Boosting Photoacoustic Effect via Intramolecular Motions Amplifying Thermal-to-Acoustic Conversion Efficiency for Adaptive Image-Guided Cancer Surgery

Article abstract of DOI:10.1002/anie.202109048

Photoacoustic (PA) imaging emerges as a promising technique for biomedical applications. The development of new strategies to boost PA conversion without depressing other properties (e.g., fluorescence) is highly desirable for multifunctional imaging but difficult to realize. Here, we report a new phenomenon that active intramolecular motions could promote PA signal by specifically increasing thermal-to-acoustic conversion efficiency. The compound with intense intramolecular motion exhibits amplified PA signal by elevating thermal-to-acoustic conversion, and the fluorescence also increases due to aggregation-induced emission signature. The simultaneously high PA and fluorescence brightness of TPA-TQ3 NPs enable precise image-guided surgery. The preoperative fluorescence and PA imaging are capable of locating orthotopic breast tumor in a high-contrast manner, and the intraoperative fluorescence imaging delineates tiny residual tumors. This study highlights a new design guideline of intramolecular motion amplifying PA effect.

Full text of DOI:10.1002/anie.202109048

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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International Edition
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Accepted Article
Title: Boosting Photoacoustic Effect via Intramolecular Motions
Amplifying Thermal-to-Acoustic Conversion Efficiency for
Adaptive Image-Guided Cancer Surgery
Authors: Heqi Gao, Xingchen Duan, Di Jiao, Yi Zeng, Xiaoyan Zheng,
Jingtian Zhang, Hanlin Ou, Ji Qi, and Dan Ding
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202109048
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10.1002/anie.202109048
Angewandte Chemie International Edition
RESEARCH ARTICLE
Boosting Photoacoustic Effect via Intramolecular Motions
Amplifying Thermal-to-Acoustic Conversion Efficiency for
Adaptive Image-Guided Cancer Surgery
Heqi Gao,+[a] Xingchen Duan,+[a] Di Jiao,[a] Yi Zeng,[b] Xiaoyan Zheng,*[b] Jingtian Zhang,[a] Hanlin Ou,[a]
Ji Qi,*[a] and Dan Ding*[a]
[a]
[b]
[+]
Dr. H. Gao, X. Duan, D. Jiao, J. Zhang, Dr. H. Ou, Prof. J. Qi, Prof. D. Ding
State Key Laboratory of Medicinal Chemical Biology, Frontiers Science Center for Cell Responses, Key Laboratory of Bioactive Materials, Ministry of
Education, and College of Life Sciences, Nankai University, Tianjin 300071, China
E-mail: qiji@nankai.edu.cn, dingd@nankai.edu.cn.
Y. Zeng, Prof. X. Zheng
Beijing Key Laboratory of Photoelectronic/Electrophotonic Conversion Materials, Key Laboratory of Cluster Science of Ministry of Education, School of
Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, China
E-mail: xiaoyanzheng@bit.edu.cn.
These authors contributed equally to this work
Supporting information for this article is given via a link at the end of the document.
Abstract: Photoacoustic (PA) imaging emerges as a promising
technique for biomedical applications. The development of new
strategies to boost PA conversion without depressing other properties
(e.g., fluorescence) is highly desirable for multifunctional imaging but
difficult to realize. Here, we report a new phenomenon that active
intramolecular motions could promote PA signal by specifically
increasing thermal-to-acoustic conversion efficiency. The compound
with intense intramolecular motion exhibits amplified PA signal by
elevating thermal-to-acoustic conversion, and the fluorescence also
increases due to aggregation-induced emission signature. The
simultaneously high PA and fluorescence brightness of TPA-TQ3 NPs
enable precise image-guided surgery. The preoperative fluorescence
and PA imaging are capable of locating orthotopic breast tumor in a
high-contrast manner, and the intraoperative fluorescence imaging
delineates tiny residual tumors. This study highlights a new design
guideline of intramolecular motion amplifying PA effect.
radiative decay from the excited state to ground state.[18-20]
Therefore, it has been widely accepted that PA and fluorescence
are opposite to or work against each other in one agent.[21-24]
Nevertheless, the intrinsic and complementary merits of PA and
fluorescence techniques make the integration of them an optimal
option for precise in vivo imaging by making full use of their
advantages. For example, PA technique possesses high tissue
penetration and spatial resolution, but suffers from relatively low
sensitivity, while fluorescence imaging, on the other hand, has
very high sensitivity but unsatisfied penetration.[25-28] Thus, it’s in
great demand to obtain both high PA and fluorescence signals at
the same time.
