Quinoline H2S donor decorated fluorescent carbon dots: Visible light responsive H2S nanocarriers

Article abstract of DOI:10.1039/c9tb02157d

Recent studies have shown that the utility of nanocarriers for the transportation of gaseous signalling molecules to their target site in a biological environment is an effective approach. In this work, we have developed for the first time visible light responsive nanocarriers for the effective release of H₂S. Our newly developed nanocarriers for H₂S release are constructed using two main ingredients: fluorescent carbon dots and quinoline as an H₂S donor. The developed nanocarriers provided interesting properties like good solubility under physiological conditions, excellent fluorescence properties and efficient release ability of H₂S with good quantum yield upon visible light irradiation. In vitro studies revealed that our designed nanocarriers exhibited abilities like efficient cellular internalization and good biocompatibility.

Full text of DOI:10.1039/c9tb02157d

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Materials for biology and medicine
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Paul, B. K. Sahoo, T. K. Maiti and P. N.D. Singh, J. Mater. Chem. B, 2020, DOI: 10.1039/C9TB02157D.
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Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
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Page 1 of 8
J Po lue ran s ae l od fo Mn aot te rai ad l jsu Cs th em mai rs gt ri ny sB
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DOI: 10.1039/C9TB02157D
ARTICLE
Quinoline H S donor Decorated Fluorescent Carbon Dots: Visible
2
Light Responsive H S Nanocarriers
2
a
b
a
a
b
Manoranjan Bera , Somnath Maji , Amrita Paul , Bikash Kumar Sahoo , Tapas Kumar Maiti and N. D.
Pradeep Singh *a
Received 00th January 20xx,
Accepted 00th January 20xx
Recent studies have shown that utility of nanocarriers for the transportation of gaseous signalling molecules to their target
DOI: 10.1039/x0xx00000x
site in the biological environment is an effective approach. In this work, we have developed for the first time a visible light
responsive nanocarriers for the effective release of H
using two main ingredients: fluorescence carbon dots and quinoline as an H
interesting properties like good solubility in physiological condition, excellent fluorescent properties and efficient release
ability of H S with good quantum yield upon visible light irradiation . In vitro studies revealed that our designed nanocarriers
exhibited abilities like efficient cellular internalization and good biocompatability.
2
S. Our newly developed nanocarriers for H
2
S release is constructed
2
S donor. The developed nanocarriers provided
2
2
discoveries related to the enormous therapeutic effects of H S,
Introduction
which includes protection against myocardial ischemia injury,
cytoprotection against oxidative stress, mediation of
neurotransmission, inhibition of insulin signalling, regulation of
Effective transportation of gaseous signaling molecules to their
target sites in the biological environment is challenging because
of their high diffusion rate and low solubility in blood. However,
if we can aid in the transportation of these gaseous molecules
to their desired locations by using stimuli responsive
gasotransmitter donors, then we can control their release and
understand their specific functions at physiologically relevant
concentrations and explore their role as potential therapeutic
agents. Hence, several research groups are involved in
developing donors for different gasotransmitters for their
effective release at their desired locations. Among them,
gasotransmitter donors based on nanocarriers have started
gaining popularity because they enhance the transportation,
penetration and accumulation of these gaseous molecules and
thereby improve their action on the target site. Recently,
Sortino and co-workers developed light responsive
gasotransmitter donor for Nitric Oxide (NO) based on carbon
quantum dots.10 The two main advantages of their nanosystem
were (i) carbon dots exhibited excellent fluorescent properties
that was utilised for cellular mapping studies and (ii) provided
spatio-temporal control over the release of NO since light was
used as an external stimulus. Impressed with the above
strategy, we were interested to explore the utility of light
responsive nanocarriers for the effective release of other
important gaseous signaling molecules.
1
4-15
inflammation and dilation of blood vessel.
light responsive donors have been developed for the release of
S. To date, we have not found any light responsive
nanosystems in the literature for the release of H S. This
prompted us to develop a light responsive fluorescent
nanocarriers for the effective release of H S.
