Polymethine-Based Semiconducting Polymer Dots with Narrow-Band Emission and Absorption/Emission Maxima at NIR-II for Bioimaging

Article abstract of DOI:10.1002/anie.202011914

Deep-penetration fluorescence imaging in the second near-infrared (NIR-II) window heralds a new era of clinical surgery, in which high-resolution vascular/lymphatic anatomy and detailed cancerous tissues can be visualized in real time. Described here is a series of polymethine-based semiconducting polymers with intrinsic emission maxima in the NIR-IIa (1300–1400 nm) window and absorption maxima ranging from 1082 to 1290 nm. These polymers were prepared as semiconducting polymer dots (Pdots) in aqueous solutions with fluorescence quantum yields of 0.05–0.18 %, and they demonstrate promising applications in noninvasive through-skull brain imaging in live mice with remarkable spatial resolution as well as signal-to-background contrast. This study offers a platform for future design of NIR-IIa or even NIR-IIb emitting Pdots.

Full text of DOI:10.1002/anie.202011914

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
International Edition Chemie
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Accepted Article
Title: Polymethine-Based Semiconducting Polymer Dots with Narrow-
Band Emission and Absorption/Emission Maxima at NIR-II for
Bioimaging
Authors: Ming-Ho Liu, Zhe Zhang, Yu-Chi Yang, and Yang-Hsiang
Chan
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202011914
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
Polymethine-Based Semiconducting Polymer Dots with Narrow-
Band Emission and Absorption/Emission Maxima at NIR-II for
Bioimaging
a†
b†
a
ac
Ming-Ho Liu, Zhe Zhang, Yu-Chi Yang, and Yang-Hsiang Chan*
[a]
M.-H. Liu, Y.-C. Yang, Prof. Y.-H. Chan
Department of Applied Chemistry/Center for Emergent Functional Matter Science
National Chiao Tung University
Hsinchu, Taiwan 30050
[*]
E-mail: yhchan@nctu.edu.tw
Both authors contributed equally to this work
†
[b]
Z. Zhang
Department of Biomedical Engineering
Southern University of Science and Technology
Shenzhen, Guangdong, China 518055
Prof. Y.-H. Chan
[c]
Department of Medicinal and Applied Chemistry
Kaohsiung Medical University
Kaohsiung, Taiwan 80708
Supporting information for this article is given via a link at the end of the document.
Abstract: Deep-penetration fluorescence imaging in the second
near-infrared (NIR-II) window heralds a new era of clinical surgery, in
which high-resolution vascular/lymphatic anatomy and detailed
cancerous tissues can be visualized in real time. Although several
types of fluorescent agents, including inorganic nanostructures and
small organic dyes have been extensively developed recently, only a
few successful examples of NIR-II fluorescing polymers with much
enhanced fluorescence brightness and photo/colloidal stability have
been reported. Here we describe a series of polymethine-based
semiconducting polymers with intrinsic emission maxima in the NIR-
IIa (1300-1400 nm) window and absorption maxima ranging from
be highly bright, photostable, and biocompatible. Conventional
small organic fluorophores, such as FDA-approved indocyanine
green (ICG), methylene blue, and fluorescein, with emission in the
visible to NIR-I (700-900 nm) windows, suffer from limited tissue
penetration depth (< 3 mm) and strong tissue autofluorescence.
This had hindered their wide adoption for clinical use. Identified
merely several years ago, fluorescence imaging in the NIR-II
(1000-1700 nm) region has been proved to have reduced photon
absorption/scattering by biospecies and minimal background
autofluorescence to afford deeper penetration depth with
[4]
remarkable spatial resolution. Enormous activities have been
thereon focused on the development of various novel contrast
1
082 to 1290 nm. We prepared these polymers as semiconducting
[5]
polymer dots (Pdots) in aqueous solutions with fluorescence quantum
yields of 0.05-0.18 %, and demonstrated their promising applications
in noninvasive through-skull brain imaging in live mice with
agents, including single-walled carbon nanotubes, rare-earth
[
6]
[7]
nanomaterials,
inorganic quantum dots,
and organic
fluorophores.[
3b, 3c, 8]
Among these fluorescent probes, organic
remarkable spatial resolution as well as signal-to-background contrast. small molecules appear to be most attractive candidates for NIR-
This study offers a universal platform for future designing NIR-IIa or
even NIR-IIb emitting Pdots.
