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Theranostics 2024; 14(19):7589-7603. doi:10.7150/thno.100930 This issue Cite

Research Paper

A fluorogenic ROS-triggered hydrogen sulfide donor for alleviating cerebral ischemia-reperfusion injury

Huangjie Lu^1,2,#, Huiying Zeng^1,2,#, Wenlong Wei^3,#, Yuying Chen^1,2, Ziqiang Zhou^1,2, Xuyang Ning^1,2, Ping Hu^1,2,3

1. State Key Laboratory of Bioactive Molecules and Druggability Assessment, Jinan University, Guangzhou 510006, China.
2. College of Pharmacy, Jinan University, Guangzhou 510006, China.
3. Department of Burns & Plastic Surgery, Guangzhou Red Cross Hospital, Faculty of Medical Science, Jinan University, Guangzhou 510006, China.
^#: These authors contributed equally.

✉ Corresponding author: Ping Hu; Mailing address: Jinan University, No. 855, East Xingye Avenue, Panyu Districst, Guangzhou 510632, China. Tel.: +86-18581483142; E-mail: inzahucom.

Abstract

Rationale: Cerebral ischemia-reperfusion injury is a severe neurovascular disease that urgently requires effective therapeutic interventions. Recently, hydrogen sulfide (H₂S) has garnered significant attention as a potential treatment for stroke; however, the precise and targeted delivery of H₂S remains a considerable challenge for its clinical application.

Methods: We have developed HSDF-NH₂, a novel H₂S donor characterized by high selectivity, self-reporting capabilities, and the ability to penetrate the blood-brain barrier (BBB).

Results: HSDF-NH₂ effectively scavenges reactive oxygen species (ROS) while generating H₂S, with emitted fluorescence facilitating the visualization and quantification of H₂S release. This compound has demonstrated protective effects against cerebral ischemia-reperfusion (I/R) injury and contributes to the reconstruction of brain structure and function in a rat stroke model (tMCAO/R).

Conclusion: As a ROS-responsive, self-reporting, and fluorescent H₂S donor, HSDF-NH₂ holds considerable promise for the treatment of ischemic diseases beyond stroke.

Keywords: Hydrogen sulfide donor, Reactive oxygen species, Molecular imaging, Cerebral ischemia-reperfusion injury, Theranostic agent

Introduction

Stroke is an acute cerebrovascular disease and the second leading cause of death worldwide [1]. It is characterized by high mortality, morbidity, disability and recurrence [2]. Notably, ischemic stroke constitutes approximately 80% of all stroke cases. Ischemic stroke triggers a series of biochemical cascades that result in metabolic abnormalities in brain tissue, leading to a variety of pathological changes including neuronal damage, neuroinflammation and oxidative stress [3]. Currently, the main strategies for treating ischemic stroke include thrombolysis (reperfusion) and neuroprotection [4]. Among them, reperfusion therapy through the use of recombinant tissue plasminogen activator (rt-PA) [5] or mechanical means remains an urgent option for the treatment of acute ischemic stroke, but its application is limited due to the narrow therapeutic window and serious risks such as intracerebral hemorrhage [6]. In addition, oxidative stress will suddenly occur after thrombolysis and large amounts of reactive oxygen species (ROS) will be produced, leading to various oxidative damages, including the production of proinflammatory cytokines, inflammatory infiltration, glial cell activation, etc., thereby aggravating secondary cerebral ischemia-reperfusion (I/R) injury [7-9]. Thus, there is an urgent need to develop new, effective treatment strategies for ischemic stroke.

