Recent Advances in Receptor-Targeted Fluorescent Probes for In Vivo Cancer Imaging.
ABSTRACT Receptor-targeted optical imaging of cancer is emerging as an attractive strategy for early cancer diagnosis and surgical guidance. The success of such strategy depends largely upon the development of receptor-targeted fluorescent probes with high specificity and binding affinity to the target receptors. Recently, a host of such probes have been reported to target cancer-specific receptors, such as somatostatin receptors (SSTRs), integrin receptors, cholecystokinin-2 (CCK2) receptor, gastrin-releasing peptide (GRP) receptor, endothelin A (ETA) receptor, translocator protein (TSPO) receptor, epidermal growth factor (EGF) receptor, human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor (VEGF) receptor, folate receptor (FR), transferrin receptor (TFR), low-density lipoprotein (LDL) receptors, type I insulin-like growth factor receptor (IGF1R), vasoactive intestinal peptide (VIP) receptors, urokinase plasminogen activator (uPA) and estrogen receptor (ER). This review will describe the recent advances in synthetic targeting optical imaging probes and demonstrate their in vivo imaging potentials. Moreover, current status of near infrared (NIR) fluorescent dyes, targeting moieties and coupling reactions, as well as strategies for designing targeted probes, will also be discussed.
- SourceAvailable from: Khashayarsha Khazaie[show abstract] [hide abstract]
ABSTRACT: Macrophages have long been recognized as a prominent component of tumors. Activated macrophages overexpress folate receptors and we used this phenomenon to image inflammatory reactions in colon dysplasia using a fluorescent folate probe (FFP). APC(Delta468) mice injected with FFP showed fluorescent adenomas (target-to-background ratio, adenoma vs. adjacent normal mucosa, of 2.46 +/- 0.41), significantly higher (p < .001) than adenomas in animals injected with a non-folate-containing control probe. Fluorescence-activated cell-sorting analysis revealed a 3-fold higher content of Mac1-positive cells in colonic adenomas compared with normal adjacent mucosa (6.8% vs. 2.2%), and confirmed the source of FFP-positive cells to be primarily an F4/80-positive macrophage subpopulation. Taken together, these results indicate that probe potentially can be used to image dysplastic intestinal adenomas in vivo.Molecular Imaging 4(1):67-74. · 3.41 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The purpose of this study is to synthesize a folate-polyethylene glycol (PEG) conjugated near-infrared fluorescence probe (fPI-01) for diagnosis of folate receptor (FR)-overexpressed tumors with high sensitivity and specificity. fPI-01 was synthesized, purified, and characterized. Its cytotoxicity and affinity to tumor cells were determined in vitro. The dynamics and biodistribution of the probe was monitored in normal nude mice. And the tumor-targeting capability was investigated in nude mice bearing different tumor xenograft. fPI-01 was successfully synthesized with strengthened optical properties. Cells experiments showed the probe had high FR affinity and without apparent cytotoxicity. Animal experiments indicated the probe excreted through urine by kidney. And its tumor-targeting ability was demonstrated on different tumor-bearing mice, with high sensitivity and tumor-to-normal tissue contrast ratio (10:1). fPI-01 is a promising optical agent for diagnosis of FR-positive tumors, especially in their early stage.Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging 04/2010; 12(6):595-607. · 2.47 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The folate receptor (FR) is a valuable therapeutic target that is highly expressed on a variety of cancers. The current development of folate-targeted cancer therapies has created the need for quantitating functional FRs in clinical specimens. In this article, we report on the creation of a highly sensitive radioactive binding method for quantitatively measuring FR expression in frozen tissue homogenates. Expression was positive in approximately 89% of human ovarian carcinomas but was negligible in both mucinous ovarian carcinomas and normal ovary. Expression was also significant in carcinomas of the kidney, endometrium, lung, breast, bladder, and pancreas. Normal tissues from humans and six different laboratory species were also analyzed; surprisingly, some interspecies variability in FR expression (especially in kidney, spleen, and lung tissue) was found. Interestingly, normal human lung tissue displayed high expression levels, whereas expression in normal lung of the other species was negligible. However, considering that folate-drug conjugates fail to accumulate in the lungs of patients, the consequence of this finding was not considered to be of clinical concern. Overall, this new methodology is reliable for determining functional FR expression levels in tissues, and it could possibly be a useful clinical test to determine patient candidacy for FR-targeted therapeutics.Analytical Biochemistry 04/2005; 338(2):284-93. · 2.58 Impact Factor
Current Medicinal Chemistry, 2012, 19, ????-???? 1
0929-8673/12 $58.00+.00 © 2012 Bentham Science Publishers
Recent Advances in Receptor-Targeted Fluorescent Probes for In Vivo Cancer
M. Bai*,1 and D.J. Bornhop2
1Department of Radiology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA
15219, USA; 2Department of Chemistry and The Vanderbilt Institute for Chemical Biology, Vanderbilt University, Nashville,
Tennessee 37232, USA
Abstract: Receptor-targeted optical imaging of cancer is emerging as an attractive strategy for early cancer diagnosis and surgical
guidance. The success of such strategy depends largely upon the development of receptor-targeted fluorescent probes with high
specificity and binding affinity to the target receptors. Recently, a host of such probes have been reported to target cancer-specific
receptors, such as somatostatin receptors (SSTRs), integrin receptors, cholecystokinin-2 (CCK2) receptor, gastrin-releasing peptide (GRP)
receptor, endothelin A (ETA) receptor, translocator protein (TSPO) receptor, epidermal growth factor (EGF) receptor, human epidermal
growth factor receptor 2 (HER2), vascular endothelial growth factor (VEGF) receptor, folate receptor (FR), transferrin receptor (TFR),
low-density lipoprotein (LDL) receptors, type I insulin-like growth factor receptor (IGF1R), vasoactive intestinal peptide (VIP) receptors,
urokinase plasminogen activator (uPA) and estrogen receptor (ER). This review will describe the recent advances in synthetic targeting
optical imaging probes and demonstrate their in vivo imaging potentials. Moreover, current status of near infrared (NIR) fluorescent dyes,
targeting moieties and coupling reactions, as well as strategies for designing targeted probes, will also be discussed.
Keywords: Cancer, fluorescence, in vivo, molecular imaging, near infrared, optical imaging, receptor, targeted probes.
In the US, nearly 1 of every 4 deaths is caused by cancer. In
2012, about 1,638,910 new cancer cases are expected to be
diagnosed in the US, and about 577,190 Americans are expected to
die of cancer, more than 1,500 people a day (www.cancer.org). In
general, current state-of-the-art therapeutic methods used in cancer
treatment are inefficient. Cancerous tissues can be missed
(undertreatment) and healthy tissues can be severely damaged
(overtreatment). Imaging tools that can specifically locate diseased
areas and precisely diagnose cancer at early stage are thus
Molecular imaging (MI) aims to visualize, characterize, and
measure biological processes at the molecular and cellular levels in
humans and other living systems . An increasing amount of
effort has been invested in developing MI tools that target cancers.
Such cancer imaging tools are critical in early diagnosis, surgical
guidance, therapeutic effect monitoring and prognosis. The most
common imaging modalities in MI research are optical imaging,
positron emission tomography (PET), single photon emission
computed tomography (SPECT), magnetic resonance imaging
(MRI), and ultrasound. Nuclear imaging, including PET and
SPECT, uses ? ray as the signal source and is therefore highly
sensitive and not limited to tissue penetration. However, nuclear
imaging suffers from relatively low resolution, high instrument cost
and injection of radioactive agents. Furthermore, the most
commonly used radioisotopes, such as 11C and 18F, have rather
short half-lives (20 and 109 minutes respectively), which limits the
imaging time. MRI has high resolution, no tissue penetration
limitation and medium instrumentation costs, but low sensitivity.
