Precise Labeling and Tracking of Lipid Droplets in Adipocytes Using a Luminescent ZnSalen Complex

Article abstract of DOI:10.1002/asia.201701010

Designing two-photon probes for precise labeling of lipid droplets (LDs) and monitoring LD dynamics in adipocytes is of great significance to understand LD homeostasis. We herein report that a luminescent metal complex LD-TPZn can specifically image LDs in adipose cells and tissue using one- or two-photon fluorescence microscopy. Importantly, LD-TPZn exhibited higher specificity to LDs than the commercial dyes Nile Red and Bodipy 493/503, probably due to different cellular uptake pathways, that is, clathrin-mediated endocytosis and non-selectively passive diffusion, respectively. More importantly, LD-TPZn can be applied as a two-photon LD probe to image adipose tissue, one of the most challengeable tissues for traditional one-photon fluorescence microscope imaging due to the strong light scattering. Most importantly, LD-TPZn can be used to monitor LD growth during adipogenesis of preadipocytes, which is highly desirable to unravel the relationship between LD homeostasis and metabolic diseases.

Full text of DOI:10.1002/asia.201701010

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Precise Labelling and Tracking Lipid Droplet in Adipocytes Using
a Luminescent ZnSalen Complex
Authors: Juan Tang, Yanfei Zhang, Hao-Yan Yin, Guoheng Xu, and
Jun-Long Zhang
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Asian J. 10.1002/asia.201701010
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Precise Labelling and Tracking Lipid Droplet in Adipocytes Using
a Luminescent ZnSalen Complex
Juan Tang,a+ Yanfei Zhang,b+ Hao-Yan Yin,a Guoheng Xub* and Jun-Long Zhanga*
Dedicated to Professor Chi-Ming Che on the occasion of his 60th birthday
from low specificity probably due to non-selectively passive
diffusion.[2b, 4] [10] Oil Red and Sudan Black only stain fixed cells.[7,
Abstract: Designing two-photon probes for precise labelling lipid
droplet (LD) and monitoring LD dynamics in adipocytes is of great
significance to understand LD homeostasis. We herein report that a
luminescent metal complex LD-TPZn can specifically image LDs in
adipose cells and tissue using one- or two-photon fluorescence
microscopy. Importantly, LD-TPZn exhibited higher specificity to LD
than commercial Nile Red and Bodipy 493/503, probably due to
different cellular uptake pathways, clathrin-mediated endocytosis
and non-selectively passive diffusion, respectively. More importantly,
LD-TPZn can be applied as a two-photon LD probe to image
adipose tissue, one of the most challengeable tissues for traditional
one-photon fluorescence microscope imaging due to the strong light
scattering. Most importantly, LD-TPZn can be used to monitor LD
growth during adipogenesis of preadipocytes, which is highly
desirable to unravel the relationship between LD homeostasis and
metabolic diseases.
11]
Moreover, recently reported LD probes such as LD-540[6],
LipidGreen,[9] monodansylpentane,[12] Seoul-Fluor,[13] FLUN-
550,[14] DPH[15] and AIE luminogens,[16] used short-wavelength
excitations (<500 nm), which have not been applied to image
adipose tissue for its strong light scattering with severe refractive
index mismatches.[17] Only recently, Rendina et al. reported a
new carborane-containing coumarin which could selectively
staine lipid droplets in live adipocytes.[18] To address these
issues, we herein report the design of a luminescent ZnSalen
complex specific to stain LD and monitor the dynamics, and
capable of two-photon excitation to circumvent the strong light
scattering.[19]
Luminescent metal complexes are emerging biological
probes for the intriguing photophysical properties and unique
chemical structures.[20] For LD imaging,
a Re tetrazolato
complex[21] and a alkynyl gold(I) cluster[22] have previously been
reported, yet suffering from poor photostability and potential
cytotoxicity arising from heavy metals. In this work, we chose
bioavailable Zn2+ ion as metal centre supported by luminescent
Salen ligand, which has been applied as fluorescent metal
probes in cell imaging.[23] A notable bonus to such metal probes
is the opportunity to modulate cellular uptake pathway and
subcellular localization by fine-tuning intermolecular interaction
between metal and ligand.[23b-d] To stain the hydrophobic interior
of LD, we used aliphatic chain modified N-substituent