II imaging because of facile chemical synthesis, low cytotoxicity,
and fast renal clearance in the body. However, there are still
several unmet challenges of small molecule-based NIR-II probes
including poor water dispersibility, low photostability, difficult
functionalization, and more importantly, low fluorescence
brightness (i.e., low extinction coefficient). To circumvent the
aforementioned issues, usually phospholipids were employed to
encapsulate these small dyes to generate lipid-based
Introduction
Biological imaging with various modalities can provide detailed
observation of therapeutic and pathological processes to
elucidate complicated molecular mechanisms underlying human
diseases.[1] In particular, noninvasive fluorescence imaging
appears to be the most versatile yet simple techniques and is able
to offer unprecedented spatial and temporal resolutions with real-
time monitoring ability.[2] The huge progress in clinical technology
and instrumentation for fluorescence image-guided surgery in
human patients in these years also makes this field, especially the
development of highly emissive and biocompatible fluorophores,
considerably highlighted and urgently demanded.[3]
[4b]
[9]
nanomicelles, while the known dye leaking phenomenon and
long-term in vivo optical stability remain a serious issue for further
clinical translation. Additionally, it is synthetically difficult to obtain
small organic molecules with absorption over 1100 nm and
emission beyond 1300 nm owing to the extended conjugation
scaffolds which are vulnerable especially in polar solvents.
Therefore, serval reported works smartly utilized the emission
tails (off-peak emission) of these NIR-II emitters to realize NIR-IIa
1300-1400 nm) or NIR-IIb (1500-1700 nm) imaging[3b, 3c, 10] but
(
the spatiotemporal resolution must greatly rely on the CCD
efficiency and adequate optical filters due to the low fluorescence
brightness. Another strategy cleverly exploited J-aggregation of
As a key component of fluorescence imaging in clinical
applications, the selection of contrast agent directly affects the
precision of disease diagnosis and the treatment outcomes of
patients. Fluorescence probes for in vivo studies are required to
small dyes to bathochromically shift both their maximal absorption
[4o, 11]
and emission into the desired windows.
In this scenario,
1
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
however, the significant aggregation-cased quenching (ACQ)
behavior by 1-2 orders of magnitude is inevitable,[4o] which might
constrain their widespread adoption.
spectra of NIR1125 (red line), NIR1270 (green line), and NIR1380 (blue line) in
CH
Emission spectra of NIR1125 (red line), NIR1270 (green line), and NIR1380
(blue line) in CH Cl (C) Synthesis of semiconducting polymer bearing
2 2
Cl . The middle-left inset shows the photograph of the dye solutions. (B)
In an effort to fundamentally tackle the above-mentioned
2
2
.
persistent
issues
for
small
organic
fluorophores,
carboxylic acid groups for further transformation to azide groups, followed by
CuAAC click reactions with polymethine dyes.
conjugated/semiconducting polymer-based nanoparticles thereby
emerge as a unique solution for NIR-II fluorescence imaging. This
type of nanoparticles, also abbreviated as Pdots/CPNs/SPNs,
have multiple superior advantages over small organic
The difficulty in getting Pdots with both absorption and
emission in the NIR-II window has plagued almost all of the Pdot-
related research communities for years. Herein, we successfully
developed a novel yet universal platform to synthesize Pdots with
narrow-band absorption and emission in the NIR-II region by
integrating both advantages of small organic fluorophores and
conjugated polymers. Specifically, we first synthesized a new type
of semiconducting polymer with a bulky architecture to provide
anti-aggregation-caused quenching (anti-ACQ) properties (Figure
[12]
molecules: (i) ultrahigh fluorescence brightness due to the large
absorption coefficient; (ii) excellent photostability; (iii) good water
dispersibility and facile surface functionalization for targeting; (iv)
large Stokes shift to avoid optical cross-talking; and (v) high
biocompatibility. Unfortunately, to date, there is still a lack of Pdot
with both absorption and emission maxima in the NIR-II window.
