A Dual-Function Indole-Benzimidazole Hybrid: Lipid Droplet Imaging and Anticancer Potential.

Introduction
Heterocyclic
compounds represent privileged
structural motifs that
serve as crucial targets in medicinal chemistry. Among these compounds,
benzimidazole is a significant class of heterocyclic compounds in
drug discovery and design, known for its broad range of pharmacological
activities, including analgesic, anti-inflammatory, antihistaminic,
anthelmintic, antiulcer, antiamoebic, and antiprotozoal effects, while the indole moiety represents a privileged
scaffold in medicinal chemistry, frequently encountered in drug discovery.

A central objective of medicinal chemistry
is the development of
novel compounds with optimized physical, chemical, and biological
properties. Combining the structural elements of two or more bioactive
substances into a single molecule can yield synergistic effects, enhanced
potency, or modulation of individual activities. These hybrid compounds not only enhance the pharmacological
profile but also effectively address challenges related to selectivity,
drug resistance, and suboptimal pharmacokinetic properties. Given the recognized significance of nitrogen-containing
heterocyclic scaffolds such as benzimidazole, indole, and naphthalimide
as key structural motifs in numerous medicinal compounds, the design
and synthesis of their hybrid molecules have emerged as a critical
strategy in medicinal chemistry. Among these hybrid scaffolds, indole-fused
benzimidazole derivatives have attracted considerable attention due
to their diverse and promising pharmacological activities, particularly
their anticancer and antiproliferative potential across multiple human
cancer cell lines. In addition to their anticancer effects, indole-fused
benzimidazole derivatives have been reported to exhibit antimicrobial,
antioxidant, antihypertensive, and other therapeutic properties, underscoring
their structural versatility and biological relevance
−

(Figure
).
Structures incorporating benzimidazole and indole
moieties have
been reported not only for their notable pharmacological activities
but also for their potential use as fluorescent stains in biological
imaging.
,−

Fluorescence imaging has become an essential tool
for studying biological molecules, pathways, and events in living
cells, tissues, and organisms. Fluorescent
labeling of live cells enables their visualization while maintaining
cellular viability. Representative indole-
and benzimidazole-based fluorescent dyes, such as DAPI and Hoechst
33258, are widely used DNA-specific probes that selectively bind to
the minor groove of AT-rich regions in double-stranded DNA and have
found extensive applications in cell imaging, flow cytometry, and
histochemical analyses
,
(Figure
).
Benzimidazole and indole are stable
owing to their
rigid planar
structures, abundant π-electrons, and aromatic ring π–π
conjugation, which together yield excellent fluorescence performance.
A recent fluorescence imaging study using a benzimidazole derivative
(compound 1) demonstrated that it can be taken up by
MDA-MB-231 cells, enter lysosomes, alter cellular ROS levels, and
cause mitochondrial damage (Figure
). Furthermore, benzimidazole-containing
compounds are used as lipid-droplet dyes. Lipid droplets (LDs) are
the main organelles responsible for intracellular lipid storage and
play a key role in maintaining lipid homeostasis. Beyond their role
as simple lipid reservoirs, LDs are involved in multiple cellular
processes, including signaling, transient protein storage, and protein
degradation. They also contribute to
membrane formation and provide an efficient source of energy. Under conditions of nutrient deprivation, LDs
play a critical role in regulating cellular energy balance by supplying
fatty acids for mitochondrial oxidation and ATP production. However, excessive lipid accumulation has been
implicated in the pathogenesis of metabolic disorders, including atherosclerosis
and insulin resistance in obesity and diabetes, and it also promotes
tumor growth and metastasis by providing energy and proliferative
signals.
,
If unregulated, lipids can become toxic
and induce cellular dysfunction and apoptosis, a phenomenon known
as lipotoxicity. Consequently, the availability
of reliable imaging tools is crucial for investigating the roles of
lipid droplets in the pathophysiology of diverse metabolic disorders. The most widely utilized LD probes are Nile
Red (compound 2) and BODIPY 493/503 (compound 3) (Figure
). The
molecular scaffolds used in the design of LD probes primarily comprise
hydrophobic fluorophores, such as coumarin, 1,8-naphthalimide, 3-hydroxyflavone,
benzoxadiazole, and polycyclic aromatic hydrocarbons. Moreover, Yoshihara and coworkers designed lipid droplet
probes (compounds 5–7), which can be used for
imaging of LDs in organs such as livers and kidneys of living mice,
based on C30 (compound 4). The phenanthrenequinone-imidazole fluorophore core (compound 8), synthesized by Lin and colleagues, demonstrates superior
fluorescence quantum yields and photostability relative to conventional
probes and additionally enables real-color imaging and tracking of
lipid droplets in living cells and tissues. The indole moiety-containing compounds (compounds 9–13) synthesized by Yu and colleagues exhibit lipid droplet-specific
properties such as low background, high selectivity, and biocompatibility
in live-cell imaging (Figure
).
Effective use of a
dye in fluorescent imaging requires key photophysical
properties, including high fluorescence, low cytotoxicity, and good
photostability, enabling long-term imaging of living cells. Notably, despite their utility for visualizing
lipid droplets in vitro, LD probes suffer from several inherent drawbacks,
including low sensitivity, inadequate selectivity, a small Stokes
shift, poor photostability, and high background interference. For example, Nile Red exhibits pronounced nonspecific
labeling of cellular lipid organelles, particularly intracellular
membranes. Furthermore, its broad absorption and emission spectra
produce significant cross-talk in the red channel, limiting its suitability
for multicolor imaging alongside other dyes or fluorescent proteins. Because it is nonfluorogenic, BODIPY 493/503
tends to generate background signals during imaging; it also exhibits
limited photostability, and its small Stokes shift can cause cross-talk
between the excitation source and the fluorescence emission.

