Non-Covalently Pre-Assembled High-Performance Near-Infrared Fluorescent Molecular Probes for Cancer Imaging

Article abstract of DOI:10.1002/chem.201801825

New fluorescent molecular probes, which can selectively target specific cell surface receptors, are needed for microscopy, in vivo imaging, and image guided surgery. The preparation of multivalent probes using standard synthetic chemistry can be a laborious process due to low reaction yields caused by steric effects. In this study, fluorescent molecular probes were prepared by a programmed non-covalent pre-assembly process that used a near-infrared fluorescent squaraine dye to thread a macrocycle bearing a cyclic arginine-glycine-aspartate peptide antagonist (cRGDfK) as a cancer targeting unit. Cell microscopy studies using OVCAR-4 (ovarian cancer) and A549 (lung cancer) cells that express high levels of the integrin αvβ3 or αvβ5 receptors, respectively, revealed a multivalent cell targeting effect. That is, there was comparatively more cell uptake of a pre-assembled probe equipped with two copies of the cRGDfK antagonist than a pre-assembled probe with only one appended cRGDfK antagonist. The remarkably high photostability and low phototoxicity of these near-infrared probes allowed for acquisition of long-term fluorescence movies showing endosome trafficking in living cells. In vivo near-infrared fluorescence imaging experiments compared the biodistribution of a targeted and untargeted probe in a xenograft mouse tumor model. The average tumor-to-muscle ratio for the pre-assembled targeted probe was 3.6 which matches the tumor targeting performance reported for analogous cRGDfK-based probes that were prepared entirely by covalent synthesis. The capability to excite these pre-assembled near-infrared fluorescent probes with blue or deep-red excitation light makes it possible to determine if a target site is located superficially or buried in tissue, a probe performance feature that is likely to be very helpful for eventual applications such as fluorescence guided surgery.

Full text of DOI:10.1002/chem.201801825

A Journal of
Accepted Article
Title: Non-Covalently Pre-Assembled High-Performance Near-Infrared
Fluorescent Molecular Probes for Cancer Imaging
Authors: Scott Shaw, Wenqi Liu, César Fernando Azael Gómez Durán,
Cynthia Schreiber, María de Lourdes Betancourt Mendiola,
Canjia Zhai, Felicia Roland, Simon Padanilam, and Bradley D.
Smith
This manuscript has been accepted after peer review and appears as an
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using the Digital Object Identifier (DOI) given below. The VoR will be
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201801825
Link to VoR: http://dx.doi.org/10.1002/chem.201801825
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Non-Covalently Pre-Assembled High-Performance Near-Infrared
Fluorescent Molecular Probes for Cancer Imaging
Scott K. Shaw, Wenqi Liu, César Fernando Azael Gómez Durán, Cynthia L. Schreiber, María de
Lourdes Betancourt Mendiola, Canjia Zhai, Felicia M. Roland, Simon J. Padanilam, and Bradley D.
Smith*[a]
Abstract: New fluorescent molecular probes, which can selectively
target specific cell surface receptors, are needed for microscopy, in
vivo imaging, and image guided surgery. The preparation of
multivalent probes using standard synthetic chemistry can be a
laborious process due to low reaction yields caused by steric effects.
In this study, fluorescent molecular probes were prepared by a
programmed non-covalent pre-assembly process that used a near-
infrared fluorescent squaraine dye to thread a macrocycle bearing a
cyclic arginine-glycine-aspartate peptide antagonist (cRGDfK) as a
cancer targeting unit. Cell microscopy studies using OVCAR-4
(ovarian cancer) and A549 (lung cancer) cells that express high levels
of the integrin αvβ3 or αvβ5 receptors, respectively, revealed a
multivalent cell targeting effect. That is, there was comparatively more
cell uptake of a pre-assembled probe equipped with two copies of the
cRGDfK antagonist than a pre-assembled probe with only one
appended cRGDfK antagonist. The remarkably high photostability
and low phototoxicity of these near-infrared probes allowed
acquisition of long-term fluorescence movies showing endosome
trafficking in living cells. In vivo near-infrared fluorescence imaging
inherent advantages for in vivo imaging due to deeper tissue
penetration of the light and reduced background signal caused by
autofluorescence.[10] Almost all in vivo fluorescent probe designs
can be divided into two distinct strategies: targeted probes that
accumulate over time at a region of interest in the living subject,
or activatable probes that “switch on” fluorescence emission at
the target site.[10] Our focus is on the former, and we are
especially interested in developing multivalent fluorescent probes
that selectively target the exterior surface of cancer cells.
Multivalent targeting of cell surface biomarkers has the potential
of achieving high affinity and high selectivity for cancer cells which
overexpress the biomarker.[11,12] But preparing multivalent probes
using standard covalent chemistry can be a laborious process,
especially when there is a large number of appended targeting
units because steric effects can lower the reaction yields.[13] An
alternative approach to probe fabrication is to employ the
principles of supramolecular chemistry and non-covalently self-
assemble
a collection of molecular components that are
strategically equipped with targeting and reporting units.[14] There
is a growing number of self-assembly methods that generate
nanoscale objects such as vesicles, micelles, and related
nanoparticles, but the relatively large size and statistical structure
of these objects is a potential drawback in some imaging
applications.[15,16] A refined version of this approach is to employ
highly complementary host/guest partners that associate with
strong affinity in water under very mild conditions and create
discrete molecular objects with precise structures and well-
defined stoichiometries. At present there are very few synthetic
host/guest pairs that have the requisite supramolecular and
optical properties to enable self-assembly of multivalent
fluorescent probes for effective in vivo imaging. Literature studies
have examined cyclodextrins, calixarenes, pillararenes, or
cucurbiturils as the host partner.[14] But most of these host
systems are limited by either insufficient host/guest affinity, or the
need to append near-infrared fluorophores by additional covalent
synthesis steps.
