Activatable Small-Molecule Photoacoustic Probes that Cross the Blood–Brain Barrier for Visualization of Copper(II) in Mice with Alzheimer's Disease

Article abstract of DOI:10.1002/anie.201904047

Copper enrichment in the brain is highly related to Alzheimer's disease (AD) pathogenesis, but in vivo tracing of Cu²⁺ in the brain by imaging techniques is still a great challenge. In this work, we developed a series of activatable photoacoustic (PA) probes with low molecular weights (less than 438 Da), RPS1–RPS4, which can specifically chelate with Cu²⁺ to form radicals with turn-on PA signals in the near-infrared (NIR) region. Introducing the electron-donating group N,N-dimethylaniline into the probe was found to significantly enhance the radical stability and PA intensity. The best probe in the series, RPS1, showed a fast response (within seconds) to Cu²⁺ with high selectivity and a low PA detection limit of 90.9 nm. Owing to the low molecular weight and amphiphilic structure, RPS1 could effectively cross the blood–brain barrier (BBB) and thus allowed us, for the first time, to visualize Cu²⁺ in vivo via PA imaging in the brains of AD mice.

Full text of DOI:10.1002/anie.201904047

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: Activatable Small-Molecule Photoacoustic Probes that Cross the
Blood-Brain Barrier for Visualization of Copper(II) in Mice with
Alzheimer’s Disease
Authors: Xuanjun Zhang, Shichao Wang, Zonghai Sheng, Zhenguo
Yang, Dehong Hu, Xiaojing Long, Gang Feng, Yubin Liu,
Zhen Yuan, Jingjing Zhang, and Hairong Zheng
This manuscript has been accepted after peer review and appears as an
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the VoR from the journal website shown below when it is published
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201904047
Angew. Chem. 10.1002/ange.201904047
Link to VoR: http://dx.doi.org/10.1002/anie.201904047
http://dx.doi.org/10.1002/ange.201904047

10.1002/anie.201904047
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Activatable Small-Molecule Photoacoustic Probes that Cross the
Blood-Brain Barrier for Visualization of Copper(II) in Mice with
Alzheimer’s Disease
Shichao Wang, Zonghai Sheng,* Zhenguo Yang, Dehong Hu, Xiaojing Long, Gang Feng, Yubin Liu,
Zhen Yuan, Jingjing Zhang, Hairong Zheng, Xuanjun Zhang*
Abstract: Copper enrichment in the brain is highly related to
Alzheimer’s disease (AD) pathogenesis, but in vivo tracing of Cu2+ in
the brain by imaging techniques is still a great challenge. In this work,
we developed a series of activatable photoacoustic (PA) probes with
low molecular weights (< 438 Da), RPS1RPS4, which can
specifically chelate with Cu2+ to form radicals with turn-on PA signals
in the near-infrared (NIR) region. Introducing the electron-donating
group N,N-dimethylaniline into the probe was found to significantly
enhance the radical stability and PA intensity. The best probe in the
series, RPS1, showed a fast response (within seconds) to Cu2+ with
high selectivity and a low PA detection limit of 90.9 nM. Owing to the
low molecular weight and amphiphilic structure, RPS1 could
effectively cross the blood-brain barrier (BBB) and thus allowed us,
for the first time, to visualize Cu2+ in vivo via PA imaging in the brains
of AD mice.
Copper is the third-most abundant trace metal in the human body
and plays a vital role in physiological and pathological activity.
Abnormal alterations in cellular copper homeostasis are
connected to serious diseases, including cardiovascular disorders,
Wilson disease, and Alzheimer’s disease (AD).[1] Some studies
have noted that Cu2+ enrichment in the brain has a close
relationship with AD pathogenesis.[2] The redox-active Cu2+ in the
brain was implicated in the generation of reactive oxygen species
(ROS), leading to the high oxidative stress that is a proposed
factor in accelerating the assembly and neurotoxicity of AD
Scheme 1. Rational design of activatable PA probes for Cu2+ detection in
amyloid-
(A)
fibrils.[3]
Therefore,
highly
sensitive
AD mouse brain. (a) Generally designed platform of small-molecule PA
probes. (b) Previous compounds with Cu2+ chelating moiety for AD therapy.
