A cyanine based fluorophore emitting both single photon near-infrared fluorescence and two-photon deep red fluorescence in aqueous solution

Article abstract of DOI:10.1039/c2ob25619c

Optical imaging provides an indispensable way to locate tumors in their early stages with high sensitivity and signal to background ratio. A heptamethine cyanine based fluorophore that emits both single photon near-infrared fluorescence and two-photon deep red fluorescence under physiological conditions was developed. Linear and nonlinear photophysical properties of this fluorophore were investigated and it demonstrated the capability to label lysosomes in cancer cells. The advantages of this fluorophore, including tolerable cytotoxicity, high fluorescence quantum yield, and the ability to emit both near-infrared single photon fluorescence and deep red two photon fluorescence in aqueous solution, give it potential to be used in intra-operatively optical image-guided tumor excision followed by two-photon fluorescence microscopy biopsy analysis after a single administration.

Full text of DOI:10.1039/c2ob25619c

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PAPER
A cyanine based fluorophore emitting both single photon near-infrared
fluorescence and two-photon deep red fluorescence in aqueous solution†
Lu Wang,‡a Jiefu Jin,‡b Xishan Chen,a Hai-Hua Fan,c Billy King Fai Li,c Kok-Wai Cheah,c Ning Ding,a
Shenghong Ju,*d Wing-Tak Wong*e and Cong Li*a
Received 26th March 2012, Accepted 30th May 2012
DOI: 10.1039/c2ob25619c
Optical imaging provides an indispensable way to locate tumors in their early stages with high sensitivity
and signal to background ratio. A heptamethine cyanine based fluorophore that emits both single photon
near-infrared fluorescence and two-photon deep red fluorescence under physiological conditions was
developed. Linear and nonlinear photophysical properties of this fluorophore were investigated and it
demonstrated the capability to label lysosomes in cancer cells. The advantages of this fluorophore,
including tolerable cytotoxicity, high fluorescence quantum yield, and the ability to emit both near-
infrared single photon fluorescence and deep red two photon fluorescence in aqueous solution, give it
potential to be used in intra-operatively optical image-guided tumor excision followed by two-photon
fluorescence microscopy biopsy analysis after a single administration.
very difficult to precisely delineate the margin of a malignant
Introduction
brain tumor, which results in a median survival of below
15 months for patients.4 Therefore, any improvements in com-
Surgery is a primary treatment strategy for most solid tumors.
The completeness of tumor removal during surgery is usually
considered of the most importance to achieve a satisfactory prog-
nosis.1 Currently, the differentiation of neoplastic tissue from
normal tissue relies on the surgeon’s ability and experience
based on subjective criteria that are not easily quantifiable.
However, the absence of a clear and distinctive boundary of
malignant tumor combined with the lack of a screening tool
often results in a compromised therapeutic response.2,3 For
example, due to the heterogeneous and infiltrative nature, it is
plete tumor removal will benefit the therapeutic efficacy for
cancer patients.
Image-guided surgery is the most promising way to remove
the neoplastic tissue while minimizing the damage to the neigh-
boring normal tissues.5,6 Among numerous imaging modalities,
fluorescence-based optical imaging affords advantages in intra-
operative tumor resection due to its superior sensitivity, non-
ionized radiation, short acquisition time and convenience for
manipulation.7 Tsien et al. developed a matrix metalloproteinase
(MMP) responsive fluorescence probe to guide tumor excision
during surgery.8 The experimental results showed that animals
whose tumors were resected under image guidance had better
long-term survival than animals whose tumors were resected
with traditional bright-field illumination.1 Most recently, Ntzia-
christos et al. for the first time demonstrated the feasibility of
optical imaging guided tumor resection in humans.2 After intra-
venous injection of a fluorecein labeled folic acid to ovarian
cancer patients, tumors with a diameter as low as 1.0 mm can be
clearly identified and excised completely. All the excised tissues
were confirmed to be malignant by histopathological analysis.