PA effect is closely related to two processes: photothermal
conversion and subsequent thermal-to-acoustic transformation of
a light-absorbing material.[29] These processes can be described
by the PA equation: p = ΓηH, where p is the PA pressure, Γ is the
Grüneisen parameter, η is the energy supplied by a laser pulse,
and H is the conversion efficiency from light into heat, or
photothermal conversion efficiency (PCE).[30-33] So far, intensive
investigations have focused on elevating PCE to improve PA
effect, yet the thermal-to-acoustic conversion efficiency or
Grüneisen parameter is seldom considered. The investigation on
thermal-to-acoustic conversion process may represent a new way
for maximizing PA signal. Grüneisen parameter is a key factor
responsible for the thermoelastic efficiency of materials, which
decidedly links to the molecular structure.[34,35] The understanding
and manipulation of Grüneisen parameter from molecular level is
thus of critical significance to boost PA effect and PA imaging
performance. To the best of our knowledge, nevertheless, such
studies are rare.
Introduction
Photoacoustic (PA) imaging has attracted tremendous attention
for biomedical applications as it offers centimeter-depth
penetration and high spatial resolution (~100 µm) simultaneously
by integrating the high contrast of optical imaging with the good
penetration of ultrasound.[1-4] For PA technique, a pulsed laser
beam usually irradiates on the light-absorbing molecules or
biospecies to produce localized thermal expansion, and then
generate pressure changes as ultrasound wave.[5-7] PA technique
has been adopted for preclinical diagnosis of many diseases in
vivo, for example, tumor imaging, inflammation and infection
monitoring, and metastatic lymph node detection.[8-13] It is highly
desirable to develop high-contrast agents that are able to
efficiently detect diseased site. Recently, organic/polymer
chromophores have been used for PA imaging thanks to the facile
synthesis and tunable material properties.[14-17] However, how to
realize maximal PA signal output is still a big challenge.
Molecular motion determines the basic properties of matter as
all macroscopic nature can be traced back to the microscopic
molecular motion.[36,37] How to tune and utilize the active
molecular motion is vitally important for both fundamental
researches and practical applications. For instance, molecular
machine has become a hot research topic in recent years
because it is expected to bring revolutions in many aspects of
According to the Jablonski diagram, PA signal is mainly related
to the thermal deactivation of a chromophore, while fluorescence
emission derives from another energy dissipation pathway, i.e.,
technology and medicine.[38-41]
A basic thermal expansion
principle that most matter expands when heated and contracts
when cooled can also be traced back to microcosmic molecular
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10.1002/anie.202109048
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RESEARCH ARTICLE
motion, in which the thermal expansion process is related to
Grüneisen parameter.[42,43] Therefore, manipulation of the
intramolecular motion is of critical importance as it greatly impacts
the photophysical energy transformation processes. Recently, we
and other groups reported that intramolecular motion could
effectively enhance PCE, which favors the related applications
such as photothermal therapy.[44-47] Nevertheless, the influence of
intramolecular motion on thermal-to-acoustic transformation
process has never been explored thus far, which may represent
a new way to tune PA property.
Moreover, the intraoperative fluorescence imaging helps to
delineate tiny residual tumors in a sensitive and fast manner,
greatly improving the surgery outcome. This work brings up a new
concept that the intense intramolecular motion helps to boost
thermal-to-acoustic transition and thus advances PA imaging and
complicated biomedical applications.
Results and Discussion
In this contribution, we demonstrate for the first time that
intramolecular motion could promote PA effect by amplifying
thermal-to-acoustic conversion, significantly benefitting the
biomedical applications (Scheme 1). By comparing the PA signal
of several analogs with different molecular rotors, we find that
both PA and fluorescence intensity enhance by increasing active
intramolecular motion, which is quite different from previous point
of view that fluorescence and PA are competitive processes.
