Imaging and Quantitative Detection of Lipid Droplets by Yellow Fluorescent Probes in Liver Sections of Plasmodium Infected Mice and Third Stage Human Cervical Cancer Tissues

Article abstract of DOI:10.1021/acs.bioconjchem.8b00552

The diagnosis and prognosis of the disease associated with lipid irregularity are areas of extreme significance. In this direction, fluoranthene based yellow fluorescent probes (FLUN-550, FLUN-552, FLUN-547) were designed and synthesized by conjugating th

Full text of DOI:10.1021/acs.bioconjchem.8b00552

Subscriber access provided by University of Sunderland
Article
Imaging and Quantitative Detection of Lipid Droplets by
Yellow Fluorescent Probes in Liver Sections of Plasmodium
Infected Mice and Third Stage Human Cervical Cancer Tissues
Ashutosh Sharma, Ajay Kumar Jha, shachi mishra, Ankita Jain, Bhavana
S Chauhan, Manoj Kathuria, Kundan Singh Rawat, Neeraj Mohan Gupta,
Renu Tripathi, Kalyan Mitra, Monika Sachdev, Madan L Bhatt, and Atul Goel
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00552 • Publication Date (Web): 24 Sep 2018
Downloaded from http://pubs.acs.org on September 25, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 22
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
Imaging and Quantitative Detection of Lipid Droplets by Yellow
Fluorescent Probes in Liver Sections of Plasmodium Infected
Mice and Third Stage Human Cervical Cancer Tissues
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
a
a
b
c
Ashutosh Sharma, Ajay K. Jha, Shachi Mishra, Ankita Jain, Bhavana S. Chauhan, Manoj
d
a,f
a
c
d
b
Kathuria, Kundan S. Rawat, Neeraj M. Gupta, Renu Tripathi, Kalyan Mitra, Monika Sachdev,
e
,a,f
Madan L.B. Bhatt, Atul Goel*
a
Fluorescent Chemistry Lab, Medicinal and Process Chemistry Division, CSIRꢀCentral Drug
Research Institute, Lucknow 226031, India
b
Endocrinology Division, CSIRꢀCentral Drug Research Institute, Lucknow 226031 (India).
c
Parasitology Division, CSIRꢀCentral Drug Research Institute, Lucknow 226031 (India).
Electron Microscopy Unit, CSIRꢀCentral Drug Research Institute, Lucknow 226031 (India).
Department of Radiotherapy, King George's Medical University , Lucknow, 226003 (India).
d
e
f
Academy of Scientific and Innovative Research, Ghaziabad 201 002 (India)
Eꢀmail: atul_goel@cdri.res.in
KEYWORDS: Fluorescent probes • Lipid droplets • Cervical cancer • Quantitative detection
Tissue Imaging
•
ABSTRACT: The diagnosis and prognosis of the disease associated with lipid irregularity is
an area of extreme significance. In this direction, fluoranthene based yellow fluorescent
probes (FLUN-550, FLUN-552, FLUN-547) were designed and synthesized by conjugating
ethanolamine head group of the phospholipid phosphatidylꢀethanolamine present in
biological membranes. Owing to unique photophysical properties and aqueous compatibility,
these probes were successfully employed for staining lipid droplets (LDs) in preꢀadipocytes
and Leishmania donovani promastigotes. Furthermore, using the fluorescent probes FLUN-
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 2 of 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
550 and FLUN-552 we successfully imaged and quantitatively detected the excess
accumulation of lipids in liver section of Plasmodium yoelii MDR infected mouse (3ꢀ to 4ꢀ
fold) and the tissue sections of third stage human cervical cancer patients (1.5ꢀ to 2ꢀfold)
compared to normal tissues. To the best of our knowledge, this is the first report of yellow
fluorescent probes for imaging and quantitative detection of LDs in human cervical cancer
tissues. These new yellow fluorescent lipid probes (FLUN-550 and FLUN-552) showed
great potential for early diagnosis of cervical cancer patients.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ
INTRODUCTION
Lipid droplets (LDs) are dynamic organelles in the cells which are considered as a place for
1,2
energy storage and are significantly involved in the lipid metabolic events.
The
abnormalities in the function of LDs have significant effects on the pathogenesis of diabetes,
obesity and cardiovascular diseases. Liver is the main organ of the human body that controls
3,4
the lipid metabolism. Literature reports revealed that LDs play a key role in the production
2,5
and assembly of hepatitis C virus, a causative agent of chronic liver diseases. The methods
generally used to access the LDs level are ultrasound and computerized tomography scanning
6
but these analyses are not precise and can detect only ~25% of the fat content in the liver.
Transhepatic biopsy is the only method to diagnose the early stage liver disease with high
sensitivity. Hence, the direct monitoring and localizing the accumulation of abnormal level of
LDs in the liver tissue is a thrust area of research. Further cancerous tissues have been
recognized to exhibit specific alterations in their metabolic activity which is thought to
facilitate the rapid proliferation of transformed cells. Earlier reports suggest increased rate of
lipid biosynthesis in cancerous tissues that are comparable to liver tissues, which have a high
7,8
rate of fatty acid biosynthesis. Despite of the growing evidence demonstrating deregulated
lipid biosynthesis as features of cancer, the mechanism of these metabolic alterations in the
development of the disease is not fully understood. Therefore, diagnostic tools for detection,
ACS Paragon Plus Environment

Page 3 of 22
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
imaging and quantification of lipid droplets in infected/cancerous and normal tissues are
highly desirable for understanding and unravelling the mysteries associated with lipid
9
structure, biosynthesis and metabolism.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Designing strategy of newly synthesized Fluoranthene based LDs staining dyes.