To produce significant contrast at the target site, either a high local
concentration of a paramagnetic (e.g. Gd3+ chelators for T1
weighted imaging) or relatively large superparamagnetic contrast
agent (e.g. iron oxide, for T2 weighted imaging) is needed.
Targeted ultrasound usually uses microbubbles as the contrast
agent. These microbubbles have difficulties in targeting receptors
present in the tumor tissues because they are too large (1-10 μm) to
exit the vasculature . In addition, targeted ultrasound imaging is
challenged by the short circulation time of microbubbles .
*Address correspondence to this author at the Molecular Imaging Laboratory,
Department of Radiology, University of Pittsburgh School of Medicine, 100
Technology Drive Suite 452G, Pittsburgh, PA 15219; USA; Tel: 1-412-624-2565; Fax:
1-412-624-2598; E-mail: email@example.com
Optical imaging is a relatively low-cost imaging method with high
sensitivity and resolution. The detection limit for optical imaging
may reach picomolar or even femtomolar concentrations of optical
probes . The major disadvantage of optical imaging is limited
tissue penetration caused by tissue absorption and scattering. This
problem can be partially resolved by adapting near infrared (NIR)
light (650-900 nm) under which tissue absorption and scattering is
relatively low . NIR light can penetrate up to 10 cm deep into
tissues. Moreover, tissue autofluorescence is negligible in the
NIR region, allowing high-contrast optical imaging. Therefore, NIR
optical imaging is commonly used in in vivo studies . In the
clinical setting, optical imaging has emerged as an attractive
approach to facilitate identification of tumors and sentinel lymph
node metastases through endoscopy or during intraoperative
visualization [8, 9]. For example, in a clinical study, the only FDA
approved NIR fluorescent dye, indocyanine green (ICG), was used
for intraoperative guidance through breast cancer lymph node
Cancer imaging research is largely driven by the development
of MI probes. A typical MI probe consists of a signaling moiety,
such as a fluorescent dye; a targeting moiety, such as a receptor
ligand; and a linker that bridges these two moieties Fig. (1). Ideally,
such MI probe should have high binding affinity and selectivity at
the target sites, quick clearance from non-target tissues, as well as
good biocompatibility and stability. Receptors that are uniquely
overexpressed in tumors are often selected as the target sites.
Within the last several years, many MI probes have been developed
to image cancers through targeting cancer-specific receptors, such
as somatostatin receptors, integrin receptors, cholecystokinin-2
receptor, gastrin-releasing peptide receptor, endothelin A receptor,
translocator protein receptor, epidermal growth factor receptor,
human epidermal growth factor receptor 2, vascular endothelial
growth factor receptor, folate receptor, and transferrin receptor.
Several recent reviews have summarized such MI probes focusing
on nuclear imaging [11-13], multimodal imaging[14, 15], optical
imaging with nanoparticles[16, 17], peptide-based probes [12, 18,
19], and dual-targeted imaging probes . In this review, we will
summarize the recently developed receptor-targeted fluorescent
probes for in vivo cancer imaging, focusing on small molecules. We
will discuss the strategies for designing receptor targeted
fluorescent probes and describe the receptors that have been
targeted in fluorescent imaging of cancers. The recently developed
imaging probes will be grouped based on their target receptors and
the in vivo imaging results using these probes will be discussed. It is
2 Current Medicinal Chemistry, 2012 Vol. 19, No. 1 Bai and Bornhop
noteworthy that the combination of optical imaging with other
imaging modalities has become an attractive strategy to overcome
the tissue penetration limitation of optical imaging, however,
multimodal imaging probes have been recently reviewed [14, 15,
18] and are beyond the scope of this review.
Fig. (1). Molecular imaging probe.
TARGETED FLUORESCENT PROBES
STRATEGIES FOR DESIGNING RECEPTOR
When designing a receptor targeted fluorescent probe, several
concerns usually arise, such as (1) what fluorescent dye and
targeting moiety should be used? (2) What types of coupling
reactions are needed to synthesize the probe? (3) What is the overall
design of the probe (one to one coupling of dye to targeting moiety,
activatable probe or multi-receptor targeted probe)? This section
reviews the key components of receptor-targeted fluorescent
probes, including fluorescent dye, targeting moiety and linker,
coupling chemistry, as well as the overall design of constructing the
2.1. Fluorescent Dyes and Targeting Moieties
For in vivo cancer imaging, usually NIR fluorescent dyes are
selected as the signaling moieties, due to the relatively deep tissue
penetration and low scattering of NIR light. Several types of NIR
fluorescent dyes have been developed for biomedical imaging Fig.
(2), including (a) carbocyanine dyes, such as ICG; (b)
phthalocyanines and porphyrin derivatives, such as P2-Suc ; (c)
squaraine derivatives, such as KSQ-4-H; and (d) BODIPY
analogs, such as KFL-4. These dyes have been summarized in
two recent review papers [24, 25] and the details of each type of
dye will not be included in this review. Among them, carbocyanine
dyes are the most widely used in biomedical imaging, because
many carbocyanine dyes have high molar extinction coefficient,
good hydrophilicity, low toxicity and functional groups allowing
bioconjugation to targeting moieties. In
carbocyanine dyes are in clinical trials or in clinical use. The most
well-known carbocyanine dye is probably ICG, which has been
approved by FDA for almost half a century. However, in general,
carbocyanine dyes suffer from photobleaching, poor stability, and
low quantum yield. Recently, some organic dyes originally
developed for solar cells, such as diketopyrroropyrole dyes; have
been modified for biomedical imaging [26-28]. Such dyes have
enhanced chemical stability; photostability and quantum yield, and
appear promising for biomedical imaging. The in vivo imaging
applications of these dyes need to be further explored. Other than
organic fluorescent dyes, quantum dots (QDs) are often used as the
fluorescent motifs for optical imaging. Quantum dots are inorganic
fluorescent semiconductor nanoparticles with many desirable
spectroscopic properties, such as high molar extinction coefficients
and quantum yields, resistance to photobleaching, and large stoke
shifts . However, in vivo imaging with QDs is challenged by the
relatively large size and short circulation time, as well as potential
toxicity [29, 30]. Other nanoparticles-based fluorescent molecules,
such as gold nanoparticles and carbon nanotubes, are challenged by
purity, dispersibility and stability in physiological environments.
Moreover, the absorption, distribution, metabolism and excretion
characteristics are highly variable for NIR fluorescent nanoparticles
because of the wide variation in the physicochemical properties of
nanomaterials . This review will mainly focus on probes based
on fluorescent dyes.
Targeting moieties allow specific binding of imaging probes to
target sites, therefore serving as a key component in probe designs.
Common targeting moieties include small receptor ligands,
peptides, proteins, antibodies, and antibody fragments. Large
targeting moieties such as antibodies have high specificity to targets
and low antigenicity. In addition, attaching signaling molecules to
the antibodies usually does not affect the pharmacokinetics and
biodistribution. However, imaging with antibody-based probes
suffers from the long blood half-life of antibodies. To address the
long blood half-life issue, great efforts have been spent in
developing antibody fragments, such as single variable domain
fragment (Fv), single chain Fv (scFv), diabody, minibody, and
scFv-Fc. Although compelling preclinical data have been presented
using these antibody fragments-based probes, the applications of
these probes in targeted imaging need to be further investigated, as
antibody fragments are challenged by several issues, such as
stability, labeling chemistry, and whether critical residues are
modified . Small targeting moieties, such as receptor ligands
and peptides, are highly specific to targets, cleared rapidly, feasible
for conjugation with signaling moieties, and usually easy for
synthesis and characterizations. The major disadvantage is that the
pharmacokinetics and biodistribution may be largely affected after
the signaling moieties are attached.
