Coumarin-benzothiazole-chlorambucil (Cou-Benz-Cbl) conjugate: An ESIPT based pH sensitive photoresponsive drug delivery system

Article abstract of DOI:10.1039/c4tb02081b

We have developed an ESIPT based drug delivery system (DDS), Cou-Benz-Cbl conjugate, by incorporating a benzothiazole group at the 8th position of the 7-hydroxy-coumarin moiety for pH sensitive fluorescence properties and photocontrolled release of the anticancer drug chlorambucil. The Cou-Benz-Cbl conjugate exhibited unique photophysical properties like good absorbance at around 350 nm, a large Stokes shift (～151 nm) and pH sensitive fluorescence properties. The pH sensitive fluorescence properties of the Cou-Benz-Cbl conjugate can be ascribed to an ESIPT turn "on and off" mechanism. At physiological pH, the ESIPT gets turned "off" and a blue fluorescence of the coumarin moiety was observed, but at acidic pH, the ESIPT gets turned "on" and a green fluorescence was noted. Photolysis of the Cou-Benz-Cbl conjugate using UV light of wavelength ≥365 nm resulted in the efficient release of the anticancer drug chlorambucil. Cellular uptake studies revealed that the Cou-Benz-Cbl conjugate was easily internalized inside the cancer cells. Further, an MTT assay showed that the Cou-Benz-Cbl conjugate has a good biocompatibility and low cytotoxicity towards the MDA-MB-231 cell line, whereas upon exposure to UV light, the Cou-Benz-Cbl conjugate exhibited enhanced cytotoxicity compared to the free drug due to the effective release of the anticancer drug chlorambucil inside the cancer cell.

Full text of DOI:10.1039/c4tb02081b

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Coumarin–benzothiazole–chlorambucil
(Cou–Benz–Cbl) conjugate: an ESIPT based pH
sensitive photoresponsive drug delivery system†
Cite this: J. Mater. Chem. B, 2015,
3, 3490
Shrabani Barman,a Sourav K. Mukhopadhyay,b Moumita Gangopadhyay,a
Sandipan Biswas,a Satyahari Dey*b and N. D. Pradeep Singh*a
We have developed an ESIPT based drug delivery system (DDS), Cou–Benz–Cbl conjugate, by incorporating
a benzothiazole group at the 8th position of the 7-hydroxy-coumarin moiety for pH sensitive fluorescence
properties and photocontrolled release of the anticancer drug chlorambucil. The Cou–Benz–Cbl conjugate
exhibited unique photophysical properties like good absorbance at around 350 nm, a large Stokes shift
(B151 nm) and pH sensitive fluorescence properties. The pH sensitive fluorescence properties of the
Cou–Benz–Cbl conjugate can be ascribed to an ESIPT turn ‘‘on and off’’ mechanism. At physiological pH,
the ESIPT gets turned ‘‘off’’ and a blue fluorescence of the coumarin moiety was observed, but at acidic
pH, the ESIPT gets turned ‘‘on’’ and a green fluorescence was noted. Photolysis of the Cou–Benz–Cbl
conjugate using UV light of wavelength Z365 nm resulted in the efficient release of the anticancer drug
chlorambucil. Cellular uptake studies revealed that the Cou–Benz–Cbl conjugate was easily internalized
inside the cancer cells. Further, an MTT assay showed that the Cou–Benz–Cbl conjugate has a good bio-
compatibility and low cytotoxicity towards the MDA-MB-231 cell line, whereas upon exposure to UV light,
the Cou–Benz–Cbl conjugate exhibited enhanced cytotoxicity compared to the free drug due to the
effective release of the anticancer drug chlorambucil inside the cancer cell.
Received 17th December 2014,
Accepted 12th March 2015
DOI: 10.1039/c4tb02081b
www.rsc.org/MaterialsB
small Stokes shift resulting in self-absorption (inner-filter
effects),3 an enhancement of the signal to noise ratio in bio-
Introduction
Photoresponsive drug delivery systems (DDSs) have recently imaging applications,4 and fluorescence properties which are
received much attention for their applications, especially in the insensitive to the intracellular pH environment.5 Such kinds of
area of cancer treatment, since they allow precise control over limitations prevent coumarin based phototriggers from broad
the release, including the location, timing and dosage.1,2 One acceptance in the areas of cell imaging and drug delivery. As a
of the main ingredients used for the construction of photo- result, several chemical modifications of these coumarin photo-
responsive DDSs is a phototrigger. Several phototriggers have triggers have been attempted to overcome the above mentioned
been reported so far in the literature. Among them, coumarin limitations, but their synthesis remains a challenging task.
