Thermoresponsive Semiconducting Polymer Nanoparticles for Contrast-Enhanced Photoacoustic Imaging

Article abstract of DOI:10.1002/adfm.201903461

Photoacoustic (PA) agents with biomarker-activated signals are developed to enhance the signal-to-background ratios (SBRs) for in vivo imaging; however, their SBRs still heavily rely on the concentration difference of biomarkers between diseased and normal tissues. By contrast, external stimuli can provide a remote way to noninvasively control the signal generation from the PA agents and in turn enhance SBR, which are less exploited. This study reports the development of thermoresponsive semiconducting polymer brush with poly(N,N-dimethylacrylamide)-r-(hydroxypropyl acrylate) (PDMA-r-HPA) grafts for contrast-enhanced in vivo imaging. Such a polymer is amphiphilic and can self-assemble into the nanoparticle (termed as SPNph1) in an aqueous medium, and has lower critical solution temperatures (LCSTs) at 48 °C. Thus, SPNph1 not only has higher photothermal conversion efficiency than the control polymer without PDMA-r-HPA grafts, but also can undergo phase separation to form large nanoparticles, leading to enhanced PA signals above LCST. The thermoresponsive PA property of SPNph1 enables in situ remote manipulation of PA signals by photoirradiation to further enhance the tumor SBR. Thus, this study introduces a new generation of organic PA agents with thermoresponsive signal for high-contrast in vivo imaging.

Full text of DOI:10.1002/adfm.201903461

FULL PAPER
Photoacoustic Polymer Nanoparticles
www.afm-journal.de
Thermoresponsive Semiconducting Polymer Nanoparticles
for Contrast-Enhanced Photoacoustic Imaging
Dong Cui, Pengcheng Li, Xu Zhen, Jingchao Li, Yuyan Jiang, Aixi Yu,* Xiang Hu,*
and Kanyi Pu*
when passing through the tissues.[2] As
tissues have native chromophores such
Photoacoustic (PA) agents with biomarker-activated signals are developed
to enhance the signal-to-background ratios (SBRs) for in vivo imaging;
however, their SBRs still heavily rely on the concentration difference of
biomarkers between diseased and normal tissues. By contrast, external
stimuli can provide a remote way to noninvasively control the signal
generation from the PA agents and in turn enhance SBR, which are less
exploited. This study reports the development of thermoresponsive
semiconducting polymer brush with poly(N,N-dimethylacrylamide)-r-
as hemoglobin and melanin that can
efficiently absorb the photoenergy and
generate ineluctable background signals,
molecular strategies to enhance the
signal-to-background ratios (SBRs) have
long been desired for PA imaging. For
instance, activatable PA probes that
change their signals in response to the
biomolecule of interest have been devel-
(
hydroxypropyl acrylate) (PDMA-r-HPA) grafts for contrast-enhanced in
[
3]
oped for sensitive imaging of enzymes,
small molecule mediators, and pH
vivo imaging. Such a polymer is amphiphilic and can self-assemble into
the nanoparticle (termed as SPNph1) in an aqueous medium, and has
lower critical solution temperatures (LCSTs) at 48 °C. Thus, SPNph1
not only has higher photothermal conversion efficiency than the control
polymer without PDMA-r-HPA grafts, but also can undergo phase
separation to form large nanoparticles, leading to enhanced PA signals
above LCST. The thermoresponsive PA property of SPNph1 enables in
situ remote manipulation of PA signals by photoirradiation to further
enhance the tumor SBR. Thus, this study introduces a new generation of
organic PA agents with thermoresponsive signal for high-contrast in vivo
imaging.
[
4]
[5]
in living mice. However, the SBRs for
such probes still heavily rely on the con-
centration difference of endogenous bio-
markers between diseased and normal
tissues. By contrast, external stimulus
is irrelevant to the location and status
of PA agents, and can provide a manual
way to noninvasively control the signal
generation from the PA imaging agent,
offering extra opportunities to enhance
the imaging sensitivity. However, stim-
uli-responsive agents have been rarely
exploited for PA imaging.