Further investigation on the photothermal and PA transition
suggests that the active intramolecular motion is capable of
elevating Grüneisen parameter and thermal-to-acoustic
conversion. As a proof-of-concept, the optimal agent has been
used for precise image-guided surgery in orthotopic 4T1 breast
tumor-bearing mice. The preoperative fluorescence imaging
helps to localize tumor site in a fast way, and PA imaging further
provides detailed information such as tumor geometry and depth,
which offer unambiguous guidance for conducting surgery.
Design, Synthesis and Calculation. To obtain low-bandgap
chromophore with near-infrared (NIR) absorption, donor-acceptor
(D-A) strategy was employed, in which triphenylamine (TPA) and
thiadiazoloquinoxaline (TQ) were used as the D and A moieties,
respectively. In order to investigate the influence of intramolecular
motion on the PA property of organic chromophore, a series of
compounds based on the structure of “TPA-TQ-TPA” with
different substituted groups in TQ core were synthesized. As
shown in Figure 1a, the substituted groups in TPA-TQ1‒3 were
hydrogen, phenyl, and tetraphenylethylene (TPE), respectively.
From TPA-TQ1 to TPA-TQ3, the intramolecular motion was
expected to increase in order. TPE is a popularly used building
block for constructing aggregation-induced emission (AIE)
luminogens due to its highly twisted structure.[48-50] Furthermore,
the free rotation and vibration nature of TPE would also allow
more space for the molecular motions in solid/aggregate state.
The synthetic route of these compounds was depicted in Scheme
S1, and the detailed synthetic processes and characterizations of
the intermediates and final products were presented in Supporting
Information (Figures S1-S12).
Density functional theory (DFT) calculation was employed to
investigate the molecular geometry and electron cloud distribution.
As shown in Figure 1b, the optimized molecular geometry became
more twisted from TPA-TQ1 to TPA-TQ3 due to the introduction
of molecular rotors. Noteworthy, TPA-TQ3 possessed a twisted
3D geometry with a severely distorted structure, which enabled
more intense intramolecular motions. The lowest unoccupied
molecular orbital (LUMO) energy level was located in the A part,
while the highest occupied molecular orbital (HOMO) distributed
in both D and A moieties (Figure S13), suggesting efficient
intramolecular charge transfer. Moreover, the distribution of
electron cloud in the phenyl rings attached to pyrazine of TPA-
TQ2 and TPA-TQ3 indicated extended conjugation. As revealed
by the X-ray diffraction (XRD) analysis (Figure 1c), the powder
crystallinity decreased from TPA-TQ1 to TPA-TQ3, indicating that
the molecular geometry had great impact on the intermolecular
interaction in aggregate state. The increased intermolecular
distance and space of TPA-TQ3 (Figure 1d) would allow more
intense intramolecular motions, which was expected to alter the
photophysical properties and optical imaging performance.
Photophysical Properties. The absorption spectra of TPA-
TQ1‒3 in THF were shown in Figure S14, and the absorption
maxima were located at 587, 609, and 614 nm (Table S1),
respectively. The gradual bathochromic shift originated from the
contribution of phenyl and TPE to the molecular conjugation.
Similarly, the photoluminescence (PL) profiles also exhibited red
shift (Figure S15), but not as pronounced as the absorption. In
order to gain in-depth understanding about the photophysical
properties, the PL spectra in different aggregate states were
studied by adding water (poor solvent) into THF (good solvent)
solution. The PL intensity decreased gradually from 0% to 50% or
Scheme 1. Schematic illustrations of (a) intramolecular motions (iMM)
amplifying fluorescence (FL) as well as thermal-to-acoustic conversion (TAC)
and PA effect, benefitting for (b) adaptive image-guided cancer surgery by
preoperative PA imaging and intraoperative FL imaging. PTC: photothermal
conversion; ROS: reactive oxygen species.
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10.1002/anie.202109048
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Figure 1. (a) Chemical structures, (b) optimized molecular geometry, (c) XRD diagrams of the powders, and (d) schematic intermolecular distance of TPA-TQ1‒3.
