An aggregation-induced emission dye-powered afterglow luminogen for tumor imaging

Article abstract of DOI:10.1039/c9sc04901k

Semiconducting polymer (SP)-based afterglow luminogens are showing increasing potential for in vivo imaging because of their long-life luminescence and the associated benefits (e.g., zero-autofluorescence background and high signal-to-noise ratio). Howeve

Full text of DOI:10.1039/c9sc04901k

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An aggregation-induced emission dye-powered
afterglow luminogen for tumor imaging†
Cite this: Chem. Sci., 2020, 11, 419
Yan Xu,‡a Weitao Yang,‡a Defan Yao,b Kexin Bian,a Weiwei Zeng,a Kai Liu,a
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
Dengbin Wangb and Bingbo Zhang
*
Semiconducting polymer (SP)-based afterglow luminogens are showing increasing potential for in vivo
imaging because of their long-life luminescence and the associated benefits (e.g., zero-
autofluorescence background and high signal-to-noise ratio). However, such organic afterglow
luminescence agents are still rare and their application is usually limited by their relatively low afterglow
intensity and short afterglow duration. Herein, we report an aggregation-induced emission (AIE) dye-
powered SP afterglow luminogen by leveraging on the unique characteristics of an AIE dye to
circumvent the concentration-quenching effect, enhance afterglow intensity and prolong afterglow
duration. The underlying working mechanism is investigated by a series of experiments and it is found
that the AIE dye provides sufficient 1O2 to excite SPs and form massive amounts of high-energy
1
intermediates, and then the SP intermediates emit photons that can activate the AIE dye to generate O2
and simultaneously trigger the energy transfer process between the SPs and AIE dye, resulting in a deep-
red emission. It is this closed-loop of “photon–1O2–SP intermediates–photon” that provides the
afterglow emission even after the cessation of the excitation light. The as-prepared luminogen shows
good performance in in vivo tumour imaging. This study demonstrates the advantages of AIE-facilitated
afterglow luminescence and discloses its mechanism, and hopefully it could inspire the development of
other innovative designs for cancer theranostics.
Received 29th September 2019
Accepted 8th November 2019
DOI: 10.1039/c9sc04901k
rsc.li/chemical-science
in the form of photonic emission.13,14 It is clear from the liter-
ature that inorganic aerglow materials are usually synthesized
Introduction
via an annealing reaction method at high temperatures (>800
ꢀC), showing good luminance performance but easily resulting
in agglomeration and particularly biosafety concerns (heavy
metal toxicity, poor biodegradability and difficulty of surface
modication).15–18 In contrast, organic aerglow materials are
considered to be superior to inorganic aerglow materials in
terms of biocompatibility, biodegradability and surface func-
tionalization, which are conducive to bioimaging applica-
tions.19–22 However, compared with inorganic aerglow
materials, organic aerglow ones are relatively under-
developed. Recently, Pu et al. reported a category of semi-
conducting polymers (SPs), namely poly[2-methoxy-5-(2-
ethylhexyloxy)-1,4-phenylenevinylene] (MEHPPV) and its
analogues, showing effective aerglow photons aer removal of
external light irradiation.23,24 Different from the inorganic
aerglow mechanism, the SP-based aerglow is more like
a light-induced chemiluminescence which has been demon-
strated by Pu et al. as follows: the vinyl bonds (C]C) of
MEHPPV are oxidized by 1O2 under light excitation to yield
unstable intermediates (1,2-dioxetane), followed by a radiative
relaxation of the excited species from degraded intermediates to
their ground state accompanied by light emission.23,25,26
Unfortunately, these SPs have poor performance in aerglow
Aerglow is a sustained self-luminescence process aer the
cessation of external light excitation.1,2 Thanks to its zero-
autouorescence background, aerglow imaging can provide
high signal-to-noise ratio images and thus it is attracting
increasing attention in the elds of biosensing, bioimaging and
imaging-guided surgery or therapy.3–6 Nevertheless, this
advanced imaging technique suffers from a severe shortage of
high-quality aerglow materials.7–10
To date, only two kinds of aerglow materials have been
reported for aerglow imaging applications, which are rare-
earth heavy metal ion (e.g., europium, dysprosium and praseo-
dymium) doped inorganic nanoparticles and SP-based organic
aerglow nanoparticles.11,12 The luminescence mechanism of
inorganic aerglow materials can be described as a physical
process of de-trapping stored photo energy in intrinsic defects
aDepartment of Medical Ultrasound, Shanghai Tenth People's Hospital, Tongji
University Cancer Center, Tongji University School of Medicine, Shanghai 200072,
China. E-mail: bingbozhang@tongji.edu.cn
bDepartment of Radiology, Xinhua Hospital, Shanghai Jiao Tong University School of
Medicine, Shanghai 200092, China
† Electronic supplementary information (ESI) available: Additional gures. See
DOI: 10.1039/c9sc04901k
‡ These authors contributed equally to this study.
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emission. Selection of a suitable SP is of great importance to
aerglow imaging. Compared with other reported SPs (such as
MEHPPV and its analogues), poly[(9,9-di(2-ethylhexyl)-9H-uo-
rene-2,7-vinylene)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-
phenylenevinylene)] (PFPV) is considered better as an aerglow
substrate since its highest occupied molecular orbital (HOMO)
is close to the lowest unoccupied molecular orbital (LUMO) of
dioxetane intermediates, and thus the intermolecular electron
transfer between them can be facilitated, improving the
1
quantum yield.27,28 However, the light-induced O2 generation
of PFPV is relatively low which results in unsatisfactory aer-
glow intensity and duration, and the short emission wavelength
(<600 nm) hinders its in vivo imaging applications.29,30 There-
1
fore, increasing the amount of O2 generation and red-shiing
of the emission are urgently needed for bioimaging.