1
. Introduction
Semiconducting polymer nanoparticles (SPNs) made of
organic ingredients have attracted increasing attention as a
new class of phototheranostic agents due to their excellent
Photoacoustic (PA) imaging is
a hybrid nonionizing
[6]
imaging technique that detects the conversion of light
energy into ultrasound.[1] Compared with traditional optical
imaging modalities, PA imaging can provide deeper tissue
penetration, because ultrasound scatters much less than light
optical properties and ideal biocompatibility. Dependent on
the molecular structures of semiconducting polymers (SPs),
[7]
SPNs have been diversified for optical imaging, photo-
[8]
therapy, and light regulation of biological events including
[
9]
[10]
enzymatic activity, protein ion channels,
and gene tran-
[11]
scription. In particular, we and others have developed several
SPN-based PA imaging agents for in vivo imaging of lymph
D. Cui, Dr. X. Zhen, Dr. J. Li, Y. Jiang, Prof. K. Pu
School of Chemical and Biomedical Engineering
Nanyang Technological University
[12]
[13]
[14]
nodes,
species (ROS),
tumors,
protein sulfenic acids,
reactive oxygen
[15]
pH,[16] and glucose.
[17]
To enhance the sen-
7
0 Nanyang Drive, Singapore 637457, Singapore
sitivity of PA imaging, we doped the SPNs with (6,6)-phenyl-
C71-butyric acid methyl ester (PC70BM) to create intrapar-
ticle photoinduced electron transfer process to increase the
E-mail: kypu@ntu.edu.sg
P. Li, Prof. A. Yu, Prof. X. Hu
Department of Trauma and Orthopaedic Microsurgery
Clinical Microsurgery Research Center of Wuhan City
Zhongnan Hospital of Wuhan University
No. 169, East Lake Road, Wuhan, Hubei 430071, China
E-mail: yuaixi@whu.edu.cn; shawnhu2002@whu.edu.cn
[
18]
heat generation and thus enhance the PA brightness.
We also incorporated vinylene bonds into the polymers to
facilitate the electron delocalization and increase the mass
absorption coefficients, allowing SPNs to absorb more photons
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adfm.201903461.
[
19]
and thus emit stronger PA signals relative to the counterpart.
More interestingly, we found that coating of SPNs with a silica
shell can reduce the interfacial heat transfer resistance and
DOI: 10.1002/adfm.201903461
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thus promote heat dissipation, eventually leading to amplified
PA signals.[ However, SPN-based PA agents responding to
external stimulus have been rarely exploited.
N,N-dimethylacrylamide (DMA) and 2-hydroxypropyl acrylate
(HPA) as monomers (the molar ratio between DMA and
HPA was optimized to be 4:6), copper (I) bromide (CuBr)
20]
In this work, we designed and synthesize thermorespon-
sive SPNs for in vivo PA imaging of tumor. These PA agents
are self-assembled from the semiconducting polymer brushes
composed of poly(N,N-dimethylacrylamide)-r-(hydroxypropyl
acrylate) (PDMA-r-HPA) grafts. PDMA-r-HPA is thermorespon-
sive and can undergo reversible phase transition in response to
the change in environmental temperature. Thus, the resulted
SPNs (SPNph1 and SPNph2) had lower critical solution tem-
peratures (LCSTs), above which phase separation occurs to
induce the aggregation of these nanoparticles. When the size
increases, the heat dissipation is promoted and thus the PA
signal is amplified. As such, the PA signals of thermorespon-
sive SPNs can be enhanced by heating, providing a way to
remotely enhance the SBRs of PA imaging.