To overcome these limitations, various
fluorescence phenomena have
been investigated and exploited in designing new and effective probes
for lipid droplet imaging. Although several commercial lipid droplet
(LD) probes have been developed, structurally diverse fluorophores
that combine real-color imaging capability with biocompatibility remain
limited; in particular, indole-benzimidazole-based probes specifically
targeting lipid droplets have not yet been explored. To address this
gap, we have designed a new LD probe, 5-methyl-2-(3-methyl-1H-indol-2-yl)-1H-benzo­[d]­imidazole, by hybridizing benzimidazole and indole moieties, inspired
by the fluorescence properties of DAPI and Hoechst 33258 (Figure
). Given the limited
investigation of its antitumor properties, the compound was evaluated
for activity against the human breast cancer cell line MDA-MB-231
using MTT assays, and its intracellular localization was examined
via fluorescence tracking. The findings not only provide a theoretical
basis for the anticarcinogenic and fluorescent properties but also
offer new insights into the rational design of next-generation indole-benzimidazole
derivatives.

Experimental Section

Chemistry
All
reagents were used as purchased from
commercial suppliers without further purification. Proton (1H) and carbon-13 (13C) nuclear magnetic resonance (NMR)
spectra were acquired on an Agilent 400 MHz spectrometer at 400 and
100 MHz, respectively, with tetramethylsilane (Me4Si) as
the internal standard. TLC was performed on 0.2 mm silica gel 60 F254
analytical aluminum plates. Liquid chromatography-tandem mass spectrometry
(LC-MS/MS) analysis was performed using a Thermo Scientific Q Exactive
spectrometer. Melting points were measured with a Stuart Melting Point
(SMP30) apparatus. All spectroscopic analyses were conducted at the
Science Research and Application Center of Van Yüzüncü
Yıl University.

General Procedure for the Preparation of
Compound 15
3-Methylindole (1 g, 7.63 mmol,
1 equiv) was added to a
mixture of phosphorus oxychloride (0.79 mL, 8.39 mmol, 1.1 equiv)
in anhydrous N,N-dimethylformamide (2.60 mL, 33.57
mmol, 4.4 equiv) after stirring for 20 min at room temperature. The
reaction mixture was then heated under reflux for 3 h. After monitoring
the reaction by thin-layer chromatography, the mixture was cooled
to room temperature and hydrolyzed with 10 mL of an aqueous sodium
acetate solution (15%). After stirring for 30 min, the mixture was
extracted with dichloromethane (3 × 10 mL). The combined extracts
were dried over MgSO4 and filtered. The solvent was evaporated, and
the crude product was purified over silica gel, eluting with hexane/EtOAc
(5:1), to give 15.

3-Methyl-1H-indole-2-carbaldehyde (15)
Pale-yellow solid. Yield:
30%. Mp. 138–139 °C. 1H NMR (400 MHz, CDCl3) δ 10.04 (s, 1H, CHO), 8.80 (bs, 1H, −NH), 7.71
(d, J = 8.2 Hz, 1H, Ar–H), 7.42–7.37
(m, 2H, Ar–H), 7.18–7.14 (m, 1H, Ar–H), 2.65
(s, 3H, −CH3). 13C NMR (100 MHz, CDCl3) δ 180.7, 137.6, 132.2, 128.3, 127.8, 125.1, 121.5,
120.5, 112.4, 8.6.