Cytosolic lipid droplet (LD) is a well-known organelle for cellular
lipids storage, featured with a hydrophobic lipid core surrounded
by a phospholipid monolayer.[1] LD has long been considered to
be much less important than other organelles such as Golgi
apparatus, mitochondria, etc., until recent studies suggested the
close association of LD homeostasis with many diseases such
as Alzheimer’s disease, type 2 diabetes and cardiovascular
diseases.[2] Thus, to conveniently visualize and monitor the
dynamics of LDs in living cells in real time by fluorescence
microscopy attracts increasing attention.[3] Generally, fluorescent
LD probes were constructed by attachment of lipophilic
conjugates, which have a high affinity to the hydrophobic lipid
core, to organic fluorophores. Following this strategy, several LD
probes have been reported such as Nile Red,[4] Bodipy
493/503,[5] LD-540,[6] Oil Red O[7] and other Sudan stains,[8] and
LipidGreen,[9] contributing to unravel the underlying complexities
and regulatory mechanisms of intracellular LDs. However, some
important issues such as low LD specificity and strong light
scattering for the adipose tissue imaging, still remained to be
addressed during monitoring of LD dynamics. For instance, the
widely used LD trackers Nile Red and Bodipy 493/503 suffered
salicylaldehyde, and synthesized
a
luminescent ZnSalen
(Salen= 2,3-bis[(1-butyl-5-hydroxy-1,2,3,4-tetrahydroquinoline-6-
methyl ene)amino]but-2-enedinitrile) complex (LD-TPZn), as
shown in Scheme 1. This complex was fully characterized by 1H-
NMR, HR-ESI, and IR spectroscopes and spectrometers
(detailed synthesis and characterization were listed in supporting
information). Photophysical data were shown in Fig. 1a and
Table S1. In DMSO, LD-TPZn showed a sharp band centred at
[a]
Dr. J. Tang,+ H. Y. Yin, Prof. J. L. Zhang
Beijing National Laboratory for Molecular Sciences, State Key
Laboratory of Rare Earth Materials Chemistry and Applications,
College of Chemistry and Molecular Engineering, Peking University.
Beijing 100871, P.R. China, Fax: +86-10-62767034.
E-mail: zhangjunlong@pku.edu.cn.
[b]
[+]
Y. F. Zhang,+ Prof. G. H Xu*
Department of Physiology and Pathophysiology, School of Basic
Medical Sciences, Peking University.
Beijing 100191, P.R. China, Fax: +86-10-62767034.
E-mail: xug@bjmu.edu.cn.
These authors contributed equally to this work.
Scheme 1. Synthetic route for LD specific LD-TPZn. (i) 1-butyl bromide,
KHCO3, CH3CN, 95℃, 16 h, 60%; (ii) BrCH2CH2CH2Br, KHCO3, CH3CN, 95℃,
16 h, 30%; (iii) POCl3, DMF, 0℃ to r.t., 30 min; followed by hydrolysis in icy
water, 90 min, 43%; (iv) BBr3, dry CH2Cl2, -78℃ to r.t., 2 h, 90%; (v) 2,3-
diaminomaleonitrile, Zn(OAc)22H2O, ethanol, 90℃, 24 h, 70%.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/asia.201701010
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390 nm with a shoulder at 440 nm assigned to π-π* transition,
and a low-energy band centred at 599 nm. It displayed an
intense red fluorescence with emission (600-700nm) centred at
637 nm and a quantum yield of 0.44. LD-TPZn exhibited a two-
photon absorption cross section (δ) of ca. 110 GM at 880 nm,
using Rhodamine B as the reference (Fig. S1). To estimate the
lipophilicity of LD-TPZn, we carried out inductively coupled
plasma-atomic emission spectrometer (ICP-AES) to access the
logarithm of the n-octanol/water partition coefficient (Log P),
giving a value of 1.8±0.1. The calculated cLog P[24] value is 8.2,
resulted
a
gradual fluorescence increase (up to 5-fold
enhancement after addition of 20 L emulsion). Furthermore, 3D
reconstructed imaging by confocal laser scanning microscopy
(CLSM) (Fig. 1d-e) showed that red fluorescence of LD-TPZn
distributed homogeneously inside the emulsion, giving a red
solid structure. To better elucidate the ability of LD-TPZn to stain
hydrophobic component, we prepared a liposome system with
an aqueous interior surrounded by an amphiphilic phospholipid
bilayer and observed LD-TPZn only staining the phospholipid
bilayers. No fluorescence was found in the aqueous interior,
leading to a red hollow structure (Fig. 1f-g). Finally, we applied
LD-TPZn to stain freshly extracted lipid droplets from mature
adipose cells. 2D fluorescence imaging showed the uniform red-
color round lipid droplets (Fig. S2). Thus, these cell-free studies
clearly suggested that LD-TPZn could accumulate in the
hydrophobic interior of lipid droplets accompanied with a “turn-
on” fluorescence driven by its lipophilicity.
