A Pyridoindole-Based Multifunctional Bioprobe: pH-Induced Fluorescence Switching and Specific Targeting of Lipid Droplets

Article abstract of DOI:10.1002/asia.201700898

A versatile fluorescent probe, PITE, based on alkyl-substituted pyridoindole (PI) and tetraphenylethylene (TE), which exhibits facile pH-induced fluorescence switching in solution, as nanoparticles, and in the solid state, is presented. Strong fluorescenc

Full text of DOI:10.1002/asia.201700898

CHEMISTRY
AN ASIAN JOURNAL
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Accepted Article
Title: A pyridoindole based multifunctional bioprobe: pH-induced
fluorescence switching and specific targeting of lipid droplets
Authors: Bahadur Sk, Pilendra Thakre, Raghuvir Singh Tomar, and
Abhijit Patra
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10.1002/asia.201700898
Chemistry - An Asian Journal
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A pyridoindole based multifunctional bioprobe: pH-induced
fluorescence switching and specific targeting of lipid droplets
Bahadur Sk,‡,[a] Pilendra Kumar Thakre,‡,[b] Raghuvir Singh Tomar,*,[b] and Abhijit Patra*,[a]
Abstract: A versatile fluorescent probe, PITE, based on alkyl
substituted pyridoindole (PI) and tetraphenylethylene (TE) exhibiting
facile pH-induced fluorescence switching in solution, nanoparticles
and solid state is presented. Strong fluorescence in the solid state
besides solution and aggregated state allow sensing of toxic acid
vapor. Fluorescence “off-on” switching of PITE by exposing to
trifluoracetic acid and triethylamine vapor is visualized through naked
eyes. A unified picture of switchable fluorescence of PITE is obtained
by comprehensive spectroscopic investigations coupled with quantum
mechanical calculations. Strong fluorescence, large Stokes shift, high
photostability and biocompatibility of PITE makes it a viable probe for
sub-cellular imaging. Extensive fluorescence microscopic studies
employing organisms including lower and higher eukaryotes reveal
specific localization of PITE to lipid droplets (LDs). It has been known
that LDs are dynamic sub-cellular organelles linked to various
physiological processes and human diseases. Hence, the specific
detection of LDs in diverse organisms is important to biomedical
research and health care. Isolation of LDs and subsequent
colocalization studies ascertain selective targeting of LDs by easily
affordable, lipophilic bioprobe, PITE. Thus, PITE turns out to be a
promising multifunctional probe for chemo-sensing as well as
selective tracking of LDs.
emerged as attractive platforms for level-free chemo/
biosensing.[4]
The rapid growth of chemical industries, production of
pharmaceuticals, pesticides raises pollution due to organic
acids.[5] Detection of acid vapor under ambient conditions is
challenging. So, the development of pH-responsive fluorescent
probe for the detection of the organic acid is crucial. Such probes
are mostly based on the phenomenon of photo-induced electron
transfer (PET), excited-state intramolecular proton transfer
(ESIPT), fluorescence resonance energy transfer (FRET) and so
forth.[6] However, reversibility and repeatability of the process for
efficient sensing remain
a critical issue. Exploration of
synthetically easily accessible molecular probe exhibiting a sharp
reversible change in fluorescence signals upon variation of pH of
the medium induced by organic acids/ bases is an interesting
problem to address. The importance of such a fluorescent probe
would further enhance if it also demonstrates selective detection
of biological species in a cellular medium besides chemical
sensing. Owing to the physiological importance of lipid droplets
(LDs), the development of LD-specific suitable bioprobe attracts
considerable attention.[7]
LDs are known as lipid-rich, highly dynamic cellular organelles
found in almost all kinds of cells.[8] Although the LDs are
heterogeneous with respect to their structure, chemical
composition and distribution in tissues but they mainly consist of
neutral lipids such as triacylglycerols or cholesteryl esters
surrounded by a phospholipid monolayer.[9] The surface of LDs is
also coated with a number of proteins.[10] The major function of
LDs is to store lipids for a variety of cellular functions such as
membrane biogenesis, protein modification, synthesis of
lipoproteins and steroids.[11] Furthermore, LDs have been known
to modulate host-pathogen interactions and immune response.[12]
The dysfunction of LDs has been linked with a variety of
pathogenic and non-pathogenic diseases.[13] Addressing the
important issues of localization and concentration variation of LDs
in cells, fluorescent probes such as Nile red and BODIPY dyes
have been commercialized. However, these probes have one or
more of serious disadvantages like photobleaching, high
background noise, small Stokes shift leading to self-absorption,
aggregation-caused-quenching (ACQ: quenching of fluorescence
in solutions of high concentrations or aggregated state) and
tedious synthetic and purification procedures.[14] Thus, new
multifunctional chemical probes are in high demand that can
provide an accurate assessment of cellular lipid content in diverse
physiological conditions.