The key step to advance the clinical translation of optical
image-guided surgery is the development of a probe with higher
sensitivity, targeting specificity and lower toxicity.9,10 Compared
to fluorescein with visible green fluorescence, probes that absorb
and emit in a wavelength range from deep red to near-infrared
(NIR) (650–950 nm) attract much attention because absorbance
and autofluorescence of tissues in this range are low, which
benefits the visualization of lesions with high sensitivity and
aKey Laboratory of Smart Drug Delivery, Ministry of Education & PLA,
School of Pharmacy, Fudan University, Shanghai 201203, China.
E-mail: congli@fudan.edu.cn; Fax: +86 21 5198 0100;
Tel: +86 21 5198 0100
bDepartment of Chemistry, The University of Hong Kong, Pokfulam
Road, Hong Kong
cCentre of Advanced Luminescence Materials, Department of Physics,
Hong Kong Baptist University, Kowloon Tong, Hong Kong, China
dJiangsu Key Laboratory of Molecular and Functional Imaging,
Department of Radiology, Zhongda Hospital, Medical School, Southeast
University, Nanjing 210009, China. E-mail: jsh0836@hotmail.com;
Fax: +86 25 8327 2121; Tel: +86 25 8331 1083
eDepartment of Applied Biology and Chemical Technology, The Hong
Kong Polytechnic University Shenzhen Research Institute, The Hong
Kong Polytechnic University, Hunghom, Kowloon, Hong Kong.
E-mail: bcwtwong@polyu.edu.hk; Fax: +852 2364 9932;
Tel: +852 3440 8789
†Electronic supplementary information (ESI) available: Detailed syn-
thesis and characterization of the fluorophores, photospectroscopy of
1a–1c, in vitro cytotoxicity, SP and TP fluorescence microscopic studies,
Fig. S1–S4, NMR and MS spectra. See DOI: 10.1039/c2ob25619c
‡These authors contributed equally to this work.
5366 | Org. Biomol. Chem., 2012, 10, 5366–5370
This journal is © The Royal Society of Chemistry 2012

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target to background signal ratio.11,12 Additionally, suspected
neoplastic tissues showing high fluorescence during image-
guided surgery need to be pathologically verified. Fluorescence
microscopy is not only helpful to identify the pathological status
of excised tissues, but also useful to delineate the boundary
between the neoplastic and normal tissues with a spatial resol-
ution at a sub-micrometer scale.13 Compared to traditional
single-photon fluorescence microscopy (SPFM), two-photon fluo-
rescence microscopy (TPFM) shows deeper penetration depth
(>500 μm), lower tissue auto-fluorescence and self-absorption,
reduced photodamage and photobleaching, which allows
imaging of deep loci embedded in the tissue sample for a long
period of time without signal attenuation.14 Therefore, a fluoro-
phore that emits both single photon (SP) NIR fluorescence and
two-photon (TP) deep red fluorescence will provide great con-
venience for optical image-guided tumor excision followed by
biopsy examination.
Results and discussion
Design and synthesis of the fluorophores
Scheme 1 General synthetic procedure for the cyanine based
fluorophores.
In this work, heptamethine cyanine derivatives were chosen as a
prototype of the dual functional fluorophores because of their
high extinction coefficient and optimal emission wavelength.15
For example, indocyanine green (ICG) has been approved by
FDA as a diagnostic medicine to determine cardiac output,
hepatic function, liver blood flow, and ophthalmic angiography
for over 30 years with an excellent safety record.16 Heptamethine
cyanine based NIR fluorophores and their derivatives have been
extensively investigated to image pH,17 inorganic ions,18
endogenous reactive nitrogen species,19 oxidative stress20 and
protease activity.21 Previous reports showed that substituents at
the 5-position of the indole ring can mediate the optical proper-
ties of heptamethine cyanine derivatives.22 Meanwhile, photo-
physical properties of cyanine derivatives were also closely
related to their aggregation states in aqueous solution, which
depends on their charge density, hydrophilicity and polarity.23
Bearing these in mind, six cyanine derivatives including IR783,
a well-known NIR fluorophore were prepared, in which the
5-position of the indole ring was functionalized with proton,
methyl as an electron-donor group or chloride as an electron-
withdrawing group. Meanwhile, sulfate or carboxylic acid was
modified at the N-alkylated carbon chain terminals to adjust the
charge density and hydrophilicity of the resulting fluorophores.
