pH-Amplified CRET Nanoparticles for In Vivo Imaging of Tumor Metastatic Lymph Nodes

Article abstract of DOI:10.1002/anie.202102044

Noninvasive imaging strategies have been extensively investigated for in vivo mapping of sentinel lymph nodes (SLNs). However, the current imaging strategies fail to accurately assess tumor metastatic status in SLNs with high sensitivity. Here we report p

Full text of DOI:10.1002/anie.202102044

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Accepted Article
Title: pH-amplified CRET nanoparticles for in vivo imaging of tumor
metastatic lymph nodes
Authors: Zenghui Wang, Heming Xia, Binlong Chen, Yaoqi Wang,
Qingqing yin, Yue Yan, Ye Yang, Mingmei Tang, Jianxiong
Liu, Ruiyang Zhao, Wenzhe Li, Qiang Zhang, and Yiguang
Wang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202102044
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
pH-amplified CRET nanoparticles for in vivo imaging of tumor
metastatic lymph nodes
[
a, b]
[a, b]
[a, b]
[b]
[b]
[b]
Zenghui Wang,
Heming Xia,
Binlong Chen,
Yaoqi Wang,
Qingqing Yin,
Yue Yan,
[
b]
[b]
[b]
[b]
[a]
[a, b]
Ye Yang,
Mingmei Tang,
Jianxiong Liu,
Ruiyang Zhao,
Wenzhe Li,
Qiang Zhang
and
[
a, b]
Yiguang Wang
*
[
[
a]
b]
Z. Wang, H. Xia, Dr. B. Chen, Dr. W Li, Prof. Q. Zhang, Prof. Y. Wang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University
Beijing 100191, China
E-mail: yiguang.wang@pku.edu.cn
Z. Wang, H. Xia, Dr. B. Chen, Y. Wang, Q. Yin, Y. Yan, Y. Yang, M, Tang, J. Liu, R. Zhao, Prof. Q. Zhang, Prof. Y. Wang
Beijing Key Laboratory of Molecular Pharmaceutics, School of Pharmaceutical Sciences, Peking University
Beijing 100191, China
Supporting information for this article is given via a link at the end of the document.
Abstract: Noninvasive imaging strategies have been extensively
investigated for in vivo mapping of sentinel lymph nodes (SLNs).
However, the current imaging strategies fail to accurately assess
tumor metastatic status in SLNs with high sensitivity. Here we report
pH-amplified self-illuminating near-infrared nanoparticles, which
integrate chemiluminescence resonance energy transfer (CRET) and
signal amplification strategy, enabling accurate identification of
metastatic SLNs. After draining into lymph nodes, the nanoparticles
were phagocytosed and dissociated in acidic phagosomes of
inflammatory macrophages to emit near-infrared luminescent light.
Using these nanoparticles, we successfully differentiated tumor
metastatic lymph nodes from benign ones. These nanoparticles also
exhibited excellent imaging capability for early detection of metastatic
It has been demonstrated that the phagocytes reside in SLNs
are activated regardless of tumor type during tumor lymphatic
metastasis.[8] Thus, accurate detection of over-activation signals
of phagocytes may be a potential strategy for the universal and
early diagnosis of tumor metastasis.[9] Myeloperoxidase (MPO)
activity is highly upregulated in inflammatory diseases, such as
cancer and atherosclerosis.[10] Several luminol-mediated
bioluminescence imaging (BLI) of MPO activity have been
reported for the visualization of inflammatory diseases.[11] In the
BLI imaging modality, luminol acts as an MPO-specific probe,
emitting a dazzling blue light (λmax ≈ 425 nm) when exposed to the
MPO, a biomarker of activated phagocytes.[12] To further enhance
the penetration depth of blue luminescence signals, nanoparticle-
based chemiluminescence resonance energy transfer (CRET) or
bioluminescence resonance energy transfer (BRET) imaging
methodologies have been proposed.[13] However, most nano-
systems are designed to be non-responsive, which may cause
SLNs in diverse animal tumor models with small tumor volume (100-
3
2
00 mm ).
signal attenuation due to aggregation-caused quenching
Introduction
[14]
(ACQ).
