Semiconducting Polymer Dots with Dual-Enhanced NIR-IIa Fluorescence for Through-Skull Mouse-Brain Imaging

Article abstract of DOI:10.1002/anie.201914397

Fluorescence probes in the NIR-IIa region show drastically improved imaging owing to the reduced photon scattering and autofluorescence in biological tissues. Now, NIR-IIa polymer dots (Pdots) are developed with a dual fluorescence enhancement mechanism.

Full text of DOI:10.1002/anie.201914397

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
International Edition Chemie
www.angewandte.org
Accepted Article
Title: Semiconducting Polymer Dots with Dually Enhanced NIR-IIa
Fluorescence for Through-Skull Mouse Brain Imaging
Authors: Zhe Zhang, Xiaofeng Fang, Zhihe Liu, Haichao Liu, Dandan
Chen, Shuqing He, Jie Zheng, Bing Yang, Weiping Qin,
Xuanjun Zhang, and Changfeng Wu
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201914397
Angew. Chem. 10.1002/ange.201914397
Link to VoR: http://dx.doi.org/10.1002/anie.201914397
http://dx.doi.org/10.1002/ange.201914397

Angewandte Chemie International Edition
10.1002/anie.201914397
RESEARCH ARTICLE
Semiconducting Polymer Dots with Dually Enhanced NIR-IIa
Fluorescence for Through-Skull Mouse Brain Imaging
[
a, b]
[a]
[a]
[b]
[a]
[a]
Zhe Zhang,
Xiaofeng Fang, Zhihe Liu, Haichao Liu, Dandan Chen, Shuqing He, Jie
[b]
[b]
[b]
[c]
[a]
Zheng, Bing Yang, Weiping Qin, Xuanjun Zhang, Changfeng Wu*
Abstract: Fluorescence probes in NIR-IIa region (1300–1400 nm)
show drastically improved imaging owing to the reduced photon
scattering and autofluorescence in biological tissues. However,
organic fluorophores in this region are largely unexplored. Here, we
develop NIR-IIa polymer dots (Pdots) by a dual fluorescence
enhancement mechanism. First, we used the aggregation induced
emission of phenothiazine to reduce the nonradiative decay pathways
of the polymers in condensed states. Second, we minimized the
fluorescence quenching by different levels of steric hindrance to
further boost the fluorescence. The resulting Pdots displayed a
fluorescence QY of ~1.7% in aqueous solution, suggesting an
enhancement of ~21 times in comparison with the original polymer in
tetrahydrofuran (THF) solution. Small animal imaging by using the
NIR-IIa Pdots exhibited a remarkable improvement in penetration
depth and signal to background ratio, as confirmed by through-skull
and through-scalp fluorescent imaging of the cerebral vasculature of
live mice. This study indicates the promising potential of NIR-IIa Pdots
for in vivo fluorescence imaging.
instance, Dai and coworkers developed a series of NIR-II
fluorescent probes and demonstrated the superior performance
of in vivo NIR-II imaging.[5] Cheng et al. synthesized
a
diketopyrrolopyrrole polymer exhibiting NIR-II fluorescence, and
the corresponding nanoparticles were capable of assisting
accurate image-guided tumor surgery.[6] Among those, the NIR-
IIa (1300–1400 nm) window is particularly attractive because the
reduced scattering and avoidance of water absorption above
1400 nm allow for through-skull fluorescence imaging of cerebral
vasculature with unprecedented levels of penetration and
resolution.[7] However, organic fluorophores in this wavelength
region have been scarcely explored so far.
Semiconducting polymer dots (Pdots) have demonstrated
practical utilities in biological imaging and biosensing owing to
their
tunable
optical
properties.[1a;1d;8]
However,
the
semiconducting polymers in nanoparticle forms typically exhibit
fluorescence quenching in comparison with the original polymers
in organic solvents. The quenching can be attributed to intense
inter- and intra-chain π-π stacking interactions, often resulting in
the formation of non-emissive excimers and exciplexes.[9] This
phenomenon is known as the aggregation-caused quenching
Introduction
Fluorescent probes play a versatile role in biological imaging
and various bioassays. The development of in vivo fluorescence
imaging has shown significant potential for a wide variety of
molecular diagnostics and therapeutic applications.[1] However,
the majority of organic fluorophores show emissions in the visible
region or the first near-infrared window (NIR-I, 700–900 nm),
where the imaging suffers from substantial photon scattering and
autofluorescence in biological tissues.[2] Recent research efforts
reveal a promising imaging window in the second near-infrared
wavelength range (NIR-II, 1000–1700 nm), resulting in large
penetration depth and high signal-to-noise ratio (SNR) for in vivo
fluorescence imaging.[3] Accordingly, NIR-II fluorescent probes
have attracted considerable attention in the past few years.[4] For
(ACQ) effect. In the visible wavelength region, fluorescence
quenching in Pdots is moderate and can be comprised because
they possess extremely large absorption coefficients and per-
particle brightness is determined by the product of absorption
cross section and fluorescence quantum yield (QY). Therefore,
Pdots exhibit high brightness that is valuable for fluorescence
imaging and biosensing applications.[10] In many cases, the large
absorption cross sections of Pdots are also favorable for
photoacoustic imaging and photothermal therapy.[11] However,
semiconducting polymers are nearly non-fluorescent in the NIR-II
region. The energy gap law predicts that the vibrational overlap
between the ground and excited states largely determines the
nonradiative decay rate.[12] The nonradiative decay rate increases
significantly with a decrease in the emission energy, making it
difficult to obtain NIR-II fluorophores with a high QY. Energy
migration in the large π-conjugated systems of semiconducting
polymers may also lead to energy funneling to the non-emissive
species, amplifying the fluorescence quenching effect. Therefore,
the development of semiconducting polymers with strong
fluorescence in the NIR-II region remains challenging.