Additionally, the modified carboxylic acids provide convenience
to label the fluorophore with biomacromolecules such as anti-
bodies through an amide bond to achieve the targeted imaging
purpose.
Table 1 Photophysical parameters of fluorophores in PBS (pH 7.4)
d
e
Amax SP
εmax SP (cm−1
mol−1 b
λmax SP (TP) ΦSP /TP
f
Dye (nm)a
)
(nm)c
(%)
σTP
1a
2a
1b
2b
1c
2c
775
775
787
788
791
789
120 000
56 000
61 500
38 200
49 800
16 900
797 (n.d.)
796 (642)
808 (656)
807 (n.d.)
806 (n.d.)
807 (n.d.)
6.5/0.033
30/0.982
8.5/0.747
7.5/0.845
1.2/0.0833 1.30
13/1.28 0.389
2.71
33.9
10.4
0.538
a Maximum absorbance wavelength of fluorophores. b Extinction
coefficients at Amax.
c Maximum SP and TP emission wavelength.
d Quantum yields (QY) of the fluorophores correlated to ICG. e QYs of
two-photon fluorescence of the fluorophores referenced to Rhodamine
6G (λex = 450 nm, QYR6G = 95% in MeOH). f Two-photon absorption
cross section of fluorophores (10−4 M) at λex = 900 nm referenced to
R6G (σR6G = 2.1 GM, 1 GM = 10−50 cm4 s photon−1 molecule−1).
cyclohexanecarboxaldehyde 6, which condensed with 4a–c or
5a–c to give the target heptamethine cyanines 1a–c or 2a–c with
yields in a range of 72–90%. During the final condensation step,
addition of 6 to N-alkylated indolium in acetic anhydride drop-
wise substantially increased the yield of resulting cyanines. Due
to the unstable aldehyde groups at high temperature, addition of
6 in batches may minimize its unwanted decomposition during
the reaction.
All heptamethine cyanine derivatives were synthesized
from corresponding alkylated indolium salts using a stepwise
process developed by Waggoner and co-workers.24 As shown
in Scheme 1, treatment of 5-substitited-2,3,3-trimethyl-3H-
indolium with 5-bromovaleric acid or 1,4-butane sultone in
o-dichlorobenzene gave the corresponding N-alkylated indolium
derivatives 4a–c functionalized with carboxylic acid or 5a–c
functionalized with sulfate. Meanwhile, the reaction between
cyclohexanone with phosphorus oxychloride (POCl3) in the
Absorbance and SP fluorescence studies in aqueous solution
The photophysical properties of the six fluorophores were
investigated in phosphate buffered saline (PBS) at pH 7.4 and
the corresponding photospectroscopic parameters are listed in
Table 1. It is obvious that the substituents at the indole rings and
presence
of
N,N-dimethylformamide
(DMF)
offered
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Fig. 2 TP fluorescence spectra of 1a–1c, 2a–2c and rhodamine
(100 μM) after excitation at 900 nm in PBS (pH 7.4). The spectrum of
rhodamine was minimized 40 times to fit with the other fluorophores.
Fluorophores 2a and 1b demonstrated remarkable TP fluorescence in
PBS.
Fig. 1 Substituents at the indole ring and N-alkylated carbon chain
terminals significantly affect the absorbance and emission of the fluoro-
phores in PBS (pH 7.4). (A) Absorbance of 2a–2c (1.0 μM, left panel)
with different substituents at indole ring and absorbance of 1a–2a
(1.0 μM, right panel) with different groups at carbon chain terminals.
