In Vivo Chemiluminescent Imaging Agents for Nitroreductase and Tissue Oxygenation

Article abstract of DOI:10.1021/acs.analchem.6b01096

Tissue oxygenation is a driving parameter of the tumor microenvironment, and hypoxia can be a prognostic indicator of aggressiveness, metastasis, and poor response to therapy. Here, we report a chemiluminescence imaging (CLI) agent based on the oxygen-dependent reduction of a nitroaromatic spiroadamantane 1,2-dioxetane scaffold. Hypoxia ChemiLuminescent Probe 2 (HyCL-2) responds to nitroreductase with ～170-fold increase in luminescence intensity and high selectivity for enzymatic reductase versus other small molecule reductants. HyCL-2 can image exogenous nitroreductase in vitro and in vivo in living mice, and total luminescent intensity is increased by ～5-fold under low oxygen conditions. HyCL-2 is demonstrated to report on tumor oxygenation during an oxygen challenge in H1299 lung tumor xenografts grown in a murine model as independently confirmed using multispectral optoacoustic tomography (MSOT) imaging of hemoglobin oxygenation.

Full text of DOI:10.1021/acs.analchem.6b01096

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Article
In Vivo Chemiluminescent Imaging Agents
for Nitroreductase and Tissue Oxygenation
Jian Cao, James Campbell, Li Liu, Ralph P. Mason, and Alexander R. Lippert
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01096 • Publication Date (Web): 07 Apr 2016
Downloaded from http://pubs.acs.org on April 9, 2016
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Analytical Chemistry
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In Vivo Chemiluminescent Imaging Agents for Nitroreductase
and Tissue Oxygenation
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Jian Cao,†,§ James Campbell,# Li Liu,# Ralph P. Mason,# and Alexander R. Lippert*,†,§,¶
§
¶
†Department of Chemistry, Center for Drug Discovery, Design, and Delivery (CD4), and Center for Global Health
Impact (CGHI), Southern Methodist University, Dallas, TX, 75275-0314. *E-mail: alippert@smu.edu. Fax: 214-768-
4089.
#Prognostic Imaging Research Laboratory (PIRL), Pre-clinical Imaging Section, Department of Radiology, UT
Southwestern Medical Center, Dallas, TX 75390-9058, USA.
ABSTRACT: Tissue oxygenation is a driving parameter of the tumor microenvironment and hypoxia can be a prognostic
indicator of aggressiveness, metastasis, and poor response to therapy. Here we report a chemiluminescence imaging (CLI)
agent based on the oxygen-dependent reduction of a nitroaromatic spiroadamantane 1,2-dioxetane scaffold. Hypoxia
ChemiLuminescent Probe 2 (HyCL-2) responds to nitroreductase with ~170-fold increase in luminescence intensity and
high selectivity for enzymatic reductase versus other small molecule reductants. HyCL-2 can image exogenous nitrore-
ductase in vitro and in vivo in living mice and total luminescent intensity is increased by ~5-fold under low oxygen condi-
tions. HyCL-2 is demonstrated to report on tumor oxygenation during oxygen challenge in H1299 lung tumor xenografts
grown in a murine model as independently confirmed using multi-spectral optoacoustic tomography (MSOT) imaging of
hemoglobin oxygenation.
,
–
–
Introduction
hanced MRI,27 28 tissue29 32 or blood33 35 oxygen level de-
pendent (TOLD or BOLD) MRI, MRI-based oxygen imag-
ing (MOXI),36 and multispectral optoacoustic tomography
(MSOT) imaging of hemoglobin and oxygenated hemo-
Hypoxia refers to a physiological state of low tissue ox-
ygenation and is a pathogenic characteristic of solid tu-
mors that arises from absent or abnormal vasculature in
the tumor microenvironment.1 4 Cancer cell populations
–
globin.37 40 These techniques offer significant clinical op-
–
portunity, but require expensive instrumentation and
highly trained experts that increase the cost and lower the
throughput of these technologies.