Introduction
Multifunctional sensors capable of selective and sensitive
detection of chemicals as well as biological species in complex
environments are of increasing interest.[1] In this regard,
fluorescence-based sensing and imaging probes have received
significant attention due to their excellent sensitivity and
spatiotemporal resolution.[2] Detection in aggregated or solid state
besides in solution is particularly important for real-time
applications. Thus, a multifunctional fluorescent probe should
ideally possess high fluorescence quantum yields both in solution,
and solid states, good photostability, large Stokes shift, long
emission lifetime, fast permeability through the cell membrane
and low cytotoxicity.[3] Aqueous dispersion of fluorescent
nano/microparticles with definite physicochemical features also
[a]
[b]
‡
Dr. Abhijit Patra
Department of Chemistry
Indian Institute of Science Education and Research Bhopal
Address: Indore Bypass Road, Bhauri, Bhopal 462066, Madhya
Pradesh, India. E-mail: abhijit@iiserb.ac.in
Dr. Raghuvir Singh Tomar
In pursuit of development of multifunctional materials,[15] our
continuous efforts led to the realization of a broad spectrum
hydrophobic fluorescent bioprobe combining aesthetically
appealing moieties of pyridoindole and tetraphenylethylene (PITE,
Figure 1). It exhibits pH-dependent fluorescence in solution,
nanoparticles and solid states as well as selective detection of
LDs. Strong solid-state fluorescence in PITE enabled us to
Department of Biological Sciences
Indian Institute of Science Education and Research Bhopal
Address: Indore Bypass Road, Bhauri, Bhopal 462066, Madhya
Pradesh, India. E-mail: rst@iiserb.ac.in
Equal contributions
Supporting information (details of synthesis, spectroscopy, imaging)
for this article is given via a link at the end of the document.
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10.1002/asia.201700898
Chemistry - An Asian Journal
FULL PAPER
excitation wavelengths (Figure S2). Absorption and emission of
PITE do not differ with the solvent polarities (Figures S3a, b, Table
S1). The aqueous dispersion of spherical nanoparticles with
average diameter of 60-80 nm was obtained by rapid addition of
tetrahydrofuran (THF) solution of PITE in water (Figure 1). PITE
nanoaggregates, solid powder and drop-casted thin films display
dual emission with peaks at 490 and 550 nm (Figure S1, Table
S1). A range of weak intra and intermolecular interactions
restricting the molecular motion were found to be responsible for
fluorescence in the aggregated state.[18] The strong fluorescence
in solution (quantum yield, f = 50%, Table S1), nanoparticles (f
= 8%) and solid state (f = 20%) led to the foundation of the
present study with PITE as a multifunctional probe for pH-induced
fluorescence switching leading to the sensing of organic acid
vapor and selective imaging of LDs.
pH-induced fluorescence switching
Figure 1. Molecular structure of pyridoindole-tetraphenylethylene (PITE) probe
and photographs of PITE depicting strong fluorescence (quantum yield, f
indicated) in solution, nanoparticles and solid state under illumination of UV light
at 365 nm. Field emission scanning electron microscopy (FESEM) image of
nanoparticles is shown (scale bar = 100 nm).