The Oil Red O (ORO) (nonꢀfluorescent dye), Nile Red and BODIPY dyes are commonly
1
0ꢀ16
used commercial dyes to measure LDs in cells and tissues.
However, these dyes have
some limitations, like for ORO staining fixation is required thus preventing its use in live
cells; solubility issues and stability problems on prolonged usage further limit its scope. LDs
probe Nile Red also stains other hydrophobic structures in the cell resulting in low LD
selectivity and has small Stokes shifts and thus induce nonradiative energy transfers and
show interference from scattered light ultimately leading to unfavorable signalꢀtoꢀnoise ratio
1
7ꢀ19
20ꢀ36
and background artifacts.
In recent past, several new fluorescent probes
such as
monodansylpentane, AFN, NPBDP, LipidTOX red, SF44, FAS, FLUNꢀ550, TPAꢀBI and
LD540 for staining LDs in live and fixed cells have been developed, however they have not
been explored for quantitative detection of LDs in infected/cancerous specimens.
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 4 of 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
LDs are primarily composed of a cluster of lipid esters (triacylglycerol, steryl esters) covered
9
with a phospholipid monolayer. The phospholipids that are predominantly found in the
plasma
membrane
of
mammalian
cells
are
mainly
Phosphatidylcholine,
Phosphatidylethanolamine, Phosphatidyl-serine, and Sphingomyelin (Figure 1). In this
manuscript, we designed and synthesized fluoranthene based fluorescent dyes by anchoring
ethanolamine head group of the phospholipid phosphatidylꢀethanolamine for specific staining
of LDs in preꢀadipocyte, L. donovani, plasmodium infected liver sections and the tissues of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
37
third stage of human cervical cancer patients. Recently we have discovered a new
fluorescent probe FLUN-550 with good biocomapatibility and cellular uptake for selective
24
staining and quantification of LDs in preꢀadipocyte, L. donovani and C. elegans. Taking
into account the ampiphillic nature and composition of LDs, new derivatives of fluoranthene
(FLUN) with monoꢀ or diꢀethanolamine moieties were designed and synthesized. The
adopted synthetic strategy is represented in the Scheme 1.
Scheme 1. Synthesis of FLUN derivatives with methylꢀethanolamine and diethanolamine as a donor
group.
ACS Paragon Plus Environment

Page 5 of 22
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
ꢀ
RESULTS AND DISCUSSION
Molecular design and synthesis
The aryl ketones 2a,b on reaction with ketene dithioacetal 1 in presence of base led to the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
formation of 6ꢀarylꢀ4ꢀ(methylthio)ꢀ2ꢀoxoꢀ2Hꢀpyranꢀ3ꢀcarbonitriles (3a,b) which were
substituted with Nꢀmethylethanolamine (4a) and bisꢀ2ꢀhydroxyethylamine (4b) to give 4ꢀ((2ꢀ
hydroxyethyl)(methyl)amino)ꢀ6ꢀ(4ꢀmethoxyphenyl)ꢀ2ꢀoxoꢀ2Hꢀpyranꢀ3ꢀcarbonitrile (5) and
38
4ꢀ(bis(2ꢀhydroxyethyl)amino)ꢀ2ꢀoxoꢀ6ꢀphenylꢀ2Hꢀpyranꢀ3ꢀcarbonitrile (8) respectively.
The compounds 5 and 8 on reaction with acenaphthylenꢀ1(2H)ꢀone (6) in the presence of a
base resulted in the formation of 8ꢀ((2ꢀhydroxyethyl)(methyl)amino)ꢀ10ꢀ(4ꢀmethoxyphenyl)ꢀ
fluorantheneꢀ7ꢀcarbonitrile (7) and 8ꢀ(bis(2ꢀhydroxyethyl)ꢀamino)ꢀ10ꢀphenylꢀfluorantheneꢀ7ꢀ
carbonitrile (9) respectively in good yields.
Photophysical studies of synthesized fluorescent probes
The photophysical properties of the synthesized FLUN derivatives 7 and 9 in PBS buffer (pH
7.2) showed absorption maxima at 342 and 335 nm (π→π*) along with low intensity charge
transfer band at 445 and 440 nm (n→π*) respectively. Photoluminescence (PL) spectra of 7
and 9 showed maxima at 552 and 547 nm respectively with good quantum yield in DMSO
and PBS (Figure 2, Table S1). These FLUN derivative 7 (FLUN-552) and 9 (FLUN-547)
exhibited high Stokes shifts of 210 nm and 212 nm respectively in buffer solution. The
solvatochromic study in solvents of varying polarity showed the intramolecular charge
transfer characteristics of these fluorescent probes (Figure S1). The fluorescent probes
FLUN-552 and FLUN-547 were found to be highly emissive in nonꢀpolar solvents like
cyclohexane and toluene suggesting their potential application in hydrophobic cellular
environment like lipids (Figure S1 and S2). As in biochemical systems, pH plays an
important role in conducting different cellular events or processes. The fluorescence
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 6 of 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
properties of the FLUN derivatives at different pH values (~1–12) were investigated. These
probes were found to be stable at pH range ~6–8, but slight variation in the fluorescence
intensity was observed beyond these ranges (Figure S3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(a)
(b)
FLUN-552
FLUN-547
Figure 2. Excitation and fluorescence spectra of FLUN-552 and FLUN-547 in PBS buffer (pH 7.2,
ꢀ5
~10 M). Image of compounds in normal and UV light
DFT study of FLUNs
To gain insight into the electronic properties, timeꢀdependent density functional theory (TDꢀ
DFT) calculations for FLUNꢀ552 and FLUNꢀ547 were performed using B3LYP/6ꢀ311++G
(d,p) method employing Gaussian 09 package. Molecular orbitals in the ground and excited
state for FLUN- 552 and FLUN-547 are represented in Supporting information (Figure S4
and Figure S5). TDꢀDFT calculations of FLUN-552 indicated strong transition at 447 nm
with an oscillator strength of f = 0.0513 corresponding to HOMO → LUMO, while FLUN-
547 showed strong transition at 438 nm with an oscillator strength of f = 0.0464
corresponding to HOMO→ LUMO. The transition band at higher wavelength may be
attributed to an intramolecular charge transfer (ICT) which was observed from the charge
transfer from HOMO (dispersed on the hydroxyl amine donor and central aromatic ring) to
LUMO (localised on the core fluoranthene scaffold). The change in planarity of FLUN
ACS Paragon Plus Environment

Page 7 of 22
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
derivatives due to the aryl and alkylamino substituents shifts the electron density towards the
fluoranthene ring (LUMO). The band gap, transition energies, oscillator strengths, and
assignments for the most relevant excited states of FLUN-552 and FLUN-547 are presented
in Table S2.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
LDs staining in 3T3-L1 pre-adipocytes and L. donovani
Considering the lipid droplets staining property of FLUNꢀ550, we investigated the staining
pattern of newly synthesized FLUN-552 and FLUN-547 in 3T3ꢀL1 preꢀadipocytes.