Herein, we introduced
a Super-pH-Responsive CRET
For cancer patients, the sentinel lymph nodes (SLNs) are
Nanosensors (PCN) for noninvasive identification of tumor
metastatic status in sentinel lymph nodes by integrating CRET
and pH-responsive signal amplification strategy (Scheme 1). In
often the first station of tumor metastasis. Accurate identification
of metastatic SLNs at an early stage, before systematic
metastasis, is of great significance for symptomatic treatment and
prognosis.[1] Clinically, intraoperative lymphangiography and
sentinel lymph node biopsy are often used as the gold standards
for lymphatic metastasis detection.[2] However, the use of
radioactive isotopes, such as technetium-99m, will inevitably
bring up some concerns to cancer patients, although the radiation
dose is relatively low.[3] In addition, intraoperative SLN biopsy is a
this strategy, luminol and a near-infrared (NIR) fluorescent probe
pyropheophorbide a (PPa),[15] were covalently conjugated to pH-
responsive nanoparticles, which have been demonstrated to
[16]
possess excellent pH responsiveness and subcellular targeting
[17]
capability
in our previous works. In the current work, we
hypothesized that the luminol component would precisely report
the activity of MPO of macrophage in metastatic SLNs and emit
blue luminescence (Scheme S2); the PPa component can relay
[
4]
time-consuming procedure.
Recently, several antibody-
fluorophore conjugated probes targeting cancer cell specific
biomarkers have been reported for the visualization of metastatic
SLNs during surgery.[5] However, owing to the high heterogeneity
of tumor-specific biomarkers within and between different cancer
types,[6] antibody-mediated imaging agents can not accurately
identify metastatic lymph nodes in all tumor types. Therefore,
there is an urgent need to develop a noninvasive imaging strategy
with high sensitivity for identification of tumor lymphatic
metastasis.[7]
the blue luminescence to emit NIR light through CRET (λmax ≈ 730
nm);[18] PCN can respond to phagosomal acidity to amplify the
luminescence signals for visualization of metastatic SLNs. A
t
series of PCNs with different transition pH (pH ) were developed
to target distinct phagosome maturation stages upon
phagocytosis by macrophage. Under neutral and alkaline
conditions, PCNs exhibit as stable nanoparticles through
hydrophobic interactions and keep silent owing to ACQ effect of
acceptor molecule (OFF state).[19] Once PCN is drained to the
1
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Scheme 1. The schematic design and work mechanism of pH-amplified self-illuminating PCN for the detection of tumor metastasis in sentinel lymph nodes in living
mice. a) The luminescence signal of PCN is quenched at pH 7.4 due to ACQ effect. As the pH decreases, the nanoparticle is disassembled at pH < pH , and the
t
NIR luminescence signal is recovered through efficient intramolecular CRET. b) Tumor metastasis in sentinel lymph nodes can be detected by pH-amplified
luminescence imaging with high sensitivity and specificity. Tumor metastasis causes inflammation of the sentinel lymph node. After injection in the hind paws, PCN
can effectively drain to the inflamed SLN. Once phagocytosis by activated macrophages, PCN can be disassembled in acidic phagosome and then catalyzed by
overexpressed MPO to emit strong luminescence. CRET = chemiluminescence resonance energy transfer, MPO = myeloperoxidase, ACQ = Aggregation caused
quenching.