respond to hypoxia by promoting survival pathways to
suppress apoptosis5 and initiate angiogenesis,6 invasive-
ness,7 and metastasis.8 A key event initiated by low oxy-
gen levels is the activation of the transcription factor hy-
poxia inducible factor 1 (HIF1). The HIF1-α subunit re-
mains inactive through oxygen-dependent enzymatic hy-
droxylation of specific residues.9 In the absence of oxygen,
however, HIF-1α dimerizes with the HIF-1β subunit and
induces transcription of a host of proteins to support can-
cer cell survival.10 Activating prodrugs and imaging agents
in a similar oxygen-dependent manner is an attractive
Optical methods can provide a cheaper alternative to
MRI and PET imaging methods. In recent years, small
molecule fluorescence imaging agents have been devel-
oped that take advantage of the reduction of azobenzene,
nitrobenzene, nitrofuran, and nitroimidazole functional
–
groups to release a caged fluorescent dye.41 49 Despite
progress towards in vivo hypoxia imaging with near-
infrared emitting fluorophores, complications arising
from autofluorescence, background, and light scattering
remain problematic. Bioluminescent substrates for ni-
troreductase have been developed and have found appli-
cation for imaging bacterial infection and cancer cells
engineered to express bacterial nitroreductase.50 Alt-
hough they display increased signal to background in
comparison to fluorescent probes, these systems require
expression or exogenous addition of luciferase and emit
light through an oxygen-dependent process, complicating
the development of caged luciferin probes for hypoxia
imaging.
–
strategy for imaging11 and treatment of cancer. 12 14
Following this idea, the oxygen-dependent reduction of
aromatic nitro groups by reductive mammalian enzymes,
including xanthine oxidase, NADPH-cytochrome c reduc-
tases, and potentially cytosol DT diaphorase, aldehyde
–
52
–
18
oxidase, and lipoyl dehydrogenase,15 is now a well-
established method to target drugs,2,12 imaging agents like
–
FMISO,11,
and immunohistochemical stains like pi-
19 22
monidazole23 to hypoxic areas. These targeting strategies
have been cross-validated in comparison to other meth-
ods for direct measurement of tissue oxygenation includ-
ing pO2 electrodes,24 magnetic resonance imaging (MRI)
Considering these outstanding challenges for optical
imaging of the tumor microenvironment, we have begun
19
,
of F-labeled perfluorocarbons,25 26 dynamic contrast en-
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to develop chemiluminescent imaging (CLI) agents that to pH 7.4 with 10% Emerald II Enhancer. Further details
1
2
can be applied in luciferase-null organisms for imaging a
can be found in the supporting information.
,
diverse set of parameters.53 54 Chemiluminescence results
In vitro deoxygenation experiments. 10 mM PBS
buffer (pH 7.4) and Emerald II Enhancer were mixed with
a ratio of 9 to 1 and N2, air, or 100% O2 was bubbled
through it for 60 minutes. The solution was then trans-
ferred into a capped cuvette by syringe, and HyCL-2,
NADH, and nitroreductase were added using a syringe.
Time scans were performed 1 min after adding nitroreduc-
tase. For deoxygenation experiments, PBS buffer (pH 7.4)
and Emerald II Enhancer mixture was deoxygenated by
bubbling N2 for 60 minutes, and then 889 µL of this deox-
ygenated mixture was transferred into a capped cuvette
by syringe, and later 2 µL of a 5 mM HyCL-2 in DMSO, 8
µL of a 50 mM NADH in 0.01 mM NaOH solution, 1.25 µL
of nitroreductase (10 mg/mL) were added into the cuvette
using syringe. Time scans were performed 1 min after add-
ing nitroreductase. The control experiment was carried
out under ambient conditions without deoxygenation.
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from the formation of an excited state product during an
exothermic chemical reaction55 and is emerging as a use-
–
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ful preclinical in vivo imaging modality.56 Triggered
chemiluminescence emission of spiroadamantane 1,2-
–
dioxetanes has been extensively used for clinical assays60
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65 and analyte sensing66 69 and has been recently demon-
strated as an effective in vivo imaging modality for
β-
galactosidase53 and H2S.54 The high-energy 4-membered
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dioxetane ring can be sterically stabilized by synthetically
appending spiroadamantane,60,66 fenchyl,
or other
70
frameworks, allowing for purification, synthetic manipu-
lation, and long term storage. These systems can be de-
signed such that a chosen chemical trigger will initiate an
electron transfer cascade that leads to dioxetane ring
cleavage. The reaction proceeds through a high-energy
transition state that populates the excited state of the
product, releasing the energy as light upon relaxation to
the ground state.55 This energy can be further transferred
to red-shifted dye molecules more suitable for biological
In vitro chemiluminescent imaging. Imaging was
carried out with a Caliper Xenogen IVIS Spectrum (Per-
kin-Elmer, Santa Clara, CA) in black 96-well Costar
plates and all the images were analyzed using Living Im-
age 3.1 software. 1 mM HyCL-2 in DMSO, 2 mg/mL ni-
troreductase (10 µL from 1 mg NTR/100 uL DI-H2O diluted
to 50 µL by adding 40 µL DI-H2O) and 50 mM NADH in
0.01 mM NaOH solution were prepared prior to imaging.
