Biotin-decorated NIR-absorbing nanosheets for targeted photodynamic cancer therapy

Article abstract of DOI:10.1039/d1ob00002k

Targeted photodynamic therapy (PDT) is one of the promising approaches for the selective killing of cancerous cells without affecting the normal cells, and hence designing new strategies for targeted PDT is extremely important. Herein we report the design and synthesis of a new class of nanosheets derived from the self-assembly of the iodo-BODIPY-biotin conjugate as a photosensitizer for targeted PDT applications. The nanosheet exhibits a high extinction coefficient in the NIR region, high singlet oxygen efficiency, no toxicity in the dark and cell targeting ligands (biotin) on the surface, which are necessary features required for an ideal photosensitizer. Overexpression of sodium-dependent multivitamin transporters (SMVTs) in HeLa and A549 (biotin receptor positive cell lines) is explored for the selective uptake of the nanophotosensitizer through receptor mediated endocytosis (interaction between biotin and SMVT). Control experiments using a biotin receptor negative cell line (WI-38) are also carried out to confirm that the specific interaction between the SMVTs and biotin is mainly responsible for the selective uptake of the photosensitizer. Efficient killing of cancerous cells is demonstrated upon light irradiation through the generation of singlet oxygen and other reactive oxygen species around the cellular environment.

Full text of DOI:10.1039/d1ob00002k

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PAPER
Biotin-decorated NIR-absorbing nanosheets for
targeted photodynamic cancer therapy†
Cite this: Org. Biomol. Chem., 2021,
19, 2804
Devanathan Perumal, Murali Golla, Kavya S. Pillai, Gowtham Raj,
Anusree Krishna P. K. and Reji Varghese
*
Targeted photodynamic therapy (PDT) is one of the promising approaches for the selective killing of can-
cerous cells without affecting the normal cells, and hence designing new strategies for targeted PDT is
extremely important. Herein we report the design and synthesis of a new class of nanosheets derived from
the self-assembly of the iodo-BODIPY–biotin conjugate as a photosensitizer for targeted PDT applications.
The nanosheet exhibits a high extinction coefficient in the NIR region, high singlet oxygen efficiency, no
toxicity in the dark and cell targeting ligands (biotin) on the surface, which are necessary features required
for an ideal photosensitizer. Overexpression of sodium-dependent multivitamin transporters (SMVTs) in
HeLa and A549 (biotin receptor positive cell lines) is explored for the selective uptake of the nanophotosen-
sitizer through receptor mediated endocytosis (interaction between biotin and SMVT). Control experiments
using a biotin receptor negative cell line (WI-38) are also carried out to confirm that the specific interaction
between the SMVTs and biotin is mainly responsible for the selective uptake of the photosensitizer. Efficient
killing of cancerous cells is demonstrated upon light irradiation through the generation of singlet oxygen
and other reactive oxygen species around the cellular environment.
Received 1st January 2021,
Accepted 9th March 2021
DOI: 10.1039/d1ob00002k
rsc.li/obc
sensitizers for PDT applications.3 However, due to their
extended π-conjugated backbone they tend to aggregate via
1. Introduction
Treatment of cancer without affecting normal cells is extremely π-stacking during blood circulation, and hence they severely
challenging.1 Hence, there is always a growing demand for the suffer from poor water solubility. Moreover, they lack selecti-
design and development of new therapeutic approaches for vity for targeted cells and tissues. These drawbacks of mole-
targeted cancer treatment. Photodynamic therapy (PDT) is cular photosensitizers can be overcome to some extent by the
undoubtedly one of the efficient non-invasive therapeutic use of a nanocarrier as a delivery vehicle.4 Though nano-
approaches for treating cancer.2 It involves the photoexcitation carriers permit the delivery of photosensitizers at the desired
of a photosensitizer, preferably in the near infra-red (IR) location, their formulation is highly laborious. Moreover, tox-
region (700–800 nm), which then undergoes intersystem cross- icity of nanocarriers and their excretion from the body are
ing from the singlet excited state to the triplet excited state. other major concerns. Hence it is extremely challenging to
The triplet excited state of the sensitizer then acts as an energy develop a single step strategy for the designing of a photosen-
donor to the ground state of molecular oxygen (3O2) and other sitizer that exhibits (i) water solubility, (ii) excellent biocompat-
molecules present in the tissue, thereby generating highly cyto- ibility, (iii) targeted delivery, (iv) efficient singlet oxygen gene-
toxic reactive oxygen species (ROS) including singlet oxygen ration and (v) strong NIR absorption.