Herein, we have designed a visible light responsive nanocarriers
for the release of H S using two main ingredients carbon dots as
a nanocarrier and quinoline as a H S donor (Scheme 1). The two
main reasons for selecting quinoline as a H S donor for the
Though several
H
2
2
2
2
1
-4
2
2
current studies are: (i) quinoline derivatives are known as pH
sensitive fluorophores and they can be exploited for cellular
1
6
imaging application (ii) quinoline-based chromophores are
well known phototriggers. They have been demonstrated for
the controlled release of biologically active molecules through
5
-9
a
1
7
both one-photon excitation (1 PE) and 2 PE (near-IR light),
which is an optimal wavelength for tissue penetration.
Recently, research on developing light responsive
gasotransmitter donors for the effective release of H
2
S is
1
1-13
Scheme 1 Schematic representation for effective photorelease of H S from quinoline
decorated fluorescent carbon dots.
flourishing tremendously.
Because of the interesting
2
a Department of Chemistry, Indian Institute of Technology Kharagpur, 721302
Kharagpur, West Bengal, India. E-mail: ndpradeep@chem.iitkgp.ernet.in
Thus our newly developed light responsive fluorescent carbon
Kb h Da er pa ag rp t umr ,e Wn te os ft BB ei on t ge ac hl , nI no dl oi ag y, Indian Institute of Technology Kharagpur, 721302 dots decorated by quinoline H
Electronic Supplementary Information (ESI) available: [details of any supplementary
S donor provided following
advantages like (i) good solubility in biological conditions (ii)
2
information available should be included here]. See DOI: 10.1039/x0xx00000x
2
excellent fluorescent properties (iii) effective release of H S by
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J Po lue ran s ae l od fo Mn aot te rai ad l jsu Cs th em mai rs gt ri ny sB
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ARTICLE
Journal Name
1
visible light (iv) excellent cellular internalisation and (v) good °C; H NMR (500 MHz, DMSO-d
biocompatibility.
6
) δ 8.20 (d, J = 8.8 Hz, 1H), 7.62
View Article Online
DOI: 10.1039/C9TB02157D
(d, J = 8.8 Hz, 1H), 7.47 (t, J = 7.8Hz,1H), 7.34 (d, J = 8.3 Hz, 1H),
7
.20 (d, J = 7.8 Hz, 1H), 4.83 (s, 2H).
Synthesis of 2,2'-(thiobis(methylene))bis(quinolin-8-ol) (4)
Experimental section
Compound 3 (200 mg, 1 mmol) was dissolve in 10 ml acetone
and add to a stir and ice-cooled solution of sodium sulfide
nonahydrate (Na S.9H O) (240 mg, 1 mmol) in water (10 ml).
2 2
After completion of the addition, warm the mixture to room
temperature for an hour. The completion of reaction was
monitored by TLC and it was extracted with EtOAc and washed
2 4
with water. The collected organic layer dried over Na SO and
solvent was removed by rotary evaporation under reduced
pressure. The crude product was purified by column
Chemicals and starting materials
All reagents were purchased from Sigma-Aldrich and were used
without further purification. Dichloromethane was distilled
from CaH before use. All anhydrous reactions were performed
2
under a dry nitrogen atmosphere. Methods and techniques The
1
H NMR spectra were recorded on a Bruker-AC 600 MHz
spectrophotometer. The chemical shifts were reported in ppm
from tetramethylsilane with the solvent resonance as the
internal standard (deuterochloroform: 7.26 ppm, DMSO: 2.50
ppm). The data were reported as follows: chemical shifts,
multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet),
chromatography using 10% EtOAc in PET ether to give the
1
product as yellow solid (0.345 g, 70%). H NMR (600 MHz, CDCl
3
)
δ 8.06 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.43 (dd, J =
1
3
coupling constant (Hz). The C NMR (150 MHz) spectra were
recorded on a Bruker-AC 600 MHz spectrometer with complete
proton decoupling. The chemical shifts were reported in ppm
from tetramethylsilane with the solvent resonance as the
internal standard (Deuterochloroform: 77.0 ppm). The UV/Vis
absorption spectra were recorded on a Shimadzu UV-2450
UV/Vis spectrophotometer; the fluorescence emission spectra
1
4
1
6.8, 8.8 Hz, 2H), 7.28 (d, J = 7.3 Hz, 2H), 7.19 (d, J = 7.6 Hz, 2H),
1
3
3
.00 (s, 4H). C NMR (151 MHz, CDCl ) δ 156.40 (s), 151.83 (s),
37.12 (s), 127.59 (s), 127.19 (s), 121.80 (s), 117.74 (d, J = 15.4
Hz), 110.73 (s), 37.62 (s). (See Fig. S1 in the ESI†)
HRMS: calcd for C20 S [M+ H]+, 348.0943; found:
48.0932.
