Aurone Derivative Revealing the Metabolism of Lipid Droplets and Monitoring Oxidative Stress in Living Cells

Article abstract of DOI:10.1021/acs.analchem.0c00456

Lipid droplets (LDs) are closely connected to many physiological processes and abnormal LDs are related to many diseases. Herein, a family of two-photon fluorescence compounds based on the aurone skeleton were developed as efficient LDs imaging probes. They exhibit the obvious solvatochromism effect from blue to orange emission (～140 nm shift) in various solvents. The robust probes possess low toxicity to living cells, high photobleaching resistance, and superior photostability and can light up LDs with high specificity. Furthermore, the probe DMMB (aurone skeleton with dimethylamino) was carefully applied in real-time monitoring of the morphological changes of LDs and the interactions between LDs and mitochondria under specific physiological conditions (e.g., starvation). We have observed for the first time the dynamic change between mitochondria and LDs when mitochondrial damage is caused by a large excess of H₂O₂ in a short period of time.

Full text of DOI:10.1021/acs.analchem.0c00456

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Article
An Aurone Derivative Revealing the Metabolism of Lipid
Droplets and Monitoring Oxidative Stress in Living Cells
Kang-Nan Wang, Shuyue Ma, Yanyan Ma, Yuping Zhao,
Miaomiao Xing, Liyu Zhou, Duxia Cao, and Weiying Lin
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.0c00456 • Publication Date (Web): 10 Apr 2020
Downloaded from pubs.acs.org on April 10, 2020
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An Aurone Derivative Revealing the Metabolism of Lipid Droplets
and Monitoring Oxidative Stress in Living Cells
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Kangnan Wang,†, ‡ Shuyue Ma,† Yanyan Ma,† Yuping Zhao,† Miaomiao Xing,† Liyu Zhou, ‡ Duxia
Cao,*,† Weiying Lin,*,†
† School of Materials Science and Engineering, Institute of Fluorescent Probes for Biological Imaging, University of Jinan,
Jinan, 250022, Shandong, China *E-mail: duxiacao@ujn.edu.cn; weiyinglin2013@163.com.
‡
Shunde Hospital, Southern Medical University (The First People's Hospital of Shunde), Foshan, 528308, Guangdong,
China
ABSTRACT: Lipid droplets (LDs) are closely connected with many physiological processes and abnormal LDs are related to
many diseases. Herein, a family of two-photon fluorescence compounds based on the aurone skeleton were developed as efficient
LDs imaging probes. They exhibit the obvious solvatochromism effect from blue to orange emission (~140 nm shift) in various
solvents. The robust probes possess low toxicity to living cells, high photobleaching resistance and superior photostability and can
light up LDs with high specificity. Furthermore, the probe DMMB was carefully applied in real-time monitoring the
morphological changes of LDs and the interactions between LDs and mitochondria under specific physiological conditions (e.g.
starvation). We have observed for the first time the dynamic change between mitochondria and LDs when mitochondrial damage
is caused by a large excess of H2O2 in a short time.
Lipid droplets (LDs), mainly enriching various neutral lipids
including cholesteryl ester and triacylglycerol, are recognized
as inert reservoirs for energy storage and reports on the fine
function of LDs are rarely declared for a long time.1-4
Inspiringly, recent evidences indicate that LDs are closely
connected with lots of physiological processes and related to
the metabolism of lipids, signal transduction and even cell
apoptosis.5,6 LDs are neutral lipid storage organelles, which
provide accessible and fast fatty acids (FAs) sources for
energy during nutritional deprivation. Miraculously, during
long-term starvation, lipids released by autophagy/apoptosis
of membrane organelles are packaged and stored in new LDs.5
Additionally, it should be highlighted that the abnormal
accumulation of LDs is related to many diseases involving
Subcellular organelles (such as mitochondria, LDs, etc.)