(B) SP fluorescence spectra of 2a–2c (1.0 μM, left panel) and 1a–2a
(1.0 μM, right panel) after excitation at 765 nm.
those of 1a and 1c (Fig. S2†). The decreased hydrophilicity
and charge density of the fluorophores after modification with
carboxylate acids may disturb the structure of H-type aggregates
that are well-documented for their non-emissive characteristics in
the aqueous solution.23,26
N-alkylated carbon chain terminals significantly change absor-
bance and emission spectra of the fluorophores. Fig. 1A demon-
strates the absorbance of 2a–c with different substituents at
indole ring (left panel) and absorbance of 1a–2a with different
functional groups at N-alkylated carbon chain terminals (right
panel). Substitution of either an electron donating or electron
withdrawing group at the indole ring led to a red-shift of the
absorbance. This phenomenon can be explained by the partici-
pation of the lone pair electrons or σ-electrons of the substituents
in the big-π conjugation system of cyanine, which leads to the
decrease of the electron transition energy level. Notably, the
replacement of sulfates by carboxylate acid at N-alkylated
carbon chain terminals did not lead to any shape variation or
position movement of the absorbance, which implies these
groups do not belong to the chromophore system. Remarkably,
the extinction coefficient of 2a decreased 53% compared to 1a
(Table 1). The functionalization of the carboxylic acid may
decrease the polarity and negative charge of fluorophore in
aqueous solution, which increases the self-aggregation tendency
of the fluorophores and results in the decrease of extinction
coefficient. Fig. 1B showed SP emission spectra of 2a–c (left
panel) and 1a–2a (right panel) in PBS. Substitution of chloride
on the indole ring greatly reduced the fluorescence intensity.
For example, SP fluorescence of 1c and 2c in PBS were
measured as only 4.4% and 2.1% compared to that of 1a and 2a
(Fig. 1B and S1†). The above chloride dominated quenching
effect may be attributed to the internal heavy atom effect,
in which the attachment of a halogen atom on the indole ring
enhances the spin–orbit perturbation in the fluorophore and
accelerates the intercombination transition through a S1–T1 spin-
forbidden process.25 Unexpectedly, replacement of the sulfates
with carboxylate acid at the carbon chain terminal results in sub-
stantial enhancement of quantum yield (Q.Y.). For example, the
Q.Y. of 2a and 2c increased 4.6 and 10.8 times compared to
TP fluorescence studies in aqueous solution
Nonlinear photophysical properties of the fluorophores were
investigated in PBS under TP excitation provided by a femto-
second mode-locked Ti:sapphire laser system. In all six fluoro-
phores, only 2a and 1b demonstrated remarkable TP absorption
cross section values of 33.9 and 10.4 GM (1 GM = 10−50 cm4 s
photon−1) at 900 nm (Table 1). Interestingly, compared with 1a,
the replacement of sulfates by carboxylate acid endows 2a with
30 and 13 time enhancements in TP induced fluorescence
QY (ΦTP) and TP absorption cross section (σTP). Consistent with
the above data, 2a and 1b demonstrated comparable TP induced
fluorescence intensities compared to Rhodamine 6G (R6G) upon
excitation at 900 nm (Fig. 2). Importantly, the power-dependence
fluorescence curve of 2a (Fig. S3†) indicated the fitting exponent
of input power to be 1.8655, implying that 900 nm fs-pulsed
incident light indeed initiated a TP absorption process in 2a.
Recently, Achilefu et al. reported heptamethine cyanine deriva-
tives such as cypate demonstrating high TP action cross-section
and prolonged fluorescence lifetime.27 1550 nm was chosen as
the TP excitation wavelength because cypate demonstrated its
maximum absorbance at 750–800 nm, which benefits the genera-
tion of strong TP fluorescence in the NIR window. In this work,
the fluorophores were excited at 900 nm instead of 1550 nm to
generate TP fluorescence that can be visualized by the naked
eye. In this way, the emission not only possesses the inherent
advantages of TP fluorescence, but also provides great conven-
ience to distinguish the neoplastic tissue from surrounding
normal tissues for the surgeons. 2a demonstrated an obvious
absorbance at 440–500 nm that is a prerequisite to produce the
strong deep red TP fluorescence after excitation at 900 nm.
