Hydrazide–Hydrazone Small Molecules as AIEgens: Illuminating Mitochondria in Cancer Cells

Article abstract of DOI:10.1002/chem.201901074

Aggregation-induced-emission luminogens (AIEgens) have gained considerable attention as interesting tools for several biomedical applications, especially for bioimaging due to their brightness and photostability. Numerous AIEgens have been developed for lighting up the subcellular organelles to understand their forms and functions not only healthy but also unhealthy states, such as in cancer cells. However, there is lack of easily synthesizable, biocompatible small molecules for illuminating mitochondria (powerhouses) inside cells. To address this issue, an easy and short synthesis of new biocompatible hydrazide–hydrazone-based small molecules with remarkable aggregation-induced emission (AIE) properties is described. These small-molecule AIEgens showed hitherto unobserved AIE properties due to dual intramolecular H-bonding confirmed by theoretical calculation, pH- and temperature-dependent fluorescence and X-ray crystallographic studies. Confocal microscopy showed that these AIEgens were internalized into the HeLa cervical cancer cells without showing any cytotoxicity. One of the AIEgens was tagged with a triphenylphosphine (TPP) moiety, which successfully localized in the mitochondria of HeLa cells in a selective way compared to L929 noncancerous fibroblast cells. These unique hydrazide–hydrazone-based biocompatible AIEgens can serve as powerful tools to illuminate multiple subcellular organelles to elucidate their forms and functions in cancer cells for next-generation biomedical applications.

Full text of DOI:10.1002/chem.201901074

A Journal of
Accepted Article
Title: Hydrazide-hydrazone Small Molecules as AIEgens: Illuminating
Mitochondria in Cancer Cells
Authors: Sohan Patil, Shalini Pandey, Amit Singh, Mithun
Radhakrishna, and Sudipta Basu
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To be cited as: Chem. Eur. J. 10.1002/chem.201901074
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Hydrazide-hydrazone Small Molecules as AIEgens: Illuminating
Mitochondria in Cancer Cells
Sohan Patil,[a] Shalini Pandey,[a] Amit Singh,[b] Mithun Radhakrishna,[c] and Sudipta Basu* [b]
Abstract: Aggregation induced emission probes (AIEgens) have
gained lots of attention as interesting tools for ample biomedical
applications especially bio-imaging due to their brightness and
photo-stability. Numerous AIEgens have been developed for lighting
up the sub-cellular organelles to understand their forms and
functions in healthy as well as diseased states like cancer. However,
there is a serious absence of easily synthesizable, biocompatible
small molecules for illuminating mitochondria (powerhouses) inside
cells. To address this, in this report we describe an easy and short
synthesis of novel biocompatible hydrazide-hydrazone based small
molecules with remarkable aggregation-induced emission (AIE)
property. These small molecule AIEgens showed hitherto
unobserved AIE property due to dual intra-molecular H-bonding
confirmed by theoretical calculation, pH and temperature dependent
fluorescence and X-ray crystallography studies. Confocal
microscopy showed that these AIEgens were internalized into the
HeLa cervical cancer cells without showing any cytotoxicity. One of
the AIEgens was tagged with triphenylphosphine (TPP) moiety
which successfully localized into mitochondria of HeLa cells
selectively compared to L929 non-cancerous fibroblast cells. These
unique hydrazide-hydrazone based biocompatible AIEgens can
serve as powerful tools to illuminate multiple sub-cellular organelles
to elucidate their forms and functions in cancer cells for next-
generation biomedical applications.
Figure 1. (a) Synthetic scheme of hydrazide-hydrazone
molecules. (b) Fluorescence emission images of compound 4a
and 4b under UV lights and white light in solid state. (c) Images
of AIE property of compound 4a and 4b in water and in methanol.
the most important organelles which orchestrate several cellular
functions, hence implicated in different diseased states including
cancer.[22-24] As a result, visualizing mitochondria inside the
cancer cells is of utmost importance to understand their form
and function in cancer progression and management. Despite
several TPE and HPS-based AIEgens have been explored
lately,[25-30] there is still a serious lack of biocompatible small
molecule based AIEgens for visualization of mitochondria in
cancer cells.
