Hoechst-IR: An imaging agent that detects necrotic tissue in vivo by binding extracellular DNA

Article abstract of DOI:10.1021/ol100923d

(Equation Presented). Cell necrosis is central to the progression of numerous diseases, and imaging agents that can detect necrotic tissue have great clinical potential. We demonstrate here that a small molecule, termed Hoechst-IR, composed of the DNA binding dye Hoechst and the near-infrared dye IR-786, can image necrotic tissue in vivo via fluorescence imaging. Hoechst-IR detects necrosis by binding extracellular DNA released from necrotic cells and was able to image necrosis generated from a myocardial infarction and lipopolysaccharide/d-galactosamine (LPS-GalN) induced sepsis.

Full text of DOI:10.1021/ol100923d

ORGANIC
LETTERS
2010
Vol. 12, No. 15
3300-3303
Hoechst-IR: An Imaging Agent That
Detects Necrotic Tissue in Vivo by
Binding Extracellular DNA
Madhuri Dasari,† Sungmun Lee,† Jay Sy,† Dongin Kim,† Seungjun Lee,†
Milton Brown,‡ Michael Davis,†,‡ and Niren Murthy*,†
The Wallace H. Coulter Department of Biomedical Engineering and Parker H. Petit
Institute for Bioengineering and Biosciences, Georgia Institute of Technology,
Atlanta, Georgia 30332, and DiVision of Cardiology, Emory UniVersity School of
Medicine, Atlanta, Georgia 30322
niren.murthy@bme.gatech.edu
Received April 21, 2010
ABSTRACT
Cell necrosis is central to the progression of numerous diseases, and imaging agents that can detect necrotic tissue have great clinical
potential. We demonstrate here that a small molecule, termed Hoechst-IR, composed of the DNA binding dye Hoechst and the near-infrared
dye IR-786, can image necrotic tissue in vivo via fluorescence imaging. Hoechst-IR detects necrosis by binding extracellular DNA released
from necrotic cells and was able to image necrosis generated from a myocardial infarction and lipopolysaccharide/D-galactosamine (LPS-
GalN) induced sepsis.
Necrotic tissue is a central characteristic of numerous
diseases including sepsis, cancer, cardiac dysfunction, ath-
erosclerosis, and several others.1 The imaging of necrotic
tissue therefore has the potential to significantly improve the
diagnosis of a wide variety of human diseases. However,
imaging necrosis in vivo has been challenging because of a
lack of imaging contrast agents.
A molecular target with considerable potential for imaging
tissue necrosis is extracellular DNA (E-DNA).2 Cell necrosis
results in the disruption of the cell membrane, which causes
the release of the cellular contents, including genomic DNA,
into the extracellular space. The intracellular DNA concentration
is in the millimolar range, and therefore high concentrations of
E-DNA are potentially generated in the extracellular space of
tissue with high levels of necrosis. Under certain pathological
conditions, E-DNA can be detected in the blood; however,
because of the large blood volume and the presence of serum
nucleases, the E-DNA concentration in the blood should be
significantly lower than in necrotic tissue. E-DNA is also not
generated during apoptosis, the primary mechanism of cell death
in healthy tissue, and therefore the E-DNA concentration in
healthy tissue should be very low. Finally, E-DNA can
† Georgia Institute of Technology.
‡ Emory University School of Medicine.
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Pisetsky, D. S.; Fairhurst, A. M. Autoimmunity 2007, 40, 281. (d) Jahr, S.;
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10.1021/ol100923d  2010 American Chemical Society
Published on Web 07/02/2010