16 2 2
H N O
3
were recorded on
a
Hitachi F-7000 fluorescence Synthesis of bis((8-(4-bromobutoxy)quinolin-2-yl)methyl)sulfane,
spectrophotometer. The photolysis of the caged compounds (QuH S) (5)
2
was carried out using a 125 W medium-pressure Hg lamp
To a solution of 4 (350 mg, 1 mmol) in dry DMF (10 mL), K
350 mg, 2.5 mmol) was added and stirred for 15 min. After that
63 mg (4 mmol) 1,4-Dibromobutane was added and stirred to
reflux for 12 h. Solvent was removed in high vacuum and the
organic portion was extracted in EtOAc and washed with cold
brine water 3 times. The collected organic layer dried over
2 3
CO
supplied by SAIC (India). Chromatographic purification was
done with 60–120-mesh silica gel (Merck). For reaction
monitoring, precoated silica gel 60 F254 TLC sheets (Merck)
were used.
(
8
Compound 2 and 3 were synthesized and purified according to
1
8
the literature procedures.
2 4
Na SO and solvent was removed by rotary evaporation under
Synthesis of 2-(hydroxymethyl) quinolin-8-ol (2)18
reduced pressure. The crude product was purified by column
chromatography using 20% EtOAc in PET ether to give the
A stirred solution of 2-formyl-8-quinolinol (0.3 g, 1.7μol) in
1
product as yellow solid (0.145 g, 70%). H NMR (600 MHz, CDCl
3
)
CH
during 5 min at 0 °C. After the addition stirring for an additional
h at 25 °C, the light yellow solution was concentrated in vacuo,
and water (20 ml) was added. The mixture was neutralized with
4
HCl, extracted with EtOAc (20 ml), dried over anhydrous Na SO ,
3 4
OH (30 ml), NaBH (80mg, 2.1 mmol) was added slowly
δ 8.24 (d, J = 8.5 Hz, 2H), 8.18 (d, J = 8.5 Hz, 2H), 7.52 (d, J = 7.9
Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.07 (d, J = 7.7 Hz, 2H), 4.24 (t, J
2
=
8
5.8 Hz, 4H), 4.03 (s, 4H), 3.62 (t, J = 6.2 Hz, 4H), 2.21 – 2.12 (m,
H). C NMR (151 MHz, CDCl ) δ 166.05 (s), 155.41 (s), 146.59
3
1
3
2
(s), 139.64 (s), 137.15 (s), 130.62 (s), 121.49 (s), 119.37 (s),
and concentrated to give the desired product (2) (85%), as
1
09.46 (s), 68.39 (s), 53.25 (s), 33.76 (s), 29.81 (s), 27.58 (s). (See
1
colorless crystals: 0.25 g; m.p. 122 °C; H NMR (500 MHz, DMSO-
Fig. S2 in the ESI†)
HRMS: calcd for C28H30Br N O S [M+ H]+, 616.0392; found:
2 2 2
d
6
) δ 8.16 (d, J =8.3Hz, 1H), 7.46 (t, J =7.8Hz, 1H), 7.39(d, J =8.3
Hz, 1H), 7.35 (d, J =8.3Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 4.97 (s,
H).