experiments compared the biodistribution of
a targeted and
untargeted probe in a xenograft mouse tumor model. The average
tumor-to-muscle ratio for the pre-assembled targeted probe was 3.6
which matches the tumor targeting performance reported for
analogous cRGDfK-based probes that were prepared entirely by
covalent synthesis. The capability to excite these pre-assembled
near-infrared fluorescent probes with blue or deep-red excitation light
makes it possible to determine if a target site is located superficially
or buried in tissue, a probe performance feature that is likely to be
very helpful for eventual applications such as fluorescence guided
surgery.
Introduction
There is increasing demand for molecular imaging agents
which can selectively target specific cell surface receptors as
biomarkers of cancer.[1–5] In particular, fluorescent molecular
probes have broad utility in biochemical assays, cell microscopy,
flow cytometry, preclinical in vivo imaging, clinical fluorescence
guided surgery, and histopathology.[6–9] Fluorescent molecular
probes with near-infrared excitation/emission wavelengths have
We are developing a multivalent near-infrared probe pre-
assembly method using Synthavidin (synthetic avidin)
technology.[17–19] To briefly summarize, we have discovered that
suitably modified tetralactam macrocycles can be threaded in
near-quantitative yield by fluorescent squaraine dyes with
flanking polyethylene glycol (PEG) chains to produce non-
covalent complexes with sufficient stability in water to enable in
vivo imaging of living subjects.[20–22] A major attraction with this
approach is the possibility of mixing different squaraine and
macrocycle components in combinatorial fashion and producing
libraries of pre-assembled probes for subsequent testing. Our
[a]
S. K. Shaw, W. Liu, Dr. C. F. A. Gómez-Durán, C. L. Schreiber, Dr.
M. L. Betancourt-Mendiola, C. Zhai, F. M. Roland, S. J. Padanilam,
Dr. B. D. Smith
Department of Chemistry & Biochemistry
University of Notre Dame
236 Nieuwland Science Hall, University of Notre Dame, IN., 46545,
USA.
previous studies threaded
a bis(thiophene)squaraine dye
platform with two different types of functionalized macrocycles. In
one case, the macrocycle was equipped with multiple
iminodiacetate groups that permitted targeting of the threaded
Supporting information for this article is given via a link at the end of
the document
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complex to the surface of bone,[20] and in a second case, the
macrocycle contained zinc dipicolylamine groups that targeted
the anionic surfaces of bacteria and necrotic tissue.[21,22] In both
of these examples, probe targeting was driven by strong
electrostatic interactions with the surface of the in vivo target, and
the probes exhibited several necessary properties for successful
in vivo operation such as high mechanical and chemical stability.
Furthermore, the probe pharmacokinetics could be controlled in
a predictable manner by modifying the length of the appended
PEG chains. The next step in the research is to substantially
broaden the technological scope by creating pre-assembled
probes with low molecular weight antagonists as cell receptor
targeting units. From a supramolecular perspective, the utilization
of receptor antagonists for probe targeting is a more demanding
challenge, compared to our previous systems, for three reasons:
(a) the increased number and variety of functional groups within
the antagonist structure might inhibit the Synthavidin pre-
assembly process, (b) the PEG chains appended to the pre-
assembled probes might lower antagonist affinity for the receptor
target,[23] and (c) the appended antagonist(s) might alter probe
biodistribution in undesirable ways.[24]
Herein, we report two important technical and conceptual
advances that help produce high performance pre-assembled
fluorescent probes for preclinical cancer imaging and eventually
clinical fluorescence guided surgery. The first advance is a
demonstration that the Synthavidin pre-assembly process can be
used to efficiently fabricate threaded probes equipped with one or
more copies of a small peptide antagonist that has selective
affinity for a cell surface receptor. The arginine-glycine-aspartate
peptide motif was chosen as the cancer targeting unit. More
specifically we used the cyclic peptide cRGDfK, a validated high-
affinity antagonist for several types of integrin receptors which are
overexpressed in many forms of cancer.[6] The second advance
is a major improvement in the photostability of the pre-assembled
Scheme 1. Illustration of Synthavidin pre-assembly, using a macrocycle (red)
with a conjugated antagonist as targeting unit (green) and a monomeric or
linked dimeric near-infrared fluorescent squaraine dye (blue).
Results and Discussion
Shown in Scheme 2a are the individual squaraine and
macrocycle components that were used to pre-assemble the five
near-infrared fluorescent probes for this study. The synthesis and
spectral characterization of each compound is provided in the
Supporting Information (SI). In Scheme 2b is a schematic
summary of the pre-assembled probes that were prepared by
simply mixing the corresponding components in water. The three
squaraine
bis(hydroxyphenyl)squaraine
derivatives,
bis(thiophene)squaraine
H, and
T,
dimeric
bis(hydroxyphenyl)squaraine H3H, were threaded by either the
symmetric macrocycle C with six appended carboxylate groups,
or the unsymmetric macrocycle R. At one end of R is a copy of
the cRGDfK antagonist, and at the other end is an appended
tricarboxylate unit which ensured good water solubility.[28] In
Scheme 2c is a molecular model showing how the tetralactam
macrocycle encapsulates a bis(hydroxyphenyl)squaraine dye.