(c) The target PA probe RPS1 and schematic diagram of RPS1 crossing the
BBB and detecting Cu2+, as observed by PA imaging.
imaging/detection of Cu2+ in brain of the AD is esstenial to
comprehensively understand its pathological events in the brain.
Currently, many fluorescent probes have been explored and
applied to stain for Cu2+ in cells and living organisms with good
performance.[4] However, in vivo fluorescence imaging of Cu2+ in
the brain is still a great challenge due to the limited penetration
depth (~ 1 mm), the obstacle for probes to cross the blood-brain
barrier (BBB) and the fluorescence quenching resulting from the
paramagnetic effect of Cu2+.[5]
Compared with fluorescence technology, photoacoustic (PA)
imaging combines optical imaging and ultrasound imaging into a
hybrid modality, exhibiting multiscale spatial resolution with deep
tissue penetration (centimeter depths).[6] Because of these
Dr. S. Wang, G. Feng, Dr. Y. Liu, Prof. Z. Yuan, Prof. X. Zhang
Cancer Centre and Centre of Reproduction, Development and
Aging, Faculty of Health Sciences, University of Macau, Macau
SAR, P.R. China
E-mail: xuanjunzhang@um.edu.mo (X. Zhang)
Prof. Z. Sheng, Dr. D. Hu, X. Long, Prof. H. Zheng
Paul C. Lauterbur Research Center for Biomedical Imaging
Institute of Biomedical and Health Engineering, Shenzhen Institute
of Advanced Technology, Chinese Academy of Sciences, Shenzhen
518055, P.R. China
E-mail: zh.sheng@siat.ac.cn (Z. Sheng)
Z. Yang, Prof. J. Zhang
Affiliated Hospital of Guangdong Medical University, Zhanjiang
524001, P.R. China
advantages, PA imaging with functional probes has been widely
applied in biomedical imaging,
[7]
disease detection,[8] and
biosensing.[9] However, only a limited number of small-molecule
PA probes with activatable features for detecting metal ions have
been reported.[9c, 9d, 10] Moreover, to the best of our knowledge,
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201904047
Angewandte Chemie International Edition
COMMUNICATION
there has been no report of PA tracing for Cu2+ in the brain of the
animal models until now.
shown in Figure S4a, RPS4-Cu with a naphthalene structure
exhibited a fast quenching of the NIR absorption within 5 minutes.
The double fluorine substituents with intensively electron-
accepting ability had a damping effect on the lifetime of RPS3-Cu
about 1 hour. The absorbance of RPS2-Cu in water decreased
substantially within 1 hour and then remained stable over the next
a few hours. Surprisingly, the introduction of N,N-dimethylaniline
greatly increased the stability of RPS1-Cu to 30 hours, perhaps
because the electron-donating group could stabilize the inert
action of free radicals (Scheme 2, Table S1).
Theoretically, the design of small-molecule PA probes is
mainly based on extending a large -conjugated backbone, such
as boron-dipyrromethene (BODIPY), indocyanine green (ICG)
and porphyrin derivatives (Scheme 1a).[11] Even with strong PA
emission, these probes generally have a large molecular mass
that exceeds the threshold of 600 Da to cross the BBB.[12] Some
of the compounds with Cu2+ chelating moiety for AD therapy are
able to cross BBB, but their insensitive absorption variation
impede them as effective Cu2+ PA probes (Scheme 1b). As
unexplored PA contrast agents, aniline derivatives could form
anilinyl radical complexes exhibiting near-infrared (NIR)
absorption triggered by Cu2+.[13] However, these complexes
usually have a short lifetime in aqueous media because free
radicals are able to abstract hydrogen from water.[14] In this work,
inspired by the structures of AD drugs and aniline radicals, we
designed a series of activatable PA probes, RPS1-RPS4, with a
small molecular weight (< 438 Da) and sepcific NIR PA detection
totwards Cu2+. To tune the PA intensity and water stability of the
probes, we modified the molecular structure with electron-
donating and electron-accepting groups. We found that the
introduction of electron-donating groups, such as N,N-
dimethylaniline (RPS1), led to an amplified PA emission in the
NIR region and significantly improved the stability of the probes
upon detecting Cu2+ in aqueous media. RPS1 could effectively
cross the BBB and thus allowed us for the first time to visualize
Cu2+ in vivo via PA imaging in AD mouse brains (Scheme 1c).
