Light-up lipid droplets for the visualization of lipophagy and atherosclerosis by coumarin-derived bioprobe

Article abstract of DOI:10.1016/j.cclet.2021.02.059

Lipid droplets (LDs) are intracellular lipid-metabolism organelles that involved in many physiological processes, metabolic disorders as well as diseases such as atherosclerosis. However, the specific probes that can visually locate abnormal LDs-rich tissues and track LDs-associated behavior to the naked eye with adequate biosafety still are rare. Herein, we develop a new design strategy of LDs-targeted probe based on the solvatochromism of coumarin derivatives. The results revealed that the emission wavelength of coumarin fluorophores gradually red shift in different solvents with increasing polarity, while absorption wavelength almost unchanged. As a result, the enlarged stokes shift of coumarin was emerged from oil to water. Furthermore, properly reducing water solubility and adding electronic donor at the structure of coumarins can enlarge this type of solvatochromism. This discovery was utilized to develop suitable probe for the image of LDs and LDs-rich tissues with high resolution and biosafety. Therefore, LDs-associated behavior was visible to the naked eye during the process of lipophagy and atherosclerosis. We deem that the developed probe here offers a new possibility to accurately diagnosis and analyse LDs-related diseases in clinic and preclinical study.

Full text of DOI:10.1016/j.cclet.2021.02.059

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Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
Light-up lipid droplets for the visualization of lipophagy and
atherosclerosis by coumarin-derived bioprobe
Jinrong Zhenga,1, Shuheng Qina,1, Lijuan Guia, Hua Lia, Lixue Fana, Yifei Yangb,
a,
Haiyan Chena, Hui Xuc, , Zhenwei Yuan
*
*
a
Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, Nanjing 210009, China
School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation
b
Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China
c
Department of Food Quality and Safety, College of Food Science and Engineering/ Collaborative Innovation Center for Modern Grain Circulation and Safety/
Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing University of Finance and Economics, Nanjing 210023, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Lipid droplets (LDs) are intracellular lipid-metabolism organelles that involved in many physiological
processes, metabolic disorders as well as diseases such as atherosclerosis. However, the specific probes
that can visually locate abnormal LDs-rich tissues and track LDs-associated behavior to the naked eye
with adequate biosafety still are rare. Herein, we develop a new design strategy of LDs-targeted probe
based on the solvatochromism of coumarin derivatives. The results revealed that the emission
wavelength of coumarin fluorophores gradually red shift in different solvents with increasing polarity,
while absorption wavelength almost unchanged. As a result, the enlarged stokes shift of coumarin was
emerged from oil to water. Furthermore, properly reducing water solubility and adding electronic donor
at the structure of coumarins can enlarge this type of solvatochromism. This discovery was utilized to
develop suitable probe for the image of LDs and LDs-rich tissues with high resolution and biosafety.
Therefore, LDs-associated behavior was visible to the naked eye during the process of lipophagy and
atherosclerosis. We deem that the developed probe here offers a new possibility to accurately diagnosis
and analyse LDs-related diseases in clinic and preclinical study.
Received 7 January 2021
Received in revised form 24 February 2021
Accepted 25 February 2021
Available online xxx
Keywords:
Lipid droplets
Probe
Lipophagy
Atherosclerosis
© 2021 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
The lipid droplets (LDs) are ubiquitous, endoplasmic reticulum
derived and dynamic organelles that function as cellular centre of
lipid metabolism [1]. They are composed of a neutral lipids core,
mainly contains cholesterol esters and triglycerides, coated with a
phospholipid monolayer membrane in which proteins are anchored
[2]. LDs regulate various cell activities, including lipid signaling
pathway, membrane biosynthesis, acting as dynamic lipid storage
depots to provide the energy by rapid mobilization of fatty acids,
sequestering toxic lipids [3]. Notably, excess of LDs has been
implicated in the pathogenesis of numerous diseases, especially in
atherosclerosis related cardiovascular diseases [4].
atherosclerotic plaques, which invade the arterial wall in order
to remove adverse modified low‑density lipoprotein (LDL)
particles, resulting in the accumulation of lipids in cytosolic LDs
[6]. Foam cells are considered as a biomarker participate in all
pathological development stages of atherosclerotic lesions, and
characterized by an aberrant accumulation of cytosolic LDs [7].
Therefore, it may be an effective strategy to visualize atheroscle-
rotic plaques by designing fluorescent probe to image LDs in foam
cells.