60% water fraction, which could be attributed to the increased
polarity and formation of twisted intramolecular charger transfer
(TICT) state.[51,52] The PL intensified when further increasing the
water fraction (Figure S16), demonstrating obvious AIE signature.
Interestingly, the enhancement of PL intensity in aggregate state
increased from TPA-TQ1 to TPA-TQ3, which could be assigned
to the more twisted molecular structure, and therefore reduced
intermolecular interaction such as π-π stacking.
To understand the intermolecular interactions in aggregate
state, molecular dynamics (MD) simulations of TPA-TQ1 and
TPA-TQ3 were carried out using the GROMACS program.[53,54]
The snapshots were picked from the production simulations to
show the packing manner of the molecules in aggregate. As
displayed in Figure 2a,b, both TPA-TQ1 and TPA-TQ3
aggregates exhibited amorphous forms in disordered manners,
which caused relatively loose packing. Additionally, the
intramolecular motions in aggregates were still active but partially
restricted due to the surrounding loosely packed molecules. To
demonstrate the microscopic dynamics in atomic resolution,
several key dihedral angle distributions were recorded (Figure
2c,d). It was found that the distributions of dihedral angles D1, D2
and D3 of TPA-TQ1 and TPA-TQ3 were similar, in which D2 and
D3 had wider distributions than D1, implying more flexible feature
of the phenyl rings in D2 and D3. Moreover, TPA-TQ3 had
additional rotors, like D4, D5 and D6 in TPE moieties, which
possessed wide distribution range, leading to reduced
intermolecular interaction and intensive intramolecular motions.
Figure 2. Snapshots of the amorphous aggregates of (a) TPA-TQ1 and (b) TPA-
TQ3 obtained from molecular dynamics simulation. Different dihedral angles
distributions of (c) TPA-TQ1 and (d) TPA-TQ3 in aggregate state.
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Fabrication and Characterization of Nanoparticles. To
study the optical imaging property in aggregate state and explore
biomedical applications, the compounds were doped into water-
soluble nanoparticles (NPs) with a nanoprecipitation method. The
could be attributed to the conformational variation from twisted
molecular geometry.[55,56] The large Stokes shift of about 200 nm
could efficiently avoid the overlap between absorption and
emission spectra, enabling full utilization of the emission light. PL
excitation (PLE) mapping (Figure 3d and Figures S18,S19) further
revealed that both the excitation and emission of the nanoagents
were located in NIR region. The PL quantum yield (PLQY) of TPA-
TQ1‒3 NPs was measured to be 1.4%, 3.9%, and 6.8% using an
integrating sphere (Figure 3e), which were in accordance with the
differences of AIE property. The increased fluorescence
brightness from TPA-TQ1 NPs to TPA-TQ3 NPs was probably
because the large twisted molecular structure could inhibit
intermolecular quenching effect, and thus facilitate the radiative
decay. The brightness had also been confirmed by the images
from in vivo imaging system (IVIS) (Figure 3e). Noteworthy, PLQY
of TPA-TQ3 NPs (6.8%) represented a high value among organic
fluorophores with similar emission wavelength in aggregate state.
amphiphilic
polymer
1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000]
(DSPE-PEG2000) was adopted as the encapsulation matrix and
the hydrophobic organic molecules self-assembled in the core.
Dynamic light scattering (DLS) measurements indicated that the
average diameters of TPA-TQ1‒3 NPs were in sequence 73, 75,
and 80 nm, and the transmission electron microscope (TEM)
results revealed that the NPs were approximate sphere structures
with diameters of about 70 nm (Figures 3a and Figure S17). The
absorption profiles showed strong absorption in the spectral
region of 550‒750 nm (Figure 3b), which could be excited by the
pulsed laser of commercially available PA instrument. TPA-TQ1‒
3 NPs displayed PL spectra in the range of 700‒1200 nm with
maxima at about 820 nm (Figure 3c), in which the broad emission
Figure 3. (a) Representative DLS and TEM results of TPA-TQ3 NPs. (b) Absorption and (c) PL spectra of TPA-TQ1‒3 NPs, respectively. (d) PLE mapping of TPA-
TQ3 NPs. (e) PLQY values of TPA-TQ1‒3 NPs. Data are presented as mean ± s.d. (n = 3). Inset shows the brightness images of various NPs solution obtained
from IVIS. (f) Fluorescence decay curves of TPA-TQ1‒3 NPs.