In PBS, all probe solutions exhibited a transparent color with
a absorption in the ultraviolet region. After the addition of Cu2+,
the color of the solution quickly changed to green within seconds
(Supporting Video 1). Structurally, RPS1, with the electron-
donating group N,N-dimethylaniline, exhibited the longest
absorption wavelength (713 nm) and the strongest absorbance (
> 10000) among the four probes in the presence of 1 equiv. Cu2+
(Figure 1b, Table S1). In contrast, the relatively weaker
absorbance ( < 4100) and blueshifted absorption peaks at
approximately 690 nm for RPS2-RPS4 metal chelate complexes
were attributed to the smaller conjugation systems and electron
acceptor groups (Figure S1b). Likewise, upon addition of Cu2+,
RPS1 had the strongest PA emission, which was at least
approximately 2.5 times greater than that of the other probes at
700 nm excitation (Figure S1c). In contrast to their lack of a PA
Scheme 2. The structures of probes RPS1 RPS4.
We tested the response of all probes to a variety of biologically
relevant metal ions in PBS (pH 7.4, 0.02 M) and found that all
RPS1-RPS4 showed specific selectivity to Cu2+, resulting in a
unique NIR absorption (Figure S5-S8). The competition
experiment in the presence of mixed metal ions (Fe2+, Fe3+, Ni2+,
Co2+, Cu2+) also showed that RPS1 with an electron-donating
group had the most superior selectivity to Cu2+ (Figure S9). A
titration experiment was conducted on RPS1 and RPS2 by
absorption spectra in PBS and fetal bovine serum (FBS), and the
results demonstrated that probes both had a good linear fit with
Cu2+ concentration. RPS1 possessed a super low absorption limit
of detection (7.2 nM) under RPS2 (30.0 nM) and good stability in
excess Cu2+ in PBS buffer (Figures 1c, S10, S11).
Encouraged by the optimal features of RPS1, this probe was
chosen as a potential activatable probe for subsequent PA
imaging. To determine the optimal wavelength for PA imaging, the
PA spectra of RPS1 and its chelated complex were measured at
wavelengths ranging from 680 to 970 nm by a Vevo LAZR
imaging system.[15] As seen in Figure 1d, we found that RPS1 had
no PA signal in this broad region. Upon chelation with Cu2+, the
RPS1 complex possessed the strongest PA signal at
approximately 710 nm, followed by dampening of the PA signal.
PA selection contrast to Cu2+ of RPS1 was overwhelmingly
greater than that of other metal ions (Figure 1e). In PA quantitative
analysis, the PA limit of detection was calculated to be as low as
90.9 nM. (Figure 1f). This limit of detection was lower than
biological Cu2+ level and indicated that RPS1 had great potential
for detecting Cu2+ in the brain by PA imaging.[4b]
background signal, all probes displayed
a substantial PA
emission upon detecting Cu2+ except RPS4 because of the
unstable free radical formed by the RPS4-Cu complex. To verify
the formation of a -radical monoanion in the chelated complexes
that gave rise to the NIR absorption,[13a] we conducted the MS
spectra and electron paramagnetic resonance (EPR)
spectroscopy of the free RPS probes and their chelated
complexes (RPS-Cu) in solutions (Figure 1a, inserted Figure 1b,
Figure S2, S3). The results showed that all RPS-Cu complexes
exhibited corresponding masses and high-intensity EPR signals,
which indicated the existence of an unpaired electron and the
formation of radicals.