close to those of reported LD probes[13b]
.
To demonstrate the capability of LD-TPZn to bind LD, we
prepared an artificial oil•in•water emulsion system to mimic LD,
where the interior consists of hydrophobic lipid enwrapped in an
amphiphilic phospholipid monolayer. As shown in Fig. 1b-c, LD-
TPZn (10 M) has very weak emission in PBS (pH 7.4),
probably due to the aggregation of LD-TPZn. Titration of oil-in-
water emulsion (10 mgmL-1) to the above PBS solution (pH 7.4)
Figure 1. (a) Normalized UV-vis and fluorescence spectra of LD-TPZn in DMSO, λex = 450 nm. (b) Oil•in•water emulsion-enhanced fluorescence of LD-TPZn in
PBS (pH 7.4); (c) Titration of oil•in•water emulsion to LD-TPZn in PBS (pH 7.4) monitored by fluorescence spectra; (d, f) Schematic diagram of LD-TPZn staining
of (d) oil•in•water emulsion and (f) liposome; (e, g) 3D reconstructed imaging of LD-TPZn by CLSM in (e) oil•in•water emulsion (width 36.27 μm, height 36.27 μm,
depth 30.00 μm) and (g) liposome (width 33.41 μm, height 33.41 μm, depth 30.00 μm).
Figure 2. Co-localization imaging of LD-TPZn (a-e) with immunolabeled perilipin-1 in adipose cells or (f-j) with Bodipy 493/503 in HeLa cells. Cells were treated
with 2 μM of LD-TPZn for 2 h. Fluorescence images of (a) immunolabeled perilipin-1 or (f) Bodipy 493/503; (b and g) Fluorescence images of LD-TPZn; (c and h)
Merged images of (a-b) and (f-g), respectively; (e and j) Differential interference contrast (DIC) images; scale bar = 10 μm. (d and i) Enlarged images of the white
box in (c and h), respectively; scale bar = 1 μm; (k and l) Relative fluorescence intensity profile along the arrow in (d and i), respectively.
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Prior to cell imaging, the cytotoxicity and photostability of
LD-TPZn were evaluated in HeLa cells. The CCK-8 assay
showed low cytotoxicity (90% cell viability, 8 M LD-TPZn) for
24 hours (Fig. S3). Compared with commercial Nile Red, higher
photostability was observed for LD-TPZn, which shows ca. 50%
fluorescence decay in contrast to ca. 90% by Nile Red upon
continuous scanning of cells by a 543 nm laser (0.91 mW) for
900 seconds (Fig. S4).
trafficking pathway would be a desirable approach. Previously,
we reported the importance of intermolecular metal-ligand
interaction of ZnSalen on the aggregation and even cellular
uptake pathway of ZnSalen.[27] In this work, we performed
dynamic light scattering (DLS) to assess the hydrodynamic
diameters (ca. 52 nm) of LD-TPZn aggregates in aqueous
solution (Fig. S5). To understand the cellular uptake pathway of
LD-TPZn, we treated adipocytes at a low temperature to stall all
To identify the precise location of LD-TPZn, we conducted
co-localization analysis in early-differentiated adipocytes with
immunolabeled perilipin-1, which coats LDs exclusively in
adipocytes.[25] As shown in Fig. 2a-e, distinct red fluorescence
from LD-TPZn precisely located within discrete green
fluorescent ring-like structures immunostained with perilipin-1.