SLNs and taken up by activated macrophages, the acidity at
different phagosome maturation stages will immediately trigger
the disassembly of nanoparticles. Subsequently, luminol is
oxidized by MPO-catalyzed hypochlorous acid in phagosome,
thereby emitting NIR light via CRET (ON state) for self-
luminescence imaging of tumor metastasis in lymph nodes with
greatly improved specificity and sensitivity.[20] We reported a pH-
responsive CRET nanoparticle imaging strategy for the first time
to be 6.86, 6.75, 6.33, and 5.26, respectively. Next, PPa as a
CRET acceptor was conjugated to each PR-Lum polymer for the
a
preparation of PCNs. Each PCN with a distinct pK value was
denoted as PCN6.9, PCN6.8, PCN6.3, and PCN5.3, respectively. pH-
nonresponsive CRET nanosensor was abbreviated as NPCN,
which composed of PEH-Lum-PPa polymer.
We then chose PCN6.9 as a representative to evaluate the pH
responsiveness and self-luminescence properties of PCN. Under
sonication, PC7A-Lum-PPa polymer self-assembled into a core-
shell nanoparticle with a diameter of 35.2 ± 2.6 nm and
polydispersity of 0.22 at pH 7.4. At pH 5.4, the PCN6.9 was
dissociated into unimer with a diameter of about 7 nm (Figure 1b).
Transmission electron microscopy (TEM) images also
demonstrated that PCN6.9 had a spherical structure with a
diameter of 33.2 ± 5.9 nm at pH 7.4, and no nanostructure was
observed at pH 5.4 (Figure 1c). For NPCNs, the particle size and
morphology were pH-independent.
and
found
that
phagosome-targeted
self-illuminating
nanoparticles could significantly amplify the luminescence signals
for visualization of metastatic lymph nodes.
Results and Discussion
Preparation and characterization of PCNs with distinct pK
a
We initially synthesized series of luminol-encoded
a
We next explored the MPO-dependent chemiluminescence
properties of PCN in vitro.[24] Using MPO as a trigger, we collected
amphiphilic block copolymers mPEG-b-(PR-r-Lum) (abbreviated
as PR-Lum), including PC7A-Lum, PEPA-Lum, PDPA-Lum, and
PDBA-Lum via atom transfer radical polymerization (ATRP)
method[21]. A pH-nonresponsive PEH-Lum polymer was also
synthesized as a control (Scheme S1, Figure S1-S7, Table S1).[22]
the luminescence spectra of luminol and PC7A-Lum copolymer.
[25]
Results showed that both luminol and PC7A-Lum exhibited a
maximum emission wavelength at 425 nm (Figure 1d), which
proved that the polymerization did not affect the luminescence
properties of luminol. Furthermore, there was a considerable
spectral overlap between the absorption spectrum of PPa and the
luminescence spectrum of PC7A-Lum, enabling efficient
Then, pK
Figure 1a).[23] Based on the titration curves, pK
Lum, PEPA-Lum, PDPA-Lum, and PDBA-Lum were determined
a
of these polymers was measured by titration method
(
a
values for PC7A-
2
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RESEARCH ARTICLE
intramolecular CRET from luminol (donor) to PPa (acceptor).[26]
The absorption spectrum of PCN6.9 exhibited both characteristic
peaks for luminol (358 nm) and PPa (413 and 668 nm) (Figure
an efficiency of 68%), which was very high among that of CRET
nanoparticles reported previously.[29]
After demonstrating PCN can induce luminescence in the
presence of MPO, we then investigated its luminescence
mechanism. The factors affecting luminescence signal were firstly
optimized (Figure S8). Then, 4-aminobenzoic hydrazide (4-
ABAH) and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) were
used to specifically block the functions of MPO and ROS,
respectively.[11] With the increasing concentrations of Tempol or
4-ABAH, the luminescence intensity of PCN was gradually
reduced (Figure S9), proving that the generation of luminescence
were induced by MPO catalyzed oxidation.