As an important technique in modern scientific research,
fluorescence imaging enables living cells and whole organisms to
be visualized and has significantly promoted advances in biology
and medicine [8–11]. Recently, a variety of fluorophore have been
exploited todesign fluorogenic probes for imaging LDs according to
the solvatochromism, such as BODIPY [12–14], AIEgen [15,16], and
more probes were summarized in the review [17]. Although there
are many LDs probes be reported so far, the commercially available
probes still rare. The Nile-Red and BODIPY are the most commonly
used commercial probes to indicate LDs (Scheme 1A). But BODIPY
is not suitable for the visualization imaging of tissues due to
Accumulation of atherosclerotic plaques in arterial walls leads
to major cardiovascular diseases and stroke [5]. The foam cells
derived from macrophages are the central components of
* Corresponding authors.
E-mail addresses: 9120191156@nufe.edu.cn (H. Xu), yuanzhenwei@cpu.edu.cn
(Z. Yuan).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.cclet.2021.02.059
1001-8417/© 2021 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article as: J. Zheng, S. Qin, L. Gui et al., Light-up lipid droplets for the visualization of lipophagy and atherosclerosis by coumarin-
derived bioprobe, Chin. Chem. Lett., https://doi.org/10.1016/j.cclet.2021.02.059

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Scheme 1. (A) The classic LDs-targeted probes Nile Red and BODIPY 493. (B) The potential LDs-targeted probes developed in this study.
narrow stokes shift, and Nile-Red cannot be used for multicolor
imaging due to wide excitation band. Therefore, a commercially
available LDs-specific probe still needs to be developed for the
visualization of LDs-involved pathological process such as lip-
ophagy and atherosclerosis. In addition, the naked eye recognition
of pathological tissue requires a visible fluorescent probe with
shorter excitation wavelength at near-UV region to avoid the
interference of excited light on human-eye resolution. Further-
more, the larger Stokes shift can avoid the interference from
biological background. Coumarin is a kind of classical fluorescent
dyes, which has the characteristics of low biological toxicity, large
stokes shift and intramolecular charge transfer (ICT) effect [18,19].
Inspired by this, we focused on the exploration of structural factors
affect the solvatochromism of coumarins to establish a new design
strategy, and then develop suitable fluorogenic probe for the
visualization of LDs-associated behavior and LDs-rich tissue in
atherosclerotic plaque (Scheme 1B).
At present, most of the LDs-specific fluorescent probes are
designed according to the solvatochromism effect. Therefore, we
investigated the solvatochromism effect of coumarin fluorophores.
We considered different water solubility and electron cloud
density of the structure when selected the candidate coumarin
derivatives. The corresponding optical properties in different
solvents were summarized in Table 1 and Table S2 (Supporting
information), and the corresponding spectrums were showed at
Figs. S1 and S2 (Supporting information). It can be seen that the
solvents with different polarity have little effect on the maximum
absorption wavelength of most coumarin derived lipid droplets
probes (COLDs) which contain COLD-1, -2, -5 and -6, but the
enhanced polarity lead to significant red shift of the maximum
emission wavelength. This transformation markedly extends the
Stokes shift of the compounds from organic solvents to water. The
compound COLD-4 was no observed similar solvatochromism
effect may due to the poor electron-donating capacity of 7-
glucuronide. The COLD-2 possesses more electron donor compare
with COLD-1, which indicated that the solvatochromism effect of
coumarin can be enhanced by modifying electron donor on the
conjugate system. Therefore, we can infer that lower water
solubility and more electron donor are advantageous to the
solvatochromism of coumarins. In addition, a gradual process of
solvatochromism of the coumarins is observed by THF/water
solvent system from 1% to 99% (Figs. S3 and S4 in Supporting
information). In addition, the result showed that the COLD dyes are
not AIE (Figs. S5 and S6 in Supporting information). So we can infer
that this solvatochromism of the dyes may be caused by other
reasons. The theoretical calculation of the dyes was conducted by
density functional theory (DFT). As Figs. S7 and 8 (Supporting
information) showed, the electron cloud in the coumarin dyes
transfer from donor to conjugated core, which was especially
obvious in the case of COLD-5 and COLD-6. Furthermore, to gain
insight into the solvatochromism of the dyes, we carried out time-
dependent density functionaltheory (TD-DFT) calculations of
COLD-4, COLD-5 and COLD-6 at DCM and water solvent
respectively. The results were similar to that observed in actual
experiments (Figs. S9-S11 in Supporting information).
To explore the visual performance of fluorescence from COLDs,
a 365 nm UV laser is used to excite coumarin molecules in different
polar solvents. As shown in Fig. 1, COLD-6 shows a gratifying
property, which emerged intensive green fluorescence in all
organic solvents while no visible fluorescence in the water solvent.
It indicated that COLD-6 can efficiently distinguish organic phase
from water phase by the naked-eye observation. Furthermore,
COLD-6 emit strong visible green light in oil and almost no light in
water (Fig. S12 in Supporting information), but COLD-1 and COLD-
2 shows the opposite characteristics (Fig. S13 in Supporting
information). This means that COLD-6 may distinguish LDs from
other cellular components or adipose tissue from other tissues.
