Synthesis and in vivo fate of zwitterionic near-infrared fluorophores

Article abstract of DOI:10.1002/anie.201102459

To address two fundamental and unsolved problems in optical imaging (nonspecific uptake of near-infrared fluorophores by normal tissues and organs and incomplete elimination of unbound targeted fluorophores from the body), novel zwitterionic near-infrared fluorophores (e.g., ZW800-1) were synthesized and their performance compared in vivo to conventional molecules (e.g., ICG) as a function of charge, charge distribution, and hydrophobicity (see picture). Copyright

Full text of DOI:10.1002/anie.201102459

Communications
DOI: 10.1002/anie.201102459
Fluorescent Probes
Synthesis and In Vivo Fate of Zwitterionic Near-Infrared
Fluorophores**
Hak Soo Choi, Khaled Nasr, Sergey Alyabyev, Dina Feith, Jeong Heon Lee, Soon Hee Kim,
Yoshitomo Ashitate, Hoon Hyun, Gabor Patonay, Lucjan Strekowski, Maged Henary,* and
John V. Frangioni*
A longstanding problem in the field of image-guided surgery
is the development of ideal near-infrared (NIR) fluorophores.
The heptamethine NIR fluorophore indocyanine green (ICG)
has been used extensively for image-guided surgery because
Previously, our group showed that rigid spherical nano-
particles, such as quantum dots, with a hydrodynamic
diameter(HD) ꢀ 5.5 nm could be rapidly cleared by the
kidneys, and exhibit low background binding to normal
tissues and organs, but only when the surface charge was
neutral, geometrically balanced, and polyionic (referred to
[
1–3]
of its clinical availability and safety.
However, ICG is far
from ideal because it exhibits high uptake in the liver,
contaminates the gastrointestinal (GI) tract, provides mod-
[
14–19]
herein as zwitterionic for simplicity).
In this study, we
[4]
[3,5]
erate optical properties, is unstable in aqueous media,
and is unable to conjugate covalently to targeting ligands.
explored the hypothesis that NIR fluorescent small molecules
would exhibit improved in vitro and in vivo performance if
synthesized with zwitterionic charges that are evenly spaced
over the molecule to shield the underlying hydrophobicity of
the relatively large heptamethine core.
[2]
Although several classes of novel molecules have been
[
6–13]
described,
none to date exhibit simultaneous low back-
ground binding, bifunctionality, excellent optical properties,
low protein binding, and high serum stability. Although it is
intuitive that physicochemical properties, that is, positive/
negative charge density, hydrophilicity/lipophilicity, and
charge distribution, will impact in vivo performance, chemical
structures that exhibit ideal characteristics have not yet been
defined.
We describe two complementary molecules, termed
ZW800 Æ i where Æ i is the charge of the conjugated targeting
ligand that will render the final molecule with a net charge of
zero (i.e., zwitterionic). ZW800-1 has a net charge = 0 prior to
targeting ligand conjugation, and a net charge = 0 after
conjugation to a targeting ligand that has a net charge of À1
(that is, a targeting ligand with a net charge of 0 prior to
conjugation). ZW800-3a has a net charge = + 2 prior to
conjugation, and a net charge = 0 after conjugation to a
targeting ligand that has a net charge of À3 (that is, a targeting
ligand with a net charge of À2 prior to conjugation). ZW800-1
and ZW800-3a were engineered for high hydrophilicity, with
logD (distribution coefficient) at pH 7.4 of À3.56 and À6.95,
respectively. Importantly, these molecules have also been
engineered with sulfonate groups to impart negative charge
and quaternary ammonium cations (quats) to impart positive
charge because preliminary results showed that the weaker,
more common natural analogues (carboxylic acids and
primary amines, respectively) did not exhibit the desired
properties.