[
a]
Dr. Z. Zhang, Dr. X. Fang, Dr. Z. Liu, Dr. D. Chen, Prof. S. He,
Prof. C. Wu
Department of Biomedical Engineering, Southern University of
Science and Technology, Shenzhen, Guangdong 518055 (China)
E-mail: wucf@sustech.edu.cn
[
b]
c]
Dr. H. Liu, Prof. J. Zheng, Prof. B. Yang, Prof. W. Qin
State Key Laboratory of Integrated Optoelectronics, College of
Electronic Science and Engineering, Jilin University, Changchun,
Jilin 130012 (China)
Aggregation-induced emission (AIE) is a phenomenon wherein
the fluorophore shows an emission boost when it transforms from
an isolated molecule state to aggregated state.[13] Mechanistic
[
Prof. X. Zhang
Faculty of Health Science, University of Macau, Taipa, Macau SAR
studies have suggested that, for an isolated AIE molecule in the
999078 (China)
excited state, energy is consumed by the free intramolecular
motion, thereby quenching the emission.[14] Such a nonradiative
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201914397
RESEARCH ARTICLE
transition would be prevented in the aggregated state, wherein
the free rotations are inhibited.[9;15] Based on this mechanism, AIE
molecules can be constructed by incorporating typical AIEgens,
conjugated polymers.[20] Third, phenothiazine also possesses
multiple reaction sites and excellent chemical reactivity, which
facilitate the synthesis of the polymers and modification of the
side-chain groups. More importantly, recent studies indicate that
phenothiazine based small molecules show an apparent AIE
feature.[21] The heterocyclic ring in phenothiazine presents a
such
as
tetraphenylethene,
hexaphenylsilole,
and
distyreneanthracene, into the molecular structure.[16] The strong
fluorescence intensity in the aggregated state and the variety of
potential synthetic routes make AIE molecules one of the most
important classes of fluorophores. The AIE fluorophores in
nanoparticles provide promising platforms for fluorescence
imaging.[1a] Several reports have demonstrated the development
of AIE nanoparticles for NIR-II imaging applications.[17] Tang and
coworkers developed short-wave infrared AIE dots, denoting real-
time high-resolution imaging in live mice.[18] However, fluorescent
probes in the NIR-IIa window are still highly preferable owing to
the superior imaging quality in this region.
3
dihedral angle of 153° because of the sp -hybridized sulfur and
nitrogen atoms.[22] This geometric configuration guarantees the
flexibility of the polymer, which implies that phenothiazine might
induce AIE for the resulting polymers in aggregation state.
Therefore, we attempt to use phenothiazine as the donor unit in
the polymer synthesis to enhance the NIR-II fluorescence.
Furthermore, benzothiazole and its derivatives (benzothiazole,
BT;
naphtho[2,3-c][1,2,5]thiadiazole,
NT;
and
benzobisthiadiazole, BBTD) with different electron affinities were
employed as acceptors to endow the polymers with highly shifted
fluorescence wavelengths. Particularly, we anticipate to drive the
fluorescence emission to the NIR-IIa window by using the
extremely strong acceptor BBTD.
Herein, we describe a molecular engineering strategy towards
the development of semiconducting polymers in the NIR-II region.
We designed nine polymers to propose a dual fluorescence
enhancement mechanism to boost the NIR-II fluorescence of
Pdots. On one hand, we use the AIE character of phenothiazine
unit to reduce the nonradiative decay pathways of the polymer in
aggregated states. On the other hand, we introduce bulky side-
chain groups to weaken the intense inter- and intra-chain π–π
stacking interactions via steric hindrance, further enhancing the
fluorescence QY. Based on this dual enhancement strategy, the
resulting NIR-II Pdots displayed a fluorescence QY of ~1.7% in
aqueous solution, suggesting an enhancement of approximately
The large π-conjugated backbones of semiconducting
polymers tend to have strong intermolecular interactions, inducing
the formation of non-emissive excimers and exciplexes (ACQ
phenomenon).[9;23] It is worth noting that AIE and ACQ can exist
simultaneously in one polymer owing to their different
mechanisms. The intense intermolecular interactions may still
attenuate the fluorescence in the aggregated state, even in AIE
molecules. Therefore, the fluorescence intensity of AIE Pdots
could be further improved by suppressing the ACQ effect in the
semiconducting polymers. Toward this end, we introduce side
groups on the nitrogen atoms of phenothiazine via nucleophilic
substitution. The steric hindrance generated by the bulky side
groups weakens the inter- and intra-chain interactions in the
aggregated state, which is referred to as anti-ACQ. To examine
the effectiveness of anti-ACQ, we choose three side groups that
provided different levels of steric hindrance in polymer synthesis.