176.4 µL, 176.1 µL, 175.9 µL, 175.5 µL 175.4 µL and 175.1 µL of
10 mM PBS buffer (pH 7.4) were added into wells from A1
to A6 in sequence, 1.6 µL NADH was pipetted into each
well and then different volume of nitroreductase solution
(0 µL, 0.25 µL, 0.5 µL, 0.75 µL, 1.0µL, 1.25 µL) were added
into each well. 2 µL HyCL-2 was injected into the mixture
and 20 µL Emerald II Enhancer was pipetted into each
well, and imaging was performed 3.45 minutes after add-
ing reagents into the 96 well plate using an open filter. All
images were acquired with f-stop 1, medium binning, auto
exposure and the chamber set to 37 °C. The detection
limit was determined from the linear plot of NTR concen-
tration versus total photon flux as the amount of nitrore-
ductase required to give a chemiluminescent signal above
three times the standard deviation of at least 3 independ-
ent experiments with 0 µg/mL nitroreductase. These pro-
vided detection limits of 9.6 ng/mL with measurements
taken 30 sec after reagent addition and 1.9 ng/mL with
measurements taken 30 min after reagent addition.
–
imaging.71 74 Direct production of light from the energy of
a chemical bond offers important advantages over fluo-
rescence imaging by eliminating light excitation of a con-
trast agent, thereby providing reduced autofluorescence
and light scattering. On the basis of these considerations
and expanding the toolbox of chemiluminescent imaging
methods for nitroreductase and hypoxia, we report the
development of a spiroadamantane 1,2-dixoetane-based
chemiluminescent platform, Hypoxia ChemiLuminescent
Probes 1 and 2 (HyCL-1 and HyCL-2) for imaging tumor
oxygenation in living animals.
Experimental section
Spectroscopic Measurements. UV/Vis spectra were
acquired on a Beckman-Coulter DU 800 Spectrometer
(Beckman Coulter, Inc., Brea, CA). Chemiluminescent
responses were acquired using a Hitachi F-7000 Fluores-
cence Spectrophotometer (Hitachi, Tokyo, Japan) using
the luminescence detection module and setting emission
wavelength to 545 nm. 889 µL of a 10 mM PBS buffered to
pH 7.4, 2 µL of a 5 mM HyCL-1 or HyCL-2 in DMSO, 8 µL
of a 50 mM NADH in 0.01 mM NaOH solution, 1.4 µL of
nitroreductase from Escherichia coli (NTR, Sigma-Aldrich
N9284-1MG) in DI-H2O (1 mg NTR dissolved in 100 µL DI-
H2O, 10 mg/mL), and 100 µL Emerald II Enhancer (Life
Technologies, Carlsbad, CA) were added to an Eppendorf
tube and samples were shaken gently to assure mixing.
The mixed solution was then transferred to a quartz cu-
vette (Starna, Atascadero, CA). Time scans were acquired
using the time scan module 1 min after adding probes. For
the dose-dependent response to nitroreductase, 10 μM
HyCL-1 or HyCL-2 were treated with 8 µL of a 50 mM
NADH and 0, 0.5, 0.75, 1.0, 1.25, 1.4 μL 10 mg/mL nitrore-
ductase and 20 min (Figure 1, S9) or 120 min (Figure S3)
time scans were measured 1 min after adding probes. Se-
lectivity for HyCL-1 and HyCL-2 was measured by moni-
toring the time-dependent chemiluminescent emission at
545 nm. All assays were performed in 10 mM PBS buffered
General animal protocols. The UT Southwestern In-
stitutional Animal Care and Use Committee approved
these investigations under Animal Protocol Number (APN
#2009-0150). Hair was removed from area surrounding
the tumor using Nair (Church & Dwight Co, Inc, Ewing,
NJ), but the skin was left unbroken before injection. Mice
were anesthetized and maintained using inhalation of
1.6% isoflurane in 16% oxygen gas.