and 1,4,8,11-tetraazacyclotetradecane (Me Cyclam) as catalyst
4
and ligand, respectively. The proton nuclear magnetic reso-
1
nance spectrum ( H NMR) showed that the characteristic
proton resonance peaks of initiator 1 were found at 4.77, 2.51,
1
and 1.96 ppm (Figure S1, Supporting Information). The H
NMR spectrum (Figure S2, Supporting Information) showed
that the characteristic proton resonance peaks of DMA was
found at 3.05, 2.35, and 1.89 ppm, while those of HPA were
found at 3.99, 3.59, 2.94, 1.69, and 1.21 ppm. The gel permea-
tion chromatography (GPC) showed that the number-average
molecular weight of polymer 2 was 10 393 with a good poly-
dispersity, which was close to the designed molecular weight
(11 954). Polymer 6 was synthesized by grafting PDMA-r-HPA
(2) and mPEG-alkyne (4) on azide-modified polymer (3) (the
molar ratio of 2 to 4 was 9:1) via copper (I)-catalyzed alkyne–
azide click reaction. Similarly, polymer 7 was synthesis via
click reaction of polymer 2 and 3, while the control polymer
2
. Results and Discussion
1
5
was obtained via click reaction of polymer 3 and 4. The H
The thermoresponsive semiconducting polymer brushes
NMR spectrum (Figure S3, Supporting Information) showed
that the characteristic proton resonance peaks of the grafted
polymer 2 were found at 3.99, 3.59, 3.05, 2.94, 2.35, 1.89-
1.69, and 1.21 ppm, OCH CH , OCH CH 
(
(
6 and 7) and the control polymer with only PEG grafts
5) were synthesized via a graft-on approach (Figure 1a).
To synthesis PDMA-r-HPA (2), the initiator prop-2-yn-
-yl 2-bromo-2-methylpropanoate (1) was first synthesized
Figure S1, Supporting Information) via alkynyl modifica-
tion of α-bromoisobutyryl bromide, followed by compound
initiated atom transfer radical polymerization using
2
2
2
2
1
(
in PEG were found at 3.66 ppm and the peaks of polymer 3
were found between 8.5 and 7.0 ppm, implying the correct
structure of SPNph1. The chemical structures of SPNph2
1
1
and SPNp were also confirmed by H NMR. The GPC data
Figure 1. a) Synthetic routes toward the thermoresponsive polymer brushes (6 and 7) and the control polymer (5) and then self-assmebly into the
corresponding nanoparticles, SPNph1, SPNph2, and SPNp. Reagents and conditions: (i) α-bromoisobutyryl bromide, 2-propyn-1-ol, triethylamine
(
(
TEA), dichloromethane (DCM), 0 °C, room temperature, 24 h; (ii) N,N-dimethylacrylamide (DMA), 2-hydroxypropyl acrylate (HPA), copper
I) bromide (CuBr), 1,4,8,11-tetraazacyclotetradecane (Me Cyclam), toluene/dimethylformamide (DMF) (v/v, 9/1); (iii) CuBr, N,N,N″,N″,N″-
4
pentamethyldiethylenetriamine (PMEDTA), tetrahydrofuran (THF), 25 °C, 24 h. (iv) CuBr, PMEDTA, tetrahydrofuran (THF), 25 °C, 24 h; (v) copper
(
(
II) sulfate (CuSO ), sodium ascorbate, DMF, 25 °C, 24 h. b) Schematic illustration of heat-amplified PA signals of the thermoresponsive SPNs
4
SPNph1 and SPNph2) in comparison with the control nanoparticle (SPNp).
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Figure 2. In vitro characterization. a) UV−vis absorption and b) fluorescence spectra of SPNs. c) DLS data of different SPNs as a function of incuba-
tion temperature in PBS (pH = 7.4). d) TEM images of SPNph1 and SPNp after incubation at 25 or 50 °C. e) ζ-Potentials of SPNph1 and SPNp after
incubation at 25 or 50 °C. f) Cell viability of 4T1 cells after incubation with SPNph1 or SPNp solutions at various concentrations. Error bars represented
standard deviations of three separate measurements.
(
Table S1, Supporting Information) showed that the number-
average molecular weights of polymer 5, 6, and 7 were 20 000,
0 000, and 120 000, respectively. The polymers 5, 6, and 7
As the higher LCST could potentially result in larger signal
enhancement, SPNph1 was used for the further studies along
with the control (SPNp). The transmission electron microscopy
(TEM) images confirmed that both SPNph1 and SPNp had uni-
form spherical morphology with an average diameter of ≈30 nm
at 25 °C (Figure 2d), while the aggregation was only observed
for SPNph1 at elevated temperature. In addition, SPNph1 and
SPNp had the ζ-potentials of −4.2 and −1.9 mV, respectively
(Figure 2e). When the solution temperature was above 50 °C,
the ζ-potential of SPNph1 showed nonsignificant changing.