(1O2). This leads to cell death at the photoirradiated region.
Halogenated boron dipyrromethene (BODIPY) derivatives
Since the photosensitizer is typically non-toxic under darkness, are one of the most explored organic photosensitizers for PDT
PDT allows selective killing of photoexposed cancer cells, applications because of their desirable optical properties.5
leaving the unexposed healthy cells intact. This makes PDT a Traditionally, tethering of photosensitizers with a specific cell
superior therapeutic approach to others. Molecular systems targeting moiety allows the effective attack of targeted cells
based on organic dyes have been traditionally used as photo- leaving the normal cells unaffected, and this technique has
been successfully applied for targeted photodynamic therapy.6
More beneficial would be the functionalization of the hydro-
School of Chemistry, Indian Institute of Science Education and Research (IISER)
phobic photosensitizer with a hydrophilic cell targeting moiety
Thiruvananthapuram, Trivandrum-695551, Kerala, India.
so that the photosensitizer transforms into an amphiphile,
E-mail: reji@iisertvm.ac.in
which then spontaneously self-assembles in aqueous medium
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
into a supramolecular nanostructure.7,8 The shell of this kind
d1ob00002k
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Scheme 1 Synthesis of amphiphile 1 by using the CuAAC reaction and a schematic illustration showing the selective uptake of 1 nanosheet through
receptor mediated endocytosis resulting in targeted killing of cancer cells via PDT.
of nanostructure typically consists of hydrophilic cell targeting a strong NIR absorption is observed for the photosensitizer.
moieties and the core is made of a hydrophobic photosensiti- Biotin (vitamin H or vitamin B7) was selected as the hydro-
zer. Accordingly, such nanostructures not only offer excellent philic cell targeting moiety because of its known interaction
water solubility and cell targeting capability, but also provide with sodium-dependent multivitamin transporters (SMVTs),
the desired properties required for a photosensitizer including which are overexpressed in many cancer cells including HeLa
efficient singlet oxygen generation and high extinction coeffi- and A549.10 Self-assembly of the amphiphile in aqueous
cient in the NIR region. This suggests that amphiphilicity- medium resulted in the formation of biotin decorated
driven self-assembly is a simple yet efficient strategy for the nanosheets. The sheet formation is attributed to the extremely
design of nanophotosensitizers that afford all the features strong π-stacking of BODIPY in aqueous medium. The sheet
required for an ideal photosensitizer in a single step self- structure consists of π-stacked BODIPY as the hydrophobic
assembly process.
membrane, which is densely decorated on both sides with
Herein, we report the self-assembly of biotin tethered iodo- hydrophilic biotin. Very efficient generation of singlet oxygen
BODIPY amphiphile (1) into biotin-decorated nanosheets and is observed upon photoexcitation of the nanosheet with a red
their application in PDT for targeted cancer treatment. The light-emitting diode (LED). The overexpression of SMVTs on
design principle of the amphiphile 1 involves the covalent con- A549 and HeLa cell lines is explored for the selective uptake of
jugation of hydrophilic biotin into the hydrophobic iodo- the nanosheet into A549 and HeLa cells through specific inter-
BODIPY through a triazole linkage. Iodo-BODIPY was selected action of biotin and SMVTs. Targeted killing of the A549 cell
as the hydrophobic part of the amphiphile due to its desirable line is also demonstrated using the PDT approach (Scheme 1).
optical properties required for a photosensitizer including
absorption in the NIR region with a high extinction coefficient,
efficient singlet oxygen generation and excellent photostability.