To address this, in this manuscript, for the first time, we have
developed hydrazide-hydrazone based small molecules through
simple and concise synthetic steps, which showed remarkable
aggregation-induced emission (AIE) properties in water as well
as in solid state by dual intra-molecular H-bonding. This dual H-
bonding mediated restriction in intra-molecular motions (RIM) for
AIE properties was confirmed by temperature and pH-dependent
fluorescence quenching studies, 1H NMR spectroscopy and
theoretical calculations as well as X-ray crystallography. One of
the hydrazide-hydrazone based AIEgens was decorated with
positively charged triphenylphosphine (TPP) to illuminate
mitochondria in HeLa cervical cancer cells compared to L929
non-cancerous fibroblast cells. These hydrazide-hydrazone
small molecule AIEgens showed remarkable biocompatibility by
demonstrating negligible cell killing even at significantly high
concentrations. This novel biocompatible AIEgens can act as
platform probes to further illuminate several intra-cellular
organelles and bio-targets.
In last decade, aggregation-induced emission luminogens
(AIEgens) have emerged as powerful tools to develop chemical
sensors, optoelectronic devices and theranostic probes for
biomedical applications due to their brightness and excellent
photo-stability.[1-6] However, till date, tetraphenylethylene (TPE)
and hexaphenylsilole (HPS) moieties are extensively explored
as effective AIEgens despite their tedious synthetic steps and
highly hydrophobic nature leading to incompatibility for biological
applications.[7-10] To introduce biocompatibility for imaging and
therapy, majority of the AIEgens was tagged with hydrophilic
moieties to decorate hydrophobic AIE core through multiple
synthetic steps.[11-15] Recently, numerous biocompatible AIEgens
have been developed for in vitro cellular imaging, especially to
visualize intra-cellular organelles.[16-21] Mitochondrion is one of
[a]
[b]
S. Patil, S. Pandey
Department of Chemistry
Indian Institute of Science Education and Research (IISER)-Pune,
Dr. Homi Bhabha Road, Pashan, Pune, Maharashtra, 411008, India.
A. Singh, Prof. S. Basu
Discipline of Chemistry
Indian Institute of Technology (IIT)-Gandhinagar, Palaj,
Gandhinagar, Gujarat, 382355, India.
Email: Sudipta.basu@iitgn.ac.in
Prof. M. Radhakrishna
Discipline of Chemical Engineering, Indian Institute of Technology
(IIT)-Gandhinagar, Palaj, Gandhinagar, Gujarat, 382355, India.
The synthetic scheme of hydrazide-hydrazone derivatives (4) is
shown in Figure 1a. 2,6-dihydroxybenzoic acid was reacted with
dimethyl sulfate in presence of potassium carbonate as base at
55 °C in acetone for 24 h to obtain 2,6-dihydroxy benzoic acid
methyl ester which was subsequently reacted with hydrazine
monohydrate in methanol at refluxing condition to afford 2,6-
dihydroxy benzohydrazide (2) in 80% overall yield. Finally,
[c]
Supporting information for this article is given via a link at the end of
the document.
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Figure 2. (a) Fluorescence emission spectra of compound 4a in
different methanol: water ratios. (b) Fluorescence emission
intensity (λmax = 502 nm) versus water fraction graph of
compound 4a in different methanol: water mixture. (c) FESEM
images of compound 4a in water. (d) Schematic diagram of the
energy level difference between the HOMOs and LUMOs of
compound 4a, estimated from DFT computation and the
isodensity surface plots of the frontier molecular orbitals (FMOs)
with isovalue of 0.02 au.
Figure 3. (a) Scheme of H-bonding disruption of compound 4a
in temperature and pH change. (b) Optimized geometry of
compound 4a obtained from B3LYP/6-31G* computation. Black
zig-zag lines showing values for the respective dihedral angles
(in degree) whereas hydrogen bonding is shown in green dotted
lines. Atoms of C, O, N, Br and H are shown in cyan, red, blue,
orange and white colors, respectively. (c) Temperature
dependent change in fluorescence emission spectra of
compound 4a in water. (d, e) Images and fluorescence emission
spectra of compound 4a in different pH.
different 4-substituted benzaldehydes (3a, b) were reacted with
compound 2 in presence of catalytic p-toluenesulfonic acid (p-
TsOH) to afford hydrazide-hydrazone derivatives (4a, b) in 95%
yield. All the intermediates and final compounds were
characterized by NMR (1H and 13C) and mass spectroscopy
(HR-
MS) (Figure S1-S9).[31] Interestingly,
all
the
hydrazidhydrazone derivatives (4a, b) showed remarkable
fluorescence emission under long range of UV light in solid state
(Figure 1b). However, no fluorescence emission was observed
under white visible light. Similarly, compounds 4a, b showed no
fluorescence emission in methanol. On the other hand, both the
hydrazide-hydrazone derivatives (4a, b) showed remarkable
fluorescence emission in water (Figure 1c). This observation
clearly indicated the aggregation-induced emission (AIE)
properties of compound 4.