This fact is dictated by the energy gap law that the nonradiative
decays are accelerated by the vibrational overlap between the
ground state and excited state due to the small energy gap.[13]
Because of this intrinsic limitation, there is only a paucity of
successful examples to acquire donor-acceptor type Pdots with
emission in the NIR-II region although their absorption maxima
are still in the NIR-I window and their fluorescence quantum yields
are very low.[4h, 14]
[15]
1).
The rigid three-dimensional Pttc moieties as well as the
bulky SeBTa units both contributed to effective steric hindrance to
prevent ACQ phenomenon of post-linked NIR-II monomers. Here
the fluorene derivatives on the polymer backbones simply offer
azide functional groups to chemically bind alkyne-functionalized
polymethine-cyanine dyes through copper(I)-catalyzed azide-
alkyne cycloaddition (CuAAC) under mild conditions. In addition
to the implantation of ani-ACQ characteristics to NIR-II dyes, the
semiconducting polymers can at the same time strongly anchor
the NIR-II fluorophores via robust chemical binding to preclude
from the dye leaching issue encountered by small-dye doped
polymeric[9] or lipidic nanoparticles (vide infra). The dye leaking
problem could result in inconsistent optical signals and potential
cytotoxicity, particularly for in vivo imaging applications. Besides
the anti-ACQ function of the -bridge, SeBTa with the absorption
peak at ~820 nm, can also serve as a light-harvesting fragment
for in vitro cellular studies whereas the penetration depth is less
concerned in cellular levels. For in vivo NIR-II imaging, the direct
excitation on the emitters is competent to offer high image clarity
and quality of deep anatomical features.
Results and Discussion
We first synthesized three types of NIR-II fluorescent polymethine
dyes of different conjugation lengths of intermediate, NIR1125,
NIR1270, and NIR1380 (see Supporting Information), with
narrow-band emissions. The narrow-band emissions are
advantageous for multiplexed imaging. These polymethine
cyanine-dyes were endowed with activable alkynes for
conjugation with polymers. Their absorption and emission spectra
are shown in Figure 1A and 1B, respectively, in which all of
absorption and emission maxima are beyond 1000 nm. It is
noticeable that the absorption maximal peak of NIR1380 locates
at 1270 nm with its emission at 1380 nm (summarized in Table 1),
which is beneficial for in vivo deep-tissue vascular imaging. We
then synthesized a very bulky semiconducting polymer with anti-
ACQ characteristics, Pttc-SeBTa-PFCOOH, to chemically bind
with the polymethine dyes by click reactions (Figure 1C). This
platform is universal that can be virtually used to integrate with all
kinds of fluorophores with reactive alkynes.
Table 1. Summary of the Spectral Data of Polymethine Dyes in DCM
Figure 1. Chemical structures of polymethine dye-conjugated semiconducting
polymers. Three types of NIR-II fluorescent polymethine dyes (NIR1125,
NIR1270 and NIR1380) were synthesized with activable alkyne functional
groups for further conjugation with the semiconducting polymer. (A) Absorption
abs
a
em
b
d
Monomer dyes
NIR1125

max (nm)
max (nm)
(%)
936, 1066
1125
1270
1380
1.2
NIR1270
NIR1380
1050, 1188
1140, 1270
0.52
0.25
2
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
a
b
c
Absorption maximum. Emission maximum. Fluorescnece Quantum
A0.8
B
Before centrifugal filtration
After centrifugal filtration
Filtrate
Before centrifugal filtration
After centrifugal filtration
Filtrate
yield.