The functionalization of the BODIPY core with iodine is to
facilitate the singlet oxygen generation through the “heavy
2. Results and discussion
2.1 Synthesis of the amphiphile
atom” effect.9 In order to extend the absorption of the photo- Synthesis of 1 was achieved through the Cu(I)-catalysed
sensitizer to the NIR region, the BODIPY core is extended with alkyne–azide cycloaddition (CuAAC) reaction as the key step
additional benzene rings through double bonds. Accordingly, (Scheme 1). Towards this end, the azide functionalized
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BODIPY core was initially synthesized by the reaction of 2,4-di- absorbance with broadening was observed with the addition of
methylpyrrole with 4-(3-azidopropoxy) benzaldehyde in the water to the DMSO solution (0→95%) of the amphiphile.
presence of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) Fluorescence analyses of 1 showed an intense emission of
and boron trifluoride ethyl etherate (BF3·OEt2) with trifluoroa- BODIPY at 700 nm (λexc = 635 nm) with a fluorescence
cetic acid (TFA) as the catalyst. Subsequently, the BODIPY core quantum yield (ϕf) of 20%. In accordance with the absorbance
was iodinated at the second and sixth positions using changes, a drastic quenching of fluorescence was observed
N-iodosuccinimide (NIS). The incorporation of triethylene with the gradual addition of water, and a complete quenching
glycol monomethyl ether was achieved by treating the iodo- of fluorescence (ϕf = ∼0%) was observed with the addition of
BODIPY core with 4-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy} 95% water (Fig. 1b). These optical changes clearly reveal that
benzaldehyde in the presence of p-toluenesulfonic acid and the amphiphile undergoes self-assembly into supramolecular
piperidine via the Knoevenagel condensation reaction. Alkyne nanostructures mainly driven by the π-stacking of the BODIPY
functionalized biotin was synthesized by treating biotin with core. Subsequently, detailed microscopic analyses were carried
N-hydroxysuccinimide in the presence of 1-ethyl-3-(3-dimethyl- out to understand the morphology of the nanostructure of 1.
aminopropyl)carbodiimide (EDC·HCl) followed by the reaction All microscopic analyses were carried out in a H2O : DMSO
with propargylamine. Finally, the CuAAC reaction between 1a (95 : 5) mixture. Atomic force microscopy (AFM) analyses of the
and 1b furnished 1 in good yield. All the compounds were pur- aggregated species of 1 showed the formation of nanosheets
ified by using column chromatography and characterized by (Fig. 1c). Section analyses of the nanosheets disclosed that the
1H, 13C spectroscopic and mass spectrometric analyses. Details height of sheets ranges from 2 to 200 nm, indicating that the
of the synthesis and characterization of 1 and its intermediates sheets are mono- to multilayered in nature. In support of this,
are provided in the ESI.†
transmission electron microscopy (TEM) analyses also showed
the formation of transparent nanosheets (Fig. 1d). The lateral
dimension of the sheets is in the order of several hundred
nanometers to a few micrometers. These results unequivocally
indicate that 1 undergoes amphiphilicity-driven self-assembly
mainly driven by the π-stacking of BODIPY to produce biotin-
decorated nanosheets. Since both biotin and triethylene glycol
moieties are hydrophilic in nature (bolaamphiphile), different
types of packings are possible for the amphiphile including
the following: (i) the sheet is decorated with biotin on one side
and triethylene glycol on the other side (monolayer), (ii) both
sides of the sheet are decorated with biotin (bilayer) or (iii) the
sheets can also be multi-layered. As a representative structure,
a bilayer sheet decorated with biotin on both sides are schema-
tically shown in Scheme 1.
