Fluorination Enhances NIR-II Fluorescence of Polymer Dots for Quantitative Brain Tumor Imaging

Article abstract of DOI:10.1002/anie.202007886

Here, we describe a fluorination strategy for semiconducting polymers for the development of highly bright second near-infrared region (NIR-II) probes. Tetrafluorination yielded a fluorescence QY of 3.2 % for the polymer dots (Pdots), over a 3-fold enhanc

Full text of DOI:10.1002/anie.202007886

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Fluorination Chemistry Enhances NIR-II Fluorescence of Polymer
Dots for Quantitative Brain Tumor Imaging
Authors: Ye Liu, Jinfeng Liu, Dandan Chen, Xiaosha Wang, Zhe
Zhang, Yicheng Yang, Lihui Jiang, Weizhi Qi, Ziyuan Ye,
Shuqing He, Quanying Liu, Lei Xi, Yingping Zou, and
Changfeng Wu
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202007886
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Angewandte Chemie International Edition
10.1002/anie.202007886
RESEARCH ARTICLE
Fluorination Chemistry Enhances NIR-II Fluorescence of Polymer
Dots for Quantitative Brain Tumor Imaging
[
a]
[a]
[b]
[a]
[b]
[b]
[a]
Ye Liu, Jinfeng Liu, Dandan Chen, Xiaosha Wang, Zhe Zhang, Yicheng Yang, Lihui Jiang,
[
b]
[b]
[b]
[b]
[b]
[a]
[b]
Weizhi Qi, Ziyuan Ye, Shuqing He, Quanying Liu, Lei Xi, Yingping Zou,* Changfeng Wu*
[
a]
Dr. Y. Liu, J. Liu, X. Wang, Prof. L. Jiang, Prof. Y. Zou
College of Chemistry and Chemical Engineering, Molecular Imaging Research Center
Central South University
Changsha 410083, China
E-mail: yingpingzou@csu.edu.cn
[
b]
Dr. D. Chen, Z. Zhang, Y. Yang, W. Qi, Z. Ye, Prof. S. He, Prof. Q. Liu, Prof. L. Xi, Prof. C. Wu
Department of Biomedical Engineering
Southern University of Science and Technology
Shenzhen, Guangdong 518055, China
E-mail: wucf@sustech.edu.cn
Supporting information for this article is given via a link at the end of the document.
Abstract: Fluorescent probes in the second near-infrared region
NIR-II, 1000~1700 nm) have resulted in unprecedented imaging
performance. Here, we describe fluorination strategy in
intrinsic limitations of the current NIR-II probes, such as low
absorptivity and poor quantum yields (QYs) have become the
bottleneck in further development of high-contrast in vivo
imaging techniques.
(
a
semiconducting polymers for development of highly bright NIR-II
probes. Notably, tetrafluorination yielded a fluorescence QY of 3.2%
for the polymer dots (Pdots), over 3-fold enhancement as compared
to non-fluorinated counterparts. The fluorescence enhancement was
attributable to nanoscale fluorous effect in the Pdots that maintained
the molecular planarity and minimized the structure distortion
between the excited state and ground state, thus reducing the
nonradiative relaxations. Followed by through-skull and through-
scalp imaging of the brain vasculature of live mice, we quantitatively
analyzed the vascular morphology of transgenic brain tumors in
terms of the vessel lengths, vessel branches, and vessel symmetry,
which showed statistically significant differences from the wild type
animals. The bright NIR-II Pdots achieved by fluorination chemistry
provide insightful information for precise diagnosis of the malignancy
of the brain tumor.