Specifically, nine polymers were designed, namely P1a, P1b, P1c,
P2a, P2b, P2c, P3a, P3b, and P3c, as shown in Figure S1. In the
naming convention, the number 1, 2, or 3 distinguishes the main
chain structures, whereas the letter a, b, or c represents the side
chain groups.
21
times in comparison with the original polymer in
tetrahydrofuran (THF) solution. One of the resulting Pdots exhibits
a fluorescence spectrum that strides the NIR-IIa region. Small
animal imaging showed significant improvement in through-skull-
scalp fluorescent imaging of the cerebral vasculature of live mice,
indicating the promising potential of such a dual enhancement
strategy for designing NIR-II fluorophores for in vivo fluorescence
imaging.
Results and Discussion
We design the semiconducting polymers according to a classic
donor-acceptor (D–A) structure. In D–A polymers, the highest
occupied molecular orbital (HOMO) and the lowest unoccupied
molecular orbital (LUMO) after orbital hybridization are mainly
Density functional theory calculations were first performed to
predict the energy levels (Figure S14). The LUMOs were mainly
located at the donor and acceptor units, respectively.[19] Therefore, distributed on the acceptors. As the electron affinity increased
a narrow bandgap between the HOMO and LUMO could be
realized by using strong donor and acceptor units. The specific
molecular engineering strategy is illustrated in Figure 1.
from P1c to P3c, the LUMO energy level decreased obviously.
The combination of the lower LUMO energy level and constant
HOMO energy level resulted in a narrow bandgap, suggesting a
red-shift in the emission peak of the polymer. The HOMOs
exhibited major delocalization on phenothiazine units with
minimal overlap with the LUMOs, indicating typical intramolecular
charge transfer within the polymer. All side chain groups exhibited
almost no molecular orbital distribution, indicating that the side
groups would not interfere with the fluorescence radiation
transition.
We choose phenothiazine as the donor because of several
properties of this chromophore. First, the sulfur and nitrogen
atoms endow phenothiazine with superior electron-donating
ability, suggesting a suitable HOMO energy level. Second, the
phenothiazine ring is an excellent nonplanar building block that
can impede π-stacking aggregation and excimer formation in
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201914397
RESEARCH ARTICLE
Figure 1. a) Synthetic route and conditions: i
) 2-bromo-9,10-diphenylanthracene, HPtBu
KOAc, dioxane, 80℃; and iv) Pd(PPh , toluene, K
a
) DMSO/NaOH, 1-bromooctane, RT; i
BF , NaOtBu, Pd (dba) , toluene, reflux; ii) NBS, toluene, acetic acid, RT; iii) Bis(pinacolato)diboron, Pd(dppf)Cl
CO , 85°C. b) Schematic of the general approach of molecular engineering for NIR fluorescence enhancement:
b 3 4 2 3
) 4-bromo-N,N-di-p-tolylaniline, HPtBu BF , NaOtBu, Pd (dba) , toluene, reflux;
i
c
3
4
2
3
2
,
)
3 4
2
3
i) acceptors with different electron affinities were employed to adjust the emission wavelength; ii) flexible phenothiazine enabled the generation of AIE character;
and iii) fluorescence QY was further considerably improved by varying the side-chain groups. c) Spatial distributions of HOMO and LUMO along with their orbital
energies for P1c, P2c, and P3c polymers.
Based on the molecular engineering strategy, the synthetic
route was devised and implemented (Figure 1). The target side
group was attached to phenothiazine via nucleophilic substitution
ratio of the fluorescence peak intensity of the polymer in the
THF/water mixture to that of the polymer in pure THF (I/I ) was
0
calculated. As an example, for P3c, the fluorescence intensity
decreased slightly when 10 vol% of water was added to the THF
solution. This effect was attributed to the interaction between
water and the polymers and the change of the hydrophobic
(M2), and this intermediate product was brominated (M3) and
boronized via Miyaura reaction (M4). Finally, M4 and brominated
acceptors were polymerized by the Suzuki reaction. Detailed
synthetic information and nuclear magnetic resonance
spectroscopy data can be found in the Supporting Information.
The number-average molecular weight (Mn) and polymer
dispersity index (PDI) were evaluated by gel permeation
chromatography, and the results are listed in Table 1.
environment. However, as f
w
increased further, the fluorescence
= 80 vol%, revealing that
intensity increased continually until f
w
the AIE signature prevails in the system.
Another key factor in dual fluorescence enhancement is the
anti-ACQ effect. The fluorescence images of the three NIR-II
polymers (P3a, P3b and P3c) and small dye molecule IR26 were
collected in different states under the same imaging conditions
(Figure 2c). The anti-ACQ effect was verified through the
comparison of the samples from the same row in Figure 2c. By
increasing the steric hindrance of side-chain groups, the mean
fluorescence intensity was noticeably enhanced among the three
polymers in the THF solution, whereas the AIE character was
ineffective. Other effects of the side groups on the radiative
transition could be excluded based on the results of the density
functional theory calculations and the same backbones of the
polymers possessing the similar spectra (e.g., P3a, P3b, and P3c).
