Simultaneous imaging of mitochondrial viscosity and hydrogen peroxide in Alzheimer's disease by a single near-infrared fluorescent probe with a large Stokes shift

Article abstract of DOI:10.1039/c9cc08267k

It has been speculated that both the intracellular viscosity and H₂O₂ level in Alzheimer's disease (AD) brains are higher than that in healthy brains, but direct evidence from living beings is scarce. Herein, we report a NIR emissive

Full text of DOI:10.1039/c9cc08267k

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Simultaneous imaging of mitochondrial viscosity
and hydrogen peroxide in Alzheimer’s disease
by a single near-infrared fluorescent probe with
a large Stokes shift†
Cite this:DOI: 10.1039/c9cc08267k
Received 22nd October 2019,
Accepted 12th December 2019
Songjiao Li, Peipei Wang, Wenqi Feng, Yunhui Xiang, Kun Dou and Zhihong Liu
*
DOI: 10.1039/c9cc08267k
rsc.li/chemcomm
It has been speculated that both the intracellular viscosity and H2O2 viscosity and H2O2 in mitochondria individually, but they are
level in Alzheimer’s disease (AD) brains are higher than that in unable to give accurate information on the two markers at the
healthy brains, but direct evidence from living beings is scarce. same time.8 Compared with the combination of several probes
Herein, we report a NIR emissive fluorescent probe with a large in one system, a single probe with the ability of sensing multiple
Stokes shift for the associated detection of mitochondrial viscosity targets has obvious advantages.9 It is able to circumvent the
and H2O2 in live rat brains with AD for the first time.
uncertainties of measurement caused by the joint use of different
probes, such as the spectral cross-talk, the difference in probe
Alzheimer’s disease (AD) is the most prevalent progressive localizations, and the different metabolism of the probes. As far as
neurodegenerative disorder and seriously affects human health we know, only two recent fluorescent probes have been reported
and the quality of life.1 It has long been speculated that AD is for sensing both viscosity and H2O2 in mitochondria.10 Never-
tightly associated with oxidative stress and reactive oxygen theless, these probes suffered from rather short emission wave-
species (ROS) in the brain.2 But comprehensive evidence from lengths in the ultraviolet or visible range, and hence they were
living beings for this speculation is rare. Intracellular viscosity able to detect viscosity and H2O2 only in cultured cells.
is known to play an important role in many diffusion mediated
As stated above, it remains a challenge to simultaneously
biological processes including signal transduction, mass trans- monitor intracellular viscosity and H2O2 content in vivo, especially
portation and so on.3 Abnormal changes in cellular viscosity are in AD brains. To achieve this goal, the probe needs to address
closely related to diseases and malfunctions.4 At the subcellular several intractable issues: (1) the ability to cross the blood–brain
level, mitochondria play critical roles in cell differentiation, barrier (BBB) so as to image the brain region;11 (2) long emission
information transmission and apoptosis.5 Previous findings wavelengths in the near-infrared (NIR) region to get deeper tissue
suggest that both the viscosity of the mitochondrial membrane penetration and less interference from background fluores-
and the abnormality of H2O2 production in mitochondria are cence;12 and (3) large Stokes shifts are desirable to overcome
related to Ab accumulation.6 To gain a better understanding on the ‘‘self-absorption’’ effect.13 Herein, we designed a single NIR
the relationship between the development of AD and the levels emissive fluorescent probe with a large Stokes shift that can
of mitochondrial viscosity and H2O2, it is necessary to monitor simultaneously detect mitochondrial viscosity and H2O2 in the
these markers at the same time. However, methods for simulta- AD brain (Scheme 1). A p-pinacolborylbenzyl moiety was chosen
neously measuring H2O2 and viscosity in AD brains are cur- as the reaction site for H2O2, which guarantees high selectivity of
rently absent.
