In vivo targeting of hydrogen peroxide by activatable cell-penetrating peptides

Article abstract of DOI:10.1021/ja411547j

A hydrogen peroxide (H₂O₂)-activated cell-penetrating peptide was developed through incorporation of a boronic acid-containing cleavable linker between polycationic cell-penetrating peptide and polyanionic fragments. Fluorescence labeling of the two ends of the molecule enabled monitoring its reaction with H₂O₂ through release of the highly adhesive cell-penetrating peptide and disruption of fluorescence resonance energy transfer. The H₂O₂ sensor selectively reacts with endogenous H₂O₂ in cell culture to monitor the oxidative burst of promyelocytes and in vivo to image lung inflammation. Targeting H₂O₂ has potential applications in imaging and therapy of diseases related to oxidative stress.

Full text of DOI:10.1021/ja411547j

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Communication
In Vivo Targeting of Hydrogen Peroxide
by Activatable Cell-Penetrating Peptides
Roy Weinstain, Elamprakash N. Savariar, Csilla N. Felsen, and Roger Y. Tsien
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 30 Dec 2013
Downloaded from http://pubs.acs.org on January 6, 2014
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In Vivo Targeting of Hydrogen Peroxide by Activatable Cell-
Penetrating Peptides
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Roy Weinstain,† Elamprakash N. Savariar,† Csilla N. Felsen,† and Roger Y. Tsien*,†,‡,§
†Department of Pharmacology, ‡Department of Chemistry and Biochemistry, and §Howard Hughes Medical Institute, Uni-
versity of California San Diego, La Jolla, California 92093, United States
Supporting Information Placeholder
have been directed towards extracellular enzymes such as matrix
metalloproteinases,16 elastases17 and thrombin,18 enabling in vivo
detection of their spatially localized enzymatic activity by various
imaging modalities. Thus, ACPPs are broadly applicable tools for
concentrating cargo of interest at the site of its activation.
ABSTRACT:
A hydrogen peroxide (H2O2)-activated cell-
penetrating peptide was developed through incorporation of a
boronic acid-containing cleavable linker between polycationic
cell-penetrating peptide and polyanionic fragments. Fluorescence
labeling of the two ends of the molecule enabled monitoring its
reaction with H2O2 through release of the highly adhesive cell-
penetrating peptide and disruption of fluorescence resonance en-
ergy transfer. The H2O2 sensor selectively reacts with endogenous
H2O2, in cell culture to monitor the oxidative burst of promyelo-
cytes, and in vivo to image lung inflammation. Targeting H2O2
has potential applications in imaging and therapy of diseases re-
lated to oxidative stress.
Here, we report the development of H2O2 targeting agents
based on ACPPs and demonstrate their ability to selectively image
endogenous levels of H2O2 in live cells and in vivo. We envi-
sioned making an ACPP reactive towards H2O2 by incorporation
of 4-boronic mandelic acid as a keystone in its linker architecture
(Figure 1A). The reaction of a phenylboronic acid with H2O2 to
form a phenol19-22 has been extensively utilized to generate a wide
23
range of small-molecule sensors for H2O2
.
Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS)
endogenously produced in living organisms. A growing body of
evidence suggests that H2O2 plays an active role in the regulation
of various physiological processes.1,2 Nevertheless, its overabun-
dance results in oxidative stress that can lead to extensive cellular
damage. Indeed, high levels of H2O2 have been implicated in
many pathological conditions including diabetes,3 cardiovascular
diseases,4,5 neurodegenerative disorders6 and cancer.7 Conse-
quently, there is increased interest in the role of H2O2 in normal
and pathological conditions, as well as in its potential as a target
in directed therapeutics delivery for oxidative stress related dis-
eases. Traditionally, these objectives are pursued separately,
through the development of dedicated molecular imaging probes8-
11 or drug delivery vehicles.12-14 Current H2O2 imaging agents that
are in vivo compatible can only be applied to transgenic animals8,9
or through localized administration10,11, while drug delivery vehi-
cles aimed at H2O2 require its presence at supranatural concentra-
tions to achieve sufficient activation12,13. Therefore, progress
could be made towards both objectives by developing molecular
targeting agents that respond to physiological levels of H2O2 in
intact animals, and that could be harnessed with interchangeable
cargo according to need.
Figure 1. Schematic illustration of H2O2-ACPP structure and its
H2O2-triggered fragmentation process. A) Fluorescence labeling
of H2O2-ACPP peptide domains enables visualization of its cleav-
age through FRET disruption. Shown are the fluorescence emis-
sions of (B) ACPP 1 and (C) ACPP 2 (1 ꢀM each) before (purple)
and 20 min after (green) reaction with H2O2 (2 mM).