different wavelengths from 680 to 800 nm. As shown in Figure 4a,
In order to gain in-depth understanding about the photophysical
transition processes, we measured the fluorescence lifetime of
various NPs. The lifetime (τ) increased from TPA-TQ1 NPs to
TPA-TQ3 NPs, which was in the same trend as PLQY (Figure 3f).
The fluorescence properties were closely related to the radiative
and non-radiative decay rates (kr and knr).[57-59] According to the
equations: τ = 1/(kr + knr), ΦF = kr/(kr + knr), the kr/knr values of TPA-
TQ1‒3 NPs were calculated to be 0.136/9.57 × 108, 0.215/5.31 ×
the PA spectra matched well with the absorption profile, which
suggested that the PA signal originated from the absorption of the
NIR chromophores. Interestingly, TPA-TQ3 NPs exhibited the
highest PA intensity, which was more than two-fold higher than
TPA-TQ1 NPs and 1.7 times higher than TPA-TQ2 NPs. Thus,
TPA-TQ3 NPs displayed both high fluorescence and PA signal,
which was different from previous reports that the PA and
fluorescence transition processes of one agent work against each
108 and 0.287/3.93 × 108 s‒1 (Table S1), respectively. Interestingly, other.[60,61] To further validate this phenomenon, the PA
the radiative decay increased gradually from TPA-TQ1 to TPA-
TQ3 NPs, meanwhile the non-radiative decay decreased. This
result revealed that the non-radiative decay process of the highly
twisted structure was greatly inhibited and the radiative decay was
enhancive in aggregate form.
amplitudes of the NPs in different concentrations were measured.
As displayed in Figure 4b,c, the PA intensity also increased in the
order of TPA-TQ3 NPs > TPA-TQ2 NPs > TPA-TQ1 NPs, and the
PA amplitude showed very good linear relationship with the
concentration, indicating good stability and great potential for
quantitative analysis. Furthermore, the comparison of PA
amplitude of TPA-TQ1‒3 NPs and the popularly used methylene
Photothermal and PA Properties. The PA spectra of TPA-
TQ1‒3 NPs were measured by recording the PA intensity at
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Figure 4. (a) PA spectra of TPA-TQ1‒3 NPs. (b) PA images and (c) corresponding PA amplitude versus the concentration of NPs. (d) PA amplitude of TPA-TQ1‒
3 NPs and methylene blue in the same concentration (50 µM) at 680 nm. (e) Photothermal heating−cooling process and (f) PCE of TPA-TQ1‒3 NPs under continuous
irradiation of 660 nm light (0.4 W/cm2). (g) ROS generation property of TPA-TQ1‒3 NPs and chlorin e6 (Ce6) under light irradiation. (h) Relative Grüneisen
parameters of TPA-TQ1‒3 NPs solution. (i) Relative Grüneisen parameters of TPA-TQ1‒3 in DMF/glycerol mixture with different glycerol fractions. Data are
presented as the means ± s.d. (n = 3).
both the photothermal conversion and subsequent thermal-to-
blue (MB) in the same concentration (50 µM) with 680 nm pulsed
laser excitation (Figure 4d) indicated that the PA intensity of TPA-
TQ1‒3 NPs were in sequence 1.6, 2.1, and 3.5 times higher than
that of MB, illustrating that TPA-TQ3 NPs were a highly potent PA
contrast agent.
acoustic transformation processes, and the decreased PCE could
not explain the high PA signal of TPA-TQ3, thus the thermal-to-
acoustic conversion property of TPA-TQ1‒3 NPs solution was
studied. Since the same excitation pulsed laser was used,
Grüneisen parameters could be estimated from the
aforementioned PA equation. As displayed in Figure 4h, the
relative Grüneisen parameters of TPA-TQ2 NPs and TPA-TQ3
NPs were about 1.5- and 4-fold higher than that of TPA-TQ1 NPs.