[
*] Dr. H. S. Choi, Dr. K. Nasr, D. Feith, J. H. Lee, Dr. S. H. Kim,
Y. Ashitate, Dr. H. Hyun, Dr. J. V. Frangioni
Division of Hematology/Oncology, Department of Medicine and
Department of Radiology, Beth Israel Deaconess Medical Center
330 Brookline Avenue, SLB-05, Boston, MA 02215 (USA)
Fax: (+1)617-667-0981
E-mail: jfrangio@bidmc.harvard.edu
Dr. S. Alyabyev, Dr. G. Patonay, Dr. L. Strekowski, Dr. M. Henary
Department of Chemistry, Georgia State University
Atlanta, GA 30303 (USA)
Fax: (+1)404-413-5505
E-mail: chemmh@langate.gsu.edu
Dr. S. H. Kim
As depicted in Figure 1a, chloro-substituted NIR fluoro-
phores 8 and 9 were synthesized by employing quats and/or
sulfonates (5 or 6) on the indocyanine backbone. Vilsmeier–
Haack reagent 7 was synthesized and used for the condensa-
tion reaction with prepared intermediate indolium salts in
anhydrous sodium acetate. Finally, to permit subsequent
conjugation of targeting ligands and biomolecules, a bifunc-
tional phenoxypropionic acid linkage was introduced on the
meso-chlorine atom. Using microwave synthesis, we were able
to achieve an extremely high conversion ratio (> 98%) and
yield (> 85%) of the final reaction (Table S1). The crude
product was washed with diethyl ether three times and was
precipitated with methanol and diethyl ether (20 mL, 1:4) to
give the final compounds 10 (ZW800-1) and 11 (ZW800-3a)
as a dark green solid. Chemical purity of ZW800-1 and
WCU Department of BIN Fusion Technology
Chonbuk National University, Jeonju 561-756 (South Korea)
[
**] This study was supported by the following grants from the National
Institutes of Health: NCI BRP grant R01-CA-115296 (J.V.F.), NIBIB
grant R01-EB-010022 (J.V.F. and H.S.C.), and NIBIB grant R01-EB-
011523 (H.S.C. and J.V.F.). We thank Lindsey Gendall and Lorissa A.
Moffitt for proofreading, and Linda Keys and Eugenia Trabucchi for
administrative assistance. All FLARE technology is owned by Beth
Israel Deaconess Medical Center, a teaching hospital of Harvard
Medical School. As inventor, J. V. Frangioni may someday receive
royalties if products are commercialized. J. V. Frangioni is the
founder and unpaid director of The FLARE Foundation, a nonprofit
organization focused on promoting the dissemination of medical
imaging technology for research and clinical use.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201102459.
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258
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Angew. Chem. Int. Ed. 2011, 50, 6258 –6263

The physicochemical and
optical properties of these
fluorophores are detailed
in Table 1, Figure 2b, S3–
S4. Although net charge
and logD at pH 7.4 varied
widely, most optical param-
eters were similar and all
compounds were stable (>
9
4%) in warm serum for
up to 4 h.
Serum protein binding
was measured by gel filtra-
tion chromatography
GFC) after incubation in
(
1
00% serum (Figure S5).
ICG, which is known to
bind serum proteins such
as a -lipoprotein, g-globu-
1
[
4,20]
lin, and serum albumin,
showed a significant HD
shift after 4 h of incubation
in FBS due to its hydro-
phobic character (logD =
7
.88) and net negative
charge
(+ 1/À2,
net
However,
charge = À1).
ZW800-1, with high hydro-
philicity
(logD = À3.56)
and balanced, alternating
charge
charge = 0)
(+ 3/À3,
net
distributed
evenly over its surface, did
not adsorb to serum pro-
teins. Although its logD
Figure 1. Synthesis and characterization of 800 nm zwitterionic heptamethine indocyanine NIR fluorophores
ZW dyes). a) Synthetic scheme for zwitterionic heptamethine indocyanine NIR fluorophores. b) LC–MS and
elemental analysis: absorbance at 770 nm (top left), fluorescence (l =770 nm and l =790 nm; top right),
(
value
at
pH 7.4
is
ex
em
extremely low (À6.95),
ELSD (bottom left), and total ion chromatogram (TIC; bottom right). Insert: ESI-TOF mass spectrum of
.6 min peak for ZW800-1 and 6.9 min peak for ZW800-3a.
ZW800-3a showed low
5
levels of protein adsorption
when exposed to 100%
ZW800-3a was 97.1% and 98.7%, respectively (Figure 1b).