[3-(4,5-Dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium] (MTS) assay showed that both
SPNs were noncytotoxic to 4T1 cells even at a high concentra-
8
could be dissolved in aqueous solutions and spontaneously
self-assemble into the corresponding nanoparticles termed as
SPNp, SPNph1, and SPNph2, respectively.
The optical properties of SPNph1, SPNph2, and SPNp were
tested in PBS solution. All SPNs had similar absorption peak
ranging from 500 to 900 nm with the maximum at ≈700 nm
(Figure 2a). The fluorescence maxima of SPNp, SPNph1, and
SPNph2 were different, which were at 820, 830, and 840 nm,
respectively (Figure 2b). This should be attributed to the dif-
ferent conformations of semiconducting polymer backbones
governed by the different hydrophobicity/hydrophilicity
between PEG and PDMA-r-HPA in aqueous solution. The
temperature dependent dynamic light scattering (DLS) meas-
urements showed that all SPNs had similar hydrodynamic dia-
meters (≈35 nm) at low temperature (25 °C) (Figure 2c). Upon
increasing the solution temperature, a sharp increase in size
was observed for both SPNph1 and SPNph2, which was fol-
lowed by the saturation at the size of 820 nm. By contrast, SPNp
did not show such a phase transition behavior. Accordingly, the
LCSTs of SPNph1 and SPNph2 were at 48, 42 °C respectively.
The lower LCST for SPNph2 relative to SPNph1 was attributed
to the higher PDMA-r-HPA grafting density.
−
1
tion of 100 µg mL (Figure 2f).
Under continuous laser irradiations at 808 nm, SPNph1
and SPNp showed increased solution temperature and
reached the respective maximum photothermal temperature
at 60.0 and 55 °C at 8 min (Figure 3a,b). The photothermal
conversion efficiency of SPNph1 was calculated as 34.9%,
higher than that of SPNp (26.7%). Under reversibly heating
and natural cooling for five times, the maximum temperature
of SPNph1 and SPNp remained nearly the same, showing
good photothermal stability (Figure 3c). The PA spectra of
SPNph1 are close to its absorption, showing the maximum
intensities at 680 nm (Figure S5, Supporting Information).
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Figure 3. In vitro photothermal and PA characterization of SPNph1 and SPNp. a) Photothermal temperatures of SPNph1 and SPNp solutions as a
function of laser irradiating time. b) Thermal images of SPNph1 and SPNp at their respective maximum temperatures. c) Stability study of SPNph1
−
1
and SPNp under photothermal heating and cooling cycles. The concentrations of the optical components of both SPs were 30 µg mL in PBS
−1
(
pH = 7.4). The laser irradiation wavelength was 808 nm with a power of 0.8 W cm−2. d) PA intensity of SPNph1 and SPNp (50 µg mL ) solutions
as a function of surrounding temperatures. e) PA images of SPNph1 and SPNp solutions at different concentrations with or without laser irradiation
808 nm, 0.8 W cm ). PA intensity as a function of different concentrations of f) SPNp and g) SPNph1 with or without laser irradiation (808 nm,
−
2
(
0
−
2
.8 W cm ).