2.5
2.0
0
0
0
.6
.4
.2
1.5
1.0
0.5
0
0
4
00 600 800 1000
400 600 800 1000
Wavelength (nm)
C100
D 100
8
6
4
2
0
0
0
0
80
Pttc-SeBTa-NIR1125
Pttc-SeBTa-NIR1380
IR-1061
ICG
60
40
20
0
Pttc-SeBTa-NIR1125
Pttc-SeBTa-NIR1380
NIR1125 nanoparticles
NIR1380 nanoparticles
00
20
40 60 80 100 120
0
20 40 60 80 100 120
Time (min)
Figure 3. Evaluation of optical stability. (A) Absorption spectra of Pttc-SeBTa-
NIR1125 Pdots before (solid blue line) and after (dashed red line) centrifugal
filtration. The absorption spectrum of the filtrate (solid green line) indicates that
no dye leaching phenomenon can be observed in the Pdot system. (B)
Absorption spectra of small NIR1125 dye-doped lipid-based nanoparticles
before (solid green line) and after (dashed purple line) centrifugal filtration. The
absorption peaks of the filtrate (solid green line) suggests the significant dye
leaching problem in the physical doping system. (C) Photostability (normalized
fluorescence intensity vs irradiation time) of Pttc-SeBTa-NIR1125 Pdots in
water (blue line), Pttc-SeBTa-NIR1380 Pdots in water (green line), IR-1061 dyes
in CH Cl (brown line), and ICG dyes in water (red line) under continuous 808
Figure 2. (A) Schematic showing the preparation of lipid-protected Pdots for in
vivo bioimaging. (B) Absorption (solid lines) and fluorescence (dashed lines)
spectra of Pttc-SeBTa-NIR1125 (red line), Pttc-SeBTa-NIR1270 (green lines),
and Pttc-SeBTa-NIR1380 (blue lines) Pdots in aqueous solutions. The
fluorescence data were obtained under 980 nm light excitation. (C)
Representative hydrodynamic sizes of Pttc-SeBTa-NIR1125 Pdots measured
by dynamic light scattering. The upper-right inset represents their corresponding
TEM image with a scale bar of 50 nm.
2
2
2
nm light irradiation (0.8 W/cm ) at the same concentration of 200 pM. (D)
Colloidal stability of Pttc-SeBTa-NIR1125 Pdots (solid green line), Pttc-SeBTa-
NIR1380 Pdots (solid blue line), NIR1125 dye-doped lipid nanoparticles (solid
pink line), and NIR1380 dye-embedded lipid nanoparticles (dashed red line) in
The preparation of NIR-II Pdots was illustrated in Figure 2A
where we used traditional nanoprecipitation method by co-
precipitating amphiphilic lipids into Pdots to provide COOH
functional groups for further surface engineering if needed. We
found that Pttc-SeBTa-NIR1270 Pdots were optically unstable in
aqueous solutions especially under light excitation while the Pttc-
o
DMEM cell culture media at 37 C.
We further assessed the optical stability of the resulting
Pdots. As shown in Figure 3A, a centrifugal filtration device with a
100 kDa molecular weight cut-off was used to concentrate Pttc-
SeBTa-NIR1270 polymers were very stable in most organic
solvents. This phenomenon is very similar to indocyanine green[16]
SeBTa-NIR1125 Pdots. The centrifugal filtration processes (for
concentration or purification) were used to examine the optical
stability of Pdots because these processes are necessary during
the bioconjugation of Pdots for versatile biological applications.