General Procedure for the Preparation of I-BZ
4-Methylbenzene-1,2-diamine (16) (0.278 g, 2.28 mmol,
1 equiv) was added to a stirred mixture of 3-methyl-1H-indole-2-carbaldehyde (15) (0.36 g, 2.28 mmol, 1 equiv)
and 39% NaHSO3 (1.83 g, 6.84 mmol, 3 equiv) in ethanol
(10 mL) at room temperature. The mixture was heated under reflux for
3 h. After monitoring the reaction by thin-layer chromatography, the
mixture was cooled to room temperature. The reaction mixture was then
transferred to 10 mL of ice water and stirred at room temperature
for 30 min. The resulting solid was filtered and air-dried. The crude
product was purified by silica gel chromatography, eluting with hexane/EtOAc
(5:1), to give I-BZ.

5-Methyl-2-(3-methyl-1H-indol-2-yl)-1H-benzo­[d]­imidazole (I-BZ)
White solid. Yield: 67%.
Mp. 140–142 °C. 1H NMR (400 MHz, CDCl3) δ 10.71 (s, 1H, indole-NH,
H-7), 7.60 (d, J
16,17 = 7.9 Hz, 1H, Ar–H,
H-16), 7.48 (d, J
17,16 = 7.9 Hz, 1H, Ar–H,
H-17), 7.36 (s, 1H, Ar–H, H-19), 7.19–7.03 (m, 4H, Ar–H,
H-1, H-2, H-3, and H-6), 2.69 (s, 3H, −CH3), 2.43
(s, 3H, −CH3). 13C NMR (100 MHz, CDCl3) δ 146.2, 136.5, 133.1, 129.0, 127.6, 124.6, 124.2,
123.9, 122.8, 121.6, 119.6, 119.2, 116.2, 111.6, 111.2, 21.7, 10.2.

1H NMR (400 MHz, DMSO-d
6) δ 12.18 (bs, 1H, indole-NH, H-7), 11.39 (bs, 1H, benzimidazole-NH,
H-15), 7.57 (d, J = 7.8 Hz, 1H, Ar–H), 7.53–7.35
(m, 3H, Ar–H), 7.14 (t, J = 7.3 Hz, 1H, Ar–H), 7.01
(t, J = 7.2 Hz, 2H, Ar–H), 2.62 (s, 3H, −CH3), 2.41 (s, 3H, −CH3). LC-MS/MS [M + H];
Calculated for C17H15N3: 262.1339;
Found: 262.1339.

Solvent-Dependent Absorption and Fluorescence
Measurements
The absorption spectra of I-BZ were
recorded in ten
solvents with varying polarity, hydrogen-bonding ability, and acid–base
character (acetonitrile, methanol, ethanol, dichloromethane, chloroform,
acetone, tetrahydrofuran, water, acetic acid, and triethylamine) using
a BMG ClarioStar UV–vis spectrophotometer. Solutions of I-BZ (10 μM) were prepared fresh before each measurement.
Fluorescence emission spectra were measured under identical conditions
using the BMG ClarioStar plate reader. Excitation wavelengths were
scanned from 320 ± 10 nm to 400 ± 10 nm, and emission spectra
were recorded over the corresponding range. All measurements were
performed at room temperature. The spectra were corrected for solvent
refractive index when applicable.

Cell Studies

Cell Culture
The human breast cancer cell line MDA-MB-231
was obtained from the American Type Culture Collection (ATCC). MDA-MB-231
cells were cultured as a monolayer in Dulbecco’s Modified Eagle
Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS,
Gibco), 4.5 g/L l-glutamine, 1 mM sodium pyruvate, 100 U/mL
penicillin-streptomycin, and 1% Amphotericin B. Noncancerous human
breast epithelial cells, MCF-10A (ATCC, Manassas, VA), from our lab
stock were used. The MCF-10A cell line was cultured in DMEM F-12 (Sartorius,
Germany) supplemented with insulin (10 μg mL–1), hydrocortisone (500 μg mL–1), and EGF
(20 ng mL–1) 36 Cultures were maintained in a humidified
incubator at 37 °C with 5% CO2. Cell morphology and
general condition were monitored daily using phase-contrast microscopy.
When cultures reached 70–80% confluence, cells were detached
with 0.25% trypsin-EDTA and passaged for continued growth.

MTT Cell Viability Test
The cytotoxic effect of the I-BZ on the MDA-MB-231 and MCF-10A cell lines was evaluated
using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] assay. Cells were seeded into 96-well plates at 5 ×
103 cells per well and incubated overnight to ensure viability.
Subsequently, cells were treated with various concentrations of the
compound (5–80 μM) for 24 and 48 h. After incubation,
10 μL of MTT (5 mg/mL) was added to each well, and plates were
incubated at 37 °C for 4 h. The
resulting formazan crystals were dissolved in 50 μL of DMSO,
and absorbance was measured at 570 nm using a microplate reader. Cell
viability was expressed as a percentage relative to the untreated
control, which was considered 100%.