Just recently, Pu and others developed strategies to amplify
and red-shi the aerglow emission by doping photosensitizers
to enhance 1O2 generation and optimize the emission
pattern.31–34 Although a number of exciting results have been
obtained, most of the photosensitizer molecules suffer from
aggregation-caused quenching (ACQ) side-effects, resulting in
limited enhancements in luminescence intensity and 1O2
generation.23,29 Yet, aggregation-induced emission (AIE) dyes
with anti-ACQ characteristics provide an opportunity to
circumvent the concentration-quenching effect, achieving high
emission brightness in the aggregated state.35–39 Moreover, it
has been reported that AIE dyes can also efficiently generate 1O2
under irradiation.40–44 To our knowledge, there are few reports
on AIE dye-facilitated aerglow materials and particularly the
study on their working mechanism is severely lacking. What is
more, the study of these aerglow nanomaterials for in vivo
tumour imaging via intravenous injection in animal models is
somehow rather scarce.45–47 It should be noted that intravenous
injection is one of the most important administration routes in
clinics.
Scheme 1 (a) Illustration of the synthesis of P-TNPs and their in vivo
afterglow imaging application. PFPV: semiconducting polymer, TTMN:
AIE dye, F127: amphiphilic triblock copolymer. (b) The mechanism of
amplified afterglow luminescence of P-TNPs.
experimentally investigated and conrmed by a series of means.
By integrating with an AIE dye, the as-prepared P-TNPs were
found to have stronger aerglow emission and longer lumi-
nescence time than PFPV. Finally, we demonstrated the supe-
rior performance of P-TNPs for aerglow imaging on tumour-
bearing mice via intravenous injection.
Results and discussion
Synthesis and characterization of P-TNPs
In this study, we are thus motivated to develop a novel
aerglow luminogen to address the above mentioned issues for
in vivo tumor imaging. To achieve this purpose, the reported In this work, we synthesized a high 1O2 generation AIE dye
PFPV was used as a light-induced aerglow matrix, and 2-((5-(4- (TTMN) according to Wang's work, with the aim of powering the
(diphenylamino)phenyl)thiophen-2-yl)methylene)malononitrile PFPV for aerglow emission.48,49 Aer purication by silica-gel
(TTMN) was synthesized as a photosensitizer with AIE charac- column chromatography, the structure and the purity of
1
teristics and efficient O2 generation ability to power the aer- TTMN were identied by 1H nuclear magnetic resonance (1H
glow emission of PFPV. These two functional chemicals were NMR) and a liquid chromatograph-mass spectrometer (LC-MS),
conned in a homogeneous nanostructure (P-TNPs) by an which are shown in Fig. S1 and S2.† In order to achieve efficient
amphiphilic triblock copolymer (F127), allowing an efficient energy transfer between the PFPV and the AIE dye (TTMN),
energy transfer process for aerglow emission. PFPV was a nanoprecipitation method was used to co-encapsulate PFPV
selected because of its reported stronger luminescence intensity and TTMN into a conned nanostructure with a commercially
than other SPs and TTMN was selected because its absorption available amphiphilic triblock copolymer F127 (PEG-b-PPG-b-
window (400 nm–600 nm) signicantly overlaps with the PEG) which has good water solubility and biocompatibility.50,51
emission of PFPV (500 nm–700 nm) which is of great impor-
In order to obtain a high content of the PFPV matrix and also
tance to facilitating resonance energy transfer. Aer the cessa- a stable enough solution, the loading capability of PFPV in F127
tion of white LED irradiation (Scheme 1), the aerglow emitted (namely the PNP formation capability) was rst investigated. It
from the decomposition of PFPV-dioxetane intermediates is found that the aerglow brightness and duration of PNPs
1
continuously excites TTMN to generate O2 and red-shis the increase with the loading content of PFPV in the range of 10 mg
aerglow wavelength, in turn, the delayed 1O2 continuously mLꢁ1–150 mg mLꢁ1 (in these cases, no TTMM was used)
oxidizes PFPV and therefore forms PFPV-dioxetane intermedi- (Fig. S3a†). However, the solution becomes turbid (Fig. S3b†)
ates for self-circulating aerglow. This working mechanism was and the particles seem to aggregate (Fig. S3c†) when the loading
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content of PFPV is 150 mg mLꢁ1. Therefore, 100 mg mLꢁ1 of PFPV PFPV component to the AIE dye TTMN. Fig. 1f shows an overlap
was selected for subsequent studies. of the absorption of TNPs (400 nm–600 nm) and the emission of
Aer PFPV and TTMN were co-encapsulated by F127, namely PNPs (500 nm–700 nm), which explains the FRET process in the
P-TNPs, the main physiochemical properties were investigated. P-TNPs. Moreover, no obvious size alteration or UV-vis absorp-
Dynamic light scattering (DLS) measurements indicate that P- tion changes of P-TNPs were observed aer storage (Fig. 1g and
TNPs have an average hydrodynamic diameter of 120 nm h), and the digital photographs in Fig. 1i show no macroscopic
(Fig. 1a), which is slightly larger than that measured by trans- aggregation aer storage in DMEM and 10% FBS solutions for 7
mission electron microscopy (TEM) (Fig. 1b) due to the days, suggesting the excellent stability of P-TNPs under physi-
shrinkage effect during TEM sample observation. The TEM ological conditions.
