MIP transverse images at
all the time points for both targeted and nontargeted nanocon-
struct are shown in Figures S12 and S13, Supporting Informa-
tion, while corresponding intensity values at a given region of
interest (ROI) in the various organs are plotted in Figure 4B
(targeted) and Figure 4C (nontargeted). The accumulation of
the targeted probe at the tumor site during the first few hours
after injection could be attributed to the active antibody-medi-
ated targeting instead of the EPR effect. Moreover, the PA
signal at the tumor site was stronger (≈3 times) for targeted
probes. Probe signal at tumor site in test group animals showed
gradual reduction after 6 h and negligible signal was observed
at 96 h. On the other hand, nontargeted nanoconstruct showed
relatively shorter retention of the particles at the tumor site,
when compared to targeted probe. Moreover, we also observed
that the peak tumor-to-muscle ratio of the PA signal was ≈5 for
the targeted probe at the 6 h time point.
Semi-quantitative biodistribution and clearance study of the
NPs in the liver and spleen was also successfully studied and
evaluated using PAI. PA signal from the probe could be detected
in the liver and spleen immediately after the injection in both
the test and control group animals. PA signal in the liver and
spleen was found to be much stronger than that from the tumor
location, which is well in agreement with reported in vivo studies
of NPs having size more than 20 nm.[41] Maximum signal was
observed at ≈4–6 h and after that it gradually decreased. In both
test and control group animals, signal was barely detectable
from these passively targeted particles by ≈72 h. We could not
measure any considerable signal from the kidney as well, which
indicates that the clearance of the particle may be via the retic-
uloendothelial system (RES). This is primarily due to the size
of the particle and in typical cases, in order to have clearance
through kidney, the size should be less than 10 nm,[42] while in
our case, the conjugated NPs have a size of ≈60 nm.
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Figure 5. SERS spectra from the tumor site using A) a targeted probe and B) a nontargeted probe at various time points. SERS intensity of 523 cm−1
peak of nanoconstruct is marked. Targeted probe show the maximum signal in tumor at 6 h while for the nontargeted probe it is at 24 h followed by
gradual decrease. Semi-quantitative biodistribution of the nanoconstruct measured by monitoring the intensity of 523 cm−1 peak at various time points
using C) targeted and D) nontargeted probes.
2.7.2. SERS Detection
and might have been excreted through the fecal pathway, which
is in agreement with the report on the excretion of the biocon-
jugated Au-NPs.[43,44] In addition, reticuloendothelial system of
the animal can also phagocyte these types of particles, which can
later be moved into the liver and excreted as feces with bile.[44]
The ultrasensitive sensing capability of SERS is quite useful
in the trace detection of biomarkers. In this context, we used
SERS to compliment the PAI to understand the probe localiza-
tion and retention in the targeted location in vivo. As a molec-
ular sensing tool, SERS could provide better sensitivity than
any imaging modality such as PA. As shown in Figure 5A,B,
actively targeted NPs in the tumor showed the highest signal at
≈6 h, while nontargeted NPs in control group animals showed
the maximum signal at ≈24 h followed by gradual decrease
(SERS spectra are acquired in the 400–1000 cm−1 region). Semi-
quantitative longitudinal SERS intensity response of both the
targeted and nontargeted probes are shown in Figure 5C,D, by
measuring the intensity of 523 cm−1 peak of the nanoconstruct.
In test group animals, probe signal at tumor was ≈2.5 times
stronger than that in control animals. Since in vivo SERS detec-
tion was achieved through point measurement, there may be a
variation in absolute intensity at different depths of the tumor.
As we observed in PAI, SERS signals in tumor also showed
similar trend with longer retention in the case of targeted
probes, when compared to that in the nontargeted probes.
Semi-quantitative biodistribution of the NPs in the liver and
spleen followed exactly similar trend as that observed in PAI.