Plotting the intensity along with the arrow in Fig. 2d gave a
complete inverse correlation between red fluorescence of LD-
TPZn and green fluorescence of immunolabeled perilipin-1 (Fig.
2k). These results strongly suggested that LD-TPZn specifically
localize in the hydrophobic core of LDs. We then examined
subcellular localization of LD-TPZn in HeLa cells using
commercial Bodipy 493/503 as a control LD tracker. As shown in
Fig. 2f-j, LD-TPZn displayed the globular red fluorescence with a
size ranged from 300 to 600 nm, which was included in green
fluorescence region of Bodipy 493/503. However, Bodipy
energy-dependent active transport and applied sucrose[28]
,
genistein[29] or cytochalasin D[30] as endocytosis inhibitors to
selectively block clathrin-mediated endocytosis, caveolae-
dependent endocytosis or macropinocytosis, respectively. As
shown in Fig. 3, each adipocyte has multiple lipid droplets which
look like spots on the cell in the bright field images. CLSM
imaging showed that 4°C-treated cells displayed weak
intracellular fluorescence and the uptake level was as low as 1%
of that in the 37°C-treated cells (the positive control). Sucrose-
treated cells exhibited distinct weak fluorescence and the uptake
was lowered to 40% of the positive control. However, both
genistein and Cytochalasin D exerted no effect on the uptake of
LD-TPZn, giving comparable intracellular fluorescence (90%
and 100%, respectively) to the positive control. Since there is no
possibiity to form the sucrose-LD-TPZn adduct (Fig. S6), the
above results indicated that cellular uptake of LD-TPZn is highly
energy-dependent and via a clathrin-mediated endocytosis
pathway. It is of importance on LD specificity since it could
prevent possible contact of LD-TPZn with lipophilic structures of
other organelles such as mitochondria and ER.[10]
493/503 showed
a more diffused fluorescence over the
cytoplasm, not only limited to LDs (yellow spots) but also to
other reticular structures (green emission) of intracellular
membranes and organelles (Fig. 2h). As shown in Fig. 2l, LD-
TPZn displayed low background, whereas the control Bodipy
493/503 showed high-baseline intensity profile. Thus, the above
results demonstrated a higher specificity of LD-TPZn toward
LDs than Bodipy 493/503.
As obesity is a growing global health problem directly related
to the number and size of the adipocytes,[31] monitoring the
processes of adipocytes differentiation is of pathological and
Cellular uptake is important to the post-internalization fate
and intracellular location of the cargoes.[26] The classical
commercial LD probes (Nile Red and Bodipy 493/503) suffer
poor LD selectivity since they passively diffuse into cells, non-
selectively interact with intracellular hydrophobic components
and accumulate in LDs and other hydrophobic structures such
as intracellular membranes and membranous organelles.[10]
Thus, directing a probe to LDs through a particular endocytosis
Figure 3. Cellular uptake of LD-TPZn by adipocytes. (a-e) Merged images of
fluorescence images of LD-TPZn and differential interference contrast (DIC)
images. (a) at 37℃; (b) at 4℃; (c) 1 μgmL-1 cytochalasin D; (d) 100 M
genistein; (e) 450 mM sucrose. Cytochalasin D, genistein and sucrose were
preincubated with cells for 30 min; scale bar: 10 μm. (f) Mean fluorescence
intensity of internalized LD-TPZn by adipose cell. (n=30, *P < 0.001, NS:
nonsignificant). Data were obtained by confocal imaging and calculated by
ImageJ.
Figure 4. LD formation during preadipocyte differentiation: (a) day 0; (b) day 1;
(c) day 2; (d) day 3; (e) day 4. (1) Images of preadipocyte in full field; (2-4)
Enlarged images of white box in (1); (2) Fluorescence images of Bodipy
493/503; (3) Fluorescence images of LD-TPZn; (4) Merged images of (2) and
(3); scale bar: 10 μm.