2.2 Self-assembly of the amphiphile
Detailed photophysical studies were carried out to understand
the self-assembly behaviors of the amphiphile in aqueous
medium. Amphiphile 1 exists as monomeric species in DMSO
and exhibits a strong absorption peak in the near IR region
with the maximum centered at 664 nm (ε664 = 7.6 × 104 cm−1
M−1, Fig. 1a). Any photosensitizer having absorption in the
NIR region with a high extinction coefficient is highly desir-
able for PDT as NIR radiation is typically non-toxic and has an
efficient skin penetrability. Interestingly, a drastic decrease in
2.3 Singlet oxygen (1O2) generation studies of the nanosheet
The singlet oxygen production of the nanosheet was studied
1
using 1,3-diphenylisobenzofuran (DPBF) as a O2 scavenger as
reported in the literature.11 A sample of 1 : 40 molar solutions
of 1 nanosheet (1 equi.) and DPBF (40 equi.) in a mixture of
water and DMSO (H2O : DMSO = 95 : 5) was irradiated with red
LED light at 30 second intervals for 5 minutes (635 5 nm,
20–25 W) and the changes in the absorbance of DPBF at
419 nm were monitored as a function of time (Fig. 2). UV-spec-
tral analyses showed an almost complete disappearance of the
DPBF absorption peak at 419 nm within 3 minutes of
irradiation of the nanosheet solution, suggesting the photo-
induced production of singlet oxygen from the nanosheet as
observed with similar systems.12 It is also important to note
that no significant change was observed in the absorption
region corresponding to the nanosheet at 664 nm, indicating
its excellent photostability (Fig. 2 inset). The photostability of
the photosensitizer is one of the desired features required for
an ideal photosensitizer for PDT applications and hence our
results suggest that the nanosheet would be a potential candi-
date for PDT applications.
Fig. 1 (a) Absorption and (b) emission (λexc = 635 nm) spectral changes
with the addition of water to the DMSO solution of 1. (c) AFM height
(z-scale = 10 nm) and (d) TEM images of 1 nanosheet. Inset of (c) shows
section analysis of the sheet along the line drawn on the image.
Concentration of 1 in all the experiments is 20 μM.
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into the biotin receptor negative cell line WI-38 (Fig. 3c),
which shows almost no internalization. These results reveal
that the nanosheets internalize selectively into HeLa and A549
cells through the receptor-mediated endocytosis mechanism
and are distributed both in the nucleus and cytoplasm.13
Furthermore, flow cytometry studies were undertaken to quan-
tify the cellular uptake of the nanosheet, and the experiments
were carried out on HeLa, A549 and WI-38 cell lines by incu-
bating them with the nanosheets for different time periods.
The flow cytometry analyses show that the significant shift in
fluorescence is associated with both HeLa (Fig. 3d) and A549
cell lines (Fig. 3e) with respect to the control. On the other
hand, no fluorescence shift is observed for the WI-38 cell line
with respect to the control (Fig. 3f), indicating no uptake of
Fig. 2 Absorption spectral changes of DPBF at 419 nm treated with 1
nanosheet upon light exposure at 30 second intervals. [1] = 2 μM and the nanosheet by WI-38. Mean fluorescence intensities (MFIs)
[DPBF] = 80 μM.
of 551 and 456 were observed for A549 and HeLa when com-
pared to the corresponding untreated samples (control), which
have MFIs of 5 and 4, respectively. Confocal and flow cytome-
try analyses collectively suggest that the nanosheet exhibits a
2.4 Targeted cell internalization studies
magnificent selective permeability into HeLa and A549 cells
One of the major challenges in PDT is the selective targeting
whereas no internalization takes place for the biotin receptor
of cancerous cells without damaging healthy cells.
negative cell line WI-38.
Photodynamic therapy involves systematic administration of
the photosensitizer, followed by irradiation with light.
Photosensitizers are admirably harmless under darkness. If
targeted accumulation of the photosensitizer is achieved
within the cancer cells, then PDT could be a safe and promis-
ing modality for cancer treatment. One of the most remarkable
structural features of the nanosheet is the dense decoration of
its surface with cell targeting biotin moieties, and hence it
offers a unique opportunity for the targeted attack of biotin
receptor overexpressed cancerous cells. We explored the poten-
tial of the nanosheet as a photosensitizer for the targeted
photodynamic therapy. We chose A549, HeLa and WI-38 cell
lines as representative cell lines in our investigation. A549 and
HeLa cells are known to be biotin receptor positive cell lines
due to the overexpression of SMVTs. However, WI-38 has no
overexpression of SMVT and hence is considered as a biotin
receptor negative cell line. In order to investigate the selective
cell uptake of the nanosheet into HeLa and A549 cell lines, the
nanosheet was incubated with HeLa, A549 and WI-38 cell lines
for 24 h and analysed by CLSM analyses. Though nanosheets
of 1 was nearly non-fluorescent (ϕf = ∼0%), interestingly an
increase in fluorescence intensity was observed with the
addition of streptavidin into the nanosheet solution. This
suggests that the interaction of biotin with a receptor such as
streptavidin leads to the partial disassembly of the nanosheet
and leads to the increase in fluorescence intensity (Fig. S1†).