The biological imaging performance of COLD-6 was further
tested. The results showed that COLD-6 no have biological toxicity
even at 100 mmol/L (Fig. S14 in Supporting information). In
Table 1
Structure and solvatochromism of coumarins.
Solvent
COLD-1 (a/b)
COLD-2 (a/b)
COLD-3 (a/b)
COLD-4 (a/b)
COLD-5 (a/b)
COLD-6 (a/b)
Toluene
Dioxane
EtOAc
CHCl3
Acetone
ACN
DMSO
Isopropyl alcohol
EtOH
MeOH
PBS
227/375
249/375
320/375
325/373
322/382
320/378
325/378
323/373
325/371
325/372
327/420
319/386
314/386
316/386
321/383
288/396
316/401
323/392
217/416
325/384
325/398
326/485
240/376
245/374
267/383
272/384
276/396
270/435
247/422
272/385
272/397
271/391
290/396
316/402
313/390
312/403
315/402
299/372
312/402
316/402
313/397
316/399
316/399
314/392
302/343
296/425
295/451
300/442
307/442
297/425
299/437
299/472
300/522
300/492
303/570
444/492
445/491
447/492
455/499
453/502
454/501
456/509
453/503
455/499
463/501
450/547
a: The longest absorption maxima are shown (nm).
b: Excited at the maximum excitation wavelength (nm).
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determined as: lex = 405 nm, lem = 500À560 nm. To verify the LDs
targeting ability of COLD-6, the A549 cells were incubated with
COLD-6 (green channel) and LDs-Red (Nile-Red, red channel)
simultaneously. The green particles were all located in the red area
(Fig. 2C). This indicated that COLD-6 has sensitivity and selectivity
to LDs.
To assess the performance of COLD-6 on monitoring behavior of
LDs in cells, a model of starvation-induced autophagy was
established to observe the process of lipophagy of LDs (Fig. 3A).
If LDs and lysosomes are fused in treated cells, more distinguish-
able yellow fluorescence arising from co-localization of lysosomes
(green) and LDs (red) will be observed. The situation of cells
starved for 0, 1, 2, 5 h were performed to observe the whole
autophagy process (Fig. S16 in Supporting information). Because
lipophagy is a gradual process, thus the distribution of LDs and
lysosomes in one cell may show three different states, exist alone
(Ⅰ, Ⅱ), gradually fuse (Ⅲ) and completely devour (Ⅳ), during
starvation. Which were represented by the results showed at
Fig. 3B. This indicates COLD-6 can track the movement of LDs with
excellent resolution and selectivity.
In the atherosclerosis plaque, the infiltrative macrophages are
continuously stimulated by ox-LDL and then transforming into
LDs-enriched foam cells which are the central component of the
plaque (Fig. 4A). Therefore, we sequentially explored the capacity
of COLD-6 in visualization of LDs in macrophages after stimulating
by ox-LDL. As Fig. 4B and Fig. S17A (Supporting information)
Fig. 1. Photograph of coumarins (100 mmol/L) in various solvents under UV lamp
(365 nm): Toluene, dioxane, EtOAc, CHCl3, acetone, ACN, DMSO, isopropyl alcohol,
EtOH, MeOH, and PBS (1-11).
addition, the fluorescence intensity increases gradually until 2 h,
therefore we select 2 h as the optimal staining time of cells in vitro
(Fig. S15 in Supporting information). The maximum absorption
wavelength of COLD-6 is near 450 nm, but the available excitation
channels of fluorescence microscope are 405 nm (DAPI) and
488 nm (FITC). Therefore, the applicability of these two channels
on COLD-6 imaging in cells was discussed. As the Fig. 2A showed,
the fluorescence of COLD-6 granulated in A549 cells under DAPI
channel while distributed near the cell membrane under FITC
channel. Furthermore, the fluorescence emission of COLD-6 was
collected in 410À480 nm (green) and 500À560 nm (purple) under
the excitation wavelength of 405 nm. As a result, the fluorescence
of different collection scope overlap in white in A549 cells, but the
green channel displayed some interferences that do not overlap
with the purple channel may due to the potential interference
from biological short-wavelength emission (Fig. 2B). Thus, the
tested parameter of COLD-6 at fluorescence microscope can be
showed, 2 mg/mL of ox-LDL can lead to the maximum accumula-
tion of LDs macrophage (Raw264.7). Moreover, the fluorescent
intensity of COLD-6 increasing 10.5-fold in Raw264.7 after ox-LDL
stimulating 48 h (Fig. 4C and Fig. S17B in Supporting information).