The temperature stability of RPS1-Cu was monitored by
absorption in PBS. As shown in Figure S12, RPS1-Cu had no
obvious absorption change in the range of 30 to 46 °C . Light
irradiation of RPS1 and RPS1-Cu within 3 hours indicated that
both had good stability under intensely ambient light (Figure S13).
RPS1 also held the admired selectivity toward Cu2+ in the
presence of ROS/reactive nitrogen species (RNS) and principally
reductive biomolecules (ascorbic acid, methylglyoxal, glutathione
and cysteine) (Figure S14). At different pH ranges from 6.0 to 8.5,
RPS1-Cu complex also showed a desired stability (Figure S15).
For further imaging application, the stability of these radicals
in aqueous media should be evaluated because the single
electron has a strong proton binding capacity and may abstract
hydrogen from water molecules to quench the radical.[14] As
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Figure 1. (a) Mass spectra of RPS1 and RPS1-Cu. (b) The absorption spectra of RPS1, Cu2+ and RPS1-Cu complex (all are 50 M). The inset shows the EPR
spectra of RPS1 (2.5 mM) and RPS1-Cu in acetonitrile and PBS (v:v = 1:1) at room temperature. The color change of RPS1 form transparent to deep green after
treating with 1 equiv. Cu2+. (c) Changes in the absorption spectra of RPS1 (50 M) after reaction with different concentrations of Cu2+ for 10 minutes. The inset
shows the color of RPS1 solution and calibration curve of the absorbance at 713 nm for detection of Cu2+ in the range of 0  35 M. (d) PA spectra of 50 M RPS1
and RPS1-Cu from 680 to 970 nm. The inset shows the PA image of an RPS1 and RPS1-Cu solution upon excitation at 710 nm. (e) PA selectivity tests for RPS1
(50 M, 1) upon addition of metal ions (2: Li+, 3: Na+, 4: K+, 5: Ca2+, 6: Mg2+, 7: Al3+, 8: Fe2+, 9: Fe3+, 10: Cu2+, 11: Zn2+, 12: Ni2+, 13: Mn2+, 14: In3+, 15: Co2+, 16:
Cd2+, 17: Pb2+, 18: Hg2+, 19: Cu+; 500 M for Na+, K+, and Ca2+ and 50 M for other metal ions) for 10 minutes. (f) Calibration curve of the PA intensity at 710 nm
for detection for Cu2+ in the range of 0  35 M. Above tests were performed in 0.02 M PBS solution at pH 7.4 unless otherwise specified. (g) Schematic overview
of the construction of an in vitro BBB model. (h) The relative transcytosis amount of RPS1 and EB in vitro within 1.5 hours. Experiments were repeated three times,
and the data are presented as the mean  standard deviation (c, d, e, f, h).
In summary, these tests indicated that free radical of RPS1-Cu
complex remained relatively inert toward possible in vivo
surrounding factors. The binding affinity of RPS1 was evaluated
as 1.18 × 104 M1 using Benesi-Hildebrand method (Figure S16),
which was in the range of affinity of the second Cu(II) binding site
of A.[16] To confirm RPS1 was able to detect Cu2+ in A fibrillar
aggregates, we applied the RPS1 to Cu-A aggregates and found
that it has a good linear detetction towards Cu2+ in A aggregates
(Figure S17).
Prior to in vivo PA imaging, the cytotoxicity of RPS1 was
examined in brain vascular endothelial (bEnd.3) cells by viability
assay (Figure S18). The results showed that RPS1 had negligible
cytotoxicity toward bEnd.3 cells after incubation for 24 hours
(probe concentration, 0-200 µM). The hemolytic analysis
exhibited a low hemolysis percentage (0.5%) of red blood cells
even at 40 µM RPS1 (Figure S19). The mouse blood panel
parameters showed that the values of the three groups of mice
presented slight or mild variations, which indicated that RPS1 had
the advantage of low in vivo cytotoxicity and adequate
hemocompatibility (Figure S20). Furthermore, hematoxylin and
eosin (H&E) staining images of the major organs demonstrated
that the probe-treated group had no distinct tissue damage
compared to the control group (Figure S21). The physiological
stability of the probes in nude mice was evaluated by injecting a
freshly prepared RPS1-Cu solution (50 M × 100 L) into the leg
muscle. Following laser irradiation (750 nm), the PA signal of the
injection site was detected (Figure S22). PA imaging showed that
the strong PA intensity of RPS1-Cu at the injection sites was
much higher than that of the surrounding tissue, even after 200
minutes, indicating high in vivo stability.