result reveals that P-TNPs have
a
uniform spherical
morphology. And particularly the contrast of every single
particle indicates a homogeneous loading. Zeta potential
measurements show a negatively charged surface of P-TNPs
with a value of ꢁ14.5 mV (Fig. 1c), favouring the dispersion of
P-TNPs in solution. To verify the co-encapsulation of PFPV and
TTMN in P-TNPs, UV-vis and uorescence spectra of PFPV
nanoparticles (PNPs) and TTMN nanoparticles (TNPs) and P-
TNPs were, respectively, recorded and are shown in Fig. 1d
and e. The maximum absorption of TNPs is located at about
500 nm. Aer co-encapsulating TTMN, a 10 nm-redshi was
found in the UV-vis absorption spectrum of P-TNPs. This
change is not very big because the main composition of P-TNPs
is the semiconducting polymer and not the TTMN. For better
illustrating this difference, we used dotted lines to label the
maximum absorption in Fig. 1d. This absorption pattern
change suggests successful incorporation of PFPV and TTMN
Aerglow property studies of P-TNPs
Above we optimized the loading content of PFPV which acts as
the aerglow matrix. We further investigated the doping
content of TTMN which acts as the aerglow enhancer. For
comparison, different doping contents of TTMN were selected
from 0% to 20% (w/w) with the same PFPV concentration
(0.1 mg mLꢁ1) for preparation of P-TNPs. As shown in Fig. 2a,
aer exposure to white LED lamp (2000 lm) irradiation for
1 min, AIE dye-doped P-TNPs emit much stronger aerglow
luminescence even at a low AIE concentration (5%), compared
with AIE free P-TNPs (0% sample) and the pure H2O as
a control. To be specic, by increasing the doping content of
TTMN, the aerglow intensity and the emission duration of P-
TNPS (5%, 10%, 15% and 20%) signicantly increase and
reach their maximum at the content value of 10% (Fig. 2b).
Different from the previous reports,23,29 the saturated aerglow
into
a whole nanoparticle. Similarly, the uorescence
measurements further verify this co-encapsulation. It should be
worth noting that by doping with the AIE dye, a signicant red-
shi of the emission of PFPV was achieved, thanks to the effi-
cient uorescence resonance energy transfer (FRET) from the
Fig. 2 (a) Afterglow luminescence images and (b) quantitative after-
glow intensity of P-TNPs in H2O (PFPV concentration, 0.1 mg mLꢁ1) at
Fig. 1 (a) Size distribution of P-TNPs in water measured by DLS. (b) different doping contents of TTMN acquired using a CCD camera after
TEM image of P-TNPs. (c) Zeta potential values of PNPs (PFPV nano- irradiation (2000 lm) with a white LED lamp for 1 min (pure H2O as
particles), TNPs (TTMN nanoparticles) and P-TNPs. (d) UV-vis a control). The error bars represent the s.d. (n ¼ 3, *p < 0.05, **p < 0.01,
absorption spectra of PNPs, TNPs and P-TNPs. (e) Fluorescence ***p < 0.001). (c) Quantitative afterglow intensity of PNPs, P-TNPs and
spectra of PNPs, TNPs and P-TNPs. (f) Normalized absorption spec- P-Ce6NPs. The top inset is their respective afterglow luminescence
trum of TNPs and emission spectrum of PNPs. (g) Average hydrody- images. (d) The afterglow intensity and duration of PNPs, TNPs and P-
namic diameters and (h) UV-vis absorption changes of P-TNPs after TNPs. (e) Quantitative afterglow intensity of P-TNPs re-excited with
storage in PBS for 7 days. (i) Digital photographs of P-TNPs after a white LED lamp (2000 lm) for 1 min. The top inset is the respective
storage in H2O, PBS, DMEM and 10% FBS for 7 days.
afterglow luminescence images.
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intensity of P-TNPs (10% sample) doesn't present a sharp
decrease when the doping content further increases to higher
values even at 20% doping amount. This extraordinary aer-
glow emission should be mainly attributed to the unique
features of the AIE effect. To explain this phenomenon more
intuitively, a typical ACQ photosensitizer chlorin e6 (Ce6) was
used to prepare PFPV-based aerglow nanoparticles (P-Ce6NPs)
with the same molar mass of TTMN. As shown in Fig. 2c, the
aerglow intensity of P-TNPs is 1.75 times that of P-Ce6NPs. The
improved doping content of AIE dyes can signicantly enhance
1O2 generation and photon emission, facilitating aerglow
emission. As for the slight decrease of aerglow intensity in the
other two samples with the doping contents of 15% and 20%, it
could be associated with the insufficient energy supply of the
matrix of the PFPV component. Therefore, we selected 10% as
the doping content of TTMN in P-TNPs.