derivatives have been widely used in DDSs mainly due to their
Recently, excited state intramolecular proton transfer (ESIPT)
strong fluorescence nature and faster release ability. However, has attracted great attention due to its applications in major areas
coumarin based phototriggers still face disadvantages like a like laser dyes,6 photostabilisers,7 radiation scintillators,8 lumines-
cent materials,9 molecular probes,10 sensing ions,11–14 and photo-
a Department of Chemistry, Indian Institute of Technology, Kharagpur 721302,
switching of polymorphs.15 Most of the ESIPT reactions involve
enol–keto photo-tautomerisation16 in the excited state of the ESIPT
molecule leading to an initial enol form and a photo-tautomer keto
form. As a result, this gives rise to dual fluorescence, one originat-
ing from the enol form and another largely red shifted fluores-
West Bengal, India. E-mail: ndpradeep@chem.iitkgp.ernet.in
b Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302,
West Bengal, India
† Electronic supplementary information (ESI) available: General experimental
procedures, characterization of compounds 3, C4 and C5, the naked eye color of a
solution of C5 in different solvents, the naked eye color of a solution of C5 in cence from the tautomeric keto form in the excited state. In
different solvents under a fluorescent lamp, fluorescence quantum yield calcula-
particular, the emission maximum and the fluorescence quantum
tions of C4, C5 and 4-methyl-7-hydroxycoumarin, fluorescence lifetime calcula-
yield of the enol and tautomeric keto forms are highly sensitive to
tions of C5 and C4 in different solvents (lmax = 516 nm and lmax = 406 nm), time-
the environment conditions, like pH,17 solvent,18 etc. The above
resolved decay curves of C5 and C4 in different solvents (lmax = 516 nm and lmax
=
characteristic features, like a large Stokes shift and environ-
mentally sensitive fluorescence properties, of ESIPT molecules
406 nm), photochemical quantum yield calculation of C5. See DOI: 10.1039/
c4tb02081b
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in trifluoroacetic acid (TFA) were refluxed for 2 h. After cooling,
the reaction mixture was extracted with dichloromethane (DCM),
and the combined organic layers were dried over MgSO4 and
evaporated under reduced pressure. A pale yellow solid of
8-formyl-7-hydroxy-4-(hydroxymethyl)-coumarin was obtained.
The crude material was purified by column chromatography
through silica gel using EtOAc/petroleum ether (2: 8) to obtain a
light yellow powder (yield 20%); mp: 180–182 1C; 1H NMR
(CDCl3, 200 MHz): d = 12.27 (s, 1H), 10.63 (s, 1H), 8.24 (s, 1H),
7.68–7.63 (d, J = 9.2 Hz, 1H), 6.96–6.92 (d, J = 9.2 Hz, 1H), 6.45
(s, 1H), 5.35 (s, 1H); 13C NMR (CDCl3, 50 MHz): d = 193.1 (1C),
165.5 (1C), 159.7 (1C), 158.6 (1C), 148.3 (1C), 131.7 (1C), 130.6
(1C), 114.8 (1C), 110.7 (1C), 108.9 (1C), 60.4 (1C); FTIR (KBr,
cmꢀ1): 3210, 2800, 1756, 1703; MS cal. for C11H9O5 [MH+]:
221.0450, found: 221.0441.
8-Benzothiazoyl-7-hydroxyl-4-(hydroxymethyl)-coumarin
(Cou–Benz–OH) (C4).
A mixture of 8-formyl-7-hydroxy-4-
(hydroxymethyl)-coumarin (1 equiv.) and o-amino-thiophenol
(1 equiv.) in dry DMSO was refluxed for 1 h. After cooling, the
reaction mixture was extracted with ethyl acetate (10 ꢁ 3 =
30 mL). The combined organic layers were dried over MgSO4
and evaporated under reduced pressure. A pale yellow solid of
8-benzothiazoyl-7-hydroxy-4-(hydroxymethyl)-coumarin (C4) was
obtained. The crude material was purified by column chromato-
graphy through silica gel using EtOAc/petroleum ether (3: 7) to
Scheme 1 ESIPT process: enol and keto forms of the DDS.
encouraged us to utilize them for the first time in the design of a
pH sensitive coumarin based photoresponsive DDS.
Among the various ESIPT molecules, 2-(20-hydroxyphenyl)-
benzothiazole (HBT) is a compound of interest due to its interesting
properties, such as (i) its simple structure, (ii) its facile chemical
modification, and (iii) the large Stokes shift arising from excited-
state intramolecular proton transfer (ESIPT).19 In addition, the HBT
molecule also shows dual emission, which is highly dependent on
the solution pH20 and proticity.21 As a result, HBT is utilized in
various applications including chemical sensors,22 OLEDs,23 laser
dyes,24 polymer modifiers25 and polymeric materials.26
1
obtain an off white powder (yield 50%); mp: 4200 1C; H NMR
(DMSO-d6, 200 MHz): d = 14.43 (bs, –OH), 8.48–8.44 (dd, J = 8 Hz,
1H), 8.34–8.31 (dd, J = 7.4 Hz, 1H), 8.22–8.18 (d, J = 8.4 Hz, 1H),
7.67–7.52 (m, 2H), 7.12–7.10 (d, J = 8.8 Hz, 1H), 6.41 (s, 1H), 4.8
(s, 2H); 13C NMR (DMSO-d6, 50 MHz): d = 162.9 (1C), 161.4 (1C),
158.9 (1C), 157.3 (1C), 151.8 (1C), 148.4 (1C), 133.0 (1C), 128.0
(1C), 127.1 (1C), 125.9 (1C), 124.2 (1C), 122.1 (1C), 121.6 (1C),
114.0 (1C), 109.3 (1C), 106.6 (1C), 59.2 (1C); FTIR (KBr, cmꢀ1):
3250 (broad), 1754, 1703; MS cal. for C17H12NO4S [MH+]:
326.0487, found: 326.0498.