This anti-ACQ effect becomes even more pronounced in the
Pdots forms because the bulky side group of P3c effectively
prevents π-stacking aggregation and excimer/exciplex formation
in the Pdots with densely packed polymer chains (Figure 2d). As
a further evidence, solid polymer powders demonstrated even
brighter NIR-II fluorescence than the Pdots (Figure 2c), revealing
great potential for applications requiring solid-state fluorescent
The absorption and fluorescence spectra of the semiconducting
polymers in organic solvent were investigated (Figure 2a). The
emission of the polymers was successfully shifted from red (~600
nm) to NIR-II (~1300 nm) by varying the electron affinity of the
acceptors. Polymers P3a, P3b, and P3c in THF exhibited two
emission peaks at approximately 1130 and 1270 nm and
absorption peak at approximately 740 nm (Figure S11). The
fluorescence emission of P3c polymer strides the spectral range
from 1000 to 1450 nm, indicating their great potential in NIR-IIa
fluorescence imaging.
In our design, AIE is one of two key factors in realizing high
fluorescence QY of the Pdots. To examine the AIE feature of the
polymers, fluorescence images and spectra of the polymers in
THF/water mixtures with different water volume fractions (f
collected (Figure S13). In the fluorescence images, the solutions
with higher f showed brighter signals (Figure 2b), indicating
apparent AIE character. For a quantitative demonstration, the
w
) were
w
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201914397
RESEARCH ARTICLE
materials. Again, P3c polymer powder exhibit stronger NIR-II
fluorescence than P3a and P3b, further confirming the anti-ACQ
effect by engineering the side chain groups.
Figure 2. a) Absorption and fluorescence spectra of P1c, P2c, and P3c polymers in THF. b) Fluorescence intensity changes of P3a, P3b, and P3c in THF/water
mixtures containing different water fractions. The 100% water fraction indicates the bare Pdots (without encapsulation) in water. I/I is the ratio of the fluorescence
peak intensity of the polymer in the THF/water mixture to that of the polymer in pure THF. The inset shows the fluorescence images of P3c in THF/water mixtures
100 μg/mL) containing different water fractions. The images were obtained using a 1250-nm long-pass filter under laser excitation at 808 nm (50 mW·cm−2). c)
0
(
Fluorescence images of IR26 and Pdots in different states. Bottom row: IR26 in DCE and P3a, P3b, and P3c in THF (100 μg/mL); Middle row: Pdots of P3a, P3b,
and P3c in water and IR26-doped polystyrene nanoparticles in water (100 μg/mL); Top row: IR26, P3a, P3b, and P3c powders. The images were recorded by using
a 1250-nm long-pass filter under laser excitation at 808 nm (30 mW·cm−2). d) Schematic illustration of the formation of Pdots. A functional polymer PS-PEG was
used for surface PEGylation that can increase blood circulation time for in vivo fluorescence imaging. The greater steric hindrance in P3c weakened the π–π
stacking interaction, resulting in the anti-ACQ effect and a higher fluorescence QY.
Quantitative optical characterizations were performed to
investigate the fluorescence dual enhancement in Pdots. The
NIR-II fluorescence QYs were measured by using IR-26 in 1,2-
dichloroethane (DCE) as a reference (QY = 0.5%).[24] Table 1
shows the optical parameters of the semiconducting polymers
and corresponding Pdots. Owing to the AIE character, all the QYs
of Pdots are higher than that of the corresponding polymer in THF
solution, with most of them exhibiting a superior increase of
approximately two times. In THF solution, the anti-ACQ effect of
the side groups was not fully active because of the weak
aggregation. In contrast, the tighter aggregation of Pdots
accentuated the effect of fluorescence enhancement by the side-
chain groups. The QYs of Pdots increased obviously from side
a c
group R to side group R , confirming the function of the side
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201914397
RESEARCH ARTICLE
groups. Among the three side groups, diphenylanthracene
significantly weakened the intermolecular interactions, resulting in
the best anti-ACQ effect.
Table 1. Optical Properties of Semiconducting Polymers and the Resulting Pdots.
ΦPdots/ΦTHF
2.6
Pdots
[nm]a
em(Φ) THF [nm, %] b
em(Φ) Pdots [nm, %]
[eV] c
M
n
[kDa]d
PDIe
3.21
3.11
4.17
1.57
1.55
2.58
3.51
1.56
1.68
Size [nm]f
λ
abs
λ
λ
E
g
P1a
P1b
P1c
P2a
P2b
P2c
P3a
P3b
P3c
456
509
482
537
522
538
736
759
746
674 (9)
699 (23)
687 (25)
685 (25)
740 (11)
732 (17)
731 (19)
1090 (0.1)
1088 (0.6)
1083 (1.7)
1.53
1.53
1.55
1.36
1.46
1.46
0.90
0.89
0.89
5.0
6.3
4.4
4.4
4.5
5.2
3.2
4.9
2.8
16
18
18
14
16
17
24
14
16
649 (15)
1.7
669 (11)
2.3
683 (10)
1.1
659 (8)
2.1
717 (6)
3.2
1113, 1284 (0.08)
1115, 1279 (0.3)
1123, 1272 (0.6)
1.3
2.0
2.2
a) Absorption maximum; b) emission maximum and fluorescence quantum efficiency; c) the optical bandgap was calculated from the intersection of the absorption
and emission spectra; d) number-average molecular weight; e) polymer dispersity index; f) sizes of Pdots in water.