Neurons in the penumbra of the stroke-affected brain retain salvageable bio-functional activity, making the use of neuroprotective drugs beneficial for reducing oxidative stress and neuroimmune inflammation to rescue damaged cells [10]. Unfortunately, many neuroprotective agents face significant limitations such as poor solubility, short half-lives, and inadequate penetration of the BBB, hindering their ability to accumulate and achieve effective therapeutic outcomes at the lesion site [11, 12]. Hydrogen sulfide (H₂S) acts as a gaseous signaling molecule, exerting significant biological effects in various physiological and pathological processes [13]. It also holds substantial therapeutic potential. Research studies in recent years have confirmed the positive effect of suitable concentrations of H₂S in the treatment of ischemic stroke [14]. H₂S can mitigate ischemic brain injury by increasing superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-PX) expression, lowering malondialdehyde (MDA) levels, and selectively eliminating excessive ROS [15-17]. H₂S can ameliorate brain tissue oedema by reducing the production of TNF-α and some other inflammatory mediators [18], as well as attenuating local inflammatory responses by inhibiting NF-κB nuclear translocation [19]. In addition, also H₂S can play a crucial protective role against cerebral ischemia/reperfusion injury by attenuating cerebrovascular endothelial cell injury, suppressing apoptosis inhibition, and mitochondrial calcium overloading [20]. However, this bioactive gas is characterized by rapid evaporation and high reactivity, and achieving precise dose and in vivo distribution tracking of H₂S is very difficult. These challenges seriously hinder the clinical translation of H₂S-based therapies and the mechanistic understanding of their biological effects. Therefore, it is crucial to develop novel hydrogen sulfide donors to overcome the aforementioned application issues.

To investigate the physiological and pathophysiological properties of H₂S, numerous H₂S donors have been developed and reported. Inorganic H₂S donors (e.g. NaHS and Na₂S) are the most commonly used hydrolysis-mediated donors for basic research, but hydrolysis results in a rapid release of H₂S and H₂S escape is observed and does not realistically mimic the biological effects of endogenous H₂S [21]. Lawesson's Reagent (LR) was originally developed as a sulfurizing agent capable of releasing H₂S more slowly than sulfide salts in aqueous solution, but it was gradually phased out due to its low aqueous solubility, limited release kinetic properties and unclear release mechanism. GYY4137, as an improved derivative of water-soluble Lawson's reagent, not only releases low concentrations of free H₂S molecules in a sustained manner, but also has good biocompatibility. However, it is typically synthesized and marketed as a dichloromethane complex, and metabolizes another signaling molecule (i.e., CO) in vivo, affecting the biological effects of H₂S [22]. Recent studies have shown that the applications of H₂S donors including NaHS, Na₂S, GYY4137, etc. have spanned a wide range of fields such as cardiovascular, neurological, anti-inflammatory, and anticancer fields, and have great potential for a wide range of medical and biological studies such as nanomedicine, medical research, clinical trials, and drug design and optimization [23]. In recent years, researchers have often introduced H₂S releasing fractions into the parent body in order to improve the pharmacological/therapeutic properties of clinical drugs [24-26]. H₂S release from these donors can be achieved by a variety of mechanisms, including photoactivation [27-31], enzymolysis [32-34], free thiol (cysteine and reduced glutathione) activation [35-44], ROS response [45-47], and other response mechanisms. Notably, ROS-responsive H₂S donors have garnered significant attention in recent years. Pluth et al. reported in 2016 that caged carbonyl sulfide (COS) binds to a ROS-responsive aryl borate as a COS/H₂S donor, which specifically responds to and depletes cellular ROS and then releases an equivalent amount of H₂S [45].

Considering these factors, we previously designed a novel fluorescent H₂S donor (HSD-B), which enabled the visualization and quantification of H₂S release in vitro [48]. On this basis, in order to improve the oil-water partition coefficient of HSD-B, a novel H₂S donor (named as HSDF-NH₂) was designed using hydrophilic amino groups instead of triphenylphosphine groups (Scheme 1).