In vivo CLI of nitroreductase. A stock solution of 5
mM HyCL-2 in DMSO, 1 mg nitroreductase dissolved in
100 µL DI-H2O and 50 mM NADH in 0.01 mM NaOH so-
lution were prepared in advance. A 30 µL aliquot of this
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Scheme 1. Spiroadamantane 1,2-dioxetanes for
solution was taken out from a mixture of 437 µL 10 mM
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PBS, 4 µL HyCL-2, 8 µL NADH, 1.25 µL NTR and 50 µL
Emerald II Enhancer and images were acquired 30 sec
after administering subcutaneous injections to anesthe-
tized (1.6% isoflurane) SCID/BALB-C mice. A vehicle con-
trol was conducted in the same way except replacing ni-
troreductase and NADH with H2O and 0.01 M NaOH,
respectively.
chemiluminescent nitroreductase (NTR) detection at
neutral pH.
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9
In vivo CLI of hypoxia in tumor xenografts. A 100 µL
solution was taken out from a mixture of 446 µL 10 mM
PBS, 50 µL Emerald II Enhancer and 4 µL 5 mM HyCL-2
and images were acquired immediately and every minute
after administering intratumoral injections to H1299 lung
tumor xenografts on mice while the animals breathed
ambient air. A similar experiment was performed in the
same mice, but while breathing 100% oxygen 5 minutes
before injection and 1.6% isoflurane and 98.4% oxygen
throughout the course of the imaging experiment. These
experiments were reproduced on two different mice (n =
2). Tumor sizes were 9.9 mm x 6.4 mm x 5.5 mm and 9.5
mm x 7.6 mm x 6.0 mm.
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Scheme 2. Synthesis of HyCL-1 and HyCL-2.
Multispectral optoacoustic tomography (MSOT). A
thin layer of ultrasound coupling gel provided contact
with the membrane and the animal holder was placed in
the imaging chamber of the MSOT 256-TF device (iThera
Medical Gmbh, Munich, Germany). Multi-spectral optoa-
coustic tomography (MSOT) imaging was performed in a
transaxial section through the center of the tumor using
five wavelengths: 715, 730, 760, 800, and 850 nm. Twenty
frames per wavelength were acquired and averaged. Ini-
tially, the tumor region was imaged while breathing 16%
oxygen, which was switched to 100% oxygen. A model-
based reconstruction was used prior to multispectral pro-
cessing to provide relative concentrations of oxyhemoglo-
bin and deoxyhemoglobin. These experiments were re-
produced on two different mice (n = 2).
photogenerated singlet oxygen using rose bengal as a sen-
sitizer to provide HyCL-1 and HyCL-2 after purification
by silica chromatography. Under hypoxic conditions or in
the presence of nitroreductase, the para-nitrobenzyl moi-
ety will be reduced, and then chemiluminescent emission
will be initiated by self-immolative carbonate or ether
cleavage to release the free phenolate bearing the 1,2-
dioxetane (Scheme 1). The negatively charged phenol unit
spontaneously triggers dioxetane cleavage and light emis-
sion through a series of electron transfer events.76
Results and Discussion
A spiroadamantane 1,2-dioxetane scaffold was selected
for the chemiluminescent-based probe design because it
(1) displays instantaneous light production under biologi-
cally compatible conditions, (2) does not require the addi-
tion of oxidative species like hydrogen peroxide, and (3)
the scaffold has demonstrated potential for in vivo imag-
ing.53,54 Our strategy for designing these reaction-based
chemiluminescent probes relies on tethering a para-
nitrobenzyl moiety to a 1,2-dioxetane chemiluminescent
scaffold via carbonate or ether linkage (Scheme 1). The
nitroaromatic group is sensitive to nitroreductase en-
zymes and has been frequently used in prodrugs and hy-
poxia detection. The synthesis of HyCL-1 proceeds from
the previously reported phenol 154 by dimethylamino-
pyridine-catalyzed coupling with the mixed N-
hydroxysuccinimidyl carbonate 2 to form the enol ether
precursor 3 (Scheme 2). For HyCL-2, the ether linkage of
the precursor was prepared by a Mitsunobu reaction75
between the phenol 1 and para-nitrobenzyl alcohol 4 to
form the enol ether precursor 5 (Scheme 2). Both enol
ethers 3 and 5 were subjected to [2+2] cycloaddition with
After obtaining HyCL-1 and HyCL-2, we proceeded to
collect their absorption (Figure S1) and emission (Figure
S2) spectra and measure their luminescent responses to
bacterial nitroreductase in the presence of reduced nico-
tinamide adenosine dinucleotide (NADH) as a cofactor, a
commonly employed model for reductase-activated
probes.41 Nitroreductase-dependent chemiluminescence
was first evaluated in PBS (10 mM, pH 7.4) buffer contain-
ing 10% Emerald II Enhancer using an F-7000 Hitachi
spectrophotometer. The Emerald II Enhancer is a com-
mercial aqueous solution that contains a proprietary cati-
onic polymer and a dye with optical properties similar to
fluorescein, with a peak absorption at 505 nm (Figure S1).