The aerglow intensity and duration of P-TNPs over time
were recorded. As shown in Fig. 2d, neither TNPs nor H2O
exhibits aerglow signals aer light excitation. Compared with
PNPs (without AIE doping) whose aerglow signal almost
disappears 0.5 hours aer the cessation of excitation, P-TNPs
can emit persistent luminescence at least for 1 hour. Particu-
larly, the aerglow intensity of P-TNPs is much higher than that
of PNPs. In order to differentiate the providers of the emitted
photons, we used a 630 nm lter to further record the aerglow
signals. The calculated results in Fig. S4a† indicate that the
aerglow intensity of P-TNPs is 2.02-fold that of PNPs aer
ltration, conrming the occurrence of FRET from PFPV to
TTMN. These data collectively indicate that doping TTMN is
able to efficiently red-shi the emission of PFPV to the red
region and also enhance the aerglow intensity. Another
important property of P-TNPs in our work is the re-excitability;
as shown in Fig. 2e, the aerglow intensity of P-TNPs still
remains above half the initial intensity even aer the 4th re-
excitation, indicating their capability for longitudinal imaging
if necessary. It should be noted that this recharging aerglow
performance is not as good as that of inorganic aerglow
materials, mainly due to the different aerglow mechanism.
Fig. 3 (a) Normalized absorption decay rate of ABDA (20 mM, final
concentration) at 375 nm and (b) normalized fluorescence increase
rate of SOSG (2.4 mM, final concentration) at 528 nm in the presence of
H2O, PNP, TNP and P-TNP solutions upon white light irradiation every
2 min. (c) Fluorescence spectra of SOSG in P-TNP solution and that
added after P-TNP irradiation for 2 min. (d) Afterglow luminescence
images and quantitative afterglow intensity of PNPs and P-TNPs with
pre-added DABCO (250 mM, final concentration) and H2O, respec-
tively, obtained after irradiation for 1 min. (e) Afterglow luminescence
images and quantitative afterglow intensity of PNPs and P-TNPs irra-
diated for 1 min, and then addition of DABCO (250 mM, final
concentration) and H2O, respectively. All the mentioned white light
source is a white LED lamp (2000 lm). The error bars represent the s.d.
(n ¼ 3).
indicator, singlet oxygen sensor green (SOSG),57–59 was further
1
applied to test the generation of O2 by measuring the uores-
cence intensity of SOSG at 528 nm (SOSG was added before
white LED light irradiation). As shown in Fig. 3b and S6,† PNPs
and TNPs exhibit slight 1O2 generation, but P-TNPs show much
higher 1O2 generation ability. This result is consistent with the
results of ABDA measurements. Interestingly, we noticed that P-
TNPs provide more 1O2 than TNPs (the pure AIE dye nano-
1
particles). It can be ascribed to the cumulative effect of O2 in
Aerglow mechanism studies of P-TNPs
the sample of P-TNPs. To be specic, in addition to the direct
1O2 generation from TTMN upon light irradiation, the energy
transfer from the degradation of the formed PFPV-dioxetane
It is reported that the vinyl bonds of SPs can be oxidized by
1
several ROS (e.g., O2, H2O2 and ClOꢁ) for chemiluminescence
or aerglow imaging,23,27–29,52,53 which is also evaluated in this
study and the result is shown in Fig. S4b.† However, there are
few systematic studies reported on disclosing this process. The
results we obtained above together with published literature
1
intermediates to TTMN further promotes O2 generation. This
1
portion of O2 generation is considered delayed since its exci-
tation energy is from the aerglow. And this delayed 1O2 in turn
accelerates the formation of PFPV-dioxetane intermediates
which can be inferred from the absorption decrease rate of
PFPV at ꢂ450 nm by comparing Fig. S5b and d.†
1
point out that the generation of O2 is a crucial factor for SP-
based aerglow luminescence systems.
To prove the delayed 1O2 generation in the sample of P-TNPs,
a solution of P-TNPs was pre-treated with white LED light irra-
diation (2000 lm, 2 min), and aer removing the LED lamp,
SOSG was added. It can be seen that a prominent increase in the
uorescence intensity of SOSG at 528 nm occurs in the P-TNP
sample (Fig. 3c), while no change was observed in the sample
of H2O, or PNPs, or TNPs (Fig. S7†). It should be noted that the
In this study, 9,10-anthracenediyl-bis(methylene) dimalonic
acid (ABDA),54–56 a typical 1O2 indicator, was used to investigate
1
the O2 generation ability of P-TNPs. It shows that the absorp-
tion of ABDA at 375 nm decreases signicantly in the sample of
P-TNPs (Fig. 3a and S5d†), revealing efficient 1O2 generation of
P-TNPs upon white LED light irradiation (2000 lm), while there
is no remarkable decrease in the sample of PNPs or TNPs
1
(Fig. 3a, S5b and c†). On the other hand, a more sensitive O2
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life-time of 1O2 in water is less than 10 microseconds. Thus, the Unfortunately, there is no obvious decrease of the 4T1 cell
SOSG uorescence increase in P-TNPs is not caused by the viability rate. The possible reason may be that the generated 1O2
attenuating 1O2 but the delayed generation of 1O2 in the sample was mainly consumed in the aerglow process of P-TNPs.
1
of P-TNPs even aer removing the LED irradiation.
