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Multiresponsive Nanoprobes for Turn-On Fluorescence/19F MRI

Dual-Modal Imaging
Yawei Li, Hecheng Zhang, Chang Guo, Gaofei Hu,* and Leyu Wang*
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ABSTRACT: Multiresponsive nanoprobes are highly desirable for low

background and highly sensitive imaging in biomedical applications. Herein,
we design a glutathione (GSH)/pH dual-responsive nanoprobe capable of both
Downloaded via POLITECHNIKA SLASKA on March 10, 2021 at 10:00:55 (UTC).

fluorescence imaging in cells and 19F magnetic resonance imaging (19F MRI) in
deep tissue, by encapsulating manganese oleate (Mn(OA)2) on the surface of
fluorinated fluorescent quantum dots (F-ZnS:Mn2+). In this approach,
Mn(OA)2 serves as an efficient quencher of both fluorescence and 19F MRI
signal. Both the fluorescence and 19F MRI signal can be turned on by
introducing glutathione (GSH) that breaks up the Mn−O bonds within
Mn(OA)2 under weak acidity conditions (e.g., pH 6.0). The imaging results in cells and mice suggest that this novel strategy can
offer a promising nanoprobe for turn-on fluorescence/19F MRI dual-modal tumor imaging.

■ INTRODUCTION
Stimuli-responsive nanoparticle-based agents have attracted
considerable interest in both medical imaging1−3 and
therapeutics4−6 in recent decades. Stimuli-responsive nanop-
robes can greatly improve the contrast between physiological
and abnormal conditions due to their good targeting ability,
reduced side effects, and capability of being triggered by
specific aspects such as pH,7,8 redox level,9 and enzymatic
activity.2,10 It is well known that hypoxia, acidosis, and
overexpression of glutathione (GSH) are characteristics of
tumor microenvironment. In view of these differentials from
normal tissue, stimuli-responsive contrast agents in the
cancerous lesions would display different imaging signals
such as “off/on”, providing higher signal-to-noise ratios and
better sensitivity, and even a real-time reflection of the
evolution of pathological process. Many previous attempts
have been made to develop smart nanosystems based on the
response to single11−13 and multiple14 tumor microenviron-
ment elements. Generally, multiresponsive nanoprobes could
achieve higher specificity and accuracy for imaging diagnosis
and enhanced efficacy for treatment due to the synergistic
elimination of tumor abnormalities.15
Magnetic resonance imaging (MRI) has been recognized as
one of the most important clinically and commonly used
medical imaging modalities owing to its advances in high
penetration depth, noninvasion, and exemption from harmful
ionizing radiation.16−18 Conventional 1H MRI generally
provides anatomical images of organs and soft tissue based
on the proton signal of water inside the organs, usually using
contrast agents (T1-weighted or T2-weighted) to improve the
imaging contrast. While for 19F MRI, 19F signal is directly
detected and proportional to the concentration of the
exogenous fluorinated probes, without background interfer-
ence owing to the absence of endogenous 19F element (except
little in bones and teeth). Therefore, 19F MRI can offer
intuitive and accurate images in pathological imaging by virtue
of anatomical location of 1H MRI, which makes it a promising
complementary technique to 1H MRI.19,20 With respect to
sensitivity, the MRI technique including 1H/19F MRI was
generally considered not comparable to other modalities such
as fluorescence imaging and positron emission computed
tomography (PET); however, 19F MRI signal can be greatly
improved by developing ultrahigh 19F-loaded nanoprobes to
overcome the limitation in sensitivity.21−23
Recently, the paramagnetic relaxation enhancement (PRE)
effect has been employed as a potent strategy for designing and
synthesizing stimuli-responsive MRI probes in which 1H/19F
signal can be modulated by the neighboring paramagnetic
metal center (such as Mn2+, Co2+, Fe3+, and Gd3+) to quench/
activate upon exposure to a specific stimulus that changes the
paramagnetic relaxivity of the metals or the structure of
probes.24−29 To date, various manganese complexes and oxides
have been used for the construction of PRE contrast agents,
relying on either their capability of reversible switching
between Mn(II) and Mn(III) toward redox activity30,31 or
the breakage of the Mn−O bonds within the probes under
reductive and/or weakly acidic conditions causing to weaken
and even vanish the PRE effect, which requires a close
Received: April 25, 2020
Accepted: July 31, 2020
Published: July 31, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.analchem.0c01786

11739 Anal. Chem. 2020, 92, 11739−11746
Analytical Chemistry
proximity between the nuclei (1H/19F) and metal cen-
ter.13,32−34 For example, Caravan’s group35 made systematic
studies on the structure−redox−relaxivity relationships for
Mn-based MRI probes and found that the ligand structural and
electronic modifications play an important role in tuning and
optimizing Mn(II) versus Mn(III) relaxivity (r1) differentials
and Mn(II/III) redox potentials, which are vital for the
responsiveness of the probes. Kim et al.36 reported a Mn3O4-
coated Fe3O4 hybrid nanocrystal, which consists of a
superparamagnetic core and a GSH-responsive paramagnetic
shell, accomplishing activatable T1/T2 relaxation switch MR
imaging (from bright to dark) under an intracellular reducing
environment so as to greatly enhance the MR contrast in
targeted locations.
In addition, as is known that each imaging technique may
have its own limitations such as sensitivity, resolution, or
interference from matrixes, combining MRI with a secondary
imaging modality would be helpful to compensate each other
for more accurate tumor imaging and diagnosis.37−39
Currently, fluorescent imaging is considered as an ultra-
sensitive technique and numerous fluorescent agents including
organic dyes,40 polymeric molecules,41 inorganic nanomateri-
als,42 and nanocomposites43 have been developed and widely
applied in cell tracking and tissue imaging. Especially as a
vector, inorganic nanomaterials with intrinsic excellent
fluorescent property like quantum dots (QDs) and upconver-
sion nanocrystals are promising for loading/assembling
fluorinated moieties to construct 19F MRI/fluorescence
multifunctional nanosystems.5,8,44 Therefore, it is essential
and challenging to develop inventive imaging strategies based
on multiresponsive (e.g., GSH/pH dual-responsive) and
multimodal nanoplatforms that can be specifically triggered
to simultaneously “turn-on” different signals, thus enabling
accurate imaging of biological targets.