Semiconducting polymer dots (Pdots) exhibit optical
properties such as high fluorescence brightness, fast emission
rate, good photostability, and nontoxic features.[8] The superior
properties of Pdots over other fluorescent probes have
established their potential in biology and medicine as very bright
in vitro and in vivo probes.[9] However, semiconducting polymers
are weakly fluorescent in the NIR-II region. Moreover, the Pdots
in aqueous solutions exhibit severe fluorescence quenching
because the strong interchain interactions lead to energy
funneling to low-energy chromophores.[10] Recently, we reported
a molecular engineering strategy towards the development of
NIR-II fluorescent Pdots by managing aggregation-induced
emission (AIE) of the polymer backbone and minimizing
aggregation-caused quenching (ACQ) with steric hindrance of
side-chain groups.[11] Despite the progresses, the development
of semiconducting polymers with bright fluorescence in the NIR-
II window remains challenging.
Introduction
Fluorination of semiconducting polymers has emerged as a
powerful strategy for fine-tuning their physical and chemical
properties.[12] With large Pauling electronegativity and a small
Fluorescence techniques are leading to a rapid proliferation of
advanced diagnostic tools in biomedicine.[1] In particular, in vivo
fluorescence imaging has resulted in significant advances in our
understanding of various physiological processes and disease
van der Waals radius, fluorine as a substituent in π-conjugated
backbones can exert a great impact on the morphology and
optical properties of the fluorinated semiconducting polymers.
Significant effects discovered include lowering the molecular
energy levels, enhancing intermolecular charge transfer (ICT),
development.[2] Recent research efforts have revealed
a
promising imaging window in the second near-infrared
wavelength region (NIR-II, 1000~1700 nm). Reduced photon
scattering in this spectral region allows fluorescence imaging at
a significantly enhanced penetration depth and high signal-to-
noise ratio as compared to the traditional wave-length region
improving the molecular planarity, and affecting the orientation
of polymer chains in the thin films.[13] Because of these important
factors, many peculiar properties of such fluorinated
semiconducting polymers have been disclosed.[14] The Pdots
(
400~900 nm).[3] In a pioneer study, Dai and co-workers utilized
consist of densely packed polymeric chromophores, thus
exhibiting chain-chain interactions and photophysical properties
that resemble those in thin-film devices. The fluorination strategy
in organic electronics is indicative of viable opportunities for
enhancing the fluorescence of Pdot probes. For instance, Chiu
and co-workers developed a highly fluorescent Pdots with a QY
the NIR-II photoluminescence of single-walled carbon nanotubes
in a non-invasive through-skull brain imaging, which resolved
cerebral vasculatures at a high spatial resolution of sub-10 μm
and a depth of >2 mm in an epifluorescence imaging mode.[4]
Because of the unprecedented imaging quality, there is a great
deal of attention in the development of various NIR-II
fluorophores, including quantum dots (QDs),[5] lanthanide
nanoparticles,[6] and organic fluorophores.[7] However, the
up to 49% in the visible region, which was 8 times brighter than
its non-fluorinated counterpart.[15] However, such effects are
largely unexplored for semiconducting polymers in NIR-II region.
1
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RESEARCH ARTICLE
Figure 1. (a) Synthetic routes and fluorination strategy of semiconducting polymers. Fluorination was performed by varying the number and position of the fluorine
substituent on the TQ acceptor of the semiconducting polymers. (b) Schematic illustration of nanoscale fluorous effect to maintain hydrophobic interior and
minimize structure distortion of the Pdots. The fluorination redshifts the optical spectra and enhances the fluorescence quantum yield.
Here, we describe a fluorination strategy for development of
highly fluorescent polymers in the NIR-II window. We designed
two sets of fluorine substituted semiconducting polymers by
using benzodithiophene (BDT) and triazole[4,5-g]-quinoxaline
ground-state, thus reducing the nonradiative relaxations. Finally,
we use the Pdots to demonstrate in vivo fluorescence imaging of
the brain-tumor vasculature through the mouse skull, which
showed a remarkable improvement in penetration depth and
signal to background ratio (SBR). These results indicate that the
fluorination strategy holds promise for development of highly
fluorescent NIR-II fluorophores.