The association constant for threading is close to 108 M-1 in water
and the complex is stabilized by a combination of four hydrogen
bonds between the squaraine oxygens and the macrocycle NH
residues, and hydrophobic stacking of the dye’s aromatic
probes,
which
was
achieved
by
replacing
the
bis(thiophene)squaraine dye platform with a more photostable
bis(hydroxy)phenyl squaraine platform.[25–27] At the project’s
outset there was some literature precedence supporting the
feasibility of each structural change as a separate modification to
the Synthavidin pre-assembly process, but it was not certain if
both modifications could be incorporated simultaneously.
Schematically, the project goal was to prepare the two targeted
pre-assembled probes shown in Scheme 1. One probe has a
surfaces against the macrcocycle’s anthracene side walls.[26,27]
A
key stuctural feature within H and H3H is the terminal N-propyl
group at each end of the bis(hydroxyphenyl)squaraine
chromophores which we knew would act as a “steric speed bump”
and slow the kinetics for threading/dethreading but not alter the
association constant.[29] Thus, we expected that probe pre-
assembly could be achieved in very high yield and short time-
frame if the squaraine and macrocycle components were mixed
in water at micromolar concentration. Indeed, preliminary
threading studies showed that near-quantitative threading
occurred when H was mixed with one molar equivalent of R or C,
or H3H was mixed with two equivalents of R or C. In Figure 1a-b
are representative absorption spectra of the squaraine dyes
before and after probe pre-assembly. The absorption profile for
free dimeric H3H is extremely broad due to folding of the structure
and stacking of its two squaraine chromophores.[20,22] But after
addition of macrocycle, near-quantitative threading and probe
pre-assembly was indicated by the appearance of diagnostic
narrow and red-shifted squaraine absorbance maxima. In Figure
1c-d are two representative sets of absorption spectra showing
the spectral changes that occur during the probe pre-assembly
single peptide
antagonist
threaded onto
a
single
bis(hydroxy)phenyl squaraine dye, and the other has two copies
of the antagonist threaded on to a linked bis(hydroxy)phenyl
squaraine dimer. We also prepared control versions of these pre-
assembled probes that lacked targeting units. The first step in the
project was to synthesize the requisite squaraine and macrocycle
building blocks and then demonstrate that they can be pre-
assembled under mild conditions to make stable fluorescent
probes in near-quantitative yield. The next step was to assess the
performance of these new integrin-targeted near-infrared
fluorescent probes for microscopic imaging of cancer cells and in
vivo imaging of tumors in a mouse model of cancer.
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Scheme 2. (a) Compounds used to pre-assemble the different fluorescent probes. (b) Molecular model of tetralactam macrocycle threaded by
bis(hydroxyphenyl)squaraine dye illustrating the important hydrogen bonding and hydrophobic stacking interactions. (c) Descriptors of the pre-assembled
fluorescent probes used in this study.
process. The threaded probes exhibit sharp squaraine absorption
maxima bands indicating little propensity for intermolecular
aggregation, a conclusion that was supported by Dynamic Light
Scattering analysis.
As a standard procedure, a 15 hour incubation period was
employed to pre-assemble a 50 µM stock solution of each probe
in water. In Table 1 is a summary of the photophysical properties
of the free squaraine dyes and the pre-assembled probes in water.
In agreement with expectations, the quantum yield for free
squaraine dye H in water was relatively high but it was extremely
low for the free dimeric squaraine H3H due to its folded and self-
quenched structure.[20,22] Likewise, the relatively low quantum
yields for the dimeric threaded probes 2(C) ⊃ H3H and 2(R) ⊃
H3H are also attributed to their partially folded and quenched
structures in water.
Additional spectral proof that macrocycle threading had
occurred was gained by observing efficient internal energy
transfer after excitation of the anthracene sidewalls in the
macrocycle with 350 nm light (Figure S1-S2). In the cases of C⊃
H and R ⊃ H, excitation of the anthracene sidewalls produced a
quenched anthracene emission band (relative to the emission
spectrum of free macrocycle) and strong squaraine emission at
710 nm. In the cases of 2(C) ⊃ H3H and 2(R)⊃ H3H, the
emission band for each threaded probe was partially quenched
but the squaraine emission was also weak due to the probe’s
partially folded and self-quenched structure.
Figure 1. Absorption spectra in water for: (a) free H and threaded probes C ⊃
H and R ⊃ H, (b) free H3H and threaded probes 2(C) ⊃ H3H and 2(R) ⊃
H3H. For all samples, the molecular concentration was 5 µM. Representative
absorption spectra showing spectral changes that occur over time due to
macrocycle threading and probe pre-assembly in water and 25 °C; (c) mixing H
and R (1:1 molar ratio) to make R ⊃ H, monitored over 140 minutes, (d) mixing
H3H and R (1:2 molar ratio) to make R ⊃ H, monitored over 200 minutes.