2.2. Coupling Reactions
When developing an imaging probe, coupling reactions are
critical in coupling a fluorescent dye and a targeting moiety
together through a linker Fig. (1). The linker is usually an alkyl
chain with a functional group on each side. Peptides are also
commonly used as linkers, especially in enzyme-activatable probes
. (Table 1) summarizes the typical coupling reactions for
imaging probe development, including amide coupling, acylation,
Michael addition, Huisgen cycloaddition, hydrazone and oxime
Amide coupling between a primary amino group (-NH2) and a
carboxylic acid (-COOH) is widely used in peptide synthesis and
perhaps the most commonly used strategy to develop imaging
probes, as these functional groups are found in many targeting and
signaling molecules, such as peptides, proteins, antibodies,
chelators, and certain fluorescent dyes. Amide coupling is typically
conducted at room temperature with an organic base and a coupling
reagent, such as O-(Benzotriazol-1-yl)-N,N,N?,N?-tetra methyl-
luronium hexafluorophosphate (HBTU). To allow amide formation
under even milder conditions without base or coupling reagent, N-
acylation between N-hydroxysuccinimide
carboxylic acid and primary amino group is also widely used. Many
commercially available NIR dyes have an NHS ester group for this
purpose, such as Cy5.5 NHS ester, IRDye800CW NHS ester, and
Cy7 NHS ester. An alternate mode of reactivity for primary amino
group is its participation in N-acylation with isocyanate (-N=C=O)
to form ureas. Isocyanate is a common functional group found in
commercially available products, including certain fluorescent
Recent Advances in Receptor-Targeted Fluorescent Probes Current Medicinal Chemistry, 2012 Vol. 19, No. 1 3
Carbocyanine Dye (ICG)
Porphyrin derivatives (P2-Suc)
Squaraine Derivatives (KSQ-4-H)
BODIPY Dyes (KFL-4)
Pyrrolopyrrole Dyes (BF2-PPCy8)
Fig. (2). Examples of NIR fluorescent dyes.
Common Coupling Reactions for Imaging Probe Synthesis
Or Oxime Formation?
dyes, such as NIR797 isocyanate. The reaction of isocyanates with
amines can proceed at ambient temperature without the need for a
Michael addition of thiols onto activated alkenes such as
maleimides, is another commonly used strategy to construct
imaging probes, especially for peptide-based probes with cysteine.
4 Current Medicinal Chemistry, 2012 Vol. 19, No. 1 Bai and Bornhop
This type of reaction is allowed to occur under physiological
conditions and the reaction progress can be monitored by the
decrease of 233 nm absorption associated with the alkene groups,
assuming that no additional underlying absorption bands are present
The concept of 1,3-dipolar cycloaddition involving azide (-N3)
and alkyne (
CCH) was introduced by Rolf Huisgen almost half
a century ago, however, high temperature or pressure was required
to promote the reaction [35, 36]. In 2001, Sharpless and co-workers
discovered that Huisgen azide-alkyne cycloaddition can be
effectively catalyzed by copper(I) under physiological conditions
. After that, Huisgen cycloaddition has become a widely used
coupling strategy and is often referred to “click chemistry”.
However, the use of copper (I) catalyzed Huisgen cycloaddition in
living systems has been hindered due to the toxicity of copper(I)
. To improve the biocompatibility of Huisgen azide-alkyne
cycloaddition, copper-free click chemistry involving strained
cyclooctyne and azide has recently been reported .
Another coupling strategy involves aldehydes (-CHO), which
can react with hydrazides (-CO-NH-NH2) and alkoxyamine
(-C-O-NH2) to form hydrazones and oximes respectively under
physiological conditions. Although aldehydes can also react with
regular amine groups, the imine bond formed is hydrolytically
labile. The hydrazones and oximes, however, are hydrolytically
stable from pH 5-7 and 2-7 respectively, although decompose
rapidly above pH 9.0.
2.3. Design of Targeted Fluorescent Probes
In general, there are three types of strategies in designing
targeted fluorescent probes Fig. (3): (1) One to one coupling of a
fluorophore to a targeting moiety. This is the simplest, but most
common approach for cancer imaging, because there are many
overexpressed surface receptors in cancer cells. One disadvantage is
that unbound probes can also emit signal, therefore, rapid clearance
of unbound probes is essential to provide good contrast using this
strategy. (2) Activatable probes. These probes are usually designed
to target enhanced enzyme activity in cancer cells [41, 42]. In an
activatable probe, usually two fluorescent dyes in close proximity
are linked by a small enzyme specific peptide. Due to the
fluorescence quenching or resonance energy transfer, the probe has
little fluorescence emission. Upon cleavage of the peptide linker by
the target enzyme, the two fluorescent dyes are separated and
fluorescence emission is recovered. This strategy allows imaging
with low background, since unbound probes do not emit any
fluorescence signal. (3) Dual targeted molecular probes. Many
cancer cell types overexpress more than one type of receptor,
therefore, it is possible to develop probes that have two different
targeting moieties. In contrast to single receptor targeted probes,
dual targeted molecular probes have two binding ligands, and are
therefore able to bind to two different targets simultaneously.
Although this strategy cannot be widely applied in cancer imaging
due to the limited cancer cells with two different and adjacent
overexpressed receptors, it allows more accurate and specific
labeling of cancer cells. Several dual targeted molecular probes
have been reported and summarized in a recent review .
3. RECEPTORS FOR CANCER IMAGING AND TARGETED
Receptors that are specifically over-expressed in tumor cells
have become the primary targets for cancer imaging. Recent
advances in molecular biology have revealed a host of these cancer
targets, such as somatostatin receptors, integrin receptors,
cholecystokinin-2 receptor, gastrin-releasing peptide receptor,
endothelin receptor, translocator protein receptor, growth factors,
folate receptor, and transferrin receptor. In this section, we will
review the recently reported fluorescent probes that target these
receptors for in vivo cancer imaging. The important criteria for
evaluating these probes include (1) Does the targeting moiety retain
its binding affinity and specificity to the target sites after being
coupled to a fluorescent dye? (2) Does the fluorescent dye have
absorption and emission in the NIR region? Does it have high molar
extinction coefficient and quantum yield, as well as low
photobleaching? Is the probe hydrophilic, biocompatible and stable
in vivo? (3) Is the tumor to control fluorescence signal ratio high in
vitro and in vivo? Can the fluorescence signal in tumor cells be
blocked? (4) Is the probe rapidly cleared from non-target tissues?
3.1. Somatostatin Receptors
Somatostatin receptors (SSTRs) are G protein-coupled
receptors (GPCRs) with five distinct subtypes (SSTR1-SSTR5).
SSTRs have been extensively targeted for cancer imaging and are
over-expressed in various cancers such as neuroendocrine cancer
[43, 44], small-cell lung cancer , colorectal cancer , and
breast cancer . Because endogenous somatostatins (SST-14 and
SST-28) have rather short in vivo half-lives (<3 mins) due to
enzymatic degradation, many synthetic somatostatin analogues with
enhanced resistance to in vivo enzymatic degradation have been
developed, such as octreotide (D-Phe-cyclo(Cys-Phe-D-Trp-Lys-
Thr-Cys)-Threol) and octreotate (D-Phe-cyclo(Cys-Phe-D-Trp-Lys-
Thr-Cys)-Thr-OH) . These two peptides have low-nanomolar
binding affinities to SSTR with longer plasma half-lives (~1.5 h)
Many imaging probes have been developed to target SSTRs for
cancer imaging applications. SSTR targeted fluorescent probes are
usually developed by attaching a fluorescent dye to octreotide,
octreotate or their analogues. For example, as one of the early
studies, Licha et al. developed two SSTR fluorescent probes by
coupling two monocarboxylated cyanine dyes to octreotate (1 and 2
in Fig. (4)) . These two probes have emissions at 670 nm and
780 nm respectively. As a follow up study, Becker et al. reported in
vivo imaging of mouse SSTR2 tumor model using compound 2.