Coumarin–benzothiazoyl–chlorambucil conjugate (Cou–Benz–
Cbl) (C5). Chlorambucil (1 equiv.) was dissolved in 1 mL of oxalyl
chloride and was stirred for 1 h at 60 1C. Then, the oxalyl chloride
was removed under vacuum to afford the acid chloride of
chlorambucil as a brown oil. Then, the acid chloride was
dissolved in dry DMF (5 mL) and the esterification reaction was
carried out without further purification. To the solution of the
acid chloride in DMF (5 mL), C4 (1.2 equiv.) was added, followed
by triethylamine (62 mL, 0.45 mmol). The mixture was stirred at
55 1C for 8 h. After cooling, the reaction mixture was extracted
with ethyl acetate (10 ꢁ 3 = 30 mL). The combined organic layers
were dried over MgSO4 and evaporated under reduced pressure.
A pale yellow solid of C5 was obtained. The crude material was
purified by column chromatography through silica gel using
EtOAc/petroleum ether (2 : 8) to obtain a white solid (yield 90%);
mp: 4200 1C; 1H NMR (CDCl3, 200 MHz): d = 8.09–7.99 (m, 2H),
7.61–7.48 (m, 3H), 7.36 (s, 1H), 7.13–7.12 (d, J = 3 Hz, 1H), 7.09–
7.07 (d, J = 3.4 Hz, 1H), 6.81–6.76 (d, J = 8.6 Hz, 2H), 6.4 (s, 1H), 5.3
(s, 2H), 3.74–3.61 (m, 8H), 2.66–2.59 (t, J = 6.6 Hz, 2H), 2.52–2.44
Hence, we thought about incorporating the HBT molecule with
the 7-hydroxy coumarin phototrigger so that we can have a DDS
with a larger Stokes shift27 and pH sensitive fluorescence proper-
ties.28 The pH sensitive fluorescence properties of the DDS can be
ascribed to an ESIPT ‘‘on and off’’ mechanism. At physiological pH,
the ESIPT will be switched ‘‘off’’ (deprotonation of the 7-hydroxy
group in coumarin will occur since the pH of the coumarin
phenolic –OH group is 6.8) and a blue fluorescence of the
coumarin moiety will be observed, but at acidic pH, the ESIPT
will be turned ‘‘on’’ and we can observe a green fluorescence
from the tautomeric keto form resulting from the intramolecular
proton transfer (Scheme 1). Further, we wanted to incorporate a
benzothiazole group at the 8th position of the coumarin moiety
so that the H-chelated ring forming distance between the
H-donor (7-hydroxy group of the coumarin moiety) and the
acceptor (nitrogen atom of HBT) are at the optimum distance.29
Keeping this in mind for the current study, we have synthesized
a new pH sensitive benzothiazole–coumarin based photorespon-
sive DDS for the delivery of the anticancer drug chlorambucil.
Experimental
Synthesis of Cou–Benz–Cbl conjugate (DDS)
8-Formyl-7-hydroxyl-4-(hydroxymethyl)-coumarin (3). 7-Hydroxy- (t, J = 7.4 Hz, 2H), 2.07–1.96 (m, 2H); 13C NMR (DMSO-d6,
4-(hydroxymethyl)-coumarin (1 equiv.) and hexamine (4 equiv.) 50 MHz): d = 175.1 (1C), 163.6 (1C), 162.1 (1C), 159.7 (1C), 158.0
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(1C), 152.5 (1C), 149.1(1C), 145.1 (1C), 133.8 (1C), 130.4 (1C), 130.0 aseptic conditions. After UV irradiation, the cells were again
(2C), 126.6 (1C), 125.2 (1C), 124.8 (1C), 122.8 (1C), 122.3 (1C), 114.7 incubated for 48 h and the cell viability was measured by an
(1C), 112.6 (2C), 110.0 (1C), 107.3 (1C), 105.8 (1C), 59.9 (1C), 52.9 MTT assay (Mosmann, 1983).30 The cell viability was calculated as
(2C), 41.8 (2C), 33.7 (1C), 27.2 (1C), 22.7 (1C); FTIR (KBr, cmꢀ1): described above.
3250 (broad), 1754, 1710; MS cal. for C31H29Cl2N2O5S [MH+]:
611.1174, found: 611.1155.