1
e). The luminescence spectrum of PCN6.9 was further collected
under optimized conditions (Figure 1f).[25] Different from PC7A-
Lum, which had single luminescence peak (~ 425 nm), PCN6.9
displayed two MPO-triggered luminescence peaks: one peak at ~
4
50 nm from the typical luminescence of luminol, and the other
peak at ~ 730 nm corresponding to the fluorescence emission of
PPa. The luminescence signal of luminol at 450 nm in PCN6.9 was
significantly reduced as compared with that of PC7A-Lum due to
the efficient CRET from luminol to PPa molecules that generated
strong NIR luminescence signals. To maximize the luminescence
efficiency of PCN6.9, the CRET ratio of luminol to PPa in each
polymer chain was systemically investigated. A series of PC7A-
Lum-PPa polymers with different molar ratios of luminol to PPa
were synthesized (Table S1 and S2), and the corresponding self-
luminescence curves were shown in Figure 1g. The slight red-
shift of the PPa peak may be resulted from the J-aggregate
formation in the polymeric micelles with higher PPa contents.[27]
The CRET ratios between luminol and PPa were calculated to
To determine whether the luminescence of PCN could exhibit
a good pH responsiveness, we captured luminescence images of
PCN6.9 at different pH values (Figure 1h). At pH higher than 7.0,
the luminescence signal of PCN6.9 was significantly diminished
duo to ACQ effect of PPa molecules at micelle state. Whereas at
the pH lower than 6.8, the luminescence signal was dramatically
amplified by more than 10-fold, which demonstrated that pH could
precisely regulate the luminescence ON/OFF of PCN. In contrast,
NPCN nanoparticles exhibited undetectable luminescence signal
as a result of pH-independent nanostructure. These results
revealed that the micelle dissociation is a prerequisite for the
amplification of luminescence signals. To further investigate the
[
28]
assess the efficiency of energy transfer. Results showed that
the CRET ratio in PCN6.9 can reach up to 2.12 (corresponding to
Figure 1. pH-responsiveness and self-luminescence properties of PCNs. a) pH titration curves of PC7A-Lum, PEPA-Lum, PDPA-Lum, and PDBA-Lum polymers.
b) Size distribution of PCN6.9 and NPCN nanoparticles in pH 5.4 and pH 7.4. c) TEM images of PCN6.9 (left) and NPCN (right) in pH 5.4 and pH 7.4. Scale bar = 100
nm. d) The normalized absorption spectrum of PPa in methanol (black) and normalized luminescence spectra of luminol (blue) and PC7A-Lum nanoparticles (red)
in PBS. The luminescence spectra were recorded with a spectrophotometer in the presence of 1 U/mL of MPO, 1.25 mM of 4-indophenol, and 50 μM of hydrogen
peroxide in PBS. e) The normalized absorption spectra of PPa (black), luminol (blue), and PCN6.9 (red) in methanol. f) Luminescence spectra of PC7A-PPa
nanoparticle (black), PC7A-Lum nanoparticle (blue), and PCN6.9 (red). g) Normalized luminescence spectra of PCN6.9 with different ratios of luminol to PPa. Peaks
around 425 nm were normalized for comparsion. h) Luminescence images of PCN6.9, NPCN, PC7A-PPa and PC7A-Lum nanoparticles in the presence of MPO, 4-
indophenol, and hydrogen peroxide and fluorescence images of PCN6.9, NPCN and PC7A-PPa nanoparticles. The luminescence sigal of PC7A-Lum is significant
lower than that of PCN6.9. i) luminescence intensity as a function of pH values for PCN, NPCN, and PC7A-PPa nanoparticles (n = 3). j) Normalized luminescence
intensity (N.L.I.) as a function of pH for PCNs. N.L.I. was defined as the value (L-Lmin)/(Lmax-Lmin) (n = 3). k) Maximum luminescence intensity (Lmax) and ON/OFF
L
ratio (R ) for each PCN. All data are represented as mean ± s.d.
