Development of a mitochondrial-targeted two-photon fluorescence turn-on probe for formaldehyde and its bio-imaging applications in living cells and tissues

Article abstract of DOI:10.1039/c8nj01240g

A mitochondrial-targeted two-photon fluorescent formaldehyde probe (MT-FA) with a large emission enhancement was developed. MT-FA could image formaldehyde in the mitochondria of living cells for the first time, and also could image formaldehyde in living liver tissue slices.

Full text of DOI:10.1039/c8nj01240g

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Development of a mitochondrial-targeted two-photon
fluorescence turn-on probe for formaldehyde and its bio-imaging
applications in living cells and tissue
Received 00th January 20xx,
Accepted 00th January 20xx
An Xu, Yonghe Tang, and Weiying Lin*
DOI: 10.1039/x0xx00000x
www.rsc.org/
A mitochondrial-targeted two-photon fluorescent formaldehyde FA with some complicated operation process and destructive
probe (MT-FA) with large emission enhancement was irreversible damage, such as electronic method, high
developed. MT-FA could image formaldehyde in mitochondria of performance liquid chromatography (HPLC), gas
living cells for the first time, and also could image formaldehyde in chromatography and colorimetric method.7 Compared with
a
living liver tissue slices.
these methods, the fluorescence imaging techniques serve as a
powerful tool to monitor target biomolecules in organisms.8
The techniques provide real-time and in situ monitoring of
biological activated molecules with the non-invasive mode. In
recent years, although a few FA fluorescent probes have been
developed,9 the organelle-targeted fluorescent FA probes are
still scarce. And most of these probes are limited by one-
photon (OP) excitation with short wavelengths which are not
able to detect FA in living tissue. By contrast, the two-photon
microscopy (TPM) has the deep tissue penetration ability and
could provide improved three-dimensional imaging long
wavelength excitation.10 To our best knowledge, there is still
no mitochondrial-target two-photon fluorescence probe for FA
has been revealed up to date. Therefore, it is necessary to
develop the fluorescent probes for detecting FA in
mitochondria of living cells and living tissue.
Formaldehyde (FA),
a common environmental toxin,
identified as one of most the dangerous carcinogens by the
World Health Organization (WHO). Prolonged exposure to FA
will increase the risk of developing some malignant diseases,
such as respiratory diseases, cancer, Alzheimer's disease.1
Although FA is a harmful substance, endogenous FA exists in
organisms. The metabolism of methylated amines by the
enzymatic reaction of Semicarbazide-sensitive amine oxidases
(SSAO) may produce FA in living systems.2 The endogenous FA
plays an important role in cells,3 especially organelles, such as
the one carbon metabolic cycle in Mitochondrion.4 However,
the role of FA in living organisms is still not well-defined. It is
urgent to develop a method to effectively detect FA in living
organisms.
In eukaryotic cells, mitochondria, as the main energy supply
organelles, play the pivotal role in the metabolic processes and
cell death pathways of living cells. When the mitochondrial
dysfunction caused by oxidative stress, it may lead to a series
of diseases, including neurodegenerative and cardiovascular
diseases.5 Excessive FA may induce mitochondria mediated
caspase-released apotosis.6 The dualism of the role
(signal/toxicology) that FA played in mitochondria is still
ambiguous. Therefore, it is highly essential to develop a
mitochondrial-target fluorescence probe for constantly
monitoring the fluctuations of the FA level in mitochondria to
further disentangle physiological and pathological effects of
FA.
There are some ideas for targeting mitochondria, such as
using of carrier to migrate to mitochondria,11a Compared with
this indirect targeting method, the positively charged lipophilic
compounds, such as triphenylphosphine (TPP), based on
membrane potential are more frequently applied for targeting
mitochondria.11b, 11c Herein, we linked hydrazine group, the FA
response site, and TPP, the mitochondrial-target functional
group, directly to 1,8-naphthalimide fluorescence platform,
the classical two-photon (TP) fluorescence platforms,12 to
construct the first mitochondrial-target TP fluorescent probe
MT-FA. Due to the photo-induced electron transfer (PET)
pathway from hydrazine group to 1,8-naphthalimide
fluorescence platform, the probe displayed almost no
fluorescence. When FA was introduced, the PET pathway was
suppressed by the condensation reaction between hydrazine
group and FA, and the strong fluorescent signal was detected
(Scheme 1).9b
Nowadays, there are lots of traditional methods for monitoring
Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and
Chemical Engineering, School of Materials Science and Engineering, University of
Jinan, Jinan, Shandong 250022, P. R. China. E-mail: weiyinglin2013@163.com
†Electronic Supplementary Informaꢀon (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
The probe MT-FA was conveniently synthetized in a four-
step reaction (Scheme S1). Triphenylphosphine (TPP)
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underwent
bromopropylamine hydrobromide yielding the compound
Bromo-1,8-naphthalic anhydride and β-alanine underwent compound MT-Na proposed in scheme 1.