SERS measurement revealed that signal from the spleen was
slightly higher than that from liver. SERS signal from these
organs in test and control groups showed the highest signal
intensities at ≈4–6 h and then decreased gradually with no detect-
able signal by ≈72 h (Figure 5C,D). Our observation in PAI and
SERS suggests that these NPs could be taken up by Kupffer cells
3. Conclusions
We designed and synthesized a group of NIR active cyanine
molecules, which concurrently exhibit both strong Raman
and PA activities. Among these three molecules, Cy7-Cl pro-
duced the highest PA and SERS signal. Subsequently this mol-
ecule was modified with LA moiety and used to construct the
nanoprobe to be simultaneously used in both PAI and SERS
sensing. This nanoconstruct showed excellent stability in terms
of SERS and PA signal up to 3 weeks. Antibody was conjugated
to this nanoconstruct for targeted detection of the biomarker
in vivo. This bioconjugated nanoconstruct exhibited excellent
cell viability. As a proof-of-concept, in vivo noninvasive molec-
ular imaging and sensing of EGFR biomarker was carried out
in a mouse xenograft model. Our study showed that targeted
probes exhibited the maximum accumulation in tumor by ≈6 h
post injection and led to longer retention. On the other hand,
nontargeted probes showed the maximum signal in tumor
by ≈24 h, which is mainly due to the passive localization via
EPR. Moreover, these particles exhibited shorter retention
compared to the targeted probe. This trend was validated and
confirmed by both SERS sensing and PAI. Semi-quantitative
biodistribution of the NPs in liver and spleen showed that these
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bioconjugated nanoconstruct was stored at 4 °C. On average, there are
≈500 antibodies anchored to each AuNP.
particles might have been cleared from the mouse body by
≈72 h, possibly via fecal pathway.
Cell Culture and Cell Viability: Human metastatic oral squamous
carcinoma cell line (OSCC) was maintained in DMEM supplemented
with 10% fetal bovine serum (FBS) and 1% antibiotic – antimycotic
solution. Cells were used for cytotoxicity assay at 80% confluence. For
cell viability study, 1 × 104 cells were seeded per well in a 96 well plate.
100 µL of 1 OD unit of AuNPs and PEG encapsulated nanoconstruct
formulations with and without antibody conjugation were added in at
least triplicates and incubated for 3 h. After discarding the free nanotags,
cells were replenished with complete DMEM, incubated for 24 and
48 h and measured for cell viability using Cell Counting Kit – 8 (CCK-8,
Sigma). In this study, cells incubated with AuNPs were used as control.
Mouse Xenograft: Balb/c nude female mice (4–6 weeks old) obtained
from the Biological Resource Centre (Biomedical Sciences Institute,
A*STAR) were inoculated subcutaneously on the right flank with
5 × 106 OSCC cells mixed in 1:1 ratio with Matrigel (BD Biosciences) in
a volume of 100 µL. After 2 weeks, when the tumors grew to a palpable
size, 200 µL of the antibody conjugated (test group, targeted) and
nonantibody conjugated (control group, nontargeted) nanoconstruct
were injected into the tail vein of the mouse. PAI and SERS experiments
were conducted in total of 4 animals. All animal experimental procedures
were performed in accordance with protocol #120774 approved by the
Institutional Animal Care and Use Committee (IACUC).
PA Imaging (Instrumentation): All PA imaging experiments were
performed using a commercial 128-channel real-time multispectral
optoacoustic tomographic (MSOT) imaging system (iThera Medical
GmbH). Optical excitation was provided by an optical parametric
oscillator with a tunable NIR wavelength range from 680 to 980 nm,
which was in turn pumped by a Q-switched Nd: YAG laser with a pulse
duration of 10 ns and repetition rate of 10 Hz. Light was delivered by a
fiber bundle divided into 10 output arms to illuminate the sample from
multiple angles around the imaging plane. PA signals were acquired
using a 128-element concave transducer array spanning a circular arc
of 270°. This transducer array has a central frequency of 5 MHz, which
gives a transverse spatial resolution of ≈150–200 µm. One transverse
image slice was acquired from each laser pulse, resulting in a frame-rate
of 10 Hz. During image acquisition, the sample was translated through
the transducer array along its axis across the volume ROI, in order
to capture the corresponding transverse image slices. All the image
reconstruction and post processing of data was carried out using the
associated View MSOT software version 3.2.