(TQ) derivatives used as donor and acceptors, respectively. We
examined the effect of fluorination on the NIR-II fluorescence of
the polymers as a function of the number and position of
fluorination on the acceptor unit, aiming to outline a clear
understanding of the benefits of the fluorine substitution. We
discovered that fluorination significantly enhanced the Results and Discussion
fluorescence of Pdots in aqueous solution, which can be
attributable to the nanoscale fluorous effect in the fluorinated
Pdots. Density functional theory reveals that fluorination
minimizes the structure distortion between the excited-state and
Molecular design and fluorination of semiconducting
polymers. There have been recent efforts in the development of
NIR-II organic fluorophores by judicious selection of building
2
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RESEARCH ARTICLE
blocks and side-chain engineering to form π-conjugated
molecules.[16] In this study, we explore the fluorination strategy in
semiconducting polymers to obtain bright NIR-II probes. We
start from two polymers with a donor-acceptor structure, in which
BDT and TQ derivatives were used as donor and acceptors,
respectively. The semiconducting polymers synthesized from
BDT and TQ monomers can emit weak fluorescence in the NIR-
II region.[9b] The large π-conjugated backbones in these
polymers provide several optional positions for fluorine
substituents in the acceptor units. As fluorination of the acceptor
unit is the most adopted strategy for donor-acceptor polymers,[13]
we examine the effect of fluorination on NIR-II fluorescence by
varying the number and position of the fluorine substituent on
the TQ acceptor. In the TQ unit, the alkoxyl chains anchored on
the meta-position of benzene can provide different levels of
hydrophobicity and steric hindrance as compared to the alkoxyl
chains anchored on the para-position of benzene. Therefore, we
performed fluorination in the two types of acceptor structures. As
shown in Figure 1a, we designed and synthesized six polymers,
namely m-PBTQ, m-PBTQ2F, m-PBTQ4F polymers with the
alkoxyl chains anchored on the meta-position of benzene, and
p-PBTQ, p-PBTQ2F, p-PBTQ4F polymers with alkoxyl chains
anchored on the para-position of benzene.
Figure 2. (a) Absorption spectra of the semiconducting polymers in THF solutions. (b) Fluorescence emission spectra of the polymers in THF. (c) Integrated
fluorescence intensity plotted as a function of OD at 808 nm for the polymers in THF and IR-26 in 1,2-dichloroethane. (d) Quantum yields of the semiconducting
polymers in THF solutions.
We rationalize that the fluorination of PBTQ polymers can
effectively modulate the optical properties of the resulting Pdots,
including the energy level lowering and fluorescence
enhancement, in multiple manners (Figure 1b). Because of the
strength of the C-F bond, fluorinated compounds show a high
thermal and oxidative stability, high hydrophobicity, weak
intermolecular interactions, and a small surface tension as
compared to their non-fluorinated counterparts.[17] These factors
can enable the Pdots to maintain a hydrophobic interior and
reduce chain-chain interactions. Because fluorination also
increases the planarity of the conjugated backbone, thus
suppressing the formation of bending or kinking of the polymer
chains. Taken together, the fluorination strategy can provide a
viable approach for enhancing the NIR-II fluorescence of Pdots.
The fluorination of the semiconducting polymers were
designed and implemented according to the synthetic strategy
described in Scheme S1. The diketone compounds
(b1/b2/b3/e1/e2/e3) were synthesized by non-fluorine (a1/d1) or
fluorine-substituted (a2/a3/d2/d3) benzene derivatives via
Grignard
reaction.
Then,
the
TQ
acceptors
(M1/M2/M3/M4/M5/M6) were synthesized from the diketone
compounds and organic amine (c) dehydration that formed a
pyrazine ring. Finally, TQ acceptors and BDT were polymerized
by the Stille coupling reaction. The detailed synthesis of
monomers and polymers, general characterizations including
optical spectroscopy (Figure S1-S7), cellular assays (Figure
S8), imaging data (Figure S9-S11), and NMR spectroscopy
(Figure S12-S39) were provided in Supporting Information. The
n
number average molecular weight (M ) and polydispersity index
(PDI) of the semiconducting polymers were measured by gel
permeation chromatography (Table S1).