Based on this dual enhancement strategy, the P3c Pdots
exhibited a NIR-II fluorescence QY of ~1.7% in aqueous solution,
corresponding to an enhancement of approximately 21 times as
compared to P3a in THF. It represents the highest QY reported
so far for NIR-II Pdots. It is worth noting that the brightness of the
fluorescent probes is determined by the product of absorption
cross section and fluorescence quantum yield.[1d] UV-Vis-NIR
absorption measurements were performed for P3c Pdots, IR26,
and Indocyanine Green (ICG) to compare their brightness. The
results indicated that the peak absorption cross section of P3c
Pdots (~16-nm-diameter) at 746 nm was ~180 times larger than
that of IR26 at 935 nm, and ~60 times higher than that of ICG at
The particle size, morphology, photostability, and
biocompatibility of the Pdots were further characterized to
evaluate their application potential. P3c Pdots was selected for
further investigations based on their outstanding fluorescence
QYs. The fluorescence spectrum of the P3c Pdots in aqueous
solution was somewhat different from that of P3c in THF because
of aggregated conformation (Figure 3a). Still, the emission profile
strides the broad wavelength range from 1000 to 1400 nm. The
emission intensity in the NIR-IIa region was relatively decreased
as compared to that of the polymer in THF solution (Figure 2a).
Dynamic light scattering demonstrated that the Pdots were well
dispersed in water with an average hydrodynamic diameter of ~16
nm. Transmission electron microscopy (TEM) imaging indicated
the spherical morphologies of the well-dispersed nanoparticles
(Figure 3b). Photostability was examined by monitoring the
fluorescence intensity under continuous laser illumination (808
7
80 nm, respectively. Considering the same excitation
wavelength at 808 nm, the absorption cross section of P3c Pdots
~16 nm) was ~190 times higher than that of IR26, and ~100 times
(
higher than that of ICG, respectively. Combined with the QY
values, the NIR-II fluorescence brightness of single P3c Pdot is
over 500 times higher than single dye fluorophores. We also
compared the fluorescence brightness of the three probes at the
same weight concentration in solutions under 808 nm excitation.
As indicated in Figure S16, the P3c Pdots in water is ~2 times
brighter than IR26 in DMSO and ~15 times brighter than ICG in
water for the emission collected by a 1250 nm long-pass filter.
Based on these results, the Pdots outperform the fluorescent dyes
at both single probe level and bulk solutions.
2
nm, 1.0 W/cm ). The fluorescence of the P3c Pdots remained
nearly 70% of the original intensity after 120 min of laser exposure,
while the IR26 and ICG dyes were completely photobleached in
10 min (Figure 3c). This comparison clearly indicated the superior
photostability of Pdots as compared to the small dye molecules.
Biocompatibility is a vital consideration for biological applications.
As shown in Figure 3d, the Pdots showed low cytotoxicity, even
at high concentrations (100 μg/mL), indicating their biocompatible
feature. Collectively, these results indicate that the Pc3-Pdots are
an excellent NIR-II fluorophore for bioimaging.
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201914397
RESEARCH ARTICLE
Figure 3. a) Absorption and fluorescence spectra of P1c, P2c and P3c Pdots in water. b) Distribution of hydrodynamic diameter of the P3c Pdots determined by
dynamic light scattering. The inset shows a representative TEM image of the P3c Pdots. c) Photostability of bare P3c-Pdots(without encapsulation) in water, ICG
2
in water, and IR26 in dimethyl sulfoxide under continuous 808 nm radiation (1.0 W/cm ). I/I
0
is the ratio of the mean fluorescence intensity of the samples after
radiation to that of the original mean fluorescence intensity. The inset shows the corresponding fluorescence images, using a 1250-nm long-pass filter under laser
excitation at 808 nm (30 mW·cm−2). d) Results of a cellular toxicity study in which HeLa cells were incubated with different concentrations of Pdots.
Optical imaging of the brain in the traditional wavelength region
400-900 nm) is severely constrained by the light penetration
Broad NIR-II emission spectrum of the P3c Pdots gives multiple
choices on the fluorescence acquisition range. Despite the
relatively small emission profile of Pdots beyond 1300 nm,
fluoprescence imaging by a 1319 nm long-pass filter produces
much sharper image than that by a 1250 nm long-pass filter
(Figure 4a). The comparison of intensity profiles of cerebral
vessels collected by the two filters confirms the high signal to
background ratio (SBR) in the NIR-IIa region (Figure 4b, 5c). The
P3c Pdots allowed clear visualization of the cerebrovascular
structures under the intact skull and scalp (Figure 4a), indicating
the large penetration depth enabled by the unique NIR-IIa
emission. Thanks to the distinct fluorescence of Pdots and low
autofluorescence in this region, high-quality imaging yielded well-
resolved distance between cerebral blood vessels (Figure 4c). As
seen from time-lapse imaging, the high SBR remains relatively
(
depth in biological tissues.[3;25] Recent studies have demonstrated
that fluorescence collection in the 1.3–1.4 μm NIR-IIa window led
to drastically improved imaging of mouse cerebral vasculature
because of the minimal scattering by rejecting photons
wavelengths shorter than 1.3 μm while avoiding an increase in
light absorption from water vibrational overtone modes above
∼
1.4 μm.[7] We performed in vivo non-invasive fluorescence
imaging of live mouse brain with the PEGylated P3c Pdots. A
solution of the Pdots was injected into a mouse via the tail vein,
and the head of the mouse with intact skull and scalp (hair shaved
off only) was imaged by a small animal imaging setup. Low
−2
excitation power density (70 mW·cm , 808 nm) was used to
minimize damage to the mouse while maintaining image quality.