To further understand the influence of intramolecular motion on
PA generation process, the PA amplitude, PCE and Grüneisen
parameter of TPA-TQ1-3 in the mixture of N,N-
dimethylformamide (DMF) and glycerol with different glycerol
fractions were measured. The increase of glycerol fraction could
result in viscous environment and restrict the intramolecular
motions. The results revealed that the relative Grüneisen
To study the origination of the highest PA signal of TPA-TQ3
NPs among all NPs, we first investigated their photothermal
property. Under the same light irradiation for 3 min, the highest
temperatures that TPA-TQ1-3 NPs solution could be reached
o
were 56.2, 52.3 and 47.6 C (Figure 4e), respectively. The PCE
values of TPA-TQ1‒3 NPs were calculated to be 33.8%, 27.8%,
and 18.7% (Figure 4f), respectively. The decrease of PCE from
TPA-TQ1 NPs to TPA-TQ3 NPs was consistent with the reduced
non-radiative decay. This result also manifested that PLQY and
PCE were competitive as they directly corresponded to the
radiative and non-radiative decay processes of Jablonski diagram. parameters of TPA-TQ1 and TPA-TQ2 hardly changed with
We also studied other possible energy dissipation pathways such
as phosphorescence and photosensitization in the triplet excited
state, but no obvious related processes could be detected for
TPA-TQ1‒3 NPs (Figure 4g). As PA effect is closely related to
viscosity. While the relative Grüneisen parameters of TPA-TQ3
decreased for about 50% in the solution with 60% glycerol (Figure
4i), probably due to the highly restricted molecular motion in high-
viscosity environment. This result suggested that the Grüneisen
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10.1002/anie.202109048
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parameter and related thermal-to-acoustic transformation were
greatly impacted by the intramolecular motions. The free
molecular rotations and vibrations were able to promote the PA
signal via amplifying Grüneisen parameter or thermal-to-acoustic
conversion. This finding also demonstrated a new strategy to
simultaneously obtain high PA and fluorescence brightness in one
chromophore.
improve both surgical outcomes and patient safety, different
imaging modalities usually need to be combined to gain
comprehensive information of the lesion in different stages.[64,65]
The excellent PA and fluorescence properties of TPA-TQ3 NPs
make it a suitable candidate for in vivo biomedical applications.
To test their potential use for image-guided surgery, the TPA-
TQ1-3 nanoprobes were injected into the mice bearing orthotopic
4T1 breast tumor through tail veins, and the florescence and PA
images were recorded at designed time intervals. The live-animal
fluorescence and PA imaging were then performed for pre-
surgery diagnosis. As presented in Figure 6a,b, the in vivo
fluorescence intensity of tumor sites gradually intensified as time
After demonstrating that active intramolecular motion could
promote thermo-to-acoustic conversion, we next sought to
validate whether this molecular design could be extended to other
system. In this case, two D-A type compounds based on TPA and
thienothiadiazole (TT) were designed and synthesized (Scheme
S2, Figures S21-S26), in which either hydrogen or TPE was
chosen as the substituted group. As expected, the TPE-
containing compound (TPE-TPA-TT) exhibited more twisted
molecular geometry, and it also possessed more pronounced AIE
feature and reduced crystallinity, when compared with its analog
(TPA-TT) (Figure 5a,b and Figures S27,S28). The fluorescence
and PA properties of TPA-TT and TPE-TAP-TT NPs were further
compared. The results showed that TPE-TPA-TT NPs exhibited
much higher PLQY and stronger PA signal than TPA-TT NPs. And
the calculated relative Grüneisen parameter of TPE-TPA-TT NPs
solution was nearly three times greater than that of TPA-TT NPs
(Figure 5c,d and Figures S29,S30). This result suggested that
TPE-TPA-TT with violent intramolecular motion showed higher
thermo-to-acoustic conversion efficiency thanks to the violent
intramolecular motion. Accordingly, the introduction of
intramolecular motion is an effective strategy to promote thermal-
to-acoustic conversion and thus PA signal.
elapse and became highest at about
8 h post-injection,
suggesting the optimal time to perform in vivo imaging.