6
16.0395.
2
Synthesis of 2-(chloromethyl) quinolin-8-ol (3)18
Synthesis of nitrogen doped carbon dots (CDs)
Fluorescent nitrogen containing carbon dots were synthesized
To 2 (0.13 g, 0.7 mmol), redistilled SOCl
added. The mixture was stirred for 1 h, then excess SOCl
removed in vacuum to give the crude yellow hydrochloride,
which was dissolved in CH Cl (10ml), basified slightly with
saturated aqueous Na CO , dried over anhydrous Na SO , and
lastly concentrated in vacuum. The residue was purified by
column chromatography on silica gel eluting it with PE/EA (5:1)
to give the desired product, which was recrystallized from
PE/EA to give (3) (80%), as colourless crystals: 0.11 g; m.p. 56
2
(3 ml) at 0 °C was slowly
1
9
according to the literature procedure. First, citric acid (1.5 g)
and urea (1.5 g) were added to distilled water (5 mL) to form a
transparent solution. The solution was then heated in a
domestic 750 W microwave oven for 4 mins, during which the
solution changed from being a colorless liquid to a brown and
finally dark-brown clustered solid, indicating the formation of
carbon dots. This solid was then transferred to a vacuum oven
and heated at 600°C for 1 h to remove the residual small
2
was
2
2
2
3
2
4
2
| J. Name., 2012, 00, 1-3
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J Po lue ran s ae l od fo Mn aot te rai ad l jsu Cs th em mai rs gt ri ny sB
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molecules. An aqueous solution of the carbon dots was purified Cytotoxicity of QuH
ARTICLE
2
S-CDs using HeLa cell line
View Article Online
DOI: 10.1039/C9TB02157D
-1
in a centrifuge (3000 r min , 20 min) to remove large or
agglomerated particles. Carbon dots were precipitated and
rinsed with anhydrous ethanol twice, and vacuum-dried at
ambient temperature to collect 0.98 g of carbon dots.
Cytotoxicity before photolysis:
The cytotoxicity in vitro was measured using the MTT assay on
the HeLa cell line. Briefly, cells growing in log phase were
seeded into a 96-well cell culture plate at 1х104 cells/mL.
QuH
added into the wells with an equal volume of PBS in the control
wells. The cells were then incubated at 37 ˚C in 5 % CO for 48
2
S-CDs (5-20 μg/mL) with different concentrations were
Synthesis of quinoline H
QuH S-CDs) (6)
The C dots (100 mg) was dissolved in dry THF (3 mL) under a N
atmosphere and potassium tertiary butoxide (75 mg) was
added into the solution. After stirring for 1 h at room
temperature, compound 5 (100 mg), dissolved in 3 mL dry THF
was added dropwise to the mixture and the resulting solution
was stirred for overnight at room temperature in the dark. The
2
S donor decorated carbon dots
(
2
2
2
-1
h. Thereafter, fresh media containing MTT (0.40 mg mL ) were
added to the 96-well plate and incubated at 37 ˚C in 5 % CO for
2
an additional 4 h. Formazan crystals formed were dissolved in
DMSO after decanting the media, and then absorbance was
recorded at 595 nm.
2
solvent was removed by rotary evaporator and QuH S-CDs thus
Cytotoxicity after photolysis
formed was precipitated and rinsed with anhydrous ethanol
twice, and vacuum-dried at ambient temperature to collect a
final quinoline attached carbon dots (6). It was also
characterized by Transmission Electron Microscopy (TEM), FT-
IR and UV–Visible absorption.
HeLa cells in a 96-well cell culture plate at a concentration of
4
1
х10 cells/mL were maintained in MEM containing 10 % FBS.