Further independent proof of the structures for R⊃H and 2(R)
⊃ H3H was provided by high resolution mass spectroscopy with
electrospray ionization, which identified molecular ion peaks that
were emblematic of the threaded complexes (Figures S3-S4). 1H
NMR spectra of the threaded complexes were quite broad at the
relatively high millimolar concentrations needed for spectral
acquisition and thus not very informative,[20] but purity analysis by
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agarose gel electrophoresis proved to be very insightful (Figure
2). Individual samples of H, H3H, R ⊃ H, and 2(R) ⊃ H3H were
loaded onto an agarose gel and separated according to their
different charge-to-mass ratios at pH 8.3. For uncharged H and
H3H there was little or no electrophoretic migration, while the
double threaded probe 2(R) ⊃ H3H with much higher charge-to-
mass ratio migrated much further than the singly threaded, R⊃ H
(Figure 2a). A fifth well was loaded with an equal volume mixture
of the four pre-assembled probes. The well-resolved bands
observed in this “mixture” lane matched those seen in the
individual lanes, and demonstrated that H, H3H, R⊃ H, and 2(R)
⊃ H3H were all stable and that the threaded probes were formed
in near-quantitative yield. The fluorescent properties of these
compounds allowed the gels to be imaged using a deep-red
excitation/near-infrared emission filter set (Figure 2b) and also a
blue excitation/near-infrared emission filter set (Figure 2c). In the
case of deep-red excitation, both the free squaraine dyes and
threaded complexes were observed with brightness
commensurate to the quantum yields listed in Table 1 (for H3H,
this means almost no emission was observed). In the case of blue
excitation, only the threaded complexes were observed to give
near-infrared emission due to their unique capacity for internal
energy transfer.
expressed at high levels ([9.8 ± 2.5] x 104/cell) on the surface of
OVCAR-4 cells, and that the cells are amenable to molecular
targeting and imaging using covalently-conjugated fluorophores
bearing the cRGDfK antagonist.[33] In contrast A549
adenocarcinoma cells do not express αvβ3, but they do express
the integrin αvβ5 receptor, which also has good affinity for the
cRGDfK motif.[34] Expression of αvβ5 in A549 cells was
corroborated using immunocytochemistry staining with an anti-β5
FITC-conjugated antibody (Figure S6).
A quantitative flow
cytometry study, using this antibody in conjunction with a
fluorescence calibration kit, found that there was an average of
[10.2 ± 3.7] x104 β5 proteins on the surface of each A549 cell
(Figure S7). Furthermore, microscopy studies using both cell lines
showed binding and uptake of a commercially available orange
fluorescent cRGDfK-Cy3 probe, along with blocking by free
cRGDfK (structures and micrographs in Figure S8). Subsequent
fluorescence microscopy studies quantified the binding and
Table 1. Photophysical properties of free dyes and pre-
assembled probes in water.
Compound
Abs (nm)
Em (nm)
Φ[a]
T
666
677
652
671
669
644
670
678
710
675
711
707
668
712
0.04[b]
0.18[b]
0.24
C ⊃ T
H
C ⊃ H
R ⊃ H
H3H
0.14
0.18
0.005
0.027
2(C) ⊃ H3H
2(R) ⊃ H3H
671
710
0.024
[a] Relative to a standard solution of methylene blue in water (Φf = 0.02)[30,31]
[b] From reference[29]
Figure 2. Gel electrophoresis using 2% agarose gel loaded with free squaraine
dyes or threaded probes (193 µM/well). The three images show the same gel
after electrophoresis (100 V for 30 min, then 125 V for 2 h, pH = 8.3). (a) White
light image, dashed circles denote location of sample bands. (b) Fluorescence
image with ex: 640/25 nm, em: 732/38 nm. (c) Fluorescence image with ex:
468/22, em: 732/38 nm.
uptake of the pre-assembled near-infrared targeted and
untargeted probes. The microscopy experiments incubated
separate samples of the cells with one of the probes for 30 min,
then fixed the cells and washed them several times to remove
unbound probe from the cell surface. Additionally, deconvolution
microscopy techniques were employed to remove erroneous
signal emanating from the cell exterior and thus allowed the
amount of internalized probe to be quantified. Both OVCAR-4 and
A549 cells exhibited high uptake of the cRGDfK-targeted probes
R ⊃ H and 2(R) ⊃ H3H with the fluorescent signal localized to
punctate compartments inside the cell (Figure 3a). In contrast,
uptake of the analogous untargeted probes C ⊃ H and 2(C) ⊃
H3H was significantly lower. Receptor blocking experiments also
showed that internalization of the cRGDfK-targeted probes could
be inhibited by an excess of free cRGDfK. The amount of cell
internalization for monovalent R ⊃ H and divalent 2(R) ⊃ H3H
was compared under cell incubation conditions that used the
same concentration (2 µM) of cRGDfK targeting unit, and in both
cell lines there was greater uptake of the divalent targeted 2(R)⊃
Taken together, the independent spectral, mass spectrometry
and electrophoresis data prove that probe pre-assembly at 50 µM
in water occurs in near-quantitative yield. Furthermore, probe
stability experiments indicated that negligible dethreading of the
pre-assembled probes occurred over 17 hours after an aliquot of
probe sample was diluted to 2 µM in fetal bovine serum (Figure
S5). This finding is consistent with our previous work showing that
Synthavidin pre-assembled probes have sufficiently high kinetic
and thermodynamic stability to be effective biological imaging
agents.[20-22]
The cancer targeting capabilities of R ⊃ H and 2(R) ⊃ H3H
were assessed using the integrin expressing cell lines OVCAR-4
and A549. OVCAR-4 is an excellent cell model of high-grade
serous ovarian carcinoma because it has a very similar genomic
sequence and response to chemotherapeutic drugs.[32] We have
recently demonstrated that the αvβ3 integrin receptor is
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H3H (Figure 3b,c), indicating an enhanced multivalent targeting
effect.