Due to the higher photothermal conversion efficiency, the PA
intensity of SPNph1 was higher than that of SPNp at same
concentration (Figure 3d). With increased solution tempera-
ture, the PA intensity of SPNph1 was significantly increased
to the maximum at 50 °C (consistent with its LCST), which
was 1.57-fold higher than that of SPNph1 at 25 °C. How-
ever, the PA intensity of SPNp showed no obviously change
with increased solution temperature (Figure 3d). Such PA
enhancement of SPNph1 was attributed to its aggregation
and formation of large nanoparticles at elevated temperature
above LCST. The PA amplitudes and images of SPNph1 and
SPNp at 680 nm with or without laser irradiation were deter-
mined at a series of concentrations of the optical components
from 6 to 500 µg mL− (Figure 3e–g), showing a good linear
relationship between the PA signals and the polymer concen-
trations. Moreover, compared with no laser irradiation treated
SPNph1, the PA amplitudes were respectively increased to
The PA signals in the tumor areas gradually increased over
time after systemic administration of SPNph1 through intra-
venous injection (Figure S6, Supporting Information). At 24 h
postinjection, the maximum intensity projection PA images
were obtained (Figure 4a), and ex vivo biodistribution at
t = 32 h postinjection showed both SPNph1 and SPNp were
mainly accumulated in spleen, tumor, and liver. In addition,
tumor had relatively strong PA signals, which were than that of
liver (1.1-fold) (Figure 4b). To further study the thermorespon-
sive PA amplification of SPNph1, an 808 nm NIR light laser
was applied to elevate the temperature of tumors (Figure 4c).
Note that the short time (1 min) of elevated temperature
(48 °C) did not cause damage of tumor (Figure S7, Supporting
Information). Without laser irradiation, both of SPNph1
and SPNp showed relative lower PA signals of the tumors
(Figure 1a). After laser irradiation for 1 min, the PA signals of
SPNph1 treated tumor was significant enhanced, while that of
SPNp treated tumor had no obviously change. The quantita-
tion data further confirmed that the PA intensity of SPNph1
treated tumor was 1.37-fold increased with laser irradiation,
while the PA intensity of SPNp treated tumor remained nearly
unchanged (Figure 4d). Thus, the signal-to-background ratio
for SPNph1 is increased from 4.16 to 6.12 after laser-mediated
heating, showing the advantage of thermoresponsive PA agents
in sensitive PA imaging. In addition, the biosafety of SPNp
and SPNph1 was evaluated by hematoxylin and eosin (H&E)
staining. No abnormal morphologies for the major organs
(heart, liver, spleen, lung, and kidney) were observed in mice
after injection of SPNp and SPNph1 (Figure S8, Supporting
1
1
2
.49, 1.83, 1.38, and 1.55-fold at the concentration of 50, 100,
00, and 500 µg mL− after laser irradiation. By contrast, laser
1
irradiation had no obvious influence on the PA amplitude for
different concentration of SPNp. These data clearly demon-
strated that SPNph1 was a better PA agent with higher con-
version ability of light to heat relative to the control (SPNp),
and SPNph1 exhibited PA enhancement with temperature
stimuli.
SPNph1 and SPNp were evaluated for in vivo PA imaging
in the subcutaneous 4T1 xenografted tumor model. After
systemic administration of SPNph1 or SPNp into the living
mice through tail veil, the PA signal was recorded at 680 nm.
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Figure 4. In vivo PA imaging. a) PA images of tumor of a living mouse 24 h postinjection of SPNph1 or SPNp (400 µg mL−1, 200 mL) via tail vein
−
2
with or without laser irradiation (808 nm, 0.8 W cm ). Scale bar = 2 mm. b) Ex vivo quantification of PA of major organs or tumors from mice
n = 3) 32 h after systemic administration of SPNph1 or SPNp. c) Schematic illustration of amplified PA imaging of SPNph1 treated mice tumor.
(
−
1
d) PA intensities of tumors in SPNp or SPNph1 (400 µg mL , 200 mL) treated living mice (n = 3) via tail vein with or without laser irradiation
808 nm, 0.8 W cm−2).
(
Information), suggesting their good biocompatibility for PA
imaging.
new generation of organic PA nanoagents with themorespon-
sive signal for high-contrast tumor imaging.
3
. Conclusion
4. Experimental Section
We have synthesized PDMA-r-HPA grafted semiconducting
polymer brushes that self-assembled into thermoresponsive PA
agents for contrast-enhanced in vivo imaging. With the help
of PDMA-r-HPA grafting, SPNph1 had LCSTs at 48 °C, and
underwent phase separation to form large nanoparticles. Thus,
SPNph1 possessed thermoresponsive PA property, showing
Characterization: UV–vis spectra were recorded on a Shimadzu
UV-2450 spectrophotometer. Fluorescence measurements were carried
out on a Fluorlog correlated single photon counting spectrofluorometer.