Between 3 and 24 h after the injection of 2, the tumor area showed
three times higher signal than normal tissue, whereas animals
injected with non-targeting control probe showed relatively low
signal in the tumor area . The biodistribution study
demonstrated that, at 24h post-injection, the kidney and liver
showed higher contrast agent uptake than tumor, and the uptake in
brain, muscle and femur was relatively low. The high uptake in
kidney and liver is in accordance with other in vivo studies .
Another two octreotate-based NIR probes were developed by
Achilefu et al. through coupling a dicarboxylated cyanine dye to
octreotate (3 and 4 in Fig. (4)) . Since this dye has two
carboxylate groups available for conjugation, it is possible to
couple two targeting moieties to one dye molecule (compound 4).
With an additional octreotate coupled to the dye molecule, the
binding affinity increased four-fold (IC50 of 3=5.4 nM; IC50 of
4=1.1 nM). In another study, Achilefu’s group developed a
macrocyclic SSTR probe by coupling another dicarboxylated
cyanine dye (cypate) to an octreotate derivative (K9-octreotate)
from head to tail (5 in Fig. (4)) . The advantage of a
macrocyclic structure is to improve metabolic stability of the
imaging probe in vivo. Compared to octreotate (IC50=0.12 nM), the
binding affinity of K9-octreotate decreased 180-fold (IC50=21.68
nM) and that of the macrocyclic probe decreased 68-fold
(IC50=8.17 nM). The nanomolar binding affinity of 5 appears
promising; unfortunately, no in vivo imaging was reported. More
recently, the same group developed multimodal optical-PET
imaging probe based on the cypate-octreotate structure .
The first in vivo imaging of human small cell long cancer
(SCLC) in mouse model using SSTR targeted fluorescent probes
was demonstrated by Kostenich et al. . In this study, nineteen
Recent Advances in Receptor-Targeted Fluorescent Probes Current Medicinal Chemistry, 2012 Vol. 19, No. 1 5
Fig. (3). Strategies for designing targeted fluorescent probes.
Fig. (4). SSTR targeted fluorescent probes.
6 Current Medicinal Chemistry, 2012 Vol. 19, No. 1 Bai and Bornhop
Fig. (5). Integrin receptors targeted probes.
fluorescent probes differing in core peptide, length of alkyl linker
and fluorescent dye were developed. Compound 6 Fig. (4) was
selected for fluorescence imaging due to high tumor selectivity.
Unfortunately, the emission peak of 6 (~520 nm) is away from NIR
region; therefore, the in vivo application of the probe is rather
limited. In a later study, the same group reported imaging of colon
cancer in a mouse xenograft model using the same probe .
In vivo SSTR targeted optical imaging has also been
demonstrated by confocal real-time mini-microscopy in an AR42-J
carcinoma mouse model . The confocal microscopy probe was
equipped with a 488 nm laser for excitation with emission window
at 505-585 nm. The contrast agent is octreotate labeled with 5-
carboxyfluorescein. Through targeted fluorescence imaging with
the confocal endoscopy, the tumor tissue could be identified. This
indicates that confocal minimicroscopy has potential in cancer
imaging and intraoperative guidance; however, the applications
may be challenged due to the significant autofluorescence at the
3.2. Integrin Receptors
Tumor progression is largely dependent upon the growth of
new blood vessels, a process called angiogenesis. Tumor
angiogenesis is mediated by various protein receptors and enzymes,
among which are integrin receptors, a family of heterodimeric
transmembrane receptors. In mammals, 18 ? and 8 ? subunits of
integrin receptors have been identified, which assemble into 24
different receptors . Among these 24 integrins, ?v?3, which is
expressed in a number of cancers such as melanoma, glioblastoma,
ovarian, prostate and breast cancer, is the most intensively targeted
for cancer imaging.
Integrin ?v?3 targeted fluorescent probes are mostly developed
based on linear or cyclic Arg-Gly-Asp (RGD) peptide, that has high
binding specificity to the receptor [58-62]. For example, Chen et al.
developed a Cy5.5-cyclic RGDyK Fig. (5) conjugate to image ?v?3
positive U87MG glioblastoma xenografts in mice . Compared
to the cyclic RGDyK peptide c(RGDyK), attaching the Cy5.5 NIR
dye only decreased the binding affinity by less than 1-fold (IC50
values for c(RGDyK) and Cy5.5-c(RGDyK) are 37.5±3.4 nM and
58.1±5.6 nM respectively). Between 3 and 24 hours after injection
of the probe, tumor-to-normal (T/N) fluorescence signal ratio of
roughly 3 was observed. Ex vivo imaging of excised organs at 4 h
post-injection showed that the compound was predominantly taken
up by the U87MG tumor. Liver, kidney, spleen, lung, muscle, and
pancreas showed significant less fluorescence signal than the tumor.
Forni’s group reported a similar probe, DA364, which is also based
on Cy5.5 and a cyclic RGD peptide . This peptide was selected
from a small library of cyclic RGD peptides. Compared to Cy5.5-
c(RGDyK), DA364 has improved binding affinity to integrin ?v?3
(IC50 = 2.75 ± 1.37 nM) and higher T/N ratio (5.14 ± 0.88) at 4 h
post-injection of DA364 into U87MG tumor bearing mice. In the
mice injected with DA364, fluorescence in the tumor area was still
Recent Advances in Receptor-Targeted Fluorescent Probes Current Medicinal Chemistry, 2012 Vol. 19, No. 1 7
detectable after 14 days. To improve the binding affinity and T/N
signal ratio of Cy5.5-c(RGDyK), Chen’s group developed dimer
and tetramer derivatives of Cy5.5-c(RGDyK) probe by attaching
multiple c(RGDyK) peptides to the Cy5.5 dye Fig. (5). The binding
affinities of dimer and tetramer increased almost 2- and 4-fold
respectively (IC50 values for monomer, dimer and tetramer are
42.9±1.2, 27.5±1.2, and 12.1±1.3 nM respectively) . The T/N
signal ratio, however, did not improve as much. No significant
increase of T/N signal ratio was observed for dimer and less than
20% increase was observed for tetramer (T/N ratio of monomer,
dimer and tetramer are 3.18±0.16, 2.98±0.05, and 3.63±0.09,
respectively). Another limitation of these Cy5.5 and c(RGDyK)
conjugates is that the emission wavelengths of these probes (~690
nm) are on the edge of NIR region where significant
autofluorescence is usually visualized. In general, emissions close
to 800 nm, where the overall tissue absorption and autofluorescence
are at minimal, are the most preferred for in vivo imaging. To
further improve the in vivo capabilities of ?v?3 targeted NIR
fluorescent probes, Chen’s group recently developed IRDye800-
E[PEG4-c(RGDfK)]2 Fig. (5) . This probe is advantageous over
the previous probes in that (1) IRDye800 emits close to 800 nm,
therefore autofluorescence and tissue absorption is decreased
compared to Cy5.5; (2) free IRDye800 dye has less tumor non-
specific binding than Cy5.5; and (3) introduction of polyethylene
glycol PEG4 improves in vivo pharmacokinetics of the probes.