2(R)⊃ H3H or the orange emitting cRGDfK-Cy3, and analyses of
these movies are provided in Figure 4c-d. The Cy3 fluorophore is
well-known for its very high photostability,[35] but the fluorescence
micrographs of A549 cells containing cRGDfK-Cy3 were almost
completely bleached after 15 min of imaging. Remarkably, under
the same conditions, there was only a 30% drop in fluorescence
intensity for cell micrographs containing 2(R) ⊃ H3H, making it a
very rare example of a near-infrared fluorescent probe that is
more photostable than Cy3.[36]
The last stage of the project was to determine the in vivo
cancer targeting capability of divalent cRGDfK-targeted 2(R) ⊃
H3H in a xenograft mouse tumor model. Our goal was to make a
preliminary assessment of probe potential for fluorescence-
guided surgery of cancer where the desired imaging outcome is
accurate identification of a tumor within a couple of hours after
dosing the subject with a fluorescent probe.[37] Sixteen athymic
nude mice were injected subcutaneously with A549 cells in their
rear flanks and tumors were allowed to develop over one month.
The anesthetized mice were randomly split into two cohorts and
given tail vein injections of either cRGDfK-targeted 2(R)⊃H3H or
the untargeted control 2(C) ⊃ H3H. A series of live animal
fluorescence imaging experiments was conducted using an In
Vivo Imaging Station (IVIS). Whole mouse images collected at 90
and 180 minutes showed significantly higher tumor accumulation
of targeted 2(R) ⊃ H3H than untargeted 2(C) ⊃ H3H (Figure 5a).
One of the major aims of the project was to improve probe
photostability by changing the structure of the encapsulated
squaraine dyes from bis(thiophene)squaraine (T) to
bis(hydroxy)phenyl squaraine (H). Enhanced photostability was
first demonstrated by conducting simple cuvette experiments that
illuminated two separate solutions containing the threaded probes,
C ⊃ T or C ⊃ H (Figure 4a-b). The decrease in squaraine
absorption for C ⊃ T indicated moderate photobleaching
compared to no change in absorption for C⊃T. Confirmation that
C ⊃ T was a stronger oxygen photosensitizer than C ⊃ H was
gained by conducting an experiment that used the commercial
indicator dye Singlet Oxygen Sensor Green (SOSG) to trap and
quantify a higher amount of photogenerated singlet oxygen
(Figure S9). This improvement in probe photostability was
maintained during live cell microcopy experiments. For example,
separate samples of cells were incubated with C⊃ T or C⊃ H for
several hours to permit non-specific uptake, and an
epifluorescence microscope was used to collect movies of probe-
labelled endosome trafficking in each sample (see SI for movies
and Figure S10 for movie montage). The movies show significant
photobleaching for the sample containing C⊃T and much greater
stability for the sample containing C ⊃ H. Additional live cell
fluorescence microscopy studies found that the cRGDfK-targeted
probes R ⊃ H and especially 2(R) ⊃ H3H probes were also
remarkably photostable. In the Supporting Information is a movie
showing trafficking of endosomes containing either near-infrared
Figure 3. (a) Representative fluorescence micrographs showing relative amounts of near-infrared probes taken up by OVCAR-4 or A549 cells after 30 min:
2.0 µM C ⊃ H, 2.0 µM R ⊃ H, 2.0 µM R ⊃ H + 100 µM cRGDfK, 1.0 µM 2(C) ⊃ H3H, 1.0 µM 2(R) ⊃ H3H, or 1.0 µM 2(R) ⊃ H3H + 100 µM cRGDfK. Blue
fluorescence shows Hoechst nuclear stain. Scale bar = 30 µm. (b,c) Quantification of mean near-infrared intracellular fluorescence intensities as a measure of
probe internalization.
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Figure 4. Absorption spectra of: (a) C ⊃ H and (b) C ⊃ T (2 µM in H2O)
before (black line) and after (red line) irradiation with red light for 20 min. (c)
Plots showing relative photobleaching over 15 min for fluorescent micrographs
of separate samples of live A549 cells containing near-infrared 2(R) ⊃ H3H or
orange emitting cRGDfk-Cy3. The micrographs were acquired every 10 s using
2 s exposure times. (d) Representative fluorescence micrographs at time = 0
min and 15 min. The gray/white color indicates 2(R) ⊃ H3H or cRGDfk-Cy3
fluorescence, and blue shows Hoechst nuclear stain
Quantitative analysis of the live mouse images (Figure S11)
provided two conclusions. At the 90 minute time point, the tumor
could be unambiguously identified in all mice treated with the
targeted probe. In addition, there was high accumulation of the
probe in the kidneys indicating reasonably rapid renal clearance
of the probe from the blood stream, which is a desirable attribute
for high contrast imaging. The live animal images at the 180
minute time point showed that probe accumulation in tumor or
kidney had hardly changed so the mice were sacrificed and the
excised organs imaged using the IVIS (Figures 5b). The ratio of
mean pixel intensity (MPI) for near-infrared probe fluorescence in
each organ relative to that of excised thigh muscle and the data
is plotted in Figure 5c. [38] The average tumor-to-muscle ratio of
3.6 for mice that were treated with targeted 2(R) ⊃ H3H is similar
to the tumor selectivity ratios reported for analogous but
completely covalent RGD-targeted molecular probes in similar
mouse tumor models.[33,39-41] Moreover, a tumor selectivity ratio of
3 or more is a desired benchmark for effective fluorescence
guided surgery.[42,43]
A final demonstration of the potential of these pre-assembled
fluorescent probes for fluorescence guided surgery was gained
by conducting a phantom imaging experiment that showcased a
unique performance feature, namely, the capability to determine
if a target site is exposed to the camera or screened by a layer of
tissue. Two wells within a multiwall plate were filled with solutions
of pre-assembled C ⊃ H and simultaneously imaged in the IVIS
using either blue excitation (ex: 410 nm) with near-infrared
Figure 5. (a) Representative near-infrared fluorescence images of tumor-
burdened mice at -15 min, 90 min, and 180 min after intravenous injection of
2(C) ⊃ H3H or 2(R) ⊃ H3H (5 nmol/mouse). (b) Near-infrared fluorescence
images of tumors removed from the mice at 3 hours after probe injection. (c)
Biodistribution of fluorescent probes in organs removed from the mice (N = 8
for each cohort). The mean pixel intensity (MPI) for near-infrared probe
fluorescence in each organ is relative to the MPI for thigh muscle from the same
animal; error bars indicate ±SEM. Insert shows scatter plot for tumor MPI data.