General Cell Morphology
Analysis
Cells were prepared
for imaging according to a previously described protocol. Briefly, MDA-MB-231 cells were seeded onto 15
mm poly-l-lysine-coated glass coverslips in 6-well plates
and incubated for approximately 24 h to allow adhesion and recovery
of normal morphology. After incubation, cells were fixed with 4% paraformaldehyde
(PFA) for 30 min, washed with phosphate-buffered saline (PBS), and
imaged on an inverted microscope. To enhance the contrast between
the nucleus and cytoplasm, cells were stained with a standard hematoxylin
and eosin (H&E) protocol: cells were immersed in hematoxylin for
1 min, followed by eosin for 30 s. After staining, coverslips were
mounted with mounting medium and visualized under a bright-field light
microscope.

Oil Red O Staining
Intracellular
lipid droplets were
detected histologically using the Oil Red O (ORO) staining method.
The working solution was freshly prepared by diluting the ORO stock
solution (0.3% in isopropanol) with distilled water at a 3:2 ratio,
then filtered through a 0.22 μm membrane filter to remove precipitates.
Cells cultured on 15 mm glass coverslips were washed three times with
PBS (3 × 5 min), fixed with 4% paraformaldehyde for 30 min, and
washed again with PBS. After fixation, cells were incubated in 60%
isopropanol for 5 min to enhance lipid droplet retention, followed
by two washes with distilled water. Cells were stained with the Oil
Red O working solution for 15 min at room temperature. Excess stain
was removed by three consecutive washes with distilled water. To counterstain
cell nuclei, samples were incubated in hematoxylin for 1 min, followed
by two washes with tap water. Finally, the stained coverslips were
mounted with an aqueous mounting medium, and images were captured
using an Olympus BX53 bright-field microscope equipped with a DP74
digital camera.

Immunofluorescence Staining
Immunofluorescence
staining
was performed to evaluate the intracellular localization and fluorescence
characteristics of I-BZ in both fixed and live MDA-MB-231
cells. For fixed-cell analysis, MDA-MB-231 cells grown on glass coverslips
were fixed with 4% paraformaldehyde (PFA) for 20 min. The fixed cells
were then permeabilized with 0.25% Triton X-100 for 10 min and rinsed
three times with PBS. To prevent nonspecific binding, the cells were
blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at room
temperature. Following blocking, the cells were incubated with I-BZ (0.5 μM) for 30 min at room temperature and washed
thoroughly with PBS to remove unbound dye. To assess the live-cell
imaging potential of I-BZ, unfixed cells were incubated
with I-BZ under standard culture conditions for 6 h prior
to imaging.
For visualization of lipid droplets, fixed cells
were used. After staining with I-BZ as described above,
cells were washed three times with PBS and then incubated with 0.1
μg/mL Nile Red in 150 mM NaCl for 15 min at room temperature
to selectively label intracellular lipid droplets. The stained cells
were washed again with PBS to remove residual dye. Finally, coverslips
were mounted with a DAPI-containing antifade mounting medium, and
fluorescence images were acquired on an Olympus BX53 fluorescence
microscope equipped with a DP74 digital camera.

Induction
of Hypoxic Conditions
To simulate hypoxic
conditions, MDA-MB-231 cells were treated with 200 μM cobalt
chloride (CoCl2). Cells were
incubated in culture medium supplemented with CoCl2 for
48 h under standard cell culture conditions (37 °C, 5% CO2). After treatment, lipid droplet accumulation was assessed
using appropriate staining techniques.

Apoptotic Gene Expression
Analysis with QPCR
MDA-MB-231
cells were incubated with the I-BZ compound at an IC50 concentration (15 μM) for 48 h, after which total
RNA was isolated using TRIzol reagent (Invitrogen, USA) according
to the manufacturer’s instructions. cDNA synthesis was performed
from the isolated RNA samples using reverse transcriptase; then, the
expression levels of the BCL-2, BAX, and Caspase-3 genes were analyzed
by quantitative real-time PCR (qPCR). Gene expression data were normalized
to the beta-actin gene used as an internal control, and relative mRNA
changes were calculated using the 2–ΔΔCt method. For statistical comparisons, the control group expression
level was fixed at 1 (fold change), and fold changes for I-BZ application on the target genes were determined relative to this
reference value.