nucleophile substitution reaction yielding the compound It is well known that a slightly basic condition exists in
Compound and compound
underwent a condensation mitochondria (pH=8.05±0.11).13 In order to evaluate whether
reaction obtaining the compound , which reacted with MT-FA is applicable to the detection of FA in mitochondria, the
a
nucleophilic addition reaction with 3- that of compound MT-Na (the product of the react between
1. 4- MT-FA and FA), and the results of HR-MS was consistent with
2.
1
2
3
hydrazine hydrate to get the finally product MT-FA. The pH effect tests about MT-FA in the absence or presence of FA
detailed synthetic processes and the characterization data were carried out. As shown in Fig. 1c, MT-FA was stable at
(standard 1H NMR, 13C NMR and mass spectrometry) of all new wide range of pH value from 4.0 to 10.0. With the introduction
compounds were presented in supporting information (see the of FA, the strong fluorescent signal enhancement change was
ESI†).
observed, and even within the mitochondrial pH environment,
the fluorescence intensity was about 31.1-fold enhancement.
These results illustrated MT-FA is applicable to detection of FA
in mitochondria. The response rate experiments of MT-FA at
539 nm with the different concentrations (0 and 150 μM) of FA
were investigated (Fig. 1d). Upon addition of FA, the emission
intensity reached the equilibrium state at about 40 min at
ambient temperature. In contrast, the fluorescent signal of
only MT-FA almost had no changed under identical condition,
suggesting that MT-FA has potential real-time imaging
application value in living systems.
Scheme 1. The fluorescence response mechanism of the
mitochondrial-target TP fluorescent FA probe MT-FA based on
condensation reaction between hydrazine group and FA.
To evaluate the response ability of MT-FA to FA, the
absorption and fluorescence titration experiments was
conducted in 10 mM PBS buffer (pH 7.4, contain 5% DMSO as
co-solvent) at room temperature. As shown in the Fig. 1a, the
free probe MT-FA (5 μM) showed the maximum absorption at
439 nm (ε=8020 M-1 cm-1). However, upon addition of
increasing concentrations of FA (0-150 μM), the absorption
gradually increased. As designed, due to the PET pathway, MT-
FA showed almost non-fluorescent. Satisfactorily, upon
addition of amount of FA (0-150 μM) to the MT-FA solution,
the fluorescent intensity was gradually increased (up to 43.5-
fold, Fig. 1b and inset of it) at about 539 nm, and a consistent
results was shown in the two-photon (TP) fluorescence
spectrum experiments of MT-FA in presence and absence of
FA (Fig. S1). The TP fluorescence spectrum of only MT-FA
displayed a relatively weak fluorescent signal due to the PET
pathway. Upon addition of FA, a strong TP fluorescence signal
was observed at around 550 nm upon excitation at 800 nm,
and the shape of the spectrum closely resembles that of the
one-photon (OP) fluorescence spectrum. These results indicate
MT-FA is capable of detecting FA in one and two-photon mode.
In addititon, the detection limit of MT-FA was calculated to
be 4.9×10-6 M (Fig. S2), and an excellent linear relationship
between the emission intensity and concentrations of FA in
range of 0-50 μM was shown in Fig. S3 It is worthy to note that
the combination of the large switch-on signal and the low
detection limit of the probe MT-FA may render it highly
desirable for quantitative detecting the basal level of FA in the
living samples. We used fluorescence spectral (Fig. S4) and HR-
MS (Fig. S5) to verify the identification mechanism of MT-FA
for FA. When MT-FA was treated with FA, a strong emission
signal was found at around 539 nm, which closely resembles
Fig. 1. (a) The absorption titration spectra were recorded upon treatment
of MT-FA (5 μM) with FA (0-150 μM) in PBS buffer (pH 7.4, 5% DMSO) for
40 min; (b) The fluorescence titration spectra were recorded upon
treatment of MT-FA (5 μM) with FA (0-150 μM) in PBS buffer (pH 7.4, 5%
DMSO) for 40 min, Inset: Fluorescence intensity ratio (F/F0) changes at 539
nm of MT-FA with the amount of FA (0-150 μM); (c) Fluorescence intensity
changes of MT-FA (5 μM) treated or untreated with FA (150 μM) at
different pH solutions for 40 min; (d) Reaction-time profiles of MT-FA (5
μM) treated with FA (0 and 150 μM). λex = 440 nm.