Hence the florescence of BODIPY can potentially be used for
CLSM imaging. Accordingly, fluorescence of BODIPY at
660 nm was used for the CLSM analyses. In order to ascertain
the location of the nanosheet inside the cells, the nuclei of the
cells were stained with 4′,6-diamino-2′-phenylindole (DAPI,
blue florescence). The CLSM analyses show the efficient intern-
alization of the nanosheets into the biotin receptor positive
2.5 Targeted PDT applications
Further, to validate the selective PDT efficiency of the
nanosheet, cytotoxicity studies were carried out using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay (Fig. 4). In vitro cytotoxic experiments were
carried out on A549 and WI-38 cell lines by incubating the
cells with the nanosheet for 1 h and irradiating them for 4 h
with red LED light (635 nm). MTT assays were also carried out
to test the cytotoxicity of the nanosheet in dark and in light
conditions. The viability of untreated cells with the nanosheet
in the dark was taken as 100%. As expected, MTT analyses of
the cells treated with the nanosheet in the dark show an
almost 100% cell viability, indicating that the nanophotosen-
sitizer is nontoxic in the absence of light. The untreated cells
with the nanosheet in light also show an almost 100% cell via-
bility, suggesting that the light used for PDT is not harmful to
the cell lines. The MTT analyses of the A549 cell line treated
with the nanosheet and irradiated with red LED light show a
cell death of 52%. Interestingly, no significant cell death (7%)
is observed for the WI-38 cell line treated with the nanosheet
and irradiated with red LED light. These results clearly imply
that the nanosheet selectively internalize into the biotin recep-
tor positive A549 cell line through the receptor mediated
endocytosis mechanism and leads to cell death by the gene-
1
ration of O2 and other reactive oxygen species in the cellular
environment upon irradiation. However, no notable internaliz-
ation takes place in the case of the biotin negative receptor
WI-38 cell line. Our results clearly demonstrate that the
biotin-decorated BODIPY-based nanosheet is a promising
nanophotosensitizer for targeted PDT applications and in vivo
experiments in this direction are progressing in our
cells HeLa (Fig. 3a) and A549 (Fig. 3b), when compared to that laboratory.
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Fig. 3 CLSM analyses of (a) HeLa, (b) A549 and (c) WI-38 cells after their incubation with the nanosheet for 24 h. Flow cytometry analyses to
monitor the binding of the nanosheet with (d) HeLa, (e) A549 and (f) WI-38 cell lines at different time intervals.
3. Conclusions
In summary, we have reported the design and synthesis of a
novel class of NIR absorbing, biotin decorated nanosheets
from the self-assembly of biotin–BODIPY amphiphile and its
potential for targeted PDT applications. Microscopic analyses
revealed the self-assembly of the amphiphile into biotin-deco-
rated nanosheets in aqueous medium primarily driven by the
π-π stacking of the BODIPY core. UV-vis absorption and fluo-
rescence studies have shown that the nanosheet exhibited a
high extinction coefficient in the NIR region and an excellent
singlet oxygen generation efficiency upon light irradiation.
Decoration of the surface of the nanosheet with cell targeting
biotin permitted selective cellular uptake through the receptor
mediated endocytosis mechanism. Excellent selectivity was
observed for biotin receptor positive cell lines when compared
to biotin receptor negative cell lines. Irradiation of the
nanosheet efficiently generated singlet oxygen in the cellular
environment and targeted killing of cancer cells was achieved.