Treatment of 10 µM of HyCL-1 or HyCL-2 with 14 µg/ml
nitroreductase, 0.4 mM NADH, and 10% Emerald II En-
hancer resulted in instantaneous and robust luminescent
emission with two peaks centered at 455 nm and 545 nm,
which correspond to the emission wavelengths of the
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Figure 2. Chemiluminescent responses of (a) 10 µM HyCL-1
Figure 1. Time scans of the chemiluminescent emission at
545 nm from (a) 10 µM HyCL-1 and (b) 10 µM HyCL-2 and 0,
2.5, 5, 7.5, 10, 12.5, 14 μg/mL nitroreductase (NTR) in the
presence of 0.4 mM NADH in 10 mM PBS buffer (pH 7.4)
containing 10% Emerald II Enhancer.
and (b) 10 µM HyCL-2 to biologically relevant analytes in 10
mM PBS buffer (pH 7.4) containing 10% Emerald II Enhanc-
er. Bars represent chemiluminescent emission at 545 nm and
4, 8, 12, 16, 20 min after addition of reducing agents. Data
shown are for 5 mM glutathione, 1 mM cysteine and homo-
cysteine, and 200 μM for other reducing agents. (1) NTR and
NADH; (2) glutathione; (3) L-cysteine; (4) homocysteine; (5)
DTT; (6) NTR; (7) NADH; (8) H2S; (9) citrate; (10) Na2S2O5;
(11) L-ascorbic acid; (12) blank.
phenolate and the fluorescein-based Emerald II Enhancer,
respectively (Figure S2). This light production increased
over a course of 60 minutes (Figure S3) to provide a ~5-
fold and ~170-fold luminescence turn-on for HyCL-1 and
HyCL-2, respectively (Figure 1). We also found that the
chemiluminescent emission enhancement was dose-
dependent with respect to nitroreductase, demonstrating
that both probes could be applied for sensitive nitrore-
ductase detection. A linear response was found between
0–14 µg/mL nitroreductase (Figure S4). This dose-
dependence of light emission is observed immediately,
revealing an advantage of using chemiluminescence
probes for quick and direct nitroreductase detection
without extensive incubation times. Chemiluminescent
response data indicated that the ether-linked HyCL-2
displayed a much lower background emission (Figure 1b,
red scan) compared with the carbonate linked HyCL-1
(Figure 1a, red scan). The reaction between HyCL-2,
NADH, and NTR was analyzed by GC-MS, confirming the
generation of 2-adamantanone (Figure S5) and methyl 4-
chloro-3-hydroxybenzoate (Figure S6), which provided
fragmentation patterns that matched the spectra found in
the NIST database. A molecular ion at m/z = 121 was also
observed that was assigned to 4-methylenecyclohexa-2,5-
dien-1-one oxime (Figure S7). A high abundance peak at
m/z = 123 (aminobenzyl alcohol) was not observed. Taken
together these observations are consistent with previous
reports that the nitro group is initially reduced to a hy-
droxylamine,52 ,77 followed by self-immolative cleavage to
the chemiluminescent dioxetane phenolate.
Figure 3. Integrated chemiluminescent emission in the
presence (red bars) and absence (green bars) of oxygen over
20 min of 10 µM HyCL-2 and 0, 5, 10, and 12.5 µg mL–1 NTR in
10 mM PBS buffer (pH 7.4) containing 0.4 mM NADH and
10% Emerald II Enhancer (n = 3). Error bars are ±S.D.
of HyCL-1 or HyCL-2 (10 µM) to 14 µg/mL nitroreductase
and 0.4 mM NADH with 10% Emerald II Enhancer was
monitored against other biological species in PBS buffer
(10 mM, pH 7.4), by adding 5 mM reduced glutathione
(GSH), 1 mM L-cysteine and homocysteine, or 200 µM
H2S, dithiothreitol (DTT), citrate, sodium metabisulfite,
or L-ascorbic acid (Figure 2). We observed that HyCL-1
with a carbonate linker displayed poor selectivity against
cellular thiols glutathione, cysteine, homocysteine, and
DTT (Figure 2a). On the other hand, HyCL-2 with an
ether linker showed excellent selectivity, and none of the
other species tested displayed significant increases in lu-
minescence intensity over the blank control (Figure 2b).
