A mitochondria-targeted chemiluminescent probe for detection of hydrogen sulfide in cancer cells, human serum and in vivo

Hydrogen sulfide (H2S) as a critical messenger molecule plays vital roles in regular cell function. However, abnormal levels of H2S, especially mitochondrial H2S, are directly correlated with the formation of pathological states including neurodegenerative diseases, cardiovascular disorders, and cancer. Thus, monitoring fluxes of mitochondrial H2S concentrations both in vitro and in vivo with high selectivity and sensitivity is crucial. In this direction, herein we developed the first ever example of a mitochondria-targeted and H2S-responsive new generation 1,2-dioxetane-based chemiluminescent probe (MCH). Chemiluminescent probes offer unique advantages compared to conventional fluorophores as they do not require external light irradiation to emit light. MCH exhibited a dramatic turn-on response in its luminescence signal upon reacting with H2S with high selectivity. It was used to detect H2S activity in different biological systems ranging from cancerous cells to human serum and tumor-bearing mice. We anticipate that MCH will pave the way for development of new organelle-targeted chemiluminescence agents towards imaging of different analytes in various biological models.


Introduction
6][17][18] Thus, it serves as an effective cytoprotective agent as it can effectively neutralize numerous reactive monitor mitochondrial H 2 S fluxes, specifically in cancer cells, in both cell cultures and in vivo with high selectivity and sensitivity.Additionally, tools that can detect H 2 S levels in human serum are highly critical in pathology.
1][52][53][54][55][56][57] Furthermore, these methods cause damage to cells and cannot offer precise detection.To address the drawbacks of conventional techniques, fluorescent probes that can offer real-time imaging opportunities, high spatial and temporal resolution, and selectivity without destruction of cells or tissues are highly promising.3][64][65] H 2 S has also been used for prodrug activation in numerous literature examples owing to its correlation with tumorogenesis. 5,45,487][68] Although highly valuable results have been obtained with conventional fluorophores, their intrinsic problems such as the need for external light irradiation, limited penetration of the excitation light, and the autofluorescence-induced low signal-to-noise ratio limit their broader acceptance and application areas, especially in in vivo models.
9][80] Additionally, they bear a modular scaffold, which can be simply modified toward the development of activity-based probes.Accordingly, new generation 1,2-dioxetane derivatives have been developed to monitor numerous analytes ranging from enzymes to ROS and biothiols including H 2 S. [81][82][83][84][85][86] Very recently, we have introduced the first ever example of a mitochondria-targeted chemiluminescence agent by modifying the phenoxy 1,2-dioxetane core, which was responsive to the leucine aminopeptidase (LAP) enzyme, with a well-established mitochondrion targeting triphenylphosphonium (TPP) group. 87ationic nature of TPP is critical to ensure the attraction between the probe and the negatively charged membrane of mitochondria as previously shown in the literature. 87e showed that TPP modification does not affect the luminescence characteristics of dioxetanes, and the probe can be used for in vivo imaging.
In this study, we turned our attention to H 2 S visualization and developed a H 2 S-activatable and mitochondria-targeted 1,2-dioxetane-based chemiluminescence agent (MCH) (Fig. 1) for the first time to monitor H 2 S activity in cell cultures, human serum and in vivo.MCH was constructed on our TPP bearing adamantyl-phenoxy 1,2-dioxetane core and linked to a H 2 Sresponsive dinitrophenyl group 88,89 through an ether linkage as a masking unit.When the phenol was masked, MCH showed no luminescence signal.H 2 S-mediated nucleophilic aromatic substitution cleaved the dinitrophenyl group and the phenolate was generated, which was followed by the chemiexcitation process to yield a strong luminescence signal (Fig. 1).

Photophysical properties of MCH
After obtaining the MCH probe, we first recorded the absorption and fluorescence spectra of MCH in DMSO (2% PBS, pH 7.4) as these are directly correlated with the chemiluminescence properties (Fig. 2). 69,87Upon reacting MCH (10 mM) with H 2 S (2 eq.), a time-dependent increase in both absorption (450 nm) and emission (520 nm) signals of the resulting benzoate was observed, indicating that the probe was activated with H 2 S (Fig. 2(A) and (B)).The cleavage of the masking unit was shown to be very rapid and completed in 10 minutes (Fig. 2(C) and (D)).We also synthesized the benzoate (MC-benzoate), 87 which is expected to be released after H 2 Sinduced activation, for comparison purposes.Fluorescence spectra of H 2 S-treated MCH and MC-benzoate perfectly overlapped (Fig. S1, ESI †), suggesting that the MC-benzoate was released upon activation.HR-MS analyses were also conducted to further prove the MC-benzoate release and to clarify the activation mechanism.As shown in Fig. S2 (ESI †), the signal of MCH at 874.2660 m/z disappeared and a peak at 558.1604 m/z, which belongs to benzoate (calculated as 558.1595 [M] + ), was detected after treating MCH with H 2 S.