DLS and ζ-potential measurements were performed on a Malvern
Nano-ZS Particle Size at different temperature (30, 32, 34, 36, 38, 40,
4
2, 44, 46, 48, 50, and 52 °C). TEM images were obtained using a JEM
2100 PLUS transmission electron microscope with an accelerating
voltage from 100 to 200 kV. The samples were incubated at 50 °C in
a water bath for 5 min or room temperature and then dropped onto
the specimen grids. GPC was conducted by a Shimadzu LC-VP system
with HPLC grade THF as the eluent and polystyrenes as the standard.
NMR spectroscopy was recorded on a BRUKER Advance 300 NMR
1
.6-fold signal increment when temperature was above LCSTs.
By contrast, the PA intensity of the control without PDMA-r-
HPA grafting (SPNp) had no such PA behavior. In addition,
the photothermal conversion efficiency of SPNph1 was 1.3-fold
higher than that of SPNp, resulting in the higher PA intensity
at the same concentration. Due to the small size (35 nm) at
room temperature, SPNph could effectively accumulate in the
tumor site through enhanced permeability and retention (EPR)
effect after systematic administration, showing even stronger
PA signal than that in liver. The thermoresponsive PA property
of SPNph1 enabled in situ remote manipulation of PA signals
by laser-irradiation-mediated heating, leading to the 1.43-fold
enhancement in the tumor SBR. Thus, this study introduces a
(
1H, 300 MHz) system with CDCl or CD OD as the solvent. The spectrum
3 3
was internally referenced to the tetramethylsilane signal at 0 ppm.
Synthesis of prop-2-yn-1-yl 2-bromo-2-methylpropanoate (1): 2-Propyn-
1
-ol (222 mg, 4 mmol) and triethylamine (439 mg, 4.4 mmol) were
dissolved in DCM (10 mL). α-Bromoisobutyryl bromide (1 g, 4.4 mmol)
was dropwise added into the above solution at 0 °C. The mixture
was stirred at room temperature for 24 h. The resulting solution was
washed by water and brine for three times and dried over sodium
sulfate anhydrous (Na SO ). The crude product was purified via silica
2
4
chromatography column (Petroleum ether/DCM = 3:1) to the compound
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1
1
4
as colorless liquid (1.07 g, 97% yield). H NMR (300 MHz, CDCl ) δ
(inner diameter: 0.59 mm; outer diameter: 0.78 mm). The PA signals
produced in LDPE tube was coupled to the single-element ultrasound
3
.77 (d, J = 2.5 Hz, 2H), 2.51 (t, J = 2.4 Hz, 1H), 1.96 (s, 3H).
Synthesis of Poly(N,N-Dimethylacrylamide)40-r-(Hydroxypropyl
Acrylate)60 (2): N,N-Dimethylacrylamide (500 mg, 5.01 mmol),
-hydroxypropyl acrylate (984 mg, 7.56 mmol) CuBr (18.1 mg,
.13 mmol), and 1,4,8,11-Tetraazacyclotetradecane (32.3 mg,
.13 mmol) were added in a 50 mL Schlenk tube. The system was
degassed via freeze-pump-thaw cycles for three times. Then the toluene/
DMF solution of compound 1 (25.84 mg, 0.13 mmol) were added to
the tube and degassed via freeze-pump-thaw cycles for three times. The
reaction was carried out at 90 °C for 48 h. The solvent was removed
under vacuum. The crude product was precipitated into ethoxyethane.
−
1
transducer, UST (V323-SU 2.25 MHz , 13 mm active area, and 70%
nominal bandwidth, Panametrics). Both the tube and UST were
immersed in aqueous medium and incubated in a water bath to change
the temperature from 25 to 55 °C. PBS solution containing SPNph1 and
SPNp at different concentration (6, 12, 25, 50, 100, 200, and 500 µg
2
0
0
−
1
mL ) were placed inside LDPE tube with or without laser irradiation
−
2
at 808 nm (0.8 W cm ) and imaged using the Endra Nexus 128 PA
imaging system.