The lone pair of electrons on nitrogen in PITE involve in
conjugation and play a significant role directing the photophysical
properties of the probe. The protonation of nitrogen completely
quenches the fluorescence of PITE. This observation prompted
us investigating PITE as a new molecular fluorescent probe in
acidic and basic environments. As shown in Figure 2a, the
fluorescence intensity of PITE in chloroform upon addition of
trifluoroacetic acid (TFA, pKa = 0.23, pH of the solution ~ 2 as
tested by pH paper) is almost at a line parallel to the x-axis. It is
due to the very weakly emissive protonated PITE formed under
strong acidic conditions (Figures S4, S5). With the increase in pH
by the addition of triethylamine (TEA, pKb = 3.25, pH of the
solution > 4 as tested by pH paper), deprotonation occurs and the
lone pair of electrons become available for the conjugation
regaining the fluorescence. The loss of planarity upon protonation
reduces the conjugation and activates the non-radiative decay
channels. The density functional theory (DFT) calculations on
protonated PITE (ground state) clearly revealed the nonplanarity
between indole and pyridyl moieties compared to that of the
structure of PITE obtained through crystal structure analysis
(Figure S7, Table S3). At low pH protonation to carbonyl oxygen
would further contribute in fluorescence quenching.[19]
The instantaneous fluorescence switching of PITE in solution
between ‘off’ and ‘on’ state can be carried out reversibly through
several cycles upon successive addition of TFA and TEA,
respectively (Figure 2b). The variation of fluorescence intensity
was noticeable for PITE nanoparticles dispersed in different pH
buffer (Figure S6, Table S2). A sharp change in fluorescence
intensity observed between pH 3-4. The fluorescence ‘off-on’ can
also be effected by the gradual addition of TFA and TEA,
respectively (Figure 2c). The switching between the
nonfluorescent and fluorescent states by consecutive protonation
and deprotonation was found to be repeatable through several
cycles (Figure 2d).
demonstrate reversible and repeatable sensing of toxic organic
acid vapors like trifluoracetic acid (TFA). PITE is also found to be
suitable for selective imaging of LDs in lower eukaryotes such as
yeast to mammalian cells both in vitro as well as in vivo. Such a
comprehensive
investigation
encompassing
pH-induced
fluorescence switching in different physical states leading to
sensing of organic acid vapors and specific targeting of LDs by a
single molecular material, to the best of our knowledge, has
scarcely been reported.
Results and Discussion
Pyridoindoles have been emerged as a new class of fluorophores
exhibiting potential biological activities as well as promising
optoelectronic properties.[16] Alkyl substituted pyrido[1,2-a]indoles
(PI) shows strong green fluorescence in organic solvents.[16c]
However, poor solubility in aqueous medium and nearly complete
quenching of fluorescence in aggregated or solid state impede its
real-time applications in chemo and biosensing. On the contrary,
tetraphenylethylene (TE) is considered as a classic aggregation-
induced-emission (AIE)-active molecule demonstrating strong
fluorescence in the aggregated state compared to that of the
solution.[17] To circumvent the issues with fluorescence quenching
of PI in aggregated state, we coupled PI and TE through covalent
linkage leading to the development of a fascinating fluorescent
probe PITE exhibiting strong fluorescence both in solution and
solid state (Figure 1). Suzuki cross-coupling between 2-bromo
6,7,8,9-tetrapropylpyrido[1,2-a]indole-10-carbaldehyde
and
4,4,5,5-tetramethyl-2-(4-(1,2,2-triphenylvinyl)
dioxaborolane yielded PITE (Schemes S1, S2).
phenyl)-1,3,2-
We employed pH-sensitive fluorescence of PITE for the
detection of acidic volatile organic compounds in the solid state.
Nonfluorescent thin-layer chromatography (TLC) plate was
employed as a solid support. The PITE loaded TLC plate emits
The absorption of PITE in solution shows two bands centering
at 320 and 420 nm respectively due to the TE and PI moieties
(Figure S1). The emission peak at 490 nm does not vary with the
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10.1002/asia.201700898
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Figure 3. 1H-NMR spectra of PITE: (a) in CDCl3, (b) after addition of
trifluoroacetic acid (TFA) and (c) after addition of triethylamine (TEA) in the
same sample tube. The significant shifts of signals a-h refer to protons indicated
in Figure 1.
7 - 9.5 ppm. The chemical shift of the aldehyde proton (‘a’ in
Figure 1) is 10.4 ppm. Upon addition of TFA in the same solution,
protons a, b, and d are significantly shielded and protons c, e and
h are deshielded (Figure 3b). The protonation of nitrogen changes
the chemical and magnetic environments of nearby aromatic
1
protons leading to a significant shift in H NMR spectrum. The
Figure 2. (a) Fluorescence spectra of PITE in CHCl3 solution (5 M) in acidic
(addition of trifluoroacetic acid, TFA, pH ~ 2) and basic medium (addition of
triethylamine, TEA, pH ~ 8); inset: respective photographs of PITE upon
addition of TFA and TEA under illumination of UV light (ex = 365 nm). (c)
Emission spectra of aqueous dispersion of PITE nanoparticles upon addition
of TFA (pH ~ 2) and TEA (pH ~ 8); inset: respective photographs of
nanoparticle dispersions under illumination of UV light (ex = 365 nm). (e)
Fluorescence spectra of PITE in solid state (drop casted on non-fluorescent
TLC plate) upon exposure to TFA vapor (10 s) and TEA vapor (10 s); inset:
photographs of PITE spots on a TLC plate upon fuming with TFA and TEA
vapor for 10 s, respectively under UV illumination at 365 nm. pH-dependent
on-off fluorescence (em = 490 nm) switching of PITE in (b) solution, (d)
nanoparticles and (f) solid state. TFA (pKa = 0.23) and TEA (pKa = 10.67)
were used to vary the pH of the solution and aqueous dispersion of
nanoparticles and respective vapors were used for solid-state switching.