Chemiluminescence characteristics of MCH
Next, the chemiluminescence characteristics of MCH in DMSO (2% PBS, pH 7.4) were investigated.Upon treating MCH with increasing concentrations of H 2 S (0-3 eq.), a time-and concentration-dependent response was reported.The signal dramatically increased (up to 206-fold) in the first 10 minutes and then started to decrease gradually in 1 hour (Fig. 3).Chemiluminescence intensity got stronger as the H 2 S concentration increased (Fig. 3).When total luminescence signal versus H 2 S concentration graph was analyzed, a linear increase was shown up to 1 eq. of H 2 S (Fig. 3-inset).The detection limit of MCH was calculated to be 0.0028 mM.In the absence of H 2 S, no signal was detected, suggesting that MCH is stable in solution.
The selectivity of MCH was checked by treating it with different biologically relevant analytes, including biothiols, hydrogen peroxide (H 2 O 2 ) and anions.Remarkable signal intensities were only reported in the case of H 2 S and glutathione (GSH); however, the intensity of the signal was approximately 4-fold higher in the case of H 2 S even though the GSH concentration (5 mM) was 250-fold higher than that of H 2 S (20 mM).This result clearly indicates that MCH is selective towards H 2 S (Fig. S3, ESI †).

Detection of H 2 S in cancer cells
Given the promising results obtained in solution, we next sought to investigate the performance of MCH in cell cultures.To this end, SH-SY5Y (neuroblastoma) and HCT116 (colon)

RSC Chemical Biology Paper
cancer cells, in which the H 2 S concentration is high, [90][91][92][93][94] were chosen as model cell lines.Increasing doses of MCH (0-10 mM) were incubated with the cells and time-dependent luminescence measurements were taken with a plate reader.MCH exhibited a similar kinetic profile in both cell lines.Chemiluminescence signal intensity increased in the first 30 minutes in a dose-dependent manner and then decreased slowly in 2 hours (Fig. 4), which is a typical trend for activity-based 1,2-dioxetanes.Treating cells with zinc chloride (ZnCl 2 ) (300 mM), a known H 2 S quencher, 95,96 for 10 minutes, dramatically reduced the signal intensity in both cells, indicating that H 2 S is the major endogenous trigger of the chemiluminescence (Fig. 4 and Fig. S4, ESI †).MCH was also found to be non-toxic in the working dose range as evidenced from the cell viability assay results.No sign of significant cell death was detected when both SH-SY5Y and HCT116 cells were incubated with MCH (0-100 mM) (Fig. S5, ESI †).These cumulative results show that MCH can be selectively activated in a cellular environment with endogenous H 2 S and can detect varying concentrations of H 2 S. Additionally, it is safe for bio-imaging studies.
Confocal microscopy studies were performed to further evaluate the imaging potential of MCH and to investigate its subcellular localization.First, HCT116 (Fig. 5) and SH-SY5Y (Fig. S6, ESI †) cells were incubated with MCH for 1 hour, the cells were washed and then the characteristic green emission of the resulting benzoate was visualized upon irradiation of the cells with a 405 nm confocal laser.A strong fluorescence signal was detected in both cells, indicating once again that MCH can be activated with an endogenous H 2 S, and the corresponding benzoate is released.The addition of ZnCl 2 to the cells remarkably quenched the fluorescence signal coming from the cells (Fig. 5 and Fig. S6, ESI †), which is in good agreement with the results obtained from the plate reader.Time-dependent activation of MCH was also captured under a confocal microscope with HCT116 cells.The signal appeared to be detectable at 15 min and got brighter after 30 min (Fig. S7, ESI †), which also supports the time-dependent luminescence signal data.

Localization of MCH to mitochondria
Next, a commercially available mitochondria stain MitoTracker Redt (100 nM) and MCH (10 mM) were co-incubated with HCT116 cells to prove their mitochondria targeting ability.As shown in Fig. 6, the emission signals coming from the red and green channels overlapped with a high Pearson's

Paper
RSC Chemical Biology coefficient (0.87), suggesting that the probe was localized to mitochondria due to its cationic nature as expected.This also clearly supports the high mitochondrial H 2 S activity in cancer cells.

Detection of H 2 S in human serum
We also tested the response of MCH in human serums, which were collected from 10 different healthy individuals (6 females, 4 males) aged between 22-39.Initially, optimization studies were performed using a serum sample.When the serum concentration in the PBS solution was increased (0-16%, v/v), a time and concentration-dependent turn-on response was reported in the chemiluminescence signal (Fig. S8, ESI †).Additionally, a linear increase was observed in the total luminescence signal as the serum concentration increased (Fig. 7(A

In vivo tumor imaging
Finally, MCH was utilized for in vivo tumor imaging.To this end, HCT116 cells were used to generate tumors on the right and left flank areas of immunocompromised mice.Later, MCH (100 mM, 100 mL) was injected intratumorally in the left flank, and time-dependent whole-body luminescence was detected under IVIS every 5 minutes for 1 hour period.The signal intensity increased during the first 40 minutes and then decreased gradually in 1 hour (Fig. 8 and Fig. S11, ESI †).As a control experiment, a group of tumor-bearing mice was injected with PBS (100 mL) subcutaneously, which did not give any signal (Fig. 8).We also checked the cytotoxicity of high concentrations of MCH in different cell lines as 100 mM of MCH was used in animal imaging.As previously mentioned, 100 mM of MCH did not cause significant cytotoxicity in HCT 116 and SH-SY5Y cancer cells (Fig. S5, ESI †).We also investigated the cytotoxicity of high concentrations of MCH in two different normal cell lines.In this direction, Vero and HGrC1 cells were incubated with various concentrations of MCH (0-100 mM).In both cells, no signs of cytotoxicity were observed, indicating that 100 mM of MCH dose is safe for bioimaging (Fig. S5, ESI †).These results support that MCH can also be utilized for in vivo imaging of tumor.

Conclusions
In summary, we have introduced the first example of a H 2 Sresponsive and mitochondria-targeted  used to image tumors generated by HCT116 cells in animal models.Given the promising results obtained in in vitro, human serum and in vivo studies, we believe that mitochondriatargeted chemiluminescent probes can be further utilized to investigate the critical roles of mitochondrial analytes in various diseases especially cancer and neurodegenerative disorders.Furthermore, thanks to our design approach, the core structure can be designed easily to target different organelles.Our work along these directions is in progress.

Cell culture experiments
The HCT116, SH-SY5Y, Vero and HGrC1 cells were grown in a high glucose DMEM supplemented with 2% penicillin-streptomycin and 10% FBS.The cells were passaged with DPBS and Trypsin-EDTA at 70% confluency and incubated in an Eppendorf Galaxy 170S incubator at 37 1C and 5% CO 2 .
For the kinetic measurements, HCT116 and SH-SY5Y cells were seeded in dark-sided, clear-bottomed 96-well plates (10 000 cells per well) and incubated overnight.One of the groups was treated with ZnCl 2 (300 mM) for 10 minutes and washed with PBS.Then, the cells were treated with MCH (0-10 mM) in PBS (1% DMSO, v/v, pH 7.4).The inhibition group was treated with MCH (10 mM) in PBS (1% DMSO, v/v, pH 7.4).The kinetic luminescence measurement was started immediately after the treatment and the chemiluminescence signal was detected using a Biotek Synergy H1 MF microplate reader.(n = 3, technical replicates).
HCT116 cells were seeded into 35 mm glass bottom confocal plates (10 000 cell per plate) and incubated overnight.One of the groups was treated with ZnCl 2 (300 mM) for 10 minutes and washed with PBS.Following the inhibition period, the cells were treated with MCH (10 mM) and washed with PBS, after half an hour.Time-dependent confocal images of HCT116 cells were captured at fixed-interval z stack before and after the addition of MCH, and recorded for 30 minutes.For the colocalization study, cells were treated with MCH (10 mM) for half an hour and washed with PBS.Then, the cells were stained with MitoTracker Red (100 nM) for 15 minutes and washed with PBS.Finally, confocal images were collected using a Leica DMI8 SP8 Inverted Confocal Microscope.Colocalization was analyzed using Image J software.(n = 4, technical replicates).
To perform the cell viability assay, cells at confluency were seeded into a dark-sided clear-bottomed 96-well plate at a density of 1 Â 10 4 cells per well and allowed to incubate overnight.The following day, the cells were treated with various concentrations of MCH in DMEM (1% DMSO, v/v).After an hour, the MCH-containing medium was replaced with fresh DMEM, and the cells were further incubated for 23 hours.To measure cell viability, 44 mL of fresh CellTiter-Glo reagent was added to each well.(n = 3, technical replicates).