The generated O2 level during the aerglow process of P-
To further disclose the role of 1O2 during the interaction TNPs at the cellular level was detected with a typical ROS
between PFPV and TTMN, a classic 1O2 quencher 1,4-dia- probe 20,70-dichlorodihydrouorescein diacetate (DCFH-
zabicyclo[2.2.2]octane (DABCO) was used for study.60–62 As DA).63–65 As shown in Fig. 4c, no obvious uorescence is
shown in Fig. 3d, to the solutions of PNPs and P-TNPs DABCO observed in non-treated 4T1 cells (control group) no matter
(250 mM, nal concentration) and an equal volume of H2O were before or aer white LED light irradiation (2000 lm, 3 min). In
pre-added, respectively, and then they were irradiated under contrast, enhanced green uorescence signals are observed in
a white LED light for 1 min, and nally imaged using an IVIS the sample of P-TNP-treated cells which indicates that efficient
system with a bioluminescence mode. The aerglow intensities generation of intracellular ROS is induced aer light irradia-
of both the samples of PNPs and P-TNPs with DABCO added tion, although P-TNPs itself without irradiation can cause weak
decrease sharply and remarkably from the beginning compared green uorescence. Red uorescence of P-TNPs excited at
with those of the samples with H2O added. Particularly, PNPs 550 nm indicates the successful cell endocytosis of the aerglow
were found more sensitive to the DABCO quenching, showing materials. PNP and TNP-induced green uorescence intensity of
rapid aerglow vanishing due to the insufficient 1O2 self- DCF was also, respectively, measured (Fig. S8†). It was found
generation in the sample of PNPs. In contrast, the aerglow that it was hard for PNPs to induce green uorescence. But for
intensity of P-TNPs which possess sufficient 1O2 supply declines TNPs, because of the photodynamic effect of the AIE compound
1
slightly slowly aer light irradiation, further indicating the role TTMN, they can generate O2 to induce green uorescence of
1
of delayed O2 in enhancing aerglow intensity and duration.
DCF, which is in accordance with the results of SOSG tests. The
Interestingly, we just adjusted the addition time of DABCO quantitative results of green uorescence intensity were calcu-
and found different aerglow results. Specically, the DABCO lated by ow cytometry. As shown in Fig. 4d, the green uo-
added later was found to show no obvious effect on quenching rescence intensity of P-TNPs increases from 67.1% to 96.2%,
aerglow emission in the sample of PNPs (Fig. 3e), while it whereas almost no increase of the green uorescence intensity
exerts strong quenching in the sample of P-TNPs. This further is observed in the no-treatment group (control group). The
1
conrms the delayed generation of 1O2 and its crucial role results indicate the O2 generation feasibility of P-TNPs at the
during the aerglow process of P-TNPs.
intracellular level. The intracellular environment is different,
aer all, from that in water solution.
1
Cytotoxicity and O2 detection at the cellular level
Before in vivo imaging, the cytotoxicity of the as-prepared P-
Tissue penetration and bio-distribution
TNPs (concentration range from 50 mg mLꢁ1 to 500 mg mLꢁ1
)
The tissue penetration depth of aerglow generated by P-TNPs
was assessed using pork slabs of different thicknesses accord-
ing to the published protocols.1,66,67 200 mL of P-TNPs (0.1 mg
mLꢁ1) was placed in a 96-well plate along with PNPs with the
same PFPV concentration which were used as a control. Aer
irradiation for 2 min using a xenon lamp (2 W cmꢁ2) xed 10 cm
above the pork slab, the plate was transferred to an IVIS system
to collect the aerglow signals. As shown in Fig. 5a, P-TNPs
show much better tissue penetration ability than PNPs. To be
specic, the aerglow luminescence of P-TNPs covered with a 5
mm–6 mm thick pork slab is still distinct while the aerglow
signals of PNPs can hardly be observed even at the 1st signal
collection, let alone at the 2nd time collection. As expected, the
aerglow signals of P-TNPs can still be seen 4 min post ceasing
irradiation thanks to the red AIE dye doping. This experiment
further shows that the AIE dye TTMN powers PFPV to produce
strong aerglow signals, red-shied emission and also pro-
longed aerglow time.
was evaluated on 4T1 cells and DC2.4 cells by standard cell
counting kit-8 (CCK-8) assays. As shown in Fig. 4a and b, no
obvious cytotoxicity of P-TNPs is observed aer 24 h and 48 h of
incubation. The cell viability remains above 75% even at
a concentration of 500 mg mLꢁ1 aer incubation for 48 h. We
also tested the cytotoxicity of P-TNPs under light irradiation.
Before the in vivo tumour aerglow imaging study, the bio-
distribution of P-TNPs was investigated on 4T1 tumour-
bearing BALB/c nude mice. The uorescence images of tissues
including the liver, heart, spleen, lung, kidney and tumour were
captured in the IVIS system separately 6 h, 12 h and 24 h aer
intravenous injection of P-TNP solution (Fig. 5b). A bright
region is found at the liver site 6 h aer injection due to the
Fig. 4 The cytotoxicity of P-TNPs against (a) 4T1 cells and (b) DC2.4
cells after incubation for 24 h or 48 h. (c) Intracellular ROS generation
of P-TNPs against 4T1 cells upon white light irradiation (2000 lm) for
3 min. Emission: BF (bright field), green from DCF, red from the AIE dye
(TTMN). Scale bar: 20 mm. (d) Flow cytometry analysis of DCF fluo-
rescence intensity in the corresponding groups.
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Fig. 5 (a) Afterglow luminescence images of PNPs and P-TNPs (200
mL, 1 mg mLꢁ1 PFPV concentration) placed in a 96-well plate covered
with pork slabs of different thicknesses after irradiation using a xenon
lamp (2 W cmꢁ2, 2 min). Scale bar: 0.8 cm. (b) Fluorescence images
and (c) quantitative fluorescence intensity of different organs from
sacrificed 4T1 tumor-bearing mice at corresponding periods after
Fig. 6 (a) Fluorescence images (ex: 475 nm, em: 630 nm) and after-
glow images (pre-irradiated using a xenon lamp for 2 min, 2 W cmꢁ2
)
intravenous injection of P-TNPs (200 mL,
concentration).