An activatable cell-penetrating peptide (ACPP) uses a generic
targeting mechanism based on selective and local unleashing of a
cell-penetrating peptide (CPP).15 It is a hairpin shaped molecule
consisting of a polycationic CPP (D-Arg9) and an inhibitory poly-
anion (D-Glu9) connected through a cleavable linker. When intact,
the polyanion neutralizes the polycation and largely masks the
adhesiveness of the CPP. Extracellular cleavage of the linker ena-
bles dissociation of the inhibitory polyanion from the CPP, releas-
ing the CPP and associated cargo to adhere to and then penetrate
into nearby cells. Through appropriate design of linkers, ACPPs
In our design, oxidation of the boronic acid by H2O2 will form a
phenolate that will subsequently undergo a spontaneous 1,6-
elimination, resulting in fragmentation of the ACPP and release of
the CPP domain. Visualization of the ACPP’s reaction with H2O2
could be facilitated by fluorescent labeling of both of its peptide
domains. The close proximity enforced by the hairpin structure
should lead to fluorescence resonance energy transfer (FRET),
which would be disrupted by H2O2-mediated cleavage.
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In line with the design presented in Figure 1A, ACPP 1 (Figure
The changes in FRET could be monitored in real-time (Figures
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S1) was prepared through a combination of in-solution and solid-
phase synthesis (Schemes S1 and S2). The polycationic and poly-
anionic domains of 1 were labeled with fluorescein (donor) and
Cy5 (acceptor), respectively. When intact, ACPP 1 produces
strong FRET, as evident by low emission from the donor (fluores-
cein, ~524 nm) and strong re-emission from the acceptor (Cy5,
~670 nm) (Figure 1B). Cleavage of the ACPP by H2O2 leads to
disruption of the FRET, which could be visualized through the
increase in donor emission (~6-fold) and decrease in the acceptor
re-emission (~7-fold). The combined ~40-fold ratio change is
comparable with our previously reported FRET-ACPPs24 and
should provide a sufficient dynamic range to differentiate between
H2O2 levels. To test whether other donor/acceptor pairs are per-
missible in this design, we synthesized ACPP 2 (Figure S1 and
Scheme S3), where fluorescein and Cy5 were replaced with
Alexa488 and Alexa594, respectively. Similar to ACPP 1, ACPP
2 showed efficient FRET that was disrupted in the presence of
H2O2 (Figure 1C).
2B and S3) and cleavage of the ACPP was further confirmed by
SDS-polyacrylamide gel electrophoresis (PAGE) (Figure 2C) and
by high-performance liquid chromatography spectrometry
(HPLC-MS) (Figure S4). To verify that the observed FRET
change is not due to non-specific peptide cleavage or damage to
the fluorophores, we synthesized ACPP 3 (Figure S1 and Scheme
S4), an uncleavable version of 1, in which the boronic acid-
containing linker was replaced with a polyethylene glycol-6
(PEG6) molecule. No ratio change was observed when the un-
cleavable ACPP 3 (1 ꢀM) was incubated with H2O2 (2 mM) over
a period of 60 minutes (Figure S3).
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The response of ACPP 1 to H2O2 requires the occurrence of the
following three consecutive steps: reaction of the boronic acid
with H2O2 to form a phenolate, 1,6-elimination and dissociation of
the polypeptides from one another. The linear dependency of the
FRET change on H2O2 concentration suggests that the boronic
acid oxidation is the rate-limiting step. We therefore explored the
kinetics of this process by measuring its second-order rate con-
stant (1 ꢀM ACPP 1 and 1, 2.5 and 5 mM H2O2, Figure S5) to
find k = 1.34 ± 0.13 M-1 s-1, in agreement with previously reported
results.8 We then evaluated the selectivity of the H2O2-ACPP to
H2O2 over other biologically relevant extracellular ROS. For this,
ACPP 1 (1 ꢀM) was incubated with various ROS (100 ꢀM) and
the FRET ratio change was monitored for 60 minutes. A time-
dependent increase in the FRET ratio was observed when ACPP 1
was treated with H2O2 (~6-fold over 60 minutes). In contrast,
other ROS, or H2O2 in the presence of catalase, had little to no
effect on the FRET ratio (Figure 2D). Taken together, these re-
sults establish that H2O2-ACPP selectively reacts with physiologi-
cal levels of H2O2 in a concentration-dependent manner, culminat-
ing in fragmentation of the ACPP and release of its CPP domain.
Under normal physiological conditions, H2O2, although diffusi-
ble, forms a concentration gradient across cellular membranes,25,26
with an intracellular concentration estimated at 0.5-7 x 10-7 M27,28
and an extracellular concentration that is ~10-fold higher.29 In
pathological conditions, local extracellular concentrations of H2O2
are additionally elevated to as high as 10-50 ꢀM.30-35 Therefore, to
effectively target extracellular H2O2, a sensitivity level in the low
micromolar range is required. To determine its lowest detection
limit, ACPP 1 (1 ꢀM) was incubated with increasing concentra-
tion of H2O2 (0-250 ꢀM) and fluorescence emissions at 524 vs.