Results and Discussion

Design and Synthesis
The synthesis of the indole-fused
benzimidazole-based fluorescent probe I-BZ is shown in Scheme
. 3-Methyl-1H-indole-2-carbaldehyde (15) was synthesized
from 3-methylindole (14) via a Vilsmeier–Haack
reaction. The target compound, 5-methyl-2-(3-methyl-1H-indol-2-yl)-1H-benzo­[d]­imidazole
(I-BZ), was obtained by reacting compound 15 with 4-methylbenzene-1,2-diamine
(16) in the presence of sodium bisulfite (NaHSO3), affording the product in 65% yield.
The structure of I-BZ was confirmed
by NMR and LC-MS/MS
analyses. In the 1H NMR spectrum of the molecule recorded
in CDCl3, only the −NH proton signal of the indole
moiety was detected. The −NH protons of the benzimidazole ring
were not observed, likely due to rapid exchange at the nitrogen atoms
on the NMR time scale
,
(Figure S3). To address this, the spectrum was recorded in DMSO-d
6, which allowed clear observation of both the indole
and benzimidazole −NH signals (Figure S5). This behavior can be attributed to hydrogen-bonding interactions
between the −NH protons and the sulfoxide group, which slow
proton exchange. Consequently, the restricted motion of the −NH
protons enables their detection on the NMR time scale, providing insight
into the dynamic behavior of these labile protons in different solvent
environments.
,
The 13C NMR data indicate
that the aldehyde carbon signal of the starting compound 15 at 180.7 ppm disappeared, while a new methyl carbon signal emerged
in the aliphatic region, and the number of signals in the aromatic
region increased. The 1H and 13C NMR spectra
exhibited signals consistent with the proposed structure, and the
LC-MS/MS data showed a molecular ion peak corresponding to the expected
molecular weight, collectively supporting the correct structural assignment
of I-BZ.
LD probes employ mechanisms such as intramolecular
charge transfer
(ICT), aggregation-induced emission (AIE), and excited-state intramolecular
proton transfer (ESIPT) to precisely monitor changes in the polarity
and viscosity of lipid droplets (LDs), thereby enabling accurate disease
assessment. In general, a molecule can
exhibit ESIPT fluorescence if its structure features an intramolecular
hydrogen-bonding interaction between a hydrogen-bond donor (such as
a hydroxyl or amino group) and a hydrogen-bond acceptor (such as a
carbonyl oxygen or azo nitrogen). The
most widely studied ESIPT fluorophores are derivatives of 2-(2′-hydroxyphenyl)­benzimidazole
(HBI), 2-(2′-hydroxyphenyl)­benzoxazole (HBO), and 2-(2′-hydroxyphenyl)­benzothiazole
(HBT). Other reported ESIPT fluorophores include quinolines, benzophenones,
flavones, anthraquinones, benzotriazoles, N-salicylidene
aniline, and quinoxalines. ESIPT-based
probes facilitate lipid droplet imaging by undergoing proton transfer
in response to changes in lipid polarity. ESIPT molecules exhibit characteristic dual fluorescence through
a four-level photophysical cycle involving the ground and excited
states of two distinct tautomers. For instance, 2-(2′-hydroxyphenyl)­benzoxazole
(HBO) preferentially exists in the enol (E) form in the ground state,
stabilized by an intramolecular hydrogen bond. Upon photoexcitation,
the excited enol (E*) rapidly undergoes an ESIPT process to yield
the excited keto (K*) tautomer. Subsequently, the keto (K) form relaxes
to the ground state and returns to the initial enol form via a reverse
proton transfer. Based on the previous
studies, fluorescence properties of LD probe I-BZ can
be explained with the occurrence of the ESIPT process.
,−

The indole-fused benzimidazole compound in the excited state
(S1, N*) is proposed to convert to its phototautomer (S1, T*) via an ESIPT process. Accordingly, an intramolecular
proton
transfer occurs from the proton-donor indole −NH group to the
proton-acceptor benzimidazole −CN group. The tautomer
returns to the ground state (S0, T) upon emission and undergoes
GSIPT to regenerate its initial form (S0, N) (Figure
).

Photophysical Properties
of the Indole-Fused Benzimidazole (I-BZ) in Different
Solvents
To evaluate the proposed
ESIPT-based fluorescence mechanism, the absorption and emission spectra
of the indole-fused benzimidazole compound were recorded in ten solvents
spanning a range of polarity, hydrogen-bonding ability, and acid–base
character (Figures
and ). As shown in Figure
, the absorption spectra show only minor
variations across solvents, with no pronounced shifts in the absorption
maxima. This suggests that the ground-state electronic transitions
are only weakly influenced by the solvent environment.
In contrast, the fluorescence
emission spectra
show clear solvent-dependent
intensity changes (Figure
). Emission is more pronounced in acidic and protic solvents
such as acetic acid, methanol, and ethanol, whereas emission intensity
decreases significantly in aprotic and basic solvents, including dimethylformamide,
triethylamine, and pyridine. The enhanced emission in acidic media
may be associated with the preservation of intramolecular hydrogen
bonding between the indole −NH proton donor and the benzimidazole
−CN proton acceptor, which can facilitate proton-transfer-related
processes in the excited state. Conversely, quenching in basic solvents
may reflect solvent-induced disruption or weakening of the intramolecular
hydrogen bond, potentially suppressing ESIPT pathways and reducing
emission efficiency.
Additionally, the Stokes shifts between
absorption and emission
maxima support the possibility that the emission originates from a
structurally relaxed excited state. Overall, the weak solvent dependence
of absorption, combined with the strong solvent dependence of emission,
provides experimental observations consistent with the proposed ESIPT
mechanism.