Tumor Mouse Model: All animal experiments were performed in
compliance with the guidelines established by the Institutional Animal
Care and Use Committee, Singapore. To establish tumor models in
six-week-old female Ncr nude mice, two million 4T1 cells suspended
in DMEM cell culture medium were subcutaneously injected into
the shoulder of each mouse. Tumors were grown for 1 week and then
the tumor-bearing mice were used for in vivo imaging experiments.
In Vivo PA Imaging of Tumor: 4T1 tumor-bearing nude mice were
1
The polymer 2 (1.27 g, 84.6% yield) was obtained as white solid. H NMR
300 MHz, MeOD) δ 3.99 (s, 120H), 3.59 (s, 40H), 3.05 (d, J = 14.1 Hz,
20H), 2.94 (s, 180H), 2.35 (d, J = 47.0 Hz, 80H), 1.69 (t, J = 61.9 Hz,
(
1
2
20H), 1.21 (s, 180H).
Synthesis of SPNp (5): Polymer 3 (5.18 mg) was synthesized
according to the previous literature[ and then dissolved in THF. CuBr
21]
−
1
(
4
5.5 mg, 0.04 mmol), PMDETA (33.4 mg, 0.2 mmol), and compound
(Mn = 2000, 40 mg, 0.02 mmol) were added into previous solution
subsequently. The mixture was stirred at room temperature under
nitrogen atmosphere for 48 h. The solvents were removed and the
residue was dissolved into water. The resulting solution was dialysis
systemically administrated with SPNph1 or SPNp (200 µL, 400 µg mL )
(n = 3). Before (0 h) and after injection of nanoparticles at different time
point (8, 24, and 32 h), the tumors of mice were irradiated by an NIR
−
2
laser at 808 nm (0.8 W cm ) for 1 min. During laser irradiation, an IR
thermal camera was used to monitor the temperature changes in tumor
sites. The tumors of mice with or without NIR laser irradiation were
imaged using the Endra Nexus 128 PA imaging system at 680 nm. For
ex vivo PA imaging, mice were sacrificed by CO2 asphyxiation, and the
tumors and major organs were extracted and immediately imaged using
the Endra Nexus128 PA imaging system.
against DI water for 12 h. The PCDPTBT-PEG was obtained after
1
lyophilization. H NMR (300 MHz, CDCl ) δ 7.71 (s, 1H), 7.61 (s, 1H),
3
7
(
.53–7.42 (m, 2H), 4.00–3.39 (m, 320H), 1.76 (d, J = 53.7 Hz, 32H), 1.31
d, J = 33.2 Hz, 32H), 0.87 (d, J = 7.3 Hz, 24H).
Synthesis of SPNph1 (6): Polymer 2 (207.8 mg), polymer 3 (5.18 mg),
and polymer 4 (2 mg) were dissolved in THF. CuBr (5.5 mg, 0.04 mmol)
and PMDETA (33.4 mg, 0.2 mmol) were added into above solution
subsequently. The mixture was stirred at room temperature for 48 h.
The solvent was removed and the residue was dissolved into water.
The resulting solution was dialysis against DI water. The SPNph1 was
obtained after lyophilization. H NMR (300 MHz, MeOD) δ 6.18 (dd,
J = 128.0, 51.4 Hz, 4H), 3.89 (d, J = 66.9 Hz, 56H), 3.66 (s, 20H), 3.60
H&E Staining: 4T1 tumor-bearing mice after 3 days intravenous
−
1
injection of SPNp or SPNph1 (200 µL, 400 µg mL ) were euthanized and
the major organs (heart, liver, spleen, lung, and kidney) were extracted
for HE staining. The organs were fixed with 4% paraformaldehyde for
24 h, dehydrated in a series of ethanol solution, immersed in xylene,
embedded in paraffin, and then cut into sections at a thickness of
10 µm. The sections were dried overnight before staining. The dried
sections were sequentially immersed in xylene, ethanol, and water.
After that, the sections were stained with hematoxylin working solution
for 5 min and eosin working solution for 5 min. The images of stained
sections were captured using a Nikon ECLIPSE 80i microscope (Nikon
Corporation, Towa Optics, New Delhi, India).
