A dual-analyte probe: Hypoxia activated nitric oxide detection with phototriggered drug release ability

Article abstract of DOI:10.1039/c8cc01854e

A new strategy for the detection of hypoxia and NO succeeded by photocontrolled delivery of an anticancer agent has been demonstrated. The developed system is able to produce distinct responses (dual channel) upon interaction with hypoxia and NO. This probe can also release anticancer drugs upon photoirradiation acting potentially as both a dual-analyte imaging agent and a prodrug.

Full text of DOI:10.1039/c8cc01854e

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A Dual-Analyte Probe: Hypoxia Activated Nitric Oxide Detection
with Phototriggered Drug Release Ability
Sandipan Biswas,a Y. Rajesh,b Shrabani Barman,a Manoranjan Beraa, Amrita Paula, Mahitosh
Mandal,b N.D. Pradeep Singha*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A new strategy for the detection of hypoxia and NO succeeded by
photocontrolled delivery of anticancer agent has been
demonstrated. The developed system is able to produce distinct
responses (dual channel) upon interaction with hypoxia and NO.
This probe can also release anticancer drug upon photoirradiation
acting potentially as both dual-analyte imaging agent and prodrug.
Hypoxia is evolving as a primary target in the development of
2
activatable chemotherapeutic prodrugs.1, Hypoxia is one of
the unique features of tumor physiology. Lower oxygen
pressure (below 5-10 mm Hg) because of disorganized
4
vasculature causes acute hypoxia in solid tumors.3, Hypoxic
tumors are significantly resistant to the traditional
chemotherapy and because of this hypoxic tumors are seen to
have lower cure rate.5, 6 On the other hand, hypoxia induces
enhanced nitric oxide (NO) production at the tumor.7-10 NO is
the major signaling and effector molecule being produced in
Scheme 1: Schematic representation of (a) Hypoxia detection strategy by
nitroaromatics, (b) NO detection strategy of o-diamino aromatics and (c) present
study (hypoxia and NO detection). Different colour represents different response
of fluorophore.
response to hypoxia to improve blood flow and oxygen supply
12
NO produced in tumor cell is known for
reduction can only occur in the tumor cells because severe
hypoxia is rarely found in the healthy tissues.22 On another
note, the most common strategy to develop NO probes is
based on the use of o-diamino aromatics (Scheme 1b).23-25
These species react with an oxidation product of NO (N2O3) to
furnish triazole moieties, an electron deficient moiety.26, 27 This
leads to a modulation of electronic distribution of the
fluorophore thereby causing an alteration in the emission
behavior. By the detection of both the unique biochemical
alterations (hypoxia and NO) of tumor microenvironment, the
exact location of tumor and clear discrimination between
healthy and diseased cells can be achieved. However, still
there is no literature available where both hypoxia and NO
detection have been demonstrated by a single system.18
Henceforth, we thought to design a single component prodrug
system for the detection of hypoxia and NO by clubbing the
aforementioned strategies (Scheme 1c). In addition, our
designed system can be able to deliver anticancer agent upon
irradiation of light.
(vasodilation).11,
tumor progression by induction of tumour-cell invasion,
proliferation and the expression of angiogenic factors.13-15
Therefore, the design of the probe which can image two
factors specific for tumour environment (hypoxia and NO) is of
great interest for biological applications, especially as only few
dual-analyte probes have so far been successfully used in
biology.16-18 In addition, if the system is able to release active
drug on the earmarked area (identified by image-guidance by
hypoxia and NO detection) in a controlled manner (e.g. by
light), it could function as both a dual-analyte probe and a
prodrug.
For targeting hypoxia, nitroaromatics were frequently used
as a bioreductive moiety since they undergo rapid reductive
metabolism to produce aminoaromatics in hypoxic
environment (Scheme 1a).19-21 This efficient metabolic
a.Department of Chemistry, Indian Institute of technology Kharagpur,
Kharagpur-721302, India.
E-mail: ndpradeep@chem.iitkgp.ernet.in
Here, we have developed a simple coumarin based dual
channel system, which is able to serve three purposes: (i)
b.School of Medical Science and Technology, Indian Institute of Technology
Kharagpur, Kharagpur-721302, India
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Figure 1: (a) Absorption spectral change and (b) fluorescence spectral change of
(1 × 10−5 M) in MeOH-PBS buffer (30:70) during addition of Na2S2O4 (10 eqv).