broadening of peaks upon protonation is also noticeable. This
effect is likely due to the loss of planarity between indole and
pyridyl moieties upon protonation disrupting the -conjugation
leading to fluorescence quenching. (Figure S7b). The large up-
field shift for ‘a’ proton arises as a result of protonation of carbonyl
oxygen.[20] The conjugation through pyridoindole nitrogen is
regained by the deprotonation upon addition of TEA leading to the
changes of the chemical shift similar to its neutral state. (Figure
3c). The slight shift of the proton signals upon addition of TEA
compared to the native state is likely to be contributed by the
change in polarity and ionic strength of the medium.[20,21]
intense bluish green emission upon irradiation with UV light (ex.
= 365 nm). The shape of the emission spectrum is different
compared to that of the drop-casted thin films presumably due to
the preferential binding of PITE to slightly acidic silica gel (Figures
2e, S1c).6b Exposure to TFA vapor quenches the fluorescence
instantaneously due to the protonation and the dye spot turns
nearly black (Figure 2e, inset). However, the bright fluorescent
spot reappeared upon exposure to TEA vapor (Figure 2e, inset).
The respective fluorescence spectra are shown in Figure 2e. The
fast switching between nonemissive and bright green states was
demonstrated to be facile, fatigue resistant and reversible by
alternately fuming with TFA and TEA vapors (Figure 2f).
The molecular mechanism of fluorescence switching was
probed by 1H NMR spectroscopy. The 1H NMR spectrum of PITE
in CDCl3 is shown in Figure 3a. The signals of indole protons of
PITE (b, c, d as labelled in Figure 1, assigned by 2D NMR analysis
(Figures S31-S33). The protons of the phenyl ring of TE
connected to PI (e, f, g, h as labelled in Figure 1) ranges between
Cellular imaging and targeting of lipid
droplets
The pH-sensitive fluorescence quenching of PITE remains
unaffected in the presence of various electron deficient species
like different metal ions largely present in the cellular environment
(Figure S8). No significant change in fluorescence intensity of
PITE (5 M) was observed in the presence of different metal ions
(1 mM) such as Na+, Mg2+, K+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+,
Zn2+, etc. The presence of four propyl groups and multiple phenyl
rings make PITE substantially hydrophobic.[22] Such a nature of
PITE may enhance its cellular uptake through membranes. In
addition, broad range of emission (460-600 nm), no interference
between absorption and emission (Stokes shift ~ 70 nm),
compatibility to cellular pH (cytoplasmic pH = 7.4),[23] high
fluorescence quantum yield in solution, nanoparticles and solid
state suggest that PITE can be a potential bioprobe for cellular/
sub-cellular imaging. We therefore employed Saccharomyces
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10.1002/asia.201700898
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Figure 4. Saccharomyces cerevisiae (yeast cells) incubated with PITE at 30 C
for 60 min. (a) DIC image, (b) fluorescent image using DAPI filter (blue channel,

ex = 335-383 nm, em = 420-470 nm) and (c) merged image. Scale bar = 2 m.
Figure 5. Yeast cells costained with PITE and Nile red, panel A: cells grown
in complete synthetic medium, panel B: ammonium sulphate deficient
synthetic medium; (a and e) DIC images, (b and f) fluorescent images using
DAPI filter (blue channel, ex = 335-383 nm, em = 420-470 nm, PITE
emission), (c, g) fluorescent images using DsRed filter (red channel, ex = 538-
562 nm, em = 570-640 nm, Nile red emission), (d, h) merged fluorescent
images of PITE and Nile red depicting colocalization in lipid droplets. Scale
bar = 2 m.
cerevisiae (budding yeast) as a model organism to understand
cellular localization of PITE and extended the investigation further
using mammalian cell lines.