Herein, we rationally designed and synthesized a GSH/pH
dual-responsive nanoprobe capable of both fluorescence and
19
F MR imaging in both cells and deep tissue. This was
accomplished by encapsulating manganese oleate (Mn(OA)2)
and perfluoro-15-crown-5-ether (PFCE, as 19F moiety) onto
the surface of ZnS:Mn2+ fluorescent quantum dots (QDs) with
amphiphilic PSIOAm (oleylamine-modified polysuccinimide)
(Scheme 1). In this approach, Mn(OA)2 exhibits excellent
quenching effects on both fluorescence and 19F MRI signals,
Scheme 1. Schematic Illustration of Fabrication and GSH/
pH-Responsive Feature for the ZnS:Mn2+-F-Mn(OA)2@
PSIOAm Dual-Modal Imaging Nanoprobes
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owing to its optical absorption and PRE effect to the adjacent
19
F nuclei, respectively. However, both the fluorescence and
19
F MRI signals can be turned on upon encountering the acidic
conditions (e.g., pH 6.0) and high level of GSH (e.g., 10 mM)
that leads to the breakage of Mn−O bonds within Mn(OA)2.
The imaging contrast effects of the off/on dual-modal
nanoprobe and the potential of this novel strategy were
confirmed through in vitro and in vivo experiments.
■
EXPERIMENTAL SECTION
Chemicals and Reagents. General chemicals were of at
least analytical- grade and used without further purification,
unless otherwise noted. Sodium hydroxide (NaOH), cyclo-
hexane, hexane, methanol, ethanol, chloroform, N,N-dimethyl-
formamide (DMF), and hydrochloric acid were purchased
from Beijing Chemical Works. Oleic acid (OA, 90%) was
obtained from Alfa Aesar (Shanghai, China), and oleylamine
(OAm, 80−90%) was from Acros Organics (Belgium). Sodium
oleate (NaOA) was supplied by Macklin (Shanghai, China).
Zinc chloride (ZnCl2) was obtained from Tianjin Guangfu
Chemical Reagent Factory (Tianjin, China). Sodium sulfide
nonahydrate (Na2S·9H2O) was purchased from Xilong
Chemical Company (Shanghai, China). Manganese chloride
tetrahydrate (MnCl2·4H2O) was from Sinopharm (Beijing,
China). Perfluoro-15-crown-5-ether (PFCE) was obtained
from Fluorochem (U.K.). Polysuccinimide (PSI) (Mw ≈
7000) was supplied by Shijiazhuang Desai Chemical Company
(Shijiazhuang, China). Ultrapure water was produced using a
Milli-Q water purification system from Millipore (Bedford,
MA).
Characterization. 19F nuclear magnetic resonance (NMR)
experiments were carried out on a Bruker Avance-III HD 400
spectrometer at 376.47 MHz using a Bruker “single-pulse”
sequence without proton decoupling. Both in vitro and in vivo
MR imaging (1H and 19F) tests were conducted on a 7.0 T
Bruker BioSpec70/20USR MRI system, using a transmitter
and transceiver-integrated 1H/19F birdcage coil for mouse
body to detect 1H and 19F signals. The transmission electron
microscopy (TEM) images were taken on a JEOL JEM-
1200EX transmission electron microscope at an accelerating
voltage of 100 kV. Fluorescence spectra were recorded on an
F-4600 spectrophotometer (Hitachi, Japan) with an excitation
wavelength of 330 nm. Dynamic light scattering (DLS) particle
size analysis was carried out using a Zetasizer Nano-ZS90 zeta
and size analyzer from Malvern. IR spectra were collected on a
Nexus 670 Fourier transform infrared (FT-IR) spectropho-
tometer (Nicolet). The Mn composition of the samples was
detected by an inductively coupled plasma optical emission
spectrometer (ICP-OES, Thermo Scientific iCAP 6000 series).
Preparation of ZnS:Mn2+ Quantum Dots (QDs). The
ZnS:Mn2+ QDs were synthesized according to the previously
reported method with moderate modification.45 In brief,
NaOH aqueous solution (3 M, 5 mL), ethanol (8 mL, 99%),
and oleic acid (10 mL) were transferred successively into a 50
mL Teflon-lined autoclave under vigorous stirring. The
mixture of ZnCl2 aqueous solution (1 M, 1.9 mL) and
MnCl2 aqueous solution (1 M, 0.1 mL) was then added into
the above solution. After 5 min, Na2S aqueous solution (0.9 M,
2 mL) was further introduced dropwise with vigorous stirring.
And the final mixture was kept stirring vigorously for another
10 min. Then, the autoclave was sealed and heated at 160 °C
for 8 h. After cooling down to room temperature, the
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ZnS:Mn2+ powder sinking at the bottom was dispersed in
cyclohexane and then precipitated with ethanol. Finally,
ZnS:Mn2+ QDs were purified by centrifuging and washing
with ethanol for three times (7000 rpm, 10 min), then
dissolved in 5 mL of chloroform for later use.
Preparation of Oleylamine-Modified PSI (PSIOAm).
PSIOAm was prepared as our previous procedure.46 Briefly,
PSI (1.6 g) and DMF (32 mL) were added into a 100 mL
flask, maintaining at 90 °C and stirring until PSI was
completely dissolved in DMF. Then, oleylamine (1.65 mL)
was added into the solution, and the resulting solution was
kept stirring at 100 °C for 5 h. After cooling down to room
temperature, the final product was collected by precipitating
with methanol and centrifuging (8000 rpm, 5 min). The
purified PSIOAm was finally dissolved in 8 mL of chloroform for
later use.
Preparation of Mn(OA)2. Manganese oleate was synthe-
sized according to the method reported previously.47
Manganese chloride tetrahydrate (4 mmol) and sodium oleate
(8 mmol) were dissolved in a mixed solvent containing 4 mL
of ultrapure water, 3 mL of ethanol, and 7 mL of hexane. The
resultant mixture was then kept at 70 °C for 4 h under stirring.