Interestingly, confocal fluorescence microscopy imaging studies revealed that FLUN-552
and FLUN-547 selectively stained the lipid droplets (LDs) of preꢀadipocytes at 100 nM
concentration (Figure S6). To confirm LDs staining, dual staining experiments were
performed with Nile Red in 3T3ꢀL1 preꢀadipocytes, which revealed specific localization in
distinct cytoplasmic lipid droplets present in these cells (Figure 3) and clearly showed coꢀ
localization of the Nile Red with FLUN-552 (Figure 3, AꢀD) and FLUN-547 (Figure 3, EꢀH)
within LDs. These FLUN derivatives FLUN-552 and FLUN-547 at 100 nM concentration
showed bright fluorescence staining without background noise. This shows the advantage of
FLUN dyes with a high Stokes shift that can be used at nanomolar concentrations without
fluorescence bleeding. Cell viability experiments revealed that FLUNꢀ550, FLUNꢀ552 and
FLUNꢀ547 dyes have no significant cytotoxicity in preadipocytes 3T3ꢀL1 up to the
concentration of 2 ꢁM for 48 hours and 72 hours (Figure S7). Approximately 93ꢀ94% 3T3ꢀ
L1 cells were observed to be viable even at 2 ꢁM of FLUN derivatives for up to 72 hours (see
Figure S7 in supporting information).
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 8 of 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
FLUN-552
Nile Red
DIC
Merged
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
FLUN-547
Nile Red
DIC
Merged
Figure 3. Confocal fluorescence image of 3T3ꢀL1 Preꢀadipocytes stained with 100 nM of FLUN-552
o
and FLUN-547 with Nile Red (300 nM) for 30 min. at 37 C. For FLUN-552 and FLUN-547, λex
=
4
05 nm, λem =505 nmꢀ570 nm; Bar indicates 5 µm.
After selective staining of cytoplasmic LDs by FLUNs in adipocytes, we further explored
their potential for imaging LDs in live Leishmania donovani parasites, the causative
39
protozoan for the disease Visceral Leishmaniasis (KALAꢀAZAR). Studies have shown that
lysophospholipid analogues (LPAs) like Edelfosine, Ilmofosine and Miltefosine (FDA
approved antileishmanial drugs) interact with various subꢀcellular structures and enzymes
especially those associated with cellular membranes thus interfering with lipid metabolism of
40,41
Leishmania donovani promastigotes.
In this direction L. donovani promastigotes were
incubated with 100 nM of FLUN-552 and FLUN-547 for 2h at physiological conditions.
Confocal microscopic observations of L. donovani stained with FLUN-552 and FLUN-547
revealed localized fluorescence within specific spherical compartments of the cells (Figure
S8). Similar localization in L. donovani promastigotes was observed with the fluorescent LD
stain, Nile Red. Furthermore, dual staining of L. donovani promastigotes with Nile Red and
FLUN derivatives confirmed the specific localization in distinct LDs with no labelling in
ACS Paragon Plus Environment

Page 9 of 22
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
nucleus and other organelles present in these cells. Thus, it is evident that FLUN-552 and
FLUN-547 fluoresces selectively in the LDs region of the parasite with high brightness
without background noise (Figure 4). The toxicity assessment data showed that FLUN
derivatives are nonꢀtoxic to L. donovani up to 1ꢀM concentration and there was no
morphological deformity in the promastigotes during the experimental conditions.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
FLUN-552
Zoomed
Nile Red
Merged
FLUN-547
Zoomed
Nile Red
Merged
Figure 4. Confocal fluorescence image of L. donovani promastigotes stained with 100 nM of FLUN-
552 and FLUN-547 with Nile Red. Bar indicates 5 µm.
LDs staining in healthy and infected liver tissue sections
The understanding of lipid accumulation in the pathogenesis of malaria is of utmost
importance. The studies with rodent malaria parasite Plasmodium yoelii nigeriensis MDR
have shown that at high parasitaemia, this parasite induces lipid infiltration in liver of showed
42,43
scanty deposition of lipid droplets
. After deposition, lipids undergo peroxidation infected
mice. The lipid deposition start appearing early in infection but become most pronounced
with the increasing level of parasitaemia (30ꢀ45%) while liver of healthy mice and ultimately
lead to hepatic tissue damage.
