New fluoranthene FLUN-550 as a fluorescent probe for selective staining and quantification of intracellular lipid droplets

Article abstract of DOI:10.1021/ol403470d

A new class of live cell permeant, nontoxic fluoranthene-based fluorescent probe (FLUN-550) having a high Stokes shift in aqueous medium has been discovered. It showed selective staining of lipid droplets (LDs, dynamic cytoplasmic organelles) at a low con

Full text of DOI:10.1021/ol403470d

Letter
pubs.acs.org/OrgLett
New Fluoranthene FLUN-550 as a Fluorescent Probe for Selective
Staining and Quantification of Intracellular Lipid Droplets
Atul Goel,*,†,⊥ Ashutosh Sharma,† Manoj Kathuria,‡ Arindam Bhattacharjee,‡ Ashwni Verma,§
Prabhat R. Mishra,§ Aamir Nazir,∥ and Kalyan Mitra*,‡
‡
§
†Fluorescent Lab, Division of Medicinal and Process Chemistry, Electron Microscopy Unit, Division of Pharmaceutics, and
∥Division of Toxicology, CSIR-Central Drug Research Institute (CDRI), Lucknow 226031, India
⊥Academy of Scientific and Innovative Research, New Delhi 110001, India
S
* Supporting Information
ABSTRACT: A new class of live cell permeant, nontoxic fluoranthene-
based fluorescent probe (FLUN-550) having a high Stokes shift in
aqueous medium has been discovered. It showed selective staining of
lipid droplets (LDs, dynamic cytoplasmic organelles) at a low concentra-
tion without background noise in in vitro live cell imaging of 3T3-L1
preadipocytes, J774 macrophages, MCF7 breast cancer cells, and single-
celled, parasitic protozoa Leishmania donovani promastigotes and in vivo
nonparasitic soil nematode C. elegans.
luorescence imaging tools have played an indispensable role
in unequivocally unraveling the mysteries of many problems
in lipid cell biology. Therefore, the development of new
fluorescent probes for cell-based screening platforms to visualize,
quantify, and/or study the molecular dynamics of lipid droplets
(LDs) is highly rewarding for technological advancements in the
area of molecular and cell biology.1
The most widely used fluorescent lipid dyes, which stain
intracellular lipid components such as LDs, are Nile Red,2
BODIPY3 (Figure 1), and recent dyes like LD-540,4 Lipid-
Green,5 MDH,6 and Seoul-Fluor.7 Undoubtedly, these LD
markers have significantly contributed to the elucidation of the
molecular mechanisms associated with lipid biology; however,
the commonly used probes (Nile Red, BODIPY) have small
Stokes shifts and thus induce nonradiative energy transfers
causing fluorescence quenching of the excited chromophore
and interference from scattered light ultimately leading to weak
fluorescence and background artifacts.8 Researchers have
recently addressed these issues and developed BODIPY
derivatives with an as yet largest Stokes shift of about 185 nm.9
The high Stokes shifts fluorophores have key advantages in
multimodal cell imaging. Herein we report a new10 cell-
permeable solvatochromic fluoranthene-based FLUN-550
fluorescent probe with high Stokes shift (220 nm) in water and
demonstrate its potential application in selective staining and
quantification of intracellular LDs in in vitro live 3T3-L1
preadipocytes, Leishmania donovani promastigotes, and in in vivo
soil nematode C. elegans.
Our research group is involved in developing new small
molecule organic fluorescent dyes for their potential applications
in applied sciences (as electroluminescent materials).11 We
have shown the importance of positioning the electron donor−
acceptor and chromophoric moieties onto the molecular
frameworks for modulating the photophysical properties of
fluorenes,12 pyrenylarenes,13 benzo[f ]quinolines,14 and fluoran-
thenes15 leading to efficient multicolor light emitting materials
for organic electronics.
F
Owing to the interesting photophysical and optical properties
of highly fluorescent organic dyes, particularly fluoranthene
(FLUN) derivatives, we tuned them by incorporating donor and
acceptor functionalities. Several new derivatives of FLUN with
hydroxy and methoxy groups (Figure 1) were synthesized in
high yields and purities as fluorescent probes for cell-imaging
applications (see Scheme S1 in the Supporting Information).
The photophysical properties of all of the synthesized
fluoranthenes (FLUNs) 6a−f and 7 in water containing 0.2%
Figure 1. Structures of known lipid droplets markers and synthesized
new fluoranthenes (FLUNs, 6a−f, 7); A-acceptor, D-donor, R-alkyl,
aromatic group.