Real time drug release in vitro and cytotoxicity
Photophysical properties of C5. The absorption and emis-
sion spectra of a degassed solution of C5 (2 ꢁ 10ꢀ5 M) in The cytotoxicity of the time dependent controlled released drug
different solvents were recorded on a UV-vis spectrophotometer with irradiation on the MDA-MB-231 cell line was determined
and fluorescence spectrophotometer, respectively.
by a conventional MTT assay (Mosmann, 1983).30 Briefly, the
Photolysis of C5 using soft UV irradiation (Z365 nm). C5 cells in the exponential growth phase were trypsinised and
(1 mg) was dissolved in a methanol/water (1 : 1 v/v, 1 mL) seeded in 96 well culture plates (1 ꢁ 105 cells per well). After
mixture. Half of the solution was kept in the dark and the 12 h of cell seeding, the medium was replaced with fresh
remaining half was purged with nitrogen and irradiated under complete medium (DMEM) containing 10 mg mLꢀ1 of C5 and
UV light (Z365 nm, 125 W medium pressure Hg lamp) using a this was kept for 4 h at 37 1C in a CO2 incubator. The drug
suitable filter (1 M CuSO4 solution in 0.1 N H2SO4). At periodic treated cells were irradiated with UV-light (Z365 nm) for
time intervals, a small aliquot (100 mL) of the solution was 0–30 min. The assay was performed in triplicate for each time
taken out and analyzed by RP-HPLC using a mobile phase of frame (0, 5, 10, 15, 20, 25 and 30 min). Then, the plate was
methanol/water (8 : 2 v/v) at a flow rate of 0.5 mL minꢀ1
.
further incubated for 48 h at 37 1C in a CO2 incubator. The real
time drug released in vitro was measured spectrophotometri-
cally in accordance with measuring the cell viability by the
Antiproliferative activity assays
Cell lines. Human breast carcinoma (MDA-MB-231) was MTT assay.
obtained from the National Centre for Cell Science (NCCS),
Pune, India and maintained in Dulbecco’s Modified Eagle’s
Fluorescence microscopy
Medium (DMEM) medium supplemented with 10% fetal
bovine serum. The cells were cultured at 37 1C in a CO2
incubator (Thermo Fisher Scientific, USA).
The real time drug release from C5 after UV-irradiation
(Z365 nm) was also studied by fluorescence microscopy.
MDA-MB-231 cells (1 ꢁ 105 cells per well) were treated with
10 mg mLꢀ1 of C5 and put aside for 4 h at 37 1C. Then, the drug
treated cells were irradiated with UV-light (Z365 nm) for
0–30 min. Thereafter, the plate was incubated for 48 h at
37 1C in a CO2 incubator. The effect of the released drug
on the cell was visually observed by fluorescence microscopy
(1X51, Olympus) and a high-performance CCD camera with an
appropriate filter using Image-Pro Discovery 5.1 software.
Real time cellular uptake and localization studies. To study
the cellular uptake and localization of C5, MDA-MB-231 cells
were first seeded at a density of 1 ꢁ 105 cells per well in a 6 well
plate and then incubated with 10 mg mLꢀ1 of C5 for 4 h at 37 1C
in a CO2 incubator. After incubation, the cells were irradiated
with UV light (Z365 nm) using a UV lamp (Bangalore Genei Pvt.
Ltd) for 0–30 min. Thereafter, the cells were fixed using 4%
paraformaldehyde for 10 min and washed twice with PBS.
Imaging was done using confocal microscopy (CLSM; Olympus
FV 1000 attached to an inverted microscope 1X81, Japan).
Cell cycle analysis
In vitro cytotoxicity assay
The cell cycle distributions of the cells (with and without drug
treatment) were analyzed by propidium iodide (PI) DNA stain-
ing, according to the method by Pilatova et al.31 Briefly, the
MDA-MB-231 cells (1 ꢁ 106) after C5 (10 mg mLꢀ1) treatment, UV
irradiation (30 min) and incubation for 48 h were harvested for
cell cycle analysis by flow cytometry. The cells were washed
twice with PBS (pH 7.4) and fixed overnight in 70% ethanol at
ꢀ20 1C. Then, the cells were washed with phosphate buffered
saline (PBS), followed by incubation with 1 mg mLꢀ1 RNase A
and 10 mg mLꢀ1 propidium iodide at 37 1C. Ten thousand cells
were counted per analysis. PI fluorescence was detected in the
pulse-processed FL2 channel. The distributions of cells in the
different cell cycle phases were analyzed from the DNA histo-
gram using a Becton-Dickinson FACSCalibur flow cytometer
and CellQuestt pro software. The percentage of cells corre-
sponding to sub G0/G1, G0/G1, S and G2/M phases of the cell
cycle were analyzed from the DNA histogram. A sub-G0/G1
fraction of cells was identified as an apoptosis population.