After the centrifugal filtration, the concentrated Pdots were diluted
to the original volume by water and we found that the absorption
of Pdots remained almost the same, indicating negligible leaching
phenomenon for the covalently linked dyes. The minimal
absorption of the filtrate also suggested that no dye leaking
behavior could be observed. On the contrary, the commonly used
lipid-based nanoparticles in which the small organic dyes were
physically encapsulated into the lipid micelles, revealed the
significant dye leaching issue as shown in Figure 3B. In this
sample, small NIR1125 dyes were encapsulated into the DSPE
lipid nanoparticles and the absorption intensity of NIR1125 dyes
decreased after centrifugal filtration, implying the leaking of the
embedded dyes from the polymer matrix. The obvious absorption
of the filtrate further confirmed the leaching phenomenon. The
above results suggested that the covalently dye-functionalized
polymers possessed enhanced optical stability in comparison to
the physically blended counterparts. In terms of photostability, an
important photophysical property in bioimaging, Pttc-SeBTa-
NIR1125 Pdots exhibited outstanding photostability under
with resembling chemical construction in which the mechanisms
of the regioselective cleavage on the heptamethine cyanines by
exogenously generated 1
O
2
have been claimed experimentally
On the other hand, NIR1125 and
[
3a, 17]
and computationally.
NIR1380 analogues exhibited high stability in organic solvents
and in Pdot forms. We speculate that it is probably due to the
shorter intermediate of NIR1125 and rigid center of NIR1380. The
absorption
and
emission
spectra
of
Pttc-SeBTa-
NIR1125/NIR1270/NIR1380 Pdots were shown in Figure 2B. The
absorption peaks at ~450 nm were attributed to the Pttc and
polyfluorene segments. The peak at ~800 nm stemmed from the

-bridge units, SeBTa, while the broad peaks from 900 to 1400
nm were from the polymethine-cyanine monomers. It clearly
indicates that these Pdots can be readily excited by several long
wavelength lasers depending on the experimental requirement.
The resulting Pdots possess the hydrodynamic diameters of 35-
73 nm (Figure S3) and the typical distribution of hydrodynamic
diameter of Ptt-SeBTa-NIR1125 Pdots along with their
transmission electron microscope (TEM) image were displayed in
Figure 2C.
3
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RESEARCH ARTICLE
2
between two thiopyrylium rings decreased from 4.13o to 1.90o,
indicating the enhanced coplanar architectures along the p-
conjugation backbones with the lengthening polymethine chains.
Interestingly, we found that the central dimethylcyclohexene
moiety of NIR1380 extended outwards the conjugated plane to
provide the relatively large steric hindrance. The incorporation of
steric hindrance on the polymethine chain is of importance to
design stable polymethine fluorophores for bioimaging. The lack
of steric hindrance on the extended polymethine chain (e.g.,
NIR1270) would result in the serious C-C oxidative cleavage
under exposure to nucleophiles as mentioned above. This
phenomenon could also explain for the excellent photostability of
NIR1380 even with the longest conjugation length among these
fluorophores, while NIR1270 appeared to possess very low
photostability. The strategies designed to increase the stability
and decrease photobleaching of polymethine dyes in aqueous
media have recently be nicely reviewed by Zhang’s group,[18] in
which the conclusions are consistent with our results here. It is
worth mentioning that the commercially available NIR-II dyes, IR-
continuous 808 nm light illumination (0.8 W/cm ), in which their
fluorescence remained over 90 % after 120 min of 808 nm light
exposure as shown in Figure 3C. For Pttc-SeBTa-NIR1380 Pdots,
the emission signal still remained nearly 70 % of their original
intensity. On the other hand, small organic dyes such as IR-1061
and ICG, displayed significant photobleaching to less than 10 %
of their original intensities in 70 min. These results clearly
indicated the exceptional photostability of Pdots as compared to
small organic fluorophores. We also evaluated the colloidal
o
stability of Pdots in the cell culture media at 37 C to mimic the
physiological conditions. As shown in Figure 3D, both Pttc-
SeBTa-NIR1125 and Pttc-SeBTa-NIR1380 Pdots unveiled
superior stability even after 2 h of incubation, while NIR1125 dye-
embedded nanoparticles showed poor colloidal stability under the
same conditions. It is worth mentioning that no fluorescence could
be detected for NIR1380 dye encapsulated nanoparticles,
probably due to the serious ACQ behaviors. This results again
emphasized the importance of the employment of the bulky
polymer skeletons.