In modern science, the budding yeast is considered as an
excellent single-celled model organism to understand the biology
of more complex multi-cellular eukaryotes.[24] The biological
processes such as DNA replication, RNA transcription, DNA
repair, protein translation, intracellular trafficking, mitochondrial
biogenesis and metabolic pathways are highly conserved from
yeast to mammalian cell lines.[25] Due to faster growth, easy
genetic manipulations and smaller genome size, yeast serve as
an important experimental organism revealing function of genes
and contributing in exploration of the underlying mechanisms of
many of the human diseases including cancer and neurological
disorders.[26] In fact, yeast has homologs to 30% of the genes
implicated in human diseases.
The cytotoxicity of PITE in yeast was determined by spot
sensitivity assay and growth curves. PITE (75 M) was found to
be non-toxic for yeast cells (Figures S9, S10). We stained the
yeast cells with PITE for 60 min at 37 °C. The cellular imaging
was performed using Apotome fluorescence microscope. The
images were acquired using 63x oil immersion lens in cyan (DAPI
filter, ex = 335-383 nm, em = 420-470 nm, Figure 4). The
fluorescence of PITE revealed punctate appearance (dots like
structure) from all over the cytoplasm. Although yeast has very
rigid cell wall structure, PITE could enter inside the cells leading
to a punctate staining pattern. We further stained yeast with PITE
in different time periods; clear fluorescent images were obtained
after 30 min of incubation (Figure S11). It was also possible to
acquire images in green (GFP filter, ex = 450-490 nm, em = 500-
550 nm) channel (Figure S11).
and Nile red signals, as revealed by merged fluorescent images
(Figures 5d, h) confirm that PITE specifically stains the LDs in a
similar fashion as that of Nile red, rather even with less
background noise. The Pearson’s correlation coefficient,
revealing the degree of colocalization between PITE and Nile red,
was found to be 0.70 (Figure S12). Further, costaining
experiments with Mito Tracker Deep Red and Lyso Tracker DND
90 Red ruled out the possibility of PITE binding to other organelles
respectively mitochondria and lysosomes (Figures S13, S14).
To further validate the specificity of PITE for LDs, we performed
two crucial experiments. First, we stained the yeast quadruple
mutant (deletion of four genes: are1Δ, are2Δ, dga1Δ, lro1Δ)[28]
with PITE. Since quadruple mutant (lacking lipid synthesizing
enzymes) is devoid of LDs, no punctate pattern customary to LDs
Specific punctate dots like pattern in the cytoplasm led to the
assumption that hydrophobic PITE might bind to the lipid-enriched
organelles like lipid droplets (LDs). To get more insight on the sub-
cellular localization of PITE, we performed costaining of yeast
cells with PITE and the commercially available LD-specific probe
Nile red (reference dye for LDs).[14a] The costaining studies were
carried out on yeast cells having a different amount of LDs (Figure
5). The cells were grown in growth media with and without
ammonium sulfate, costained with PITE and Nile red. It has been
observed that the yeast cells grown in ammonium sulfate-
deficient media accumulate more LDs.[27] In accordance with the
existing reports, we could detect more fluorescence in cells grown
in ammonium sulfate-deficient media. The colocalization of PITE
Figure 6. Microscopy images using PITE; panel A: wild type yeast cells, panel
B: LD-deficient yeast cells and panel C: isolated lipid droplets from yeast; (a,
e, i) DIC images, fluorescent images using (b, f, j) DAPI filter (blue channel,

ex = 335-383 nm, em = 420-470 nm), (c, g, k) GFP filter (green channel, ex
= 450-490 nm, em = 500-550 nm) and (d, h, l) merged images. Scale bar = 2
m.
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Figure 7. Microscopy images of HeLa cells costained with PITE and Nile red;
panel A: HeLa cells grown in DMEM media; panel B: HeLa cells grown in the
presence of 50 µM oleic acid for 6 h; (a, e) DIC images, fluorescent images
using (b, f) DAPI filter, (c, g) DsRed filter and (d, h) merged fluorescent images
of PITE and Nile red depicting colocalization in LDs. Scale bar = 5 m.
were observed (Figure 6, Panel A and B). Secondly, we isolated
LDs from yeast following a reported procedure.[29] The isolated
LDs of average diameter 0.2 -1 m were characterized by particle
size analyzer using dynamic light scattering (DLS) and
fluorescence microscopy (Figures S15, S16).[29b] Microscopic
investigations revealed that PITE could stain all the isolated LDs
(Figure 6, Panel C). We also costained the isolated LDs with PITE
and Nile red demonstrating colocalization of both the dyes (Figure
S17). Therefore, the study with quadruple mutants and isolated
lipid droplets unambiguously ascertain the specificity of PITE to
LDs.