the probe toward H2O2. The quaternarized quinoline unit here
Fluorescence imaging is featured by real-time visualization, not only acts as a mitochondria-targeting carrier but also
high sensitivity and relatively simple operation.7 To date, there
have been quite a lot of outstanding outcomes for imaging
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of
Education), College of Chemistry and Molecular Sciences, Wuhan University,
Wuhan, Hubei 430072, China. E-mail: zhhliu@whu.edu.cn; Fax: +86-27-6875-4067;
Tel: +86-27-8721-7886
† Electronic supplementary information (ESI) available: Additional experimental
data, including characterization, photophysical properties, cytotoxicity, and
experimental details, and supplementary fluorescence images in cells and mice.
Scheme 1 The design of the probe Mito-NIRHV.
See DOI: 10.1039/c9cc08267k
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improves the water solubility of the probe. The lipophilic this case were similar to that with the 10 mM probe (Fig. S11,
p-conjugated thiophene-bridge was introduced for extension ESI†), verifying that the emission was a true emission peak
to NIR emission and also for the enhancement of BBB penetr- from the monomeric probe. Moreover, the fluorescence inten-
ability. The twistable ethylene was designed to respond to the sity at 700 nm was linearly proportional to the H2O2 concen-
viscosity of the environment. The synthetic route for the probe tration in the range of 0.5–100 mM (R2 = 0.9986, Fig. 1b), and the
(named Mito-NIRHV) is outlined in Scheme S1 (ESI†), and its detection limit was calculated (3s/slope method) as 3 nM. This
structure was confirmed by 1H NMR, 13C NMR and HRMS high sensitivity may enable the probe to detect trace amounts
(ESI†). The probe was able to sense viscosity and H2O2 with of intracellular H2O2. The molar absorptivity for Mito-NIRHV
high sensitivity, affording two emission peaks in the NIR and its reaction product are 9.5 Â 103 MÀ1 cmÀ1 and 7.4 Â
region, both with a big Stokes shift of 4200 nm. We have 103 MÀ1 cmÀ1, respectively. The fluorescence quantum yield for
demonstrated in detail its ability to monitor the fluctuations of Mito-NIRHV and its reaction product are 0.012 and 0.232,
mitochondrial viscosity and H2O2 content, either individually respectively. To test the specificity of Mito-NIRHV toward
or simultaneously, in living cells and animals. Benefiting from H2O2, we investigated various reactive oxygen/nitrogen species
its favourable photophysical properties and proper BBB penetr- and mercaptans. The fluorescence signals showed negligible
ability, the Mito-NIRHV was applied to simultaneously track the changes for all these reactive species, presenting excellent
variation of mitochondrial viscosity and H2O2 levels in AD selectivity of the probe (Fig. S12, ESI†). We then evaluated
brains for the first time.
the response time of Mito-NIRHV to H2O2. The fluorescence
With the desired probe in hand, we first tested its spectro- intensity of the probe gradually increased with reaction time,
scopic properties in response to H2O2. The absorption and and then leveled off at about 40 min in the presence of varying
fluorescence spectra of compound 3, Mito-NIRHV, in the concentrations of H2O2 (Fig. S13, ESI†). In addition, Mito-
absence and presence of H2O2 were measured (Fig. S10, ESI†). NIRHV remained weakly emissive in a wide pH range of
The UV-vis spectra of Mito-NIRHV exhibited a maximum 4.0–10.0, while the product of the reaction with H2O2 exhibited
absorption at 570 nm. After reacting with H2O2, the absorption dramatic fluorescence enhancement around pH 7.4 (Fig. S14,