26, IR-1061, and IR-1048, each has a chlorocyclohexene unit on
the center of its backbone (without the dimethyl moieties) and an
even shorter methine unit of 5. It was found that these dye-loaded
phospholipid micelles were prone to rapid degradation in polar
[19]
solvent. This behavior highlights the uniqueness of our stable
NIR1380. To better understand the stability of these fluorophores
from the aspect of charge distribution in a molecule, we performed
the maps of electrostatic potential on the molecular surfaces as
shown in Figure 4D. The oxidative cleavage process on the
polymethine backbone immediately after the formation of C(OO)C
dioxetane unit on C27/C50 carbon atoms (plotted as red and blue,
respectively) has been proved to be the major reaction
Figure 4. (A) Calculated HOMOs and LUMOs of the molecular fluorophores.
0 1
(B) Optimized S and S geometries and the dihedral angles of these NIR-II
[17]
fluorophores by density functional theory. (C) Multi-view images of optimized
molecular geometries of NIR1125/NIR1270/NIR1380 in the ground state based
on B3LYP/6-31G(d) level, showing the torsional angles between two
thiopyrylium rings. (D) Maps of electrostatic potential surfaces of these dyes
pathway.
Electrostatic potential analysis reveals that the
C27/C50 atoms of NIR1270 have the favorably negative
electrostatic potential to form the dioxetane intermediate and
subsequent cleavage. Altogether, we have experimentally and
computationally confirmed the critical role of dimethylcyclohexene
shielding segment. This information is very important for chemists
to rationally design more stable polymethine-based fluorophores
with NIR-IIa/IIb fluorescence for bioimaging.
(upper panel) and computed reaction energies of two possible sites (C27 and
C50) for cleavage reactions.
To further elucidate the influence of different intermediate
lengths on the electronic energies and geometries of the
fluorescent molecules, we executed time-dependent density
functional theory (TDDFT) calculations at the B3LYP/6-31G(d)
level using the Spartan software (the computational details were
provided in the Supporting Information). Their highest occupied
molecular orbitals (HOMOs) and lowest unoccupied molecular
orbitals (LUMOs) are illustrated in Figure 4A. We can clearly
observe that the extension of methine units effectively lowered the
energy band gaps, leading to the redshifting in the both absorption
and emission of the dyes of more than 200 nm (NIR1125 vs
NIR1380). The HOMOs and LUMOs are delocalized over the
polymethine backbones for all of the dyes. The partial separation
of the LUMOs and HOMOs on the thiopyrylium (electron-donating
group) and thiopyrylium salt (electron-withdrawing group)
moieties accounted for intramolecular charge transfer between
The fluorescence quantum yields of the resulting Pdots
were determined by using IR-1061 in CH Cl as the reference.
2 2
Instead of using IR-26 as the reference owing to the variations of
its quantum yields on different reports,[4o, 8a, 14b] we used IR-1061
,[20] in which its quantum
2 2
with a certified value of 0.59 % in CH Cl
yield was carefully corrected for reabsorption and reemission
under the excitation light at 925 nm. Pttc-SeBTa-NIR1125 Pdots
exhibited a good quantum yield of 0.18 %, while Pttc-SeBTa-
NIR1380 Pdots possessed a modest quantum yield of 0.05 % in
aqueous solutions (Table S1). Even the fluorescence quantum
yield of the Pdots might not be very high as compared to
traditional NIR small dyes, their fluorescence brightness, however,
showed 1-2 orders of magnitude higher than that of commonly
used organic dyes (Table S2). Moreover, the fluorescence
background from biological species in the NIR-II window is low to
ensure the practical implantation of these Pdots for in vivo
bioimaging.
0 1
them. The optimized ground-state (S ) and excited-state (S )
geometries of these molecules were also plotted in Figure 4B. The
dihedral angles between the thiopyrylium rings and polymethine
chain are all smaller than 4o at both S
0 1
and S states for all
molecules, suggesting the low backbone distortion of these dyes.
