A New Polymer Nanoprobe Based on Chemiluminescence Resonance Energy Transfer for Ultrasensitive Imaging of Intrinsic Superoxide Anion in Mice

Article abstract of DOI:10.1021/jacs.5b11784

Despite significant developments in optical imaging of superoxide anion (O₂^?-) as the preliminary reactive oxygen species, novel visualizing strategies that offer ultrahigh sensitivity are still imperative. This is mainly because int

Full text of DOI:10.1021/jacs.5b11784

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Communication
A New Polymer Nanoprobe Based on Chemiluminescence
Resonance Energy Transfer (CRET) for Ultrasensitive
Imaging of Intrinsic Superoxide Anion in Mice
Ping Li, Lu Liu, Haibin Xiao, Wei Zhang, Lulin Wang, and Bo Tang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b11784 • Publication Date (Web): 24 Feb 2016
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A New Polymer Nanoprobe Based on Chemiluminescence
Resonance Energy Transfer (CRET) for Ultrasensitive Imaging
of Intrinsic Superoxide Anion in Mice
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Ping Li, Lu Liu, Haibin Xiao, Wei Zhang, Lulin Wang, and Bo Tang*
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functional-
ized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Min-
istry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal
University, Jinan 250014, P. R. China
Supporting Information Placeholder
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the detection of endogenous O2 in tissues and in vivo em-
ploying molecular probes or nanoprobes without stimulation
ABSTRACT: Despite significant developments in optical
imaging of superoxide anion (O2•−) as the preliminary reac-
has not been realized so far.
tive oxygen species (ROS), novel visualizing strategies that
offer ultra-high sensitivity are still imperative. This is mainly
because intrinsic concentrations of O2 are extremely low in
Chemiluminescence (CL) with imidazopyrazinone5-7 is a
well-established technology applied to biological analysis of
O2•−. This is attributed to no external light excitation, offer-
ing many prominent advantages over traditional photon
emission-based detection methods, such as significantly re-
duced background fluorescence interference and photo-
damage, and so on. Despite these appealing features, there
remain substantial stumbling blocks to the routine use of CL
as an optical imaging strategy for O2•−, for instance, low
emission intensity, short chemiluminescence time, and short
emission wavelength. To solve the above problems, chemi-
luminescence resonance energy transfer (CRET) provides
good opportunities,8-10 because CRET can effectively prolong
luminescence time, and cause wavelength red-shift. In the
meantime, conjugated polymers (CPs)11-15 as fluorophores are
endowed with high absorptivity, extraordinary brightness,
and excellent biocompatibility, as well as easy fabrication.
Their fascinating properties bring out booming develop-
ments in imaging probes. Based on distinguished signal am-
plification along the backbone and adjustable optical per-
formance, CPs are suitable candidates as the energy recep-
tors of CRET process. Importantly, a hydrophobic environ-
ment of CPs nanoparticle interior can further boost CL in-
tensity and extend luminescence time.5,8 Moreover, unlike
fluorescence resonance energy transfer (FRET), CRET,16,17 the
nonradiative resonance energy transfer process, we suppose
that a higher efficiency can be achieved through covalent
link between the acceptor and the donor than physical inte-
gration. Furthermore, previous studies suggest that covalent
attachments not only provide a readily energy transfer, but
also avoid the leakage problem of small molecules encapsu-
lated in nanoparticles.18 Therefore, in order to highly sensi-
tive imaging intrinsic O2•− of ultra-low concentration in vivo,
development of the conjugated polymer probe based on
CRET by covalent attachment is of great value.
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living systems. Herein, we present the rational design and
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construction of a new polymer nanoprobe PCLA-O2 for
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detecting O2 based on chemiluminescence resonance ener-
gy transfer (CRET) without an external excitation source.