Before irradiation. The cytotoxicity of C4, C5 and chloram-
bucil (Cbl) were determined with and without UV irradiation on
MDA-MB-231 cells using an MTT assay according to the method
by Mosmann.30 The breast cancer cells (1 ꢁ 105 cells per well of
a 96 well plate) were treated for 48 h with different concentra-
tions (0.1–10 mg mLꢀ1) and the cell viability was determined by
an MTT assay (Mosmann, 1983),30 measuring the absorbance at
595 nm using a micro plate reader (Bio-Rad 550). The cell
viability was calculated using the formula: viability (%) = 100 ꢁ
A2/A1, where A2 is the absorbance of the treated cells and A1 is
the absorbance of the control cells.
After irradiation. The breast cancer cells (1 ꢁ 105 cells per well
of a 96 well plate) were treated with different concentrations of C4,
C5 and Cbl (0.1–10 mg mLꢀ1) and incubated for 4 h at 37 1C in a
CO2 incubator. Thereafter, the cells were irradiated with UV light
(l Z 365 nm) for 30 min (keeping the culture plate 6 cm from the
light source) using a UV lamp (Bangalore Genei Pvt. Ltd) under
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Apoptosis detection by SEM
MDA-MB-231 cells (1 ꢁ 105) were seeded on sterile cover slips,
placed in a 6 well plate and incubated overnight for cell
attachment. Then, the cells were treated with 10 mg mLꢀ1 of
C5, irradiated with UV-light as described earlier and finally
incubated for 48 h. The cells were further washed with PBS and
fixed with 4% paraformaldehyde. Thereafter, the cells were
again washed twice with PBS and dehydrated in various grades
of ethanol (30–100%). Then, the samples were dried in desic-
cators. Samples were then fixed onto a graphite stub and kept
in an auto sputter coater (E5200, Bio-Rad) under vacuum for up
to 150 s for gold coating. Images were captured using a field
emission scanning electron microscope (FESEM) from Carl
Zeiss (model SUPRATM 40) with an accelerated voltage of
5–20 kV.34
Scheme 2 Synthesis of the DDS Cou–Benz–Cbl (C5).
DDS was characterized by UV-vis spectroscopy, FT-IR spectro-
scopy, NMR (1H and 13C) spectroscopy and mass spectrometry
(see Fig. S1–S9, ESI†).
Nuclear morphology determination by PI staining
The assay was performed as described previously by Shon
et al.32 Briefly, 1 ꢁ 105 MDA-MB-231 cells after C5 treatment
(both UV non-irradiated and irradiated) for 48 h were rinsed
twice with PBS and fixed in 4% formaldehyde solution for
10 min. Thereafter, the cells were washed with PBS and
permeabilized with 0.1% Triton-X. The cells were again washed
with PBS and stained with PI (10 mg mLꢀ1) under dark condi-
tions. The nuclear morphology was observed by confocal micro-
scopy (CLSM; Olympus FV 1000 attached to an inverted
microscope 1X81, Japan).
Photophysical properties of Cou–Benz–OH (C4) and
Cou–Benz–Cbl (C5)
Fig. 1 shows the absorption and emission spectra of the
phototrigger (C4) and DDS (C5) in absolute ethanol. The absorp-
tion spectrum of C4 showed an absorption maximum at
330 nm, while the absorption spectrum of C5 exhibited a broad
range area from 200–400 nm. On the other hand, the emission
spectra of C4 and C5 were quite similar and showed resolved
dual emissions, a normal Stokes shifted emission maximum at
406 nm (enol form) along with a large Stokes shifted proton-
transfer tautomer emission maximum at 516 nm (keto form).
Further, the fluorescence quantum yield (Ff) of C5 was calcu-
lated to be 24.27%, which is slightly higher than the parent
coumarin moiety (Table S1, ESI†). The good absorbance above
350 nm and the well resolved dual emission indicate that our
synthesized C5 compound can be used for cellular imaging and
light induced drug delivery in the visible wavelength region.
DNA fragmentation assay
C5 treated MDA-MB-231 cells (both UV non-irradiated and
irradiated) were washed twice with PBS. DNA was isolated from
the cells of both the non-irradiated and irradiated groups using
a quick Apoptotic DNA Ladder Detection Kit (Invitrogen), as per
the manufacturer protocol. Test samples along with a 100 bp
DNA marker ladder were loaded on to 1.2% agarose gel and run
with 40 V for 2 h using 1XTBE buffer. DNA fragments were
visualized using a UV transilluminator (UST-20M-8R, BioStep,
Germany).33
Solvent dependent fluorescence properties of Cou–Benz–Cbl (C5)
It is well known that HBT can exhibit an ESIPT process upon
photoexcitation and the tautomeric keto form gives a strong
fluorescence emission at longer wavelength in comparison with
the enol form.34 Further, the ESIPT process exhibited by HBT is
strongly dependent on the nature of the solvent. To check this,
we examined the photophysical properties of Cou–Benz–Cbl
(C5) in different solvent systems. The UV-vis absorption and
Results and discussion
Synthesis of the coumarin–benzothiazole–chlorambucil
conjugate (Cou–Benz–Cbl)
We synthesized our DDS Cou–Benz–Cbl conjugate as shown in
Scheme 2. First, 7-hydroxy-4-(hydroxymethyl)-coumarin (2) was
synthesized from 4-(bromomethyl)-7-hydroxy-coumarin (1) by
refluxing overnight in H2O. Next, 7-hydroxy-4-(hydroxymethyl)-
coumarin and hexamine in trifluoroacetic acid (TFA) were refluxed
for 2 h to yield 8-formyl-7-hydroxy-4-(hydroxymethyl)-coumarin (3).