From the multi-view images as shown in Figure 4C, these
molecules all exhibited good coplanar geometries along the
polymethine chains. As the methine units extended from 5 (i.e.,
NIR1125) to 9 (i.e., NIR1380), the torsional angles (side view)
4
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RESEARCH ARTICLE
Figure 5. (A) Photographs of Pttc-SeBTa-NIR1125 (left) and Pttc-SeBTa-NIR1380 (right) Pdots under ambient light (first panel), and under 808 nm laser
excitation with a 1020-nm long-pass filter (second panel), 1100-nm long-pass filter (third panel), 1250-nm long-pass filter (fourth panel), and 1312-nm long-pass
filter (fifth panel). (B) Whole-body imaging of living mice intravenously injected by Pttc-SeBTa-NIR1125 Pdots in supine (left) and prone (right) positions with a
1
100-nm long-pass filter. (C) Whole-body imaging of living mice intravenously injected by Pttc-SeBTa-NIR1380 Pdots in supine (left) and prone (right) positions
with a 1250-nm long-pass filter. (D) Whole-body imaging of living mice intravenously injected by ICG in supine (left) and prone (right) positions with a 1250-nm
long-pass filter. (E) The enlarged view of mouse hindlimb vasculature in the green squares of (B) (upper panel) and (C) (bottom panel), respectively. (F) Cross-
sectional intensity profiles along the green lines in (E) for Pttc-SeBTa-NIR1125 (left) and Pttc-SeBTa-NIR1380 Pdots (right), respectively. (G) The enlarged view
of the area close to the spinal cord in the red squares of (B) (upper panel) and (C) (bottom panel), respectively. (H) Cross-sectional intensity profiles along the
red lines in (G) for Pttc-SeBTa-NIR1125 (left) and Pttc-SeBTa-NIR1380 Pdots (right), respectively. (I) NIR-II fluorescence imaging of wild-type C57BL/6 mouse
brain vasculature after intravenous injection of Pttc-SeBTa-NIR1125 (left, 1100-nm long-pass filter), Pttc-SeBTa-NIR1380 (middle, 1250-nm long-pass filter)
Pdots, and ICG (right, 1250-nm long-pass filter). (J) NIR-II fluorescence imaging of ND2:SmoA1 mouse brain vasculature after intravenous injection of Pttc-
SeBTa-NIR1380 (1250-nm long-pass filter) Pdots. (I) Accumulation of Pttc-SeBTa-NIR1125 (upper panel in the inset) and Pttc-SeBTa-NIR1380 (bottom panel
in the inset) Pdots in major excised organs at 24 h post-injection. Their corresponding quantitative mean fluorescence intensities were also plotted. The scale
2
bars are 10 and 5 mm in B/C and I, respectively. The excitation power laser density was 5 mW/cm with an exposure time of 800 ms. The mice were injected
with 200 L of Pdots (1.6 mg/mL) or ICG (200 g/mL) via intravenous injection through. NIR-II imaging was performed after 10 min of post-injection For ICG
experiments, all of the experimental conditions including laser power and filter sets were the same with Pttc-SeBTa-NIR1380 Pdots except that the acquisition
time was 5 min of post-injection due to the fast renal clearance rate of ICG.
For biological applications, we first evaluated the
cytotoxicity of Pdots by using MTT assays at different
concentrations (Figure S5). The results revealed that minimal
toxicity of Pdots in cells. We also performed the experiments of
hepatotoxicity by intravenously injecting PBS and Pdots into the
two separate mice. After 24 h post-injection, we measured the
values of AST (aspartate aminotransferase; GOT, glutamate
oxalacetate transaminase) and ALT (alanine aminotransferase;
GPT, glutamate pyruvate transaminase) in serum to evaluate the
liver function under Pdot treatment. Serum levels of AST at 24 h
after injection were measured to be 129 and 76 U/L for PBS-
treated and Pdot-treated mice, respectively. For the serum levels
of ALT at 24 h after injection, they were determined to be 42 and
1250, and 1312 nm). Although it was obvious to find that the use
of a shorter long-pass filter (i.e., 1020 nm) appeared the highest
fluorescence brightness, the spatial resolution and the signal-to-
background ratio (SBR) for in vivo imaging presented the opposite
[3b, 4o, 14b]
way due to strong background interference.