672 nm (λex = 488 nm) were monitored over 20 minutes. The
FRET ratio change (R/R0) was calculated by dividing the ratio of
524/672 nm emissions at each time-point (R) by the ratio before
H2O2 addition (R0). This assay established a linear dependence of
Next, we explored whether H2O2-ACPP could be used to detect
H2O2 in the cellular environment. Initial experiments in which
ACPP 1 (1 ꢀM) was treated with exogenous H2O2 (10-125 ꢀM) in
the presence of HL-60 cells, a human promyelocytic leukemia cell
line, demonstrated an increase in FRET ratio with linear depend-
ency on the applied H2O2 concentration (Figures 3A and S6, R2 =
0.9947).
ACPP 1’s FRET ratio change on H2O2 concentration (R2
=
0.9962) in a physiologically relevant range, with a detection limit
of ~5 ꢀM (Figures 2A and S2).
Figure 2. Selective and concentration-dependent cleavage of
ACPP 1 by H2O2. A) Fold increase in fluorescein/Cy5 emission
ratio (524/672 nm) after 20 min incubation of ACPP 1 (1 ꢀM)
with indicated concentrations of H2O2. Error bars represent ±
standard deviation. * p < 0.05. B) Time course fluorescence emis-
sion spectra of ACPP 1 (1 ꢀM) in the presence of H2O2 (1 mM).
C) H2O2-dependent cleavage of ACPP 1 (1 ꢀM) after 30 min
incubation with 1 mM H2O2. D) Fold increase in fluorescein/Cy5
emission ratio at indicated times of ACPP 1 (1 ꢀM) with indicated
ROS or their donors (100 ꢀM, catalase 0.5 mg/mL).
Figure 3. Detection of H2O2 by ACPP 1 in cellular environment.
A) Fold increase in fluorescein/Cy5 emission ratio (524/672 nm)
after 30 min of ACPP 1 (1 ꢀM) upon exogenous addition of H2O2
at indicated concentration in the presence of HL-60 cells. Error
bars represent ± standard deviation. B) Time course of fold in-
crease in fluorescein/Cy5 emission ratio (524/672 nm) of ACPP 1
(1 ꢀM) incubated with HL-60 cells at the indicated conditions
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(catalase 0.5 mg/mL, PMA 0.5 ꢀM). Error bars represent ± stand-
ard deviation. * p < 1 x 10-10, ** p < 1 x 10-4.
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We then tested the ACPP’s ability to detect endogenous levels
of H2O2. HL-60 cells express both membrane and cytoplasmic
NADPH oxidase (NOX) subunits. Exposure of these cells to
stimulants such as calcimycin, opsonized zymosan or phorbol 12-
myristate-13-acetate (PMA), triggers assembly of functional NOX
on the cellular membrane and generation of superoxide,36 which is
converted to various ROS including H2O2.37 NOX activity is the
main source of ROS generation in the oxidative burst mechanism
of immune cells. When HL-60 cells were treated with ACPP 1 (1
ꢀM) and either stimulated with PMA (0.5 ꢀM) or not, a time-
dependent increase in FRET ratio was observed in the stimulated
cells (Figure 3B).
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Using the FRET ratio change from the exogenous H2O2 appli-
cation experiment as a calibration curve (Figure S5), we calculat-
ed that under the aforementioned conditions, PMA-stimulated
HL-60 cells generate H2O2 at a rate of 0.98 ± 0.05 nmol/104
cells/hour,
in
agreement
with
previously
reported
measurements.35,38 Conversely, addition of catalase (0.5 mg/mL)
to PMA-stimulated cells completely suppressed the ratio change.
Interestingly, non-stimulated HL-60 cells exhibited a slight, but
statistically significant (p < 0.0001), increase in ratio compared to
catalase-treated cells, whether PMA-stimulated or not, suggesting
that non-stimulated cells produce a basal level of H2O2. The rate
of H2O2 production by non-stimulated cells was calculated to be
0.050 ± 0.004 nmol/104 cells/hour, about 20-fold lower than
PMA-stimulated cells. These results demonstrate that H2O2-ACPP
has sufficient sensitivity to detect endogenously produced H2O2 in
the cellular environment.