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Structurally, PCLA-O2 contains two moieties linked cova-
lently, namely imidazopyrazinone that capable of chemilu-
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minescence triggered by O2 as the energy donor, and con-
jugated polymers with light-amplifying property as the ener-
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gy acceptor. Experiment results demonstrate that PCLA-O2
exhibits ultra-high sensitivity at the picomole level, dramati-
cally prolonged luminescence time, specificity, and excellent
biocompatibility. Without exogenous stimulation, this probe
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for the first time in situ visualizes O2 level differences be-
tween normal and tumor tissues of mice. These exceptional
features ensure that PCLA-O2•− as a self-luminescing probe is
an alternative in vivo imaging approach for ultra-low level
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O2
.
Reactive oxygen species (ROS) produced in living systems
are usually tamed as cellular redox balances maintained by
enzymatic and nonenzymatic antioxidant defenses.1 Alt-
hough ROS contribute to cellular functions, any disruption
of their physiologic homeostasis could potentially lead to
serious issues,2,3 such as cancer, diabetes, neurodegeneration,
and other disorders. Noteworthily, the superoxide radical
(O2•−) is one of the primary ROS4 and acts as a sink for many
other radicals generated intracellularly. Consequently, the
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highly sensitive and specific detection of O2 in biological
systems has received growing attention. In particular, nonin-
vasive in vivo fluorescent imaging associated with easy oper-
ation molecule probes and nanoprobes becomes an attractive
avenue for elucidating its biological function. However, be-
cause of very low actual concentration in biological systems,
In this paper, we report an in vivo imaging platform for na-
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tive O2 based on CRET. As illustrated in the Scheme 1, the
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designed probe termed PCLA-O2 comprises two segments
connected through the covalent bond. Structurally, imidaz-
opyrazinone moiety (CLA for short) acts as both recognition
Subsequently, we compared the optical properties of
PCLA-O2•−, PFBT and CHO-CLA. Figure 1c showed that the
absorption peaks of PFBT were at 350 nm and 470 nm,
meanwhile the CL peak of CHO-CLA centered at 490 nm in
the presence of O2•−. Obviously, there was overlap between
absorption spectrum of the PFBT and luminescence spec-
trum of CHO-CLA, meaning distinct possibility of CRET oc-
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unit of O2 and the energy donor, while CPs (PFBT)19 with
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quaternary ammonium groups act as both the energy accep-
tor and signal amplification matrix.13 Using nanoprecipita-
tion,12 PCLA-O2 polymer chains covalently attaching the
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multiple antennas (CLA) fold to form compact spherical na-
noparticles in aqueous medium. As a result, the nanoparti-
cles can afford a hydrophobic interior, in which CLA parts
are embedded. Once the O2 is added, through a radiation-
less energy transfer process, the energy produced by specific
currence. Next, whether O2 can make PCLA-O2 self-
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emitting by CRET was examined. Upon addition of O2
,
without any external light excitation, intense luminescence
was observed at 560 nm of longer wavelength (Figure 1d),
confirming efficient CRET in PCLA-O2•− from CLA to PFBT.
To further verify the validity of CRET, a control experiment
was performed in a mixed aqueous solution of CHO-CLA and
PFBT. As illustrated in Figure S2, CL intensities of PFBT in
the control experiment remained almost unchanged under
the same condition, even with increasing amount of CHO-
CLA. This may be because a part of CHO-CLA dispersed in
the mixed solvent. Therefore, these results not only demon-
strate the successful covalent conjugation of CLA to PFBT,
but also highlight its necessary covalent linkage by control-
ling close distance between CLA and PFBT, resulting in an
efficient CRET.
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reaction between CLA and O2 is transferred to PFBT with
non-emitting of green light (λmax, 490 nm). Afterwards, the
PFBT acceptor can be excited and emits magnified light
(λmax, 560 nm). Based on the above principle, the novel na-
noprobe was fabricated. As expected, this CRET system ena-
bles successful live imaging of O2•− in mice.