After removing the aqueous solution, the oily residue was
obtained and further washed with water and dried over
anhydrous MgSO4. The final product was dried under vacuum
at 60 °C for 2 h to obtain a deep red waxy solid.
Preparation of ZnS:Mn2+-F-Mn(OA)2@PSIOAm. The as-
prepared ZnS:Mn2+ QDs (15 mg), PSIOAm (14 mg), Mn(OA)2
(8 mg), and PFCE (10 μL) were dispersed in 1.0 mL of
CHCl3. The mixture was fully oscillated and then added into
NaOH aqueous solution (5 mM, 10 mL), followed by
ultrasonication treatment for 6 min (300 W, 3 s “on” and 3
s “off” in turn). The resultant emulsion was kept stirring for
another 2 h at 50 °C to remove the chloroform, then
centrifuged (18 000 rpm, 10 min) with water twice. The final
product was dispersed in 1 mL of phosphate-buffered saline
(PBS) for further imaging experiments.
Simulation of Tumor Microenvironment (GSH/pH) for
In Vitro 19F MR Tests. In brief, 0.2 M phosphate buffer
solutions (PBS) with different pH values (5.5, 6.0, 6.5, 7.0, 7.4)
were first prepared. In a 1.5 mL centrifuge tube, 800 μL (20
mg/mL) of the as-prepared nanoprobes was mixed with GSH
at various concentrations, making up to 1.0 mL with PBS to
ensure the expected pH. Then, the resultant mixed solution
was incubated at 37 °C for 1 h, and the 19F NMR or 19F MRI
tests of the solutions were conducted.
Cytotoxicity Tests. Cytotoxicity of the fabricated hydro-
philic ZnS:Mn2+-F-Mn(OA)2@PSIOAm nanoprobes was eval-
uated via the methyl thiazolyl tetrazolium (MTT) method
against tumor cells (4T1, breast cancer cell of mouse) and
normal tissue cells (HUVECs, umbilical vein endothelial cells
of human), respectively. About 5 × 104 cells/well were seeded
in a 96-well microtiter plate; then, nanoprobes of different
concentrations (from 0 to 250 μg/mL) were added and
cultured at 37 °C under 5% CO2 and a 95% relative humidity
atmosphere. Cytotoxicity was assessed at 24 and 48 h post-
treatment, respectively. After 10 μL of sterile-filtered MTT
stock solution in PBS (4.0 mg/mL) was added into each well
of the microtiter plate, it was incubated at 37 °C for another 3
h. Only the viable cells can induce the cellular reduction of
MTT, forming the soluble colored formazan. Then, the
absorbance of the produced formazan in each well can be
measured at 490 nm on an ELISA plate reader (F50, TECAN).
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The cell viability can be calculated from the absorbance
compared to that of the control group.
Cell Imaging. 4T1 cells and MREpiC cells (kidney
epithelial cells of mouse) were seeded on a sterilized culture
flask and cultured at 37 °C in a 12-well cell culture plate
overnight. Then, the as-prepared ZnS:Mn2+-F-Mn(OA)2@
PSIOAm stock solution was added into each well, respectively,
with a final concentration of 400 μg/mL. The cells were
incubated for another 0−12 h. Then, it was transferred to
glass-bottom cell culture dishes for imaging tests. Confocal
laser scanning microscopy (CLSM) images were recorded on a
laser confocal microscope (Leica SP8). And the excitation
wavelength was set at 405 nm.
Animal Experiments. Animal experiments were per-
formed using female mice (Balb/c). All animal experiments
were approved by the local ethics review board and carried out
in accordance with the guidelines of the Animal Care and Use
Committee of Beijing University of Chemical Technology.
4T1 cells (∼107) mixed with 50% Matrigel (BD Biosciences)
suspension were injected into the right flank of 4-week-old
female mice subcutaneously. As the tumors grew to around
300 mm3, the nanoprobe solution (20 mg/mL in PBS, 150 μL)
was injected into the tumor and normal tissue (as a comparing
group) of the mice. The MRI measurements in vivo were
carried out after live mice were anesthetized with isoflurane.
The T1-RARE sequence was used for 19F MRI, and the related
parameters were set as follows: matrix size was 100 × 100; TR
and TE were 3000 and 4.64 ms, respectively; and the field of
view (FOV) was set at 50 mm × 50 mm with a slice thickness
of 20 mm. The total experiment time was 19 min.
Statistical Analysis. All quantitative data were performed
as mean ± standard deviation (SD). The statistical significance
was carried out using one-way analysis of variance (ANOVA)
with Origin 9.0 (OriginLab). Differences between groups were
tested for statistical significance with Student’s t-test, accepting
significance at p < 0.05 or p < 0.01.
■ RESULTS AND DISCUSSION
Characterization of the Multiresponsive Nanoprobes.
The transmission electron microscope (TEM) image (Figure
1a) showed that the as-prepared hydrophobic ZnS:Mn2+ QDs
exhibited as homogeneous monodisperse nanoparticles (NPs)
with an average size of 11.5 ± 0.4 nm obtained from the
dynamic light scattering (DLS) analysis (Figure 1b). The
phase structure was further confirmed by X-ray powder
diffraction (XRD) (Figure S1). Afterward, ZnS:Mn2+ QDs
were coencapsulated with PFCE (source of 19F MR signal) and
Mn(OA)2 (the quencher) by amphiphilic PSIOAm, generating
ZnS:Mn2+-F-Mn(OA)2@PSIOAm nanoprobes, which displayed
a good dispersion in water with an average DLS size of 39.4 ±
11.1 nm (Figure 1c,d). After incubation with GSH (10 mM, at
pH 6.0), the DLS size of the nanoprobes slightly decreased to
around 30 ± 11.2 nm, while the morphology was well
maintained, indicating the probable degradation of manganese
oleate surrounding the ZnS:Mn2+ core due to the breakage of
Mn−O bonds under acidic and reducing (H + /GSH)
conditions (Figure 1e,f).48,49 Moreover, the HRTEM images
of the ZnS:Mn2+-F-Mn(OA)2@PSIOAm nanoprobe before and
after reaction with GSH showed that the crystal lattice
characteristics and particle size of the core part (ZnS:Mn2+
QDs) were also well retained (Figure S2). In addition, as
shown in the FTIR spectra of ZnS:Mn2+-F-Mn(OA)2@PSIOAm
and the corresponding components (Figure S3), the bands at
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Analytical Chemistry
Figure 1. TEM images (a, c, e) and DLS size distribution (b, d, f) of
hydrophobic ZnS:Mn2+ QDs (a, b), hydrophilic ZnS:Mn2+-F-
Mn(OA)2@PSIOAm before (c, d) and after (e, f) reaction with GSH
(10 mM, at pH 6.0). Accordingly, the insets of (b, d, f) are the
photographs of the nanoparticle colloid solution under UV light (Ex.