at 570 nm declined obviously, whereas a new absorption peak ESI†), suggesting the potential applicability of Mito-NIRHV in
appeared at 440 nm. The resulting absorption spectrum was physiological conditions. To confirm the sensing mechanism
similar to that of compound 3. Meanwhile, the emission proposed in Scheme 1, a high resolution mass spectrometry
intensity at 700 nm was enhanced and the resulting fluores- (HRMS) analysis was performed on Mito-NIRHV before and
cence spectrum was the same as that of compound 3 in the after reacting with H2O2. The results showed the signal at m/z =
emission band. The fluorescence intensity at 700 nm under 357.1427 after reaction, which is nearly equal to the molecular
440 nm excitation showed a gradual increase along with the weight of compound 3 (Fig. S15, ESI†). To check whether the
increase of H2O2 concentration indicating that Mito-NIRHV probe Mito-NIRHV was responsive to viscosity, its optical
served as a ‘‘turn-on’’ fluorescent sensor for H2O2 (Fig. 1a). properties in an ethanol–glycerol system were evaluated. As
To preclude the possibility that a 10 mM probe may form displayed in Fig. S16 (ESI†), the maximal absorption of the
aggregates or particles, which could lead to a red-shift of the probe at 570 nm was slightly enhanced as the viscosity
emission peak, we also used a 2 mM probe to test its spectro- increased. Meanwhile, with the glycerol fraction increased from
scopic properties in response to H2O2. The spectra obtained in 0% to 100%, which represents a stepwise increase of viscosity,
the fluorescence intensity of Mito-NIRHV at around 800 nm
showed a remarkable increase (Fig. 1c). Moreover, the fluores-
cence intensity at 800 nm also presented a good linear relation-
ship between log(I800) and log(Iviscosity) in the range of 1.56–954 cP
(Fig. 1d). Next, a 2 mM probe was also used to test its spectroscopic
properties in response to viscosity (Fig. S17, ESI†), which again
confirmed the emission from the monomeric probe. This
viscosity-dependent fluorescence change distinctly verifies the
occurrence of a typical twisted intramolecular charge transfer
(TICT) process in the system. To preclude the possible effect of
pH on the emission, the fluorescence spectra of the probe were
measured at varying pH values. As shown in Fig. S18 (ESI†),
the fluorescence intensity of the probe at either low or high
viscosity remained unchanged for all pH values.
The cytotoxic effect of the probe was assessed with an MTT
assay. More than 85% of HeLa cells survived after 24 h even
with 40 mM Mito-NIRHV, indicating a very low cytotoxicity of
the probe (Fig. S19, ESI†). As shown in Fig. S20 (ESI†), Mito-
Fig. 1 (a) Fluorescence response of Mito-NIRHV (10 mM) to varying con-
centrations of H2O2 (0–100 mM). lex = 440 nm. (b) The linear relationship
between the fluorescence intensity at 700 nm and H2O2 concentration. (c)
The fluorescence spectra of Mito-NIRHV (10 mM) at different viscosities,
NIRHV predominantly localized in the mitochondria (Pearson’s
coefficient (PC) 0.957), as compared with other organelles such
which were recorded in ethanol–glycerol systems with various ratios. lex
570 nm. (d) The linear relationship between log(I800) and log(Iviscosity).
=
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as Lyso- (PC 0.815) and ER-Tracker (PC 0.858). Next, we evaluated the at the same time (Fig. S24b2 and b3, ESI†). These results demon-
potential ability of the probe to visualize H2O2 in living cells. strated that the probe was effective for simultaneous imaging of
As shown in Fig. S21a (ESI†), the HeLa cells treated only with H2O2 and viscosity in cellular environments.