QuH
2
S-CDs (5-20 μg/mL) with different concentrations were
incubated at 37 ˚C and 5 % CO
2
for 48 h. Then, the cells were
irradiated for 50 min using a 125 W cm- medium-pressure Hg
2
lamp as the irradiation source (≥410 nm) with NaNO
2
solution
(1 M) as a UV cut off filter by keeping the cell culture plate 5 cm
away from the light source. After irradiation, the cells were
incubated for another 48 h. Finally, the cytotoxicity was
measured using the MTT assay as described in the previous
section.
Scheme 2 Schematic representation for synthesis of the quinoline tagged carbon dots
(
QuH
2
S-CDs), 6 as the H
2
S Donor.
All the experiments were carried out in triplicates and the data
were reported as mean ± standard deviation (n = 3 per group)
and statistical analysis was carried out using one way ANOVA
with 95% confidence interval. All the data with p < 0.05 was
considered as statistically significant.
Photolysis of quinoline H
CDs) using visible light irradiation (≥ 410 nm)
These experiments were carried out using a previously
described method. 2 mg of QuH S-CDs was dissolved in 10 mL
of methanol in quartz cuvettes. They were irradiated for 50 min
under UV light by 125 W cm medium pressure Hg vapour lamp
using NaNO solution (1 M) as a UV cut off filter. At regular
interval of time (10 min), 2 mL of the aliquots was taken and
analyzed by UV- visible spectroscopy.
2 2
S donor decorated carbon dots (QuH S-
2
Result and discussion
-2
Characterisation of the quinoline H
QuH S-CDs)
We synthesised the quinoline H2S donor, bis((8-(4-
bromobutoxy)quinolin-2-yl)methyl)sulfane (QuH S), as shown
2
S donor decorated carbon dots
2
(
2
2
Cell Imaging of QuH
2
S-CDs using HeLa cell line
in the Scheme S1 (ESI†). Nitrogen doped carbon dots were
synthesised by heating citric acid and urea in distilled water in a
domestic 750 W microwave oven for 4–5 min, as reported in the
literature. Finally, the quinoline H S donor (QuH S) was
2 2
covalently attached on the surface of CDs using potassium tert-
butoxide as a base in dry THF (Scheme 2).
2
The anchoring of QuH S onto the surface of CDs was confirmed
by FT-IR (Fig. 1), TEM (Fig. 2a and b) and UV-Vis absorption
spectroscopy (Fig. 4).
Cell imaging studies were carried out using the HeLa cell line,
which was maintained in minimum essential medium (MEM)
containing 10 % FBS at 37 ˚C and 5 % CO
1
9
2
. To study the cellular
4
uptake QuH
2
S-CDs, HeLa cells (5 х 10 cells/well) were placed on
six-well plates and allowed to adhere for 4-8 h. The cells were
then incubated with QuH S-CDs 20 µg/mL in cell culture
medium at 37 ˚C and 5 % CO for 6 h. Imaging was captured by
a Nikon confocal microscope (Nikon Eclipse TE2000-E) using the
respective filter.
Then, the cells were irradiated for 50 min using a 125 W cm-2
medium-pressure Hg lamp as the irradiation source (≥410 nm)
2
2
2
with NaNO solution (1 M) as a UV cut off filter by keeping the
cell culture plate 5 cm away from the light source. Again cell
imaging was captured by a Nikon confocal microscope (Nikon
Eclipse TE2000-E) using the respective filter.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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DOI: 10.1039/C9TB02157D
2 2
Fig. 1 Overlay FT-IR spectra of carbon dots (CDs), quinoline H S donor (QuH S) and
quinoline H S donor attached carbon dots (QuH S-CDs).
2 2
In Fig. 1 , the FT-IR spectrum of CDs showed peak at 1575 cm-1
which corresponds to the N-H bending frequency of amine
functional groups present on the surface of carbon dots. On the
2 2
Fig. 3 Absorption spectra of CDs, QuH S and QuH S-CDs in methanol.