produce a variety of stress responses when living cells are
stimulated by the external environment.10,11 For example,
intracellular excess reactive oxygen species (ROS) can cause
changes in mitochondrial membrane potential (MMP) and
even induce apoptosis.12,13 When cells are in deprivation from
nutrition (starvation), they will activate related mechanisms
(such as autophagy) to synthesize a large amount of energy
substances storing in LDs to meet the needs of future
survival.14,15 In this process, there are also polarity changes in
many organelles (such as mitochondria, LDs, etc.).16,17
Recently, Tang's group reported
a
LDs-specific dye
TPAP-BB, which was used to track the dynamic autophagy
process between LDs and mitochondria in the case of ROS
inducing apoptosis.18 Anila et al. reported a fluorescent dye
DXB-NIR used to monitor the changes in LDs polarity
derived from fundamental nutrition and oxidative stress.19
According to the reports from the literatures,20,21 large excess
of H2O2 can directly stimulate cells to produce oxidative stress
1
obesity,
hyperlipidemia,
hepatocellular
carcinoma,
inflammation, atherosclerosis and cancer.7-9 Therefore, it is of
significance to monitor LDs accurately for the exploration of
their biological metabolism contributing to the early diagnosis
of related diseases.
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balanced salt solution (HBSS) without serum. More
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importantly, we have observed for the first time the dynamic
changes between mitochondria and LDs when mitochondrial
damage is caused by a large excess of H2O2 in a short time.
O
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MATERIALS AND METHODS
PMB
ECMB
DMMB
Cell culture and colocalization assay. A549 and HeLa cells
were cultured as reported method.26 A549 cells were first
incubated with 10 μM of DMMB/ECMB/PMB for 30 min,
and then further treated with BODIPY493/503 (1 μg/mL) for
another 30 min. After that, the cells were washed by using
phosphate buffered saline (PBS), and then viewed
immediately on a confocal microscope (Carl Zeiss LSM 710).
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Figure 1. Molecular structures of the probes.
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response in a short time and result in MMP collapse or even
damage. However, it is not clear what happens between
mitochondria and LDs when mitochondrial damage is caused
by a large excess of ROS in a short time.
Fluorescence microscopy imaging. A549 cells were reared
into confocal plates and and cultured for 24 h. Afterwards, the
cells were cultivated with the probes for 30 min. All the cells
were rinsed three times with PBS buffer solution ahead of
confocal images.
It is significant to track LDs accurately for exploring their
biological metabolism devoting to the early diagnosis of
related diseases. However, the common experimental methods
require cell fixation. Moreover, proteins associated with LDs
are seldomly moinored after the permeabilization.22 A widely
used method is visualizing FAs trafficking with a fluorescent
FAs probe.23,24 Fluorescent labeling of FAs is a complex
process and may be at risk of miss targeting and
photobleaching. On the other hand, despite being the
commercially available probes to staining LDs, BODIPY
493/503, Nile Red and Oil Red O face many limitations such
as specificity, photostability, small Stokes shifts, and large
background noise, etc.2 Thus, fluorescent small dyes with
excellent photophysical properties, staining the lipid core of
native LDs, and being able to be used to monitor LD-related
biological events are urgently needed.
Detection of oleic acid-induced accumulation of LDs. A549
cells were pre-treated with different concentrations (0, 100
μM, 200 μM) of oleic acid for 8 h. After that, cell media was
replaced by fresh RPMI 1640 media containing 20 µM of
DMMB. After staining for 30 min, cells were immediately
subjected to cell imaging by confocal microscope.
Monitoring the dynamics of LDs during starvation. The
starved A549 cells were incubated in HBSS for 8 h. And then,
the starved and control cells were stained with DMMB (20
µM) and Mito Tracker Red (MTR, 100 nM) in HBSS and
complete 1640 media, respectively, for 30 min. After that, the
cells were observed with a confocal microscope.