Received: November 30, 2013
Published: January 10, 2014
© 2014 American Chemical Society
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DMSO showed absorption maxima in the range of 327−340 nm
with a low intensity band (n→π*) in the range of 428−458 nm
and photoluminescence (PL) maxima in the range of 545−
555 nm (see Table S1 and Figures S1−S7 in the Supporting
Information). These FLUNs showed high Stokes shifts
in aqueous medium as shown in Figure 2 for compound 7
studies in live J774 macrophages cells and MCF7 breast cancer
cells using confocal laser scanning microscopy. After 2 h incuba-
tion with dye FLUN-550 (at 50 nM), we observed good
intracellular fluorescence with a high signal-to-noise ratio, which
was attributed to the FLUN-550 (Figure 4). These results
indicated that the dye is capable of entering into the cytoplasm
but not in the nucleus.
Figure 2. Absorption, excitation, and emission spectra of FLUN-550 dye
in 0.2% DMSO in water (∼10−5 M).
Figure 4. (A) Confocal microscopy images of live J774 cells with
intensity line plot; (B) DIC image; (C) confocal microscopy images of
live MCF7 cells with intensity line plot; (D) DIC image.
(FLUN-550, λmax, PL 550 nm; Stokes shift 220 nm) with a
quantum yield of ∼4% in water and 35% in DMSO. To study the
effect of the polarity of the solvent on the excited state of the
compounds, we examined the absorption and PL spectra of
FLUNs using solvents of varying polarity. Interestingly large
solvatochromic shifts were observed in the PL spectra for FLUN
derivatives (Figure S8 in the Supporting Information). The
positive solvatochromism indicated that the donor−acceptor
fluoranthene FLUN-550 exhibited a strongly stabilized excited
state compared to the ground state by the surrounding solvent
molecules (see Table S2 in the Supporting Information).
In order to examine the cellular permeability/uptake, fluore-
scent FLUN dyes 6a−f and 7 were incubated in populations
of J774 mouse macrophages and MCF7 cells under growth
conditions (see the Supporting Information). Flow cytometry
(FACS) analysis of J774 and MCF7 cells revealed that the
hydroxy group at the benzene periphery at position 10 (FLUN-
550) displayed nice cellular uptake as compared to other
methoxy (6d), methylenedioxyphenyl (6e), or other substitu-
ents. Cytometric results for dye-untreated (with blank medium)
J774 and MCF7 cells showed 121 and 255 au intensity at 405 nm
excitation wavelength, respectively, whereas FLUN-550-treated
J774 cells and MCF7cells showed intensities shifting from 121 au
(control) to 6804 au and from 255 au to 8818 au, respectively.
Higher emission intensities confirmed the high cell permeability
of FLUN-550 (Figure 3).
Since mammalian J774 macrophages cells store excess
unesterified cholesterol in cytoplasmic lipid droplets (LDs),16
we envisioned the fluorescent staining in cytoplasm by the
fluorescent dye FLUN-550 may be due to the staining of large
number of small LDs present in the cytoplasm as described by
Neef and Schultz.1a In order to examine the selective staining of
FLUN-550 for LDs, we further investigated the staining of the
dye FLUN-550 in 3T3-L1 preadipocyte cells, which are known
to house LDs in large numbers. Interestingly, confocal results
revealed that FLUN-550 selectively stained the lipid droplets
(LDs) of preadipocytes at the optimized concentration of
100 nM (Figure 5). Quantification of lipid droplets was achieved
using image J software, which accounted for 192 lipid droplets.
On the basis of the cellular uptake studies on fluoranthene
derivatives (6a−f and 7), FLUN-550 was chosen for selectivity
Figure 5. 3T3-L1 preadipocytes stained with 100 nM of FLUN-550 for
30 min at 37 °C/5% CO2 on confocal dishes: (A) confocal fluorescence
image; (B) phase image; (C) quantification of lipid droplets using image
J software. Total LDs quantified is 192.
To confirm these observations, dual-staining experiments
were performed with Nile Red (a known lipid droplets marker)
and Hoechst-33342 (nucleus staining dye) in 3T3-L1
preadipocytes, which revealed specific localization in distinct
cytoplasmic lipid droplets present in these cells (Figure S9 and
movie-1 in the Supporting Information) and clearly showed
colocalization of the Nile Red and FLUN-550 within LDs (see
Figure S10 in the Supporting Information). Cell viability
experiments revealed that FLUN-550 showed no cytotoxicity
in 3T3-L1 adipocytes up to a concentration of 1 μM, where
Figure 3. Flow cytometry analysis (shift in emission intensity) of
FLUN-550 (7) at 50 nM after 2 h incubation in (A) J774 cells and (B)
MCF7 cells. P3 corresponds to control cells, whereas P4 corresponds to
cells treated with FLUN-550.