-1
other hand, QuH
2
S donor showed peaks at 1311 cm and 1082
cm corresponding to the CH bending frequency of the linker
moiety and C-O bond stretching frequency, respectively. The FT-
IR spectrum of QuH S-CDs showed peaks corresponding to both
QuH S donor and CDs, which indicated that the quinoline H
donor (QuH S) was successfully attached on the surface of the
carbon dots.
The photophysical properties of CDs, QuH
2
S and QuH
2
S-CDs
S-CDs
-1
2
were investigated (Fig. 3). The UV-Vis spectrum of QuH
2
(Fig. 4a) showed a broad absorption band from 300 nm to 500
2
nm with λmax at 330 nm in methanol. They also exhibited an
emission maximum at λmax = 425 nm (Fig. 4b). Interestingly,
2
2
S
2
QuH
2
S-CDs exhibited a tunable emission from 331 to 510 nm
(
Fig. S4, ESI†), which is due to the carbon dots, as CDs are well
1
7c
known for exhibiting tunable emission property.
Fig. 2 TEM image of (a) nitrogen doped carbon dots (CDs) and (b) quinoline H
attached carbon dots (QuH S-CDs).
2
S donor
2
2
Fig. 4 (a) UV-Vis absorption spectrum and (b) emission spectrum of QuH S-CDs in MeOH
(Concentration = 0.2 mg/mL).
The physicochemical properties of QuH
morphology, size, and zeta potential have an influence on the Fluorescent quantum yields of CDs and QuH
cellular uptake. Therefore, we studied the size and shape of calculated to be 11 % and 7.8 %, respectively using quinine
nitrogen doped carbon dots (CDs) and quinoline H
S donor sulphate as the standard (quinine sulphate quantum yield:
attached carbon dots (QuH
S-CDs) by the transition electron 54%). Hence, QuH S-CDs can be explored for the image guided
microscopy (TEM). The representative TEM images of CDs and photorelease of H S. Also we performed the controlled
QuH
S-CDs are presented in Fig. 2a and 2b. Fig. 2a showed that experiment for H S release in the presence of the H S selective
the CDs were spherical in shape with an average diameter of ~3- fluorescent probe (D) to find the efficiency of our synthesized
nm and Fig. 2b revealed that the QuH
S-CDs were globular and Quinoline H S donor attached with carbon dots over simple
crystalline in nature with an average diameter of ~10 nm. The quinoline H S donor (see Fig. S5, ESI†).
size of the QuH S-CDs is larger than the CDs, which also
2
S-CDs such as
2
S-CDs were
2
2
2
2
2
2
2
5
2
2
2
2
Photochemical properties of QuH S-CDs (6)
2
indicated that the quinoline H S donor (QuH S) was anchored
2
2
on the surface of the CDs. Most importantly, the size of the
QuH S-CDs is in the preferred range for the biological
application. Furthermore, zeta potentials of the CDs and the
QuH S-CDs, were determined to be -25.0 mV and -32.9 mV,
2
2
respectively (Fig. S3, ESI†).
2 2
Scheme 3 Photorelease of H S from QuH S-CDs triggered by visible light in biological
medium.
2
Photophysical properties of CDs and QuH S-CDs
We studied the photochemical properties of QuH
2
S-CDs
(
Scheme 3) using 0.2 mg/mL solution of QuH S-CDs in PBS
2
4
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buffer (pH ~7.4). We performed the photolysis of QuH
ARTICLE
2
S-CDs in the emission spectra (Fig. 6b), hence emission intensity
View Article Online
DOI: 10.1039/C9TB02157D
under nitrogen atmosphere using visible light (λ ≥ 410 nm, 125 decreased notable after the formation of A.
W medium-pressure mercury lamp using 1 M NaNO
a UV cut-off filter) with continuous stirring for 50 min. Further, Visible and emission spectroscopy (See Fig. S7 and S8, ESI†).
the photochemical quantum yield of the release of H S from
QuH S-CDs was calculated to be 0.09 using potassium
ferrioxalate as an actinometer (calculation details in ESI†).