The donor combines the ROS-responsive motif with self-reporting fluorescence transition mechanism that operates without consuming H₂S. This design aims to simultaneously consume ROS, release H₂S, and visualize and quantify the released H₂S. We anticipated that HSDF-NH₂ would be able to penetrate the damaged BBB. In this process, ROS would react with phenylboronic acid pinacol ester to release COS, which would then be hydrolyzed to form H₂S catalyzed by the enzyme carbonic anhydrase (CA), commonly found in mammals. Concurrently, thiocarbamate-substituted naphthalimides would convert to amine-substituted naphthalimides (designated HSDG-NH₂), resulting in the restoration of the previously quenched green fluorescence. Finally, PC-12 was used as the cell model to evaluate the neuroprotective effect of HSDF-NH₂ in vitro. We used the rat transient middle cerebral artery occlusion/reperfusion model and evaluated the in vivo efficacy and explored the therapeutic mechanism by injecting HSDF-NH₂ into the tail vein.

Results and Discussion

Donor design and characterizations

Among various fluorescent dyes, naphthalimide is notable for its ease of modification and exceptional optical properties [49]. The compound HSDF-NH₂ was synthesized by linking the phenylboronic acid pinacol ester to the 4-amino-naphthalimide tethered thiocarbamate (Figure 1A). The fluorescence of 4-amino-naphthalimide is quenched by the electron-withdrawing group of thiocarbamate. The ROS-responsive phenylboronic acid pinacol ester acts as a switch for the donor. In regions of stroke infarction, HSDF-NH₂ is activated by ROS to yield COS, a precursor of H₂S. Concurrently, the unstable intermediate is converted to HSDG-NH₂, which exhibits strong fluorescence and facilitates real-time monitoring of H₂S release. The introduction of an amino group at the N-position of 1,8-naphthalimide enhances the logD value of the donor and satisfies the covalent binding requirements of various designs (Figure 1B).

The spectral properties and response of the donor HSDF-NH₂ (10 μM) were studied in phosphate buffer (10 mM, pH = 7.4) as solvent. The maximum absorption of HSDF-NH₂ is at 385 nm. Upon incubation with H₂O₂ (100 μM) at 37 ℃, the maximum absorption peak of the test solvent appeared gradually at 427 nm (Figure 1C). H₂O₂ triggers chemoselective cleavage of the boronate-based thiocarbamate protecting group of HSDF-NH₂ to deliver HSDG-NH₂ as characterized by its emission spectra (λ_em = 565 nm). As shown in Figure 1D, Figure 2A and Figure S3, in the presence of H₂O₂ and CA, the emission peak of HSDF-NH₂ at 565 nm increased concomitantly and almost reached the intensity of HSDG-NH₂ at 150 min.

 Scheme 1 

Illustration of the reaction mechanism of the hydrogen sulfide donor and the production of hydrogen sulfide and related pharmacological effect.

 Figure 1 

(A) Synthesis of HSDF-NH₂ and the structure of CODF-NH₂ (B) The mechanism of H₂S releasing and monitoring by HSDF-NH₂ in the presence of ROS. Effects of (C) Absorption spectrum of 10 μM HSDF-NH₂ reacting with 100 μM H₂O₂ at 37 ℃ for 0 min and 150 min. (D) Fluorescence response of HSDF-NH₂ (10 μM) to H₂O₂ (100 μM) and CA (10 μg/mL). HSDG-NH₂ (10 μM) were used as controls. (E) HPLC traces of the samples: HSDF-NH₂, HSDF-NH₂ after reacting with H₂O₂ in PBS buffer for 120 min, and HSDG-NH₂.