3
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RESEARCH ARTICLE
underlying mechanism of pH-responsive luminescence emission
of PCNs, the fluorescence image of PC7A-PPa and luminescence
image of PC7A-Lum were also captured. The results
demonstrated that luminescence quenching of PCN at higher
pHis due to ACQ effect of PPa rather than the quenching of self-
luminescence of luminol. To quantitatively assess the pH
responsive properties of PCN, luminescence intensity vs. pH
curve was plotted (Figure 1i). In the above curve, the pH transition
was determined to be 6.90 with a very sharp responsiveness (0.2
L
pH unit). The ratio of Lmax and Lmin (R = Lmax/Lmin) was also
measured to evaluate the luminescence recovery between ON
and OFF states, where Lmax and Lmin were the maximal and
minimal luminescence intensities among ON and OFF states. R
and Lmax of polymers with different luminol to PPa ratios were
listed in Table S2. To achieve high CRET ratio, R , and Lmax
L
L
,
PCNs with a luminol to PPa ratio of 3: 4.5 was selected for further
investigation, which presented 11.25-fold signal amplification
capability (Figure S10). Furthermore, another NIR fluorescent
t
(pH) was defined as the pH value corresponding to 50% of the
maximum self-luminescence signal is recovered. The pH
t
of PCN
Figure 2. In vitro luminescence imaging of inflammatory macrophages. a, b) subcellular distribution of MPO (a) and PCN (b) in LPS-stimulated Raw 264.7
macrophages. Red color represents the early phagosome, lysosome or PCN signals, green color represents the distribution of MPO, yellow color is an indicator for
the colocalization of red and green signals. Early phagosome and lysosome were labeled with CellLight® Early Endosomes-RFP (BacMam 2.0) or CellLight®
Lysosomes-GFP (BacMam 2.0) reagents, respecively. MPO was labeled with Alexa Fluor 488-labeled antibody. Scale bar = 10 μm. c) Luminescence images and
d) time-dependent luminescence intensity curves of Raw 264.7 cells after incubation with PBS, NPCN or PCN6.9, with LPS or without LPS stimulation (n = 3). e)
Normalized luminescence intensity (N.L.I.) of Raw 264.7 cells at 30 min after incubation with PBS, PC7A, luminol, PC7A-PPa, PC7A-Lum, NPCN, and PCN6.9,
respectively. N.L.I. was defined as the ratio of determined luminescence intensity over PBS group (n = 3). f) Luminescence images and g) corresponding
luminescence intensity of Raw 264.7 cells at predetermined time points after incubation with 250 μg/mL of PCN6.9, PCN6.8, PCN6.3, PCN5.3 or NPCNfor 15 min under
stimulation with 10 μg/mL of LPS (n = 3). h) Real-time fluorescence activation of PCNs. Fluorescence intensity of Raw 264.7 cells was obtained at predetermined
time points after incubation with PCNs for 15 min (n = 3). i) Schematic illustration of pH-responsive CRET signal amplification for PCNs and NPCN in specific
phagosome maturation stages into macrophages. All data are represented as mean ± s.d., *P<0.05, **P<0.01.
4
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probe, Ce6, which has similar spectral properties to PPa, was also
conjugated to PC7A-Lum polymer to show the universality of
CRET nanoparticle design. We also found that the synthesized
PC7A-Lum-Ce6 nanoparticle showed good luminescence
properties (Figure S11).
PCN5.3, respectively, consistent with pK
a
values of corresponding
polymers. Moreover, R and Lmax for each PCN were also obtained
L
and summarized in Figure 1k and Table S3. PCN6.8, PCN6.3, and
PCN5.3 displayed 9.56, 7.80, and 5.18 folds of luminescence
amplification capacity, respectively. As
a
control, pH-
We next evaluated the pH-dependent amplification of
luminescence for other PCNs, including PCN6.8, PCN6.3, and
PCN5.3 (Figure S12). All these nanosensors exhibited good pH-
dependent luminescence ON/OFF pattern. For quantitative
analysis, we plotted the normalized luminescence intensities
nonresponsive NPCN presented the lowest Lmax and negligible
signal enhancement (1.07-fold).