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 10 of 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[
A] Healthy Tissue
Infected Tissue
co-staining
Merged
[B]
FLUN-550
FLUN-550
NILE RED
MERGED
FLUN-552
FLUN-552
NILE RED
MERGED
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
FLUN-547
FLUN-547
NILE RED
MERGED
FLUN-550 FLUN-552 FLUN-547 NILE RED
Figure 5. Confocal fluorescence image of healthy and infected liver sections [A] Dual staining with
FLUNꢀ550, FLUN-552 and FLUN-547 (100 nM) and Nile Red (300 nM) at 63X in P. yoelii MDR
infected mouse. [B] Fluorescence signal quantification by LAS (Leica application suit). ***P 0.0004
FLUN-550), <0.0001 (FLUN-552), 0.0009 (Nile Red) & **P =0.0016 (FLUN-547), statistical
analysis by student tꢀtest; Bar indicates 50µm.
(
To examine the staining pattern of lipids, FLUN derivatives (100 nM) were incubated in P.
yoelii infected liver tissue sections for 5 minutes. Tissue sections were washed twice with
PBS buffer and mounted with 75% glycerol for confocal imaging. A large number of clusters
of lipid droplets were observed (Figure 5A, white arrows). Further the coꢀstaining
experiments with commercial LDs dye Nile Red showed similar staining pattern suggesting
the selectivity of FLUNs for LDs in tissue sections. Based on the data obtained from the
imaging of tissue sections on staining with different FLUN derivatives, the quantitative
10
detection was done following standard protocol . The results showed that FLUN-550 and
FLUN-552 significantly increased the fluorescence intensity of the Plasmodium yoelii MDR
infected specimen stained compared to healthy liver sections (Figure 5B and S9). The
quantification analysis implicated three to four fold increased level of lipid accumulation in
malaria infected liver tissues as compared to control.
LDs staining in human cancerous cervical tissue
ACS Paragon Plus Environment

Page 11 of 22
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
We next examined the potential of the probes for lipid staining during cancer cell
proliferation which requires the rapid synthesis of lipids for the generation of biological
membranes. The accumulation of energyꢀrich lipids could provide cancer cells with energy
during times of nutrient depletion. But the exact role of the deposition of lipid droplets in
cancerous tissues is not clearly understood due to the lack of potential fluorescent probes,
which could efficiently perform at tissue level. Therefore, the standardization of quick
fluorescent labeling for these lipid droplets in cancerous as well as adjacent noncancerous
tissues with these newly developed probes may help in better understanding of metabolic
alterations during cell transformation and cancer development.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. [A] Confocal fluorescence image of Cervical Cancer (IIIB Stage) vs. adjacent nonꢀ
cancerous tissue sections. Coꢀstaining of FLUN-550, FLUN-552 and FLUN-547 (200 nM) with Nile
Red (200 nM) and ToꢀPro nuclear stain in serial section of cancer tissues; [B] Analyses of average
fluorescence intensity of lipid droplets by FLUN-550, FLUN-552 and FLUN-547 assessed through
Image J and Prism 3 software demonstrated a significant difference between cancerous and adjoining
noncancerous cervix tissue sections. *P < 0.05, **P = 0.0015, Bar indicates 100 µm.
To check the potential application of the FLUN derivatives to stain lipid in tissue sections of
cervical cancer, human cervical punch biopsies from cervical cancer patients were obtained
from King George’s Medical University, Lucknow, India in accordance with Institutional
Ethics Committee and after patient’s written consent (51 E.C.M. IIa/P1). The staining pattern
of these novel probes by confocal fluorescence imaging was compared with Nile Red through
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 12 of 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the coꢀstaining experiments as shown in Figure 6. Staining of lipid droplets mostly coꢀ
localized in the cancerous tissue sections. The overall staining pattern was found to be quite
different in the cancerous tissues as compared to the nonꢀcancerous ones; as quite a few lipid
accumulation pockets (lipid clusters) were observed in the cancerous sections (Figure 6A,
white arrows) but not in the nonꢀcancerous tissues (Figure 6A and S10). Moreover, we also
quantified and compared overall lipid droplets in these stained sections through the
measurement of fluorescence intensity using Image J software. This analysis established a
significant difference in the lipid content of cancerous versus adjoining noncancerous tissues,
using FLUN derivatives (Figure 6B). Statistical evaluation was performed through the
student’s tꢀtest (p < 0.05) using Prism software. The quantitation analysis of fluorescence
intensity of serial sections of cancer tissue stained by FLUN-550 and FLUN-552 revealed a
significant increase in the lipid content of cancerous tissues as compared to the adjoining
nonꢀcancerous counterpart. Similarly in the case of Nile red staining, the fluorescence
intensities of lipid droplets between cancerous and adjoining nonꢀcancerous tissue sections
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was also enhanced but the intensity was statistically nonꢀsignificant possibly due to low LDs
17ꢀ19
selectivity by Nile Red.
As per tissue analysis after 6 days, these new probes were found
to be more stable as compare to nile red staining. To the best of our knowledge, this is the
first report of yellow fluorescent probes for simple, selective and quantitative detection of
lipid droplets in cancerous and nonꢀcancerous tissue sections of the third stage of human
cervical cancer patients.