The effects of pH and temperature on the reaction between HSDF-NH₂ and H₂O₂ were also investigated. As shown in Figures 2B-C, the reaction remains inert in acidic conditions, whereas the fluorescence intensity significantly increases with rising pH values. In the presence of H₂O₂, the aryl-bronic ester C-B bond is oxidized to phenol (C-O bond). Higher pH values are also likely to enhance the ensuing 1,6-elimination that releases COS and HSDG-NH₂ due to the reactive phenoxide (RO-) form being the dominant species in solution under more basic conditions. Although higher temperatures were favorable for the reaction, HSDF-NH₂ also demonstrated effective performance under physiological conditions (37 ℃, pH 7.4). The fluorescence intensity of HSDF-NH₂ after reacting with varying concentrations of H₂O₂ for 40 min was recorded (Figure S1). A strong linear correlation between the fluorescent signal and H₂O₂ concentration was observed over the range of 0 to 150 μM (Figure S1 inset). To evaluate the selectivity of HSDF-NH₂ for H₂O₂, various analytes, including ROS, inorganic salts, sulfur, and amino acids [50], were tested. As shown in Figures 2D-E, only the addition of H₂O₂ resulted in a significant increase in fluorescence, whereas other analytes had negligible effects. These findings indicate that HSDF-NH₂ exhibits excellent sensitivity and selectivity for H₂O₂.

Methylene blue colorimetric method for H₂S detection

To ascertain the ability of the donor to release H₂S in the presence of CA and H₂O₂, the widely recognized methylene blue method was employed to quantify H₂S generation. As shown in Figure 2F, the combination of methylene blue solution with HSDF-NH₂ solution, H₂O₂, and CA exhibited distinct absorption peaks at 670 nm and 745 nm, unequivocally indicating the release of H₂S by HSDF-NH₂. Using the established calibration curve (Figure S2), the concentration of H₂S liberated by HSDF-NH₂ was quantitatively determined and presented in Figure 2G, revealing a time-dependent release pattern with peak release occurring approximately 150 min after initiation, achieving an efficiency of approximately 30%. The underlying factors contributing to this temporal profile are likely multifaceted, involving the oxidative properties of H₂O₂ and the volatility of the gas. Notably, omission of CA from the reaction system notably suppressed the absorbance at 670 nm, underscoring the dependency of HSDF-NH₂'s H₂S release on CA during the conversion from COS to H₂S. Crucially, the robust linear correlation observed between fluorescence measurements and H₂S quantification via the methylene blue method (R² = 0.988) underscores the reliability of fluorescent readings as optical tools for monitoring COS/H₂S release dynamics from HSDF-NH₂ with high temporal resolution (Figure 2H-I).

The proposed mechanism of H₂S release from HSDF-NH₂ with self-reporting fluorescence

The proposed mechanism detailing H₂S release from HSDF-NH₂, along with its self-reporting fluorescence in the presence of ROS, is illustrated in Figure 1B. Initially, HSDF-NH₂ undergoes partial hydrolysis to boronic acid upon exposure to water, followed by oxidation triggered by hydrogen peroxide. This sequential process yields the fluorescent amino compound HSDG-NH₂ and releases COS. The caged COS subsequently undergoes conversion to H₂S in the presence of CA. The confirmation of these compounds was achieved using HPLC and high-resolution mass spectrometry (HRMS), as depicted in Figure 1E and Figure S4.

 Figure 2 

In vitro characterizations of HSDF-NH₂. (A) Time-dependent fluorescence intensities of HSDF-NH₂ at 565 nm in the presence of H₂O₂ (100 μM). (B) Temperature at pH 7.4 and (C) pH at 37 ℃ on the fluorescence of HSDG-NH₂ and HSDF-NH₂ reacting with 0 and H₂O₂ for 2 h. λex/em = 427/565 nm. (D) Fluorescence intensity at of 10 μM HSDF-NH₂ toward various species. Deionized water was used as a control. (E) Fluorescence responses of 10 μM HSDF-NH₂ to various species. (F) UV-Vis absorption spectrum of HSDF-NH₂ at different time points via MB assay. (G) H₂S release from HSDF-NH₂ upon introducing CA and H₂O₂ (■), H₂O₂ without CA (●) and CA without H₂O₂ (▲). (H) Time-dependent fluorescence turn on (red) and H₂S release (blue) of HSDF-NH₂. (I) Correlation between fluorescence measurement and MB detection.