The H NMR spectrum, the C NMR spectrum, and ESI-
TOF MS were consistent with the proposed structures
serum, which is likely due to its unbalanced charge distribu-
tion (+ 3/À1, net charge = + 2).
1
13
To investigate the effect of chemical structure and net
charge on in vivo biodistribution and clearance, the NIR
(
Figure S1–S2). Owing to the polar and hygroscopic nature
of the final compounds, both ZW800-1 and ZW800-3a
included four extra water molecules, as was analyzed by
CHN elemental analysis (see the Supporting Information for
details).
Table 1: Comparison of chemical and optical properties of variously
charged NIR fluorophores in 100% FBS.
[a]
Property
CW800 RS800 ICG
ZW800-1 ZW800-3a
The hypothesis guiding our work was that zwitterionic
small molecules might recapitulate the results seen previously
with nanoparticles, namely lower in vivo background fluores-
cence. To test this hypothesis, we analyzed a series of
heptamethine indocyanine NIR fluorophores that systemati-
cally varied in net charge as follows: À4 (CW800), À2
MW [Da]
1091
À4
887
À2
775
À1
1149
0
À3.56
945
+2
À6.95
309000
774
net Charge
logD at pH 7.4
2.51
0.11
7.88
À1
À1
e [m cm
]
237000 240000 121000 249000
lexc [nm]
lem [nm]
Stokes shift [nm]
786
800
14
784
800
16
807
822
15
772
788
16
790
16
(
RS800), À1 (ICG), 0 (ZW800-1), and + 2 (ZW800-3a). The
chemical structures and energy-minimized 3D structures of
these NIR fluorophores are shown in Figure 2a. A note
should be made of the significantly different distributions of
charge and hydrophobicity over the surface of each molecule.
F [%]
14.2
16.9
4.4
9.3
5.9
15.1
5.5
16.1
3.8
decomposition [%] 4.0
[a] CW800: IRDye800-CW carboxylic acid; ICG: indocyanine green;
RS800: IRDye800-RS carboxylic acid; ZW: zwitterionic.
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Communications
phores in real time over 4 h. As
shown in Figure 3a, the b phase
blood half-life (t =
À4), RS800 (À2), ICG (À1),
ZW800-1 (0), and ZW800-3a (+
1
2b) of CW800
(
2
3
) in CD-1 mice was 18.5, 11.2,
.6, 15.1, and 28.1 min, respec-
tively. All half-life values were
statistically different from each
other by analysis of variance
(ANOVA). Elimination of NIR
fluorophores from the body by
renal clearance also varied mark-
edly (Table 2), with ICG being
undetectable in urine (caused by
liver uptake and subsequent bili-
ary clearance) and ZW800-1 being
mostly renally cleared. The frac-
tion of the injected dose that was
eliminated from the body into
urine by 4 h postinjection in mice
was
44.0 Æ 4.6%ID,
15.5 Æ
1
1
.2%ID, 2.2 Æ 0.9%ID, 86.0 Æ
5.4%ID, and 23.2 Æ 11.4%ID,
respectively for CW800 (À4),
RS800 (À2), ICG (À1), ZW800-1
(0), and ZW800-3a (+ 2; see also
Figure S6–S8). In fact, only 1 h
after injection, 51.3 Æ 9.7%ID of
ZW800-1 was found in urine.
Uptake of NIR fluorophores
in organs and tissues of mice and
rats also varied markedly. As
shown in Figure 3b (see also Fig-
ure S6–S8 and Videos S1–S3), all
anionic and cationic NIR fluoro-
phores exhibited high nonspecific
uptake in a variety of organs, with
anionic NIR fluorophores exhibit-
ing significant excretion into bile,
resulting in high NIR fluorescent
signal throughout the GI tract. For
example, at 4 h postinjection,
uptake values for CW800 (À4)
were: intestine: 28.5 Æ 7.0%ID/g,
liver: 11.4 Æ 0.8%ID/g, and kid-
neys: 7.3 Æ 1.3%ID/g. For ICG
(
À1), uptake values were: intes-
tine: liver:
2
62.0 Æ 6.6%ID/g,
Figure 2. Physicochemical and optical properties of NIR fluorophores having systematically varying net
7.1 Æ 0.5%ID/g, and kidneys:
charge. a) All molecules shown are bifunctional, permitting conjugation through their single carboxylic
acid group, except ICG. Red: negative charge; blue: positive charge; gray: hydrophobicity. b) Absorb-
ance and fluorescence emission spectra of each fluorophore in 100% FBS (1 mm) supplemented with
5.9 Æ 1.0%ID/g, which were also
caused by the extremely high
5
0 mm HEPES, pH 7.4.