ꢀ
CONCLUSION
In conclusion, we have developed new fluoranthene based yellow fluorescent probes FLUN-
50 and FLUN-552 for selective staining of lipid droplets in live 3T3ꢀL1 preꢀadipocytes and
5
live L. donovani promastigotes. We have designed new probes by conjugating ethanolamine
moiety so as to mimic the head groups of phospholipids present in biological membrane for
ACS Paragon Plus Environment

Page 13 of 22
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
hydrophobicꢀhydrophobic interactions. These new probes showed visible excitability,
aqueous compatibility, high stokes shift (210ꢀ230 nm), good quantum yield and
photostability. The confocal fluorescence imaging and quantitative detection of lipid droplets
in liver sections of Plasmodium yoelii infected mouse and human cervical cancerous tissues
revealed significantly increased accumulation of lipids as compared to control. Such
quantitative detection studies are difficult to achieve and have not been reported in these
different tissues prior to this study. Considering these encouraging results, new yellow
fluorescent probes (FLUN-550 and FLUN-552) are useful for early diagnosis of cervical
cancer patients. According to the present study, FLUN-550 and FLUN-552 observed to be
really significant and more stable as compare to commercially available stain Nile Red. These
new findings will not only be beneficial for diagnosis but also for understanding the excess
accumulation of lipids during pathogenesis of malaria, cancer and metabolic disorders.
Further studies with these probes for significant quantitative analysis of lipid droplets can be
established as a novel cancer biomarker in different grades of cancer patient biopsy samples.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ
EXPERIMENTAL SECTION
General Information.
The chemicals and solvents of analytical grade were used without further purification.
Synthetic details of the fluorescent probes have been given in Scheme 1 and the supporting
information.
1
13
H NMR spectra were recorded at 400 and 300 MHz and C NMR spectra were recorded at
00 MHz. Chemical shifts are reported in parts per million shift (δꢀvalue) from Me Si (δ 0
1
4
1
1
3
ppm for H) or based on the middle peak of the solvent (CDCl ) (δ 7.26 ppm for H NMR
13
1
6
and δ 77.00 ppm for C NMR) or DMSOꢀd (δ 2.50 ppm for H NMR and δ 40.00 ppm for
1
3
C NMR) as an internal standard. Signal pattern are indicated as s, singlet; d, doublet; t,
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 14 of 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
triplet; m, multiplet. Coupling constant (J) is given in Hertz. Infrared (IR) spectra were
ꢀ1
recorded in KBr disc and reported in wave number (cm ). The ESIꢀMS were recorded on
MICROMASS QuadroꢀII LCMS system. The HRMS spectra were recorded as ESIꢀHRMS
on a mass analyzer system. All the reactions were monitored by TLC and visualization was
done with UV light (254 nm). Agilent Cary 100 UVꢀVis spectrophotometer was used for
absorbance measurements. A Varian Cary Eclipse spectrofluorophotometer was used for
fluorescence measurements. For cell imaging, confocal laser scanning microscope (Lieca,
Germany) was used. Further, fluorescence intensity was quantified using image J software
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Image J, National Institute of Health, Bethesda, MD). Fluorescence signal quantification was
done by LAS (Leica application suit) and Prism 3 software.
Cell culture and Staining studies (3T3-L1 pre-adipocytes and L. donovani)
Cell Culture: 3T3ꢀL1 preꢀadipocytes were cultured in DMEM with 10% heatꢀinactivated
FBS at 37 °C. After reaching 70% confluence, cells were seeded for two days before starting
experiments. Leishmania donovani (strain MHOM/IN/80/Dd8) promastigotes were cultured
as described previously. Briefly, cells were cultured in Dulbecco’s Modified Eagles Medium
(DMEM) with phenol red, Lꢀglutamine and 4.8g/L Dꢀglucose supplemented with 10% heat
inactivated FBS and sodium pyruvate. Cell were checked for their health periodically and
6
maintained in standard tissue culture flasks. After reaching an approximate density of 10
cells/ml they were prepared for incubation.
Confocal Microscopy: 3T3ꢀL1 preꢀadipocytes adhered to confocal microscopy dishes were
incubated with the dye (FLUN-552 and FLUN-547) in DMEM for 30 min at 37°C. They
were washed and stained with 1 µM Hoechst 33342 (Invitrogen) for 10 min. Samples were
analyzed under a laser scanning microscope LSM 510 META (Carl Zeiss, Jena, Germany).
ACS Paragon Plus Environment

Page 15 of 22
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
Images were acquired with a 63X Plan Apochromat Oil Phase II 1.4 objective. Lasers used
were Diode 405 nm, Ar/ML 458/477/488/514 nm and DPSS 561nm.
L. donovani cells, briefly after harvesting, were incubated with 100 nM of dye FLUN-552
and FLUN-547 in medium for 2 hrs. at room temperature (DMSO in the media was merely
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.01% v/v). Afterwards, they were washed with PBS. Cells were then adhered to polyꢀLꢀ
lysine coated cover slips and immediately processed for confocal imaging. Stacks of images
at different focal planes were acquired and merged to create a movie with AIM 4.0 software.
Analysis: Images were analyzed with the LSM Image Examiner software (Carl Zeiss). The
2.5D option was used to display the fluorescence peaks of fluorescent LDs, which are distinct
from the background (i.e. fluorescent against nonꢀfluorescent background). This mode
displays twoꢀdimensional fluorescence intensity information in a pseudoꢀ3D image. The
increase in fluorescence intensity is color coded as change in color was observed from blue to
red. The viewing angle was set at 45º at all times. Rotation at both axes was set to 0º; filled
option with rainbow palette was used.
Dual staining with Nile Red: Cells (L. donovani), briefly after harvesting, were incubated
with 100 nM FLUN-552 and FLUN-547 for 2 hrs. Afterwards, they were washed with PBS
and further incubated with 300 nM Nile Red upto 10 minutes and washed again with PBS.
Cells were then immediately adhered to polyꢀLꢀlysine coated coverslips and processed for
microscopic examination. The images were analyzed using with the LSM Image Examiner
software (Zeiss). Nile red staining showed fluorescence with excitation wavelength at 561 nm
and emission at 575 nm long pass whilst excitation wavelength of Fluorescence Probes was
405 nm and emission at band of 505 nmꢀ570 nm.