To evaluate the exact solvent ratio of aggregation induction, we
dissolved compound 4a in methanol and gradually titrated with
increased amount of water. There was no fluorescence signal
observed till methanol: water = 20:80 (v/v). However, compound
4a showed an increase in fluorescence emission intensity at
methanol: water = 10:90 (v/v) at λmax = 502 nm, which increased
significantly at 100 % water (Figure 2a,b, Figure S10a). The UV-
Vis spectra of compound 4a showed absorption maxima at λmax
= 322 nm in methanol and water (Figure S10b), indicative of a
large Stokes shift of 180 nm with negligible overlap between
absorption and emission spectra, which is highly suited for
fluorescence imaging. Similarly, compound 4b showed
demonstrated that the mean hydrodynamic diameter of
aggregated structure in water to be 706 nm (Figure S12). In
contrast, compound 4a showed very small hydrodynamic
diameter (~ 3.5 nm) in methanol indicating negligible
aggregation (Figure S12). We also visualized the aggregation of
compound 4a by scanning electron microscopy. The FESEM
images evidently showed that compound 4a aggregated into 2D-
sheet like structures in water, whereas no such significant
aggregation was found in methanol (Figure 2c). To confirm the
remarkable enhancement in the fluorescence emission intensity
of compound 4a and 4b due to the effect of aggregation, we
assessed the fluorescence emission in
a
concentration
dependent manner. As expected, the fluorescence emission
intensity of compound 4a and 4b were increased continuously
after increasing the concentration of the compounds in water
(Figure S13). We also performed fluorescence life time
experiment which revealed that compound 4a and 4b had 1.97
and 1.50 ns of fluorescence life time respectively which is little
less compared to Rhodamine 123,
a traditionally used
commercial mitochondria imaging agent (Figure S14).[32] The
fluorescence quantum yield of compound 4a and 4b were found
to be 12.2% and 5.4 % respectively which are comparable with
the structurally similar imine compounds as well as much higher
to Rhodamine 123 (~ 0.9%).[32,33] Subsequently the Frontier
molecular orbitals and the energy gap between HOMOs and
remarkable fluorescence emission intensity increment (λmax
=
492 nm) in water compared to methanol due to aggregation
(Figure S11). The aggregation of compound 4a in water was
further validated by dynamic light scattering (DLS), which
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broadening of both these peaks while increasing the
temperature from 25°C to 80°C (Figure S15). This observation is
in complete agreement with our previous study.[39] To validate
our hypothesis further, we performed temperature dependent
fluorescence spectroscopy on compound 4a in water. It was
observed that, with gradual increase in temperature from 25°C
to 80°C, the intensity of fluorescence emission was gradually
decreased by 3.5 folds (Figure 3c) due to the destruction of H-
bonding in structure 4a’. Interestingly, this fluorescence
quenching phenomena was found to be reversible.
Inter- and intra-molecular H-bonding leading to AIE property is
highly sensitive to the pH of the medium.[40] Hence, we perturbed
intra-molecular H-bonding by increasing the pH of the medium
from 7.4 to 8.5. Interestingly, it was observed that compound 4a
lost its fluorescence property completely (Figure 3d) leading to
the destruction of H-bonding in structure 4a”. On the other hand,
decreasing the pH from 8.5 to 7.4 resumed the fluorescence
property into compound 4a which even retained at much lower
pH = 4 (Figure 3d,e). As expected, compound 4b also showed
similar pH dependent fluorescence property (Figure S16). For
future mitochondrial imaging, the AIE probe should be
fluorescent at pH 7.8-8.0 which mimics the mitochondria matrix.
Hence, we assessed the AIE property of compound 4a at pH =
7.8 and 8.0. It was observed that the fluorescence intensity of
compound 4a was marginally reduced at pH = 7.8 and 8.0
compared to pH = 7.4 (Figure S17). Also 9.3% quantum yield
was calculated for compound 4a at pH = 7.8 which indicated that
compound 4a is suitable for mitochondrial bio-imaging. We
further evaluated the effect of solvents on the AIE property of
compound 4a and 4b. To our surprise, compound 4a showed
high and marginal AIE property in water and toluene respectively
(Figure S18a,b). However, compound 4a showed no AIE
property in other solvents like hexane, THF and DMSO. Similarly,
compound 4b also demonstrated AIE property in water only
(Figure S18c,d). This theoretical calculation along with
temperature and pH dependent reversible fluorescence
quenching-regaining clearly confirmed the role of intra-molecular
H-bonding in inducing AIE property in compound 4a. However,
we anticipate that the amphiphilicity of compound 4a and 4b
also played a crucial role in inducing AIE property, which needs
to be explored carefully in future to understand the exact
mechanism.