In vitro luminescence imaging of inflammatory macrophages
Macrophages are one of the most important barrier cells in
(
N.L.I.) versus pH profiles, where N.L.I. was calculated as the ratio
(Figure 1j).[30] The pH
values were
calculated to be 6.75, 6.35, and 5.45 for PCN6.8, PCN6.3, and
[
31]
lymph nodes,
which have been demonstrated to be widely
of (L-Lmin)/(Lmax-Lmin
)
t
activated, recruited and proliferated once disease is triggered.[32]
Figure 3. In vivo imaging of metastatic tumor-induced inflammatory sentinel lymph nodes. a) Schematic illustration of establishment of 4T1 popliteal lymph node
POLN) metastasis model and luminescence imaging of inflammatory lymph nodes. b, c) Fluorescence imaging (up) and luminescence imaging (down) of POLNs
(
at tumor and normal sides. The mice were subcutaneously injected with PCN6.9 (b) or NPCN (c) in the hind paws. d) Quantitative analysis of fluorescence and
luminescence intensity in POLNs treated with PCN6.9. Signals on the tumor sides were normalized to normal sides (n = 6). e, f) Full landscape scanning of POLNs
on the tumor sides (e) and normal sides (f) dissected from mice treated with TMR-labelled PCN6.9 nanoparticles. Blue, green, and red signals represent the nuclei,
MPO, and PCN6.9 nanoparticles, respectively. The distribution of macrophages was also analyzed in an adjacent section. The merged image does not contain
macrophage signal. Scale bar = 300 μm. g) Luminescence imaging of POLNs at tumor sides. The mice were subcutaneously injected with PCN6.8, PCN6.3, PCN5.3
,
and NPCN in the hind paws. h) Quantitative analysis of luminescence intensity of POLNs at tumor sides after injection with PCNs. Images were acquired with the
same intensity and exposure time. All data are represented as mean ± s.d., n.s.: no significance, *P<0.05, **P<0.01, ***P<0.001.
5
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For in vitro experiments, we firstly cultured inflammatory
macrophages, which overexpress MPO upon activation.[33]
Weexplored the subcellular distribution of MPO in LPS-induced
inflammatory macrophages.[34] Raw 264.7 macrophages were
transfected with fluorescent protein (GFP or RFP)-fused Rab5a
and Lamp1 for the fluorescently labeling of early phagosome and
lysosome, respectively. Confocal images revealed that MPO was
widely distributed in the phagosome and lysosomes of
macrophages (Figure 2a).[35] PCNs could be efficiently
luminescence signal due to the ACQ effect. Furthermore, an
additional experiment was performed to support our
understanding (Figure S16). Firstly, the luminol consumption was
imaged in real time at dissociation state of PCNs. Results
revealed that the luminol can be consumed within 30 min. Then,
the luminescence of PCN5.3 was carried out in the micelle state at
pH 6.5. As predicted, there was no luminescence emitted. After
30 min, the pH of solution was adjusted to 5.0. However, still no
luminescence signal was detected. Collectively, an efficient
CRET luminescence amplification can be achieved in PCNs with
phagocytosed by activated macrophages, and
a
good
colocalization between PCNs and MPO in these macrophages
was also observed (Figure 2b).
pH
t
ranging from 6.3-6.9, which can be dissociated in phagosome
(pH ~ 6.2) of inflammatory macrophages as shown in the
[
38]
We next explored the luminescence profile of PCN in
macrophages in vitro. As shown in Figure 2c, strong
luminescence signal was detected in LPS-stimulated
proposed mechanism (Figure 2i).
In further experiment, we
selected PCN6.9 for in vivo imaging experiments.