1
(
s, 15H), 3.02 (d, J = 40.7 Hz, 160H), 2.36 (d, J = 45.5 Hz, 32H), 1.65 (d,
J = 47.2 Hz, 80H), 1.12 (d, J = 60.9 Hz, 120H).
Synthesis of SPNph2 (7): Polymer 2 (215.2 mg) and polymer 3
(5.18 mg) were dissolved in THF, following by addition of CuBr (5.5 mg,
0
.04 mmol) and PMDETA (33.4 mg, 0.2 mmol). The mixture was
stirred at room temperature for 48 h. The solvent was removed and the
Data Analysis: PA signal amplitudes were detected by ROS analysis
using OsiriX. Results are expressed as the mean ± SD deviation unless
otherwise stated. Statistical comparisons between two groups were
determined by Student’s t-test. A p-value of 0.05 was selected as the
level of significance. And the data were classified according to their
p-values and denoted by (*) for p < 0.05.
residue was dissolved into water. The resulting solution was dialysis
1
against DI water. The SPNph2 was obtained after lyophilization.
H
NMR (300 MHz, MeOD) δ 7.83 (m, 4H), 3.99 (s, 240H), 3.59 (s, 80H),
3
4
.05-2.94 (m, 600H), 2.35 (d, J = 47.0 Hz, 160H), 1.69 (t, J = 61.9 Hz,
40H), 1.21 (s, 560H).
Cell Culture and Cytotoxicity Assay: The in vitro cytotoxicity of 4T1
cancer cells was measured using MTS viability assay. 4T1 cancer
cells were cultured in Dulbecco’s modified Eagle medium (DMEM)
containing 10% fetal bovine serum at 37 °C and 5% CO . 4T1 cells were Supporting Information
2
4
seeded in 96-well plates (Costar, IL, USA) at a density of 1 × 10 cells
Supporting Information is available from the Wiley Online Library or
from the author.
per well. After 24 h culture, the medium was replaced by fresh medium
containing SPNph1 and SPNp at different concentrations (0, 10, 20, 50,
−
1
7
5, 100 µg mL ) and the cells were then incubated for 24 h. After that,
MTS reagent was added into cell culture medium (v/v, 1/10) and the
cells were incubated for another 3 h. Then the absorbance of cell culture
medium at 490 nm was measured and used to calculate the cell viability.
In Vitro Photothermal Studies: PBS solution (1×, pH = 7.4) containing
Acknowledgements
K.P. thanks Nanyang Technological University (Start-Up grant:
NTU-SUG: M4081627.120) and Singapore Ministry of Education
Academic Research Fund Tier 1 (2017-T1-002-134-RG147/17) and Tier
2 (MOE2016-T2-1-098&MOE2018-T2-2-042) for financial support. H.X.
thanks the National Natural Science Foundation of China (81501535),
Special Funds for Basic Scientific Research Business in Central
Universities (2042018kf0216), Platform Support and Construction
Project of Hubei Health and Family Planning Commission and Zhongnan
hospital of Wuhan University, Wuhan Young and Middle-aged Medical
Talents Project. Y.A. thanks Major Technological Innovation Project in
Hubei Province (in the Field of Social Development).
−
1
SPNph1, SPNph2, and SPNp (200 µL, 30 µg mL ) was exposed to laser
−
2
irradiation at 808 nm (0.8 W cm ). The temperature was monitored
using an IR thermal camera (FLIR T420) every 20 s until reaching
maxima after ≈8 min. After laser irradiation, the temperature of solution
was continuously monitored every 20 s for 8 min. The heating and
cooling were repeated five times to test the photothermal stability of
SPNph1 and SPNp. The photothermal conversion efficiencies were
[10]
calculated according to the previous report.
In Vitro PA Studies: PBS solution containing SPNph1 or SPNp at
−
1
5
0 µg mL was placed inside a low-density polyethylene LDPE tube
Adv. Funct. Mater. 2019, 1903461
1903461 (6 of 7)
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Conflict of Interest
The authors declare no conflict of interest.
Keywords
photoacoustic imaging, polymer nanoparticles, thermoresponsive
materials, tumor imaging
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Revised: May 31, 2019
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© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim