Redox-responsive xanthene-coumarin-chlorambucil-based FRET-guided theranostics for "activatable" combination therapy with real-time monitoring

Article abstract of DOI:10.1039/c7cc03241b

A FRET donor-acceptor xanthene-coumarin conjugate has been designed for redox-regulated synergic treatment of photodynamic therapy and chemotherapy with real-time monitoring. The "locked" FRET pair was selectively "unlocked" by biological reducing thiols via rupture of a sacrificial disulfide linker. A distinct change in fluorescence color and selective cancer cell toxicity were observed in vitro.

Full text of DOI:10.1039/c7cc03241b

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Gangopadhyay, R. Mengji, A. Paul, V. Yarra , V. Venugopal, A. Jana and P. N.D. Singh, Chem. Commun.,
2017, DOI: 10.1039/C7CC03241B.
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DOI: 10.1039/C7CC03241B
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Redox-responsive Xanthene-coumarin-chlorambucil-based FRET-
guided theranostics for “activatable” combination therapy with
real-time monitoring
Received 00th January 20xx,
Accepted 00th January 20xx
Moumita Gangopadhyay,a Rakesh Mengji,b Amrita Paul,a Yarra Venkatesh,a Venugopal
DOI: 10.1039/x0xx00000x
Vangala,b,d Dr. Avijit Jana,*b,c,d Dr. N D Pradeep Singh*a
www.rsc.org/
thioredoxin (Trx) etc. owing to their higher content in cancer
cells (126 nmol/mg-protein) than normal cells (40 nmol/mg-
FRET donor-acceptor Xanthene-coumarin conjugate has been
designed for redox-regulated synergic treatment of photodynamic
therapy and chemotherapy with real-time monitoring. The
“locked” FRET pair was selectively “unlocked” by biological
reducing thiols via rupture of sacrificial disulfide linker. Distinct
change in fluorescence color and selective cancer cell toxicity were
observed in vitro.
protein).24 Thus,
a thiol-responsive prodrug containing
disulfide linkage can serve as an efficient target-specific
theranostics for cancer treatment. Though there have been
several reports on such “activatable” PDT, development of
“activatable” combination therapeutic system is still rare.25
Real-time monitoring of drug action is a prime concern for
target specific cancer treatment. To this end, various energy
and electron transfer processes viz. photoinduced electron
transfer (PET),26–28 internal charge transfer (ICT),29–31 Förster
resonance energy transfer (FRET)32–35 etc. have been
extensively explored. Among these, FRET is one of the most
promising processes, which depends on the spectral overlap of
the emission of the donor with the absorption range of the
acceptor moiety.36 Motivated by these, we aimed to design a
FRET-based theranostic, which could show “activatable”
synergic treatment of PDT and chemotherapy with real-time
monitoring of drug-action. In the present study, we designed
7-hydroxy-(4-methyl)coumarin-xanthene derivative as the
FRET donor-acceptor pair. Effective FRET process between
coumarin (donor) and xanthene (acceptor) attached through a
disulfide linker, makes the whole prodrug system “locked” to
show any therapeutic effect (scheme 1). On contact with
reducing thiols, the prodrug gets “unlocked” releasing
coumarin-chlorambucil conjugate as well as the xanthene dye,
which could result in simultaneous PDT and photoinduced
drug delivery. In addition, such FRET “ON” and “OFF” states
lead to the discrete change in fluorescence requisite for real-
time monitoring of drug release.
Multimodal therapy i.e. combination of two or more therapies
to treat a single disease has now become a well-explored field
with regard to efficient cancer healing. There have been ample
examples of multimodal therapy involving photothermal
therapy (PTT), chemotherapy, and photodynamic therapy
(PDT).1–4 However, most of such multimodal systems involved
two or more different drugs for different treatment
modalities.5 Our group has been engaged in developing single
chromophore to exhibit synergistic effect of two different
treatment modalities for profound tumor eradication.5,6 The
major restraint of previously reported single component
multimodal systems is that the chromophore is always in its
“unlocked” form to show the synergistic effect leading to
undesired side effects.7 To circumvent this issue, nowadays
researchers are developing endogenous stimuli-activated
chromophores for target specific cancer therapy.8–10 With
regard to endogenous stimuli-responsive targeting therapy,
intracellular pH, specific physiological enzymes, intracellular
hypoxia etc. showed remarkable results.11–13 Recently, redox-
responsive chemotherapy is gaining much attention.14–22 In
such cases, the chemotherapeutic drug molecule is attached to
a fluorophore by means of a sacrificing disulfide linkage.23 Such
disulfide linkages can be easily cleaved selectively in cancer
cells by biological reducing agents viz. glutathione (GSH),
a.Department of Chemistry, Indian Institute of Technology, Kharagpur 721302,
West Bengal, India, E-mail:ndpradeep@chem.iitkgp.ernet.in.
b.Division of Chemical Biology, c.Division of Natural Product Chemistry, d.Academy of
Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical
Technology Hyderabad, Hyderabad 500007, Telangana, India, E-mail:
avijit@iict.res.in
Electronic Supplementary Information (ESI) available: [1H, 13C, DEPT NMR, UV
spectra, quantum yield, competing fluorescence spectra, pH dependence of
fluorescence property of Xan-ss-Cou-Cbl are discussed in supplementary material.].
See DOI: 10.1039/x0xx00000x
Scheme 1 Redox-responsive FRET-based theranostic Xan-SS-Cou-Cbl mediated
bimodal treatment of PDT and chemotherapy.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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Xanthene-disulphide-coumarin-chlorambucil
(Xan-SS-Cou-
View Article Online
Cbl) (
chlorambucil conjugate (Cou-Cbl) (
3H-xanthen-3-one ( ) were synthesized following previous
literatures.37,38 Thereafter, compound
was afforded by
reacting with 2,2'-hydroxyethyldisulfide in presence of
triphosgene and 4-dimethylaminopyridine (DMAP) for 17 h at
room temperature.39 Finally, compound
was treated with
DMAP and triphosgene at room temperature for 5 h, followed
by addition of compound and stirring for 17 h afforded
desired compound in 35 % yield as shown in scheme 2
6
) was synthesized in three steps. First, coumarin-
DOI: 10.1039/C7CC03241B
3
) and 6-hydroxy-9-methyl-
5
4
3
4
5
Figure 1 (a) Fluorescence changes of compound
increasing concentrations of GSH (0.0-5.5 mM) [excitation wavelength (
nm], and (b) time-dependent increase of fluorescence at 408 nm upon reaction
of (15 M) with GSH (0.0-5.5 mM) [ ex = 330 nm]. All data were acquired at 37
6
(15

M) after subjecting to
6
.

ex) = 330
6


°C in HEPES buffer (pH 7.2) containing 10 % (v/v) ethanol.
Increase in fluorescence intensity at 408 nm with respect to
time was monitored in presence of varying concentration of
GSH. It was observed that maximum increase in fluorescence
intensity was observed after 20 min of addition of 5 mM GSH,
after which it reached saturation point (fig. 1b). As 5 mM is the
Scheme 2 Synthesis of Xan-SS-Cou-Cbl conjugate (6).
average GSH concentration in cancer cells,40 compound
6 could
be used as a competent redox-responsive prodrug for target-
specific cancer treatment. The change in fluorescence property
The UV-vis spectrum of compound
6
was recorded in a
HEPES buffer solution containing 10 % ethanol (pH 7.2) at
ambient temperature. It is evident from the UV-vis spectra (fig.
S1) that the coumarin moiety has absorption maximum at 330
nm and emits in the range of 400-500 nm (as shown in fig.
S1b). Interestingly, the absorption range of xanthene moiety
falls in the same region of coumarin emission (fig. S1b). Thus,
in the presence of disulfide bridge, coumarin moiety upon
excitation with 330 nm light transfers its energy to xanthene
moiety serving as a FRET donor. Xanthene, on the other hand,
exhibited its characteristic emission maximum at 516 nm upon
energy transfer from coumarin, which confirmed the FRET
process occurring from coumarin to xanthene. Comparative
of compound
metal ions viz. Na+, K+, Ca2+, Mg2+, Fe2+, Cu2+ and Zn2+
S4a) and amino acids (fig. S3b, S4b) was carried out to
establish the selectivity of compound towards biological
6
in presence of various biologically relevant
(fig. S3a,
6
reducing thiols. The change in fluorescence property was
observed only for thiol containing biological reducing agents
viz. GSH, cysteine (Cys), homo-cysteine (Hcy) and
dithiothreotol (DTT). Effect of pH (fig. S5) on the fluorescent
nature of compound
6 was also studied by gradually adding 1
M NaOH or 1 M HCl into a solution of compound
ethanol:HEPES buffer (pH 7.2).
6 in
The process (fig. S6) of redox-triggered release of Cou-Cbl
from Xan-SS-Cou-Cbl was supported by mass spectral studies
emission properties (fig. S2) of compound
xanthene at excitation wavelength 350 nm were also
monitored to further validate FRET process.
6, Cou-Cbl and
and HPLC analysis. Compound
6 showed a single peak at m/z
910.1529 (M + H+) and a single peak at retention time (tR = 3.1
min) in HPLC chromatogram implying undisturbed prodrug
system (fig. S7). However, after treatment with 5 mM GSH,
two discrete peaks were seen at m/z 478.1192 (M + H+) and
Redox-regulated emission was monitored by subjecting a 15

