Cell-specific activation of gemcitabine by endogenous H2S stimulation and tracking through simultaneous fluorescence turn-on

Article abstract of DOI:10.1039/d1cc00118c

The endogenous H₂S-driven theranosticH₂S-Gemhas been invented. The theranostic prodrugH₂S-Gemis selectively activated in cancer cells, releasing active gemcitabine with a simultaneous fluorescence turn-on.H₂S-Gemselectively inhibited cancer cell growth compared to the mother chemotherapeutic gemcitabine. Overall, it is a unique protocol for tracking and transporting chemotherapeutic agents to tumor areas without the guidance of tumor-directive ligands.

Full text of DOI:10.1039/d1cc00118c

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Cell-specific activation of gemcitabine by
endogenous H2S stimulation and tracking
Cite this: DOI: 10.1039/d1cc00118c
through simultaneous fluorescence turn-on†
Received 8th January 2021,
Accepted 23rd August 2021
Mrinmoy Maiti,‡a Shin A. Yoon,‡b Yujin Cha,b K. K. Athul,c
b
Sankarprasad Bhuniya *c and Min Hee Lee
*
DOI: 10.1039/d1cc00118c
rsc.li/chemcomm
The endogenous H2S-driven theranostic H2S-Gem has been biodistributed chemotherapeutic and therapeutic interventions
invented. The theranostic prodrug H2S-Gem is selectively activated through fluorescence imaging. Several endogenous stimulators
in cancer cells, releasing active gemcitabine with a simultaneous have been considered to develop such an all in one system.12
fluorescence turn-on. H2S-Gem selectively inhibited cancer cell
Recently, it has been observed that endogenous H2S is
growth compared to the mother chemotherapeutic gemcitabine. produced to a large extent in several cancer cells, increasing
Overall, it is a unique protocol for tracking and transporting cell proliferation activity.13 Mostly, cystathionine b-synthase
chemotherapeutic agents to tumor areas without the guidance of (CBS) and cystathionine g-lyase are the major enzymes respon-
tumor-directive ligands.
sible for producing endogenous H2S in cancer cells.13 Recently,
by considering endogenous H2S as a stimulator a few thera-
Cancer is a major disease burden that threatens people’s lives nostic molecules14 have been developed including inhibition of
worldwide, and the majority of cancer-related deaths are due to cancer cell growth selectively in cancer cells. Thus, we believe
metastasis of the original tumor.1 Current treatment options at that endogenous H2S can be a suitable stimulator for transport-
the diagnosis level are multimodal and include chemotherapy, ing chemotherapeutics to the desired area with maximum
radiotherapy, and surgical resection or combination therapy. clinical benefits without causing adverse side effects.
Although chemotherapy and radiotherapy show significant
In the present work, gemcitabine is linked through a labile
treatment modalities for cancer, they are still hindered by acute secondary amine bond with an inactive form of coumarin
side effects on neighboring healthy cells/tissues due to their (Scheme 1). Gemcitabine was used as a model drug because it
high toxicity and non-selectivity.2–9 Indeed, cancer cells are treats various cancers and has serious adverse effects.15,16 In
resistant to radiotherapy and chemotherapy.10,11 Therefore, addition, this strategy may increase the half-life of gemcitabine.
new concepts are highly desirable for the precise diagnosis Also, as gemcitabine is a non-fluorescent therapeutic, tracking
and effective treatment of cancer. One such type of modality is with a fluorescent reporter is important. The endogenous H2S
theranostics, a modern technology with a bi-functional system induced reduction of N3 to amine, triggering a secondary
that serves as both a therapy and the diagnosis. This technique amine bond cleavage that releases the gemcitabine via a self-
is particularly interesting as it provides real-time information immolative pathway is proposed in Scheme 1. In this event, the
on cancer therapy. By using this method, different cancer reduction of an azide group to an electron rich NH2 group in
targeting molecules, anticancer drugs, and fluorophores are coumarin will increase the fluorescence intensity. The detail of
covalently conjugated through chemical reactions that provide
a Department of Science, School of Engineering, Amrita Vishwa Vidyapeetham,
Coimbatore, 641112, India
b Department of Chemistry, Sookmyung Women’s University, Seoul 04310, Korea.