We tested the selectivity of HyCL-1 and HyCL-2 against
other biologically relevant molecules, particularly nucleo-
philic thiols, which are ubiquitous in physiological sys-
tems and involved in various redox cycles. The response
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When treated with nitroreductase (14 ug/mL) or NADH
(0.4 mM) alone, there was no increase in chemilumines-
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cent emission over a blank control, indicating that both
enzyme and cofactor are needed to induce a chemilumi-
nescent response from HyCL-1 and HyCL-2. To better
understand the response when treating HyCL-1 with cel-
lular relevant thiol species and decipher the poor selectiv-
ity of HyCL-1, this probe was treated with cysteine and
1
the reaction progress was monitored via H NMR (Figure
9
S8). This experiment provided two salient observations: 1.
The peak at 5.38 ppm corresponding to the -CH2– meth-
ylene peak para to the nitro group rapidly disappeared
within 5 minutes, to be replaced by a new peak at 5.32
ppm; 2. The nitroaromatic peaks at 8.18 ppm and 7.58
ppm persist even after 25 hours of reaction time. The first
observation is consistent with displacement of the phenol
by the reaction of the thiol with the carbonate functional
group, thereby initiating chemiluminescence. The second
observation confirms the stability of the nitroaromatic
group in the presence of cysteine. Taken together, these
observations indicate that the ether linkage provides an
improved response and selectivity for HyCL-2 over HyCL-
1, making it a sensitive chemiluminescent agent for imag-
ing enzymatic reductase activity without interferences
from other biological nucleophiles or small molecule re-
ductants.
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Figure 4. Imaging of nitroreductase using HyCL-2. (a) Imag-
es 30 min after adding 10 µM HyCL-2 to 0, 2.5, 5, 7.5, 10, 12.5
µg/mL NTR in 10 mM PBS buffer (pH 7.4) containing 0.4 mM
NADH and 10% Emerald II Enhancer (n = 3 wells). (b) Quan-
tification of total photon flux of the images in (a) (n = 3
wells). Error bars are ±S.D.
After demonstrating that HyCL-2 had the ability to de-
tect enzymatic reductase activity with high sensitivity and
selectivity, we moved towards testing its ability for hypox-
ia detection using an in vitro model. In order to interro-
gate luminescence response to low oxygen conditions, we
deoxygenated a solution of 10 mM PBS (pH 7.4) contain-
ing 10% Emerald II Enhancer by bubbling nitrogen gas
through the solution for 60 minutes. We then compared
the chemiluminescent response of HyCL-2 to nitroreduc-
tase under oxygen-free conditions and ambient air condi-
tions (Figure 3). At 5, 10, and 12.5 µg/mL nitroreductase a
significant increase in luminescence intensity was ob-
served under low oxygen conditions with up to a 5-fold
increase at 12.5 µg/mL nitroreductase. A control experi-
ment consisted of bubbling air through a solution of PBS
and Emerald II Enhancer for 60 minutes before probe and
enzyme incubation. No significant difference in lumines-
cence emission was observed from the experiment per-
formed under ambient conditions, confirming that the
increased response is due to deoxygenation and not from
a mechanical effect of the bubbling procedure (Figure S9).
Bubbling 100% oxygen through the buffer and Enhancer
solution provided a small, but not total, decrease in lumi-
nescence versus the air-bubbled conditions (Figure S9).
These important experiments demonstrate that the lumi-
nescent response of HyCL-2 depends sharply on the pres-
ence or absence of dissolved oxygen, a necessary trait for
imaging tumor hypoxia.
Figure 5. In vivo nitroreductase imaging. (a) Image of a living
SCID/BALB-C mouse 30 s after administering subcutaneous
injections of 30 µL of a solution containing 40 µM HyCL-2, 14
µg mL–1 NTR, 0.4 mM NADH, and 10% Emerald II Enhancer
in 10 mM PBS (pH 7.4). In the vehicle control nitroreductase
and NADH were replaced with H2O and 0.01 M NaOH. (b)
Quantification of biological replicates (n = 2 mice). Error
bars are high and low values of the two replicates.