Figure 4. (a) Scheme for the synthesis of compound 7. (b) Solid
state fluorescence images of compound 7 under white light and
UV. (c) Fluorescence spectra of compound 7 in water. (d)
Fluorescence emission intensity versus water fraction graph of
compound 7 in different methanol: water mixture.(e) Schematic
diagram of the energy level difference between the HOMOs and
LUMOs of compound 7, estimated from DFT computation and
the isodensity surface plots of the frontier molecular orbitals
(FMOs) with isovalue of 0.02 au. (f) Optimized geometry of
compound 7 obtained from B3LYP/6-31G* computation. Black
zig-zag lines showing values for the respective dihedral angles
(in degree) whereas hydrogen bonding is shown in green dotted
lines. The atoms of C, O, N, Br and H are shown in cyan, red,
blue, orange and white colors, respectively.
LUMOs of compound 4a was calculated by Density functional
theory (DFT), which revealed the energy gap was ~ 4.18 eV (~
300 nm) correlating with the wavelength of UV-Vis absorbance
(Figure 2d).
We hypothesize dual intra-molecular H-bonding as the guiding
factor for this hitherto unobserved AIE property for these novel
hydrazide-hydrazone small molecules leading to restriction in
intra-molecular motion (RIM) (Figure 3a). To confirm our
hypothesis, we performed theoretical calculation using Gaussian
09 program on compound 4a.[34] 3D coordinates of compound 4a
were generated from the crystal structure of 4-N, N-
dimethylaminoaniline salicylaldehyde reported by Feng et. al.27
with a few modifications. The geometries were then additionally
optimized by the B3LYP[35] hybrid density functional with 6-31G*
Gaussian basis set. The presence of water was mimicked with
polarizable continuum model using the integral equation
formalism variant.[36] The geometry of the optimized molecular
structure of compound 4a showed that all the atoms were in the
same plane (Figure 3b) and confirmed the proposed dual intra-
molecular H-bonding.
One of the plausible mechanisms of this photophysical property
of compound 4a, b could be exited state intramolecular proton
transfer (ESIPT).[41,42] To validate the involvement of ESIPT as
well as the role of dual intra-molecular H-bonding in triggering
AIE property in compound 4a, we have synthesized mono-
hydroxy hydrazide-hydrazone compound 7 starting from 2-
hydroxybenzoic acid (5) by the same synthetic strategy via 2-
hydroxy benzohydrazide (6) (Figure 4a). All the compounds
were chemically characterized by NMR (1H and 13C) and HR-MS
Inter- or intra-molecular H-bonding are weak supramolecular
forces which can be disrupted by increasing the temperature of
the medium.[37] Moreover, 1H NMR spectroscopy has emerged
as one of the tools to probe the presence of inter- and intra-
molecular H-bonding.[38] Hence, we performed temperature
dependent H NMR spectroscopy of compound 4a from 25°C to
80°C and monitored the H-bonded proton peaks at 12.07 (-OH)
and 11.81 ppm (-NH). Remarkably, we observed the gradual
spectroscopy (Figure S19-S24). Interestingly, compound 7
showed negligible fluorescence property in solid state as well as
in water (Figure 4b,c). We further evaluated the change in
fluorescence property in methanol/water mixture. Negligible
increase in fluorescence intensity of compound 7 was observed
in 100% methanol to 100% water (Figure 4d and Figure S25).
The Frontier molecular orbitals and the energy gap between
HOMOs and LUMOs of compound 7 were calculated by DFT
1
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To evaluate the biocompatibility in non-cancerous cells, we
incubated fibroblast L929 cells with compound 4a and 4b in a
concentration dependent manner for 24 h. The MTT assay
revealed that compound 4a induced only 97.6 ± 1.4%, 94.3 ±
1.7% and 86.3 ± 1.8% cell viability at 12.5, 25 and 50 μM
concentrations respectively (Figure S26b). Similarly, compound
4b also showed only 94.6 ± 4.8%, 91.3 ±5.6% and 83.2 ± 2.3%
cell viability at 12.5, 25 and 50 μM concentrations respectively.