3
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We investigated the optical properties of the semiconducting
polymers in organic solvents. As shown by Figure 2a and 2b,
the absorption and emission spectra of polymers in THF were
gradually red-shifted as the number of fluorine atoms on TQ
increased. The absorption spectra of these polymers stride the
traditional NIR-I window, which can be conveniently excited by
an 808 nm laser. The fluorescence of the polymers covers the
NIR-II spectral region, with their shoulder peaks extending
beyond 1300 nm. The fluorescence QYs of these polymers in
THF were determined by using IR26 dye as a reference (Figure
brightness and photostability of the m-PBTQ4F Pdots for
fluorescence imaging in the NIR-II window.
Mechanistic study on fluorination enhanced fluorescence.
The m-PBTQ and p-PBTQ polymers series in organic solvents
present relatively high fluorescence QYs. However, the Pdots
show fluorescence quenching in comparison with the original
polymers in organic solvents. The quenching can be attributed to
intense inter- and intra-chain interactions, whereas the bending
or kinking of the polymer chains in Pdots result in the formation
of non-emissive species.[19] In addition, energy migration in the
large π-conjugated systems may also lead to energy funneling
to the non-emissive species, amplifying the fluorescence
quenching effect. Both m-PBTQ and p-PBTQ polymer series
show consistent variation trend in the fluorination enhanced
fluorescence. While each polymer series in organic solvents
show only minor changes, the resulting Pdots exhibit
significantly different QYs owing to fluorination. Most probably,
fluorous effect plays an important role in the enhanced
fluorescence of the Pdots. Fluorous effect can be described as
the tendency of fluorinated compounds to segregate in order to
favor fluorine–fluorine interactions, or more appropriately to
avoid unfavored interactions with other elements.[20] Presumably,
the fluorination can lead to a hydrophobic nanoparticle interior
as well as planar polymer conformation as compared to the
nonfluorinated Pdots. Figure S6 showed the absorption and
fluorescence spectra of the p-PBTQ polymer series in THF
solutions and the related Pdots in water, respectively. For the
nonfluorianted p-PBTQ polymer, the absorption and
fluorescence spectra of the Pdots in water were
inhomogeneously broadened and the vibronic peaks were
significantly increased as compared to those of the polymer in
THF solutions. These results are consistent with previous
studies that the increased conformational disorder in conjugated
polymers increased the inhomogeneous spectral broadening
and enhanced the 0-1 intensity relative to the 0-0 intensity.[21] In
contrast, the tetrafluorinated p-PBTQ4F Pdots show slightly
redshifted absorption and fluorescence with only minor increase
in vibronic peaks as compared to those of the polymer in THF
solutions. The m-PBTQ4F Pdots showed similar spectral shape
to those of the polymer in THF solution (Figure S7). These
spectroscopic results support the presumption that the
fluorination leads to planar polymer conformation and reduce
conformational disorder during formation of the Pdots.
2c). The QYs of the six polymers varied from ~6.6% to ~9.5%,
(
Figure 2d), indicating that the fluorination has only minor effect
on the fluorescence of the polymers in organic solvents. It is
worth noting that the QY of ~9.5% was the highest among
various NIR-II fluorophores, indicate that these polymers have
great potential in the NIR-II imaging.