Effect of I-BZ Molecule on Cell
Viability in MDA-MB-231
Cells
MDA-MB-231 cells were seeded at 5000 cells per well
to assess the potential cytotoxicity of I-BZ during lipid
droplet staining. One day later, I-BZ was applied to
the cells at a dose range of 5–80 μM, and cell viability
was assessed 24 h later using the MTT method. The I-BZ molecule was used at concentrations below 0.5 μM for live-cell
staining. Our cell viability assay showed no toxicity even at 5 μM,
which is 10-fold the dose used for live staining (Figure
). This result demonstrated
that I-BZ can stain live cells for 24 h without causing
toxicity. The 24-h results indicated that this molecule exhibited
anticancer activity at high doses. In this study, cell viability was
also monitored at 48 h to more clearly demonstrate the therapeutic
efficacy of this molecule. The IC50 value of the I-BZ molecule in MDA-MB-231 breast cancer cells at 48 h decreased
significantly, reaching 15 μM (Figure
). Normal breast epithelial cells (MCF-10A)
were cocultured for 48 h to evaluate the effects of I-BZ on healthy, noncancerous cells. Cell viability was analyzed. The
48-h IC50 dose of I-BZ in MCF-10A cells was
found to be 37 μM (Figure S7). The
selectivity index of I-BZ was 2.46. These results demonstrate
that I-BZ, which is nontoxic at low doses during live
staining, exhibits anticancer properties at doses above a certain
threshold, is less toxic to normal cells, and provides therapeutic
diagnostic effects.
The effects of the I-BZ compound on
MDA-MB-231 cells
were evaluated through changes in the expression levels of BAX, BCL2,
and Caspase-3 genes, which are molecular markers of apoptosis. qRT-PCR
results revealed that I-BZ application caused a statistically
significant increase in pro-apoptotic BAX and Caspase-3 expression
compared to the control group (Figure
a–b), while significantly suppressing antiapoptotic
BCL2 expression (Figure
c). In the literature, apoptosis is defined as a programmed cell
death mechanism that cells use for homeostasis, and it is well established
that cancer cells often survive by inhibiting this pathway. Considering that the Bax protein triggers apoptosis
by increasing mitochondrial membrane permeability and that Bcl-2 plays
an inhibitory role in this process, the
increase in the Bax/Bcl-2 ratio observed in our study provides strong
evidence that I-BZ activates the intrinsic apoptotic
pathway. Furthermore, the upregulation of Caspase-3, a critical component
of the caspase cascade, confirms that I-BZ-induced cytotoxicity
occurs via a caspase-dependent cell death mechanism. Taken together,
these findings indicate that I-BZ exhibits strong antitumor
potential by overcoming apoptotic resistance in MDA-MB-231 breast
cancer cells.

Morphological Characterization of MDA-MB-231 Cells
The general
morphology of MDA-MB-231 breast cancer cells was first
examined under an inverted light microscope to establish baseline
cellular characteristics prior to fluorescence imaging studies. As
shown in Figure
,
the cells exhibited the typical elongated, spindle-like morphology
and strong adherence to the culture surface, consistent with the characteristic
phenotype of MDA-MB-231 cells. To further distinguish cellular compartments,
cells were subjected to hematoxylin-eosin (H&E) staining. To better
distinguish cellular compartments, a distinct contrast between the
nuclear and cytoplasmic regions was observed in H&E-stained cells
(Figure
). The intracellular
lipid droplet content of the cells was subsequently visualized using
Oil Red O staining (Figure
). Oil Red O staining, a well-established method for visualizing
intracellular lipid droplets, was employed
to confirm the presence and distribution of lipid-rich regions within
MDA-MB-231 cells. Numerous red, spherical lipid droplets were observed
within the cytoplasm, confirming the presence of lipid-rich domains
in MDA-MB-231 cells.