Monitoring H 2 S level in human serum
The serum experiments were performed with the approval of the local ethical committee of Koç University.Informed consent was obtained from all participants and experiments were performed according to the guidelines of the Committee on human research at Koç University, Tu ¨rkiye.10 different healthy participant serum samples have been obtained from a previous study, where methodological details were reported in detail. 97or optimization studies, serums were diluted with PBS in different concentrations (1-16%), then 10 mM MCH was added to each well.Time-dependent luminescence response was monitored for 2 hours using a Biotek Synergy H1 MF microplate reader.Later, 10 human serum samples were diluted with PBS to a final 4% concentration.For those treated with ZnCl 2 , 5 mM ZnCl 2 was added.All samples were incubated at 37 1C for 30 min, then 10 mM MCH was added to each well.Subsequently, time-dependent luminescence response was monitored 2 hours using a Biotek Synergy H1 MF microplate

In vivo imaging
The institutional ethical committee of Koç University approved all in vivo experiments.All animal experiments were performed according to the guidelines of the Animal Research Local Ethics Committee at Koç University, Tu ¨rkiye.For these experiments, 6-8-week-old non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were anesthetized using isoflurane.
Tumor formation was induced subcutaneously for the administration of HCT116 cells to the mice.Each injection consisted of 2.5 Â 10 6 cells in a 100 mL volume of PBS, and injections were made into the right and left flank area using a 23-gauge needle.
Once the tumor diameter reached approximately 0.3-0.5 cm, 100 mL of 100 mM MCH was injected intratumorally.In control experiments, healthy mice were injected 100 mL of 100 mM MCH subcutaneously.For the control experiments with tumorigenic mice, 100 mL of 100 mM PBS was injected as a vehicle control intratumorally into another group of tumor-bearing mice.The kinetic luminescence measurement was initiated immediately following the injections, and in vivo imaging was performed using the PerkinElmer IVIS Lumina Series III instrument.

Fig. 1
Fig. 1 Structure of MCH and its activation with H 2 S.

Fig. 2 (
Fig. 2 (A) Absorption and (B) fluorescence spectra of MCH (10 mM) with or without the addition of 2 eq. of H 2 S in DMSO (2% PBS, pH 7.4) and (C) corresponding absorbance at 450 nm and (D) fluorescence at 520 nm in the presence of 2 eq. of H 2 S from 0 to 10 min.l ex = 440 nm.

Fig. 3
Fig. 3 Time-dependent chemiluminescence intensity of MCH (10 mM) in the presence of various equivalents of H 2 S (0-3) (inset: plot of the total luminescence with respect to varying H 2 S concentrations in the linear range) in DMSO (2% PBS, pH 7.4).

Fig. 6 Fig. 7
Fig. 6 Confocal microscopy images of HCT116 cells treated with MCH (10 mM) (ex/em: 405/500-550) and Mitotrackert Red (100 nM) (ex/em: 561/ 600-650).(n = 4) ) and Fig.S9, ESI †).To prove that the signal was raised due to H 2 S-induced activation, we treated a 4% (v/v) serum sample with increasing concentrations of ZnCl 2 .The signal decreased dramatically upon increasing the inhibitor concentration and almost complete inhibition was detected at a 5 mM ZnCl 2 dose (Fig.7(B)).Later, we treated the same serum sample (4%, v/v) with increasing concentrations of NaHS and checked the luminescence signal.As the NaHS concentration increased, the chemiluminescence signal was enhanced remarkably (Fig.7(C)).Similarly, ZnCl 2 (5 mM) treatment quenched the luminescence signal (Fig.7(C)), proving that MCH can detect varying concentrations of H 2 S in a complex serum environment.Then, we tested MCH in 10 different serum samples to investigate its potential for extensive usage.The average signal obtained from 10 serum samples (4%, v/v) is shown in Fig.7(D).It is worth mentioning that a comparable chemiluminescence signal was detected in all samples with slight variations (Fig.7(D) and Fig. S10, ESI †), suggesting that MCH can function precisely in similar serum samples.Treating the samples with ZnCl 2 (5 mM) again resulted in a strong inhibition and the total luminescence signal dropped to a level that is correlative with the signal obtained from untreated MCH in PBS solution, proving selective activation (Fig. 7(D) and Fig. S10, ESI †).