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201914397
RESEARCH ARTICLE
constant in 180 min because of the relatively long circulation half-
life (~6.6 hours) of the PEGylated Pdots (Figure S17). In contrast,
small molecule dyes such as ICG was usually cleared in ~5
minutes,[26] which led to a very short imaging window.
Figure 4. a) In vivo NIR-II fluorescence imaging of mouse brain at designated time points after intrvenous injection of P3c Pdots (100 μL, 1 mg/mL). The top panel
shows the images collected by a 1250 nm long-pass filter, while the bottom shows those collected by a 1319 nm long-pass filter. b) Cross-sectional intensity profile
along the white line in images collected by a 1250 nm long-pass filter with the peak fitted to a Gaussian function (red curve). c) Cross-sectional intensity profile
along the white line in images collected by a 1319 nm long-pass filter with the peak fitted to a Gaussian function (red curve). d) In vivo NIR-II fluorescence images
of mouse hindlimb vasculature 5 min after intravenous injection of P3a Pdots (100 μL, 1 mg/mL) and P3c Pdots (100 μL, 1 mg/mL), respectively. e) Cross-sectional
intensity profile along the white line in c) #1 with the peak fitted to a Gaussian function (red curve). f) Cross-sectional intensity profile along the white line in c) #2
with the peak fitted to a Gaussian function (red curve). g) Mean fluorescence intensity of the heart (H), lung (Lu), kidney (Ki), spleen (Sp), blood (Bl), and liver (Li)
at 24 h after the injection of the P3a and P3c Pdots, respectively. The inset shows the fluorescence images of the heart, lung, kidney, spleen, blood, and liver of the
injected mouse. The images in d) and g) were obtained using a 1319-nm long-pass filter under 808-nm laser excitation (70 mW·cm−2).
Whole-body NIR-IIa fluorescence imaging was finally
performed to reaffirm the superior performance of the Pdots
achieved by molecular engineering. P3a and P3c Pdots were
used for whole-body imaging and compared under the same
unique optical properties of P3c Pdots are promising NIR-IIa
fluorophore for small animal studies.
conditions. In the image obtained using P3a Pdots, the mouse Conclusion
body was nearly invisible (Figure 4d). In contrast, the P3c Pdots
with AIE and stronger anti-ACQ effects resulted in much higher
SBR and more details were achieved from the image (Figure 4e,
In summary, a molecular engineering strategy was proposed to
identify new NIR-II fluorophores for bioimaging. Specifically, nine
semiconducting polymers were designed and synthesized to
validate a dual fluorescence enhancement mechanism. The
adjustment of electron affinity realized tunable excitation and
emission wavelengths, with emission in the range of 600–1400
nm. The donor unit phenothiazine endowed the polymers with a
narrow bandgap along with AIE character, leading to strong
emission in the corresponding Pdots. Side groups provided
different levels of steric hindrance to weaken the intermolecular
5f). This comparative imaging clearly demonstrated the
improvement provided by the P3c Pdots with respect to the P3a
Pdots, revealing the successful dual fluorescence enhancement
based on the AIE and anti-ACQ effects. The major organs were
dissected from the mouse 24 h after injection and quantitatively
analyzed by fluorescence imaging (Figure 4g). The analysis
results indicated that the Pdots were mainly distributed in the liver,
blood, and spleen. The in vivo imaging results reveal that the
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201914397
RESEARCH ARTICLE
Zeng, Y. Xiao, L. Tang, J. Nong, Y. Liu, H. Zhou, B. Ding, F. Xu, H. Tong,
Z. Deng, X. Hong, Chem. Sci. 2019, 10, 1219-1226; f) X. Lu, P. Yuan, W.
Zhang, Q. Wu, X. Wang, M. Zhao, P. Sun, W. Huang, Q. Fan, Polym.
Chem. 2018, 9, 3118-3126.
interactions. This anti-ACQ effect further improved the
fluorescence QY based on the AIE character. Under the dual
enhancement by AIE character and anti-ACQ effect, the NIR-II
fluorescence QY of P3c Pdots was approximately 21 times higher
than that of P3a in THF. We used the P3c Pdots for in vivo NIR-II
fluorescence imaging of mouse brain. Because the unique
emission strides the NIR-IIa region, the through-scalp and
through-skull imaging show clear visualization of cerebral
vasculature. This study reveals the great potential of dual
fluorescence enhancement based on the AIE and anti-ACQ
effects for obtaining superior NIR-II fluorophores.
[
11] a) K. Pu, S. Adam J, J. Jesse V, J. Mei, G. Sanjiv S, Z. Bao, J. Rao,
Nature Nanotech. 2014, 9, 233-239; b) Z. Wang, X. Zhen, P. K. Upputuri,
Y. Jiang, J. Lau, M. Pramanik, K. Pu, B. Xing, ACS nano 2019, 13, 5816-
5825.