hydrophobicity/lipophilicity
of
ICG (logD at pH 7.4 = 7.88). Two
positive charges of ZW800-3a (+
fluorophore series was injected intravenously into mice and
rats. Using the fluorescence-assisted resection and explora-
tion (FLARE) intraoperative image-guided surgery
2) resulted in unfavorable biodistribution, mostly found in the
liver (40.6 Æ 2.6%ID/g), intestine (11.5 Æ 3.4%ID/g), and
kidneys (9.0 Æ 1.1%ID/g). On the contrary, ZW800-1 was
exclusively cleared by the kidneys, with no appreciable
[21,22]
system,
we measured the behavior of injected fluoro-
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Angew. Chem. Int. Ed. 2011, 50, 6258 –6263

(Table 2). It should be
emphasized that no single
value, such as logD or net
charge, is predictive of
in vivo
fate.
Optimal
in vivo behavior appears to
be mediated by alternating
and balanced charge, dis-
tributed over the entire sur-
face of the molecule, which
completely shields the
underlying hydrophobicity.
The most common strat-
egy to engineer targeted
NIR fluorescent contrast
agents is to employ “modu-
[23–25]
lar” chemistry,
covalently combines an
NIR fluorophore and
which
a
high-affinity, small-mole-
cule-targeting ligand. How-
ever, commercially avail-
able NIR fluorophores are
typically hydrophobic and/
or di-/tetra-sulfonated. One
of the key results of our
study is that purely anionic
or cationic NIR fluoro-
phores exhibit significant
nonspecific
uptake
in
normal tissues and organs,
although their logD at 7.4 is
extremely low (i.e., hydro-
philic). For molecules that
have a high “hydrophobic
moment” like ICG, liver
uptake was significant and
rapid, thus reducing blood
concentration, but increas-
ing NIR fluorescence of the
[26]
liver, bile, and GI tract.
This rapid uptake contami-
nates the liver and/or lower
GI tract with high signal
and precludes many impor-
tant image-guided surgeries
of the abdomen. The blood
half-life of ICG was only 2
to 5 min because of the
rapid liver uptake. RS800
had a lower hydrophobic
moment, but still had
exposed aromatic groups
Figure 3. Biodistribution and clearance of NIR fluorophores. a) Blood concentration (%ID/g), elimination in
urine (%ID), carcass retention (%ID), and total body clearance (%ID) in CD-1 mice. Each data point is the
meanÆS.D. from N=5 animals. *P<0.01 and ***P<0.001 for pairwise comparisons of half-life using
À1
ANOVA. b) NIR fluorophores (40 pmolg ) were injected intravenously into CD-1 mice (top two panels) and
SD rats (bottom two panels), 1 h prior to imaging. Shown are color and 800 nm NIR fluorescence images of
surgically exposed organs/tissues. Abbreviations used are: AW: abdominal wall; Bl: bladder; BD: bile duct;
Du: duodenum; In: intestine; Ki: kidneys; Li: liver; Lu: lungs; Pa: pancreas; Re: rectum, Sk: skin; Ur: ureter.
Scale bars: 1 cm.
nonspecific background signal in any tissues and organs and
only 12.4 Æ 3.4%ID remaining in the carcass at 4 h post-
injection, mostly in the kidneys (9.8 Æ 2.2%ID/g) and only
negligible amounts in the liver (0.9 Æ 0.1%ID/g) and intestine
that were recognized by the liver and resulted in rapid
excretion into bile (short transit time through the liver).