Hence, we described three efficient LDs markers based on
aurone
skeleton
with
dimethylamino
(DMMB),
N-ethylcarbazolyl (ECMB) and pyrene (PMB) as the terminal
groups (Figure 1), which have been described as the
two-photon fluorescence materials by our group.25 Strikingly,
DMMB presents a dotted shape distribution in the cells and
demonstrates the accumulation around LDs. Co-stain
experiments of these probes with BODIPY493/503 reveal that
DMMB can monitor LDs with high Pearson’s correlation
efficiency up to 0.983. The applications of DMMB for
monitoring LDs in living cells have been investigated, which
include inspecting the formation of LDs under oleic acid and
transportation for free fatty acids from LDs to mitochondria in
the process of autophagy under the condition of Hank’s
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Monitoring of the dynamics of LDs during oxidative stress.
After A549 cells were cultured in 35 mm dishes for 24h,
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the cells were incubated with DMMB (10 µM) and Mito
Tracker Deep Red (MTDR, 100 nM) for 30 min. And then,
the cells were washed by using PBS and further incubated
with H2O2 (10 mM) at for 20 min to induce oxidative
stress. The treated cells were immediately observed under a
confocal microscope for DMMB (λex = 405 nm) and
MTDR (λex = 633 nm). In the comparative experiments,
only MTDR was incubated in A549 cells for 30 min and
washed with PBS. After that, A549 cells were immediately
observed under the same conditions.
Figure 2. (a) Normalized fluorescence spectra of DMMB (10
µM) in organic solvents of varying polarity. Fluorescence
spectra of DMMB in the mixture of water and 1,4-dioxane
with different proportions. (λex = 405 nm).
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To monitor LDs in cells under physiological environment,
the probe should sustain stable at a wide pH range. Thus, the
fluorescence spectra of the probes in various pH environments
were separately measured. As shown in Figure S4(a), these
probes are stable over a wide pH range (5.0-11.0), indicating
the great potential in detecting LDs for biological applications
in cells. Furthermore, an effective fluorescent probe should
possess good selectivity in a complex biological system. The
effect of various analytes on the fluorescence properties of
DMMB was also detected. As shown in Figure S4(b), upon
the excitation at 405 nm, other species have little interference
with the fluorescence of DMMB at 550 nm, indicating that the
probe is inert to these analytes.
RESULTS AND DISCUSSION
Synthesis and spectral properties of the probes. These
three aurone derivatives were synthesized through the
cyclization reaction of corresponding hydroxychalcones under
the catalyzation of mercuric acetate (Scheme S1), which have
been reported as two-photon fluorescence materials by our
group.25 The selection of aurone molecular backbone is based
on its large conjugation, potential strong fluorescence
properties and low physiological toxicity.
UV-vis absorption and fluorescence spectra of the probes in
different organic solvents (Figures 2, S1, S2 and Table S1)
indicate that these probes show obvious fluorescence peaks
red shift upon the increase of solvent polarity. Especially
DMMB exhibits maximum fluorescence emission red shift
from 450 nm, 480 nm (non-polar hexane) to 592 nm (polar
DMSO) (Figure 2a). These probes are weakly emissive in
pure water, while the fluorescence intensity grows gradually
with increasing 1,4-dioxane volume content, accompanied
with apparent blue-shifted maximum emission from 588 nm
(DMMB, water) to 521 nm (DMMB, 1,4-dioxane) (Figure 2b)
due to the intramolecular charge transfer effect. Particularly,
DMMB exhibits stronger positive solvatochromism compared
with ECMB and PMB. The lipophilicity determined by a
classical shake-flask method27 for the probes are distributed
within 1.4-1.8. The high lipophilicity and high fluorescence
emission in low polar solvents of the probes provide
opportunities for their application in LDs imaging. Density
functional theory (DFT, Figure S3) calculation exhibits that
DMMB has higher dipole moment (6.24 Debye) relative to
other two compounds (ECMB, 5.51 Debye, PMB, 2.91
Debye).