6
quantum yield (Φf) = 0.01 (Figure 1a). The responses of
6
against hypoxia and NO was monitored by UV-Vis and
fluorescence spectroscopy under physiological conditions
(methanol : PBS buffer = 30 : 70, pH = 7.4). Na2S2O4 was used
as a reducing agent. During the addition of Na2S2O4, the
absorption maxima blue shifted from 410 nm to 387 nm
Scheme 2: Working protocol of conjugate 6.
detection of hypoxic environment, (ii) act as a NO fluorescent
probe and (iii) can deliver antitumor agent upon inherent fluorescence of coumarin was rapidly recovered in
photoirradiation. When our system ( ) enters into hypoxic the due course of addition of Na2S2O4 (10 eqv), giving rise to a
(Figure 1a). An isosbestic point was noted at 407 nm. The
6
environment, it undergoes reductive metabolism to produce 55-fold fluorescence enhancement (Φf = 0.55, Figure 1b). After
the activated diaminocoumarin (A6). A6 exhibits green addition of Na2S2O4, green fluorescence was observed with an
fluorescence and acts as fluorescent probe for NO. Then, A6 emission maxima at around 535 nm. The same observation
reacts with the NO present in the tumor cell and produces the was noted in case of emission behaviour, when 6 was treated
new fluorescent phototrigger triazolocoumarin (T6), which with bacterial nitroreductase enzyme (Figure S11). According
exhibits blue fluorescence. Then, blue colour can be used as to the well-established reaction mechanism,29 the observed
guiding mark of the hypoxia and NO positive areas, which spectral change might be attributable to the formation of A6
.
could then be irradiated leading to a photorelease of the drug
(Scheme 2).
Figure 2: (a) Absorption spectral change and (b) fluorescence spectral change of
A6 (pretreated 6 with Na2S2O4, 1 × 10−5 M) in MeOH-PBS buffer (30:70) during
the addition of NO.
Next, introduction of an excess amount (500 eqv.) of NO
donor (DEA-NANOate) resulted in a blue shift of the
absorption maxima of A6 from 387 nm to 355 nm with an
isosbestic point at 372 nm (Figure 2a). The emission behaviour
Scheme 3: Synthetic procedure of 6.
also showed a gradual blue shift of the 533 nm maxima with
Synthesis of the compound
6
was carried out according to increase in the intensity (Figure 2b). This might be attributable
the procedure depicted in scheme 3. Compound
3 was to the formation of the blue triazolocoumarin (T6). The
prepared by following the procedure described in our earlier formation of A6 and T6 were confirmed by RP-HPLC (reverse
work.28 Nitration of
aluminium nitrate and acetic anhydride at room temperature and ESI-MS analysis (Figure S13 to S15).
to produce . The deprotection of in presence of conc. H2SO4 The selective fluorescence activation of
and glacial acetic acid led to compound
of with anticancer drug chlorambucil furnished coumarin- glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and β-
chlorambucil conjugate . The products described in each step Nicotinamide adenine dinucleotide (NADH) was also examined.
were characterized by NMR (1H & 13C) spectroscopy and mass Significant fluorescence modulation (Figure S16) was noted
spectrometry (Figure S1 to S9). only upon addition of Na2S2O4, indicating clear selectivity of
The UV spectrum shown in the Figure S10 reveals that toward hypoxia in the cellular milieu. Further, we evaluated
3
was carried out by treating
3
with phase high performance liquid chromatography, Figure S12
)
4
4
6
by hypoxia over
5. Finally, esterification other biological reductants like dithiothreitol (DTT),
5
6
6
conjugate
6
has absorption maxima at 410 nm. Conjugate
6
the specificity of A6 for NO. We screened a wide variety of
exhibits very weak fluorescence intensity with a fluorescence
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Now, we intended to investigate the hypoxia activated
fluorescence activation, NO detection and photoregulated
drug release ability of
was internalized into the cancer cells and kept 6 h for
hypoxic incubation. The green fluorescent cells in Figure 4b
showed the clear activation of under hypoxia condition.
6 in vitro using HeLa cell line. Conjugate
6
6
Next, the 6-pretreated hypoxic HeLa cells were incubated with
NO for another 6 h. After the treatment of NO, the cells
exhibited blue coloured fluorescence indicating the formation
of T6 in the cellular milieu (Figure 4c). Then, the blue
fluorescent cells were irradiated with visible light (≥ 410 nm) to
release the anticancer drug chlorambucil. Increased round cell
population after 30 min of photoirradiation indicates the cell
death (Figure 4d & S23). The cellular fluorescence intensities
at the above mentioned conditions were also quantified
Figure 3: HPLC overlay chromatogram recorded during the photolysis of T6 (1 ×
10-4 M) using visible light (≥ 410 nm) ACN-water mixture. a = T6, b = HO-T6 and c
= released chlorambucil.
possible competitive species, reactive oxygen and nitrogen
-
.