Later, a mixture of compound 3 and o-aminothiophenol in dry
DMSO were heated for 1 h to afford 8-benzothiazoyl-7-hydroxyl-
4-(hydroxymethyl)-coumarin (Cou–Benz–OH) (C4). Finally, the
DDS coumarin–benzothiazole–chlorambucil conjugate (Cou–
Benz–Cbl) (C5) was synthesized by coupling C4 and the acid
chloride of chlorambucil in dry DMF for 8 h. The resulting
Fig. 1 The absorption (a) and emission (b) spectra of Cou–Benz–OH (C4)
and Cou–Benz–Cbl (C5).
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Fig. 2 (a) Absorption spectra of Cou–Benz–Cbl conjugate in different
solvent system. (b) Emission spectra of Cou–Benz–Cbl (C5) in different
solvents.
Fig. 3 Emission spectra of C5 in aqueous binary systems: (a) MeOH–H2O
and (b) THF–H2O.
around 520 nm. In the case of a binary mixture of MeOH–H2O,
we observed that as we increased the percentage of water in
MeOH, the strong intensity band (410 nm) corresponding to
the enol form of C5 gradually diminished and the band at
520 nm of C5 corresponding to the keto form became promi-
nent. This is mainly because the H-bond formation between
MeOH and H2O becomes more prominent over MeOH and C5,
releasing C5 from the hydrogen bonding and enabling the
ESIPT process (Fig. 3a). On the other hand, C5 exhibited only
an enol emission at 400 nm in pure THF (polar aprotic solvent),
which showed a red shift to 500 nm as we increased the amount
of water in THF (Fig. 3b). The presence of only an enol emission
at 400 nm in THF is because of the stable H-bond formation
between the hydroxyl group (–OH) of C5 and THF that restricts
the ESIPT process. With the gradual addition of water, the
H-bond formation between THF and H2O enables the ESIPT
process of C5, leading to a keto emission at 500 nm.
emission of C5 were recorded in different solvents at a concen-
tration of 2 ꢁ 10ꢀ5 M.
Fig. 2b clearly shows only one emission maxima at 516 nm,
corresponding to the keto form of C5 in non H-bonding
solvents (cyclohexane, benzene and chloroform). The existence
of the keto form of C5 in non H-bonding solvents can be
attributed to ultrafast proton-transfer at intrinsically rapid rates
from the hydroxyl group (–OH) of coumarin to the nitrogen
atom of HBT (ESIPT). While in the case of polar aprotic solvents
(ACN and THF) and polar protic solvents (EtOH and MeOH), we
noted two emission maxima, one around 406 nm and another
at 516 nm (keto form). The new emission maxima at 406 nm
exhibited by C5 in H-bonding solvents corresponds to the enol
form, which arises due to the restricted ESIPT process. In
addition, we also noted that as we increase the H-bonding
donating ability of the solvent, the intensity of the enol emis-
sion becomes stronger compared to the keto form. Moreover,
no change in the UV-vis absorption of C5 was observed in
different solvents (Fig. 2a), which indicates the presence of
only one form in the ground state. We also show the naked eye
colour of C5 in different solvents under normal conditions and
a fluorescent lamp in Fig. S10 and S11, ESI†.
pH dependent fluorescence properties of Cou–Benz–Cbl (C5)
As ESIPT molecules are sensitive to the pH of the medium,
we studied the pH dependent emission properties of C5 in
different pH solutions ranging from 4.4–9.4 (ACN : HEPES
(30 : 70) buffer solution). Fig. 4a shows that the emission
maximum of C5 at 527 nm undergoes a blue shift as the pH
value increases from 4.4 to 9.4. Interestingly, we also observed a
distinct fluorescence colour change from green to blue with the
increase in the pH of the solution from acidic to basic (Fig. 4b).
The above result can be attributed to the availability of the
phenolic –OH of the coumarin moiety in C5 at different pH
values. The pKa value of the phenolic –OH of coumarin in C5 is
around 7.4. Hence, at physiological pH and above, the phenolic
–OH of the coumarin moiety remains as a phenoxide ion
We have determined the lifetime of C4 and C5 in different
solvents (Tables S2–S5, ESI†) from the biexponential fitting of
the fluorescence decays (Fig. S12–S15, ESI†). The results
revealed that as we move from benzene to THF, the lifetime
of the keto form for C5 gradually decreases from 3.21 ns to
1.28 ns. The higher stability of the keto form in a non-polar
solvent can be attributed to the decrease of the non-radiative
deactivation mechanism in the excited state. On the other
hand, we also noted that as we move from methanol to THF,
the lifetime of the enol form corresponding to C5 also decreases,
which may be due to the strong H-bonding ability of MeOH
compared to THF. Further, the above observation was also noted
for compound C4.