emission (enabled by energy transfer from the excited anthracene
sidewalls of the surrounding macrocycle to the encapsulated
squaraine dye within C ⊃ H) or direct deep-red excitation of the
encapsulated squaraine (ex: 670 nm). As illustrated in Figure 6,
the light path of one of the wells was screened with a 0.5 cm thick
slice of chicken breast. Inspection of the near-infrared
fluorescence images shows that screening of a cuvette with
tissue barely decreased emission intensity when the samples
were illuminated with deep-red (670 nm) light, but there was 70%
decrease in emission intensity for a screened cuvette when it was
illuminated with blue (410 nm) light whose intensity was strongly
attenuated by the tissue. Thus, by simply switching between blue
and deep-red illumination light, an observer can rapidly and
conveniently determine if a fluorescence signal is radiating from
an exposed target site or from a site that is buried underneath
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tissue. This is a key question that has to be answered often during
fluorescence guided surgery of cancer.[37]
Experimental Section
Materials
Antibodies were purchased from ThermoFischer: Anti-Human
CD51/CD61 (Integrin αvβ3) FITC, catalog number 11-0519-42; Integrin β5
Monoclonal Antibody (KN52) FITC, 11-0497-4; Mouse IgG1 kappa Isotype
Control FITC, catalog number 11-4714-42. cRGDfk-Cy3 was purchased
from Molecular Targeting Technologies, catalog number RG-1002.
Agarose gel and Tris/Borate/EDTA (TBE) Buffer materials were
purchased from Sigma Aldrich. Singlet Oxygen Sensor Green (SOSG)
was purchased from Thermo Fischer.
Synthesis
Figure 6. (a) Illustration of phantom imaging experiment to assess fluorescence
screening by tissue. (b) Fluorescence images of two wells, each containing a
solution of C ⊃ H (5 µM) with ex: 410/10 nm or 670/10 nm and em: 700/17.5
nm. A 0.5 cm thick slice of chicken breast screened the light path of the upper
well, while the lower well was exposed to the camera. (c) Quantification of the
two sets of images (ex: 410 nm or 670 nm) as a ratio of fluorescence mean pixel
intensity (MPI) for the screened well divided by the MPI for the exposed well.
The synthetic methods used to prepare the squaraine dyes and
macrocycles as well as the compound characterization are described in
the Supporting Information.
Stock Preparation
Stock solutions (1 mM) of squaraine and macrocycle components C, R, T,
or H were prepared in water. A stock solution (1 mM) of H3H was prepared
in DMSO. Stock solutions (50 µM) of pre-assembled probes, C ⊃ T, C ⊃
H, R ⊃ H, 2(R) ⊃ H3H, or 2(R) ⊃ H3H were prepared by mixing the
macrocycle and squaraine components in water at appropriate
stoichiometries (1:1 or 2:1) and allowing the sample to shake gently for 15
hours at room temperature. After spectral confirmation that each pre-
assembled structure was formed in near-quantitative yield, the stock
solutions of pre-assembled probe were used for bioimaging experiments.
Conclusions
A new class of non-covalently pre-assembled near-infrared
molecular probes was prepared with appended cRGDfK
antagonist(s) that have selective affinity for integrin receptors that
are often overexpressed in many forms of cancer. The probes
were pre-assembled by threading a functionalized tetralactam
macrocycle with a squaraine dye that had flanking PEG chains.
Quantitative probe pre-assembly in water was confirmed by a
collection of independent fluorescence, mass spectral, and
electrophoresis data. The new pre-assembled probes are based
on bis(hydroxy)phenyl squaraine dyes which are much more
photostable and generate less singlet oxygen than analogous
probes based on bis(thiophene)squaraine dyes. This significant
improvement made it possible to acquire long-term microcopy
movies of probe-labeled living cells. Fluorescence microscopy
studies using OVCAR-4 (ovarian cancer) and A549 (lung cancer)
cells that expressed high levels of the integrin αvβ3 or αvβ5
receptors, respectively, found a multivalent cell targeting effect.
That is, there was comparatively more cell uptake of a pre-
assembled probe equipped with two copies of the cRGDfK
antagonist than a pre-assembled probe with only one appended
cRGDfK antagonist. In vivo near-infrared fluorescence imaging
experiments compared the biodistribution of targeted 2(R)⊃ H3H
and untargeted 2(C) ⊃ H3H in a xenograft mouse tumor model.