E-mail: minheelee@sookmyung.ac.kr
c Centre for Interdisciplinary Science, JIS Institute of Advanced Studies and
Research, JIS University, Salt Lake, Kolkata, 700091, India.
E-mail: spbhuniya@jisiasr.org
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1cc00118c
Scheme 1 Activation mechanism of H2S-Gem for H2S.
‡ These authors equally contributed to this work.
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Fig. 1 (a) UV-absorption and (b) fluorescence spectra of H2S-Gem (5 mM) for various concentrations of NaHS (0–200 mM) after 1 h incubation at 37 1C.
(c) Plot of fluorescence intensity at 507 nm in the presence of NaHS (0–50 mM). Fluorescence changes of H2S-Gem (5 mM) with NaHS (200 mM) at
different time intervals (d) 0–35 min and (e) 40–180 min. (f) Time-dependent fluorescence changes of H2S-Gem (5 mM) in the absence (black) and
presence (red) of NaHS (200 mM). All data were obtained in a PBS (10 mM, pH 7.4) solution containing 1% (v/v) of DMSO using an excitation wavelength
of 410 nm.
the synthetic route for H2S-Gem is available in the ESI† includ- 507 nm (Fig. 1e). The fluorescence intensity reached satura-
ing spectroscopic analysis data (Scheme S1 and Fig. S1–S22).
To continue to prove our concept, initially, we have
tion within 120 min (Fig. 1f).
Further, we tested the chemoselectivity between H2S-Gem
examined the exogenous H2S response of H2S-Gem by record- and H2S in comparison with other biologically relevant analytes
ing UV-absorption changes in H2S (NaHS) treated H2S-Gem such as various metal ions, ROS, Cys, Hcy, GSH, NaNO2,
under physiological conditions (Fig. 1a and Fig. S23, ESI†). Na2CO3 and ascorbic acid. It was noticed that H2S-Gem
We observed that the UV-absorption of H2S-Gem showed a remains inactive in altering the fluorescence signal in all cases
bathochromic shift from 350 nm to 410 nm with increasing (Fig. S24, ESI†). In addition, the kinetic stack plot of H2S-Gem
doses of H2S (Fig. 1a). Similarly, in the H2S dose-dependent with 1 mM of various thiols such as Cys, Hcy, GSH and NaHS
titration, we observed the fluorescence intensity at 507 nm firmly showed that NaHS dominantly increases the fluores-
increased compared to the shoulder peak at 450 nm (Fig. 1b). cence of H2S-Gem, unlike the other thiols (Fig. S25, ESI†).
The fluorescence increment with different doses of H2S These results proved that H2S-Gem is chemoselective towards
showed perfect linearity (R2 = 0.99) with a low detection limit H2S. Moreover, the pH-dependent chemoselective reaction
(LOD) of 37 nM (Fig. 1c).17 The time-course study initially between H2S-Gem and H2S has been investigated. The result
(0–35 min) showed increments in fluorescence at 450 nm and in Fig. S26 (ESI†) indicated that within a biologically relevant
507 nm (Fig. 1d); however, as time progressed (after 35 min), pH, H2S-Gem reacted with H2S. Nonetheless, the reactivity
it showed a ratiometric fluorescence change at 450 nm and between H2S-Gem and H2S is highest in the pH 6 to 8 range.
Next, the H2S-driven structural changes in H2S-Gem that
cause the fluorescence signal were confirmed using mass
analysis of H2S pretreated H2S-Gem. It was observed that
mass peaks for gemcitabine and the fluorophore appeared
(Fig. S27, ESI†).
After confirming the H2S triggered gemcitabine release, we
investigated the in vitro cell labeling to determine the cell-
specific gemcitabine release from H2S-Gem. In
a time-
dependent fluorescence study, we observed that the extent of
fluorescence labeling in a couple of cancer cell lines (HeLa and
A549) increased with the incubation period (Fig. 2). It indicated
that both cervical cancer cells (HeLa) and adenocarcinoma
human alveolar basal epithelial cells (A549) have sufficient
endogenous H2S that released active gemcitabine from
H2S-Gem. On the contrary, H2S-Gem pretreated human normal
fibroblast WI38 cells did not show such fluorescence. Whereas
Fig. 2 Time-dependent cancer cell labeling. Cells were administered with
H2S-Gem (15 mM) for 0–80 min. Emission collected in the 505–600 nm
region with the excitation wavelength set at 405 nm. Scale bar: 20 mm.