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 16 of 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Tissue preparation and staining studies (Healthy and Infected Liver Tissue)
Tissue preparation: To evaluate fat deposition, liver specimen from healthy mice and P.
yoelii MDR infected mice (at 50% parasitaemia) were embedded using tissue OCTꢀfreeze
medium and sliced into 10 µmꢀthick sections using a cryostat microtome (LEICA).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Nile Red staining: A stock solution of Nile red (10 mM) in acetone was prepared and stored
chilled and protected from light. A fresh staining solution of Nile red (300 nM) was made by
diluting the stock in 75% glycerol followed by brisk vortexing. The glycerolꢀdye solution was
then briefly degassed by vacuum to remove bubbles. To stain frozen sections of liver, a drop
of glycerol staining solution was added to each section and incubated for 5 min. After
2
7
incubation, the sections were examined by confocal microscopy.
Fluorescent probes (FLUN-550, FLUN-552 and FLUN-547) staining: A stock solution of
each probe (10 mM) namely FLUNꢀ550, FLUNꢀ552 and FLUN-547 in PBS buffer (DMSO
in the media was merely 0.01% v/v) was prepared and stored, chilled and protected from
light. A fresh staining solution of probe (100 nM) was made by diluting the stock in PBS
buffer. To stain frozen section of liver, a drop of probe was added to each section, incubated
for 5 min. After washing two times with PBS buffer, tissue sections were mounted with 75%
glycerol and examined.
Dual Staining with Nile Red and Probes: Frozen liver sections were incubated for 5 min
with Nile red and probe, washed two times with PBS and mounted with 75% glycerol.
Confocal microscopy: The sections were examined with Leica SP8 confocal microscope at
63X with immersion oil. Excitation wavelength of Fluorescence Probes was 405 nm and
ACS Paragon Plus Environment

Page 17 of 22
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
emission at band of 505 nmꢀ570 nm and Nile red staining showed fluorescence in red
channel.
Tissue preparation and staining studies (Cancerous Cervical Tissue)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Tissue sections of cancer samples: Human cervical punch biopsies from cervical cancer
patients were obtained from King George’s Medical University, Lucknow, India in
accordance with institutional Ethics Committee and after patient’s written consent (51 E.C.M.
IIa/P1). Tissues were immediately fixed in 4% neutral buffered formalin and then embedded
into paraffin wax to make the blocks of these tissues. Now 5 ꢁm serial sections were cut from
these tissue blocks and placed on polylysine coated glass slides. These slides were stored at
room temperature for further experiments.
Fluorescent probes (FLUN-550, FLUN-552 and 547) co-staining with Nile Red: Stock (5mM) solutions
of probe FLUN-550 and its derivatives (FLUN-552 and FLUN-547) were prepared in analytical grade
DMSO (DMSO in the media was merely 0.01% v/v); whereas stock solution of Nile Red (5mM) was
prepared in acetone. All the stocks were stored chilled and protected from light. Fresh dilutions (200
nM) of FLUN-550 and Nile Red were made in sterile PBS buffer. Now the slides of the tissue sections
were heated at 60°C for 20 min, deparaffinized with xylene and rehydrated with various grades of
ethanol. Then, these tissue sections were incubated with FLUN derivatives or Nile Red (200 nM) for
1
5 minutes and washed twice with PBS buffer. The sections were then incubated with the nuclear
stain To-Pro (1 µM) for 20 minutes and washed twice with PBS buffer. Stained slides were mounted
with a glass cover slip using 75% glycerol.
ꢀ
ACKNOWLEDGMENTS
AG thanks Department of Atomic Energy (DAEꢀSRC) for Outstanding Investigator Award
(21/13/2015ꢀBRNS/35029). Authors thank CSIR, New Delhi for research fellowships. We
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 18 of 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
acknowledge Mrs. Rima A. Sarkar for confocal imaging of tissue sections and SAIFꢀCDRI
for providing spectral data. The CDRI communication number is 9674.
ꢀ
ASSOCIATED CONTENT
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Supporting Information
1
13
Spectroscopic data, H, C NMR and UVꢀFL spectra of all the compounds are given in
supporting. This material is available free of charge via the internet at http://pubs.acs.org
ꢀ
REFERENCES
[
1]
Martin, S., and Parton, R. G. (2006) Lipid droplets: a unified view of a dynamic
organelle. Nat. Rev. Mol. Cell Biol. 7, 373ꢀ378.
[2]
Zhang, H., Wang, Y., Li, J., Yu, J., Pu, J., Li, L., Zhang, H., Zhang, S., Peng, G.,
Yang, F. and Liu, P. (2011) Proteome of Skeletal Muscle Lipid Droplet Reveals
Association with Mitochondria and Apolipoprotein AꢀI. J. Proteome Res. 10, 4757ꢀ
4768.
[
[
3]
4]
Boulant, S. P., Adams, T. and McLauchlan, J. (2007) Disrupting the association of
hepatitis C virus core protein with lipid droplets correlates with a loss in production of
infectious virus. J. Gen. Virol., 88, 2204ꢀ2213.
Miyanari, Y., Atsuzawa, K., Usuda, N., Watashi, K., Hishiki, T., Zayas, M.,
Bartenschlager, R., Wakita, T., Hijikata, M. and Shimotohno, K. (2007) The lipid
droplet is an important organelle for hepatitis C virus production. Nat. Cell Biol., 9,
1089ꢀ1097.
[5]
Davis, J., Crunk, A. E., Monks, J., Murakami, A., Jackman, M., MacLean, P. S.,
Ladinsky, M., Bales, E., Cain, S., Orlicky, S. D. J. and McManaman, J. L. (2013)
Dynamic regulation of hepatic lipid droplet properties by diet. PLoS One, 8 e67631.