However, it also had enough charge to keep it in the
bloodstream long enough to undergo approximately 20%
renal filtration. For highly anionic CW800, the hydrophobic
moment was compensated by the charge (more hydrophilic)
(
0.7 Æ 0.4%ID/g). Similar results were observed in rats, but
excretion rates were slightly slower than those in mice
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6261

Communications
[
a]
Table 2: In vivo organ/tissue distribution of variously charged NIR fluorophores 4 h postinjection into mice (N=5) and rats (N=5).
Fluorophore
CW800
Species
Ki
Sk/Mu
He/Lu
Sp
Li
In
Re
Bl
Carcass
Eliminated
mouse
rat
mouse
rat
mouse
rat
mouse
rat
+
++
+
+
+
À
À
À
À
À
À
À
À
À
+
À
À
À
À
À
À
À
À
À
+
À
+++
+++
++
++
+
+++
+++
++
52.4Æ9.6%ID
N.D.
44.0Æ4.6%ID
N.D.
+
À
À
+++
++
+
79.0Æ4.1%ID
15.5Æ1.2%ID
RS800
+
À
À
+++
+++
+++
À
À
N.D.
N.D.
À
À
++
++
À
À
97.1Æ9.4%ID
N.D.
12.4Æ3.4%ID
14.8Æ6.5%ID
68.6Æ8.6%ID
68.3Æ15.7%ID
2.2Æ0.9%ID
N.D.
86.0Æ15.4%ID
83.4Æ15.9%ID
23.2Æ11.4%ID
28.7Æ16.3%ID
ICG
À
À
+
À
À
À
À
+++
+++
+++
+++
ZW800-1
ZW800-3a
+
À
À
À
À
mouse
rat
+
++
+
++
++
+++
+++
++
+
À
[a] Abbreviations used are: Bl: bladder; He: heart; In: intestine; Ki: kidneys; Li: liver; Lu: lungs; Mu: muscle; Re: rectum; Sk: skin; Sp: spleen; and
N.D.: not done. All mouse values were measured directly. Shown for each measurement is meanÆS.D. The CBR of each organ/tissue relative to the
abdominal wall was quantified and labeled as À: 0 to 1; +: 1 to 2; ++: 2 to 5; and +++: >5.
[
27]
that resulted in approximately 55% renal filtration, but
zwitterionic NIR fluorophores to complement those we now
have at 800 nm. When used with the two NIR channel
capabilities of the FLARE imaging system, it should be
possible to simultaneously image two independent targeted
NIR fluorophores during surgery. This approach is especially
important in cancer surgery when resection of the tumor (i.e.,
using one NIR channel) needs to be performed, while
avoiding critical structures such as vessels and nerves (i.e.,
using the second NIR channel).
[
28]
also significant liver clearance, GI tract contamination, and
nonspecific uptake in normal tissues and organs. The high
cationic charge of ZW800-3a caused the molecule to exhibit
high nonspecific uptake in almost every tissue and organ in
the body, thus resulting in only approximately 20% renal
excretion. Only a true zwitterionic molecule, such as ZW800-
1
, with charge balanced over the entire surface and hydro-
phobicity well shielded by this charge, does not exhibit
protein binding, nonspecific tissue/organ uptake, or liver
clearance. Although the precise mechanism for this observa-
tion is not yet known, zwitterionic molecules likely minimize
the interaction with serum proteins by charge shielding,
and minimize penetration of cellular membranes by extreme
[29]
Experimental Section
Synthesis of ZW800–1 (10) and ZW800–3a (11). As depicted in
Figure 1a, starting materials 1–4 were used to prepare the 800 nm
emitting, zwitterionic heptamethine indocyanine fluorophores 10
(ZW800-1) and 11 (ZW800-3a). All products 5–11 were obtained in
reasonably high purity as indicated by TLC analyses by using C18
[
30,31]
polarity.