[
36]
macrophages
incubated with PCN6.9. In contrast, almost no
In vivo luminescence imaging of tumor metastatic lymph
luminescence signal was observed in a series of control groups,
including PBS, PC7A, and PC7A-PPa polymers. For PC7A-Lum
group, without the energy transfer to PPa, the short emission
wavelength hampered the acquisition of signals due to poor
transmission of blue light (Figure S13). In particular, since NPCN
could not be disassembled after phagocytosis, no obvious self-
luminescence signal could be detected as a result of the
consumption of luminol in the ACQ state of PPa. For the
macrophages without LPS stimulation, the luminescence signal
was also significantly suppressed in the absence of activated
MPO. Quantitative results indicated that MPO in activated
nodes
Having proved the luminescence imaging potential of PCN6.9
for inflammatory macrophages in vitro, we next investigated its
imaging efficacy for tumor metastatic lymph nodes, which have a
mass of MPO-overexpressed macrophages regardless of tumor
types.[39] A lymphatic metastasis model was established by
subcutaneous inoculation of 4T1 tumor cells into the right flank of
mice (Figure 3a).[29b] Efficient drainage of PCN6.9 to popliteal
lymph nodes (POLN) was verified through intradermal injection of
PCN6.9 in the hind paws of mice (Figure S17).[40] Tumor-bearing
mice were intradermally injected with PCN6.9 in the left and right
hind paws. Fluorescence images exhibited that PCN6.9 can
efficiently delineate the areas of the POLN in both sides (Figure
macrophages caused
a
significant and time-dependent
luminescence signal enhancement with peaking time at
approximately 10-15 min after incubation (Figure 2d). In addition,
PCN exhibited significantly stronger luminescence signal for
about 30 min as compared with other groups, with more than 10
times stronger than that of PBS group at 30 min post-incubation,
providing sufficient time window for in vivo imaging (Figure 2e).
We also cultured 4T1 cells, a mouse breast cancer cell line
without MPO expression, as a negative control (Figure S14). We
only detected a baseline-level signal from the beginning to the end
after incubating with PCN, and the intensity was almost the same
as the non-luminescent PC7A-PPa, although fluorescence
images indicated that PCN can be efficiently taken up by 4T1 cells.
These results demonstrated that PCN can specifically amplify the
pathological MPO signals for the luminescence imaging of
inflammatory macrophages rather than tumor cells.
3
b). However, quantitative results showed no significant
difference between the tumor side and normal side (Figure 3d).
We also performed luminescence imaging of these POLNs.
Surprisingly, luminescence imaging of POLNs displayed a black
and white pattern in both sides (Figure 3b). In tumor side, a
significant dazzling luminescence was detected, which was
colocalized with the fluorescence signals. In contrast, no
luminescence signal was detected in the normal site, even though
preferential accumulation and activation of PCN6.9 in this area.
The luminescence signal for POLN in tumor site was up to 10-fold
higher than that of normal site (Figure 3d). Additionally, pH-
insensitive NPCN failed to luminescently image SLNs regardless
of tumor metastasis or not (Figure 3c). It is noteworthy that PCN6.9
was injected into the hind paw of tumor bearing mice at a luminol
dose of 12 μg per mouse for luminescence imaging of tumor
metastatic lymph nodes, which is a relatively low dose as
compared with other luminol-contained system.[13b, 41]
Since PCNs can be efficiently taken up by macrophages, the
activation of those nanoparticles during phagosome maturation
[
37]
process would be closely related to the signal amplification.
Therefore, we then performed luminescence imaging of PCNs
with different pH in inflammatory macrophages. As shown in
After in vivo imaging, both sides of POLNs were collected and
frozen sectioned. The landscape images of POLNs in the tumor
side indicated a significant increase in MPO expression as
compared with normal side (Figure 3e and 3f). Interestingly, PCNs
t
Figure 2f, PCN6.9, PCN6.8, and PCN6.3 emitted a relatively dazzling
luminescence for about 30 min. Whereas, PCN5.3 only released
negligible luminescence signals within 2 h incubation (Figure 2g).