Neuroprotective effect of HSDF-NH₂ in tMCAO/R rats

The protective effect of HSDF-NH₂ on I/R injury in the hypoxia/reoxygenation (H/R) model of PC-12 cells was investigated. The cell viability assay demonstrated that HSDF-NH₂ exhibited a dose-dependent protective effect on OGD-insulted PC-12 cells (Figure 3A). Excessive ROS accumulation in the infarct microenvironment exacerbates injury [51]; therefore, intracellular ROS levels can serve as an indirect indicator of oxidative stress following cellular damage. In this study, we utilized the ROS probe dihydroethidium (DHE) to monitor and analyze intracellular ROS levels in PC-12 cells after various treatments (Figure 3B and S7). Compared to untreated control cells, OGD/R-treated cells exhibited significantly enhanced DHE red fluorescence signals, indicating an abnormal increase in ROS levels. Treatment with HSDF-NH₂ and edaravone significantly inhibited the ROS elevation, confirming the synthesized donors' effective ROS scavenging capability at the cellular level. Flow cytometry analysis further elucidated the protective mechanisms of HSDF-NH₂ against I/R injury (Figure 3C). HSDF-NH₂ significantly reduced the percentage of apoptotic and necrotic PC-12 cells induced by H/R injury, demonstrating an anti-apoptotic effect comparable to that of Pro. These results provide compelling evidence that HSDF-NH₂ is a potent H₂S donor, offering cellular protection from oxidative stress.

 Figure 3 

(A) Cell viabilities of OGD-treated PC-12 cells incubated with HSDF-NH₂ for 24 h. Pretreated PC-12 cells with different concentrations of HSDF-NH₂ (0, 1, 5, 10, 20, and 50 μM), CODF-NH₂ (COD, 10 μM), or EDR (edaravone, 10 μM, positive control). Data are presented as means ± SD, n = 3. **P < 0.01; ***P < 0.001. (B) Representative images of the DHE fluorescence in PC-12 cells treated with OGD/R or different groups (Scale bar: 100 μm). (C) Flow cytometry analysis of hypoxia-induced apoptosis of PC-12 cells pretreated with PBS, various doses of HSDF-NH₂, CODF-NH₂ or Pro. (D) Schematic diagram of the model of tMCAO/R established by the currently accepted suture embolic method (ECA: External carotid artery; ICA: Internal carotid artery; PPA: Paramedian pontine arteries; PCA: Posterior cerebral artery; MCA: Middle cerebral artery; ACA: Anterior cerebral artery). (E) The infarct area of tMCAO/R rats with different treatment, brain slices were stained with TTC at 72 h post reperfusion. (F) The infarct area of tMCAO/R rats treated with different drugs, monitored by MRI at 4th day post reperfusion. (G) The quantified results of TTC staining in E (G1: Sham; G2: Model; G3: CODF-NH₂; G4: HSDF-NH₂). Data are presented as means ± SD, n = 3, ***P < 0.001. (H) The neurological assessment of tMCAO/R rats treated with different drugs. Data are presented as means ± SD, n = 5, **P < 0.01.

The neuroprotective effect of HSDF-NH₂ was assessed using the rat transient middle cerebral artery occlusion/reperfusion (tMCAO/R) model, established via the widely accepted suture embolic method (Figure 3D) [52]. Primary neurological scores [53] were evaluated 24 h post-treatment, revealing that the HSDF-NH₂ group scored 1.6 points, in contrast to the CODF-NH₂ and model groups, which scored 2.4 and 2.8 points, respectively. This indicates that the HSDF-NH₂ group experienced the most significant recovery in neurological function (Figure 3H). TTC staining was employed to distinguish between infarcted and normal brain tissue, with infarcted regions appearing white and normal regions red. On day 3 post-treatment, TTC staining demonstrated substantial brain recovery in the HSDF-NH₂ group (Figure 3E). Specifically, the infarcted area was reduced to 21.93% in the HSDF-NH₂ group, compared to 45.7% in the model group (Figure 3G). These findings were corroborated by MRI imaging, which also indicated a reduction in infarct size (Figure 3F).