Our strategy allows for the crafting of nanophotosentizers that
offer all the structural features necessary for a photosensitizer,
including (i) water solubility, (ii) excellent biocompatibility,
(iii) strong NIR absorption, (iv) efficient generation of singlet
oxygen and other reactive oxygen species and (v) targeted
Fig. 4 MTT assay of (a) WI-38 and A549 cells treated with 1 nanosheet
attack in a single-step self-assembly approach. Our results
clearly imply that the tethering of hydrophilic cell targeting
in the dark condition and (b) WI-38 and A549 cells treated with 1
nanosheet in the light condition.
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moieties to a hydrophobic photosensitiser of interest would be times. The cells were further permeabilised using 0.01%
a promising strategy for the design of nanophotosensitizers Triton X for 10 min and after washing with PBS three times,
for targeted PDT applications.
nuclear stain DAPI (10 μg mL−1) was added for 30 s and then
washed off. The cover slips were then mounted on a glass slide
with Prolong Gold Antifade Reagent for confocal imaging
using a SP5 Leica confocal microscope (Leica, Germany) with a
100× oil immersion object. Excitation and collection wave-
length ranges for DAPI are 405 nm and 420–500 nm. For 1
4. Experimental section
4.1 General methods
All chemicals used for the organic syntheses were purchased nanosheet 635 nm laser was used for excitation with the collec-
from Sigma Aldrich, Alfa Aesar or TCI Chemicals and were tion wavelength range of 650–750 nm. For FACS analysis,
used as received. Solvents were dried following the standard HeLa, A549 and WI-38 cells were placed in 6 well plates at a
procedures. TLC analyses were performed on aluminium seeding density of 0.6 × 106 and incubated for 24 h at 37 °C in
plates coated with the silica gel 60 F254. Column chromato- a 5.0% CO2 atmosphere to allow cell adhesion. The cells were
graphy was performed on 200–400 mesh silica gel. 1H and treated with 1 at 20 μM concentration and after 6 h of incu-
13C-NMR spectra were recorded on a 500 MHz Bruker Avance bation, FACS analyses were carried out on a FACS LSR Fortessa
spectrometer using tetramethylsilane (TMS) as internal stan- flow cytometer (BD, USA). For the MTT assay, A549 and WI-38
dard. Milli Q deionised water (18.2 MΩ cm) was used for all cells were seeded in 96-well plates at a seeding density of 3000
studies. GC-MS analyses were performed on a Shimadzu cells per well in complete media (100 μL) and incubated for
GC-MS, QP-2010. HRMS measurements were performed on an 24 h at 37 °C in a 5.0% CO2 atmosphere to allow cell adhesion.
Agilent Technologies 6538 Q-TOF-LCMS system. Absorption Prior to the treatment with 1 nanosheet, 100 μL of media was
spectra were recorded using a quartz cuvette of 10 mm path prepared and the concentration of 1 nanosheet was main-
length on a Shimadzu UV-3600 vis-NIR spectrophotometer. tained at 20 μM. Singlet oxygen generation experiments were
Steady state fluorescence spectra were recorded by using a carried out by irradiating 1 nanosheet and DPBF (1 : 40 molar
quartz cuvette of 10 mm path length on a Horiba Jobin Yvon ratio) in the DMSO : H2O mixture using a Rayonet photoreactor
Fluorometer. TEM analyses were carried out on an FEI Tecnai equipped with a 635 5 nm lamp (20–25 W) for different time
G2 F20 HR-TEM. Imaging was performed at 200 kV. Samples periods. For the MTT analysis, the cells incubated with 1
were prepared by depositing 2 μL of the sample on a 400-mesh nanosheet were irradiated with red LED light (635 5 nm,
carbon coated copper grid (Ted Pella, Inc.). The samples were 20–25 W) for 4 h.
allowed to adsorb on to the grid for 2 min, excess samples
were wicked with a piece of filter paper and then the grid was
dried under air. AFM analyses were carried out on Multimode
8. Samples were prepared by drop-casting 2 μL solution of the
sample on a freshly cleaved mica surface and dried under air.