This cell viability assay confirmed that compound 4a and 4b
were not at all toxic to the cancer cells as well as non-cancerous
fibroblast cells, hence can be used for bio-imaging.
To evaluate the effectiveness of compound 4a and 4b in sub-
cellular mitochondria imaging in cancer cells, we treated HeLa
cells with both the compounds at 10 μM concentrations for 12 h.
Mitochondria were co-stained by MitoTracker Red dye and the
live cells were visualized by fluorescence confocal microscopy.
Confocal images in Figure S26c evidently showed that both
compounds 4a and 4b were internalized into the cells
successfully and localized into mitochondria leading to the
generation of yellow regions by overlapping green and red
fluorescence signals. However, both the compounds were also
localized into nucleus of the cells significantly. In fact, compound
4a and 4b showed non-specific sub-cellular localization which
was non-effective in specific mitochondria labeling. Furthermore,
to estimate the cancer cell specificity over non-cancerous cells,
we incubated fibroblast L929 cells with compound 4a and 4b at
20 μM concentrations for 12 h. The nuclei of the cells were
counter stained with blue fluorescence DAPI. The cells were
then fixed and visualized by confocal microscopy. Surprisingly,
the fluorescence microscopy images (Figure S27) hardly
showed any sub-cellular green fluorescence signals, indicating
that compound 4a and 4b barely internalized into L929 cells
even after 12h. These confocal images confirmed that these
novel AIE active hydrazide-hydrazone derivatives specifically
internalized into HeLa cancer cells over L929 non-cancerous
cells.
To overcome this challenge, we have synthesized hydrazide-
hydrazone based molecule tagged with positively charged
triphenylphosphonium (TPP) moiety for mitochondria targeting.
4-hydroxybenzaldehyde (8) was first reacted with excess 1,4-
dibromobutane in presence of potassium carbonate as base in
acetone in refluxing condition to obtain compound 9 in 77 %
yield (Figure 5a). The bromine moiety was further replaced by
triphenylphosphine in refluxing condition to obtain compound 10
in 92% yield. Finally, compound 10 was coupled with 2,6-
dihydroxy benzohydrazide (2) in presence of p-TsOH as catalyst
to obtain positively charged triphenylphosphonium tagged
hydrazide-hydrazone (11) in 95% yield. NMR (1H, 13C and 31P)
and HR-MS were used to characterize all the compounds
(Figure S28-S38). We also characterized the structure of
compound 11 by X-ray crystallography (Figure 5b) which clearly
confirmed the presence of proposed dual intra-molecular H-
bonding correlating with the theoretical electronic structure in
Figure 3b.We further assessed the photo-physical properties of
Figure 5. (a) Synthetic scheme of mitochondria targeted
hydrazide-hydrazone molecule (11). (b) ORTEP diagram of
compound 11 with 50% thermal ellipsoids. Solvent and counter
ions are removed for clarity. (c) Fluorescence emission spectra
of compound 11 in water. (d) Fluorescence emission image of
compound 11 in solid state under UV light.
which showed the energy gap was ~ 4.21 eV (~ 295 nm)(Figure
4e) comparable to the energy gap in compound 4a. Finally, we
executed theoretical calculations using Gaussian 09 program on
compound 7. Most interestingly, the optimized molecular
geometry of compound 7 showed the dihedral angle of the
mono-intramolecular H-bonded moiety was increased to ~12.3º
(Figure 4f) compared to nearly 0º dihedral angle found in
compound 4a. This clearly indicated that single intra-molecular
H-bonding is not sufficient to maintain coplanarity along the
largest molecular axis in compound 7 leading to lack of
aggregated structure with negligible AIE property. Moreover, in
our previous study, we synthesized O-phenyl-hydrazide-
hydrazone analogue of compound 7, which showed no AIE
property in solid state as well as in solution.[43] These solid state
fluorescence, water titration studies and theoretical structure
optimization clearly confirmed that (a) ESIPT is not the
mechanism involved and (b) dual intra-molecular H-bonding is
critical to maintain the planarity in compound 4a, b to induce
aggregation to show AIE property. However, at this point, the
exact mechanism of this photophysical phenomena in these
novel hydrazide-hydrazone based small molecules is elusive
and we are exploring this area currently.