1
mg mLꢁ1 PFPV
of 4T1 tumor-bearing mice at corresponding periods after intravenous
injection of PNPs and P-TNPs (200 mL, 1 mg mLꢁ1 PFPV concentra-
tion). (b) Quantitative fluorescence and afterglow intensity of the
mentioned groups. The error bars represent the s.d. (n ¼ 3). (c)
Afterglow and the afterglow decay images of 4T1 tumor-bearing mice
acquired after the first and second excitation in the mentioned groups.
uptake of nanomaterials in the hepatic reticuloendothelial
system. The uorescence intensity of the tumour reaches the
highest level at 12 h post injection which might be ascribed to
the enhanced permeability and retention effect (EPR), whereas
the uorescence intensity of the liver gradually decreases
because of its metabolic function. Quantitative analysis of the
mean uorescence intensity (Fig. 5c) in collected tissues reveals
that the accumulation of P-TNPs in the tumour reaches the apex
at 12 h post injection which is about 1.59-fold that at 6 h post
injection, while the signal of the liver at 24 h reduces to ꢂ0.53-
fold that at 6 h post injection. The bio-distribution data suggest
that P-TNPs can target the tumour and aer that undergo
hepatic clearance from the body.
TNPs at 24 h post injection is higher than that at 12 h post
injection, which is different from that of PNPs.
In contrast, the aerglow imaging modality possesses much
lower tissue background noise than the uorescence imaging
modality. As shown in Fig. 6a, the aerglow signals are much
easier to detect at the tumour site aer xenon lamp irradiation
(2 W cmꢁ2, 2 min). The most intense signal appears at 12 h post
intravenous injection, which is consistent with the results of the
bio-distribution test. Moreover, the mean aerglow intensity of
the tumour site in the P-TNP group is 2.75-fold, 2.89-fold and
2.67-fold, respectively, higher than that of the PNP group aer
quantitative calculation (Fig. 6b). We can see that both the
uorescence and aerglow signals of P-TNPs at tumour sites are
much higher than those of PNPs.
It is important to note that the aerglow of P-TNPs can be re-
excited in vivo. As shown in Fig. 6c, the re-excited aerglow
signals of P-TNPs are still strong enough to detect. These data
suggest the superior performance of P-TNP mediated aerglow
imaging over uorescence imaging, and the excellent re-
excitable property of P-TNPs makes them more suitable for
tumour diagnosis.
In vivo aerglow imaging on tumour-bearing mice
Aer verifying the cytocompatibility and bio-distribution of P-
TNPs, the aerglow performance of P-TNPs was evaluated on
tumour-bearing mice. 4T1 tumour-bearing nude mice were
divided into three groups corresponding to three time points of
6 h, 12 h and 24 h post intravenous injection. The PNP (without
AIE doping) group was used as a control to the P-TNP group. It
can be seen from the uorescence modality that the uorescent
signals are obvious at the tumour site (Fig. 6a), and the signal
intensities of the P-TNP group are 2.87-fold, 1.60-fold and 3.45-
fold, respectively, higher than those of the PNP group at three
time points (Fig. 6b) due to the FRET, resulting in the
In vivo toxicology analysis
enhancement of TTMN red uorescence emission. Although Blood routine analysis and serum biochemical analysis were
the tumour sites can be visualized by uorescence imaging aer carried out aer the intravenous injection of P-TNPs on the 1st,
accumulation of P-TNPs or PNPs in tumour lesions, the tumour 7th and 14th day post injection. As shown in Fig. 7, seven
boundary is difficult to identify on account of the interference markers of blood routine including red blood cells (RBCs),
from the mouse body-induced autouorescence. Thus it can hemoglobin (HGB), hematocrit (HCT), platelets (PLT), mean
cause a phenomenon in which the uorescence intensity of P- corpuscular hemoglobin (MCH), mean corpuscular volume
424 | Chem. Sci., 2020, 11, 419–428
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Conclusions
In summary, an AIE dye-powered organic aerglow luminogen
(P-TNPs) is reported and it shows superior aerglow perfor-
mance to the current SP-based aerglow materials and ACQ
photosensitizer-amplied aerglow materials. The resultant P-
TNPs possess much stronger aerglow emission and a pro-
longed aerglow time compared with those of PNPs. Particu-
larly, the underlying aerglow mechanism especially the
delayed generation of 1O2 is clearly demonstrated through
various experiments, which is not achieved by the previous
reports. We found that the delayed 1O2 promotes the self-excited
aerglow effect and thus prolongs the aerglow time and
intensity. Thanks to the improved aerglow properties, P-TNPs
have shown good performance in in vivo tumour imaging.
Overall, this work proposes an effective approach to improve the
aerglow performance of SP materials and circumvent the
issues of ACQ dye-doped aerglow materials. The disclosed
mechanism we believe can inspire the development of other
effective AIE-facilitated aerglow materials for tumour imaging
and even therapy.
Fig. 7 Blood routine and blood biochemical tests on mice after
intravenous injection of P-TNPs (200 mL, 1 mg mLꢁ1 PFPV concen-
tration) or saline as the control: (a) red blood cells, (b) white blood cells,
(c) hemoglobin, (d) hematocrit, (e) platelets, (f) mean corpuscular
hemoglobin, (g) mean corpuscular volume, (h) mean corpuscular
hemoglobin concentration, (i) aminotransferase, (j) alanine amino-
transferase, (k) alkaline phosphatase, (l) alanine albumin, (m) globulin,
(n) total protein, (o) ratio of albumin to globulin, and (p) urea.