Fluorescence properties of Cou–Benz–Cbl (C5) in aqueous
binary mixtures
To further support the presence of an ESIPT process, we
studied the emission properties of C5 in aqueous binary mix-
tures of polar protic and polar aprotic solvents. In pure MeOH
(polar protic solvent), C5 exhibited two emission bands, one
with high intensity at 410 nm and another with weak intensity
Fig. 4 (a) Emission spectra of Cou–Benz–Cbl (C5) at different pH values.
(b) Different pH solutions of C5 under a fluorescent lamp.
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(m, Fig. 4a) and the ESIPT process ceases, resulting in a blue
fluorescence (lmax 480 nm), as exhibited by the 7-hydroxy-
coumarin moiety. While in acidic medium (pH o 7.4), the
phenolic –OH of the coumarin moiety remains protonated
(n, Fig. 4a), thus enabling the ESIPT process and resulting in
a strong green fluorescence (lmax 528 nm) corresponding to the
keto form of C5.
Photochemical properties of Cou–Benz–Cbl (C5)
Fig. 6 (a) Amount of chlorambucil released by Cou–Benz–Cbl (C5) at
To investigate the drug release ability, Cou–Benz–Cbl (C5) was different irradiation times using UV light (Z365 nm), (b) progress of the
release of chlorambucil under bright and dark conditions (‘‘ON’’ indicates
dissolved in MeOH : H2O (1 : 1) and exposed to UV light
(Z365 nm) using a 125 W medium pressure Hg lamp as the
source with continuous stirring. The release of the anticancer
the start of UV light irradiation and ‘‘OFF’’ indicates the end of UV light
irradiation.
drug, chlorambucil, was monitored by RP-HPLC. The HPLC
results showed that as the irradiation time increased, there was
a gradual decrease in the intensity of the peak at tR 8.89 min,
suggesting the photocleavage of C5. Meanwhile, we also
observed a gradual appearance of two additional new peaks
at tR 6.96 min and tR 8.12 min, corresponding to the photo-
products C4 and chlorambucil, respectively (Fig. 5).
The light induced release of the anticancer drug was then
quantified by plotting the HPLC peak area obtained for chlor-
ambucil vs. different irradiation times (Fig. 6a). The photolysis
was followed until 90% of chlorambucil was released by C5.
Further, to demonstrate the precise control over the drug
release by light, we monitored the release of chlorambucil by
periodically switching the UV light source on and off.35,36
Fig. 6b clearly indicates that whenever the light source was
Fig. 7 Confocal images from the cellular uptake study: (A–C) control cell;
switched off, the drug release stopped, which clearly indicates
(D–F) cell + Cou–Benz–Cbl (C5) after 4 h incubation. (A and D) bright field;
(B and E) fluorescence field; (C and F) overlay images of the bright and
that external stimulus light only induces the anticancer drug
release.37 Further, we calculated the rate constant and the
fluorescence fields (scale bar = 50 mm).
photochemical quantum yield for the drug release and found
the values to be k = 1.15 ꢁ 10ꢀ3 minꢀ1 and Fp = 0.006,
the cell (Fig. 7D–F). At the same time, no fluorescence was
observed in the control cell (Fig. 7A–C) (drug treated).
respectively (Table S6, ESI†).
Cellular internalization of Cou–Benz–Cbl (C5)
Anticancer efficacy of C5 before and after UV light irradiation
To establish C5 as an efficient photoresponsive DDS, the
The anticancer activity of C5 was evaluated against human
cellular uptake capability was studied against human breast
breast cancer cell line MDA-MB-231 using an MTT assay.
cancer cell line MDA-MB-231 using confocal microscopy (Fig. 7).
The cell viability tests of C4, C5 and chlorambucil (Cbl)
After 4 h of C5 incubation, the images of the cell showed cyan
(0.1–10 mg mLꢀ1) are shown in Fig. 8A (before photolysis) and
blue fluorescence in the cytoplasm and the nucleus portion of
Fig. 8B (after photolysis). The cytotoxicity of C5 was much lower
than chlorambucil at any given concentration (Fig. 8A). How-
ever, after irradiation, C5 showed a higher cytotoxicity than free
chlorambucil (Fig. 8B). This may be due to the efficient release
of the anticancer drug38 chlorambucil inside the cancer cell.
On the other hand, cells incubated with Cou–Benz–OH (Fig. 8A
and B) showed insignificant cell death, indicating that the
photoreleased anticancer drug chlorambucil is responsible
for the cytotoxicity.