Fluorination enhanced fluorescence in Pdots. The Pdots
were prepared from the semiconducting polymers according to a
nanoprecipitation method described in previous reports.[8a,18]
A
functional amphiphilic polystyrene polymer (PS-PEG-COOH)
was used to form PEGylated Pdots. The resulting Pdots have a
hydrodynamic diameter of ~22 nm, as determined by dynamic
light scattering. The particle size and spherical morphologies of
the Pdots were confirmed by transmission electron microscopy
(
Figure 3a). All the Pdots exhibited good colloidal stability in
aqueous solutions for months. As shown by the optical
spectroscopy (Figure 3b and 3c), fluorination red-shifted the
absorption and emission spectra of the Pdots, following the
same trend as those for the polymers in THF. The absorption of
the Pdots were normalized according to an optical density value
of 0.1 at 808 nm (Figure 3b). Under the same excitation density
at 808 nm, the fluorescence spectra of the Pdots were shown in
Figure 3c. In contrast with the similar fluorescence intensities
for the polymers in THF solutions, the fluorinated Pdots showed
significantly enhanced fluorescence as compared to the non-
fluorinated counterparts. Impressively, the m-PBTQ Pdots series
exhibited higher QYs than the p-PBTQ Pdots series, and in
each series, the QYs of Pdots were greatly improved as the
number of fluorine atoms on the acceptor increased. The
tetrafluorinated m-PBTQ4F Pdots achieved a QY of ~3.2%,
which was the highest reported so far for NIR-II Pdots. This
value was over 3-fold higher than that of the non-fluorinated m-
PBTQ Pdots (~1.0%) and 5-fold higher than that of non-
fluorinated p-PBTQ Pdots (~0.6%).
The QY ratios of the Pdots relative to the polymers in THF
were plotted in Figure 3f, which measures the extent of
fluorescence quenching in Pdots. As indicated, the value of m-
PBTQ4F (~0.38) was over 3-fold higher than that of m-PBTQ
(~0.12), whereas the value of p-PBTQ4F (~0.21) was over 2-
fold higher than that of p-PBTQ (~0.09), indicating that the
fluorescence of the m-PBTQ4F polymer was largely retained in
the Pdots with only moderate quenching. It is surprising to
observe the QY of the m-PBTQ4F Pdots (~3.2%) was over 2
times higher than that of the p-PBTQ4F (~1.5%) despite their
similar QYs for the polymers in THF solution. This is an
additional evidence for fluorous effect as the four fluorine atoms
in m-PBTQ4F are in proximity to each other, thus collectively
We compared the imaging performance of the fluorinated
Pdots with two organic dyes such as Indocyanine Green (ICG)
and IR26. As shown in Figure 3d, NIR-II fluorescent images of
IR26 (in DCE), ICG in water, and m-PBTQ4F Pdots in water
were taken with the same weight concentration (100 μg/mL),
which indicated the fluorescence signal of m-PBTQ4F Pdots
was ~5 times brighter than that of ICG and ~4 times than IR26,
respectively. Furthermore, the photostability of ICG in water,
IR26 in dimethyl sulfoxide, and m-PBTQ4F Pdots in water with
the same weight concentration (100 μg/mL) was examined
2
under continuous laser excitation (808-nm laser, 1 W/cm ) for
over 120 min (Figure 3e). In contrast to the poor photostability
of ICG and IR26, which exhibited severe photodegradation and
fluorescence decrease to ~20% in 20 min, the fluorescence of
m-PBTQ4F Pdots retained ~80% of the initial intensity after 120-
min laser illumination. These results indicate the superior
enabling
a
strong repulsion to water and chain-chain
aggregation. Because of the fluorous effect, the hydrophobicity
and molecular planarity of the polymer are largely retained in the
Pdots, suppressing the quenching by the inter-chain interactions
and non-emissive species.
4
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Figure 3. (a) Hydrodynamic diameter distribution of the m-PBTQ4F Pdots measured by dynamic light scattering. The insets show a typical transmission electron
microscope image of the m-PBTQ4F Pdots and photograph for m-PBTQ4F Pdots solution at 100 μg/mL concentration. (b) Absorption spectra of the Pdots with
the optical density of 0.1 at 808 nm. (c) Fluorescence spectra of the Pdots measured with the same excitation density at 808 nm. (d) The mean fluorescence
2
intensity of ICG in water, IR-26 in 1,2-dichloroethane, m-PBTQ4F Pdots in water with the same mass concentration (100 μg/mL) (30 mW/cm , 808 nm laser, 1000
nm LP filter). The insets show the corresponding photograph and NIR-II fluorescent images. (e) Photostability of ICG in water, IR-26 in dimethyl sulfoxide, and m-
2
PBTQ4F Pdots in water with the same mass concentration (100 μg/mL) under continuous 808 nm radiation (1 W/cm ) for 120 min. I/I
0
represents the ratio of the
fluorescence intensity of the samples relative to the initial intensity. The inset shows the corresponding NIR-II fluorescent images. (f) The ratios of the quantum
yields of the Pdots relative to the polymer in THF solutions. (g) Theoretical calculation results of the HOMOs and LUMOs of m-PBTQ series. (h) Optimized S and
geometries and the dihedral angles for the m-PBTQ series by density functional theory.