365 nm).
1158 and 1228 cm−1 correspond to the C−F and C−O
stretching vibrations in PFCE; the bands at 1554 and 1413
cm−1 are ascribed to the antistretching and stretching
vibrations of carboxylate group in Mn(OA)2, respectively,
manifesting that PFCE and Mn(OA)2 were encapsulated into
PSIOAm successfully.
Furthermore, the photographs (insets of Figure 1b,d,f)
clearly showed that the fluorescence of ZnS:Mn2+-F-Mn-
(OA)2@PSIOAm solution was quenched (turn-off) compared to
that of ZnS:Mn2+ QDs and recovered (turn-on) after exposure
to GSH (10 mM) at pH 6.0 because the encapsulated
Mn(OA)2 has strong absorption at the excitation wavelength
of ZnS:Mn2+-F-Mn(OA)2@PSIOAm (Figures S4 and S5).
Accordingly, the 19F NMR signal of ZnS:Mn2+-F-Mn(OA)2@
PSIOAm solution also displayed obviously from off to on
(Figure S6), due to PRE effects of Mn(II) from the adjacent
Mn(OA)2 on 19F nuclei and thereafter greatly weakened along
with the degradation of Mn(OA)2 under H+/GSH conditions
(significantly increased distance between Mn(II) and 19F).
These observations proved Mn(OA)2 an efficient quencher for
both fluorescence and 19F MR signal, endowing the as-
prepared ZnS:Mn2+-F-Mn(OA)2@PSIOAm with desired multi-
responsive (GSH/pH) property for enhanced dual-modal
imaging.
Evaluation of Response to GSH/H+. As the quencher for
both fluorescence and 19F MR modalities, the dosage of
manganese complexes was optimized to attain superior
responsiveness for the as-prepared nanoprobes. According to
extensive reports32,50−53 on the features of physiological and
tumor microenvironment, three pH levels (7.4, 6.5, and 6.0)
and concentrations of GSH in the range of 1.0−10 mM were
set for the in vitro investigations. Figure 2 indicates that the
quenching effect on 19F MR signal continuously improved with
an increase in the amount of Mn(OA)2, while upon exposure
to GSH/H+ conditions (10 mM of GSH, pH 6.0), the signals
recovered to almost an equivalent level (normalized SNR
around 0.6) until the dosage of Mn(OA)2 exceeds 9.6 μmol
and then gradually downward to about 0.2 at the dosage of
16.0 μmol of Mn(OA)2. However, it can be seen that the
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Figure 2. Normalized 19F NMR SNR (signal-to-noise ratio) as a
function of dosage of Mn(OA)2 for the responsiveness of ZnS:Mn2+-
F-Mn(OA)2@PSIOAm nanoprobes. CGSH = 10 mM, at pH 6.0. (*p <
0.05, **p < 0.01, n = 3).
recovery efficiency (ratio of I to I0, where I and I0 represent the
19
F MR signal intensity after and before exposure to GSH/H+,
respectively) is the indicator to attain the optimal contrast
imaging between physiological and tumor conditions. There-
fore, 12.8 μmol of Mn(OA)2 was finally utilized in view of the
highest recovery efficiency of around 15.0.
The fluorescence and 19F MR signal evolution of ZnS:Mn2+-
F-Mn(OA)2@PSIOAm colloidal solution toward GSH at
different pH values (Figure 3) showed that the recovery
Figure 3. Fluorescence (a) and 19F MR signal (b) recovery as a
function of GSH concentration under different pH conditions. The
nanoprobes were incubated at 37 °C for 1 h. For each point, three
independent parallel experiments were carried out (n = 3).
efficiency of both fluorescence and 19F MR signal increased
along with the increase of GSH concentration at acidic media
(pH 6.5 and 6.0), and the lower the pH, the more efficient the
recovery (faster and higher). While at physiological pH of 7.4,
both of the fluorescence and MR signals recovered very slightly
even at the highest concentration of GSH (10 mM).
Furthermore, the fluorescence and 19F MR signal variation
versus time at different pH and GSH combining conditions
was investigated (Figure 4). Although it is shown that the
response time for fluorescence was longer than that for 19F MR
(Figure 4a,d), 15 min was sufficient for both 19F MR and
fluorescence turn-on. It is also displayed that only weak acidity
in the absence of GSH led to no recovery of 19F MR (Figure
4b) and recovery to a certain extent for fluorescence (Figure
4e); meanwhile, only GSH (under pH 7.4) could not result in
recovery for both 19F MR and fluorescence (Figure 4c,f),
suggesting that the desirable dual-responsive property was
based on the decomposition of loaded Mn(OA)2 under GSH/
H+ combining conditions.
Besides, it is noteworthy that some recovery of fluorescence
toward acidity (without GSH) might be ascribed to the
decrease of the absorbance within the range of excitation
wavelength for fluorescence, which can be proved by the
absorption spectra of ZnS-Mn(OA)2@PSIOAm under various
pH and GSH combining conditions (Figure S7a). Additionally,
the ICP-OES measurements of the Mn content in the
supernatants of ZnS-Mn(OA)2@PSIOAm colloidal solution
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Analytical Chemistry
Figure 4. 19F MR signal (a, b, c) and fluorescence (d, e, f) evolution
versus time at different pH values with CGSH = 10 mM (a, d), without
GSH (b, e), and with different GSH concentrations at pH 7.4 (c, f).
For each point, two independent parallel experiments were carried
out.