To be successful in bio-imaging, the hydrazide-hydrazone AIE
probes should be biocompatible. Hence, we evaluated the in
vitro cytotoxicity of compound 4a and 4b in HeLa cervical cancer
cells at 48 h post-incubation in a dose dependent manner. The
cell viability was measured by MTT assay which showed
compound 4a induced only 94.2 ± 4.5 %, 97.4 ± 2.2 % and 91.8
± 5.6 % cell viability at 12.5, 25 and 50 μM concentrations
respectively (Figure S26a). Similarly, compound 4b also
demonstrated 99.5 ± 0.4 %, 95.3 ± 2.7 % and 94.1 ± 2.2 % cell
viabilities at 12.5, 25 and 50 μM concentrations (Figure S26a).
compound 11 which showed fluorescence emission at λmax
=
500 nm in water as well as AIE in solid state under UV light
(Figure 5c,d).
The viability of HeLa cells in presence of compound 11 after 48
h was determined by MTT assay in a dose dependent manner.
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bonding was responsible for restricting the intra-molecular
motion to show this hitherto unobserved AIE property. A
triphenylphosphonium labeled hydrazide-hydrazone AIEgen was
synthesized to illuminate mitochondria in HeLa cells selectively.
These novel biocompatible hydrazide-hydrazone small
molecules can be used as platform to develop innovative
AIEgens to image different sub-cellular organelles and targets in
cancer cells as future theranostic strategies by incorporating
photothermal and photo-dynamic probes.
Acknowledgements
Figure 6. (a) Viability of HeLa cells after treatment with
compound 11 in a dose dependent manner for 48 h by MTT
assay. (b) CLSM images of HeLa cells after treatment with
compound 11 for 12 h. Mitochondria were stained by
MitoTracker Red. Scale bar = 10 μm.
S.B. thanks Department of Biotechnology for Ramalingaswami
Fellowship (BT/RLF/Re-entry/13/2011) and other financial
supports
(BT/PR9918/NNT/28/692/2013,
and
BT/PR14724/NNT/28/831/2015). S. Patil and S. Pandey
acknowledge CSIR-UGC and IISER-Pune for doctoral fellowship
respectively. We sincerely thank Mr. Ravindra Raut of IISER-
Pune for helping in X-ray crystallography analysis. A.S and M.R
thank the High Performance Computing facility at IIT
Gandhinagar for computational resources.
This cell viability assay showed that compound 11 induced only
5.5± 1.8, 15.9±3.7 and 23.8±3.6 % cell death at 12.5, 25 and 50
μM concentrations respectively (Figure 6a). We also evaluated
the biocompatibility of compound 11 in non-cancerous fibroblast
L929 cells at 24 h post-incubation. The MTT assay revealed that
compound 11 induced 92.0± 3.5%, 87.6 ± 1.4% and 76.1 ±1.9%
cell viability at 12.5, 25 and 50 μM concentrations respectively
(Figure S39a). These cell viability assays indicated that
compound 11 is biocompatible for cervical cancer cells as well
as non-cancerous fibroblast cells for imaging. Furthermore,
compound 11 showed much better biocompatibility compared to
the traditionally used Rhodamine 123 as mitochondria staining
red fluorescent dye.[44,45]
Keywords: Hydrazide-hydrazone • aggregation-induced
emission • H-bonding • mitochondria • cancer
[1] J. Mei, N. L. C. Leung, R. T. K. Kwok, J. W. Y. Lam, B. Z. Tang! Chem. Rev.
2015, 115, 11718-11940.
[2] B. Liu, R. Zhang, Faraday Discussion 2017, 196, 461-472.
[3] X. Jiang, H. Gao, X. Zhang, J. Pang, Y. Li, K. Li, Y. Wu, S. Li, J. Zhu, Y.
Wei, L. Jiang, Nat. Commun. 2018, 9, 3799.
[4] Y. Chen, X. Min, X, Zhang, F. Zhang, S. Lu. L. Xu, X. Lou, F. Xia, X. Zhang,
S. Wang, Biosens. Bioelectron. 2018, 111, 124-130.
[5] J. Qi, C. Chen, D. Ding, B. Z. Tang, Adv. Healthc. Mater. 2018, 7,
e1800477.