[
[
6]
7]
Yonezawa, T., Kurata, R., Kimura, M. and Inoko, H. (2011) Which CIDE are you on?
Apoptosis and energy metabolism. Mol. BioSyst., 7, 91ꢀ100.
Medes, G., Thomas, A. and Weinhouse, S. (1953) Metabolism of neoplastic tissue.
IV. A study of lipid synthesis in neoplastic tissue slices in vitro. Cancer research, 13,
27ꢀ29.
[
8]
Baenke, F., Peck, B. and Miess, H. (2013) A. Schulze, Hooked on fat: the role of lipid
synthesis in cancer metabolism and tumour development. Disease Models and
Mechanisms, 6, 1353ꢀ1363.
ACS Paragon Plus Environment

Page 19 of 22
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
[9]
Ohsaki, Y., Suzuki, M. and Fujimoto, T. (2014) Open Questions in Lipid Droplet
Biology. Chem. Biol. 21, 86ꢀ96.
[
[
[
[
10] Mehlem, A., Hagberg, C. E., Muhl, L. and Eriksson, U. (2013) A. Falkevall, Imaging
of neutral lipids by oil red O for analyzing the metabolic status in health and disease.
Nat Protoc., 8, 1149ꢀ1154.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
11] Fowler, S. D. and Greenspan, P. (1985) Application of Nile red, a fluorescent
hydrophobic probe, for the detection of neutral lipid deposits in tissue sections:
comparison with oil red O. J. Histochem. Cytochem., 33, 833ꢀ836.
12] Bonilla, E. and Prelle, A. (1987) Application of nile blue and nile red, two fluorescent
probes, for detection of lipid droplets in human skeletal muscle. J. Histochem.
Cytochem., 35, 619–621.
13] De Bock, K., Dresselaers, T., Kiens, B., Richter, E. A., Van Hecke, P. and Hespel, P.
(2007) Evaluation of intramyocellular lipid breakdown during exercise by
biochemical assay, NMR spectroscopy, and Oil Red O staining. Am. J. Physiol., 293,
E428ꢀE434.
[
14] Shaw, C. S., Jones, D. A. and Wagenmakers, A. J. M. (2008) Network distribution of
mitochondria and lipid droplets in human muscle fibres. Histochem. Cell. Biol., 129
65ꢀ72.
[15] Shaw, C. S., Sherlock, M., Stewart, P. M. and Wagenmakers, A. J. M. (2009)
Adipophilin distribution and colocalization with lipid droplets in skeletal muscle.
Histochem. Cell Biol., 131, 575ꢀ581.
[
16] Zietkowski, D., deSouza, N. M., Davidson, R. L. and Payne, G. S. (2013)
Characterisation of mobile lipid resonances in tissue biopsies from patients with
cervical cancer and correlation with cytoplasmic lipid droplets. NMR Biomed. 26,
1096ꢀ1102.
[
[
[
[
17] Listenberger,L. L. and Brown, D. A. (2007) Fluorescent detection of lipid droplets
and associated proteins. Current Protocols in Cell Biology, Wiley & Sons, Inc.,
chapter 24, unit 24.2.1.
18] Cosgrave, L., Devocelle, M., Forster, R. J. and Keyes, T. E. (2010) Multimodal cell
imaging by ruthenium polypyridyl labelled cell penetrating peptides. Chem. Commun.
46, 103.
19] Rumin, J., Bonnefond, H., SaintꢀJean, B., Rouxel, C., Sciandra, A., Bernard, O.,
Cadoret, J.ꢀP. and Bougaran, G. (2015) The use of fluorescent Nile red and BODIPY
for lipid measurement in microalgae. Biotechnol. Biofuels, 8, 42.
20]
Spandl, J., White, D. J., Peychl, J. and Thiele, C. (2009) Live cell multicolor imaging
of lipid droplets with a new dye, LD540. Traffic, 10, 1579–1584.
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 20 of 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[21] Neef, A. B. and Schultz, C. (2009) Selective Fluorescence Labeling of Lipids in
Living Cells. Angew. Chem., Int. Ed., 48, 1498–1500.
[
[
[
22] Lee,Y., Na, S., Lee, S., Jeon, N. L. and Park, S. B. (2013) Optimization of Seoulꢀ
Fluorꢀbased lipid droplet bioprobes and their application in microalgae for bioꢀfuel
study. Mol. BioSyst., 9, 952ꢀ956.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
23] Lee, J. H., So, J. H., Jeon, J. H., Choi, E. B., Lee, Y. R., Chang, Y. T., Kim, C. H.,
Bae, M. A. and Ahn, J. H. (2011) Synthesis of a new fluorescent small molecule
probe and its use for in vivo lipid imaging. Chem. Commun.,47, 7500–7502.
24] Goel, A., Sharma, A., Kathuria, M., Bhattacharjee, A., Verma, A., Mishra, P. R.,
Nazir, A. and K. Mitra, (2014) New Fluoranthene FLUNꢀ550 as a Fluorescent Probe
for Selective Staining and Quantification of Intracellular Lipid Droplets. Org.
Lett.,16, 756−759.
[
[
25] Sk, B., Thakre, P. K., Tomar, R. S. and Patra, A. (2017) A Pyridoindole‐Based
Multifunctional Bioprobe: pH‐Induced Fluorescence Switching and Specific
Targeting of Lipid Droplets Chem. – Asian J., 12, 2501ꢀ2509.
26] Wu, A., Kolanowski, J. L., Boumelhem, B. B., Yang, K., Lee, R., Kaur, A., Fraser, S.