0
S
1
To elucidate the fluorination enhanced fluorescence, we
effectively lower the HOMO and LUMO energy levels of the
polymers. The energy gaps (E ) calculated from the HOMO and
carried out theoretical calculations regarding the energy levels
and molecular geometries of m-PBTQ series by using density
functional theory (DFT) and time-dependent density functional
theory (TDDFT). In the calculation, we calculate only one
repeating-unit (A-D-A) in water owing to the limit of computation
power. As shown in Figure 3g, all the HOMOs are mainly
located on the BDT donor, and the LUMOs are mainly localized
on the TQ acceptor. Additionally, both the HOMO and LUMO
energy levels were reduced by increasing the number of fluorine
atoms on the TQ unit. It indicates that the fluorination could
g
LUMO were 2.00 eV for m-PBTQ, 1.95 eV for m-PBTQ2F, and
1.91 eV for m-PBTQ4F, respectively. This is consistent with the
experimental results by spectroscopy that the fluorination could
narrow the energy gap and red-shift both the absorption and
emission spectra.
We further analyzed the molecular geometry structures to
better understand the fluorination enhanced fluorescence. The
energy gap law dictates that as the emission wavelength shifts
to the red, the nonradiative decay of the excited state becomes
5
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RESEARCH ARTICLE
more dominant.[22] To decrease the nonradiative decay rate, the
structural distortion between the emitting excited-state and the
ground-state must be minimized.[23] Besides the probable
suppression of non-emissive species, fluorination may reduce
the structure distortion between the excited and ground states to
enhance the emission QY of the Pdots. We computed the
the right acceptor and middle donor) were computed and
summarized in Table S2. As seen clearly, the differences (Δ
0 1
values) in the dihedral angles between S and S were
determined to be 4.3° and 4.5° for m-PBTQ, 4.2° and 3.0° for m-
PBTQ2F, and 0.8° and 0.7° for m-PBTQ4F, respectively. As the
number of fluorine atoms increased, the Δ values apparently
ground-state (S
0
) and first singlet excited-state (S
1
) geometries
decreased, indicating reduced structure distortion between S
and S state for the tetrafluorinated m-PBTQ4F as compared to
0
of the m-PBTQ series (Figure 3h). Considering A-D-A unit, two
1
dihedral angels were calculated in terms of the left and the right
non-fluorinated m-PBTQ. Taken together, the structural
distortion between the excited-state and the ground-state was
minimized by fluorination, thereby decreasing the nonradiative
decay rates and enhancing the emission QY.
acceptor relative to the middle donor. For both S
geometries, the dihedral angles Ф (dihedral angle between the
left acceptor and middle donor) and Ф (dihedral angle between
0 1
and S
L
R
Figure 4. (a) In vivo NIR-II whole-body fluorescence imaging of C57BL/6 mice in prone and supine positions after tail-vein injection of 100 μL m-PBTQ4F Pdots
200 μg/mL). (b) and (c) show the cross-sectional fluorescence intensity profile measured along the white line and Gaussian fits (red) in (a)-1 and (b)-2,
(
respectively. (d) In vivo NIR-II fluorescence imaging of cerebral vasculature of C57BL/6 mice injected with 100 μL ICG or m-PBTQ4F Pdots (200 μg/mL) at certain
2
time intervals from 1 to 120 min. (70 mW/cm , 808 nm laser, 1319 nm LP filter)
2
Quantitative through-skull imaging of brain vasculature.