To explain the above phenomenon, fluorescence activation of
PCNs in macrophages was imaged in real time (Figure S15). As
shown in Figure 2h, it took more than 60 min for PCN5.3 to entirely
activate the fluorescence signal. In comparison, the other three
nanosensors only need about 15-20 min for fully activation. These
results indicated that once PCNs were phagocytosed into
macrophages, MPO in the phagosome will immediately catalyze
the luminescence emission of luminol regardless of PCNs
disassembly or not. Thus, luminol in PCN5.3 was fully consumed
before nanoparticle dissociation, resulting in diminished PPa
[
42]
were mainly distributed at the afferent area of the lymph nodes,
and exhibited a good co-localization with MPO and macrophages.
These results demonstrated that the presence of tumors indeed
induced the inflammation of SLNs.
We next parallelly compared the SLNs imaging efficacy for a
series of PCNs, including PCN6.9, PCN6.8, PCN6.3, PCN5.3, and
NPCN (Figure 3g). In accordance with the luminescence imaging
of inflammatory macrophages in vitro (Figure 2f), the imaging
efficacy of these five PCNs for SLNs was also highly correlated
with pH
t t
. The quantitative results showed that the higher the pH
of PCNs, the stronger the luminescence intensity in SLNs, and
6
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3
thus resulting in more sensitive detection of metastatic lymph
nodes (Figure 3h). These results further demonstrated the
importance of prompt disassmbly of PCN in acidic phagosome for
the amplification of luminescence signals after phagocytosis by
macrophages.
bearing different volumes of 4T1-GFP tumors (50-1000 mm )
were established for luminescence imaging experiments. The
mice were intradermally injected in the right hind paws with PCN6.9
at a luminol dose of 12 μg per mouse. Then, the fluorescence and
luminescence images were simultaneously captured by IVIS
imaging system. The tumor size, fluorescence, and luminescence
images of several representative mice were shown in Figure 4a
and 4b. Strong luminescence signals from lymph nodes were
monitored when the 4T1 tumor volume reached up to 200 mm3.
Furthermore, PCN6.9 exhibited excellent correlation between
luminescence intensity in SLNs and primary tumor volumes. In
contrast, for all the tumor-bearing mice, high fluorescence signals
PCNs profiles lymphatic metastasis in tumor progression
Having proved the ability of PCNs to specifically and
sensitively image metastatic lymph nodes, we then examine
whether the PCNs can report the early lymphatic metastasis
during tumor progression (Figure 4). A series of balb/c mice
Figure 4. PCN identifies lymphatic metastasis status in tumor progression. a) Appearance images of mice bearing different volumes of 4T1-GFP subcutaneous
tumors. The dotted line indicates the area of the tumors. b) Luminescence and fluorescence imaging of the POLNs in Balb/c mice corresponding to a). The mice
were intradermally treated with PCN6.9 in the hind paws. c) Immunofluorescence staining of POLNs dissected from the mice corresponding to a). Blue, green, and
red signals represent the nuclei, metastatic 4T1-GFP tumor cells, and MPO expression, respectively. Scale bar = 100 μm. d) Full landscape scanning of POLNs
3
dissected from mice bearing large 4T1-GFP tumors (~1000 mm ). Blue, green, and red signals represent the nuclei, 4T1-GFP tumor cells, and MPO expression,
respectively. Scale bar = 200 μm. e) and f) Correlation analysis between tumor volume and luminescence intensity of POLNs in PCN6.9 treated e) 4T1 tumor bearing
mice and f) CT26 tumor bearing mice (n = 16). The tumor size was measured with a digital vernier caliper, and the tumor volume was calculated as Volume (mm3)
=
length × width2 × 0.5. Both images were acquired with the same intensity and exposure time.
7
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
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Entry for the Table of Contents
A series of pH-amplified self-illuminating nanoprobes dually respond to acidity and myeloperoxidase of phagosome in inflammatory
macrophages were synthesized. The nanoparticles exhibit excellent imaging capability for identification of metastatic sentinel lymph
nodes in diverse animal tumor models.
1
0
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