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physiological significance. In this work, we investigated the
capability of LD-TPZn to monitor LD growth during adipogenesis
of rat preadipocytes and used Bodipy 493/503 as the positive
control. At the initiation day (day 0, Fig. 4a), LD-TPZn treated-
fibroblast-like cells display negligible intracellular fluorescence,
in addition to the occasional few red fluorescent LDs. From day
1 to day 3 (Fig. 4b-d), cells proliferated by cell division and
differentiated into spherical adipocytes. The LDs that
accumulated in each adipocyte grew from less than 1 μm to
more than 5 μm in diameter during this period. However, at day
4 (Fig. 4e), the large LDs fragmented into smaller size LDs with
diameter of 1-2 μm and filled the whole cells. As shown in Fig. 4,
LD-TPZn overlapped well with Bodipy 493/503 during the
process. These results highlight the potential application of LD-
TPZn in tracking LD biogenesis during adipogenesis of
preadipocytes.
image LDs within adipose tissue for the less sensitivity to light
scattering, making high-resolution deep imaging possible.[19] Due
to moderate two-photon absorptions cross sections across 790-
880 nm spectral window (up to 110 GM) (Fig. S1), LD-TPZn was
firstly investigated at cell level in two-photon confocal imaging,
using a 790 nm femtosecond laser and a LP 650 filter. As shown
in Fig. S7, in HeLa cells, two-photon excited LD-TPZn displayed
apparent punctate red fluorescence. Then, we performed Z-
stack two-photon confocal imaging of rat subcutaneous
addipose tissues using LD-TPZn as a bioprobe. As shown in Fig.
6a, LDs in a 130 m-thick adipose tissue slice emerged as many
red globular structures. To demonstrate the globular structures
are LDs rather than mature adipocytes, one-photon 3D
reconstructed imaging was carried out using Hoechst 33342 to
stain the nuclei. As shown in Fig. 6b, every mature adipocyte
contained a red single globular LD with ca. 50 - 100 m in
diameter clinging to a relative little punctate nucleus with ca. 10
m in diameter. Thus, two-photon imaging of LDs at adipose cell
and tissue level is achievable via LD-TPZn.
Adipose tissue is one of the most challenging tissues for
traditional fluorescence imaging due to its highly scattering
feature with severe refractive index mismatches.[17] To address
this issue, two-photon fluorescence microscopy is alternative to
Figure 6. (a) Two-photon fluorescence imaging of LDs in adipose tissue slide incubated with LD-TPZn (2 M) for 6 h; a 790 nm femtosecond laser and a LP 650
filter were used for detection of LD-TPZn; scale bar: 100 m. Labels from 0-115 m indicate scanning depths of the tissue slices. (b) One-photon 3D
reconstructed imaging of adipose tissue stained with LD-TPZn (red fluorescence) and Hoechst 33342 (blue fluorescence), width 638.05 μm, height 638.05 μm,
depth 130.50 μm.
In conclusion, we have synthesized a new luminescent
ZnSalen complex (LD-TPZn) and demonstrated the capability to
stain LD precisely in living cells. Importantly, compared with the
commercial tracker (Bodipy 493/503), LD-TPZn exhibited higher
specificity via clathrin-mediated endocytosis pathway, which is
related to the intendancy to self-aggregation in aqueous media,
arising from the intermolecular metal-ligand interaction. More
importantly, large two-photon absorption cross section (up to
110 GM) at 790-880 nm spectral window renders LD-TPZn a
two-photon bioprobe for imaging LDs at even tissue level. Most
importantly, LD-TPZn could be used for dynamic adipogenesis
of preadipocytes, which demonstrated the potential application
in further understanding the relationship between LD
homeostasis and some metabolic diseases at cellular or even
tissue levels.
and the National Scientific Foundation of China (Grants
21571007, 21271013, 21321001, 81570791).
Keywords: Luminescent metal complex • lipid droplets •
fluorescence microscopy • cell imaging • chemical biology
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10.1002/asia.201701010
Chemistry - An Asian Journal
COMMUNICATION
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COMMUNICATION
A two-photon
Author(s), Corresponding
Author(s)*
luminescent ZnSalen
complex LD-TPZn
precisely images LDs in
adipose cells and tissues
due to its clathrin-
mediated endocytosis
pathway.
Page No. – Page No.
Title
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