The average tumor-to-muscle ratio for the targeted probe was 3.6
which reaches the tumor selectivity benchmark that is needed for
effective fluorescence guided surgery. A unique performance
feature of these pre-assembled probes is the capability to excite
the near-infrared fluorescence with blue or deep-red excitation
light. It should be possible to exploit this feature, as a convenient
means to determine if a fluorescent target site is located
superficially or buried in tissue.
Photophysical studies
Fluorescence spectra were taken and analyzed on a Horiba Fluoromax-4
Fluorometer with FluorEssence software. Absorption spectra were
collected on an Evolution 201 UV/Vis Spectrometer with Thermo Insight
software. All spectra were obtained using spectrophotometric grade
solvent at 20 ˚C with either a glass cuvette or quartz cuvette (1 mL, 10 mm
path length). Quantum yield measurements used methylene blue (Φf =
0.02 in H2O) as a standard. The sample concentrations were adjusted to
have an absorption of 0.08 at 600 nm. The fluorescence spectrum of each
solution was obtained by excitation at 600 nm and the integrated area was
used to calculate the quantum yield. The estimated error of this method is
±10%.[30]
Photobleaching and singlet oxygen photogeneration studies examined
separate 2 µM solutions of C ⊃ T or C ⊃ H in water, with or without SOSG
(30 µg/mL). The samples were exposed to air and irradiated with a Xenon
lamp (150 W, 620 nm long-pass filter) for 20 min. The photobleaching
experiment monitored loss of the squaraine absorbance maxima band.
The singlet oxygen photogeneration experiment monitored fluorescence
emission between 500-650 nm (ex: 490 nm) with fluorescence increase
indicating singlet oxygen photogeneration.
Gel Electrophoresis
Separate solutions of pre-assembled probes H, R ⊃ H, H3H, and 2(R) ⊃
H3H (193 µM) were added (20 µL) to separate wells in a 2% agarose gel.
A mixture of all four probe solutions (5 µL each) was added into a fifth well.
Cold TBE buffer (pH 8.3, 8.9 mM Tris, 8.9 mM borate, 200 µM) was filled
to the top of the gel (but not covering the wells, to prevent the compounds
from leaking into the buffer). The gel was run for at 100 V for 30 min to
load the compounds into the gel. The entire gel was subsequently covered
with TBE buffer and run at 125 V for 2 hr. The gel was photographed in
ambient light, and a fluorescent image was acquired using an In Vivo
Imaging Station with the following parameters: Cy5.5 filter set (ex: 640/25
nm, em: 732/38 nm, exposure: 7 s, F-stop: 2, binning: small), or
GFP/Cy5.5 (ex: 468/22 nm, em: 732/38 nm, exposure: 7 s, F-stop: 2
binning: small). Overlaid images were created using ImageJ software.
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photobleaching: (1) a plot of the micrograph MFI vs time was generated,
(2) the plot was fit with a one phase decay and the baseline of the fit was
used to approximate the MFI of the fully photobleached sample, (3) this
baseline MFI was subtracted from the MFI of each micrograph, (4) the
baseline adjusted MFI was normalized to the baseline adjusted MFI at time
= 0 min and plotted.
Cell Culture
OVCAR-4 human ovarian carcinoma cells (DCTD Tumor Repository,
National Cancer Institute at Frederick, Maryland) and A549 human lung
carcinoma cells (ATCC CCL-185) were cultured in RPMI-1640 or F-12K
media (Thermo Fischer) supplemented with 10% (w/w) fetal bovine serum,
1% streptomycin-penicillin, 5% L-Glutamine (only in RPMI media), and 0.1
mM NEAA in a humidified incubator at 37 °C, 5% CO2 over air.
In vivo Imaging
All mouse experiments were approved by the University of Notre Dame
Institutional Animal Care and Use Committee. A549 cells were grown to
confluency in complete F12K growth medium supplemented with 10% fetal
bovine serum. Female Foxn1 nude mice (Charles River Laboratories)
were injected subcutaneously on the right rear flank with A549 cells (1×106
cells/mouse, 100 μL) in 1:1 Matrigel(Corning):media. Approximately, one
month later, the mice were randomly divided into two cohorts of eight, and
each mouse received an intravenous injection of either 2(C)⊃ H3H or 2(R)
⊃ H3H (170 µL, 5 nmol/mouse). The mice were then anesthetized with 2-
3% isoflurane (oxygen rate of 2 L/min) for imaging. A preliminary
fluorescence image was acquired in an In vivo Imaging System (IVIS)
using a Cy5.5 filter set (ex: 640/25 nm, em: 732/38 nm, exposure: 3 s, F-
stop: 2, binning: small). The mice were imaged again at 90 min and 180
min and the images analyzed using ImageJ software to calculate tumor-
to-background ratio (TBR) and kidney-to-background ratio (KBR). After
180 min, the mice were anesthetized, sacrificed via cervical dislocation,
and the major organs were removed (liver, heart, lungs, spleen, and
kidneys, along with thigh muscle, blood, and tumor). The organs were
imaged following the above protocol. Images were processed and
analyzed using ImageJ software. For whole mouse images and tumor
images, a 1000 pnt. rolling ball radius was applied for background
subtraction and all images were scaled against the highest intensity in the
set. The biodistribution analysis involved: (1) subtracting a 1000 point
rolling background, (2) creating a region of interest (ROI) around each
organ using an intensity threshold, (3) measuring the mean pixel intensity
(MPI), (4) normalizing the MPI to that of the thigh muscle, (5) averaging
and plotting the normalized MPI with its SEM. It is important to realize that
the threaded probe has to be the source of the near-infrared fluorescence
signal in the mouse images because the IVIS does not detect the blue-
shifted and very weakly fluorescent squaraine dye H3H that is produced
by probe unthreading (Figure S12).