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Figure 3. (A) The confocal images of the probes (20 μM) in living A549 cells co-stained with BODIPY 493/503 (1 μg/mL) for
30 min. λex = 405 (OPM) and 850 nm (TPM) for the probes (green); λex = 488 nm for BODIPY 493/503 (red). Overlay 1: Merged
images of BODIPY493/503 signal and OPM signals of the probes. Overlay 2: Merged images of the BODIPY493/503 signal and
TPM signals of the probes. Scale bar: 20 μm. (B) The LDs confocal images of DMMB (20 μM) with MTDR (a)/LTDR (b) (λex/em
= 633/650±10 nm) in living cells. Scale bar: 20 μm. (C) 3D images of A549 cells after incubation with DMMB (20 μM) and Syto
59 (100 ng/mL) for the LDs (green) and the nucleus (red).
Cell cytotoxicity assay and cell imaging. The cytotoxicity
of the probes was firstly studied by standard MTT assay in
A549 and HeLa cells according to a reported procedure.26 As
shown in Figure S5, the viability of cells was higher than 80%
after incubation with 50 μM of probes for 24 h, demonstrating
that the probes are biocompatible to living cells.
BODIPY493/503 was performed to confirm the staining
location of the probes. As shown in Figure 3A, the co-location
images of the three probes for LDs present a point shape
distribution in the cells, and the corresponding colocation
probes stained cells well with strong fluorescence at
one-photon mode (OPM) and two-photon mode (TPM)
(Figure 3A). Notably, DMMB presents a dotted shape
distribution in cells and mainly accumulates on LDs.
Compared with OPM, TPM has a better signal-to-noise ratio,
which avoids the cell autofluorescence and background
interference caused by short-wavelength excitation.16 The
TPM co-localization data presented in Figure 3A further
indicates that the probes could be enriched in LDs instead of
the fluorescence of BODIPY 493/503 being re-excited. To
detect the penetrating depth of DMMB, the analysis data of
A549 cells treated with DMMB were recorded at both OPM
and TPM. As shown in Figure S6, the A549 cells incubated
coefficients
of
green-emissive
the
probes
with
BODIPY493/503 are calculated as 0.983 (DMMB), 0.832
(ECMB) and 0.887 (PMB), respectively, which indicates that
the location of LDs with the probes is satisfactory. All of the
with DMMB displays larger fluorescence emissive
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penetration into the depth at TPM than that of OPM,
illustrating that DMMB can track the spatial accumulation of
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LDs and their dynamic movement in living cells with good
penetration by TPM.
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To further investigate whether probe DMMB could target
other organelles in cellular environment, we explored the
co-location experiment of DMMB with three typical cellular
markers: (i) MTDR for mitochondria, (ii) Lyso-Tracker Deep
Red (LTDR) for Lysosome and (iii) Syto 59 for nucleic acid.
As shown in Figure 3B and Figure S7, the dotted green
fluorescence of DMMB barely merged with the red emission
of MTDR, LTDR and Syto 59, suggesting DMMB was not
targeted to mitochondria, lysosomes and nuclei. The
photostability is a significant evaluation criterion for probes.
Thus, the light bleaching properties of the probes together
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with
commercially
available
LD-specific
dye
BODIPY493/503 were then investigated. As shown in Figure
S8, it could be observed that the fluorescence of the cells
stained with BODIPY493/503 is drastically lost, while the
emissive signal of the aurone derivative probes is retained
under the same conditions of 20 scans. The line series analysis
for each group further verifies photostable character of the
aurone probes (Figure S8D). The above research implies that
the aurone probes have excellent photobleaching resistance
and could be utilized for mapping LDs with good
photostability in living cells. The 3D imaging of confocal
microscope is usually obtained by Z-stack scanning, and
therefore the sample need be scanned continuously for many
times, which usually leads to the photobleaching of the
fluorophore.4 Therefore, high quality 3D images can be
obtained only when the probe has good fluorescence intensity
and photostability. Here, DMMB allows excellent 3D imaging
of LDs, which shows the cellular localization of LDs and the
interrelations with nucleus more clearly (Figure 3C).