We extended the application of PITE for LD staining in the
human cell line. The HeLa cell viability was estimated by 3-(4, 5-
dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide (MTT)
assay revealing biocompatibility (250 M in THF, Figure S18).
HeLa cells cultured in the absence and presence of oleic acid for
varying time periods and concentrations were stained with PITE
(Figures S19, S20) and costained with PITE and Nile red (Figure
7). The accumulation of LDs is increased when cells are grown in
media supplemented with oleic acid compared to that in normal
growth media. The sensitivity of PITE towards the different levels
of LDs in HeLa cells are clearly revealed in Figures 7 and S19,
S20. The colocalization with Nile red as indicated in the merged
fluorescent images (Figures 7d, h) suggest the specificity of PITE
to LDs in HeLa cells. The specificity was further confirmed by
confocal laser scanning microscopy (CLSM, Figure 8). The
fluorescence intensity profiles of PITE and Nile red obtained from
HeLa cells costained with both the dyes vary in a close synchrony
Figure 9. The percentage decrease of fluorescence intensity of PITE in HeLa
cells with the increasing scanning time under the constant laser irradiation (0.3
mW, 1% of 30 mW laser power). Inset: CLSM images of HeLa cells stained
with PITE with increasing time of irradiation. Excitation Laser: 405 nm,
emission filter: 415–585 nm, scale bar = 5 µm.
(Figure S21). The Pearson’s correlation coefficient was found to
be 0.70 (Figure S21c). Even though Pearson’s correlation
coefficient is unambiguously suggesting colocalization,[30] the
deviation from unity is due to the back-ground fluorescence of Nile
red in red channel compared to specific localization of PITE to
LDs revealed in the blue channel (Figures 8, S21). Since the
alteration in expression of LDs is associated with several diseases
including cancer,[13] PITE can be a suitable bioprobe acting as a
marker for specific and selective detection of LDs in diverse
organisms.
The fluorescent probe should be highly photostable for real-
time applications in cellular imaging. Continuous scanning of
PITE solution (5 M) by irradiation with 450 W xenon lamp (ex
=
415 nm) shows no significant decrease in fluorescence intensity
with time (Figure S22). The photostability of PITE in cellular
imaging was ascertained through CLSM. HeLa cells stained with
PITE were exposed to continuous irradiation of 30 mW laser
power at 405 nm (LSM 780, Zeiss). The fluorescence intensity of
PITE in HeLa cells was found to be good enough for imaging even
after 900 scans with a total irradiation time of 15 min (Figures 9,
S23). The strong fluorescence of PITE allows us carrying out the
imaging respectively with only 1% (0.3 mW) and even 0.2 % of
laser power (0.06 mW) for untreated (grown in normal growth
media, Figure 9, inset) and oleic acid-treated HeLa cells (Figure
S23). Thus, PITE is highly photostable and would be a suitable
probe to investigate dynamic behavior of LDs.
The next interesting question to address is the molecular level
picture of cellular uptake of PITE. It could be uptaken in the form
of molecules or nanoparticles, both states are sufficiently
fluorescent for cellular imaging. PITE solution in acetonitrile was
mixed with PBS buffer following identical conditions used for
cellular staining. The resultant aqueous dispersion (100 µM PITE)
was found to have nanoparticles with average diameter of 120-
150 nm as revealed by FESEM (Figure S24a). The dynamic light
scattering (DLS) measurements also supported the formation of
Figure 8. Specific targeting of LDs by PITE in HeLa cells probed by confocal
laser scanning microscopy (CLSM). HeLa cells costained with PITE and Nile
red: (a) DIC image, fluorescent images using (b) DAPI filter (blue channel, ex
= 405 nm, em = 415–585 nm, PITE emission), (c) TRITC filter (red channel, ex
= 561 nm, em = 570–750 nm, Nile red emission) and (d) merged fluorescent
image of PITE and Nile red depicting colocalization in LDs. Scale bar = 10 µm.
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Coulter Delso Nano C instrument was used for the dynamic light scattering
measurements. The morphology and size distribution of nanoparticles
were studied using a Carl Zeiss (Ultraplus) field emission scanning
electron microscope (FESEM).