To estimate its potential in mitochondria imaging, we treated
HeLa cells with compound 11 at 10 μM concentration for 12 h,
followed by staining mitochondria by MitoTracker Red. The
confocal images of the live cells (Figure 6b) evidently showed
that compound 11 homed into mitochondria of HeLa cells
leading to yellow overlapping regions. Interestingly, hardly any
green fluorescence signal was observed into nucleus of the
HeLa cells, which confirmed the specific mitochondria imaging
by compound 11. We also visualized the specificity of compound
11 to illuminate mitochondria in cancer cells over non-cancerous
cells by confocal microscopy. Non-cancerous fibroblast L929
cells were treated with compound 11 at 20 μM concentration
followed by staining nuclei and mitochondria by DAPI (blue) and
MitoTracker Red (red). The confocal microscopy images (Figure
39b) barely showed any green or yellow fluorescence signal
inside the cells or in mitochondria respectively. This microscopy
experiment established that compound 11 is highly specific for
mitochondria in HeLa cancer cells compared to non-cancerous
L929 cells at much lower concentration compared to Rhodamine
123.[46]
[6] C. Zhu, R. T. K. Kwok, J. W. Y. Lam, B. Z. Tang, ACS Appl. Bio. Mater.
2018, 1, 1768-1786.
[7] Y. Hong, J. W. Y. Lam, B. Z. Tang, Chem. Commun. 2009, 0, 4332-4353.
[8] Y. Hong, J. W. Y. Lam, B. Z. Tang, Chem. Soc. Rev. 2011, 40, 5361-5388.
[9] D. Ding, K. Li, B. Liu, B. Z. Tang, Acc. Chem. Res. 2013, 46, 2441-2453.
[10] J. Mei, Y. Hong, J. W. Y. Lam, A. Qin, Y. Tang, B. Z. Tang, Adv. Mater.
2014, 26, 5429-5479.
[11] Y. Chen, W. Zhang, Z. Zhao, Y. Cai, J. Gong, R. T. K. Kwok, J. W. Y. Lam,
H. H. Y. Sung, I. D. Williams, B. Z. Tang, Angew Chem. Int. Ed. 2018, 57,
5011-5015.
[12] Y. Hong, S. Chen, C. Wai, T. Leung, J. Wing, Y. Lam, B. Z. Tang, Chem.
Asian J. 2013, 8, 1806-1812.
[13] H. Shi, R. T. K. Kwok, J. Liu, B. Xing, B. Z. Tang, B. Liu, J. Am. Chem.
Soc. 2012, 134, 17972-17981.
[14] G. Jiang, G. Zeng, W. Zhu, Y. Li, X. Dong, G. Zhang, X. Fan, J. Wang, Y.
Wu, B. Z. Tang, Chem. Commun. 2017, 53, 4505-4508.
[15] F. Hu, D. Mao, Kenry, X. Cai, W. Wu, D. Kong, B. Liu, Angew Chem. Int.
Ed. 2018, 57, 10182-10186.
[16] F. Hu, B. Liu, Org. Biomol. Chem. 2016, 14, 9931-9944.
[17] J. Qian, B. Z. Tang, Chem 2017, 3, 56-91.
In conclusion, we have synthesized hydrazide-hydrazone based
small molecules in concise and easy steps in high yield, which
demonstrated for the first time remarkable aggregation-induced
emission (AIE) property in water as well as in solid state.
[18] G. Niu, R. Zhang, J. P. C. Kwong, J. W. Y. Lam, C. Chen, J. Wang, Y.
Chen, X. Feng, R. T. K. Kwok, H. H.-Y. Sung, I. D. Williams, M. R. J. Elsegood,
J. Qu, C. Ma, K. S. Wong, X. Yu, B. Z. Tang, Chem. Mater. 2018, 30, 4778–
4787.
Theoretical
calculation,
crystal
structure,
proton-NMR
[19] Y. Cai, C. Gui, K. Samedov, H. Su, X. Gu, S. Li, W. Luo, H. H. Y. Sung, J.
W. Y. Lam, R. T. K. Kwok, I. D. Williams, A. Qin, B. Z. Tang, Chem. Sci. 2017,
8, 7593-7603.
spectroscopy along with reversible temperature and pH
dependent studies confirmed that dual intra-molecular H-
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[20] Y. Li, Y. Wu, J. Chang, M. Chen, R. Liu, F. Li, Chem. Commun. 2013, 49,
11335-11337.
R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson,
Gaussain 09, Revision D. 01; Gaussian. Inc.: Wallingford, CT, USA, 2013.
[35] P. J. Stephens, F. J. Devlin, C. F. N. Chabalowski, M. J. Frisch, J. Phys.
Chem. 1994, 98, 11623-11627.
[21] C. Y. Y. Yu, W. Zhang, R. T. K. Kwok, C. W. T. Leung, J. W. Y. Lam, B. Z.
Tang, J. Mater. Chem. B 2016, 4, 2614-2619.
[22] D. C. Wallace, Nat. Rev. Cancer 2012, 12, 685-698.