We performed in vivo fluorescence imaging in the NIR-II window
by using the m-PBTQ4F Pdots in view of their high brightness
and good photostability. We first examined the cytotoxicity of
Pdots by measuring cell viabilities at various concentrations (0-
excitation density (70 mW/cm ) by a 808 nm laser was adopted
to reduce damage to the mice while maintaining image quality.
C57BL/6 mice were administered with the Pdots through the tail
vein (100 μL, 200 μg/mL). We identified that the imaging with a
1319 nm long-pass filter resulted in the best signal to noise ratio
(Figure S9). As indicated in Figure 4a, the whole-body imaging
of a mice in both supine and prone positions clearly show the
vascular structures throughout the body. As expected, these
images with tetrafluorinated m-PBTQ4F Pdots exhibited much
higher signal-to-noise ratio (SNR) than those with non-
100 μg/mL) in MTT assays. As shown in Figure S8, the cell
viability after 24 h incubation was over 90% even in the high
Pdot concentration (100 μg/mL), indicating good biocompatibility
of Pdots with cells. Next, we evaluated the in vivo whole-body
fluorescence imaging of m-PBTQ4F Pdots in live mice. Low
6
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RESEARCH ARTICLE
fluorinated m-PBTQ Pdots (Figure S10), confirming the superior
performance achieved by the fluorination strategy. In the prone
position, the images of the blood vessels in the mice’s back
C57BL/6 mice through the tail vein. Fluorescence images were
taken through-scalp and through-skull of the mice at certain time
intervals for 120 min (Figure 4d). As indicated, the fluorescence
signal of the cerebral vasculature with ICG declined very fast,
and almost disappeared in 5 min. In contrast, there was much
higher SNR and resolution in the imaging of the vasculature with
m-PBTQ4F Pdots, and it persisted for more than 2 h after Pdots
injection because of their long blood circulation time. This could
be advantageous for vascular imaging as well as imaging-
guided surgery. At the first minute post injection, the
fluorescence signal of m-PBTQ4F Pdots in the cerebral
vasculature was much stronger than that of ICG. These results
demonstrate that the performance of m-PBTQ4F Pdots, with
long imaging duration, high SNR and spatial resolution, is
superior to the clinically-approved imaging by ICG in the NIR-II
window.
(SBR=1.8) could be clearly observed (Figure 4b). The images of
the blood vessels of the hindlimbs can reach a high SBR of 5.5,
which is over 2-fold higher SBR (5.5 vs 2.4) than that of m-
PBTQ Pdots. These results demonstrate that the fluorinated
Pdots could offer high SBR and resolution in whole-body
fluorescence imaging.
Recent studies demonstrated that fluorescence detection in
the 1300~1400 nm region resulted in drastically improved SNR
and penetration depth in deep-tissue imaging in vivo.[4,11] We
explore the application of the fluorinated Pdots for through-scalp
and through-skull imaging of mice brain. We compared the
performance of m-PBTQ4F Pdots and ICG dye in the imaging of
mouse cerebral vasculature. The Pdots and ICG solution at the
same injection dose (200 μg/mL, 100 μL) were administered into
Figure 5. (a) In vivo NIR-II fluorescence images (top panel) and Hessian-matrix-enhanced images (bottom panel) of cerebral vasculature of wild-type C57BL/6
mice (n=5). (b) In vivo NIR-II fluorescence images (top panel) and Hessian-matrix-enhanced images (bottom panel) of cerebral vasculature of ND2:SmoA1 mice
(n=5). (c-f) Quantitative imaging of vascular morphology of the mouse brain by using a vascular segmentation and quantification algorithm. The data are analyzed
by two-sided Student’s t test. Red data are reported as mean ± standard deviation.