To test the selectivity of MT-FA for FA, MT-FA was treated
with various aldehydes and biological relevant analytes in PBS
buffer (pH 7.4, 5% DMSO) at room temperature, including
amino acids, 1 mM for GSH, Hcy, Cys; inorganic salts, 1 mM for
CaCl2, MgCl2, KNO3, NaHSO3, Na2SO4, NaNO2, Na2S; reactive
oxygen and nitrogen species, 100 μM for NaClO, H2O2, di-tert-
butyl peroxide (DTBP), tert-butyl hydroperoxide (TBHP), NO;
ketone and aldehydes, 150 μM for FA, glyoxal, methylglyoxal,
sodium pyruvate, 4-dimethylamino bezaldehyde, acetone,
trichloro-acetaldehyde, acetaldehyde, 4-nitro-benzaldehyde.
The above species induced nearly no fluorescent signal
changed except of glyoxal (2.9-fold enhancement), Ca2+ (2.8-
2 | J. Name., 2012, 00, 1-3
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fold enhancement), TBHP (3.9-fold enhancement), while upon stimulator9l to conduct endogenous FA imaging experiments.
addition of FA, a 43.5-fold enhanced fluorescent signal was As shown in the Fig. 3, the HeLa cells that were treated with 15
obtained (Fig. 2). In the anti-interference experiments, only μM TG for 30 min only showed no fluorescence whether by OP
NaHSO3 caused a violent decrease of fluorescence intensity mode or TP mode (Fig. 3, a2 and a4). While the HeLa cells were
-
due to FA was consumed by HSO3 via nucleophilic addition pre-treated with 15 μM TG for 30 min, then treated with 5 μM
reaction, other interfering species has a small influence on MT- MT-FA for another 40 min, the strong fluorescent signals was
FA response to FA. (Fig. S6) The property of high selectivity and observed by OP and TP modes (Fig. 3, b2 and b4). While the
anti-interference of the probe indicated the probe is critical to cells were pre-treated with 15 μM TG and 300 μM NaHSO3 for
its potential biological applications. Furthermore, we also 30 min, then treated with 5 μM MT-FA for more 40 min, there
investigated the photo-stability of the probe. After MT-FA was was almost no fluorescence in green channel was observed
irradiated or non-irradiated by the short wavelength light (Fig. 3, c2 and c4). These studies showed that MT-FA is capable
source (UV light, the wavelength is 365 nm), the fluorescence of tracking endogenous FA in living cells.
intensity of the probe were almost no change (Fig. S7),
indicating that MT-FA would not be affected by the UV light
and it may have potential imaging application value.
Fig. 3. Fluorescence imaging of the endogenous FA in the HeLa cells by one
and two photon mode. a1)-a4) The image of the HeLa cells treated with TG
Fig. 2. Selectivity profiles: the fluorescence intensity of MT-FA (5 μM, at 539
nm) responding to FA in presence of various interfering species: amino
acids, 1 mM; inorganic salts, 1 mM; reactive oxygen and nitrogen species,
100 μM; ketone and aldehydes, 150 μM. Legend: (1) MT-FA; (2) glyoxal; (3)
(20 μM); b1)-b4) The image of the HeLa cells treated with TG (20 μM) for 30
min and MT-FA (5 μM) for further 20 min; c1)-c4) The image of the HeLa
cells treated with TG (20 μM), NaHSO3 (300 μM) for 30 min and MT-FA (5
methylglyoxal; (4) sodium pyruvate; (5) 4-dimethyl-aminobezaldehyde; (6)
μM) for further 20 min. OP-excitation was at 488 nm, TP-excitation was at
trichloroacetaldehyde; (7) acetaldehyde; (8) 4-nitro-benzaldehyde; (9)
800 nm and emission collection was from 500 - 550 nm. Scale bar: 20 μm.
acetone; (10) NaClO; (11) H2O2; (12) DTBP; (13) TBHP; (14) NO; (15) CaCl2;
(16) MgCl2; (17) KNO3; (18) NaHSO3; (19) Na2SO4; (20) NaNO2 (21) Na2S; (22)
GSH; (23) Hcy; (24) Cys; (25) FA. λex = 440 nm.
As an imaging reagent, MT-FA should have no significant
cytotoxicity. Then the cytotoxicity of the probe was
investigated by the MTT assay. As shown in the Fig. S8, the
MTT results showed that MT-FA has little cytotoxicity at low
dose level (0-10 μM), indicating that MT-FA could be used in
Fig. 4. The images of the living HeLa cells treated with TG (15 μM) 30 min,
MT-FA (5 μM) for further 40min and then treated with Mito-Tracker Deep
imaging experiments.