Figure 4. In vivo targeting of H2O2 by ACPPs 1 and 2. A) Repre-
sentative ratiometric fluorescent images of fluorescein/Cy5
(ACPP 1) or Alexa488/594 (ACPP 2) emission ratios of mouse
lungs in the indicated conditions treated with ACPP 1 (10 nmol)
or ACPP 2 (5 nmol) for 6 h. Right; scales of appropriate emission
ratios. B) Mean change in ratios of images presented in Figure 4A
with additional animals (n = 5). Error bars represent ± standard
deviation. * p < 0.05, ** p < 0.005. C) SDS-PAGE analysis of
lung extracts from LPS-mice treated with ACPPs 1-3 for 6 h.
Bands were pseudo-colored according to their emission spectra
(Figure S9): intact ACPP (purple) or cleaved ACPP (green).
Finally, we investigated the potential of H2O2-ACPP to detect
H2O2 endogenously produced by activated macrophages and neu-
trophils, in a lipopolysaccharide (LPS) model of lung inflamma-
tion. For this, C57BL/6 mice were either treated (LPS) or not
(control) with 10 ꢀg LPS by intranasal (i.n.) administration for
four consecutive days.39 Recruitment of inflammatory cells to the
airways of mice treated with LPS was confirmed by hematoxylin
In conclusion, we developed a H2O2 targeting mechanism based
on activatable cell-penetrating peptides (H2O2-ACPP). Fluores-
cence labeling of the H2O2-ACPP enabled visualization of its
reaction with H2O2 through FRET disruption. The H2O2-ACPP
reacts selectively and in a concentration-dependent manner with
H2O2 to release its CPP domain, whose adhesiveness and nondif-
fusibility preserve spatial resolution. Its low micromolar sensi-
tivity enabled detection and quantification of H2O2 secreted by
activated HL-60 cells. Moreover, H2O2-ACPP was sensitive
enough to react with endogenous levels of H2O2 in an in vivo
model of lung inflammation. Developing ACPPs for H2O2 target-
ing will potentially enable its imaging by a variety of modalities,
including fluorescence, magnetic resonance and radioactive tech-
niques. Importantly, a similar targeting mechanism could be fur-
ther used for directed delivery of therapeutics to local sites of
oxidative stress related diseases.
and eosin staining (Fig. S7). Mice were then administered ACPP
1 i.n. (10 nmol) and, after 6 hours, lungs were harvested, inflated
and imaged for the fluorescein/Cy5 emission ratio. Lungs of LPS-
treated mice presented ~2-fold increase in fluorescein/Cy5 emis-
sion ratio compared to control (Figures 4A,B).
To test whether H2O2 contributed to the observed increase in
ratio, LPS-treated mice were given D-penicillamine (DPA, 1
ꢀmol), a H2O2 scavenger,40-42 five minutes prior to ACPP 1 ad-
ministration. In our in vitro studies, DPA completely inhibited the
reaction between ACPP 1 and H2O2 (Figure S8). Lungs of LPS-
and DPA-treated mice showed a decreased fluorescein/Cy5 emis-
sion ratio, at least as low as control mouse lungs. Cleavage of
ACPP 1 in lungs of LPS-treated mice was confirmed by SDS-
PAGE analysis of homogenized lung extracts (Figure 4C). In
contrast, when similar mice were treated with uncleavable ACPP
3, no cleavage products were observed. We repeated the experi-
ments described above with ACPP 2. Results were consistent with
those observed with ACPP 1, i.e. lungs of LPS-treated mice
showed ~2-fold increase in ratio (Alexa488/Alexa594 emission)
that was completely suppressed by DPA (Figures 4A-C), suggest-
ing that H2O2-ACPP is a platform that can accommodate a range
of fluorophores. Taken together, these results suggest that H2O2-
ACPP can target and respond to endogenous levels of H2O2 pro-
duced in a model of lung inflammation in vivo.
ASSOCIATED CONTENT
Supporting Information
Experimental details, synthetic procedures, characterization meth-
ods, molecules structures, and figures. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
rtsien@ucsd.edu
Notes
R. Y. Tsien is a scientific advisor to Avelas Biosiences, which has
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(34) Suematsu, M.; Schmid-Schonbein, G. W.; Chavez-Chavez, R. H.;
Yee, T. T.; Tamatani, T.; Miyasaka, M.; Delano, F. A.; Zweifach, B. W.
Am. J. Physiol. 1993, 264, H881.
licensed the ACPP technology from University of California Rea-
gents. All the other authors declare no competing financial inter-
ests.
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ACKNOWLEDGMENT
The authors thank Qing Xiong and Larry Gross for technical as-
sistance and to all other lab members for helpful discussions. This
work was supported by the Howard Hughes Medical Institute,
National Institute of Health (NIH) grant 5R01CA158448-02 and
Department of Defense (DoD) grant W81XWH-09-1-0699 (to R.
Y. T.), NIH training grant 5R25CA153915-03 (to R. W. and C. N.
F.), American Asthma Foundation grant 11-0321 and NIH
1F30HL118998-01 (to C. N. F.).
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