Assessment of Fluorescent Staining with I-BZ in
Fixed and Live Cells
Fluorescence-based dyes are indispensable
tools in cell imaging, offering high sensitivity, spatial resolution,
and molecular specificity that enable visualization of intricate cellular
structures and dynamic biological processes. The design and discovery of novel fluorescent dyes with enhanced
photostability, biocompatibility, and selective organelle targeting
are therefore of great significance for advancing bioimaging research.
In particular, the development of lipid droplet (LD)-targeting probes
has attracted increasing attention because of the emerging roles of
LDs in cancer biology and metabolic regulation. In this context, we
examined the fluorescence properties and cell-staining performance
of I-BZ to assess its potential as a fluorescent dye
in both fixed- and live-cell models.
Initially, we aimed to
investigate the potential of I-BZ in fluorescence-based
bioimaging. For this purpose, the staining activity of I-BZ was evaluated by using a fluorescence microscope in both fixed and
live MDA-MB-231 cells. Initially, staining of fixed cells revealed
bright, punctate fluorescence localized in the cytoplasm, a staining
pattern characteristic of lipid droplet-targeting probes rather than
diffusely distributed cytosolic or nuclear dyes, indicating that the
compound stains intracellular lipid droplets (Figure
). This punctate cytoplasmic distribution
is consistent with previously reported LD probes, such as Nile Red
and BODIPY derivatives, which preferentially accumulate in neutral
lipid-rich compartments. It is well-documented that alcohol-based
fixation or solvent treatment can induce morphological alterations
of lipid droplets structures. To further
confirm the localization of I-BZ within lipid droplets,
cells were treated with ethanol (EtOH), which is known to perturb
lipid-rich structures and disrupt lipid droplet integrity. Upon ethanol
treatment, the characteristic punctate fluorescence pattern of the
probe was significantly reduced or dispersed, consistent with LD disruption.
This observation supports the fact that the compound selectively accumulates
in lipid-rich compartments within the cytoplasm. Unlike typical membrane-bound
organelles enclosed by phospholipid bilayers, lipid droplets are surrounded
by a single phospholipid monolayer that stabilizes the neutral lipid
core. The relatively fragile nature of this monolayer makes lipid
droplets particularly susceptible to disruption by alcohols and other
organic solvents. Previous studies have shown that ethanol increases
lipid disorder and permeability in phospholipid membranes and can deform or fuse lipid droplets in fixed
cell structures. Therefore, the loss
or dispersion of punctate fluorescence observed after EtOH treatment
supports the localization of I-BZ within lipid droplets
(Figure
).
To assess the duration of fluorescent staining
activity, stored
stained samples were visualized using a fluorescence microscope after
1 week. Although fluorescence intensity decreased relative to the
initial staining, lipid droplets in the cell cytoplasm remained stained.
This residual fluorescence suggests reasonable photostability and
retention within LDs, which are critical parameters for prolonged
imaging experiments. After demonstrating fluorescence staining in
fixed cells, I-BZ was added to MDA-MB-231 cell culture
medium and incubated for 6 h to evaluate its imaging properties in
living cells. The images we obtained demonstrated that I-BZ has the potential to stain lipid droplets in living cells (Figure
), indicating sufficient
cellular permeability and minimal acute cytotoxic effects during imaging.
Nile Red is a widely used fluorescent dye with a selective affinity
for intracellular lipid droplets, making it a valuable tool for their
visualization and quantitative analysis in cellular studies. To substantiate the intracellular localization
of I-BZ, MDA-MB-231 cells were costained with Nile Red,
a well-established fluorescent marker for lipid droplets. I-BZ (green) and Nile Red (red) exhibited overlapping fluorescence patterns
within the cytoplasm, and the merged images revealed extensive colocalization
(Figure
). This
strong spatial overlap demonstrates that I-BZ targets
the same intracellular lipid-rich compartments as Nile Red, positioning
it among established LD probes. Compared with Nile Red, which exhibits
environment-dependent spectral shifts and background fluorescence
in less hydrophobic regions, the well-defined punctate staining pattern
observed for I-BZ suggests a favorable selectivity for
neutral lipid cores. To further enhance visualization, a 3D cell-surface
plot was generated from the immunofluorescence images, enabling a
clear distinction of lipid-droplet-stained regions within the cytoplasm
(Figure
). The observed
spatial concordance of the two signals provides compelling evidence
that I-BZ selectively accumulates in lipid droplets,
thereby confirming its specificity for these lipid-rich organelles
and reinforcing its potential as a targeted fluorescent dye for lipid
droplet imaging in cells. Collectively, these findings suggest that
the indole-benzimidazole hybrid scaffold of I-BZ represents
a structurally simple and promising platform for lipid droplet imaging,
with the potential to complement established LD probes such as Nile
Red and BODIPY, while supporting further exploration of indole-benzimidazole-based
LD-targeting fluorophores.