The course of photolysis of QuH S-CDs was studied by UV-vis
2
solution as We isolated the adduct A and characterised by HRMS, UV-
2
2
2
0
2
absorption (Fig. 5a) and fluorescence spectroscopy (Fig. 5b). As
we increase the irradiation time, we noted increase in the
intensity of the absorption maximum (λ = 330 nm) and emission
maximum (λ = 425 nm). The above observation can be
attributed due to the increase in the hydrophilicity of our given
system because of the formation of the photoproduct, quinolin-
Fig. 6 Detection of released H
spectra using the probe D.
2 2
S from QuH S-CDs by (a) UV-vis and (b) fluorescence
2
-ylmethanol-CDs (QuOH-CDs). Also we performed
2
experiments to check the hydrolytic stability of QuH S-CDs (see
Fig. S6, ESI†).
2
Quantitative H S detection using methylene blue assay
To quantify the amount of H
2
S released from QuH S-CDs, we
2
used the standard methylene blue assay (see Fig. S9, ESI†). In
this study, upon irradiation of light (λ ≥ 410 nm) on 0.2 mg/mL
solution of QuH S-CDs in pH~7.4 PBS buffer, exhibited an
2
increase in absorption at 663 nm at different time intervals,
corresponding to the formation of methylene blue. The above
study confirms the ability of QuH
We found that the concentrations of released H
CDs reached a maximum of ~ 40 μM in about 50 min. Also we
2
S-CDs to produce H
2
S (Fig. 7).
2
S from QuH
2
S-
2
Fig. 5 (a) UV-Vis absorption spectra and (b) emission spectra of QuH S-CDs at regular
time intervals (0–50 min) of irradiation with light (λ ≥ 410 nm) in PBS buffer (C = 0.2
mg/mL).
2 2
calculated 60% of H S release from QuH S-CDs (see Fig. S10,
ESI†).
Qualitative H
dimethylaniline−hemicyanine dye
To detect H S release from QuH
dimethylaniline−hemicyanine dye (D) as a H
N,N-dimethylaniline−hemicyanine dye reacts with H
adduct A21 (Scheme 4). The dye D shows an intense red colour
2
S detection using fluorescent probe N,N-
2
2
S-CDs, we used the N,N-
S−sensitive probe.
S to form
2
2
through naked eye and bright red fluorescence.
Fig. 7 Spectra of methylene blue assay. black line: Na
2 2
S (50 μM). Other lines: H S release
from QuH S-CDs upon irradiation at different time intervals.
2
Scheme 4 H
2 2 2
S detection from QuH S-CDs using H S−sensitive fluorescent probe
Mechanism for the photorelease of H
On the basis of literature reports, we proposed the mechanism
for the photorelease of H S from the QuH S-CDs as shown in
Scheme 5. On irradiation, quinoline moiety of QuH S-CDs gets
2 2
S from the QuH S-CDs
dye D.
The dye (10 M) was irradiated by visible light (λ ≥ 410 nm) in
the presence of QuH S-CDs (0.2 mg/mL) in PBS buffer (pH~7.4)
2
2
2
2
with continuous stirring. UV-Vis absorption spectrum of dye
displayed an absorption maximum at 521 nm. As we increase
the irradiation time from 0 min to 50 min, we observed the
intensity of absorption peak at 521 nm decreases with a
concomitant increase in new absorption maximum at around
excited to its singlet state (6*). In the singlet state it acts as a
photoremovable protecting group and undergoes heterolytic C-
S bond cleavage to produce ion pair intermediate, i.e quinoline
carbocation (7) and disulphide anion in the solvent cage. Then,
these ion pairs in the presence of water generates H S along
2
with the formation of photoproduct, quinolin-2-ylmethanol-
4
31 nm (Fig. 6a). This hypsochromic shift in absorption spectra
was due to the loss in the conjugation of D, because of the
reaction with the released H S to form A. Similarly when D
reacts with H S to form A, the lost in conjugation also reflected
CDs (QuOH-CDs) (8). Again we characterized the photoproduct
2
(QuOH-CDs) via a parallel experiment (see Scheme 2, ESI†).