potentially be imaged with small molecule based imaging
agents, because numerous small molecules have been developed
that can bind DNA with nanomolar affinity and high specific-
ity.3 However, despite its potential, the development of an
imaging agent that can image E-DNA in vivo still remains a
major challenge.3c In this report, we demonstrate that a small
molecule, termed Hoechst-IR, can image necrotic tissue in vivo
via fluorescence imaging. Hoechst-IR detects necrosis by
binding E-DNA released from necrotic cells and was able to
image necrosis generated from a myocardial infarction and
lipopolysaccharide/D-galactosamine (LPS-GalN) induced sepsis.
Scheme 2. Synthesis of Hoechst Amine (2)
Scheme 1. Structure and Retrosynthesis of Hoechst-IR (1)
synthesized using modified literature procedures.7 We chose
this route, after unsuccessfully trying to alkylate the com-
mercially available Hoechst 33258 at its phenol group. The
compound 2 was obtained in 10 steps starting from com-
mercially available 4-hydroxy benzonitrile (4). Nucleophilic
substitution of 4 with bromo-PEG-phthalimide 58 provided
the ether 6 in 92% yield, which upon acidification generated
the iminium ether 7. Condensation of 7 with piperazyl
diamine 89 gave the phthalimide protected Hoechst 9 in a
38% yield. Finally, deprotection of 9 with hydrazine hydrate
and acidification with aqueous HCl generated the Hoechst
amine salt 2 in quantitative yield.
The molecular design of Hoechst-IR (1) is shown in
Scheme 1 and is composed of two moieties, namely, Hoechst,
a DNA binding agent, and the near-infrared (NIR) dye IR-
786. Hoechst was chosen as the E-DNA binding component
of Hoechst-IR because of its nanomolar affinity for DNA
and well-documented biocompatibility in humans,4 and also
because it can be modified at its terminal phenol without
losing DNA binding activity.5 An 11-unit PEG linker was
incorporated into Hoechst-IR to increase its hydrophilicity
and prevent it from crossing the cell membrane and binding
intracellular DNA. Lastly, Hoechst-IR contains the NIR
fluorophore IR-786, which allows for imaging of E-DNA,
using near IR wavelengths.6
Scheme 3. Synthesis of Hoechst-IR (1)
The retrosynthesis of 1 is shown in Scheme 1 and was
accomplished by coupling the two synthetic building blocks
2 and 3 via a carbamate linkage. The compound 2 (Scheme
2) is a derivative of Hoechst with a terminal amine, and was
(3) (a) Strekowski, L.; Wilson, B. Mutat. Res. 2007, 623, 3. (b) Telford,
W. G.; King, L. E.; Fraker, P. J. Cytometry 1992, 13, 137. (c) Garanger,
E.; Hilderbrand, S. A.; Blois, J. T.; Sosnovik, D. E.; Weissleder, R.;
Josephson, L. Chem. Commun. 2009, 4444.
The second synthetic building block 3 (Scheme 3) is a
derivative of the cyanine dye IR-786 iodide (10) and was
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B. C.; MacDonald, J. InVest. New Drugs 1991, 9, 95. (b) Patel, S. R.; Kvols,
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Org. Lett., Vol. 12, No. 15, 2010
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synthesized in three steps. Treatment of 10 with PEG8-thiol
11 generated the thioether 12 in 60% yield via a nucleophilic
addition/elimination reaction.10 Activation of the hydroxyl
group in 12 with 4-nitrophenyl chloroformate generated the
carbonate ester 3 in 78% yield, which upon coupling with 2
afforded the final conjugate 1 in 31% yield. We verified that
Hoechst-IR binds DNA with high affinity by titrating it with
a 22 bp double-stranded oligonucleotide (sense strand, 5′-
AGTTGAGGGGACTTTCCCAGGC-3′;complementarystrand,
3′-TCAACTCCCCTGAAAGGG TCCG-5′) and determined
that it has a dissociation constant (Kd) of 0.2 nM (see
Supporting Information).11 Interestingly, this binding affinity
is a little higher than that of free Hoechst (Kd ) 1-3 nM).3a
We anticipate that this additional increase in binding affinity
may be due to the affinity of cyanine dyes for DNA, which
bind DNA with moderate affinity.12
A key issue in developing molecules that can image
E-DNA is ensuring that they are membrane-impermeable.
Hoechst-based compounds are potentially problematic for
imaging E-DNA because commercially available Hoechst
33258 is membrane-permeable. We therefore evaluated the
membrane permeability of Hoechst-IR in vitro in live cells
and cells that were fixed and permeabilized with methanol.
of intracellular fluorescence (Figure 1B). This further con-
firms that the low levels of fluorescence seen in Figure 1A
were due to the low membrane permeability of Hoechst-IR
and not due to a reduction in its DNA binding capability.
Additionally, live cells incubated with Hoechst 33258
generate high levels of intracellular fluorescence, demonstrat-
ing that Hoechst 33258 can easily cross cell membranes and
bind intracellular DNA (Figure 1C).
We examined the ability of Hoechst-IR to target E-DNA
generated by tissue necrosis after a myocardial infarction
(MI). Myocardial infarction generates large amounts of tissue
necrosis because of reperfusion injury. There is great clinical
interest in identifying necrotic tissue in patients suffering
from MI because it helps identify infarcted tissue and also
the development of congestive heart failure, which usually
follows after an MI.13 A mouse ischemia-reperfusion model
of MI was used to evaluate the efficacy of Hoechst-IR.
Figure 2. Hoechst-IR targets tissue necrosis in vivo after a