Scheme 1. Schematic illustration of nanoparticles
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preparation by “nanoprecipitation” and O2 sensing
Figure 1. a) HRTEM image of PCLA-O2•− in water. Scale
bar=20 nm. b) DLS image of PCLA-O2•−. c) Absorption spec-
trum of PFBT (black line) and CL spectrum of CHO-CLA
(green line). d) Excitation/emission spectra (black lines) and
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of PCLA- O2 , and the structure of PCLA- O2 .
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PCLA-O2 was synthesized from PFBT with quaternary
ammonium groups and CHO-CLA with imidazopyrazinone
(Scheme S1 and S2). The specific synthetic routes and charac-
CL spectrum (red line) of PCLA-O2•− in PBS (10 mM, pH=7.4).
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CL is detected in the presence of 10 μM O2
.
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terization of PFBT, CHO-CLA and PCLA-O2 are showed in
Supporting Information. FT-IR spectroscopy in Figure S1
showed that 1723 cm-1 strong peak is assigned to C=N bond,
indicating the covalent conjugation of CLA to PFBT. Finally,
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nanoparticles of PCLA-O2 were prepared by nanoprecipita-
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tion, after addition of PCLA-O2 solution in tetrahydrofuran
(THF) into excessive water. The physical properties of this
nanoprobe were investigated by the high-resolution trans-
mission electronmicroscopy (HRTEM) and dynamic light
scattering (DLS). As is clearly visible from the HRTEM imag-
es (Figure 1a), spherical morphology of nanoparticles was
confirmed. Their average diameters were about 20 nm, con-
sistent with the results obtained in DLS experiments (Figure
1b). Moreover, potentiometric analysis displayed that Zpotential
was +49 mV, which should be owing to hydrophilic quater-
nary ammonium groups on the surface of the nanoparticles.
Together, these data manifest that PCLA-O2•− nanoprobe has
been constructed successfully in full accordance with the
design.
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Figure 2. The linear relationship between CL of PCLA-O2
and [O2•−] (0-950 pM) (PCLA-O2•−=0.45 mg/mL, PBS=10 mM,
•−
pH=7.4, 10% THF). Inset, CL images of PCLA-O2 in re-
sponse to indicated concentrations of O2•− by an IVIS Lumina
II in vivo imaging system with open filter (570 ± 10 nm).
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Then, the CL responses of PCLA-O2•− toward different con-
color) of PCLA-O2 toward O2 was carried out under four
different conditions. The experiments results summarized in
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centrations of O2 were examined in PBS buffer (pH 7.4).
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Figure 2 revealed, when O2 level was elevated, the CL sig-
Figure S7a showed that PCLA-O2 strongly emitted CL in
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nals were pronouncedly intensified. The output of images
showed CL intensities increased linearly with the concentra-
tions of O2 in the range from 0 to 950 pM (Figure 2). The
extracts from PMA-stimulated macrophages. The CL intensi-
ty increased by 3.6 times, as a comparison with the control
without PMA treatment. In the same time, superoxide dis-
mutases (SOD) and Tiron,21 the free-radical (O2•−) scaven-
gers, were used to treat macrophages pre-incubated with
PMA respectively. We found that CL signals were markedly
suppressed, indicating the successful scavenging of O2•−. Fur-
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detection limit was estimated to be 19.3 pM, as calculated by
equation LOD=3S0/K. To the best our knowledge, up to now,
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this is the most sensitive luminescence probe for O2 com-
pared with nanomolar level of reported probes. Furthermore,
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upon addition of various other reactive species (H2O2, O2,
thermore, PCLA-O2 was used to monitor intrinsic O2 in a
nonmetastatic human mammary epithelial cell line (MCF-
10A) and mouse mammary carcinoma cells (4T1), respective-
ly. As shown in Figure S7b, CL intensity in the 4T1 extracts
was 2.1-times higher than that of MCF-10A extracts, reflecting
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NO, ascorbic acid, ClO−, TBHP, ·OH, GSH, ONOO−), there
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were negligible CL responses of PCLA-O2 (Figure S3). And
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also, the CL of PCLA-O2 in response to O2 was independ-
ent of pH in the physiological range (Figure S4) and serum
(Figure S5). Meanwhile, Figure S6 reflects the excellent sta-
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higher level O2 in tumor cells. These in vitro data clearly
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bility of PCLA-O2 without O2 over time. Thus, PCLA-O2
substantiate that PCLA-O2 has the capability of specifically
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turns out to be extremely sensitive and selective to O2
.