The UV-Vis absorption spectra were recorded on an Agilent Cary 100
spectrophotometer. Diffuse Reflectance UV-Vis spectra were recorded
using integrating sphere attached to the spectrometer. Steady-state
fluorescence measurements were carried out on a Jobin Yvon Horiba
Model Fluorolog-3-21. All the fluorescence spectra were corrected with
respect to the excitation light intensity and photomultiplier tube (PMT)
response using the correction files available with Horiba software. Optical
density of the samples was maintained very low (≤ 0.1) by diluting with the
required amount of solvent to avoid any inner filter effect.
spherical aggregates (Figure S24b). Thus, PITE circumvents the
ACQ effect providing a facile platform of cellular imaging in the
form of nanoparticles. The broader absorption of the
nanoparticles allows imaging both in blue and green channels
(Figures 6, S11, S19). The spherical nanoaggregates of organic
hydrophobic dye with multiple alkyl substituents e.g., PITE is likely
to be more hydrophobic in nature.[31] The lipid droplets are
composed of neutral lipids (core region) and surrounded by the
phospholipids bilayer and dispersed in the aqueous cytoplasm
(pH 7.4).[23] Hydrophobic PITE nanoparticles being fluorescent at
physiological pH turn out to be a suitable probe for LD staining.
PITE nanoparticles localize at the most hydrophobic part of the
cell,[32] i.e.; LDs by hydrophobic interactions making the process
highly specific. A comparison with the reported LD-specific dyes
(Table S4) further ascertains the utility of PITE in selective
imaging of LDs.
Zeiss Axio Imager M2 Apotome.2 upright fluorescence microscope was
used for imaging of yeast and HeLa cells as well as isolated lipid droplets.
Zeiss Confocal Laser Scanning Microscope LSM780 was used for
fluorescence imaging and photostability experiments.
Growth conditions for yeast and HeLa cells:
Haploid yeast strains BY4741 (genotype: MATa his3Δ1 leu2Δ0 met15Δ0
ura3Δ0) and BY4742 (genotype: MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0)
were used for growth curve, spot assay and fluorescence microscopic
studies. Lipid droplets deficient yeast mutant strain; H1246 (genotype:
MATα are1Δ::HIS3 are2Δ::LEU2 dga1Δ::KanMX4 lro1Δ::TRP1 ADE2) and
its isogenic wild type SCY62 (genotype: MATa ADE2) were used.[28]
HeLa cells were grown on the coverslip, kept inside 35 mm sterile Petri
dish in DMEM media containing 10% FBS, antimycotic and antibiotic
(Thermo Fisher Scientific) in an incubator with 5% CO2 at 37 C.
Conclusions
A multifunctional bioprobe, PITE, based on pyridoindole and
tetraphenylethylene was shown to exhibit strong pH-dependent
fluorescence in solution, nanoparticles and solid state and in vitro
as well as in vivo specific targeting of LDs. The fluorescence
switching with the variation of pH was found to be reversible and
repeatable through several cycles. Detailed spectroscopic
investigations supported by 1H NMR studies and quantum
mechanical calculations provide a molecular level picture of the
fluorescence switching behavior. The pH-induced fluorescence
switching was further explored for the vapor-phase detection of
volatile organic acids like trifluoroacetic acid. The strong
fluorescence in different physical states coupled with
hydrophobicity, biocompatibility and high photostability render
PITE suitable for sub-cellular imaging. Comprehensive bio-
chemical and fluorescence microscopic investigations along with
isolation of LDs and subsequent colocalization studies
established the selective detection of LDs in lower and higher
eukaryotes by hydrophobic PITE nanoparticles. Employing PITE
for real-time tracking of LDs under various pathophysiological
conditions would be an interesting future scope to be pursued.
Another intriguing question, whether the specificity of PITE to LDs
is due to the binding to certain lipids or proteins present in LDs to
be explored further. One possible way would be to utilize gene
deletion mutants of yeast that encode LD-associated proteins and
lipids. Thus, self-assembly of PITE into supramolecular
nanoprobes with definite physicochemical characteristics paves
the way for the development of a promising molecular optical
material for pH-based sensing and lipid droplets tracking.