myocardial infarction. (A) Micrograph of a heart isolated from a
mouse subjected to ischemia-reperfusion injury and treated with
Hoechst-IR. Hoechst-IR accumulates in the necrotic zone of the
infarct. (B) Micrographs of mouse hearts subjected to ischemia-
reperfusion surgery + Hoechst-IR, sham surgery + Hoechst-IR,
or sham surgery + saline injection. (C) Mouse hearts subjected to
ischemia-reperfusion surgery + Hoechst-IR have a 2-fold increase
in IR-786 fluorescence compared with mouse hearts subjected to
sham surgery + Hoechst-IR (*P < 0.05, n ) 3).
Figure 1. Hoechst-IR is membrane-impermeable. RAW 264.7
macrophage cells were incubated for 30 min with either Hoechst-
IR (20 µM) or Hoechst 33258 (20 µM) and imaged by fluorescence
microscopy, using an excitation of 340 nm. (A) Live cells treated
with Hoechst-IR have negligible fluorescence, demonstrating that
Hoechst-IR is membrane-impermeable. (B) Fixed cells treated with
Hoechst-IR and (C) live cells treated with Hoechst 33258 have high
fluorescence
Briefly, the left anterior descending coronary artery was
ligated with a suture, generating ischemia for 30 min and
then opened to allow for reperfusion. Two hours after the
suture was removed, Hoechst-IR (10 nmol) was administered
intravenously via a retro-orbital injection and allowed to
circulate for 1 h. The mice were then sacrificed, perfusion
fixed with paraformaldehyde, and the organs were harvested
and imaged using an in vivo imager. Figure 2A-C demon-
strates that Hoechst-IR can detect necrosis in the myocardium
after an MI. For example, Figure 2A shows a representative
image of the heart of a mouse that received Hoechst-IR, after
ischemia-reperfusion surgery, and demonstrates that Hoechst-
IR accumulates in the vicinity of the necrotic zone of the
infarct. Figure 2B and C also demonstrates that Hoechst-IR
accumulates to a higher level in infarcted hearts than in sham
operated hearts, further suggesting again that Hoechst-IR can
detect E-DNA in necrotic tissue.
RAW 264.7 macrophages and methanol fixed macroph-
ages were incubated with Hoechst-IR and imaged by
fluorescence microscopy. Figure 1 demonstrates that Ho-
echst-IR has very low cell permeability. For example, live
cells incubated with Hoechst-IR generate low levels of
intracellular fluorescence, indicating that Hoechst-IR has very
low cell permeability, presumably because of its 11 unit PEG
chain (Figure 1A). In contrast, methanol-treated permeabi-
lized cells incubated with Hoechst-IR generate high levels
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an IVIS imaging system, and the total fluorescence from the
lung area was integrated.
Figure 3A and B demonstrates that mice treated with LPS-
GalN and Hoechst-IR have a 4-fold increase in fluorescence
over mice treated with saline and Hoechst-IR, demonstrating
that Hoechst-IR can detect necrosis caused by sepsis.
Surprisingly, the fluorescence intensities from the ip cavity
of LPS-GalN treated and control mice were both very low,
suggesting that the E-DNA generated from the organs of the
ip cavity are either rapidly degraded or cleared by phagocytic
cells.
In summary, we demonstrate that Hoechst-IR can image
necrotic tissue in vivo via fluorescence imaging. Hoechst-
IR was synthesized by the coupling of hoechst and IR
derivatives via a carbamate linkage. Hoechst-IR detects
necrosis by binding E-DNA and was able to image necrosis
generated from a myocardial infarction and LPS-GalN
induced sepsis. We anticipate numerous applications of
Hoechst-IR given the large number of diseases associated
with tissue necrosis.
Figure 3. Hoechst-IR can image tissue necrosis in vivo caused by
LPS-GalN induced sepsis. (A) (i) Representative fluorescent image
of mouse treated with an ip injection of LPS (2.5 µg/kg) and GalN
(700 mg/kg), followed by an iv injection of Hoechst-IR (5 mg/kg),
(ii) representative fluorescent image of mouse treated with an ip
injection of saline, followed by an iv injection of Hoechst-IR (5
mg/kg). (B) Quantification of Hoechst-IR fluorescence intensities
of mice treated with LPS-GalN and saline. Mice treated with LPS-
GalN have a 3- to 4-fold increase in fluorescence over mice treated
with saline (*P < 0.05, n ) 5).
Acknowledgment. This work was supported by NSF-
BES-0546962 Career Award (N.M.), NIH UO1HL80711-
01 (N.M.), and NIH RO1 HL80711-01 (N.M.).
Supporting Information Available: Experimental pro-
cedures, characterization data for all compounds, and DNA
binding isotherm for Hoechst-IR. This material is available
free of charge via the Internet at http://pubs.acs.org.
Finally, Hoechst-IR was evaluated for its ability to image
necrosis in vivo during sepsis. Sepsis is a leading cause of
death worldwide and generates severe necrosis in many vital
organs, such as in the lungs.14 Sepsis was induced in mice
by an intraperitoneal (ip) injection of LPS-GalN, and 24 h
later Hoechst-IR was injected via the jugular vein and
allowed to circulate for 24 h. The mice were then imaged in
OL100923D
(14) (a) Rivers, E.; Nguyen, B.; Havstad, S.; Ressler, J.; Muzzin, A.;
Knoblich, B.; Peterson, E.; Tomlanovich, M. N. Engl. J. Med. 2001, 345,
1368. (b) Huber-Lang, M.; Sarma, V. J.; Lu, K. T.; McGuire, S. R.;
Padgaonkar, V. A.; Guo, R. F.; Younkin, E. M.; Kunkel, R. G.; Ding, J.;
Erickson, R.; Curnutte, J. T.; Ward, P. A. J. Immunol. 2001, 166, 1193.
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