Imaging was performed under ambient conditions in the
tapping mode. The probe used for imaging was an antimony
doped silicon cantilever with a resonant frequency of 300 kHz
and a spring constant of 40 N m−1. HeLa (human cervical
cancer) and WI-38 (human lung fibroblast) cells were cultured
in DMEM (HiMedia Laboratories Pvt. Ltd, India) sup-
plemented with 10% FBS (PAN-Biotech GmbH, Germany) and
Antibiotic Solution 100× liquid with 1000U penicillin and
10 mg streptomycin per mL (HiMedia Laboratories). A549
(human lung adenocarcinoma) cells were cultured in RPMI
(HiMedia Laboratories Pvt. Ltd, India) supplemented with
10% FBS (PAN-Biotech GmbH, Germany) and Antibiotic
4.2 Synthesis and characterization of 1
Solution 100× Liquid with 1000U penicillin and 10 mg strepto- In a two-neck round bottomed flask, 0.025 g (0.02 mmol) of
mycin per mL (HiMedia Laboratories). Cell lines were bor- compound 1a and 0.0126 g (0.024 mmol) of compound 1b
rowed from NCCS, Pune, and were obtained from the were dissolved in 5 mL of freshly distilled tetrahydrofuran and
American Type Culture Collection (ATCC). For confocal micro- the solution was degassed for 15 min. To this solution,
scopic analysis, HeLa, A549 and WI-38 cells were plated in 0.0076 g (0.04 mmol) of CuI and 0.01026 g (0.08 mmol) of
12 mm plates with coverslips (HiMedia Laboratories) at a DIPEA were added and the reaction mixture was stirred for
seeding density of 0.05 × 106 and incubated for 24 h at 37 °C 24 h at 60 °C. After the completion of the reaction, the residue
in a 5.0% CO2 atmosphere to allow cell adhesion. The cells was purified by using column chromatography with 10%
were treated with the nanosheet (20 μM) and then 24 h post methanol in dichloromethane to get the desired product 1 as a
incubation, the cells were fixed using 4% paraformaldehyde greenish solid (80%). TLC (methanol : dichloromethane,
solution for 10 min followed by washing with 1× PBS three 10 : 90), Rf = 0.3. 1H NMR (DMSO-d6, 500 MHz): δ (ppm) = 1.23
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(s, 6H), 1.46 (s, 6H), 2.09 (t, J = 7 Hz, 2H), 2.32 (t, J = 6 Hz, 2H),
2.57 (d, J = 12 Hz, 1H), 2.63 (s, 1H), 2.72 (s, 1H), 2.80 (q, J = 5
Hz, 2H), 2.88 (s, 1H), 3.07 (s, 1H) 3.23 (s, 6H), 3.43 (m, 4H),
3.52 (m, 3H), 3.54 (m, 3H), 3.59 (m, 3H), 3.76 (t, J = 4.5 Hz,
4H), 4.09 (m, 3H), 4.16 (t, J = 4 Hz, 4H), 4.29 (d, J = 5.5 Hz, 3H),
4.55 (t, J = 7 Hz, 2H), 6.35 (s, 1H), 6.41 (s, 1H), 7.07 (d, J = 8.5
Hz, 4H), 7.13 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.44
(d, J = 17 Hz, 2H), 7.58 (d, J = 8.5 Hz, 3H), 7.96 (s, 1H), 8.08
(d, J = 16.5 Hz, 2H), 8.29 (t, J = 5.5 Hz, 1H); 13C NMR
(DMSO-d6, 125 MHz): δ (ppm) = 17.73, 28.57, 48.21, 60.67,
60.70, 67.92, 69.32, 70.23, 70.27, 70.44, 72.80, 115.83,
123.38, 139.10, 146.03, 160.41; HR-MS (m/z): [M + Na]+ of
C63H77BF2I2N8O11SNa: 1479.23 (cal.), 1479.35 (expt.). Not all
the peaks of 1 were fully resolved in the 13C-NMR spectrum
due to its poor solubility.
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Financial supports from DBT (BT/PR30172/NNT/28/1593/-
2018) and CSIR are gratefully acknowledged.
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