Experimental
Materials
(MCV) and mean corpuscular hemoglobin concentration
(MCHC) in the P-TNP treated group remain within the normal
ranges, and there is no signicant difference from those of the
control group. For serum biochemical analysis, renal function
markers (such as urea) and hepatic function markers including
aspartate aminotransferase (AST), alanine aminotransferase
(ALT), alkaline phosphatase (ALP), alanine albumin (ALB) and
total protein show no signicant difference between the treated
group and the control group.
Hematoxylin and eosin (H&E) staining was further carried
out to evaluate potential tissue lesion or inammation. The
results presented in Fig. 8 reveal that liver cell edema occurs in
the treated group on day 7, but recovers to the normal state on
day 14 and no obvious abnormalities are found in other organs.
These results suggest low toxicity of P-TNPs.
4-Bromotriphenylamine, 4-formylthiophene-2-boronic acid,
malononitrile, 9,10-anthracenediyl-bis(methylene) dima-
lonic acid (ABDA), 1,4-diazabicyclo[2.2.2]octane (DABCO) and
F127 were purchased from Aladdin Industrial Inc. Poly[(9,9-
di(2-ethylhexyl)-9H-uorene-2,7-vinylene)-co-(1-methoxy-4-(2-
ethylhexyloxy)-2,5-phenylenevinylene)] (PFPV) was obtained
from Sigma-Aldrich. Singlet oxygen sensor green (SOSG),
20,70-dichlorodihydrouorescein diacetate (DCFH-DA) and
cell counting kit-8 (CCK-8) were purchased from Beyotime
Biotechnology. Analytical-grade reagents, including toluene,
methanol, ethanol, tetrahydrofuran (THF) and anhydrous
K2CO3, were provided by Sinopharm Chemical Reagent
Company. Deionized (DI) water (18.2 MU cm) was used
throughout the experiments. All other chemicals, if not
mentioned, were used without further purication.
Synthesis of TTMN
4-Bromotriphenylamine (0.97 g, 3 mmol), 4-formylthiophene-2-
boronic acid (0.70 g, 4.5 mmol) and anhydrous K2CO3 (2.07 g,
15 mmol) were added to a mixture of toluene (15 mL) and
methanol (15 mL), purged with nitrogen and stirred for 2 min,
and then PdCl2(dppf) (0.07 g, 0.01 mmol) was added and purged
with nitrogen for another two times. The whole mixture was
stirred at 75 ꢀC for 12 h. Aer cooling down to room temperature,
the mixture was evaporated, then dissolved in dichloromethane
and washed with brine and water, subsequently dried with
anhydrous Na2SO4, and further puried by silica-gel column
chromatography using petroleum ether and ethyl acetate (from
40 : 1 to 10 : 1) to yield a yellow solid, 0.58 g. The obtained solid
(150 mg, 0.42 mmol) and malononitrile (36.24 mg, 0.55 mmol)
were added to ethanol (10 mL) and stirred at 78 ꢀC for 48 h. Aer
Fig. 8 H&E staining images of major organs (heart, liver, spleen, lung
and kidney) of the mice collected from control, 1 day, 7 days and 14
days after intravenous injection of P-TNPs (200 mL, 1 mg mLꢁ1 PFPV
concentration). Scale bar: 50 mm.
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cooling down to room temperature, the mixture was evaporated interval. For comparison, the concentrations of PNPs and TNPs
and puried by silica-gel column chromatography using petro- were prepared the same as P-TNPs (PFPV 0.1 mg mLꢁ1, TTMN
leum ether and ethyl acetate (from 30 : 1 to 5 : 1) to yield a red 0.01 mg mLꢁ1). The absorption of ABDA at 375 nm was
solid, TTMN (140 mg, 82%).
normalized to calculate the decomposition rate of ABDA. For
another indicator, SOSG, the measurement was conducted in
a similar way. 100 mL of SOSG (50 mM) was added to 2 mL of P-
TNP solution, and irradiated as the previous method. Then, the
uorescence intensity of SOSG (excitation at 504 nm, emission
at 525 nm) was normalized to calculate the increase rate.
Another way is to only set the addition time of SOSG aer the
irradiation. To demonstrate the 1O2 generation in another way,
a classic 1O2 quencher DABCO was employed. One method is to
add 20 mL of DABCO (2.5 M) or H2O to PNPs and P-TNPs (180 mL)
in 96-well plates, respectively. The prepared samples were irra-
diated with a white LED lamp (2000 lm) for 1 min, and then the
aerglow signals were collected using an IVIS spectrum
imaging system as before. The other method is to only set the
addition time of DABCO aer the irradiation.
Synthesis of P-TNPs
PFPV (1 mg), TTMN (from 0 to 200 mg) and F127 (20 mg) were
dissolved in 1 mL of THF to form a clear solution, and then
rapidly injected into DI water (9 mL) under sonication for 3 min
with a sonicator (Scientz, JY92-2D). Aer that, the mixture was
stirred in the dark for 12 h at room temperature to evaporate
THF. The resulting solution was ltered with a syringe-lter
(0.22 mm) and concentrated by ultraltration with DI water or
PBS under centrifugation at 6000 rpm for 20 min. PFPV-Ce6 NPs
were prepared by the same method, except that the addition
amount of Ce6 was according to the molar mass of corre-
sponding TTMN. All the prepared nanoparticles were stored in
ꢀ
the dark at 4 C.