The exogenous FA fluorescence imaging experiments were
carried out by OP and TP modes, and the results were shown
in the Fig. S9. The HeLa cells that treated with 5 μM MT-FA for
40 min only showed almost invisible fluorescence whether by
the OP mode or TP mode (Fig. S9, a2 and a4). While the HeLa
cells were pre-treated with 150 μM FA for 20 min, then
treated with 5 μM MT-FA for another 40 min, the marked
enhanced green fluorescence was observed at both OP and TP
modes (Fig. S9, b2 and b4). We used NaHSO3 for negative
control experiments because it is a commonly used FA
inhibitor.9b When the cells pre-treated with 150 μM FA and
300 μM NaHSO3 for 20 min, then treated with 5 μM MT-FA for
more 40 min, the green fluorescence became very faint and
almost invisible (Fig. S9, c2 and c4). In addition, we had
reached the consistent conclusion from the spectrum (Fig. S10).
These studies showed that MT-FA has good membrane
permeability and may monitor exogenous FA in living cells.
Encourage by exogenous imaging experiments, we
continued to conduct endogenous imaging experiments. We
chose thapsigargin (TG) as the endogenous formaldehyde
Red (500 nM) for 5 min. a) Bright-field image of the HeLa cells; b) The
fluorescence image of the green channel; c) The fluorescence image of the
red channel; d) The merged image of a, b, and c; e) Intensity scatter plot of
the green and red channels. f) Intensity profile of linear region of interest
across the cell costained with red channel of Mito Tracker Deep Red and green
channel of MT-FA of a). The green channel: λex = 440 nm, λem= 500-550 nm;
The red channel: λex = 647 nm, λem= 663-738 nm Scale bar: 20 μm.
The co-localization experiment was carried out to
investigate the mitochondrial-target ability of MT-FA
.
Colocalization results obtained using a confocal laser
microscope with 5 μM MT-FA and 500 nM Mito-tracker deep
red (the commercial mitochondrial location dye, Thermo Fisher
Scientific.) in presence of 15 μM TG and 150 μM FA separately.
As shown in Fig. 4 and Fig. S11, the red channel of Mito-tracker
deep red overlapped well with the green channel of MT-FA
treated with endogenous and exogenous FA separately. The
intensity profile of the linear regions of interest in green
channel of MT-FA and red channels of Mito-tracker deep red
displayed a tendency toward synchrony. The corres-ponding
Pearson’s correlation coefficient was calculated as 0.91 and
0.90 separately, indicating the location of MT-FA may
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accumulate in the mitochondria and could detect the FA in the
mitochondria.
Encouraged by the satisfactory results of cell imaging
experiments, the tissue imaging experiments were also
investigated. Consistent with expectations, the results of the
tissue imaging experiments were consistent with the results of
cell imaging. When the mice liver tissue slices were soaked in
FA solution (300 μM) for 1 h, and then soaked in the MT-FA
solution (10 μM) for another 40 min, the strong fluorescent
signals was observed with the penetration depth up to about
80 µm (Fig. 5). By contrast, the mice liver tissue slices were
only soaked in MT-FA solution (10 μM) for 40 min showed no
fluorescence signal in green channel (Fig. S12). These data
implied that the probe is capable of detecting FA in the liver
tissue slides.
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In conclusion, the first mitochondrial-targeted TP
fluorescent probe, named as MT-FA, has been rationally
designed and synthesized. Upon reaction with FA, the PET
pathway which inhibits the fluorescence emission of the probe
is suppressed, and the probe shows significant fluorescence
enhancement than the other analytes. Importantly, the co-
localization experiments indicate that MT-FA may effectively
accumulate in the mitochondria for monitoring of FA in living
cells. Moreover, MT-FA could also successfully detect FA in
living mice liver tissue slices with the penetration depth up to
about 80 µm. We believe the MT-FA is conducive to further
disentangle physiological and pathological effects of FA in
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This work was financially supported by NSFC (21472067,
21672083), the Taishan Scholar Foundation (TS 201511041),
and the start-up fund of the University of Jinan (309-10004).
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Conflicts of interest
There are no conflicts to declare.
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Graphic content entry
The first mitochondrial-targeted two-photon fluorescent probe (MT-FA) for
formaldehyde (FA) was engineered for monitoring FA in mitochondria of the living
cells and liver tissue by one- and two-photon modes.