[
[
12] R. Englman, J. Jortner, Mol. Phys. 1970, 18, 145-164.
13] J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu, H. S. Kwok, X.
Zhan, Y. Liu, D. Zhu, B. Z. Tang, Chem. Commun. 2001, 1740-1741.
[14] a) E. P. J. Parrott, N. Y. Tan, R. Hu, J. A. Zeitler, B. Z. Tang, E. Pickwell-
MacPherson, Mater. Horiz. 2014, 1, 251-258; b) G. Yu, S. Yin, Y. Liu, J.
Chen, X. Xu, X. Sun, D. Ma, X. Zhan, Q. Peng, Z. Shuai, B. Tang, D. Zhu,
W. Fang, Y. Luo, J. Am. Chem. Soc. 2005, 127, 6335-6346; c) Q. Peng,
Y. Yi, Z. Shuai, J. Shao, J. Am. Chem. Soc. 2007, 129, 9333-9339.
Acknowledgements
[
15] a) F. Bu, R. Duan, Y. Xie, Y. Yi, Q. Peng, R. Hu, A. Qin, Z. Zhao, B. Z.
Tang, Angew. Chem. Int. Ed. 2016, 127, 14700-14705; b) N. L. C. Leung,
X. Ni, Y. Wangzhang, L. Yang, W. Qunyan, P. Qian, M. Qian, J. W. Y.
Lam, T. Ben Zhong, Chem. 2015, 20, 15349-15353; c) J. Chen, C. C. W.
Law, J. W. Y. Lam, Y. Dong, S. M. F. Lo, I. D. Williams, D. Zhu, B. Z.
Tang, Chem. Mater. 2003, 15, 1535-1546.
C. Wu acknowledges financial support from the National Natural
Science Foundation of China (NSFC) (81771930) and Shenzhen
Science
JCYJ20170307110157501). The authors declare no competing
financial interest.
and
Technology
Innovation
Commission
(
[
[
16] a) R. Hu, N. L. C. Leung, B. Z. Tang, Chem. Soc. Rev. 2014, 43, 4494-
4562; b) S. Xu, Y. Duan, B. Liu, Adv. Mater., 0, 1903530.
17] a) Z. Sheng, B. Guo, D. Hu, S. Xu, W. Wu, W. H. Liew, K. Yao, J. Jiang,
C. Liu, H. Zheng, Adv. Mater. 2018, 1800766; b) J. Qi, C. Sun, A.
Zebibula, H. Zhang, R. T. K. Kwok, X. Zhao, W. Xi, J. W. Y. Lam, J. Qian,
B. Z. Tang, Adv. Mater. 2018, 30, e1706856; c) N. Alifu, A. Zebibula, J.
Qi, H. Zhang, C. Sun, X. Yu, D. Xue, J. W. Y. Lam, G. Li, J. Qian, B. Z.
Tang, ACS nano 2018, 12, 11282-11293.
Keywords: molecular engineering • NIR-II fluorescence imaging
semiconducting polymers • aggregation-induced emission •
polymer dots
•
[
[
1]
2]
a) H.-S. Peng, D. T. Chiu, Chem. Soc. Rev. 2015, 44, 4699-4722; b) J.
N. Liu, W. Bu, J. Shi, Chem. Rev. 2017, 117, 6160-6224; c) L. Yan, K.
Pu, Adv. Sci. 2017, 4, 1600481; d) C. Wu, D. T. Chiu, Angew. Chem. Int.
Ed. 2013, 52, 3086-3109.
[18] W. Chen, C. A. Cheng, E. D. Cosco, S. Ramakrishnan, J. G. P. Lingg, O.
T. Bruns, J. I. Zink, E. M. Sletten, J. Am. Chem. Soc. 2019, 141, 12475-
12480.
[19] Z. G. Zhang, J. Wang, J. Mater. Chem. 2012, 22, 4178-4187.
[20] A. C. B. Rodrigues, J. Pina, W. Dong, M. Forster, U. Scherf, J. S. Seixas
de Melo, Macromol. 2018, 51, 8501-8512.
a) G. Hong, S. Diao, A. L. Antaris, H. Dai, Chem. Rev. 2015, 115, 10816-
10906; b) J. Huang, C. Xie, X. Zhang, Y. Jiang, J. Li, Q. Fan, K. Pu,
Angew. Chem. Int. Ed. 2019, 58, 15120-15127.
[21] X. Jiang, H. Gao, X. Zhang, J. Pang, Y. Li, K. Li, Y. Wu, S. Li, J. Zhu, Y.
Wei, L. Jiang, Nat. Commun. 2018, 9, 3799.
[
[
3]
4]
G. Hong, A. L. Antaris, H. Dai, Nat. Biomed. Eng. 2017, 1, 0010.
a) Y. Cai, Z. Wei, C. Song, C. Tang, W. Han, X. Dong, Chem. Soc. Rev.
[22] A. P. Kulkarni, Y. Zhu, A. Babel, P. T. Wu, S. A. Jenekhe, Chem. Mater.
2008, 20, 4212-4223.
2019, 48, 22-37; b) C. Li, Q. Wang, ACS nano 2018, 12, 9654-9659; c)
Y. Tang, Y. Li, X. Lu, X. Hu, H. Zhao, W. Hu, F. Lu, Q. Fan, W. Huang,
Adv. Funct. Mater. 2019, 29, 1807376.