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cells were pretreated with exogenous H2S (NaHS), the fluores-
cence images in the green channel increased because of
H2S-Gem. Further, the exogenous addition of a substrate
(cysteine) produces excess H2S,13 which is well reflected in
the fluorescence images by an increment in fluorescence
intensity. On the other hand, cystathionine b-synthase and
cystathionine g-lyase inhibitor i.e. aminooxyacetic acid (AOAA)
and N-propargyl glycine (PAG)13,18 pretreated cancer cells
showed a decreased fluorescence signal in the green channel.
This suggested that inhibition of the H2S producing enzymes
reduces the release of gemcitabine from the theranostic
H2S-Gem and the formation of the fluorophore.
Finally, we evaluated the anticancer activity of the theranos-
tic H2S-Gem using both the dose- and time-dependent MTT
assay methods. We observed that about 50% of the cell growth
was inhibited upon incubation (48–72 h) with 5 mM of H2S-Gem
with A549 and HeLa cells, respectively (Fig. 5a, b and Fig. S31a,
ESI†). Concentrations of up to 60 mM of H2S-Gem failed to show
considerable cell growth inhibition in normal human fibroblast
WI38 cells (Fig. 5c and Fig. S31a, ESI†). While the theranostic
H2S-Gem has shown cancer cell specific activation of gemcita-
bine, gemcitabine alone has shown nonspecific cell growth
inhibition irrespective of cancer cells and normal WI38 cells
(Fig. S31b, ESI†). Additionally, the anti-proliferative activity of
H2S-Gem was monitored in NaHS and PAG-treated HeLa cells.
Compared to the untreated HeLa cells, for H2S-Gem, the
NaHS-treated cells showed a decreased cell viability whereas
the PAG-treated cells exhibited an increased cell viability
(Fig. S32, ESI†). Additionally, the high cell viability of the
released fluorophore pretreated HeLa cells indicates that the
growth inhibition of H2S-Gem incubated cancer cells is
because of the release of nascent gemcitabine from H2S-Gem
(Fig. S33, ESI†). Such specific cell growth inhibition toward cancer
cells indicated that endogenous H2S in cancer cells allows H2S-Gem
to release active chemotherapeutic gemcitabine. In contrast,
Fig. 3 Dose-dependent cancer cell labelling. Cells were incubated with
H2S-Gem (0–20 mM) for 60 min prior to imaging using a confocal
microscope. Emission collected in the 505–600 nm region with the
excitation wavelength set at 405 nm. Scale bar: 20 mm.
WI38 cells treated with exogenous H2S were labeled with green
fluorescence (Fig. S28, ESI†). This implied that the theranostic
H2S-Gem entered into the WI38 cells, but due to insufficient
endogenous H2S it failed to activate the H2S-Gem to provide
fluorescence images.
Further, in a concentration-dependent study, we observed
that the extent of fluorescence labeling in cancer cells (HeLa
and A549 cells) increased with the dose of H2S-Gem (Fig. 3);
however, normal fibroblast cells (WI38 cells) did not show any
fluorescence under identical conditions. Moreover, the fluores-
cence intensity also increased according to the concentration of
NaHS (Fig. S29, ESI†). This indicated that the endogenous H2S
level in cancer cells is higher than that in normal cells.
Next, we clarified whether such fluorescence images in the
H2S-Gem pretreated cancer cells were due to endogenous H2S
or other thiol species. Thus, we recorded fluorescence images
of living cells by altering the formation of endogenous H2S in
living cells. In Fig. 4 and Fig. S30 (ESI†), we noticed that while
Fig. 4 Confocal microscopy images of H2S-Gem in HeLa cells. Cells were incubated with (a) gemcitabine (15 mM) and (b) H2S-Gem (15 mM) for 1 h,
respectively. (c) Cells were pretreated with H2S-Gem for 1 h then incubated with NaHS (600 mM) for 30 min. (d) Cells were pretreated with cysteine
(200 mM) for 1 h then incubated with H2S-Gem for 1 h. (e) Cells were pretreated with AOAA/PAG (2 mM) for 1 h then incubated with H2S-Gem for 1 h.