[23] S. Fulda, L. Galluzzi, G. Kroemer, Nat. Rev. Drug Discovery 2010, 9, 447-
464.
[36] S. Miertuš, E. Scrocco, J. Tomasi, Chem. Phys. 1981, 55, 117-129.
[37] Q. Yuan, T. Zhou, L. Li, J. Zhang, X. Liu, X. Ke, A. Zhang, RSC Adv. 2015,
5, 31153-31165.
[24] J. Nunnari, A. Suomalainen, Cell 2012, 148, 1145-1159.
[25] N. Zhao, M. Li, Y. Yan, J. W. Y. Lam, Y. L. Zhang, Y. S. Zhao, K. S. Wong,
B. Z. Tang, J. Mater. Chem. C 2013, 1, 4640-4646.
[38] N. Avakyan, A. A. Greschner, F. Aldaye, C. J. Serpell, V. Toader, A.
Petitjean, H. F. Sleiman, Nat. Chem. 2016, 8, 368-376
[39] C. Ghosh, A. Nandi, S, Basu, Nanoscale 2019, 11, 3326-3335
[40] M. S. Mathew, K. Sreenivasan, K. Joseph, RSC Adv. 2015, 5, 100176.
[41] J. Zhao, S. Ji, Y.Chen, H. Guo P.Yang, Phys. Chem. Chem. Phys.
2012, 14, 8803-8817.
[26] C. W. T. Leung, Y. Hong, S. Chen, E. Zhao, J. W. Y. Lam, B. Z. Tang, J.
Am. Chem. Soc. 2013, 135, 62-65.
[27] M. Gao, C. K. Sim, C. W. T. Leung, Q. Hu, G. Feng, F. Xu, B. Z. Tang, B.
Liu, Chem. Commun. 2014, 50, 8312-8315.
[42] A. C. Sedgwick, L. Wu, H-H. Han, S. D. Bull, X-P. He, T. D. James, J. L.
Sessler, B. Z. Tang, H. Tian, J. Yoon, Chem. Soc. Rev. 2018, 47, 8842- 8880
[43] S. Patil, M. M. Kuman, S. Palvai, P. Sengupta, S. Basu, ACS Omega,
2018, 3, 1470-1481.
[28] W. Zhang, R. T. K. Kwok, Y. Chen, S. Chen, E. Zhao, C. Y. Y. Yu, J. W. Y.
Lam, Q. Zheng, B. Z. Tang, Chem. Commun. 2015, 51, 9022-9025.
[29] Q. Hu, M. Gao, G. Feng, B. Liu, Angew. Chem. Int. Ed. 2014, 53, 14225-
14229.
[44] J. S. Modica-Napolitano, J. R. Aprille, Cancer Res. 1987, 47, 4361-4365.
[45] W. H. Goffney, J. H. Wong, D. H. Kern, D. Chase, D. N. Krag, F. K. Storm,
Cancer Res. 1990, 50, 459-463.
[30] G. Yu, D. Wu, Y. Li, Z. Zhang, L. Shao, J. Zhou, Q. Hu, G. Tang, F.
Huang, Chem. Sci. 2016, 7, 3017-3024.
[31] O. G. Tsay, S. T. Manjare, H. Kim, K. M. Lee, Y. S. Lee, D. G. Churchill,
Inorg. Chem. 2013, 52, 10052−10061
[46] L. V. Johnson, M. L. Walsh, and L. B. Chen, Proc. Natl. Acad. Sci. USA,
1980, 77, 990-994.
[32] M. Savarese, A. Aliberti, I. D. Santo, E, Battista, F, Causa, P, A. Netti, N.
Rega, J. Phys. Chem. A. 2012, 116, 7491-7497.
[33] Q. Feng, Y. Li, L. Wang, C. Li, J. Wang, Y. Liu, K. Li, H, Hou, Chem.
Commun. 2016, 52, 3123-3126.
[34] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J.
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Entry for the Table of Contents (Please choose one layout)
COMMUNICATION
Light up the
powerhouse:
Biocompatible
Sohan Patil,[a] Shalini
Pandey,[a] Amit Singh,[b]
Mithun Radhakrishna,[c]
and Sudipta Basu* [b]
hydrazide-hydrazones
showed remarkable
aggregation-induced
emission (AIE)
through dual intra-
molecular H-bonding
to illuminate the
powerhouse
Page No. – Page No.
Hydrazide-hydrazone
Small Molecules as
AIEgens: Illuminating
Mitochondria in
Cancer Cells
(mitochondrion) in
cancer cells.
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