Compared to IRDye800-E[c(RGDfK)]2 without PEG4, the binding
affinity of IRDye800-E[PEG4-c(RGDfK)]2
94.31±7.14 nM to 56.76±3.41 nM. It is possible that the insertion of
two PEG4 groups makes the two RGD motifs more flexible for
receptor binding. In addition, in vivo imaging using IRDye800-
E[PEG4-c(RGDfK)]2 showed significantly higher T/N signal ratio
(4.63±0.28 vs 3.31±0.22) than IRDye800-E[c(RGDfK)]2.
Li et al. reported NIR fluorescent probes based on a non-
peptidic ?v?3 antagonist [67, 68]. One probe, bivalent-IA-Cy5.5 Fig.
(5), binds to ?v?3 with sub-nanomolar binding affinity
(IC50=0.13±0.02 nM). The cellular imaging studies showed
integrin-mediated endocytosis of bivalent-IA-Cy5.5, which was
effectively blocked by nonfluorescent bivalent IA. However, when
the probe was injected in glioblastoma mouse xenografts, only
modest T/N signal ratio (~1.84) was observed .
An interesting discovery in developing ?v?3 targeted probes is
that conjugating a presumably inactive linear hexapeptide GRDSPK
with an NIR dye (cypate) yielded Cyp-GRD Fig. (5) that turned out
to target ?v?3 integrin positive tumors instead . The cell
internalization and in vivo uptake of the compound could be
blocked by a cyclic RGD peptide.?Further studies revealed that the
internalization of Cyp-GRD was mediated by ?3 integrin instead of
the heterodimer ?v?3 .
Other than ?v?3 integrin, other integrins have also been targeted
for cancer imaging. For example, Huang et al. reported ?2?1
targeted prostate cancer imaging using a peptide based NIR probe,
Cy5.5-DGEA . Although in vivo images showed higher
fluorescence signal in the tumor area than normal tissues, the
specific binding of the probe is questionable, as the binding of the
probe to ?2?1 could not be completely blocked by unconjugated
DGEA peptide. In another study, Xiao et al. imaged glioblastoma
mouse xenograft by targeting ?3 integrin. The targeting probe,
Streptavidin-Cy5.5, appears to have high specificity to the target
receptor, but with a relatively low affinity (Kd=0.5±0.1 μM) .
It is noteworthy that quantum dots [73-76], gold nanoparticles
 and single walled carbon nanotubes (SWNTs)  have also
been adapted as the fluorescent motifs in developing integrin
targeted fluorescent probes. Furthermore, great efforts have been
invested in combining optical imaging with other imaging
modalities, such as PET [79, 80], SPECT [81-83] an MRI [84, 85],
for multimodal integrin receptors targeted cancer imaging.
3.3. Cholecystokinin-2 (CCK2) Receptor
Similar to somatostatin receptors, the CCK2 receptor is a
member of the GPCR family. CCK2 receptor is expressed in several
cancers, such as medullary thyroid carcinomas, small-cell lung
cancer, gastroenteropancreatic neuroendocrine, stromal ovarian
cancer, astrocytomas, and gastrointestinal stromal cancer [43, 86].
The known peptides that bind to CCK2 include cholecystokinin,
gastrin, and gastrin derivatives. CCK2 receptor targeted
fluorescence imaging has not been attempted until recently. Laabs
and co-workers reported a CCK2 receptor targeted NIR probe DY-
676-DGlu1-minigastrin Fig. (6), which consists of a NIR dye, DY-
676 (emits at 699 nm), and a 13 amino acids gastrin derivative,
minigastrin . In cellular imaging studies, CCK2 positive HT-29
colorectal carcinoma cells incubated with the probe showed higher
fluorescence signal than CCK2 negative A-375 melanoma cells. In
vivo mouse imaging demonstrated a clear depiction of HT-29
tumors, which was blocked by nonlabeled minigastrin. The T/N
signal ratio, however, remained low (< 2) after injection of the
probe, and high uptake in organs such as liver, gut, stomach, lung
and kidney was observed. In addition, the emission of the probe
was at about 700 nm where the autofluorscence was still high.
Probes with close to 800 nm emission and fast clearance from
organs will significantly enhance the in vivo imaging outcomes.
3.4. Gastrin-Releasing Peptide (GRP) Receptor
Gastrin-releasing peptide (GRP) receptor has great potential as
the targets for cancer imaging, because GRP receptor is massively
overexpressed in breast, prostate, small cell lung, ovarian,
gastrointestinal stromal and some endometrial cancers, whereas the
expression levels in normal tissues are relatively low . The
native GRP receptor ligand, bombesin (BBN), is a 14 amino acid
peptide, in which the last eight residues are the most important for
binding . This octapeptide, Gln-Trp-Ala-Val-Gly-His-Leu-Met
is named bombesin (7-14), and its derivatives have been
fluorescently labeled for in vivo cancer imaging.
In an early study, Achilefu et al. reported a NIR fluorescence
probe (Cypate-Cybesin, Fig. (7)) which consists of the NIR Cypate
dye and a bombesin (7-14) derivative (Gly-Ser-Gly-Gln-Trp-Ala-
Val-Gly-His-Leu-Met) . This probe has a surprisingly higher
binding affinity (IC50 = 1.8 nM) than the parent peptide (IC50 = 7.6
nM), and showed tumor uptake in AR42-J rat pancreatic cancer
model, although the T/N ratio was not quantified and blocking
experiments were not demonstrated . In 2007, Ma et al.
developed another GRP receptor targeted probe by coupling Alexa
fluor 680 dye with bombesin (7-14) through a gly-gly-gly linker
Fig. (7) . This probe has nanomolar binding affinity to GRP
receptor (IC50=7.7 nM) and demonstrated specific binding to the
receptor in vitro and in T-47D breast tumor bearing mice. The T/N
signal ratio, however, was not quantified and the in vivo imaging
outcome can be improved by replacing the Alexa fluor 680 dye
(emission at 702 nm) with an 800 nm emitting dye. In another
study, viral nanoparticles (VNPs) were used as the platform to
attach bombesin (7-14) and Alexa Fluor 647 dye .
Accumulation of both targeted and untargeted nanoparticles in the
tumor in chicken chorioallantoic membrane tumor model was
observed, probably due to the enhanced permeation and retention
(EPR) effect. Recently, bombesin (7-14) was dual-labeled with
Cy5.5 and Gd3+ chelators for bimodal MR/optical imaging of GRP
3.5. Endothelin A (ETA) Receptor
The endothelin A (ETA) receptor mediates tumorigenesis and
tumor progression by activation of tumor proliferation, invasion,
angiogenesis and inhibition of apoptosis . Accordingly, ETA
receptor is over-expressed in various cancers such as breast ,
8 Current Medicinal Chemistry, 2012 Vol. 19, No. 1 Bai and Bornhop
Fig. (6). Structure of DY-676-DGlu1-minigastrin.
Alexa Fluor 680
Alexa Fluor 680-G-G-G-BBN(7-14)
Fig. (7). GRP receptor targeted probes.
ovarian , oral , and prostate cancer . Based on the
nonpeptidyl, high-affinity and selective ETA receptor antagonist,
PD 156707, Holtke et al. developed an ETA receptor targeted probe
(ETA-Cy5.5) by attaching Cy5.5 dye to the antagonist through a
short PEG spacer Fig. (8) . The specific binding of the probe
was demonstrated by cellular imaging of cancer cell lines with
different degrees of ETA receptor expression levels. The ETA
receptor positive human fibrosarcoma HT-1080 cells and human
breast cancer MCF-7 cells showed significant fluorescence signal,
whereas ETA-negative MDA-MB-435 human breast carcinoma
cells showed no accumulation of the probe. In a follow-up study,
Holtke and co-workers reported the biodistribution of the ETA-
Cy5.5 probe in wild-type CD-1 mice . The probe demonstrated
specific uptake in ETA receptor expressing tissues, such as heart
and lung; however, no in vivo tumor imaging was reported.