Compared to the reported TP fluorophores such as DTTC and
cypate,28 2a showed additional advantages including high water
solubility and uncompromised TP fluorescence in aqueous
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Fig. 3 Cyanine based fluorophores demonstrated the capabilities to
label lysosomes in HeLa cells. (A) SPFM images of cells at 24 h post-
incubation with 1a (1.0 μM, upper panel) or 2a (lower panel). (B)
SPFM images of cells pre-stained with lysosomal marker: Dextran-FITC
followed the incubation with 1.0 μM 1a or 2a for 24 h. SP NIR fluor-
escence is displayed in red, FITC fluorescence is displayed in green,
nuclei stained by DAPI are displayed in blue. Bar: 25 μm.
Fig. 4 Fluorophore 2a internalized into live HeLa cells can be success-
fully visualized by TPFM after excitation at 900 nm. (A) White light
(W. L.) and TPFM images of live cells upon incubation with 1a
(100 μM, upper panel) or 2a (100 μM, lower panel) for 24 h. TP Fluo.
signals were collected by the PMT channel ranging at 580–660 nm.
Scale bar: 20 mm; (B) Mean cellular TP fluorescence intensities (with
nearby background subtracted) after treatment of the fluorophore for
24 h. The values represent mean SD (n = 12).
solution. As far as we know, 2a is the first fluorophore that pos-
sesses the capability to visualize the physiological events with
both SP NIR fluorescence and TP deep red fluorescence.
In vitro fluorescence microscopic studies
lysosomes and the colocalization coefficient was calculated
above 90% by using an Image J software with Pearson’s plugin.
The potential of 2a as a TPFM fluorophore was further investi-
gated in HeLa cells. Fig. 4A demonstrates the white light and TP
fluorescence images of the cells treated with 100 μM 1a or 2a
for 24 h upon excitation at 900 nm. While there was not any
detectable TP intracellular fluorescence after treatment with 1a,
remarkable TP induced red fluorescence signals of 2a were
observed as vesicular structures in the perinuclear area. Quantifi-
cation analysis showed that the average cellular TP fluorescence
intensity of 2a in a range of 580–660 nm was about 8.9 times
higher than 1a (Fig. 4B).
Tolerable cytotoxicity is a pre-requirement for the clinical trans-
lation of a fluorophore. To address this issue, cell viabilities of
2a and 1a (control fluorophore) were tested in human epithelial
cervical cancer HeLa cells via MTT assay. Fig. S4† shows the
viabilities of HeLa cells after treatment with a series concen-
tration of fluorophores for 24 h. Interestingly, the replacement of
sulfates by carboxylate acids in carbon chain terminals signifi-
cantly reduced the cytotoxicity of fluorophore. IC50 value of 2a
was measured as 140 μM that is about 3.5 times higher than that
of 1a. The above experimental results indicate the tolerable cyto-
toxicity of 2a, which benefits its applications under in vitro as
well as in vivo conditions. Subsequently, the cellular uptakes of
1a and 2a were evaluated by SPFM in HeLa cells (Fig. 3A).
SPFM images showed that both fluorophores can be effectively
taken up by HeLa cells, and these fluorophores predominately
located in the perinuclear region as vesicular structures after
treatment for 24 h. Previous reports demonstrated the lysosomal
delivery of the cyanine derivatives in cancer cells.29–31 To deter-
mine the subcellular location of the fluorophores, HeLa cells
were pre-stained by lysosomal marker: Dextran-FITC, which
was used as a long-term lysosomal fluorescence marker in live
cells.30 As shown in Fig. 3B, following the treatment of 1a or 2a
for 24 h, both of them colocalized well with pre-stained
Conclusions
We developed a heptamethine cyanine based fluorophore that
emits both SP NIR fluorescence and TP deep red fluorescence
under physiological conditions. The advantages of this fluoro-
phore, including tolerable cytotoxicity, high water solubility and
strong single-photon/two-photon fluorescence, make it promising
for intra-operatively optical image-guided tumor excision
followed by TPFM biopsy analysis after a single administration.
Additionally, the high lysosomal specificity of this fluorophore
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Acknowledgements
This work was supported by the National Basic Research
Program of China (973 Program, No. 2011CB910404), National
Natural Science Foundation of China (Nos. 81171384,
30900353, 30830039) and Shenzhen Bio-industry Development
Fund-Basic Research Program (JC201005280607A).
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This journal is © The Royal Society of Chemistry 2012