Synthesis of PITE
Precursor compounds 2-bromo-6,7,8,9-tetrapropylpyrido[1,2-a]indole-10-
carbaldehyde and 4,4,5,5-tetramethyl-2-(4-(1,2,2-triphenylvinyl)-phenyl)-
1,3,2-dioxaborolane were synthesized following the procedure established
earlier in our group (Scheme S1).[18] PITE was synthesized following a
minor modification of the reported procedure.[18] Details of synthesis and
characterizations of PITE was mentioned in the supporting information.
pH-induced fluorescence switching
The effect of pH on the fluorescence of PITE nanoparticles (Figure S6)
was probed by measuring the fluorescence intensity of nanoparticle
dispersion prepared using different pH buffer (Table S2). Nanoparticles
were fabricated by rapid injection of 60 μL of PITE solution in THF (1.4
mM) to 3 mL of the standard pH buffer solution.
Computational investigations
Ground-state geometry optimizations of PITE were carried out using
Gaussian 09 program package. The molecular structure obtained from
crystal structure analysis was taken as the input geometry. The same was
optimized at the B3LYP/6-31G(d) level. Further, the geometry was
optimized upon protonation of pyridoindole nitrogen.
Biocompatibility of PITE in yeast and mammalian cells
The effect of PITE on the growth of yeast cells was determined by spot
assay and growth curve analysis. Growth was determined by plating 10-
fold serial dilutions of wild type cells of two opposite mating types (BY4741
a and BY4742 α) on a synthetic complete agar plate. Plates were
incubated at 30C for 2-3 days, subsequent scanning of the plates
revealed the growth of the cells. The cell growth analysis of wild type yeast
cells (BY4741) in liquid media containing various concentrations of PITE
was performed using a 96-well plate. Optical density at 600 nm was
measured continuously at a regular interval (30 min) by microtiter
spectrophotometer (Eon BioTek).
Experimental Section
General Methods
Solution-state 1H and 13C NMR spectra were recorded on Bruker Avance
III 500 MHz NMR spectrometers. Fourier transform infrared spectroscopic
measurements were carried on PerkinElmer Model 2000 FTIR. Beckman
The viability of Hela cells in the presence of PITE was determined by
MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay.
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10.1002/asia.201700898
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Cells were first treated with different concentration of PITE for 1 h. For
control, equivalent numbers of cells were treated with respective solvents.
Subsequently, MTT (0.3 mg/mL) reagent was added to cells and incubated
for 3 h. After that DMSO was added and optical density of formazan
crystals (indicative of live cells) was measured at 570 nm.
Biomacromolecules 2016, 17, 148-153; f) L. Zhou, Q. Wang, Y. Tan, M.
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LD imaging in Yeast cells
Yeast cells were grown till stationary phase. 1 mL of the culture was
harvested by centrifugation at 5000 rpm for 5 min and washed twice with
distilled water. The cells were resuspended in solution containing PITE
(100 L, 100 µM) and were incubated at 30 C for 30-60 minutes.
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L 100-K). Isolated LDs were
characterized by DLS and fluorescence microscopy.
LD imaging in mammalian cells
HeLa cells were grown with or without oleic acid (optimal confluency 70-
80%) on the cover slip in 35 mm Petri dish. Cellular fixation was done using
3.7% of formaldehyde for 5 min and washed twice with PBS buffer (pH
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Acknowledgements
Financial support from BRNS, DAE (No. 37(2)/14/06/2016-
BRNS/37020), DST, SERB (EMR/2015/001797), New Delhi and
infrastructural support from IISERB are gratefully acknowledged.
BS thanks IISERB and PKT thank UGC for fellowship. We thank
Sten Stymne for providing LD-deficient quadruple mutant and
Saumitra Dey Choudhury with the help of CLSM.
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Keywords: pyridoindole • tetraphenylethylene • fluorescence •
imaging agents • lipid droplets • multifunctional • cellular imaging
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Entry for the Table of Contents
FULL PAPER
Multifunctional Fluorescent
Agents: We present a fluorescent
bioprobe based on pyridoindole and
tetraphenylethylene, PITE, showing
pH-responsive fluorescence
Bahadur Sk, Pilendra Kumar Thakre,
Raghuvir Singh Tomar,* Abhijit Patra*
Page No. – Page No.
A pyridoindole based
switching in solution, nanoparticle
and solid states leading to the
sensing of toxic organic acid vapor.
PITE is found to be highly specific for
lipid droplets (LDs) imaging in
diverse organisms under various
physiological conditions.
multifunctional bioprobe: pH-
induced fluorescence switching and
specific targeting of lipid droplets
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