(Figure S7b) indicated a remarkable release of Mn ions at pH
6.0 and 10 mM of GSH compared to other conditions, further
confirming that breakage of Mn−O bonds under GSH/H+
conditions rather than individual GSH or acidic environment.
Moreover, the selectivity of ZnS:Mn2+-F-Mn(OA)2@PSIOAm
toward GSH over potential interferents was investigated under
both pH 7.4 and 6.0 (Figure S8). As expected, the nanoprobe
had slight 19F NMR response to GSH under pH 7.4 (Figure 3)
and similar response to the interferents (Figure S8a). While
under pH 6.0, highly desirable selectivity of the nanoprobe
toward GSH was shown over all of the interferents (Figure
S8b), indicating that the probe was highly specific to GSH/H+
combined condition.
In Vitro Dual-Modal Imaging Investigation. Biocom-
patibility and stability are fundamental and vital for the as-
prepared ZnS:Mn2+-F-Mn(OA)2@PSIOAm nanoprobes to
achieve reliable biological applications. The cytotoxicity
evaluation of ZnS:Mn 2+ -F-Mn(OA) 2 @PSI OAm was first
checked on different types of cells via the MTT assay. As
shown in Figure S9, after incubating with ZnS:Mn2+-F-
Mn(OA)2@PSIOAm even at the highest level (250 μg/mL)
for 48 h, normal cells (HUVECs) still had a viability over 85%;
in contrast, only less than 40% of the tumor cells (4T1)
survived. This demonstrated a good biocompatibility of
ZnS:Mn2+-F-Mn(OA)2@PSIOAm under normal cellular envi-
ronments and some certain toxicity toward tumor cells, that is,
available chemotherapeutic effects on tumor cells. The ICP-
OES results showed that the concentration of Mn in the
supernatant of ZnS:Mn2+-F-Mn(OA)2@PSIOAm aqueous sol-
ution (pH 7.4) increased from 0.473 to 1.98 μg/mL after the
addition of GSH (10 mM, pH 6.0), which might ascribe the
higher toxicity in tumor cells to the release of Mn2+ under
tumor microenvironment. The flow cytometry analysis of both
tumor (4T1) and normal (MREpiC) cells incubated with the
Nile red (NLR)-loaded nanoprobe, ZnS:Mn2+-F-NLR-Mn-
(OA)2@PSIOAm, further suggested that the nanoprobe was
more sensitive to tumor cells due to stripping off the Mn(OA)2
shell under tumor environment (Figure S10). Additionally, as
shown in Figure S11, 19F MR signal, fluorescence intensity, as
well as average DLS size of ZnS:Mn2+-F-Mn(OA)2@PSIOAm
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colloidal solution showed negligible changes over 5 weeks.
While upon exposing the nanoprobe solution to GSH (10
mM) at pH 6.0 for a long term, the changes in 19F MR signal,
fluorescence intensity, and DLS size were consistent with those
of the freshly prepared nanoprobes. All of these observations
clearly indicated that these multifunctional nanoprobes have
excellent biocompatibility and stability for further applications.
To ensure a sufficient responsiveness of the nanoprobes for
19
F MRI in vivo, the phantom studies were performed in
advance. The 19F MR imaging of the nanoprobe solution with
different pH values and GSH concentrations (Figure 5)
Figure 5. 1H and 19F MR images of ZnS:Mn2+-F-Mn(OA)2@PSIOAm
colloidal solution under different pH values and GSH concentrations
(0, 0.5, 1.0, 5.0, 10.0 mM). 1H MRI was carried out with a T1-
weighted imaging method.
obviously showed that very high-contrast images can be
acquired in an environment with a high concentration of GSH
(over 5.0 mM) under weak acidity conditions (especially at pH
6.0), which precisely simulated the tumor microenvironment,
suggesting the great potential of ZnS:Mn2+-F-Mn(OA)2@
PSIOAm nanoprobes for in vivo 19F MRI application. As for the
1
H MR images, the darkening phenomenon occurred due to
the influence of relatively high concentration of the nanop-
robes producing Mn (either complexed or free) on the
surrounding protons, as confirmed by the T1-weighted and T2-
weighted 1 H MRI of the nanoprobes with different
concentrations diluted from samples of different pH values
and GSH conditions (Figure S12a,b). Moreover, the released
(free) Mn ions might have a greater impact on protons since
the sample of pH 6.0 and 10 mM GSH indicated a higher
relaxation rate than that under pH 7.4 and absence of GSH
(Figure S12c,d).
Accordingly, fluorescence imaging of tumor cells (4T1) and
normal cells (MREpiC) incubated with ZnS:Mn2+-F-Mn-
(OA)2@PSIOAm was evaluated. The confocal laser scanning
microscopy (CLSM) imaging conditions were first optimized
using 4T1 cells, and the incubation time of 4 h was
consequently selected as it observed an unobvious increase
of fluorescence (brightness) after 4 h and even up to 12 h
(Figure S13). As shown in Figure 6, 4T1 cells incubated with
the as-prepared nanoprobes displayed a satisfactory fluores-
cence image; meanwhile, no fluorescence could be observed in
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Anal. Chem. 2020, 92, 11739−11746
Analytical Chemistry
Figure 6. CLSM fluorescence images of 4T1 (a) and MREpiC cells
(b) incubated with ZnS:Mn2+-F-Mn(OA)2@PSIOAm for 4 h. The
concentration of the nanoprobe was 400 μg/mL. The excitation
wavelength was set at 405 nm. All of the scale bars are 25 μm.
MREpiC cells, demonstrating that ZnS:Mn2+-F-Mn(OA)2@
PSIOAm nanoprobes have desirable responsiveness to tumor
microenvironment for fluorescence imaging in live cells.