Fig. (8). ETA receptor targeted probe.
3.6. Translocator Protein (TSPO) Receptor
The translocator protein (18 kDa) (TSPO), previously named
peripheral benzodiazepine receptor (PBR), is a five transmembrane
domain protein that is localized primarily in the outer mitochondrial
membrane and is expressed predominantly in steroid-synthesizing
tissues, including the brain . TSPO is significantly
overexpressed in breast, prostate, colon, and brain cancer, with
protein expression linked to cancer progression and poor survival
rates, suggesting that the protein may be an attractive target for
cancer imaging .
We developed the first NIR fluorescent TSPO targeted probe by
coupling a NIR dye (IRDye 800CW) to a conjugable analog of
PK11195, a well known TSPO ligand with nanomolar binding
affinity to TSPO. This TSPO targeted NIR probe, NIR-
conPK11195, has been tested in colonic tumors in Smad3-/- mice
. NIR-conPK11195 localized and was retained in target
colonic adenomas and carcinomas but not in non-neoplastic
hamartomas or chronically inflamed colonic tissue. The colonic
tumors were detected with a sensitivity of 67% and a specificity of
86% in a cohort of 37 Smad3-/- mice and control littermates. These
results are comparable to 18FDG-PET data in humans reported at
91% to 93% sensitivity and 63% to 83% specificity [102, 103].
NIR-conPK11195 appears to be a promising optical molecular
imaging tool to rapidly screen for colonic tumors in mice and to
discriminate inflammation from cancer. In another study, NIR-
conPK11195 was used to image athymic nude mice bearing MDA-
MB-231 breast cancer xenografts . The in vivo biodistribution
and accumulation of TSPO-targeted NIR-conPK11195 and non-
targeting free NIR dye are demonstrated in Fig. (10).
Approximately 1 h post-injection, both compounds exhibit fairly
uniform distribution throughout the mice. Over time, the free NIR
dye clears primarily through the kidneys with a much more rapid
clearance profile than NIR-conPK11195, which is cleared through
both the renal and hepatobiliary systems. After 4 h, NIR-
conPK11195 preferentially accumulates in the tumor regions to a
significantly greater extent than the free NIR dye. The contrast
enhancement steadily increases to 11-fold (p<0.001) and 7-fold
Recent Advances in Receptor-Targeted Fluorescent Probes Current Medicinal Chemistry, 2012 Vol. 19, No. 1 9
Fig. (9). TSPO targeted probes.
Fig. (10). Direct comparison of the biodistribution and accumulation of the TSPO-targeted NIR-conPK11195 (a) and free NIR dye (b) in tumor-bearing mice
demonstrates significantly different clearance profiles and enhanced preferential labeling of MDA-MB-231 tumors in vivo by NIR-conPK11195. Fluorescence
images were normalized to laser power and integration time, overlaid onto the corresponding white light images, and displayed in terms of normalized photon
counts over 48 h postinjection (pi). Images are representative of n=3 mice per group. Note: These images are all displayed on the same scale (0 to 12,149
counts) pre-injection, 1, 4, 6, 12, 24, and 48 h pi (left to right). Reprint from .
(p<0.001) over normal tissue and unconjugated NIR dye
respectively at 48 h postinjection Fig. (10). These results indicate
that NIR-conPK11195 is a promising TSPO-targeted molecular
imaging agent for visualization and quantification of breast cancer
cells in vivo.
In an effort to improve the micromolar binding affinity of NIR-
conPK11195 (Ki=1 μM), we synthesized a series of functional
phenoxyphenyl-acetamide molecules, n-TSPOmbb732 (n=3-9)
. The selected targeting molecule, 6-TSPOmbb732 (6T), was
labeled with IRDye 800CW. The resulting imaging probe, NIR6T
(Ki=42±23 nM), displayed nanomolar binding affinities to TSPO
and labeled MDA-MB-231 and C6 cells specifically. The in vivo
imaging studies using these probes have not been reported yet.
3.7. Growth Factor Receptors
Along with the increased metabolism rate and tumor
angiogenesis to supply sufficient oxygen and nutrients, the
increased growth factor receptors are essential for the limitless
replicative potential of tumor cells . These features can be
exploited by targeted fluorescent probes to image cancers. Several
groups have reported such probes by coupling fluorophores to
growth factor receptor ligands or monoclonal antibodies for
imaging of epidermal growth factor (EGF) receptor, human
epidermal growth factor receptor 2 (HER2) and vascular
endothelial growth factor (VEGF) receptor in different tumors.
10 Current Medicinal Chemistry, 2012 Vol. 19, No. 1 Bai and Bornhop
3.7.1. EGF Receptor
EGF receptor (170 kDa) is a transmembrane glycoprotein that
is involved in the regulation of cell proliferation and promotes
tumor invasion and metastasis [106, 107]. Over-expression of this
receptor is associated with brain, breast, colon, lung, head and neck,
ovarian, pancreas, prostate and skin cancer [108-113]. Among the
three well-known EGF receptor ligands, epidermal growth factor
(EGF), amphiregulin (AR) and transforming growth factor-?
(TGF?), EGF (a 6 kDa polypeptide) is the most widely used as the
targeting moiety for cancer imaging. For example, Ke et al. labeled
EGF with Cy5.5 and imaged MDA-MB-468 breast tumor
xenografts . The fluorescence signal from Cy5.5-EGF in
MDA-MB-468 tumors was blocked by EGF or anti-EGF receptor
antibody. In addition, relatively low signal was observed in EGF
receptor negative MDA-MB-465 tumor xenografts. Similarly, EGF
was coupled to IRDye 800CW to image orthotopic prostate tumors
in mice [115, 116], human colorectal cancer xenografts  and
murine glioma . Due to the longer excitation and emission
wavelengths, IRDye 800CW-EGF showed better tissue penetration
and signal to background ratio (SBR) than Cy5.5-EGF .
Besides EGF, anti-EGF receptor monoclonal antibodies are also
commonly used to develop EGF receptor targeted fluorescent
probes [120-124]. As an example, Wang et al. labeled an anti-EGF
receptor monoclonal antibody, Erbitux, with Cy5.5 dye for in vivo
imaging of breast cancer xenografts . In flow cytometry probe
specificity assay, Erbitux-Cy5.5 showed a 9.32-fold higher affinity
for high EGF receptor expressing MDB-MB-231 than low EGF
receptor expressing MCF-7 cells. In vivo, the maximum probe
uptake was 1.65-fold higher in the MDA-MB-231 tumor than in the
MCF-7 tumor. Anti-EGF receptor monoclonal antibody based
probe has also been used in colorectal cancer imaging with confocal
endomicroscopy . In this study, Goetz et al. imaged colon
cancer xenografts in vivo with a handheld confocal laser
endomicroscopy (CLE) probe after injection of FITC dye labeled
anti-EGF receptor antibody. As a newly developed technology,
CLE enables endoscopists to collect real-time in vivo histological
images or “virtual biopsies” of the gastrointestinal (GI) mucosa
during endoscopy [126, 127]. Although this probe (emission at 516
nm) does not have NIR emission, as a fluorescent probe for
endomicroscopy, it clearly showed specific binding to target sites in
a xenograft model of human colorectal cancer and ex vivo on
human tissue .