Mito-NIRHV exhibited weak fluorescence in the NIR channel
In order to ultimately expand the application scope of the
(668–726 nm). However, when cells were pretreated with exogenous probe to mammals, we applied it for in vivo imaging of the
H2O2 (50 and 100 mM) followed by incubation with the probe, the targets in living mice. The first trial was to sense exogenous
fluorescence in this channel remarkably increased (Fig. S21b and c, H2O2 in mice. Before that, the cytotoxicity of Mito-NIRHV
ESI†). The results suggested that Mito-NIRHV had the potential to in vivo was examined by hematoxylin and eosin (H&E) staining
sense the intracellular H2O2. Next, we further investigated whether of organs after intravenous injection of the probe. The images of
Mito-NIRHV could be used to track endogenous H2O2 in living cells. the major organs showed no noticeable sign of organ damage
According to the literature, phorbol myristate acetate (PMA) is after inserting the probe (Fig. S25, ESI†), implying the negligible
an effective stimulant to promote cellular ROS levels. Therefore, toxicity of Mito-NIRHV in the body. For the detection of H2O2, an
the HeLa cells were pretreated with PMA (1 mg mLÀ1) and then aqueous solution of H2O2 in PBS was injected into the peritoneal
incubated with the probe. As expected, the emission intensity cavity of BALB/c mice, followed by intraperitoneal injection of
exhibited a remarkable increase in this channel (Fig. S21d, ESI†). Mito-NIRHV. As shown in Fig. S26a (ESI†), the control group (with
In contrast, when the cells were pretreated with NAC, an efficient no H2O2 injected) showed the weakest fluorescence, while the
ROS scavenger, the intracellular fluorescence was almost completely group with external H2O2 injection showed much brighter fluores-
inhibited (Fig. S21e, ESI†), verifying that the fluorescence enhance- cence (Fig. S26b, ESI†). Next, the feasibility of the probe to track
ment upon stimulation with PMA was due to the reaction of Mito- endogenous H2O2 in mice was examined. To this end, the BALB/c
NIRHV with intracellular H2O2. All these results indicated that Mito- mice were firstly injected with rotenone (a documented reagent to
NIRHV was capable of detecting both exogenous and endogenous induce the production of H2O2 in the body), followed by the
H2O2 in living cells.
injection of Mito-NIRHV. As seen in Fig. S26c (ESI†), rotenone
To verify that Mito-NIRHV is also a mitochondria targetable treated mice also exhibited obviously enhanced fluorescence as
probe for viscosity, we performed another set of fluorescence compared to the control. The above experimental results show
co-localization experiments employing organelle trackers (Mito-, that Mito-NIRHV can be used to measure H2O2 in mice.
ER-, and Lyso-Tracker Green). Fig. S22 (ESI†) also showed that
Subsequently, the in vivo tracking of viscosity changes was
Mito-NIRHV was predominantly distributed in the mitochondria performed in the mice models. The variation of viscosity level in
(Pearson’s coefficient (PC) 0.968) compared with other organelles the body was also induced by monensin and nystatin. After
such as Lyso- (PC 0.832) and ER-Tracker (PC 0.868). This strongly the abdominal injection of Mito-NIRHV, the normal mice
indicates that Mito-NIRHV localizes at the mitochondria at vis- emitted inconspicuous fluorescence (Fig. S27a, ESI†). However,
cous cellular conditions. To identify the effectiveness of Mito- when triggered by either monensin (Fig. S27b, ESI†) or nystatin
NIRHV in mitochondrial viscosity imaging, confocal laser fluores- (Fig. S27c, ESI†), the mice exhibited obviously enhanced fluores-
cence images of HeLa cells with Mito-NIRHV loaded were cence. The fluorescence intensities of the three groups were calcu-
acquired after the treatment of two ionophores (nystatin and lated and presented in Fig. S27d (ESI†). These results coincided well
monensin). When the cells were loaded only with the probe, weak with the findings in cells, suggesting that Mito-NIRHV can also be a
fluorescence was observed in the NIR channel (754–816 nm, competent tool for the imaging of viscosity in living mice.