19
F MR Imaging Assessment in Mice. Encouraged by the
desirable results from in vitro tests, we tried to explore the
feasibility of applying these nanoprobes for in vivo studies. The
nanoprobe solution was injected into both the left and right
limbs of the tumor-bearing mice, but only the right limb was
inoculated with 4T1 cells. As shown in Figure 7, 1H MRI was
Figure 7. 1H (a) and 19F (b) MR and overlay (c) images of 4T1
tumor-bearing mice after intratumoral injection of ZnS:Mn2+-F-
Mn(OA)2@PSIOAm colloidal solution (20 mg/mL, 150 μL). The
same dosage of nanoprobe was subcutaneously injected into normal
tissue for comparison. 1H MRI was conducted with a T1-weighted
imaging method.
performed to show the anatomical structure of the mice and
the tumor profiles, and 19F MRI exhibited extremely a high-
contrast (bright) image only at the tumor site, as expected. In
normal physiological tissue, it is known as a weakly alkaline
environment with low concentration of GSH, in which the
nanoprobes displayed negligible 19F MR images because of the
PRE effect from the effective quencher (Mn (OA)2) within the
nanoprobe. Compared to traditional 1H MRI, the ZnS:Mn2+-F-
Mn(OA)2@PSIOAm-based 19F MRI could be more sensitive
and intuitive to distinguish between tumor and normal tissue,
thus providing a promisingly potential strategy for cancer
diagnosis and treatment.
To explore the fate of 19F imaging signal in the tumor along
with its progression, time-dependent 1H/19F MRI was further
conducted (Figure 8). As expected, 19F MRI displayed high
contrast in tumor site, while a negligible signal in normal tissue
shortly after injection of the nanoprobe (0.5 h). Afterward, 19F
images of the tumor weakened obviously at 4 h and gradually
decayed but were still observable at 24 h, indicating that the
probe (19F signal) had a relative long retention time and would
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pubs.acs.org/ac Article
Figure 8. 1H/19F MR images of 4T1 tumor-bearing mice after
intratumoral injection of ZnS:Mn2+-F-Mn(OA)2@PSIOAm colloidal
solution (20 mg/mL, 150 μL) at 0.5 h (a1−a3), 4 h (b1−b3), and 24 h
(c1−c3). The same dosage of nanoprobe was subcutaneously injected
into normal tissue for comparison. 1H MRI was conducted with a T1-
weighted imaging method.
be excreted in the long run, which meets expectations in
biomedical applications. In contrast, the 19F signal in normal
tissue was greatly weaker than that in the tumor during the
whole period, demonstrating the stimuli-responsive and low
background imaging properties of the as-prepared nanoprobe.
■ CONCLUSIONS
In summary, a smart GSH/pH dual-responsive imaging
nanoplatform, ZnS:Mn2+-F-Mn(OA)2@PSIOAm, was success-
fully fabricated for turn-on 19F MRI/fluorescence dual-modal
imaging in live cells and tumor tissue. Of this nanoplatform,
the core is ZnS:Mn2+ QDs, which provides excellent
fluorescence, and the 19F MR signal stems from the
encapsulated PFCE. The coencapsulated Mn(OA)2 serves as
an efficient quencher, which can be decomposed by
responding to GSH and acidic tumor microenvironment,
thus activating both fluorescence and 19F MR imaging. Both in
vitro and in vivo imaging results demonstrated desirable
effectiveness (high contrast between physiological and cancer-
ous conditions) of the as-prepared nanoprobes. This work also
demonstrated that incorporation of multiple stimuli-respon-
siveness into multimodal nanoprobes would inspire new
interests for the design of smart nanoprobes and provide a
promising strategy in biomedical applications.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.analchem.0c01786.
XRD pattern of ZnS:Mn2+ QDs (Figure S1); HRTEM
images of ZnS:Mn2+-F-Mn(OA)2@PSIOAm before and
after reaction with GSH (Figure S2); FT-IR spectra of
ZnS:Mn2+ QDs (the ligand is oleic acid), Mn(OA)2,
PFCE, PSIOAm, and ZnS:Mn2+-F-Mn(OA)2@PSIOAm
(Figure S3); UV−Vis absorption spectrum of Mn(OA)2
and fluorescence spectra of ZnS:Mn2+@PSIOAm and
ZnS:Mn2+-Mn(OA)2@PSIOAm (Figure S4); UV−Vis
https://dx.doi.org/10.1021/acs.analchem.0c01786
Anal. Chem. 2020, 92, 11739−11746
Analytical Chemistry
absorption of ZnS:Mn2+-F@PSIOAm and ZnS:Mn2+-F-
Mn(OA)2@PSIOAm (Figure S5); 19F NMR spectra of
ZnS:Mn2+-F-Mn(OA)2@PSIOAm aqueous solution be-
fore and after reaction with GSH (10 mM) at pH 6.0
(Figure S6); UV−Vis absorption spectra and ICP-OES
measurements of the Mn content in the supernatants of
ZnS-Mn(OA)2@PSIOAm colloidal solution under differ-
ent conditions (Figure S7); selectivity of ZnS:Mn2+-F-
Mn(OA)2@PSIOAm toward GSH (Figure S8); cytotox-
icity tests of ZnS:Mn2+-F-Mn(OA)2@PSIOAm colloids
after incubation with HUVECs and 4T1 cell lines
(Figure S9); flow cytometry analysis of MREpiC and
4T1 cells incubated with ZnS:Mn2+-F-NLR-Mn(OA)2@
PSIOAm (Figure S10); stability evaluation of ZnS:Mn2+-
F-Mn(OA)2@PSIOAm colloid solution (Figure S11); T1-
weighted and T 2 -weighted 1 H MR imaging of
ZnS:Mn2+-F-Mn(OA)2@PSIOAm under different condi-
tions (Figure S12); and fluorescence images of 4T1 cells
cultured with ZnS:Mn2+-F-Mn(OA)2@PSIOAm nanop-
robes for different time (Figure S13) (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
Gaofei Hu − State Key Laboratory of Chemical Resource
Engineering, Beijing Advanced Innovation Center for Soft
Matter Science and Engineering, Beijing University of Chemical
Technology, Beijing 100029, China; orcid.org/0000-0002-
7823-2966; Email: hugf@mail.buct.edu.cn
Leyu Wang − State Key Laboratory of Chemical Resource
Engineering, Beijing Advanced Innovation Center for Soft
Matter Science and Engineering, Beijing University of Chemical
Technology, Beijing 100029, China; orcid.org/0000-0002-
5961-7764; Email: lywang@mail.buct.edu.cn
Authors
Yawei Li − State Key Laboratory of Chemical Resource
Engineering, Beijing Advanced Innovation Center for Soft
Matter Science and Engineering, Beijing University of Chemical
Technology, Beijing 100029, China
Hecheng Zhang − Dongzhimen Hospital, Beijing University of
Chinese Medicine, Beijing 100700, China
Chang Guo − State Key Laboratory of Chemical Resource
Engineering, Beijing Advanced Innovation Center for Soft
Matter Science and Engineering, Beijing University of Chemical
Technology, Beijing 100029, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.analchem.0c01786
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors gratefully acknowledge financial support from the
National Natural Science Foundation of China (21675009 and
21725501) and the Fundamental Research Funds for the
Central Universities (XK1901, buctrc201812, and
buctrc201815).