This means
that the selection of a longer long-pass filter while keeping the
high brightness is prerequisite. As a trade-off, we used a 1100-
nm long-pass filter for Pttc-SeBTa-NIR1125 Pdots (traditional
NIR-II) and a 1250-nm long-pass filter for Pttc-SeBTa-NIR1380
Pdots (NIR-IIb), respectively. We further executed whole-body
fluorescence imaging in living mice by intravenously injecting
these two Pdots separately into mice via the tail vein and
compared their performance (Figure 5B-E). From Figure 5B-C, it
can be clearly seen that the fluorescence imaging performed by
Pttc-SeBTa-NIR1380 Pdots equipped with a 1250-nm long-pass
filter revealed a significantly higher SBR as compared to the 1125
ones. On the contrary, the imaging resolution obtained by ICG
appears to be very poor as shown in Figure 5D. From the
33 U/L for PBS-treated and Pdot-treated mice, respectively. Both
values were in the normal ranges for PBS-treated or Pdot-treated
mouse, indicating that the livers were still in normal function for
both mice. We further carried out histological examination of livers
by analyzing the H&E staining of liver tissues for both mice (Figure
S6). The results suggest that Pdots have negligible hepatotoxicity. enlarged view of the mouse hindlimb vasculature (Figure 5E),
Before the in vivo non-invasive fluorescence imaging in mice, we
examined the optical parameters of two types of Pdots with high
these two Pdots exhibited the similar spatial resolution of 0.53-
0.61 mm while the Pttc-SeBTa-NIR1380 Pdots showed an
enhanced SBR of 2.32, about 1.2 times higher than that of Pttc-
SeBTa-NIR1125 Pdots (Figure 5F). These results further
confirmed the advantages of NIR-IIb imaging with nearly zero
autofluorescence and minimal photon scattering. Similarly, the
stability which are suitable for bioimaging. As displayed in Figure
2
5
A, we used a 808-nm laser with low excitation power (5 mW/cm )
to minimize the photo-damage to mice as well as Pdots, and
equipped with four different types of long-pass filters (1020, 1100,
5
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
blood vessels nearby the spinal cord could also be easily
visualized but the Pttc-SeBTa-NIR1380 Pdots offered a much
higher resolution due to the low background interference (Figure
Areas Research Center Program within the framework of the
Higher Education Sprout Project by the Ministry of Education
(MOE) in Taiwan.
5G-H). It should be noted that the direct comparison of spatial
resolution and SBR with other imaging platforms might be less
scientifically important because these values depend highly on
the quantum efficiency of CCD camera, the concentration of
probes, imaging software, and the power/wavelength of the
excitation light; rather than simply on the fluorescence brightness
of the probes used. The cerebral blood vessels of mouse through
intact scalp and skull could be distinctly delineated with fine
vascular structures (Figure 5I) by taking the advantages of high
penetration depth and minimized background interference in the
NIR-II window. The sharp resolution of NIR-IIb imaging were
again highlighted by the use of Pttc-SeBTa-NIR1380 Pdots
Keywords: semiconducting polymers • NIR-II • narrow-band
fluorescence • synthesis of Pdots • deep-tissue imaging
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RESEARCH ARTICLE
Entry for the Table of Contents
A novel series of polymethine-based semiconducting polymers were synthesized for the first time and prepared as Pdots in water with
both absorption and emission in the NIR-II window. These Pdots can be further applied for deep-tissue non-invasive fluorescence
imaging.
8
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