Noteworthy, the fluorescence intensity of the tumor site of TPA-
TQ3 NPs-treated mice was about 3.1-fold and 1.9-fold stronger
than that of TPA-TQ2 NPs and TPA-TQ1 NPs, respectively, which
was in line with the PLQY results. With the high fluorescence
brightness, TPA-TQ3 NPs significantly lighted up the tumor tissue
and clearly delineated tumor from surrounding normal tissues.
The ex vivo imaging of the main organs and tumor resected from
the mice injected with TPA-TQ3 NPs for 24 h (Figure S31) also
revealed that the tumor tissue displayed very high fluorescence
signal, which benefited from the high PLQY of TPA-TQ3 NPs as
well as its potent tumor accumulation medicated by the enhanced
permeability and retention (EPR) effect.[66,67] In the meantime, the
mice were also imaged with PA instruments under 700 nm pulsed
laser excitation. After intravenous administration of TPA-TQ3 NPs,
the PA signal of tumor site could be clearly observed at 3 h post
injection and reached highest at 8 h post injection. The PA signal
of tumor at 8 h was elevated by 7.3 folds when compared with that
at 0 h. Such result was even better than most reported high-
performance PA agents.[60,68] By making full use of the high
sensitivity and facile equipment of fluorescence technique and
good penetration ability and spatial resolution of PA imaging, the
TPA-TQ3 NPs-based combined preoperative imaging strategy
provided comprehensive information about tumor, rendering the
surgeon advantageous guidance on surgical plan.
Figure 5. (a) Plot of PL peak intensity of TPA-TT and TPE-TPA-TT versus water
fraction (fw) in THF/water mixtures (2 × 10‒5 M). I0 and I represent the PL
intensities of the compounds in pure THF (fw = 0) and THF/water mixtures with
specific fws. (b) XRD diagrams of TPA-TT and TPE-TPA-TT powders. (c) PA
amplitude of TPA-TT NPs and TPE-TPA-TT NPs in the same concentration (50
µM) at 680 nm. (d) Relative Grüneisen parameters of TPA-TT NPs and TPE-
TPA-TT NPs solution. Data are presented as the means ± s.d. (n = 3)
Figure 6. (a) Fluorescence intensity of the tumor site as a function of post-
injection time and representative fluorescence images of mice bearing
orthotopic 4T1 breast tumor at 8 h post injection of TPA-TQ1‒3 NPs. Data are
presented as the means ± s.d. (n = 3 mice per group). Representative (b)
fluorescence and (c) PA images of mice bearing orthotopic 4T1 breast tumor
after intravenous injection of TPA-TQ3 NPs at different time intervals as
indicated.
In Vivo Image-Guided Tumor Surgery. Precise image-guided
surgery calls for high-performance imaging techniques that meet
the requirements during the whole operation process.[62,63] To
Tumor resection was then conducted by a surgeon from Tianjin
First Central Hospital (Tianjin, China) with the information
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provided by preoperative fluorescence and PA imaging. The most
challenging problem during cancer surgery is the differentiation of
tiny residual tumors as they are really difficult to recognize by
naked eyes.[69] The high sensitivity and equipment flexibility of
fluorescence imaging make it an ideal option for rapid
intraoperative imaging, which has also been used in clinic.[70,71] As
indicated in Figure 7a, the intraoperative NIR fluorescence
imaging enabled unambiguous detection of tiny tumor tissues in
real time. The fluorescence signal was found to be in good
accordance with the bioluminescence from the luciferase-
expressed tumor cells, which confirmed that the fluorescently
labelled areas were indeed tumor tissue. The residual tumor
nodules identified by fluorescence imaging were also examined
with hematoxylin and eosin (H&E) histological analysis (Figure
S33), implying those were tiny tumors of < 500 µm in size. With
the guidance of fluorescence imaging, the second surgery was
carried out to resect all residual orthotopic breast tumor nodules
until no NIR fluorescence signal could be observed. To examine
the validity of this integrated surgery imaging strategy, survival
rate study was conducted on several groups: the orthotopic 4T1
breast tumor-bearing mice without any surgery (Control), with
residual tumors from intraoperative NIR fluorescence imaging
(S1), and excision of residual tumors until no intraoperative NIR
fluorescence signal (S2). The mice in the Control and S1 groups
were all died within 40 days. In contrast, only one mouse in S2
group was died on day 31 post-surgery, and all other mice were
survival in the 45-days duration (Figure 7b). The significantly
increased survival rate in S2 group manifested the great
advantage of TPA-TQ3 NPs for integrated imaging in different
surgical stages. This work validated that the optical imaging agent
developed here with both excellent fluorescence and PA
properties could serve as a potent probe for tumor resection
guidance by combining the preoperative fluorescence/PA and
intraoperative fluorescence imaging.