Having well characterized the in vitro spectroscopy, we
then proceeded to investigate the ability of HyCL-2 as a
CLI agent for nitroreductase activity at physiological pH
using an IVIS Spectrum. An opaque 96-well plate was
loaded with 10 µM HyCL-2, 0.4 mM NADH and 0, 2.5, 7.5,
10, 12.5 µg/mL nitroreductase in PBS buffer (pH 7.4) con-
taining 10% Emerald II Enhancer. Imaging light pro-
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Figure 6. Images of living H1299 lung tumor xenografts on
SCID/BALB-C mice 1.5 min after administering intratumoral
injections of 100 µL 40 µM HyCL-2 and 10% Emerald II En-
hancer in 10 mM PBS (pH 7.4) while breathing (a) air or (b)
100% oxygen. (c) Quantification of biological replicates (n = 2
mice). Error bars are high and low values of the two repli-
cates.
Figure 7. Multi-spectral optoacoustic tomography (MSOT)
images of a living H1299 bearing SCID/BALB-C mouse while
breathing (a) 16% oxygen or (b) 100% oxygen. Scale bars are
3 mm.
duction indicated a clear increase in luminescence inten-
sity with increasing nitroreductase concentrations (Figure
4a). The emitted light displayed good linearity with in-
creasing concentrations of nitroreductase (Figure 4b),
demonstrating the ability of HyCL-2 to accurately and
sensitively image reductase activity, with an estimated
detection limit (3 σ) between 2–10 ng/mL. Encouraged by
the robust chemiluminescent light generated from the
multi-well plate experiment, we next tested if light pro-
duction from HyCL-2 would be sufficient for nitroreduc-
tase imaging in living animal tissue. Anesthetized
SCID/BALB-C mice were administered subcutaneous in-
jections on their back. On the upper back, a solution of
HyCL-2, NADH, nitroreductase, and Emerald II Enhancer
was injected (Figure 5a, NTR+NADH), and a vehicle con-
trol solution with HyCL-2 and no nitroreductase or
NADH was injected on the lower back (Figure 5a, Vehi-
cle). The injected area on the upper back displayed a
dramatic increase in chemiluminescent emission com-
pared with the vehicle injection without nitroreductase or
NADH on the lower side (Figure 5b). These experiments
show that HyCL-2 can function as an in vivo CLI agent for
nitroreductase activity.
oxygen for several minutes prior to and during imaging. A
solution of HyCL-2 in PBS buffer (pH 7.4) containing 10%
Emerald II Enhancer was injected intratumorally and then
imaged using an IVIS Spectra. While breathing air (21%
oxygen), high chemiluminescence emission was observed
(Figure 6a) that decayed over a period of 10 minutes. After
allowing time for the agent to clear, the mice were again
injected intratumorally with HyCL-2 in PBS buffer (pH
7.4) containing 10% Emerald II Enhancer, but this time
with the mice breathing 100% oxygen. In comparison,
breathing 100% oxygen caused an attenuation of the
chemiluminescent signal (Figure 6b). This indicates, in
agreement with the in vitro experiment described in Fig-
ure 3, that HyCL-2 provides increased signal in low oxy-
genation conditions found in hypoxic tumors. In order to
further support this claim, the same mice underwent the
oxygen challenge experiment while the levels of hemo-
globin (Hb) and oxygen-bound hemoglobin (HbO2) were
determined by multi-spectral optoacoustic tomography
(MSOT) imaging37 (Figure 7). Magnification of the tumor
reveals low total hemoglobin in the tumor center, charac-
teristic of a hypoxic core surrounded by blood vascula-
ture. Comparison of the images of the mouse breathing
16% oxygen (Figure 7a) to the images of the mouse
breathing 100% oxygen (Figure 7b) shows a clearly ob-
servable increase in HbO2, consistent with the data col-
lected by chemiluminescent imaging and providing a sec-
Finally, we interrogated the ability of HyCL-2 to visual-
ize tissue oxygenation and hypoxia in human tumor xen-
ograft models. H1299 lung tumors were grown subcuta-
neously on the flanks of SCID/BALB-C mice. Tumor oxy-
genation was adjusted using an oxygen challenge experi-
ment, where mice breathe either air (21% oxygen) or 100%
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will contribute towards the generalized development of
ondary confirmation. Taken together, these experiments
1
provide a preliminary validation of the CLI agent HyCL-2
chemiluminescence as a robust in vivo imaging modality.