Figure 4. (A) Confocal images of A549 cells stained with
DMMB (20 μM) after the cells were pre-treated with different
concentrations of oleic acid (0, 100 and 200 μM) for 6 h. (B)
Confocal images of A549 cells incubated in HBSS for 0 h
(control) and 8 h, followed by staining with DMMB (20 μM, 30
min λex = 405 nm) and Mito Tracker Red (100 nM, 30 min, λex
543 nm), respectively. Scale bar: 20 μm.
=
targeting ability of DMMB toward LDs in living cells. More
meaningful, when living cells are in the early stage of
starvation, they are under pressure to survive.29,30 The cells
will activate related mechanisms (such as autophagy) to
synthesize energy substances stored in LDs to meet the needs
of future survival.15,31 Therefore, in the following experiments,
the metabolism of LDs in the process of autophagy induced by
cell starvation was carefully explored. After the cells were
under the starvation of HBSS medium without serum for 8 h
and then co-stained with DMMB and mito-tracker red for 30
min, two major phenomena were observed clearly (Figure
4B). First, the fluorescence of DMMB enhances obviously
and increases intracellular distribution indicating the
generation of LDs during nutrition stress for serum. Second,
the mitochondria with red emission appears green colour from
the DMMB-stained LDs compared with the control group,
demonstrating the transportation of LDs from cytoplasm to
According to the reports from literatures,28 oleic acid is
taken up by cells and the phase splitting can occur, which
results in a significant increase in the number of neutral lipids
in cells, and cell fractionation shows that most newly
synthesized neutrophils are concentrated in LDs together with
LDs proteins. We treated the cells with nutrient rich substance
of oleic acid. As shown in Figure 4A, an apparent green
emission enhancement is observed after 6 h stimulation of
oleic acid with the increasing concentration from 0 to 200 μM.
The results reveal the accumulation of LDs and specific
mitochondria. The above interesting changes of LDs are
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consistent with the recent study for the pathway of LDs
catabolism, namely starvation-induced “lipophagy”.32-35 In the
process of autophagic degradation of intracellular membranes,
the formation of LDs in cells tends to transport the free fatty
acids against its excessive accumulation and lipotoxicity.
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LDs formation is a hallmark of cellular stress, but the
LDs do not work in isolation.10,11 Cells always try to combat
poisonous (such as, apoptosis and oxidative stress) stimuli by
transforming their metabolism from oxidative phosphorylation
to glycolysis.36 ROS produced by H2O2 can induce apoptosis
and decrease mitochondrial membrane potential (MMP).12,13,18
Large excess of H2O2 can directly stimulate cells to produce
oxidative stress response in a short time, resulting in MMP
collapse or even damage.20,21 Mitochondria-LDs contaction
promotes the transfer of FAs for β-oxidation in
mitochondria.15 But in this process, the dynamic change
between LDs and mitochondria is still unclear. Commercial
mitochondrial dyes MTR and MTDR are commonly used to
track changes in MMP in cells.37-39 During apoptosis,
mitochondrial dysfunction can also cause extensive LDs
formation.6 Currently, MTR and LDs dyes have been used to
track the fusion process of mitochondria and LDs during H2O2
inducing apoptosis in real time.18 However, to the best of our
knowledge, the real-time tracking dynamic changes between
LDs and mitochondria when the cells are under oxidative
stress have not been reported.
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Figure 5. (A) The confocal images of DMMB and
MTDR-stained A549 cells treated with 10 mM H2O2. (B)
Control experiment, MTDR-stained A549 cells were scanned
continuously under the same laser intensity. Scale bar: 20 μm.