T., New, E. J.and Rendina, L. M. (2017) A Carborane-Containing Fluorophore as a
Stain of Cellular Lipid Droplets Chem. Asian J., 12, 1704ꢀ1708.
[
[
[
[
[
[
27] Daemen, S., van Zandvoort, M. A., Parekh, S. H. and Hesselink, M. K. (2016)
Microscopy Tools for the Investigation of Intracellular Lipid Storage and Dynamics.
Mol. Metabol., 5, 153ꢀ163.
28] Wang, Z., Gui, C., Zhao, E., Wang, J., Li, X., Qin, A., Zhao, Z., Yu, Z. and Tang, B.
Z. (2016) Specific Fluorescence Probes for Lipid Droplets Based on Simple AIEgens.
ACS Appl. Mater. Interfaces 8, 10193−10200.
29] Sharma, A., Umar, S., Kar, P., Singh, K., Sachdev, M. and Goel, A. (2016) A new
type of biocompatible fluorescent probe AFN for fixed and live cell imaging of
intracellular lipid droplets. Analyst, 141, 137ꢀ143.
30] Jiang, M., Gu, X., Lam, J. W. Y., Zhang, Y., Kwok, R. T. K., Wong, K. S. and Tang,
B. Z. (2017) Twoꢀphoton AIE bioꢀprobe with large Stokes shift for specific imaging
of lipid droplets. Chem. Sci., 8, 5440ꢀ5446.
31] Appelqvist, H, Stranius, K., Boerjesson, K., Nilsson, K. P. R. and Dyrager, C. (2017)
Benzothiadiazole Derivatives as Fluorescence Imaging Probes: Beyond Classical
Scaffolds. Bioconjugate Chem., 28, 1363ꢀ1370.
32] Kang, M., Gu, X., Kwok, R. T., Leung, C. W., Lam, J. W., Li, F. and Tang, B. Z.
(2016) A nearꢀinfrared AIEgen for specific imaging of lipid droplets. Chem.
Commun., 52, 5957ꢀ5960
ACS Paragon Plus Environment

Page 21 of 22
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
[33] Lee, B., Park, B. G., Cho, W., Lee, H. Y., Olasz, A., Chen, C.ꢀH., Park, S. B. And
Lee, D. (2016) BOIMPY: Fluorescent Boron Complexes with Tunable and
Environment‐Responsive Light‐Emitting Properties Chem. Eur. J., 22, 17321 ꢀ17328
[
34] Guo, L., Tian, M., Feng, R., Zhang, G., Zhang, R., Li, X., Liu, Z., He, X., and Sun, J.
Z. and Yu, X. (2018) InterfaceꢀTargeting Strategy Enables TwoꢀPhoton Fluorescent
Lipid Droplet Probes for HighꢀFidelity Imaging of Turbid Tissues and Detecting Fatty
Liver, ACS Appl. Mater. Interfaces 10, 10706ꢀ10717.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[35] Mota, A. A. R., Correa, J. R., de Andrade, L. P., Assumpção, J. A. F., de Souza
Cintra, G. A., Junior, L.H. Fꢀ W., da Silva, de Oliveira, A. H. C. B. and Neto, B. A.
D. (2018) From Live Cells to Caenorhabditis elegans: Selective Staining and
Quantification of Lipid Structures Using a Fluorescent Hybrid Benzothiadiazole
Derivative ACS Omega 3, 3874ꢀ3881.
[36] Collot, M., Fam, T. K., Ashokkumar, P., Faklaris, O., Galli, T., Danglot, L. and
Klymchenko, A. S. (2018) Ultrabright and Fluorogenic Probes for Multicolor Imaging
and Tracking of Lipid Droplets in Cells and Tissues J. Am. Chem. Soc.140, 5401ꢀ
5411
[
37] Goel, A., Sharma, A., Mitra, K., Bhattacharjee, A. and Kathuria, M. Substituted
fluorantheneꢀ7ꢀcarbonitriles/esters as fluorescent dyes for cell imaging applications,
CSIR Pat. No.: 807DEL2013 dated 19ꢀMarꢀ2013; PCT/IN2014/000156 dated 10ꢀ
Marꢀ2014.
[
38] Goel, A., Parihar, A., Mishra, P., Varshney, S., Nag, P., Beg, M., Gaikwad, A. and
Rath, S. K. (2013) Design and synthesis of novel pyranoneꢀbased insulin sensitizers
exhibiting in vivo hepatoprotective activity. Med. Chem. Commun., 4, 1532ꢀ1536.
[39] Rakotomanga, M., Blanc, S., Gaudin, K., Chaminade, P. and Loiseau, P. M. (2007)
Miltefosine affects lipid metabolism in Leishmania donovani promastigotes.
Antimicrob. Agents Chemother., 51, 1425ꢀ1430.
[
40] Croft, S. L. and Yardley, V. (2002) Chemotherapy of leishmaniasis. Curr. Pharm.
Des. 8, 319ꢀ342.
[
41] Croft, S. L. and Coombs, G. H. (2003) Leishmaniasis– current chemotherapy and
recent advances in the search for novel drugs. Trends Parasitol., 19, 502ꢀ508.
[42] Bajpai, R. and Dutta, G. P. (1990) Role of fatty infiltration in pathology of malaria.
Life Sci., 47, 25ꢀ28.
[
43] Siddiqi, N. J., Puri, S. K., Dutta, G. P., Maheshwari, R. K. and Pandey, V. C. (1999)
Studies on hepatic oxidative stress and antioxidant defence system during
chloroquine/poly ICLC treatment of Plasmodium yoelii nigeriensis infected mice.
Mol. Cell. Biochem., 194, 179ꢀ183.
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 22 of 22
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TOC Graphics
7
0x33mm (300 x 300 DPI)
ACS Paragon Plus Environment