1
species (ROS or RNS) including H2O2, ClO-, O2 -, OH-, O2, NO3 ,
-
NO2 , ascorbic acid (AA), and dehydroascorbic acid (DHA). A6
(
Figure S24).
did not elicit any noticeable change in the emission spectrum
except for NO suggesting high selectivity of A6 for NO (Figure
S17). The detection limit of NO by conjugate A6 was found to
be 24 nM (Figure S18).
The anticancer efficacy of
6
was evaluated using MTT30
(MTT=
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, a yellow tetrazole) assay against HeLa cell line.
Percent of cell viability of 6, A6, T6, HO-T6 and chlorambucil
have been depicted in Figure S25a (before photolysis). The
cytotoxicity of T6 is lower than chlorambucil at any given
concentration. In contrast, after photolysis T6 exhibited higher
cytotoxicity than free chlorambucil indicating efficient release
of chlorambucil inside the cell (Figure S25b). Additionally, HO-
T6 showed insignificant cell death both before and after
photolysis, which clearly indicates that released chlorambucil
is solely responsible for the cytotoxicity. Moreover, increased
cytotoxicity was noted with increasing time of irradiation
The photorelease ability of in-situ generated T6 was
examined by exposing an ACN-water binary solution of T6
(1×10-4 M) in visible light (≥ 410 nm) by medium pressure Hg-
lamp using a suitable UV cut off filter (1 M NaNO2 solution).
The time course of the photolysis was monitored by RP-HPLC
(Figure 3). Continual depletion of the peak at retention time
(tR) 6.42 min with increasing irradiation time denotes the
photodecomposition of T6. On the other hand, appearance
and gradual enhancement of two new peaks at tR 4.43 min and
2.65 min indicate the photoproduct hydroxy-triazolocoumarin
(
Figure S25c), which confirms the controlled delivery of the
(HO-T6) and the released antitumor drug chlorambucil, which
anticancer agent. Further, highest level of cytotoxicity was
observed after 45 min of photoirradiation.
was further supported by ESI-MS (Figure S19 & S20).
T6 showed a great control over the photoresponsive drug
release when they were exposed to the periodic light and dark
conditions, indicating light is entirely responsible for the
release (Figure S21a). Upto 90% drug release was monitored,
which occurred within 45 min of visible (≥ 410 nm) light
irradiation (Figure S21b). Further, photochemical quantum
yield of T6 was calculated to be 0.04 (Figure S22).
While T6 is the most probable light-activated pro-drug
form in the case when hypoxia and NO are both present, in
fact both
experiments in a cuvette showed that
the drug after 27 min and 73 min of irradiation with the visible
light ((≥ 410 nm), respectively (Figure S26 which is
comparable to the efficiency of drug release by T6 (45 min).
This is also in line with the photo-induced toxicity of and A6
6
and A6 can also act as phototriggers. Photolysis
6
and A6 release 90% of
)
6
in HeLa cells (Figure S27), which is in fact similar to the one
observed for T6. This fact, however, does not interfere with
the potential practical application of
6 as both dual-analyte
probe and a photo-induced drug delivery system, as the
irradiation could be applied selectively only to the areas
stained in blue, and therefore positive for the presence of
hypoxia and NO.
In summary, our newly developed coumarin-based single
system system performed as a dual channel sensor (for
hypoxia and NO) and delivered the antitumor drug
chlorambucil in a photoregulated manner. Interestingly, good
biocompatibility, ample sensitivity towards hypoxia and NO,
great spatiotemporal precision over the drug delivery make
our system an appreciable dual channel sensor and drug
delivery vehicle. As such it also becomes one of the very few
biocompatible dual-target probes and one of the first tools to
Figure 4: (a) Cellular fluorescence image of HeLa cells after incubated with
6
, (b)
cells pretreated with
6
were kept in hypoxic condition, (c) hypoxic cells
pretreated with 6 were incubated with NO and (d) after visible light (≥ 410 nm)
irradiation. Excitation wavelength for imaging experiment was 400 nm. Each
image was collected on different samples.
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We thank DST-SERB for the financial support. Sandipan Biswas
is thankful to UGC-New Delhi and Y. Rajesh is thankful to DST
for the fellowship.
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