As our efforts focused on lowering of the background
fluorescence, it would have been counter-productive if this
lowering were achieved at the expense of the signal. This issue
is a major problem, for example, when utilizing two-photon
upconversion strategies for NIR fluorescence. Fortunately, we
demonstrate that the optical properties of zwitterionic NIR
fluorophores are excellent. In fact, the product of the
extinction coefficient and quantum yield of ZW800-1 in
serum are more than three times higher than that of the FDA-
approved NIR fluorophore ICG (Table 1). In addition, the
1
13
adsorbents and high-resolution H and C nuclear magnetic reso-
nance (NMR) spectra. Chemical purity was measured by using HPLC
combined with simultaneous evaporative light scatter detection
(ELSD), absorbance (photodiode array), fluorescence, and ESI-
TOF MS. Compounds were also analyzed by MALDI-TOF MS and
CHN elemental analysis. See Supporting Information for detailed
chemical syntheses and analyses.
Optical and physicochemical property analyses: Commercially
available NIR fluorophores included IRDye 800-CW (CW800; Li-
Cor, Lincoln, NE), IRDye 800-RS (RS800; Li-Cor), and indocyanine
green (ICG; Akorn, Decatur, IL). When only an N-hydroxysuccini-
mide (NHS) ester was available, the corresponding carboxylic acid
(CA) was formed by incubation at high concentration in borate buffer
(50 mm), pH 9.0 for 3 h followed by dilution into the desired buffer.
All optical measurements were performed at 378C in phosphate-
buffered saline (PBS), pH 7.4 or 100% fetal bovine serum (FBS)
buffered with HEPES (50 mm, pH 7.4). For fluorescence quantum
yield (F) measurements, ICG in dimethyl sulfoxide (F = 13%) was
8
00 nm zwitterionic heptamethine indocyanine NIR fluoro-
phore ZW800-1 has remarkable in vivo properties, including
no serum protein binding, rapid renal clearance, and ultralow
nonspecific tissue uptake (i.e., background). ZW800-1 also
provides a single carboxylic acid for the covalent conjugation
to targeting ligands through a stable amide bond; because of
these features, ZW800-1 has been selected by the US National
Cancer Instituteꢀs Experimental Therapeutics (NExT) Pro-
gram as a first-in-class molecule for rapid translation into
human clinical studies.
used as a calibration standard under conditions of matched absorb-
[14–19]
ance at 770 nm.
For in vitro optical property measurements,
A key unanswered question in the present study is
whether a targeted NIR fluorophore made zwitterionic by
choosing the proper ZW800 Æ i fluorophore for conjugation
requires intermingled positive and negative charges over the
entire surface of the final molecule (similar to unconjugated
ZW800-1), or whether a dipole-like zwitterionic structure is
adequate for improved in vivo performance. Ongoing studies
are also focused on the engineering of a family of 700 nm
online fiberoptic HR2000 absorbance (200–1100 nm) and
USB2000FL fluorescence (350–1000 nm) spectrometers (Ocean
Optics, Dunedin, FL) were used. NIR excitation was provided by a
770 nm NIR laser diode light source (Electro Optical Components,
Santa Rosa, CA) set to 8 mW and coupled through a 300 mm core
diameter, NA 0.22 fiber (Fiberguide Industries, Stirling, NJ). In silico
investigations of the partition coefficient (logD) and surface molec-
ular charge and hydrophobicity were carried out using MarvinSketch
5.2.1 (ChemAxon, Budapest, Hungary).
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Angew. Chem. Int. Ed. 2011, 50, 6258 –6263

In vivo biodistribution and clearance: Animals were housed in an
AAALAC-certified facility and were studied under the supervision of
an approved institutional protocol. CD-1 male mice weighing 25 to
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[
21]
intraoperative imaging system as described in detail previously. For
À2
fluorescence excitation, 14 mWcm of 745 to 779 nm-filtered light
was used, and for emission, light was filtered using a 800 to 848 nm
bandpass filter. For each experiment, camera exposure time and
image normalization was held constant. Color video images were
collected on a separate channel using custom-designed optics and
software. To quantify the blood clearance rate and urinary excretion,
intermittent sampling from the tail vein was performed over 4 h.
Approximately 20 mL of blood and urine were collected at the
following time points using glass capillary tubes: 0, 1, 2, 5, 10, 15, 30,
6
0, 90, 120, 180, and 240 min. The FLARE imaging system measured
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