seeded with bEnd.3 cellular monolayer according to a previously
reported method [17] The transwell model was separated into three
typical spaces, i.e., the apical chamber, cellular monolayer, and
basolateral compartment (Figure 1g). The integrity of the cellular
monolayer was monitored by trans-endothelial electrical
resistance (TEER) and cell staining of crystal violet. As shown in
Figure S23, TEER reached a peak at about 7 days and the bEnd.3
seeded on the surface of filter integrally. For the negative control
group, Evans blue (EB), which has been widely used as a
negative tracer to assess permeability of endothelial-type barriers
in normal brains without damage, was applied.[18] We chose EB
as a negative control to ensure that the transwell model was
reliable. After the addition of EB (10 M) and RPS1 (150 M, very
low toxicity to cells) into the apical chamber with integrally cellular
monolayer, the absorbance of the solution on the basolateral side
at 610 nm and 310 nm was measured three times every half hour,
respectively. By calculating the absorbance based on Figure S24,
we found that EB was almost completely unable to cross the cell
monolayer in the transwell model. In contrast, RPS1 had good
transport capability across the cell monolayer and reached an
appreciable permeability efficiency of more than 40% after 1.5
hours (Figure 1h).
Based on the above favorable results of the photophysical
and PA analysis, RPS1 was applied to PA imaging to further
confirm the feasibility of detecting Cu2+ in mouse brains. AD mice
and age-matched normal mice were employed for comparison
according the previous reports.[19] In one group, AD and normal
mice were all injected with RPS1 (0.5 mgkg-1) through tail-vein
intravenous treatment. In another group, AD mice were injected
with PBS for the control. As shown in Figure 2a, with the aid of
ultrasound imaging, a whole map of the brains with distinct
borders was obtained. For the brains of normal mice treated with
To assess whether RPS1 crosses the BBB, we measured the
in vitro BBB-permeability efficacy of RPS1 by transwell filters
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10.1002/anie.201904047
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were also found in this region from H&E staining AD brain slices.
These are significantly neurodegenerative characters of AD
compared with normal mice (Figure 2c). Thus, this result provided
visual evidence that RPS1 could effectively cross the BBB and
detect Cu2+ in the whole brains of normal and AD mice by the PA
technique. In addition, the study also verified the higher level of
Cu2+ in AD mice compared than in normal mice by PA imaging for
the first time.
Different from conventional PA probes with extended -
conjuation, RPS probes with low molecular weight that can
specifically chelate Cu2+ to form radicals with turn-on PA response
in the NIR region. Introducing electron-donating group N,N-
dimethylaniline was found to dramatically improve the stability of
the probe-Cu radical and enhance the NIR PA intensity. Owing to
the low molecular weight and amphiphilic structure, RPS1 could
effectively corss BBB and achieve the visualization of Cu2+ in the
whole brain of AD mice by PA imaging. This work provided a new
design of PA probes and broaden the PA sensing application in
the brain. We anticipate that this type of small-molecular-weight
probe will find a potential application in AD diagnosis, drug
screening and treatment evaluation.
Figure 2. (a) Representative PA images of from the brain of normal and AD
mice after 1.5 h of intravenous injection of RPS1 and PBS. The scale bar
represents 5 mm. The scan wavelength of the laser was 750 nm at 10
mJ·cm2. (b) The PA intensity of normal and AD mice from ROI areas. Data
presented as mean ± SD, *p < 0.05. (c) Histological staining of the brain
slices in hippocampus region from mouse brains using Congo red and H&E.