2
for imaging tumor hypoxia.
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AUTHOR INFORMATION
Corresponding Author
Conclusions
We have designed and synthesized two spiroadaman-
tane 1,2-dioxetane chemiluminescent reaction-based im-
aging probes, HyCL-1 and HyCL-2, that display immedi-
ate light emission upon reacting with nitroreductase and
NADH at physiological pH. Due to our scaffold optimiza-
tion, the ether linked chemiluminescent agent HyCL-2
responds to nitroreductase with ~170-fold increase in lu-
minescence intensity compared to a blank control, largely
due to a drastically reduced background of the more hy-
drolytically stable ether linkage. This ether linkage is also
critical to achieving high selectivity, particularly towards
abundant biological thiol species. HyCL-2 has been suc-
cessively applied for detection of nitroreductase using
spectrophotometers, and imaging nitroreductase in vivo
using an IVIS Spectra instrument.
*
E-mail: alippert@smu.edu. Fax: 214-768-4089.
Present Addresses
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
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Funding Sources
CPRIT RP120670-P3; NIH R15GM114792; P30 1CA142543;
1S10RR024757 and S10 OD018094.
Conflict of Interest
The authors declare no competing financial interest.
Studies on the oxygen dependence of the chemilumi-
nescence emission of HyCL-2 in vitro revealed increases
in luminescence in the absence of oxygen, a result that
was also observed during in vivo oxygen challenge exper-
iments in human H1299 lung tumor xenografts grown on
mice. It is unlikely that reductase expression changes
within the timeframe of the imaging experiment and the
differences in luminescent emission are most likely due to
the change in oxygen levels. While the oxygen-
dependence of the probe in vivo is consistent with other
nitroaromatic prodrugs and imaging agents,11–23 the oxy-
gen-sensitivity in vitro is striking because the nitroreduc-
tase used in this study is generally believed to be oxygen-
insensitive.78,79 This suggests a secondary mechanism of
oxygen-sensitivity for HyCL-2, potentially the quenching
of triplet state products of the chemiluminescent reac-
tion.80,81 Tumor hypoxia and changes in oxygenation dur-
ing the oxygen challenge experiments were independently
confirmed by MSOT imaging of hemoglobin and oxygen-
ated hemoglobin as a technique to directly measure in
vivo blood oxygen levels. While this application of MSOT
is incapable of direct oxygen measurements in central
tumor areas with no vasculature, it provides supportive
and complementary data to the CLI experiments per-
formed herein.
ACKNOWLEDGMENT
This work was supported by Southern Methodist University
(start-up funds to A.R.L), the Center for Drug Design, Dis-
covery, and Delivery (CD4 seed money to A.R.L.), the Na-
tional Institutes of Health R15GM114792 (A.R.L.), and CPRIT
RP120670-P3 (R.P.M). Imaging was facilitated by the SW-
SAIR, a Resource of the Simmons Cancer Center supported
in part by NIH P30 1CA142543 and used an IVIS Spectrum,
which was purchased under NIH 1S10RR024757 and iThera
MSOT purchased under S10 OD018094. We thank Maciej
Kukula of the Shimadzu Center for Advanced Analytical
Chemistry at the University of Texas at Arlington for acquir-
ing high-resolution mass spectrometry data and Ramona
Lopez for experimental assistance.
Supporting Information
Synthetic procedures, absorption and emission spectra, de-
tailed procedures for selectivity tests, supplementary proce-
dures and figures, and scanned spectra. The Supporting In-
formation is available free of charge on the ACS Publications
website. See DOI: 10.1039/b000000x/.
It should be noted that similar CLI experiments on
larger tumors failed to produce consistent results due to
decay of light intensity with imaging depth and attempts
at imaging hypoxia in cell culture models were complicat-
ed by technical challenges with adding chemilumines-
cence reagents in the absence of oxygen. Future work
aims to solve these challenges by developing single com-
ponent agents with increased light production and non-
intensity based readouts that can more accurately report
on parameters without the confounding variables of im-
aging depth and temporal dependence. With these cave-
ats in mind, HyCL-2 can find application for optical mon-
itoring of tumor oxygenation in well-controlled preclini-
cal experiments. We further anticipate that these studies
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10
ACS Paragon Plus Environment