However, the growth of LDs in endothelial cells HUVEC was not
obvious after ox-LDL stimulating (Figs. S18 and S19 in Supporting
information). This indicated that ox-LDL mainly affects macro-
phage rather than endothelium at atheromatous vascular wall,
which can be significantly visualized by COLD-6.
Atherosclerotic plaque contains LDs enriched macrophages and
extracellular lipids. To simulate the visible spectacle of these two
components, the 3D-scan model of macrophage and adipose tissue
were prepared respectively. First, COLD-6 exhibited that the dense
Fig. 2. The A549 cells were treated with COLD-6 (1 mmol/L), and (A) the
fluorescence images were obtained under 405 nm/DAPI and 488 nm/FITC channel;
(B) the fluorescence images were obtained under the receiving range of 410-480 nm
and 500-560 nm, lex = 405 nm. (C) The A549 cells were treated with COLD-6 (1
mol/L) and LDs-tracker (Nile-Red, mol/L), the fluorescence images were
obtained, green: lex = 405 nm and lem = 500-550 nm, red: lex = 555 nm and
lem = 560-620 nm. Scale bar = 10 m.
m
Fig. 3. (A) The scheme of growth, lipophagy process of the LDs in live cells. (B) The
1
m
A549 cells starved 2 h and then treated with COLD-6 (1
(1 mol/L), the fluorescence images were obtained, green: lex = 405 nm and
lem = 500-550 nm, red: lex = 555 nm and lem = 560-620 nm.
mmol/L) and Lyso-tracker
m
m
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Fig. 4. (A) ox-LDL induces macrophages to transform into LDs enriched foam cells in atherosclerotic plaque. (B) The Raw264.7 cells pre-treated with ox-LDL (0,1, 2
h and then treated with COLD-6 (10 mol/L), the fluorescence images were obtained. (C) The Raw264.7 cells pre-treated with ox-LDL (2 g/mL) 0, 24 and 48 h and then
treated with COLD-6 (1 mol/L), the fluorescence images were obtained.
mg/mL) 48
m
m
m
LDs were crowding in the 3D-scan image of single macrophage
with visual impact (Fig. 5A). Next, the adipose tissue emitted bright
green fluorescence after staining by COLD-6 ex vivo (Fig. 5B).
Importantly, COLD-6 only emitted visible green fluorescence at the
site of adipose tissue when the fat-attached kidney was staining by
COLD-6 (Fig. 5C). The results indicated that COLD-6 can be a
potential probe for visualization of atherosclerosis plaque. The
experiment of COLD-6 mediated atherosclerotic plaque visualiza-
tion and diagnosis needs to be explored by the future research.
In summary, we discussed the solvatochromism of different
coumarin structures to developed high resolution LDs probe which
is visible to the naked eye. The solvents with different polarity have
little effect on the maximum absorption wavelength of most
coumarin, but the enhanced polarity lead to significant red shift of
the emission wavelength. This transformation markedly extends
the Stokes shift of the compounds from organic solvents to water.
Moreover, this solvatochromism can be enhanced by modifying
electron donor on the conjugate system and reducing water
solubility of coumarins. Therefore, COLD-6 was selected to image
LDs, and the tested parameter was determined as: lex = 405 nm,
lem = 500À560 nm. COLD-6 can track the movement of LDs with
excellent selectivity under a lipophagy model and atherosclerosis
model in vitro. In addition, a hand-held 365 nm laser source was
selected to visually imaging COLD-6, which can distinguish normal
tissue and adipose tissue even observation with naked eyes. These
results indicated that COLD-6 may serveas a potential probe for
the visualization of atherosclerosis plaque in clinic, and the
applicability still needs to be further explored.
Declaration of competing interest
The authors have declared no conflict of interest.
Acknowledgments
We are grateful to the National Natural Science Foundation of
China (No. 82001891), the Post-doctoral Innovative Talent Support
Program (No. BX20190389), and China Postdoctoral Science
Foundation (No. 2019M662009). We thank the Cellular and
Molecular Biology Center of China Pharmaceutical University for
assistance with confocal microscopy work and we are grateful to
Xiao-Nan Ma for her technical help.
Fig. 5. (A) The 3D-scan of LDs in macrophages, the Raw264.7 cells were pre-treated
with ox-LDL (2
mg/mL) 48 h and then treated with COLD-6 (1 mmol/L) 2 h, the 3D-
scan fluorescent images were obtained by confocal. (B) The abdominal adipose
tissue was harvested and then incubated with COLD-6 (10 mmol/L) or PBS 2 h at
37 ꢀC ex vivo. The picture was obtained under 365 nm laser. (C) The kidney and
affiliated adipose tissue was harvested and then incubated with COLD-6
(10 m
mol/L) or PBS 2 h at 37 ꢀC ex vivo. The picture was obtained under 365 nm laser.
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