after NPs injection (Figure S37). Taken together, these results
revealed that the organic NPs had good biocompatibility and no
obvious side effect was observed.
Conclusion
In summary, we demonstrated for the first time that intramolecular
motion represented an efficient strategy to amplify PA signal, in
particular the thermal-to-acoustic conversion process. By
introducing molecular rotor-rich unit (e.g., TPE), both the
fluorescence and PA properties were improved. The underlying
mechanism was also investigated, which demonstrated that the
twisted conformations of the rotor-rich compounds were favorable
for AIE property and high fluorescence brightness, and
intramolecular motion promoted the thermal-to-acoustic
conversion process to achieve outstanding PA signal. The
simultaneously high PA and fluorescence imaging properties
warranted TPA-TQ3 NPs a superb probe for in vivo biomedical
applications. The preoperative NIR fluorescence/PA imaging
helped to localize tumor site, geometry and depth of orthotopic
4T1 breast tumor-bearing mice in a high-contrast manner,
providing unambiguous guidance for performing surgery.
Furthermore, the intraoperative NIR fluorescence imaging
enabled the delineation of tiny residual tumors in a fast and
sensitive way, significantly boosting the surgery outcome. This
work demonstrated that intramolecular motion represented a new
approach to amplify PA signal by increasing the thermal-to-
acoustic conversion, greatly advancing complicated biomedical
applications.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (51873092, 51961160730 and 21803007),
the National Key R&D Program of China (Intergovernmental
cooperation project, 2017YFE0132200), Tianjin Science Fund for
Distinguished Young Scholars (19JCJQJC61200), and Beijing
Institute of Technology (BIT) Research Fund Program for Young
Scholar.
Figure
7.
(a)
Representative
pre-,
intra-
and
post-surgical
Conflict of interest
fluorescence/bioluminescence images of orthotopic 4T1 breast tumors. (b)
Survival curves of orthotopic 4T1 breast tumor-bearing mice received various
treatments (n = 8 mice per group).
The authors declare no conflict of interest.
Biocompatibility was of critical significance for the in vivo
applications of optical imaging agents, so we investigated the in
vitro and in vivo safety of TPA-TQ3 NPs. The cytotoxicity was first
evaluated by co-culturing different concentrations (0, 2, 5, 10, 25,
50 µM) of TPA-TQ1-3 NPs with 4T1 cancer cells. As shown in
Figure S34, the cell viability was higher than 90% with the
treatment of a high concentration (50 µM), indicating low
cytotoxicity of the NPs. TPA-TQ3 NPs were then injected into
healthy mice to examine the in vivo biosafety. The hepatic and
renal function analyses (Figure S35) and blood routine
examination (Figure S36) of the TPA-TQ3 NPs-treated mice
showed no noticeable abnormalities. In addition, the H&E staining
of the major organs, including heart, liver, spleen, lung, and
kidney did not show any notable pathological changes 7 days
Keywords: intramolecular motion • thermal-to-acoustic
conversion • photoacoustic • aggregation-induced emission •
image-guided surgery
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RESEARCH ARTICLE
Entry for the Table of Contents
Insert graphic for Table of Contents here.
The compound with intense intramolecular motions exhibits amplified PA signal by specifically elevating the thermal-to-acoustic
conversion efficiency, and the fluorescence brightness also increases due to the aggregation-induced emission signature. Based on
the high-performance optical imaging agent, precise in vivo image-guided cancer surgery in different stages is conducted, greatly
boosting the surgery outcome.
9
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