sensing native O2 in biological samples, and also differenti-
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ating normal cells and tumor cells by variant O2 levels. Ad-
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ditionally, PCLA-O2 was found to be non-toxic to cells and
mice (Figure S8).
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Figure 3. a) CL images of CHO-CLA (λem=520±10 nm) and
Figure 4. a) The CL imaging of endogenous O2 in the LPS-
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PCLA-O2 (λem=570±10 nm) in response to O2 (0.9 μM and
0.1 μM, respectively) over time (CHO-CLA=4.2 mM, PCLA-
O2•−=0.45 mg/mL, PBS=10 mM, pH=7.4, 10% THF). b) Quan-
treated mice (n=4). (I) LPS was injected into peritoneal cavity
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of mice, after 4 h, followed by injection PCLA-O2 (0.068
mg). (II) LPS was injected into abdomen, after 3 h, followed
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titative CL intensities of CHO-CLA and PCLA-O2 as a func-
by injection Tiron (400 μL, 10 μM). 1 h later, PCLA-O2
tion of time.
(0.068 mg) was injected. (III) Saline+PCLA-O2•− (0.068 mg).
(IV) The control: saline. b) Quantitative of CL emission in-
tensities from groups (I)–(IV) at 0.5 min.
To test whether our design strategy gives rise to more lu-
minescent time, delayed luminescence (DL) of CHO-CLA
and PCLA-O2•− were investigated, respectively. After addition
of O2•−, the CL channels of the PCLA-O2•− (λem =570±10 nm)
and CHO-CLA (λem =520±10 nm) were imaged over time,
depicted as in Figure 3. It is can be seen that the CL intensi-
ties of CHO-CLA attenuated dramatically in 2.1 min (Figure
•−
Based on the above discussed results, PCLA-O2 possesses
the distinguished advantages over the existing small mole-
cule CL compounds, we consider that PCLA-O2•− may in situ
detect the fluctuations of O2•− in vivo. As reported, excessive-
ly produced O2•− is implicated in the development of numer-
ous inflammatory diseases.22 Thereby, PCLA-O2•− was used to
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3b). However, luminescence of PCLA-O2 obviously lasted
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much longer. The results suggest that a hydrophobic envi-
evaluate the endogenous O2 in mice with imflammation
ronment of CPs nanoparticle interior and effective CRET by
without an external excitation, which can remarkably im-
prove sensitivity and reduce photodamage. Different groups
of mice were treated with intraperitoneally (i.p.) adminis-
trated lipopolysaccharide (LPS)23 for inducing acute inflam-
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covalent link5,18 indeed lengthen the duration of DL for O2
.
This fascinating merit is crucial and favorable for a practical
application of biological imaging.
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mation (I), LPS+Tiron (II), saline and PCLA-O2 (III), and
All these optical and photophysical advantages of PCLA-
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O2 make it a promising nanoplatform for in vivo O2•− imag-
saline (IV), respectively. Then, CL (λem=570±10 nm) images
were acquired. We found PCLA-O2•− displayed much brighter
CL in LPS-treated mice than that of only PCLA-O2•−-treated
(Figure 4a). Notably, CL intensity in the LPS-treated group
was 7.9-times higher than that of only PCLA-O2•−-treated
group showed in Figure 4b, meaning rise of [O2•−]. In con-
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ing. With the PCLA-O2 in hand, we started to validate its
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biological feasibility for detecting O2•−. Firstly, we texted O2
levels in macrophages extracts utilizing PCLA-O2•−, phorbol
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12-myristate 13-acetate (PMA)20 as activator to elevate O2
levels. The CL (λem=570±10 nm) response imaging (pseudo-
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trast, upon the injection Tiron to the LPS-pretreated mouse,
tions. These interesting results and limited tissue penetration
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CL intensity weakened distinctly. Meanwhile, Figure 4a IV
demonstrated that there was no emission in the group with
only saline injection, indicating no background fluorescence.