Fluorescence imaging of H₂S release from HSDF-NH₂ in vitro and in vivo

The fluorescence properties of HSDF-NH₂ were further evaluated for potential biomedical imaging applications. In cell culture experiments, Rosup was used to induce the generation of H₂O₂ and subsequent release of H₂S. To monitor H₂S production within the cells, Cy-NO₂ was employed as a fluorescent probe [54]. In the absence of Rosup, no red fluorescence signal from Cy-NO₂ was detected, indicating that H₂S was not produced (Figures 4A-B). However, after 30 min of incubation with Rosup, a dose-dependent increase in fluorescence signals was observed in both the donor and probe channels. Analyzed at the cellular level, HSDF-NH₂ exerted the best effect at the level of ROS scavenging, protection of neuronal cells, and reduction of apoptosis. And as a control, CODF-NH₂ group could observe a certain but weak neuroprotective effect, which confirmed that the direct quenching of ROS by the borate structure could not maximize the realization of the protective effect, and it was obvious that the release of H₂S was the key. And we also found that Rusup-treated cells do not emit fluorescence in the Cy-NO₂ channel in the absence of HSDF-NH₂ presence, which demonstrates that Cy-NO₂ is stable under the experimental conditions without the introduction of a donor and can accurately represent H₂S release (Figure S6). These results confirm that HSDF-NH₂ successfully delivered H₂S and provided enhanced green fluorescence, enabling real-time monitoring of H₂S release in living cells.

Given that the oil-water Distribution coefficient (logD) is a critical parameter for evaluating the capacity of a small molecule to cross the blood-brain barrier [55], we have preliminarily estimated the logD value of HSDF-NH₂ to be approximately 2.35. Notably, the differential fluorescence intensities between HSDF-NH₂ and its cleaved product (HSDG-NH₂) upon H₂S release facilitated the monitoring and imaging of H₂S release in t/MCAO rats. A single dose of HSDF-NH₂ at 4 mg/kg was administered via tail vein injection in tMCAO/R rats, and subsequent fluorescence changes were observed through ex vivo imaging. The results indicated that the green fluorescence, which signifies the presence of HSDG-NH₂ in the brain after the conversion of HSDF-NH₂, increased gradually, peaking at 2 h before declining and becoming weak at 24 h (Figure 4E-F). This observation suggests that HSDG-NH₂ is metabolized and cleared from the brain over time. Furthermore, ex vivo imaging of various organs following HSDF-NH₂ administration in tMCAO/R rats revealed that HSDF-NH₂ is predominantly metabolized by the liver and kidneys (Figure 4C-D). However, HSDF-NH₂ presents certain limitations for in vivo imaging and quantification, primarily due to autofluorescence in live animals and the insufficient emission wavelength of HSDG-NH₂.

Behavior tests and mechanisms underpinning therapeutic effects of HSDF-NH₂ on tMCAO/R

As shown in Figure 5A, the tMCAO/R model was successfully established following the pretraining of rats. Behavioral assessments were conducted on days 1, 3, 5, 7, 9, 11, and 13. In the adhesive removal test (Figures 5B-C, F-G), rats in the ischemic model group exhibited severe behavioral deficits. However, 9 days post-treatment with HSDF-NH₂, this behavioral asymmetry was significantly ameliorated, indicating a substantial therapeutic effect of HSDF-NH₂. In the grid-walking test, the model groups demonstrated increased contralateral foot faults, whereas these faults were significantly reduced in the HSDF-NH₂-treated groups (Figures 5D, H). The cylinder test further revealed that tMCAO/R induced a higher rate of asymmetry, which was significantly improved with HSDF-NH₂ treatment (Figures 5E, I), underscoring the motor-functional neurological recovery facilitated by HSDF-NH₂.