[23] a) X. Cai, A. Bandla, D. Mao, G. Feng, W. Qin, L. D. Liao, N. Thakor, B.
Z. Tang, B. Liu, Adv. Mater. 2016, 28, 8760-8765; b) J. Mei, N. L. Leung,
R. T. Kwok, J. W. Lam, B. Z. Tang, Chem. Rev. 2015, 115, 11718-11940.
[24] a) J. Cornil, I. Gueli, A. Dkhissi, J. C. Sancho-Garcia, E. Hennebicq, J. P.
Calbert, V. Lemaur, D. Beljonne, J. L. Brédas, J. Chem. Phys. 2003, 118,
6615-6623; b) J. E. Murphy, M. C. Beard, A. G. Norman, A. S Phillip, J.
C. Johnson, Y. Pingrong, O. I. Mićić, R. J. Ellingson, A. J. Nozik, J. Amer.
Chem. Soc. 2006, 128, 3241-3247.
[
5]
a) G. Hong, Y. Zou, A. L. Antaris, S. Diao, D. Wu, K. Cheng, X. Zhang,
C. Chen, B. Liu, Y. He, J. Z. Wu, J. Yuan, B. Zhang, Z. Tao, C. Fukunaga,
H. Dai, Nat. Commun. 2014, 5, 4206; b) Q. Yang, Z. Hu, S. Zhu, R. Ma,
H. Ma, Z. Ma, H. Wan, T. Zhu, Z. Jiang, W. Liu, L. Jiao, H. Sun, Y. Liang,
H. Dai, J. Am. Chem. Soc. 2018, 140, 1715-1724; c) S. Zhu, S. Herraiz,
J. Yue, M. Zhang, H. Wan, Q. Yang, Z. Ma, Y. Wang, J. He, A. L. Antaris,
Y. Zhong, S. Diao, Y. Feng, Y. Zhou, K. Yu, G. Hong, Y. Liang, A. J.
Hsueh, H. Dai, Adv. Mater 2018, 30, e1705799.
[25] S. Diao, J. L. Blackburn, G. Hong, A. L. Antaris, J. Chang, J. Z. Wu, B.
Zhang, K. Cheng, C. J. Kuo, H. Dai, Angew. Chem. Int. Ed. 2015, 54,
14758-14762.
[
[
6]
7]
K. Shou, Y. Tang, H. Chen, S. Chen, L. Zhang, A. Zhang, Q. Fan, A. Yu,
Z. Cheng, Chem. Sci. 2018, 9, 3105-3110.
[26] C. Li, Y. Zhang, M. Wang, Y. Zhang, G. Chen, L. Li, D. Wu, Q. Wang,
Biomater. 2014, 35, 393-400.
G. Hong, S. Diao, J. Chang, A. L. Antaris, C. Chen, B. Zhang, S. Zhao,
D. N. Atochin, P. L. Huang, K. I. Andreasson, C. J. Kuo, H. Dai, Nat.
Photonics 2014, 8, 723-730.
[
8]
a) J. Li, J. Rao, K. Pu, Biomater. 2018, 155, 217-235; b) L. Feng, C. Zhu,
H. Yuan, L. Liu, F. Lv, S. Wang, Chem. Soc. Rev. 2013, 42, 6620-6633;
c) Y. Jiang, J. McNeill, Chem. Rev. 2017, 117, 838-859; d) Y. Jiang, M.
Novoa, T. Nongnual, R. Powell, T. Bruce, J. McNeill, Nano Lett. 2017,
17, 3896-3901.
[
[
9]
Y. Hong, J. W. Lam, B. Z. Tang, Chem. Commun. 2009, 40, 4332-4353.
10] a) C. Wu, C. Szymanski, Z. Cain, J. McNeill, J. Am. Chem. Soc. 2007,
1
29, 12904-12905; b) C. Wu, C. Szymanski, J. McNeill, Langmuir 2006,
2, 2956-2960; c) X. Chen, Z. Liu, R. Li, C. Shan, Z. Zeng, B. Xue, W.
2
Yuan, C. Mo, P. Xi, C. Wu, Y. Sun, ACS nano 2017, 11, 8084-8091; d)
K. Y. Pu, B. Liu, Biosens Bioelectron 2009, 24, 1067-1073; e) J. Lin, X.
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201914397
RESEARCH ARTICLE
RESEARCH ARTICLE
Authors: Zhe Zhang,
Xiaofeng Fang, Zhihe Liu,
Haichao Liu, Dandan
Chen, Shuqing He, Jie
Zheng, Bin Yang, Weiping
Qin, Xuanjun Zhang,
Changfeng Wu
We demonstrate a molecular engineering strategy to enhance NIR-IIa fluorescence
of polymer dots by managing the aggregation-induced emission (AIE) and
aggregation-caused quenching (ACQ) effects. The polymer dots enabled a
remarkable improvement in penetration depth and signal to background ratio in
through-skull and through-scalp fluorescent imaging of the cerebral vasculature of
live mice.
Corresponding Author*:
Changfeng Wu
E-mail address:
wucf@sustech.edu.cn
Page No.–Page No.
This article is protected by copyright. All rights reserved.