In future, we envision that the complimentary imaging and
sensing capabilities offered by PA and SERS could help in the
identification of the tumor tissues residing under the surface of
normal tissues and further to completely remove microscopic
tumor deposits. This dual imaging modality could offer both
diagnostic and prognostic imaging options along with image-
guided resection.
4. Experimental Section
Materials: All chemicals and reagents were commercially available
and used as received without further purification. Anhydrous N,N-
dimethylformamide (DMF) and acetonitrile (ACN) were purified by
Innovative Solvent Purification System. Toluene and dichloromethane
(DCM) were distilled from sodium benzophenone ketyl and calcium
hydride, respectively, under nitrogen immediately prior to use. n-Butanol
(n-BuOH) was dried by anhydrous magnesium sulfate before use.
Dicyclohexylcarbodiimide (DCC), lipoic acid (LA), and iodoethanol were
purchased from Aldrich. Phosphorus oxychloride (POCl3) and p-toluene
sulfonic acid (TsOH) monohydrate were purchased from International
Laboratory and Meryer, respectively. Commercially available MGITC (Life
technologies) and Rh6G (sigma) were purchased. Cy5 was synthesized
in the lab.[45]
Absorbance Measurement: UV–vis absorption spectra were obtained
with Hitachi U-2900 spectrometer with a double-beam optical system and
spectral band pass of 1.5 nm and the measured spectrum was used as an
input spectrum to be used for multispectral post-processing of PA signals.
TEM and DLS Measurement: TEM images were taken using the JEOL
JEM-1010 machine. Acceleration voltage was 40–100 kV and stability
was 2 ppm min−1. The magnification range of the instrument covers
from 50 to 600 000× with a resolution of 0.3000 nm. Images of the PEG
encapsulated nanoconstructs were taken at a 250 000× magnification.
DLS measurement was carried out using Zetasizer (Malvern
instruments, Nano-ZS).
Preparation of Nanoconstruct and Bioconjunction: The nanoconstruct
was realized with Cy7-lip as RM. Cy7-lip (dissolved in DMSO) solutions
at various concentrations (100 × 10−6, 50 × 10−6, 30 × 10−6, 20 × 10−6
,
10 × 10−6, 5 × 10−6, and 1 × 10−6 M) were mixed with 60 nm AuNPs
(BB International, 2.6 × 1010 particles mL−1) in 1:9 v/v ratio for 15 min.
Optimization of the concentration of RM was carried out to yield stable
and high SERS signal with minimal colloidal aggregation. LA linker
on the RM can chemisorb on to AuNPs by thiol linkage and results
in stable SERS signal.[45] Excess and unbound RMs were removed by
centrifugation. The number of RMs attached onto AuNPs was estimated
to be ≈13000, which is in accordance with previous similar reports.[29,30]
We then employed PEG encapsulation for antibody conjugation and
also for the protection of the nanoconstruct. Thiolated-carboxylated
PEG (HS-PEG-CO2H, 10 × 10−6 M, RAPP Polymere GmbH) was first
added drop by drop to the AuNP-RM conjugate and mixed for 20 min.