M of compound
reducing agent glutathione (GSH). Initially, in absence of GSH,
compound exhibited strong green fluorescence with an
6 to increasing concentration of biological
6
227.0712 (M + H+) corresponding to compounds
3
and
respectively (fig. 2a). After GSH treatment, HPLC showed two
peaks at tR = 3.9 and 6.8 min (fig. 2b) for xanthene ( ) and Cou-
Cbl ( ) respectively.
5
emission maximum at 516 nm upon excitation at 330 nm. This
is attributed to the ongoing FRET process from coumarin to
xanthene moiety. Upon gradual addition of excess GSH (0-5
mM), the emission maximum at 516 nm (excitation 330 nm)
decreased sharply with concomitant increase in the emission
intensity at 408 nm (fig. 1a). Such observation is indicative of
cleavage of disulfide linkage between Cou-Cbl and xanthene
moiety. Thus, in presence of excess GSH the fluorophores
become independent of each other showing characteristic
5
3
emission maxima of
Remarkable change in fluorescence from green to blue of
compound depending on the varying concentration of GSH
3 at 408 nm (excitation 330 nm).
6
made them useful for real-time monitoring of drug delivery
processes under biological system. The fluorescence quantum
Figure 2 (a) Mass spectrum (TOF MS ES+), and (b) HPLC chromatogram of
compound after addition of GSH.
6
yield (Φf) of compound
6 in HEPES buffer containing 10 %
ethanol was calculated to be 0.13 taking quinine sulfate in 0.1
M H2SO4 (Φf = 0.54) as reference.
Redox-responsive singlet oxygen generation from compound
was ascertained by a well-known photodegradation study of
6
1,3-diphenylisobenzofuran (DPBF).41 The decrease in the
absorption maximum of DPBF at 425 nm after 1 h treatment of
GSH on compound
6 solution with respect to time indicated
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Figure 4 HPLC overlay chromatogram (Wavelength of detection = 254 nm) of the
redox-activated singlet oxygen generation. Upon irradiation
View Article Online
with ≥ 350-nm light (20 mW/cm2) in an HEPES buffer solution
compound 6 after treatment with GSH at different time intervals of light irradiation (0–
DOI: 10.1039/C7CC03241B
60 min). Photolysis was carried out after 1 h treatment of GSH. [A = anticancer drug
chlorambucil, B = xanthene, C = photoproduct coumarin alcohol and D = Cou-Cbl].
containing 10 % EtOH, compound
6 showed singlet-oxygen
quantum yield (ΦΔ) ~ 0.38 in presence of GSH. This can be
attributed to the “unlocking” of Cou-Cbl moiety from
It was observed that upon GSH treatment, compound
6 showed
compound
6 after cleavage of disulfide linker by GSH.
almost 80 % drug release after 60 min of illumination with a
quantum yield of 0.039; however, in absence of GSH, drug release
was negligible (fig. S9a). Due to FRET, Cou-Cbl remained “locked” in
Conversely, when Cou-Cbl was attached to xanthene moiety by
disulfide linker, coumarin served as the energy donor (FRET
donor) to xanthene (FRET acceptor). Hence, coumarin was in
its “locked” state during such FRET process and was unable to
generate singlet oxygen. Without treatment with GSH,
compound
exposure to reducing thiol. This kind of “activatable” photoinduced
chemotherapeutic property of compound made them an
6 in absence of GSH and gets “unlocked” only on
6
compound
owing to weak singlet oxygen generation from 6-hydroxy-9-
methyl-3H-xanthen-3-one ( ) moiety. Comparative singlet
6 showed comparatively low ΦΔ ~ 0.15 (fig. 3)
excellent choice for targeted cancer treatment. Precise control of
the photolytic release of chlorambucil was demonstrated by
monitoring the release of chlorambucil after periods of exposure to
light and dark conditions (fig. S9b).
5
oxygen generation abilities of Xan-SS-Cou-Cbl upon treatment
with GSH, free xanthene, and free Cou-Cbl were also
monitored (fig. S8).
In vitro redox-responsiveness was monitored by incubating HeLa
cell line with compound
6
for 4 h at 37 °C.43 The cells showed
strong blue fluorescence (fig. 5(b1)) under confocal laser scanning
microscopy (CLSM) upon excitation at 330 nm attributed to
presence of free Cou-Cbl following reaction between compound
6
with GSH that is inherently present in cancerous cells like HeLa.
However, a strong green fluorescence (fig. 5(c1)) was observed
upon excitation at 450 nm for HeLa cell line owing to the presence
of free xanthene molecule. Thus, confocal images revealed
successful rupture of disulfide bond between Cou-Cbl and xanthene
upon interaction with biological reducing thiols showing their
characteristic emission properties upon excitation at 330 (blue