Cell experiments
Characterization
The 4T1 murine breast cancer cells and the DC2.4 dendritic
cells were employed to evaluate the cytotoxicity of P-TNPs. 4T1
cells were cultured in DMEM and DC2.4 were cultured in RMPI
1640 at 37 ꢀC under a humidied 5% CO2 atmosphere, both
containing 10% fetal bovine serum and 1% penicillin–strepto-
mycin. For cytotoxicity experiments, 4T1 cells and DC2.4 cells
were seeded in 96-well plates at a density of 1 ꢃ 104 cells per
well and cultured for 24 h to allow cell adherence. The medium
was then replaced by 200 mL of fresh medium containing P-
TNPs at various concentrations (0–500 mg mLꢁ1, based on
freeze-dried weight) for another 24 h and 48 h of incubation.
The medium in each well was replaced by fresh medium with
the CCK-8 agent and further incubated for 4 h. Finally, the
absorption at 450 nm was recorded on a microplate reader
(Thermo, Mutilskan MK3). Due to the absorption of P-TNPs at
440 nm, the absorption of the medium containing each
1H NMR spectra of TTMN were recorded on a Bruker Avarice TM
NMR spectrometer, and the corresponding purity and molec-
ular weight were measured using an LC-MS (Waters, UPLC H-
CLASS-SQD). An FEI Tecnai G2 F20 instrument was used to
obtain TEM images at a voltage of 200 kV. Hydrodynamic size
distribution and z-potential were measured by DLS (Malvern
Zetasizer Nano ZS). The UV-vis absorption spectra and uores-
cence spectra were, respectively, recorded on Cary 60 (Agilent)
and Cary Eclipse (Agilent) instruments. Biological uorescence
images were collected on
a
uorescence microscope
(OLYMPUS, IX73). Aerglow images and in vivo uorescence
images were obtained by using an IVIS spectrum imaging
system (Berthold, NightOWL LB 983).
Aerglow luminescence measurement
Aerglow signals were collected using an IVIS spectrum concentration of P-TNPs was also recorded.
imaging system (6 ꢃ 6 binning, exposure time 60 s) under
To evaluate the intracellular 1O2 generation of P-TNPs,
bioluminescence mode. Typically, 200 mL of P-TNPs (0.1 mg DCFH-DA was employed as an indicator. 4T1 cells were
mLꢁ1, based on the concentration of PFPV) was placed in seeded in a 35 mm cell well with a density of 4 ꢃ 105 cells per
Eppendorf (EP) tubes or 96-well plates, and then pre- well and cultured for 24 h to allow cell adherence. The medium
illuminated using a white LED lamp (2000 lm) for 1 min in was then replaced by 1 mL of fresh medium containing DCFH-
a xed position. The aerglow images were acquired with an DA (10 mM) for 0.5 h of incubation. Then, the cells were washed
open lter (full-wave reception) or a 630 nm emission lter and with DMEM without fetal bovine serum at least three times and
the decay of the aerglow was collected by continuous opera- incubated with 1 mL of fresh medium containing P-TNPs (200
tion of the imaging soware without any movement of the mg mLꢁ1, based on freeze-dried weight) for 2 h. Aer that, the
samples. The data of aerglow images were analyzed by using cells were washed with PBS three times and irradiated using
the IndiGo soware.
a white LED lamp (2000 lm) for 3 min. Finally, the cells were
observed using a uorescence microscope and analyzed by ow
cytometry (Invitrogen/A24684).
1
In vitro evaluation of O2 generation
1
The O2 generation of H2O, PNPs, TNPs and P-TNPs was eval-
uated using ABDA and SOSG as indicators, respectively. In brief,
In vitro tissue-penetration study
40 mL of ABDA (1 mM) was added to 2 mL of PFPV-TTMN NP Typically, 200 mL of PNPs and P-TNPs (PFPV concentration,
solution, and the original absorption spectrum was recorded. 0.1 mg mLꢁ1) were placed in a 96-well plate and covered with
The mixture was irradiated with a white LED lamp (2000 lm) pork slabs of different thicknesses. Subsequently, the covered
every 2 min and the absorption spectra were measured at every samples were irradiated using a xenon lamp (BETICAL, HDL-II,
426 | Chem. Sci., 2020, 11, 419–428
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2 W cmꢁ2) for 2 min. The aerglow images were acquired for 81801823, 81571742, 81871399, 81922035), and Shanghai
60 s with an open lter and ꢂ630 nm lter, respectively.
Science and Technology Biomedical Innovation Funds (Grant
No. 19441904200).
Animal model and in vivo imaging
Notes and references
All animal procedures were performed in accordance with the
Guidelines for Care and Use of Laboratory Animals published in
GB/T 35892-2018 and the experiments were approved by the
Animal Ethics Committee of Tongji University. For tumour
model establishment, 4T1 cells (1–2 ꢃ 106 cells per mouse) sus-
pended in 100 mL PBS were subcutaneously injected into the right
thigh of female BLAB/c nude mice (6 weeks old, body weight ca.
20 g). For the tissue bio-distribution study, the mice were sacri-
ced at the 6 h, 12 h and 24 h time points aer the intravenous
injection of P-TNPs (200 mL, 1 mg mLꢁ1 based on PFPV concen-
tration). Major organs including the liver, heart, spleen, lung,
kidney and tumour were collected and washed with PBS quickly.
The uorescence images of organs were acquired for 0.1 s at
630 nm with the excitation at 475 nm. The uorescence intensity
of each organ was analyzed by using the IndiGo soware.
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