Subsequently, thiolated PEG (PEG-SH, 10 × 10−6 M, RAPP Polymere
GmbH) was added slowly to the mixture and incubated for 3 h. Later, the
solution was centrifuged to remove the excess PEG and re-suspended in
PBS. To achieve antibody bioconjugation on the particles, carboxylic acid
functional group on the surface of these PEG encapsulated particles was
activated by ethyl dimethylaminopropyl carbodiimide (EDC) and sulfo-N-
hydroxysuccinimide (NHS) coupling reaction. To achieve this, 10 µL of
EDC (25 × 10−3 M in water) and NHS (25 × 10−3 M in water) were added
to the PEG encapsulated NP solution and mixed for 20 min. The excess
amount of EDC and NHS was removed by centrifugation and then the
solution was re-suspended in PBS. Finally, anti-EGFR antibody (100 µL,
200 µg mL−1, Santa Cruz, sc-120) was reacted with activated with Cy7-lip
nanoconstruct at 25 °C for 2 h and then kept for overnight incubation
at 4 °C. Finally, nonspecifically bound antibodies were removed and the
Phantom Measurement: In order to evaluate the PA activity
of the cyanine molecules, we performed the study in a phantom
(iThera Medical). This phantom is cylindrical in shape and made of
polyurethrane material, with tissue-mimicking optical properties. It has
a diameter of 2 cm and contains 2 channels in which test molecules and
control can be placed. Initially, all the three molecules (Cy7-Cl, Cy7 (S)-
Cl, and Cy7-OTPE, all dissolved in DMSO) were tested at a concentration
of 5 × 10−6 M and DMSO was used as the control. Data from multiple
transverse slices across the channel that contains the test molecule and
control were measured within an excitation wavelength scanning range
from 680 to 900 nm, with an interval of 10 nm for each transverse slice.
Averaged PA signals from 10 frames for each wavelength and position
were detected. Similarly, nanoconstruct with Cy7-lip as the reporter
molecule was also tested in the phantom by using AuNPs as the control.
Image reconstruction was carried out to evaluate the PA activity of the
molecule with respect to the control solution.
In Vivo Longitudinal Imaging: Tumor-bearing mice was anaesthetized
under isoflurane and injected with the nanoconstruct via the tail vein.
Subsequently, ultrasound gel was applied on the mouse skin surface and
in vivo imaging was carried out in a temperature-controlled water medium
for optimal acoustic coupling. An animal holder with a thin polyethylene
membrane was used to prevent direct contact between the mouse
and the water. Animal was kept under isoflurane and localization and
biodistribution of the nanoconstruct were studied over time in various
organs. Before image acquisition, a volume ROI consisting of transverse
slices through various organs with a step size of 0.3 mm spanning from
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2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Funct. Mater. 2015,
DOI: 10.1002/adfm.201404341

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the liver to the lower abdomen was selected by manual inspection of
live MSOT images. Six laser wavelengths of 680, 700, 750, 800, 850, and
900 nm were selected for excitation, based on the absorption spectra of
the nanoconstruct and endogenous absorbers such as oxy- and deoxy-
haemoglobin. Multispectral imaging was then performed with 10 signal
averages per wavelength per transverse slice at various time points
(before injection, 1, 4, 6, 24, 48, 72, and 96 h after injection).
Image Reconstruction and Multispectral Processing: Images were
reconstructed using a model-based approach for offline analysis. After
image reconstruction, spectral unmixing was performed to resolve
individual components from different absorbers in the system. For each
pixel in the image, the method fits the total measured optoacoustic
spectrum to the known absorption spectra of the individual absorbers,
based on least-squares linear regression.
Image Processing: Maximum intensity projection (MIP) images at
t = 6 and 24 h were prepared in both transverse and coronal planes
for better display of anatomy and quantification. A MIP is a volume
rendering method for 3D data that projects in the visualization plane
of the voxels with maximum intensity that fall in the way of parallel rays
traced from the viewpoint to the plane of projection. Although a MIP
is a 2D projection of 3D data which results in some information loss,
image correlation and validation between MIPs from both transverse
and coronal planes overcomes this issue and helps to identify regions of
probe localization in various organs and the tumor site.
Acknowledgements
U.S.D. and Z.S. contributed equally to this work. We thank the Singapore
Bioimaging Consortium (SBIC), A*STAR, Singapore Ministry of
Education (R279–000–391–112), Singapore NRF Investigatorship, the
Research Grants Council of Hong Kong (HKUST2/CRF/10), the Ministry
of Science and Technology of China (973 program 2013CB834701), and
Guangdong Innovative Research Team Program (201101C0105067115)
for financial support. The authors also would like to thank Mr. Yu
Yang and Miss Ruchi (Industrial attachment students from Nanyang
Technological University, Singapore) for their help in this project.
Received: December 8, 2014
Revised: February 4, 2015
Published online:
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.
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DOI: 10.1002/adfm.201404341