■
REFERENCES
(1) Xiong, K.; Huo, F.; Chao, J.; Zhang, Y.; Yin, C. Anal. Chem.
2019, 91, 1472−1478.
(2) Guo, C.; Zhang, Y.; Li, Y.; Xu, S. Y.; Wang, L. Y. Anal. Chem.
2019, 91, 8147−8153.
11745
pubs.acs.org/ac Article
(3) Xu, M. M.; Guo, C.; Hu, G. F.; Xu, S. Y.; Wang, L. Y. Chin. J.
Chem. 2018, 36, 25−30.
(4) Kong, F.; Liang, Z.; Luan, D.; Liu, X.; Xu, K.; Tang, B. Anal.
Chem. 2016, 88, 6450−6456.
(5) Cui, J. B.; Jiang, R.; Guo, C.; Bai, X.; Xu, S. Y.; Wang, L. Y. J. Am.
Chem. Soc. 2018, 140, 5890−5894.
(6) Huang, S.; Liu, J.; He, Q.; Chen, H.; Cui, J. B.; Xu, S. Y.; Zhao,
Y.; Chen, C.; Wang, L. Y. Nano Res. 2015, 8, 4038−4047.
(7) Guo, C.; Xu, S. Y.; Arshad, A.; Wang, L. Y. Chem. Commun.
2018, 54, 9853−9856.
(8) Srivastava, K.; Ferrauto, G.; Young, V. G., Jr.; Aime, S.; Pierre, V.
C. Inorg. Chem. 2017, 56, 12206−12213.
(9) Peng, C.; Xing, H.; Fan, X.; Xue, Y.; Li, J.; Wang, E. Anal. Chem.
2019, 91, 5762−5767.
(10) Akazawa, K.; Sugihara, F.; Minoshima, M.; Mizukami, S.;
Kikuchi, K. Chem. Commun. 2018, 54, 11785−11788.
(11) Nie, L.; Gao, C.; Shen, T.; Jing, J.; Zhang, S.; Zhang, X. Anal.
Chem. 2019, 91, 4451−4456.
(12) Liu, Y.; Yang, Z.; Huang, X.; Yu, G.; Wang, S.; Zhou, Z.; Shen,
Z.; Fan, W.; Liu, Y.; Davisson, M.; et al. ACS Nano 2018, 12, 8129−
8137.
(13) Deng, R.; Xie, X.; Vendrell, M.; Chang, Y.-T.; Liu, X. J. Am.
Chem. Soc. 2011, 133, 20168−20171.
(14) Choi, C. A.; Lee, J. E.; Mazrad, Z. A. I.; Kim, Y. K.; In, I.; Jeong,
J. H.; Park, S. Y. ChemMedChem 2018, 13, 1459−1468.
(15) Yu, T.; Zhuang, W.; Su, X.; Ma, B.; Hu, J.; He, H.; Li, G.;
Wang, Y. Bioconjugate Chem. 2019, 30, 2075−2087.
(16) Zhao, Z.; Fan, H.; Zhou, G.; Bai, H.; Liang, H.; Wang, R.;
Zhang, X.; Tan, W. J. Am. Chem. Soc. 2014, 136, 11220−11223.
(17) Lin, J.; Xin, P.; An, L.; Xu, Y.; Tao, C.; Tian, Q.; Zhou, Z.; Hu,
B.; Yang, S. Chem. Commun. 2019, 55, 478−481.
(18) Hill, L. K.; Frezzo, J. A.; Katyal, P.; Hoang, D. M.; Ben Youss
Gironda, Z.; Xu, C.; Xie, X.; Delgado-Fukushima, E.; Wadghiri, Y. Z.;
Montclare, J. K. ACS Nano 2019, 13, 2969−2985.
(19) Hu, G. F.; Tang, J.; Bai, X.; Xu, S. Y.; Wang, L. Y. Nano Res.
2016, 9, 1630−1638.
(20) Chen, H.; Song, M.; Tang, J.; Hu, G. F.; Xu, S. Y.; Guo, Z.; Li,
N.; Cui, J. B.; Zhang, X.; Chen, X.; Wang, L. Y. ACS Nano 2016, 10,
1355−1362.
(21) Janjic, J. M.; Srinivas, M.; Kadayakkara, D. K.; Ahrens, E. T. J.
Am. Chem. Soc. 2008, 130, 2832−2841.
(22) Rolfe, B. E.; Blakey, I.; Squires, O.; Peng, H.; Boase, N. R.;
Alexander, C.; Parsons, P. G.; Boyle, G. M.; Whittaker, A. K.;
Thurecht, K. J. J. Am. Chem. Soc. 2014, 136, 2413−2419.
(23) Janjic, J. M.; Shao, P.; Zhang, S.; Yang, X.; Patel, S. K.; Bai, M.
Biomaterials 2014, 35, 4958−4968.
(24) Kislukhin, A. A.; Xu, H.; Adams, S. R.; Narsinh, K. H.; Tsien, R.
Y.; Ahrens, E. T. Nat. Mater. 2016, 15, 662−668.
(25) Yu, M.; Bouley, B. S.; Xie, D.; Enriquez, J. S.; Que, E. L. J. Am.
Chem. Soc. 2018, 140, 10546−10552.
(26) Yu, J. X.; Kodibagkar, V. D.; Hallac, R. R.; Liu, L.; Mason, R. P.