Recently, affibody molecules are emerging as attractive
targeting moieties for cancer imaging. Affibody molecules are a
class of small (? 7 kDa) phage display-selected affinity proteins
with a three-helical bundle structure . Although affibody
molecules are small, they display binding surface as large as
antibodies and high affinities to target sites. Affibody molecules
usually show fast tumor targeting and clearance from normal
tissues. In addition, affibody molecules can be synthesized with
regular solid-phase peptide synthesis. Miao et al. reported an
affibody-based, Cy5.5 labeled probe, Cy5.5-ZEGFR:1907, for EGF
receptor targeted imaging . Attachment of Cy5.5 decreased the
binding affinity of the ZEGFR:1907 affibody about 8-fold (from 5.4 nM
to 44 nM). In A431 (EGF receptor positive) epidermoid
carcinoma tumor bearing mice, the maximum T/N ratio at
2.9±0.1 was observed 4 hours post injection of Cy5.5-
ZEGFR:1907. When Cy5.5-ZEGFR:1907 was blocked by unlabeled
ZEGFR:1907 affibody, the T/N ratio dropped to 1.8±0.4, about the
same level as in EGF receptor negative MCF tumor bearing mice
Similar to EGF receptor, HER2 is a transmembrane
glycoprotein that is involved in cell survival, proliferation,
angiogenesis and invasiveness . A number of cancers
overexpress HER2, such as breast, ovarian, salivary glad, stomach,
kidney, colon, prostate, urinary and non-small cell lung cancer
. The levels of HER2 expression are associated with
aggressiveness and poor prognosis. Although HER2 has no such
natural ligands as EGF for EGF receptor, anti-HER2 antibodies
[132-139] and affibodies [140-142] have been labeled with various
fluorescent dyes and nanoparticles for cancer imaging.
As an example, Lee et al. attached NIR Alexa Fluor 750 dye to
three different types of HER2-specific affibody molecules,
monomer ZHER2:342, dimer (ZHER2:477)2 and albumin-binding domain-
fused-(ZHER2:342)2 . The binding affinity of the ZHER2:342-Alexa
Fluor 750 was reported to be ~5 fold lower (Kd=190 pM) than
unlabeled affibody molecule ZHER2:342. After being injected into
HER2-expressing tumor xenografts, affibody monomer ZHER2:342-
Alexa Fluor 750 and dimer (ZHER2:477)2-Alexa Fluor 750 conjugates
failed to show significant tumor accumulation. Instead, fast
accumulation in the kidney was observed. To prolong the
circulation time, albumin was attached to the probe and the
conjugate showed clear signs of tumor accumulation. In follow-up
studies, this probe was used to image subcutaneous tumor
xenografts expressing different levels of HER2 , and monitor
changes of HER2 expression after therapy in breast carcinomas
tumor-bearing mice .
Kobayashi’s group reported a number of activatable fluorescent
probes based on anti-HER2 antibodies [143-147]. For example, one
of these “smart” probes, Traz-TM-Q7, consists of a monoclonal
antibody against HER2, trastuzumab; and a TAMRA (fluorophore)-
ASY7 (Quencher) pair . After internalization, Traz-TM-Q7 is
catabolized and the fluorescence resonance energy transfer (FRET)
quenching is abolished. Although this activation strategy allows
imaging with low background signal, the emission of the TAMRA
fluorophore is only at around 580 nm, thus limiting the tissue
penetration. In another study, Ogawa et al. attached five NIR ICG-
sulfo-OSu dye (an ICG derivative) molecules to trastuzumab
antibody . Tra-ICG-sulfo-OSu (1:5) conjugate exhibited no
fluorescence in PBS buffer solution. After cell binding and
internalization, the ICG-sulfo-OSu molecules dissociate from the
antibody, and fluorescence is activated. In HER2+ cells, Tra-ICG-
sulfo-OSu (1:5) became activated after 8 h of incubation. Based on
this observation, the authors proposed that Tra-ICG-sulfo-OSu (1:5)
was internalized and cut into component peptides, releasing ICG-
sulfo-OSu in the endosome-lysosome over 8 h. Similarly, uptake
within HER2+ tumors can be shown with Tra-ICG-sulfo-OSu (1:5)
conjugate with minimal background signal.
3.7.3. VEGF Receptors
Similar to integrin receptors, VEGF receptors are also involved
in regulation of tumor angiogenesis, therefore can be potentially
targeted for cancer imaging . Unfortunately, the expression
levels of VEGF receptors in cancers are much lower than many
other tumor markers, such as EGF receptor, HER2 and integrin
receptors . Therefore, the VEGF receptors are ineffective
targets for cancer diagnostic imaging. Backer et al. synthesized a
VEGF receptor targeted fluorescent probe by attach VEGF and Cy5
fluorescent dye to a boronnated dendrimer. The in vivo imaging
studies revealed that this VEGF-BD/Cy5 probe labeled tumor
vasculature endothelial cells, instead of the tumor itself, because
endothelial cells in tumor neurovasculature overexpress VEGF
receptors . In a more recent study, Backer et al. observed
similar in vivo imaging results using another probe, scVEGF/Cy,
which consists of a single-chain Cys-tagged VEGF (scVEGF) and
NIR Cy5.5 dye. In 4T1 and MDA-MB-231 breast tumor bearing
mice, scVEGF/Cy appeared to label endothelial capillary cells
between large muscle cells . Indeed, scVEGF/Cy was later on
used to image inflammation and neurovasculation [152-154].
In another study, Terwisscha van Scheltinga et al. reported
intraoperative imaging using two antibody-based NIR probes that
Recent Advances in Receptor-Targeted Fluorescent Probes Current Medicinal Chemistry, 2012 Vol. 19, No. 1 11
target VEGF receptors and HER-2 respectively . These two
probes (bevacizumab-800CW and trastuzumab-800CW) were
developed by attaching IRDye 800CW to anti-VEGF antibody
bevacizumab and anti-HER2 antibody trastuzumab respectively. In
vivo fluorescence imaging of human xenograft bearing mice
showed T/N ratio of 1.93 ± 0.40 for bevacizumab-800CW and 2.92
± 0.29 for trastuzumab-800CW on day 6 after tracer injection. In
intraoperative imaging study, submillimeter lesions could be
visualized with bevacizumab-800CW or trastuzumab-800CW.
These results indicate that VEGF or HER2 targeted antibody-based
NIR contrast agents can be used to image tumor lesions in vivo.
3.8. Folate Receptor (FR)
Folate receptor (FR) is a 38-40 kDa glycosylphosphati-
dylinositol (GPI)-linked membrane glycoprotein . While FR
expression is low to absent in most normal tissues except for
choroid plexus and placenta, high FR expression is observed in
various types of cancers, such as ovarian, cervix, brain, head and
neck, lung, kidney and endometrium cancer . This high
tumor/normal tissue FR expression ratio qualifies FR as a good
cancer-imaging target. In addition, the FR ligand, folic acid, has
high binding affinity in the picomolar range  and has
carboxylate group that can be easily coupled to signaling
molecules. Moreover, folate conjugates bind to FR and get cleared
from non-target sites rapidly . Therefore, FR has become an
attractive target for in vivo cancer imaging.
Tung et al. conjugated folate to a NIR fluorescent dye, NIR2,
through a hydrophilic diamine linker Fig. (11) . Significant
fluorescence signal from the tumor area was observed only 1 hour
after injection of the NIR2-folate probe. In a follow-up study,
NIR2-folate was injected into FR+ nasopharyngeal epidermoid
carcinoma KB and FR- human fibrosarcoma HT1080 mouse
models . KB tumors showed 2.4-fold higher signal intensity
than HT1080 tumors at 24 h post injection. A 3-fold T/N signal
ratio was observed at 4 h post injection and this tumor enhancement
persisted over 48 h. The same group also used the NIR2-folate to
image macrophages, which are associated with dysplastic intestinal
adenomas . FP+ APC?468 mice injected with NIR2-folate
showed 2.46 ± 0.41 T/N fluorescence contrast. These studies
indicate that folate-coupled NIR probes can be used to specifically
image FR+ tumors.