Flow Cytometry
A549 cells were dissociated from a confluent plate with non-enzymatic
dissociation solution (Gibco). The cells were then incubated for 30 min at
4 °C with either 0.375 μg/mL of FITC conjugated β5 antibody or 7.5 μg/mL
of FITC conjugated IgG1 kappa isotype control. After incubation, the cells
were washed and fixed with IC fixation buffer (ThermoFisher Scientific).
Using a BD LSRFortessa X-20 flow cytometer, the cells were excited at
488 nm and emission was collected with a FITC 520/530 nm filter (max:
20,000 events). The data was then analyzed using FlowJo software,
where the geometric mean fluorescence of each sample was calculated
by subtracting the isotype control fluorescence from the sample
fluorescence. The geometric mean fluorescence was then plotted to a
calibration plot created using a Quantum™ FITC-5 MESF kit. This
identified the number of FITC fluorophores per cell which was
subsequently divided by the number of FITC fluorophores per antibody
(β5: 5.5 ± 1.0). A resulting value of ([10.2 ± 3.7] x104 β5 proteins/cell) was
determined, assuming only one antibody could bind at a time. Experiments
were repeated independently three times in duplicate
Microscopic Imaging of Cancer Cells
Sample preparation
Cells were seeded onto an 8-well chambered coverglass (Lab-Tek, Nunc,
USA) and grown to 80% confluence (48 h). Prior to treatment with probes,
the media was replaced with a supplemented HEPES buffer (25 mM
HEPES, 130 mM NaCl, 3.8 mM KCl, 0.35 g/L Na2HCO3, 7.0 mM glucose,
2 mM sodium pyruvate, 1x MEM NEAA, pH 7.4) for 20 min. The cells were
then incubated with 200 µL R ⊃ H (2 µM) or 2(R) ⊃ H3H (1 µM) in buffer
at 37 °C for 30 min. For live cell experiments, cells were washed and co-
stained with 3 µM Hoescht for 10 min, then washed twice and imaged
under supplemented HEPES buffer. For fixed cell experiments,
endocytosis was stopped by the addition of 100 μL ice cold 4%
paraformaldehyde for 5 min. Blocking experiments used free cRGDfk (100
μM) applied both during the 20 min FBS starvation treatment and during
the cell staining treatment. Control experiments were conducted in an
Light Penetration through Tissue
Separate solutions of C ⊃ H (2.5 mL, 5 µM) were added to two wells in a
multi-well plate plate. A 0.5 cm thick slice of chicken breast was placed
under one of the wells to screen the excitation/emission light path. The
wells were imaged with a Bruker Xtreme with ex: 410/10 nm or 670/10 nm
and em: 700/17.5 nm. The fluorescence images were processed in
ImageJ by creating an ROI around the perimeter of each well and
measuring the MPI. The MPI for the image of the screened well was
normalized to that of the exposed well for both the 410 nm excitation and
670 nm excitation image sets.
analogous
manner
with
orange
emitting
cRGDfk-Cy3.
Immunocytochemistry experiments for αvβ5 expression on A549 cells
used FITC conjugated β5 monoclonal antibody (KN52) (0.125 μg/100 μL,
eBioscience 11-0497-41) where the antibody was administered for 20 min
in supplemented HEPES buffer followed by two wash steps, fixation as
before, and imaging under PBS.
Fluorescence microscopy
Fluorescence microscopy was conducted on a GE Healthcare DeltaVision
Deconvolution epifluorescence microscope equipped with a X-cite 120
fluorescence illumination system and the following filter sets: near-infrared
(ex: 660/20 nm, em: 700/30 nm), orange (ex: 555/28, em: 617/73), green
(ex: 490/20 nm, em: 528/38 nm), ultraviolet (ex: 360/40 nm, em: 457/50
nm). Images were collected in softWoRx using a Photometrics Cascade
II:512 EMCCD camera operating in CCD mode with 1 s acquisition times,
1 MHz readout speed. Images were deconvolved in softWoRx, and a 50
pnt. rolling background was subtracted in ImageJ. The mean fluorescence
intensity (MFI) for each micrograph was calculated from the average of 20
randomly generated 25x25 pixel extra-nuclear ROIs. Averages and SEM
were calculated and plotted in GraphPad Prism using 3 images each from
3 biological replicate experiments. Timelapse image sets of live cells were
acquired with the same settings, every 10 s, for 15 min. For plots of
Acknowledgements
This work was supported by grants from the NSF (CHE1401783
to B.D.S.), the NIH (R01GM059078 to B.D.S. and T32GM075762
to S.K.S, C.L.S, and F.M.R.) and the CONACyT (Mexico).
Keywords: Supramolecular chemistry • Host-Guest systems •
Self-assembly • Fluorescent probes • Cancer
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A macrocycle bearing a cancer targeting peptide is used to pre-assemble highly
photostable near-infrared fluorescent molecular probes for cancer imaging in cells or
living subjects.
Scott K. Shaw, Wenqi Liu, César
Fernando Azael Gómez Durán, Cynthia
L. Schreiber, María de Lourdes
Betancourt Mendiola, Canjia Zhai,
Felicia M. Roland, Simon J. Padanilam,
and Bradley D. Smith*
Page No. – Page No.
Non-Covalently Pre-Assembled High-
Performance Near-Infrared
Fluorescent Molecular Probes for
Cancer Imaging
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