7
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Malignant brain tumors are characterized by a number of
The fluorine-substituted Pdots exhibit red-shifted emission and
high fluorescence QY which are superior properties for deep-
tissue NIR-II imaging. In one case, the QY of the tetrafluorinated
m-PBTQ4F Pdots, with good photostability, reached up to 3.2%
in aqueous solution, which was over 3-fold higher than that of
the non-fluorinated counterparts and 6-fold higher than that of
IR26. We postulate that the fluorination can result in planar
polymer conformation and reduce chain-chain interactions in the
fluorinated Pdots, which are consistent with the comparative
spectroscopic studies for the polymer in THF solutions and
Pdots in water. DFT calculations indicate that fluorination also
minimize the structure distortion between the excited and ground
states to enhance the emission QY of the Pdots. The fluorinated
Pdots exhibited a remarkable improvement in penetration depth
and signal to background ratio for deep tissue imaging, as
confirmed by through-skull and through-scalp quantitative
imaging of the brain-tumor vasculature of live mice. These
results indicate that fluorination is a promising strategy for the
design of highly fluorescence NIR-II fluorophores.
histopathological
features
including
infiltrative
growth,
microvascular proliferation and pleomorphic vessels.[24] Inspired
by the imaging results of mouse cerebral vasculature, we
attempt to explore the through-skull and through-scalp imaging
of brain tumor vasculature by using the fluorinated Pdots. We
chose a transgenic mouse model, ND2:SmoA1, because it
closely resembles human medulloblastoma, the most common
malignant childhood brain tumor.[25] For comparative studies, we
used five ND2:SmoA1 mice and five wild-type C57BL/6 mice,
and two of each group was shown in Figure 5 (the images of the
other 3 mice were provided in Figure S11). As indicated by the
through-skull imaging results, the brain vasculature in
ND2:SmoA1 mice is structurally abnormal as compared to that
of the wild-type animals. We can visually identify the significant
difference between the normal and brain tumor group. Whereas
the normal vasculature is arranged in a hierarchy of evenly
spaced and well-differentiated blood vessels, the tumor
vasculature is unevenly distributed and chaotic, exhibiting
serpentine courses and irregular branches. These imaging
results clearly reveal the vascular characteristics of
medulloblastoma as compared to the wild-type animals.
Acknowledgements
Finally, we assess and quantify the vascular morphology of
the mouse brain by using
a vascular segmentation and
C. Wu acknowledges financial support from Shenzhen Science
and Technology Innovation Commission (Grant No.
KQTD20170810111314625) and the National Natural Science
Foundation of China (Grant No. 81771930), and the National
Key Research and Development Program of China (Grant No.
quantification algorithm. Quantitative imaging of vascular
morphology of the medulloblastoma not only generates multiple
parameters regarding tumor angiogenesis, but also provides
insightful information for precise diagnosis of the malignancy of
the tumor. The vascular quantification algorithm is based on a
modified Hessian matrix method.[26] First, the original
fluorescence images (Top panel in Figure 5a and 5b) were
processed to generate the Hessian-matrix-enhanced images
2018YFB0407200). Y. Zou acknowledges the National Natural
Science Foundation of China (Grant No. 51673215), and
Science Fund for Distinguished Young Scholars of Hunan
Province (2017JJ1029).
(Bottom panel in Figure 5a and 5b). Then, blood vessels were
extracted from the enhanced images and the vascular
centerlines were further identified. Finally, using the extracted
image and the identified centerlines, the vascular morphological
parameters, including the total vessel length (Figure 5c), the
vessel branches (Figure 5d), and the vessel diameter entropy
Keywords: fluorination • fluorescent probes • polymer dots •
NIR-II optical imaging • brain tumor
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9
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RESEARCH ARTICLE
Entry for the Table of Contents
Fluorination chemistry in semiconducting polymers resulted in highly bright polymer dots (QY=3.2%) in the NIR-II region. Followed by
through-skull and through-scalp imaging of the mouse brain, the vascular morphology of transgenic brain tumors was quantitatively
analyzed.
1
0
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