Additionally, Fig. 8C and 9, corresponding to the MTT assay
and fluorescence microscopy studies, revealed that cells incu-
bated with C5 showed a decrease in cell viability with an
increase in the irradiation time (0–30 min), confirming the
controlled drug release. Further, an increased round cell popu-
lation after irradiation by UV light (Z365 nm) for 30 min
Fig. 5 (a) HPLC profile of chlorambucil release by Cou–Benz–Cbl (C5)
using UV light (Z365 nm).
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Fig. 10 Cell apoptosis study: (A–C) SEM images of the control cell; (D–F)
SEM images of the treated cell (30 min irradiation); PI staining of (G) the
control cell and (H) the treated cell; cell cycle analysis by FACS (I) before
irradiation and (J) after irradiation; (K) DNA fragmentation assay. Scale
bars = 50 mm.
Fig. 8 The cell viability studies of Cou–Benz–OH, Cou–Benz–Cbl con-
jugate and Cbl (0.1–10 mg mLꢀ1) was depicted (a) before photolysis; and
(b) after photolysis; (c) cell viability test of Cou–Benz–Cbl conjugate
(10 mM mLꢀ1), at regular time intervals of irradiation.
The nuclear morphology analysis by PI staining showed char-
acteristic apoptotic changes, like nuclear fragmentation, in the
breast cancer cells after treatment for 48 h by UV irradiation
with C5 (Fig. 10H) in comparison to cells before UV treatment
(Fig. 10G). For further confirmation of apoptotic cell death by
DNA damage, a DNA fragmentation assay was performed
(Fig. 10K). Apoptosis by DNA fragmentation can appear in
two patterns on an agarose gel: as a ‘‘DNA ladder’’ because of
endonucleases and/or as a ‘‘smear’’ of randomly degraded DNA
molecules. DNA agarose gel electrophoresis demonstrated that
the MDA-MB-231 cells displayed DNA smears for apoptosis
after UV treatment (Fig. 10K; lane 3). No DNA fragmentation
was observed in the control cells (Fig. 10K; lane 2).
Fig. 9 Anticancer activity study of Cou–Benz–Cbl (C5) by fluorescence
microscopy: (A and E) control cell; (B and F) after 10 min of UV irradiation;
(C and G) after 20 min of UV irradiation; (D and H) after 30 min of UV
irradiation. (A–D) Bright field and (E–H) fluorescence field (scale bar = 100 mm).
C5 induced apoptosis in MDA-MB-231 cells was also demon-
strated by fluorescence-activated cell sorting (FACS). As shown
in Fig. 10I and J, 1.74% sub-G0/G1, 95.21% G0/G1, 0.67% S and
2.46% G2/M phase cells were found in the non-irradiated drug
indicates cell death (Fig. 9D and H). The higher cytotoxicity of treated control (Fig. 10I), whereas the UV irradiated drug
C5 compared to free chlorambucil after UV light irradiation treated cells (Fig. 10J) had 21.79% sub-G0/G1, 61.36% G0/G1,
suggests that C5 can enhance the efficiency of the intracellular 12.78% S, and 2.87% G2/M phase cells after 48 h of incubation.
delivery of the drug chlorambucil for a sustained period A rise in the sub G0/G1 cell population is indicative of apoptotic
of time.
cell death, which was clearly observed in the UV irradiated C5
treated cells (Fig. 10J). In conclusion, C5 upon irradiation
releases a drug of interest, which induces an arrest in the
Apoptosis induction by Cou–Benz–Cbl (C5)
To investigate whether the photoinduced cytotoxicity of C5 in G2/M phase of the cell cycle with an instantaneous increase
MDA-MB-231 cells proceeds through an apoptotic mechanism, in the sub-G0/G1phase of the cells and a decrease in the G0/G1
a cell and nuclear morphological study was carried out by SEM phase of the cells.
and PI staining. The SEM images (Fig. 10A–F) show morpho-
logical changes in the MDA-MB-231 cells. The control cells have
a spindle shape with intact cell membranes (Fig. 10A–C). In
contrast, the images of the cells incubated with C5 after UV
Conclusion
treatment exhibit a significant loss in the membrane rigidity In conclusion, we have successfully synthesized Cou–Benz–Cbl
and show deformation of the cell with membrane blebbing, a conjugate (C5), an ESIPT based drug delivery system. Our newly
characteristic feature of apoptotic cell death (Fig. 10D–F).
synthesised DDS showed a large Stokes shift, by which it can
Fig. 10G and H show the nuclear morphology upon drug avoid self-absorption. The photophysical properties revealed
treatment before (smooth) and after (condensed or fragmented) that our DDS is highly sensitive to the environment, in parti-
photolysis, which are easily distinguished microscopically. cular towards the polarity of the solvent and pH of the medium.
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Acknowledgements
We thank the DST (SERB) for financial support and the DST-FIST
for 400 MHz NMR spectroscopy. S. Barman, S. K. Mukhopadhyay,
and M. Gangopadhyay are grateful to the Indian Institute of
Technology Kharagpur, and S. Biswas is grateful to the UGC
(India) for their research fellowships.
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