Fig. S23a, ESI†). In contrast, upon loading nystatin (Fig. S23b, ESI†)
Our final-step examination on the performance of Mito-
or monensin (Fig. S23c, ESI†) together with the probe, obvious NIRHV was to check whether it could track the endogenously
fluorescence enhancement was seen in both cases, which can be generated H2O2 and viscosity variations in living animals. To
ascribed to the mitochondrial swelling or large-scale alteration of achieve this goal, we constructed a mouse model of inflammation
mitochondrial metabolism induced by ionophores.
induced by LPS, for simultaneous detection of the subtle changes
Through the above investigations, we have verified the of both H2O2 and viscosity in the inflammatory mice. While the
ability of Mito-NIRHV to separately sense mitochondrial H2O2 healthy mice injected with only Mito-NIRHV displayed weak
and viscosity in living cells. Then we further tried our probe in fluorescence (Fig. 2a1 and a2), the inflammatory mice showed
an inflammation model to evaluate its capability of simultaneously strong fluorescence in both channel 1 (for H2O2, Fig. 2b1) and
tracking the levels of intracellular H2O2 and viscosity. As reported channel 2 (for viscosity, Fig. 2b2). These data, together with all the
earlier, inflammation could cause an increase of both the content of above findings, solidly confirmed the power of Mito-NIRHV to
H2O2 and viscosity in cells. Lipopolysaccharide (LPS) is known as a simultaneously image H2O2 and viscosity in living animals, thus
cell wall component of Gram-negative bacteria which has been encouraging us to ultimately use the probe in investigating some
confirmed to trigger cells to engender inflammation. We therefore challenging biological issues.
applied LPS to build an inflammatory model in HeLa cells. As
In order for a molecular probe to possibly cross the BBB, its
illustrated in Fig. S24a2 and a3 (ESI†), the cells loaded with the lipophilicity index log P normally is within the range of 1.0–3.0,
probe showed faint fluorescence in both channel 1 (668–726 nm, for with the molecular weight (MW) o600 Da. The log P value of
H2O2) and channel 2 (754–816 nm, for viscosity). When cells were Mito-NIRHV was calculated as 1.57, and it also possessed an
pretreated with LPS before loading Mito-NIRHV, the fluorescence appropriate MW (573 Da). Some molecular probes with a quaternary
signals of both channel 1 and channel 2 were significantly increased salt moiety have been reported for the imaging of amyloid species
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and the viscosity channel (b2, d2). These results vividly illustrated
the elevated H2O2 level and increased viscosity of AD brains
compared to normal brains.
In summary, a dual-response fluorescent probe was designed
and synthesized for simultaneously detecting mitochondrial H2O2
and viscosity. The probe exhibited two independent emissions in
the NIR region upon responding to the two targets. It featured
high sensitivity and selectivity towards the targets, large Stokes
shift overcoming spectral cross-talk, satisfactory mitochondria-
targeting ability, appropriate BBB penetrability and low cytotoxi-
city both in vitro and in vivo. The probe was proved for its ability to
monitor the contents of H2O2 and viscosity in living cells and
animals, either individually or simultaneously. The probe was
applied to visually track the variations of H2O2 and viscosity in the
brain of AD mice. Our result provides a more comprehensive
picture of H2O2 and viscosity in AD brains, which could lead to
better understanding of AD pathology.
Fig. 2 Fluorescence imaging of H2O2 and viscosity in BALB/c mice.
(a) Only Mito-NIRHV (1 mM, 100 mL) was injected as a control. (b) Mice
pretreated with LPS for 4 h and then injected with Mito-NIRHV. (c) Relative
fluorescence intensities of (a) and (b). Channel 1: lex = 430 nm, lem
700 nm; Channel 2: lex = 570 nm, lem = 800 nm.
=
This work was financially supported by the National Natural
Science Foundation of China (No. 21575109, 21625503). All animal
procedures were performed in accordance with the Guidelines for
Care and Use of Laboratory Animals of Wuhan University and
approved by the Animal Ethics Committee of Wuhan University.
Conflicts of interest
There are no conflicts of interest to declare.
Fig. 3 Imaging and analysis of H2O2 and viscosity in a transgenic mouse Notes and references
model of AD. (A) Representative images of WT and APP/PS1 mice at 0 min
and 30 min after the injection of Mito-NIRHV. (B) Quantitative analysis of
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