to form lipid-soluble conjugates, which escapes from
mitochondria due to mitochondrial damage and the decrease
in MMP, those lipid-soluble conjugates then enter
DMMB-stained LDs.20,42 Considering that MTDR may be
bleached, comparative experiments were presented. As shown
in Figure 5B, the red fluorescence of A549 cells only staining
with MTDR does not change significantly under the same
conditions. Under oxidative stress, mitochondria collapse and
the lipid-soluble conjugates are fused by LDs, suggesting that
LDs play a protective antioxidant role in cells.43,44 At
subcellular organelle level, the direct impact of oxidative
stress on mitochondrial-lipid interactions was enriched the
content of the mitochondrial-lipid interaction network. We
have also studied the polarity changes of LDs of A549 cells in
a large amount of H2O2 in a short period of time.20 As shown
in Figure S9, the colour changes of the fluorescence in green
and red channels are similar to the red shift of DMMB caused
by the increase of the solvent polarity, which indicates that the
polarity of LDs in A549 cells increases significantly at this
case. LDs are closely connected with many physiological
processes and abnormal LDs are related to many diseases
including hepatocellular carcinoma, atherosclerosis and
cancer. Research on the effect of oxidative stress in LDs may
open up a new avenue for exploring their biological
metabolism devoting to the early diagnosis of related diseases.
As shown in Figure 5A, H2O2 (10 mM) was added to
A549 cells pretreated with MTDR and DMMB and the cells
were further incubated for 20 minutes. As time goes on, the
fluorescence of LDs stained with DMMB remains stable, and
the red fluorescence of mitochondria gradually diffuses and
disappears after 20 minutes. At the same time, in the overlay
layer, it can be clearly seen that the green and red fluorescence
gradually merge on the LDs (yellow spots). It is worth noting
that the position of the LDs does not shift significantly during
the entire process. Interestingly, this phenomenon is
significantly different from the LDs and mitochondrial fusion
observed in the case of ROS inducing apoptosis, in which,
mitochondrial morphology changes and LDs move around the
mitochondria to undergo autophagy.18,36,40,41 The main reason
may be that MMP collapses and mitochondria damages due to
the oxidative stress response of cells stimulated by high
concentrations of H2O2.20,21 The chloromethyl-containing
MTDR reacts with the thiol groups of mitochondrial proteins
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REFERENCES
CONCLUSIONS
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(1) Beller, M.; Thiel, K.; Thul, P. J.; Jäckle, H. Lipid Droplets: A
Dynamic Organelle Moves Into Focus. FEBS Lett. 2010, 584,
2176-2182.
In summary, three aurone derivatives as LDs markers
have been successfully developed. The cell imaging
experiments demonstrate that all of them could specifically
stain LDs with striking photostability. Additionally, one of the
aurone derivatives, DMMB provides a direct way to visualize
the metabolism of LDs under either nutrient-rich oleic acid or
starvation stress of serum indicating the formation and
transporting function in autophagy period of LDs. DMMB
also can be used to trace membrane integrity of mitochondria
during the oxidative stress. Notably, the dynamic change
between mitochondria and LDs when mitochondrial damage is
caused by a large excess of H2O2 in a short time was observed
for the first time by using DMMB. Thus, based on the aurone
derivative solvatochromic dye, we propose a concept of
Mitochondria-LDs interaction network for monitoring their
response to metabolism and oxidative stress, which may be
useful for the early diagnosis of related diseases.
(2) Fam, T. K.; Klymchenko, A. S.; Collot, M. Recent Advances in
Fluorescent Probes for Lipid Droplets. Materials 2018, 11, 1768.
(3) Fujimoto, T.; Parton, R. G. Not Just Fat: The Structure and
Function of the Lipid Droplet. Cold Spring Harb Perspect Biol.
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Supporting Information
The Supporting Information is available free of charge on
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Experimental details, additional UV-vis absorption,
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AUTHOR INFORMATION
Corresponding Author
* E-mail: duxiacao@ujn.edu.cn.
* E-mail: weiyinglin2013@163.com.
ORCID
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Kangnan Wang: 0000-0002-0835-8803
Yanyan Ma: 0000-0002-9358-5866
Duxia Cao: 0000-0002-9452-4045
Weiying Lin: 0000-0001-8080-4102
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ACKNOWLEDGEMENTS
This work was supported by the National Natural Science
Foundation of China (21472067, 21672083, 21877048,
21672130), the Natural Science Foundation of Shandong
Province (ZR2017MEM006), Taishan Scholar Foundation
(TS201511041), the startup fund of University of Jinan
(309-10004), and China Postdoctoral Science Foundation
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