I-BZ Prominently Stains Lipid Droplet Structures
Increased under Hypoxic Conditions
Cancer cells increase
lipid droplet accumulation under stress conditions. Treatment of MDA-MB-231 cells with CoCl2, a
hypoxia-mimicking agent, induced the formation of small, spherical
structures in the cytoplasm, observable by inverted microscopy (Figure
). Consistent with
previous reports describing hypoxia-induced lipid storage, ORO staining
confirmed a marked increase in lipid droplet content, appearing as
bright red cytoplasmic inclusions (Figure
). Importantly, I-BZ effectively
labeled lipid droplets as distinct green, spherical structures, confirming
its specificity for these organelles (Figure
). The strong concordance between ORO staining
and I-BZ fluorescence indicates that the probe reliably
tracks changes in lipid droplet abundance induced by metabolic stress.
These findings are particularly relevant in the context of earlier
studies highlighting the importance of LD-targeting fluorescent probes
for monitoring lipid metabolism in cancer cells under pathological
conditions. This behavior further underscores the suitability of I-BZ as a structurally simple, biocompatible, and selective
fluorescent probe for monitoring lipid droplet alterations associated
with dynamic cellular metabolic changes.
The fluorescence behavior of I-BZ was
first compared
with that of the ICT-based solvatochromic lipid-droplet probe Nile
Red. Its emission maximum remained nearly constant across solvents,
while intensity was highest in acidic media and lowest in basic or
aprotic environments, indicating that solvent effects are primarily
governed by ESIPT-mediated intramolecular proton transfer rather than
shifts in emission wavelength. In contrast, Nile Red shows changes
in both emission intensity and wavelength in response to solvent polarity
and is largely independent of proton transfer30, highlighting the
complementary environmental information I-BZ provides.
The comparison was then extended to the π → π*-based
fluorescent dye BODIPY, which exhibits bright, stable fluorescence
across various solvents with minimal changes in intensity or emission
wavelength and is largely insensitive to protonation or hydrogen-bonding
interactions. Finally, I-BZ was evaluated alongside classical ESIPT-based probes such as HBO,
HBI, and HBT. Like I-BZ, these probes utilize intramolecular
proton transfer in the excited state: HBO produces long-wavelength
emission sensitive to hydrogen bonding and solvent polarity, HBI exhibits
dual emission influenced by both ESIPT and intramolecular hydrogen
bonding, and HBT shows red-shifted emission reflecting microenvironmental
protonation and hydrogen bonding.
−

Compared with these
ESIPT probes, I-BZ combines pronounced solvent-dependent
intensity with a nearly constant emission maximum.
The emission
maximum of I-BZ remains essentially constant
across solvents, providing a reliable spectral reference, while its
fluorescence intensity is highly sensitive to environmental factors
and protonation states due to ESIPT-mediated intramolecular proton
transfer. This property enables mechanistic insights into microenvironmental
protonation and hydrogen-bonding interactions, which is particularly
valuable for monitoring lipid droplets and other cellular compartments.
Nevertheless, pronounced solvent-dependent intensity may complicate
quantitative analyses under uncontrolled conditions, and fluorescence
is substantially quenched in basic or aprotic media, potentially limiting
detectability. In comparison, classical dyes such as BODIPY exhibit
bright and stable fluorescence across a wide range of conditions,
highlighting the need for careful optimization when employing I-BZ to ensure consistent labeling and a reliable signal.
Overall, I-BZ provides unique environmental sensitivity
through ESIPT, but its practical application requires careful consideration
of solvent and protonation conditions to fully exploit its capabilities.

Conclusion
To summarize, we developed and characterized
a novel fluorescent
indole-fused benzimidazole probe, I-BZ, and demonstrated its strong
potential for imaging lipid droplets. I-BZ exhibits ESIPT-based
fluorescence and shows excellent biocompatibility at low staining
doses, while higher concentrations exhibit anticancer activity. Co-staining
with DAPI enabled clear nuclear visualization, providing spatial context
for I-BZ localization and confirming selective staining
of cytoplasmic lipid droplets.
Fluorescence imaging in both
fixed and live MDA-MB-231 cells confirmed
that I-BZ selectively accumulates in lipid droplets,
as supported by ethanol disruption assays and colocalization with
Nile Red. Furthermore, I-BZ effectively detected increased
LD formation under hypoxic conditions, underscoring its utility for
monitoring lipid metabolic changes in stressed cancer cells. Overall,
these results establish I-BZ as a promising ESIPT-based
fluorescent probe for selective LD visualization, offering a valuable
tool for studying lipid metabolism-related processes with potential
diagnostic and therapeutic applications.
Taken together, this
study positions I-BZ as a structurally
simple yet functionally effective indole-benzimidazole-based fluorescent
probe that complements established LD dyes such as Nile Red and BODIPY.
Although its ESIPT-based emission and selective LD localization are
key advantages, further studies on long-term photostability and broader
cell-type applicability will be valuable. Nonetheless, the present
findings expand the chemical space of LD-targeting fluorophores and
provide a foundation for developing next-generation probes to investigate
lipid metabolism-associated cellular dynamics.

Supplementary Material