fluorescence) and 450 nm (green fluorescence) respectively.
Figure 3 Photodegradation of DPBF at 425 nm by Xan-SS-Cou-Cbl before and after
treatment with GSH.
Redox-activatable photoinduced chemotherapy of compound
was screened by photolysis study in presence and absence of GSH.
Towards this end, compound was treated with GSH. Afterwards,
6
6
the solution was irradiated with light of ≥ 350 nm (1 M CuSO4 filter)
using 125 W medium pressure Hg lamp.42 The reverse phase HPLC
was carried out using 7:3 (acetonitrile:water) as the mobile phase.
Before photolysis, the reverse phase chromatogram (fig. 4) showed
two peaks at retention times (tR) 6.8 min and 3.9 min corresponding
to Cou-Cbl (D) and xanthene moiety (B) respectively, which were
formed from 6 upon treatment with GSH. The peak at 6.8 min
gradually decreased with increase in irradiation time attributed to
the steady decomposition of Cou-Cbl (D). Concomitantly,
appearance of two new peaks at tR = 4.9 and 2.6 min were
observed. The peak at retention time 2.6 min corresponded to the
successive release of anticancer drug chlorambucil (A); however
relatively less polar photoproduct coumarin-based alcohol (C)
appeared at 4.9 min. However, the peak corresponding to xanthene
moiety (tR = 3.9 min) remained same throughout the photolysis.
Such chromatogram precisely demonstrated the light-induced
chemotherapeutic application. The uncaging of Cou-Cbl proceeds
via singlet excited state of coumarin, wherefrom it undergoes
heterolytic bond scission leading to release of clorambucil.42
Figure 5 Confocal fluorescence images of compound 6 (10 M) in HeLa cell line under
(a) brightfield, (b) excitation at 330 nm [emission channel = 408 nm], (c) excitation at
450 nm [emission channel = 520 nm], and (d) overlay of b and c. [Scale bar = 50 m]
A dose-dependent study was carried out by incubating
both cell lines with compound
6 for 4 h. After 10 min of
irradiation with UV-vis light of ≥ 350 nm wavelength using 125-
W medium-pressure Hg lamp, only 12 % of the cancerous HeLa
cells survived in a 10-µM solution of compound
However, under the same conditions, compound
6
6
(
fig. 6b).
showed ~
75 % cell viability after 60 min of irradiation in the non-
cancerous HEK 293 cells. Hence, it was evident from the MTT
assay data that compound
6 showed extremely high
cytotoxicity to cancerous HeLa cells by means of combined
treatment of PDT and chemotherapeutic effect of
chlorambucil; however, it remained significantly non-toxic to a
non-cancerous cell line. When we performed the same MTT
assay in the dark (fig. 6a), compound
6 did not show any
significant cytotoxicity in either HeLa or HEK 293 cells.
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In conclusion, we have presented redox-triggered
“activatable” synergic treatment of PDT and chemotherapy
using FRET-based disulfide-linked xanthene-coumarin-
chlorambucil conjugate (Xan-SS-Cou-Cbl) enabling real-time
monitoring of drug delivery. The prodrug, Xan-SS-Cou-Cbl
containing a FRET donor-acceptor pair of coumarin and
xanthenes, showed a remarkable FRET-induced change in
fluorescence property before and after GSH treatment.
Comparative in vitro application in normal and cancerous cells
revealed the intracellular redox-responsiveness of Xan-SS-Cou-
Cbl by the prominent color change from green to blue under
confocal microscope. Photocytotoxicity assay demonstrated
the selective toxicity of Xan-SS-Cou-Cbl only in case of cancer
cells due to presence of excess GSH; however remained non-
toxic in normal cells.
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Authors are thankful to DST-SERB for financial support. M.
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DOI: 10.1039/C7CC03241B
Redox-responsive Xanthene-coumarin-chlorambucil-based FRET-guided
theranostics for “activatable” combination therapy with real-time
monitoring
Moumita Gangopadhyay,a Rakesh Mengji,b Amrita Paul,a Yarra Venkatesh,a Venugopal Vangala,b,d Dr. Avijit
Jana,*b,c,d Dr. N D Pradeep Singh*a
aDepartment of Chemistry, Indian Institute of Technology
Kharagpur 721302, West Bengal, India
E-mail: ndpradeep@chem.iitkgp.ernet.in
bDivision of Chemical Biology, cDivision of Natural Product Chemistry, dAcademy of Scientific and
Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Technology Hyderabad, Hyderabad 500007,
Telangana, India, E-mail: avijit@iict.res.in
FRET-based
theranostic,
xanthene-coumarin-chlorambucil,
exhibited
redox-responsive
“activatable” synergic treatment of PDT and chemotherapy with fluorescence-change from green
to blue.