Bioconjugate Chem. 2012, 23, 596−603.
(27) Zheng, M.; Wang, Y.; Shi, H.; Hu, Y.; Feng, L.; Luo, Z.; Zhou,
M.; He, J.; Zhou, Z.; Zhang, Y.; Ye, D. ACS Nano 2016, 10, 10075−
10085.
(28) Jahromi, A. H.; Wang, C.; Adams, S. R.; Zhu, W.; Narsinh, K.;
Xu, H.; Gray, D. L.; Tsien, R. Y.; Ahrens, E. T. ACS Nano 2019, 13,
143−151.
(29) Srivastava, K.; Weitz, E. A.; Peterson, K. L.; Marjanska, M.;
Pierre, V. C. Inorg. Chem. 2017, 56, 1546−1557.
(30) Wang, S.; Li, F.; Qiao, R.; Hu, X.; Liao, H.; Chen, L.; Wu, J.;
Wu, H.; Zhao, M.; Liu, J.; et al. ACS Nano 2018, 12, 12380−12392.
(31) Loving, G. S.; Mukherjee, S.; Caravan, P. J. Am. Chem. Soc.
2013, 135, 4620−4623.
(32) Yu, L.; Chen, Y.; Wu, M.; Cai, X.; Yao, H.; Zhang, L.; Chen, H.;
Shi, J. J. Am. Chem. Soc. 2016, 138, 9881−9894.
(33) Bhang, S. H.; Han, J.; Jang, H. K.; Noh, M. K.; La, W. G.; Yi,
M.; Kim, W. S.; Kwon, Y. K.; Yu, T.; Kim, B. S. Biomaterials 2015, 55,
33−43.
https://dx.doi.org/10.1021/acs.analchem.0c01786
Anal. Chem. 2020, 92, 11739−11746
Analytical Chemistry pubs.acs.org/ac Article

(34) Lin, L. S.; Song, J.; Song, L.; Ke, K.; Liu, Y.; Zhou, Z.; Shen, Z.;
Li, J.; Yang, Z.; Tang, W.; Niu, G.; Yang, H. H.; Chen, X. Angew.
Chem., Int. Ed. 2018, 57, 4902−4906.
(35) Gale, E. M.; Mukherjee, S.; Liu, C.; Loving, G. S.; Caravan, P.
Inorg. Chem. 2014, 53, 10748−10761.
(36) Kim, M. H.; Son, H. Y.; Kim, G. Y.; Park, K.; Huh, Y. M.;
Haam, S. Biomaterials 2016, 101, 121−130.
(37) Mizukami, S.; Takikawa, R.; Sugihara, F.; Shirakawa, M.;
Kikuchi, K. Angew. Chem., Int. Ed. 2009, 48, 3641−3643.
(38) Zhang, C.; Moonshi, S. S.; Wang, W.; Ta, H. T.; Han, Y.; Han,
F. Y.; Peng, H.; Kral, P.; Rolfe, B. E.; Gooding, J. J.; Gaus, K.;
Whittaker, A. K. ACS Nano 2018, 12, 9162−9176.
(39) Guo, C.; Xu, M.; Xu, S. Y.; Wang, L. Y. Nanoscale 2017, 9,
7163−7168.
(40) Bettucci, O.; Skaltsas, T.; Calamante, M.; Dessì, A.; Bartolini,
M.; Sinicropi, A.; Filippi, J.; Reginato, G.; Mordini, A.; Fornasiero, P.;
Zani, L. ACS Appl. Energy Mater. 2019, 2, 5600−5612.
(41) Ungati, H.; Govindaraj, V.; Mugesh, G. Angew. Chem., Int. Ed.
2018, 57, 8989−8993.
(42) Ju, Y.; Dong, B.; Yu, J.; Hou, Y. Nano Today 2019, 26, 108−
122.
(43) Monguzzi, A.; Ballabio, M.; Yanai, N.; Kimizuka, N.; Fazzi, D.;
Campione, M.; Meinardi, F. Nano Lett. 2018, 18, 528−534.
(44) Hu, G. F.; Li, N.; Tang, J.; Xu, S. Y.; Wang, L. Y. ACS Appl.
Mater. Interfaces 2016, 8, 22830−22838.
(45) Wang, X.; Zhuang, J.; Peng, Q.; Li, Y. Nature 2005, 437, 121−
124.
(46) Huang, S.; Bai, M.; Wang, L. Y. Sci. Rep. 2013, 3, No. 2023.
(47) Schladt, T. D.; Graf, T.; Tremel, W. Chem. Mater. 2009, 21,
3183−3190.
(48) Chen, Y.; Ye, D.; Wu, M.; Chen, H.; Zhang, L.; Shi, J.; Wang, L.
Adv. Mater. 2014, 26, 7019−7026.
(49) Fan, W.; Bu, W.; Shen, B.; He, Q.; Cui, Z.; Liu, Y.; Zheng, X.;
Zhao, K.; Shi, J. Adv. Mater. 2015, 27, 4155−4161.
(50) Li, J.; Huo, M.; Wang, J.; Zhou, J.; Mohammad, J. M.; Zhang,
Y.; Zhu, Q.; Waddad, A. Y.; Zhang, Q. Biomaterials 2012, 33, 2310−
2320.
(51) Li, Y.; Xiao, K.; Luo, J.; Xiao, W.; Lee, J. S.; Gonik, A. M.; Kato,
J.; Dong, T. A.; Lam, K. S. Biomaterials 2011, 32, 6633−6645.
(52) Wang, S.; Li, F.; Qiao, R.; Hu, X.; Liao, H.; Chen, L.; Wu, J.;
Wu, H.; Zhao, M.; Liu, J.; Chen, R.; Ma, X.; Kim, D.; Sun, J.; Davis, T.
P.; Chen, C.; Tian, J.; Hyeon, T.; Ling, D. ACS Nano 2018, 12,
12380−12392.
(53) Zhang, A.; Zhang, Q.; Alfranca, G.; Pan, S.; Huang, Z.; Cheng,
J.; Ma, Q.; Song, J.; Pan, Y.; Ni, J.; Ma, L.; Cui, D. Nano Res. 2020, 13,
160−172.

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