Liu et al. reported another FR targeted NIR probe, folate-PEG-
ICG-Der-01 (fPI-01), which consists of folate, a 4 kDa PEG and an
ICG analog Fig. (11). Compared to the NIR2-folate probe
Tung et al. reported, the additional 4 kDa PEG chain has the
advantages of reduced endocytosis by reticuloendothelial system
Fig. (11). Folate receptor targeted probes.
12 Current Medicinal Chemistry, 2012 Vol. 19, No. 1 Bai and Bornhop
and slow filtration by renal glomerulus. The tumor-targeting
capability of fPI-01 was demonstrated in mice bearing FR positive
Bel-7402 (human hepatoma cells) tumor xenografts. Four hours
after injection of fPI-01, T/N ratio reached as high as 20:1. The
fluorescence signal in tumor remained bright even after 48 h post-
injection. The authors also studied the biodistribution of fPI-01 in a
normal nude mouse. Unfortunately, high fluorescence signal in the
kidney was observed both in vivo and ex vivo at 8 h post-injection.
The authors argued that this was due to the FR expression in the
kidney . Other than NIR fluorescent dye-labeled folate probes,
other groups have reported quantum dots [164-166] and other
nanoparticles [167-173] labeled folate for in vivo cancer imaging.
The first in-human FR targeted real-time intraoperative
fluorescence imaging in ovarian cancer was recently reported by
van Dam and co-workers . In this study, a folate-FITC
conjugate Fig. (11) was intravenously injected into ten ovarian
cancer patients (4 with a malignant epithelial ovarian tumor, 1 with
a serous borderline tumor and 5 with a benign ovarian tumor).
Fluorescence was detectable intraoperatively in 3 out of 4 patients
with a malignant tumor and FR expression. One patient has a non-
FR expressing malignant tumor; therefore, no fluorescence was
detected. Moderate fluorescence signal was detected in the patient
with serous borderline tumor, which has moderate FR expression.
No signal was seen in any of the patients with benign lesions, which
have no FR expression. Healthy tissue did not show any
fluorescence signal either in vivo, ex vivo or on histopathological
validation. The fluorescence signals detected intraoperatively
appeared to correlate with FR expression levels. The imaging
results, however, can be improved, if the FITC dye is replaced with
a NIR dye, which allows identification of more deeply seated
tumors. This study indicates that targeted optical imaging may offer
the unique opportunity to intraoperatively identify tumors and
improve surgical outcome.
3.9. Transferrin Receptor (TFR)
Transferrin-receptor (TFR) regulates iron uptake and delivery
into the cells as demanded by metabolic need . TFR may
represent a suitable target for early detection of cancer, as the
receptor has been qualitatively described for various cancers,
presumably due to malignant transformation of normal cells .
The native TFR ligand, transferrin, is an 80 kDa glycoprotein and
serves as a good targeting moiety for TFR targeted cancer imaging.
Intriguingly, different TFR targeted in vivo imaging results were
observed from two studies. In one study, Shan et al. imaged human
head and neck squamous cell carcinomas (HNSCCs) xenografts
with an Alexa Fluor 680-transferrin conjugate . Fluorescence
signal in the tumor area was seen as early as 10 minutes, and
reached the maximum at 90-120 minutes post injection. Maximum
T/N fluorescence signal ratios, ranging from 1.42 to 4.15, appear to
correlate with tumor size. This study indicates that transferrin-based
optical imaging probes are potentially useful for cancer imaging. In
the other study, human holotranferrin (diferric transferrin) and anti-
transferrin antibody, anti-CD71, were labeled with fluorescent
calcium phosphosilicate nanocomposite particles (CPNPs) . In
vivo imaging in human breast cancer xenografts revealed that
tumors from mice receiving Anti-CD71-Avidin-CPNPs, and not
Human Holotransferrin-Avidin-CPNPs or untargeted PEGCPNPs,
were effectively targeted. The authors argued that the transferrin
receptors could be saturated with transferrin and therefore were
unable to bind the Human Holotransferrin-Avidin-CPNPs. Anti-
CD71-Avidin-CPNPs recog-nize an epitope separate from the
ligand-binding site on the transferrin receptor and therefore were
able to label the target tumor area. In several other studies,
transferrin was labeled with fluorescent nanoparticles such as
quantum dots [179-181], nanocomposites  and viral
nanoparticles , however, no in vivo studies have been reported.
3.10. Other Receptors
Targeted optical cancer imaging is not limited to the receptors
discussed above. Indeed, many probes have been reported to target
other receptors that are over-expressed in cancers, such as a SDF-1
and IRDye800CW conjugate for chemokine receptor CXCR-4 and
CXCR7 targeted imaging , a NIR fluorescent probe based on a
prostate specific membrane antigen (PSMA) ligand, GPI ,
low-density lipoprotein (LDL)-based probes that target LDL
receptors [186-188], Alexa Fluor 680-AVE-1642 for imaging type I
insulin-like growth factor receptor (IGF1R) , a cyanine dye
labeled vasoactive intestinal peptide (VIP) targeting VIP receptors
, NIR-NFP for labeling urokinase plasminogen activator
(uPA) and NIRDC1 that binds to estrogen receptor .
Such probes have demonstrated potentials in imaging breast, liver,
and gastroenteropancreatic tumors in vivo.
Recent advances in optical cancer imaging have indicated that
receptor-targeted fluorescent probes have become a promising class
of agents for imaging a wide variety of tumors. In general, the
success of such probes has depended on the following factors: (1)
emit close to 800 nm to achieve deep tissue penetration with low
tissue autofluorescence, (2) bind with high affinities to their target
receptors, (3) target receptors that are significantly overexpressed in
cancer cells, (4) clear rapidly from non-targeting tissues, and (5) be
abundantly taken up by target cells to show good contrast. Although
optical imaging has demonstrated as an outstanding imaging
modality for cell, tissue and small animal imaging, the disadvantage
of limited tissue penetration is more obvious in clinical
applications. As such, under clinical settings, optical cancer
imaging is usually only applicable in limited sites such as the
lesions close to the surface of the skin and tumors accessible by
endoscopy. However, recent development in intraoperative optical
imaging systems that allow detection of tumors during surgical
procedures provides more opportunities for clinical applications of
optical imaging. More recently, hands-free and wireless goggles for
NIR fluorescence and real-time image-guided surgery were
developed and facilitated tumor resection and biopsy procedures
. We vision that future potent receptor targeted fluorescent
probes, along with advanced optical imaging instruments, will
continue to play a critical role in cancer imaging.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no conflicts
confocal laser endomicroscopy
calcium phosphosilicate nanocomposite particle
epidermal growth factor
enhanced permeation and retention
Recent Advances in Receptor-Targeted Fluorescent Probes Current Medicinal Chemistry, 2012 Vol. 19, No. 1 13
fluorescence resonance energy transfer
G protein-coupled receptor
human epidermal growth factor receptor 2
head and neck squamous cell carcinomas
type I insulin-like growth factor receptor
magnetic resonance imaging
peripheral benzodiazepine receptor
positron emission tomography
prostate specific membrane antigen
small cell long cancer
single photon emission computed tomography
single walled carbon nanotubes
transforming growth factor-?
urokinase plasminogen activator
vascular endothelial growth factor
vasoactive intestinal peptide
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