Apparently, owing to no interference from biological auto-
fluorescence, effective CRET and interior hydrophobic condi-
tion of this nanoprobe, imaging quality and sensitivity were
significantly upgraded. Moreover, Figure S9 demonstrated
enough long luminescence time was benefit to conveniently
monitor O2•−. Altogether, we conclude that PCLA-O2•− allows
for in situ imaging of the O2•− in vivo by means of CL.
capability of 560 nm prompt us to develop the new near-
infrared probe for deep tissue imaging and clinical applica-
tions in superoxide anion related biological and medical re-
search. And also, we can investigate the similar CRET plat-
forms to visualize other active molecules in vivo.
ASSOCIATED CONTENT
Supporting Information
9
Synthesis, characterization, and experimental details. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
tangb@sdnu.edu.cn
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
Figure 5. a) Representative images (pseudocolor) of mice in
vivo tumor (I), tumor+Tiron (II) and normal (III) tissues
followed by PCLA-O2•− (n = 3). Images (λem=570±10 nm) were
acquired using an IVIS Lumina II at 30 s after PCLA-O2 ad-
ministration. b) Quantitative CL intensities of (I)-(III).
This work was supported by 973 Program (2013CB933800)
and National Natural Science Foundation of China (21390411
， 21535004，21227005, 21475079, and 21305080).
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REFERENCES
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intrinsic O2 in the absence of stimulation, we examined
•−
whether it could distinguish the difference on O2 concen-
trations between normal and tumor tissues in mice. A mouse
mammary carcinoma model was constructed by injecting 4T1
cells in forelimb armpit. After 10 days, a tumor mass was ob-
tained. Then, PCLA-O2•− (0.068 mg) was injected in tumor (I),
tumor+Tiron (II) and normal (III) tissues of the mice, respec-
•−
tively. Subsequently, we found PCLA-O2 displayed much
stronger CL in tumor tissue than that of in normal tissue
(Figure 5a). The CL intensity was 3.0-times higher than the
normal tissue (Figure 5b), indicating noticeably higher level
•−
of O2 in the tumor tissue. Meanwhile, weak CL signal in
tumor tissue was observed after injection of Tiron. The re-
sults presented here validate for the first time visualization of
the O2•− native variance without stimulation in small animals.
In summary, we describe a supersensitive imaging nano-
•−
•−
probe PCLA-O2 for O2 based on CRET between imidaz-
opyrazinone and conjugated polymers. Without an external
excitation source, the attractive probe was applied in mice to
•−
selectively visualize O2 in normal/inflammation tissues.
•−
More importantly, as the most sensitive O2 probe to our
•−
knowledge, PCLA-O2 revealed native concentration differ-
•−
ences of O2 between normal and tumor tissues without
exogenous stimulation. The above breakthroughs achieved
are attributed to several remarkable advantages of the new
imaging probe: (1) elevated CRET efficiency by covalent link
between accepter and donor, (2) substantially extended lu-
minescence time due to effective CRET process, (3) signifi-
cantly enhanced CL intensity under interior hydrophobic
environment via polymer nanoparticle formation, (4) greatly
improved sensitivity and imaging resolution owing to longer
luminescence time and no background fluorescence interfer-
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ence without external light source. We deem that PCLA-O2
would serve as a powerful tool to differentiate normal tissues
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and pathological tissues via distinguishing O2 concentra-
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