RPS1, there was a weak PA signal, possibly due to the low
amount of Cu2+ detection and endogenous hemoglobin in the
brain blood vessels. The brains of AD mice showed widely
distributed strong PA signals throughout the whole cortex region.
The AD mouse brains treated with PBS showed a weak PA signal
mainly distributed over the narrow central area. The PA signal of
PBS-treated AD mice has an approximate intensity but different
distribution in the brain compared with probe-treated normal mice,
which was possibly from the hemoglobin. The mean PA intensity
in the region of interest (ROI) of AD mice treated with probe was
9.57-fold and 8.50-fold stronger than that of probe-treated normal
mice and PBS-treated AD mice, respectively (Figure 2b). This
result was appreciable agreement with Cu2+ concentration ratio
between AD and normal mice brain that was determined by
inductively coupled plasma-atomic emission spectrometry (ICP-
AES) (Table S2). Higher resolution and deeper ex vivo PA
imaging of brains were performed on advanced three-
dimensional (3D) PA system. As shown in Figure S25, the
cerebral blood vessels (red ellipses dash) in normal and AD mice
treated with RPS1 became more visible. Along the cerebral blood
vessels, the cerebral cortex region of AD mice showed strong PA
signals, whereas the same region of normal mice displayed dim
photoacoustic signals. The green-red color of PA images
represented a higher Cu2+ concentration in cortex regions (blue
arrows). This phenomenon was more obvious in 3D PA imaging
(Supporting Video 2_3D and Video 3_3D). To determine the
biodistribution of RPS1 in living mice, ex vivo PA images of major
organs were acquired. As shown in Figure S26, compared with
control group, photoacoustic signals of the liver, kidney and brain
of RPS1-treated group were significantly enhanced after
intravenous injection of probe for 1.5 h, indicating that the probe
was mainly metabolized by liver and kidney and enriched in the
brain. After PA imaging, the brains were prepared with slices and
stained by Congo red and H&E for observation. Notably, the
Congo red staining AD brain slices showed a mass of plaques
deposition in hippocampus region. The shrunken cell nucleuses
Acknowledgements
This work was supported by the National Key Research and
Development Program of China: Scientific and technological
innovation
cooperation
of
Mainland
and
Macao
(2017YFE0120000), the Macao Science and Technology
Development Fund (Grant Nos. 019/2017/AMJ, and
082/2016/A2), the Natural Science Foundation of China
(91859117, 81571745, 81771906). Shenzhen Double Chain
Grant [2018]256 and Natural Science Foundation of Guangdong
Province (2014A030312006). We thank Shenzhen Key
Laboratory
of
Ultrasound
Imaging
and
Therapy
(ZDSYS20180206180631473). We are also grateful to Jian Su for
assistance with the EPR measurements and Prof. Rongqin Zheng
from the third people's hospital of Sun Yat-Sen University for 3D
photoacoustic imaging.
Conflict of interest
The authors declare no competing financial interests.
Keywords: photoacoustic probe • blood-brain barrier•
Alzheimer’s disease • cupric ion
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10.1002/anie.201904047
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
Layout 2:
COMMUNICATION
Shichao Wang, Zonghai Sheng,*
Zhenguo Yang, Dehong Hu, Xiaojing
Long, Gang Feng, Yubin Liu, Zhen
Yuan, Jingjing Zhang, Hairong Zheng,
Xuanjun Zhang*
Page No. – Page No.
Activatable Small-Molecule
Photoacoustic Probes that Cross the
Blood-Brain Barrier for Visualization
of Copper(II) in Mice with Alzheimer’s
Disease
Photoacoustic Imaging of Copper(II) in Brain. Small-molecule probe RPS1 can selectively bind to Cu2+ forming radical with turn-on
photoacoustic response in the NIR region. RPS1 can effectively cross blood-brain barrier and image copper(II) in AD mouse brain.
This article is protected by copyright. All rights reserved.