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93.5% cells were viable (see Figure S11 in the Supporting
Information).
throughout the worm body with specific lipid droplets
fluorescing in the head region around the pharynx, alongside
the gut region, and leading all the way to the tail region of the
worms. The staining was more intense toward the midbody,
which usually possesses high lipid content in gravid well-fed
adults (see movie-2 in the Supporting Information). The
hallmark of the staining with FLUN-550 dye was its specificity
to the body lipids within the worms. There was no nonspecific
fluorescence within the nematode gut as is seen in case of FLUN-
550 (black arrowheads, Figure 7D). The colocalization studies,
wherein FLUN-550 and Nile Red were supplemented together,
showed complete overlap of the lipid droplets stained by
either dye, with the FLUN-550 dye exhibiting rather sturdier
fluorescence (spots for comparison marked with white arrow-
heads in Figure 7A,B). Comparison of emission maxima of
commercially available dyes BODIPY (514 nm) and Nile Red
(632 nm) with FLUN-550 (550 nm) showed a clearly distinct
region with the advantage of having remarkably high Stokes shift,
which is an important parameter in cell imaging applications.
In conclusion, we have discovered a new class of live cell
permeant, fluorescent probe FLUN-550 for strong and selective
staining of intracellular lipid droplets in in vitro live 3T3-L1
preadipocytes, J774 macrophages, MCF7 breast cancer cells,
parasitic protozoa L. donovani promastigotes, and in vivo
nonparasitic soil nematode C. elegans. The live cell imaging
studies clearly demonstrated that FLUN-550 is a specific and
nontoxic lipid molecular probe for quantification of lipid droplets
with high brightness at a low concentration without background
noise. FLUN-550 exhibited clearly distinct PL from the
commercially available BODIPY and Nile Red dyes, and it may
be used as a fluorescent probe alone or in combination with other
fluorophores for multicolor imaging applications.
Considering the importance of lipid metabolism events in live
Leishmania donovani parasites, we further explored the potential
of FLUN-550 for selective staining of lipids in live L. donovani
parasites, the causative protozoan for the disease visceral
leishmaniasis (KALA-AZAR).17,18 Microscopic observations of
L. donovani stained with FLUN-550 revealed localized
fluorescence within specific spherical compartments of the cells
with 99% cell viability (see Figure 6 and Figure S12 in the
Supporting Information).
Figure 6. Confocal image of L. donovani promastigotes stained with
100 nM FLUN-550: (A) fluorescence image; (B) pseudo-3D
fluorescence intensity plot of lipid droplets; (C) phase image; (D)
merge of image A and C (bar indicates 5 μm); (E) quantification of lipid
droplets (LDs encircled) by fluorescence staining with FLUN-550; (F)
phase image.
Furthermore, dual staining of L. donovani promastigotes with
Nile Red and FLUN-550 confirmed the specific localization in
distinct LDs with no labeling in nucleus and other organelles
present in these cells (see Figure S13 in the Supporting
Information). Due to the strong and specific labeling of LDs
with FLUN-550, the quantity and size of the stained lipid
droplets served as a biotool for investigating lipid metabolism
(Figure 6E,F).
We further endeavored to study in vivo lipid specificity of
the fluorescent dye in nonparasitic soil nematode C. elegans,
which is of relevance in studying lipid biology. We observed
that treatment of these nematodes with different concentrations
of FLUN-550 dye led to a concentration-dependent staining
pattern with 1 μM concentration exhibiting the most robust
fluorescence staining pattern (Figure 7). Staining was observed
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis, 1H and 13C NMR, and UV-FL spectra of compounds
6a−f and 7. Cytotoxicity data, dual staining and colocalization in
3T3-L1 preadipocytes and L. donovani, movie-1 (LDs staining in
3T3-L1 preadipocytes), and movie-2 (LDs staining in C. elegans).
This material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: atul_goel@cdri.res.in.
*E-mail: k_mitra@cdri.res.in.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work is supported by the CSIR, New Delhi, under Network
project BSC0102 and BSC0114. We thank Drs. Y. K. Manju and
A. Gaikwad of CDRI for initially providing 3T3 preadipocytes
and Mr. A. L. Vishwakarma of CDRI for FACS instrument
facility. Nematode strains used in this work were provided by the
C. elegans Genetics Center (CGC), University of Minnesota,
which is funded by the NIH National Center for Research
Resources (NCRR). We are thankful to Lalit Kumar,
Shamsuzzama, Pooja Jadiya, and Dr. Kavita Singh of CDRI for
C. elegans experiments. This is CSIR-CDRI communication
no. 8591.
Figure 7. (A) Fluorescence microscopic image of C. elegans stained with
1 μM FLUN-550. (B) Fluorescence microscopic image of C. elegans
stained with 1 μM Nile Red. (C) Confocal fluorescence image of
C. elegans stained with 1 μM FLUN-550. (D) Merged DIC image.
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DEDICATION
■
Dedicated to Prof. T. K. Chakraborty on the occasion of his
voluntary retirement from CSIR-CDRI, Lucknow.
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