2
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Journal Name
the data was reported as mean ± standard deviation (n = 3 per group) and statistical
View Article Online
analysis was carried out using one way ANOVA with 95% confidence interval. All the data
DOI: 10.1039/C9TB02157D
with p < 0.05 was considered as statistically significant.
QuH
biocompatible. Therefore, we evaluated the cytotoxicity of
QuH S-CDs using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
2 2
S-CDs to be useful as a H S donor it need to be
2
diphenyltetrazolium bromide) assay on HeLa cells before and
after photolysis (Fig. 9a and 9b). Briefly, HeLa cells were
incubated with 20 g/mL, 10 g/mL and
concentrations of QuH S-CDs for 6 h and subjected to light (λ ≥
10 nm) for 50 min. After 72 h of incubation, MTT was added to
5
g/mL
2
4
-1
the cells at 0.4 mg mL and incubated for another 4 h. From the
results, we observed that there is no evidence for the inhibition
Scheme 5 Proposed mechanism of photorelease of H
2
S from QuH
2
S-CDs.
of proliferation of HeLa cells by QuH S-CDs before (Fig. 9a) and
2
after (Fig. 9b) photolysis, suggesting that quinoline decorated
carbon dots, QuH
concentrations.
2
S-CDs is not cytotoxic at the studied
Cellular Internalization and Cell Viability study
H
2
S releasing ability of QuH
by confocal microscopy imaging. Cervical cancer cells (HeLa)
were incubated with QuH S-CDs for 6 h followed by irradiation
2
S-CDs was studied in the live cells
Conclusions
2
In conclusion, we have demonstrated fluorescent carbon dots
decorated with quinoline H S donor as a visible light responsive
nanocarriers for the efficient release of hydrogen sulfide. Our
nanocarriers exhibited strong fluorescence and spatio-
with light (λ ≥ 410 nm) for 50 min. Before irradiation the cells
emitted weak green fluorescence due to quinoline attached
carbon dots (Fig. 8). After irradiation for 50 min, increase in the
fluorescence intensity of the cells was observed, indicating
2
2
temporally controlled release of H S on activation by visible
release of H
significant change in fluorescence in the absence of either light
or the H S donor QuH S-CDs.
Also we demonstrated the release of H
2 2
S from QuH S-CDs. The cells did not show any
light. Further our newly developed nanocarriers showed very
good solubility in biological conditions, cellular internalisation
and biocompatibility. In future, we are interested to develop a
single component nanocarriers for the dual release of two
different gaseous signalling molecules.
2
2
2
S in the cell by confocal
2
imaging at different irradiation time intervals, using H S
sensitive fluorescent probe (D) (see Fig. S10, ESI†).
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank CSIR Extramural (Sanction Letter No.
0
2(367)/19/EMR-II) for financial support and DST-FIST for 600
and 400 MHz NMR. M. Bera is thankful to IIT Kharagpur for
fellowship.
Fig. 8 Confocal microscopy images showing cellular uptake of QuH
from QuH S-CDs was monitored before and after irradiation for 50 min with light (λ ≥
10 nm).
2 2
S-CDs. Release of H S
2
4
Notes and references
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Journal of Materials Chemistry B
Page 8 of 8
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DOI: 10.1039/C9TB02157D
Quinoline H S Donor Decorated Fluorescent Carbon Dots: Visible Light
2
Responsive H S Nanocarriers
2
a
b
a
a
b
Manoranjan Bera , Somnath Maji , Amrita Paul , Bikash Kumar Sahoo , Tapas Kumar Maiti and
N. D. Pradeep Singh*a
Fluorescent Carbon Dots decorated by Quinoline H S donor has been demonstrated as a
2
Photoresponsive nanocarriers for the effective release of H S.
2