(f) Cells were pretreated with AOAA/PAG (2 mM) and cysteine (200 mM) for 1 h, respectively, then incubated with H2S-Gem for 1 h. All images were
obtained using a laser with an excitation wavelength of 405 nm and a band-path filter of 505–600 nm. Scale bar: 20 mm. (g) Quantification of the
fluorescence intensities from each cell. Error bars indicate the standard deviation (SD). Asterisks indicate statistically significant changes (***P r 0.001).
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Fig. 5 MTT assay of H2S-Gem for 24, 48 and 72 h in (a) HeLa, (b) A549 and (c) WI38 cells. Error bars indicate SD, n = 5.
6 R. Mahato, W. Tai and K. Cheng, Adv. Drug Delivery Rev., 2011, 63,
659–670.
7 S. Zhang, K. H. Chan, R. K. Prud’Homme and A. Link, Mol.
Pharmaceutics, 2012, 9, 2228–2236.
8 E. Kim, D. Kim, H. Jung, J. Lee, S. Paul, N. Selvapalam, Y. Yang,
N. Lim, C. G. Park and K. Kim, Angew. Chem., Int. Ed., 2010, 49,
4405–4408.
9 G. Cafeo, G. Carbotti, A. Cuzzola, M. Fabbi, S. Ferrini, F. H. Kohnke,
G. Papanikolaou, M. R. Plutino, C. Rosano and A. J. P. White, J. Am.
Chem. Soc., 2013, 135, 2544–2551.
insufficient endogenous H2S formation in WI38 cells disfavors H2S-
Gem to show a considerable extent of anti-proliferative activity.
In conclusion, we demonstrated the endogenous H2S stimu-
lated theranostic H2S-Gem for selectively ending the progres-
sion of cancer via activation of chemotherapeutic gemcitabine.
The theranostic prodrug H2S-Gem constituted of a combination
of coumarin and gemcitabine. It showed UV-absorption at
400 nm and emission at 505 nm in the presence of exogenous
H2S. This theranostic H2S-Gem was even activated in the
presence of a minimal quantity (37 nM) of H2S. The gemcita-
bine release process is highly selective toward H2S, and such a
specific activation allows gemcitabine release exclusively in
cancer cells. In vitro cellular images indicated that the endo-
genous H2S level in cancer cells is relatively high compared to
that in the normal WI38 cells. The inhibition assay study
indicated that cystathionine b-synthase and cystathionine g-
lyase are sources of H2S formation in cancer cells. It showed
selective antiproliferative activity exclusively in the cancer cells
(HeLa and A549) over the human normal fibroblast cells
(WI38). All of these facts advocated that it is a unique strategy
for releasing cargo and selectively tracking therapeutic events
in cancer cells without the support of any tumor/cancer cell-
specific ligands.
10 C. Sawyers, Nature, 2004, 432, 294–297.
11 I. Brigger, C. Dubernet and P. Couvreur, Adv. Drug Delivery Rev.,
2012, 64, 24–36.
12 (a) Y. Zhou, M. Maiti, A. Sharma, L. Yu, X. W. Lv, J. Shin, M. Won,
J. Han, A. Podder, K. N. Bobba, S. Bhuniya and J. S. Kim, J. Controlled
Release, 2018, 288, 14–22; (b) A. Sharma, M.-G. Lee, H. Shi, M. Won,
J. F. Arambula, J. L. Sessler, J. Y. Lee, S.-G. Chi and J. S. Kim, Chem,
2018, 4, 2370–2383; (c) S. Barman, S. K. Mukhopadhyay, S. Biswas,
S. Nandi, M. Gangopadhyay, S. Dey, A. Anoop and N. D. Pradeep
Singh, Angew. Chem., 2016, 128, 4266–4270; (d) S. Maiti, N. Park,
J. H. Han, H. M. Jeon, J. H. Lee, S. Bhuniya, C. Kang and J. S. Kim,
J. Am. Chem. Soc., 2013, 135, 4567–4572; (e) R. Kumar, E.-J. Kim,
J. Han, H. Lee, W. S. Shin, H. M. Kim, S. Bhuniya, J. S. Kim and
K. S. Hong, Biomaterials, 2016, 104, 119–128; ( f ) F. Kong, Z. Liang,
D. Luan, X. Liu, K. Xu and B. A. Tang, Anal. Chem., 2016, 88,
6450–6456; (g) T. Kim, H. M. Jeon, H. T. Le, T. W. Kim, C. Kang
and J. S. Kim, Chem. Commun., 2014, 50, 7690–7693; (h) P. Liu,
D. Yan, P. Zhang, F. Zeng, B. Li and S. A. Wu, Chem. Commun., 2015,
51, 9567–9570; (i) D. Dutta, S. M. Alex, K. N. Bobba, K. K. Maiti and
S. Bhuniya, ACS Appl. Mater. Interfaces, 2016, 8, 33430–33438.
´
13 C. Szabo, C. Coletta, C. Chao, K. Modis, B. Szczesny,
This research was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korea govern-
ment (MSIT) (2021R1F1A1045790). Also, this work is supported
by the SERB, India (Grant No. CRG/2019/002068, SB), CSIR-
India (Grant No. 02(0386)19/EMR II, SB), and the DST (INT/
RUS/RFBR/392, SB).
A. Papapetropoulos and M. R. Hellmich, Proc. Natl. Acad. Sci.
U. S. A., 2013, 110, 12474–12479.
14 (a) H. Zhang, X. Kong, Y. Tang and W. Lin, ACS Appl. Mater.
Interfaces, 2016, 8, 16227–16239; (b) D. Zheng, Z. Gao, T. Xu,
C. Liang, Y. Shi, L. Wang and Z. Yang, Nanoscale, 2018, 10,
21459–21465; (c) K. N. Bobba, G. Saranya, P. T. Sujai,
M. M. Joseph, N. Velusamy, A. Podder, K. K. Maiti and S. Bhuniya,
ACS Appl. Bio Mater., 2019, 2, 1322–1330; (d) K. N. Bobba, A. Binoy,
S. Koo, A. Podder, N. Mishra, J. S. Kim and S. Bhuniya, Chem.
Commun., 2019, 55, 6429–6432; (e) B. Shi, N. Ren, L. Gu, G. Xu,
R. Wang, T. Zhu, Y. Zhu, C. Fan, C. Zhao and H. Tian, Angew. Chem.,
Int. Ed., 2019, 58, 16826–16830.
Conflicts of interest
There are no conflicts to declare.
15 A. Henthorn and M. D. Pluth, J. Am. Chem. Soc., 2015, 137,
15330–15336.
16 H. C. Zhu, C. X. Liang, X. X. Cai, H. M. Zhang, C. Y. Liu, P. Jia,
Z. L. Li, Y. M. Yu, X. Zhang, W. L. Sheng and B. C. Zhu, Anal. Chem.,
2019, 92, 1883–1889.
17 S. Y. Park, W. Kim, S. H. Park, J. Han, J. Lee, C. Kang and M. H. Lee,
Chem. Commun., 2017, 53, 4457–4460.
Notes and references
1 R. L. Siegel, K. D. Miller and A. Jemal, Ca-Cancer J. Clin., 2019, 69, 7–34.
2 J. H. Atkins and L. J. Gershell, Nat. Rev. Cancer, 2002, 2, 645–646.
3 D. J. Irvine, Nat. Mater., 2011, 10, 342–343.
4 J. A. Hubbell and R. Langer, Nat. Mater., 2013, 12, 963–966.
5 S. Mura, J. Nicolas and P. Couvreur, Nat. Mater., 2013, 12, 991–1003.
18 N. Velusamy, A. Binoy, K. N. Bobba, D. Nedungadi, N. Mishra and
S. Bhuniya, Chem. Commun., 2017, 53, 8802.
Chem. Commun.
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