We then investigated the capability of HSDF-NH₂ to modulate the proinflammatory microenvironment and rescue damaged neurons. HSDF-NH₂ significantly reduced the expression of pro-inflammatory cytokines TNF-α and IL-1β (Figures 6C, E). To assess oxidative stress levels in the lesion, we utilized the DHE probe to detect changes in ROS content (Figure 6B). The results demonstrated that HSDF-NH₂ administration markedly decreased ROS levels in the ischemic semi-dark band, thereby reducing neuronal oxidative stress compared to the model group. Additionally, we examined the therapeutic effects of HSDF-NH₂ on glial scar formation. 14 days post-treatment, we observed reduced GFAP expression in the infarcted hemisphere in the HSDF-NH₂ group (Figure 6D). Given the additional ROS generated during reperfusion, studies utilizing reperfusion models of brain injury could yield more clinically relevant results. Whether HSDF-NH₂ can exert beneficial effects in I/R models warrants further investigation.

 Figure 4 

(A) 20× and (B) 63× Confocal microscopy images of PC-12 cells after different treatments. Cells were first incubated with HSDF-NH₂ (10 μM) and Cy-NO₂ (10 μM) for 60 min. After removal of excess HSDF-NH2 and Cy-NO2, PBS (control), low-dose Rosup at 50 μg/mL (Rosup (L)), or high-dose Rosup at 100 μg/mL (Rosup (H)) was added. Fluorescence images were acquired after 30 min (20× mirror scale bar: 100 µm; 63× mirror scale bar: 20 µm). Representative IVIS images (C) and quantification (D) of main organs after intravenous administration of different drugs for 2 h. Ex vivo fluorescence images (E) and quantification (F) of H₂S release in brains collected from t/MCAO rats at different time points after treatment with the same dose of HSDF-NH₂. Data are presented as means ± SD, n = 3.

 Figure 5 

(A) Schematic illustration of the timeline of behavioral test. Functional recovery was evaluated by (B-C) the adhesive test, (D) grid-walking test and (E) the cylinder test at a series of time points after tMCAO/R. Results of the (F-G) adhesion test, (H) grid walking test, and (I) cylinder test on day 9 after tMCAO/R. Data are presented as means ± SD, n = 5.

In vitro and in vivo biocompatibility of HSDF-NH₂

HSDF-NH₂ did not exhibit significant cytotoxicity at concentrations up to 20 μM in normal PC-12 cells (Figure S5), indicating high cytocompatibility. Furthermore, blood routine examinations of both sham and model rats post-injection of HSDF-NH₂ revealed no noticeable abnormalities (Figure S8), demonstrating its low toxicity in vivo. Additionally, histological H&E staining analyses of the heart, kidney, liver, lung, and spleen tissues showed no apparent variations (Figures 6A and S9), further underscoring the favorable biological safety profile of HSDF-NH₂.

Conclusions

In conclusion, we have designed and synthesized a novel ROS-responsive H₂S donor, HSDF-NH₂, which not only scavenges ROS, but also releases H₂S and activates fluorescence. This compound significantly increased the viability of OGD/R-injured PC-12 cells in vitro. Furthermore, its cytoprotective effects were successfully translated to an in vivo tMCAO/R rat model. HSDF-NH₂ effectively penetrates the blood-brain barrier and exhibits therapeutic effects by reducing infarct volume, decreasing apoptosis, and mitigating oxidative stress. As a result, neurological function was notably improved in rats treated with HSDF-NH₂. Overall, HSDF-NH₂ introduces new strategies for the treatment of ischemic stroke.

Experimental section

Materials and instruments

All chemicals were obtained from commercial suppliers and used without additional purification. Roswell Park Memorial Institute (RPMI-1640) medium, penicillin and streptomycin were purchased from Thermo Fisher Scientific (Massachusetts, U.S.A). Trypsin was purchased from NCM Biotech (Suzhou, China). ROS Assay Kit was purchased from BIOESN (Shanghai, China). Fetal bovine serum (FBS) and Annexin V-FITC apoptosis detection kit was purchased from Pricella (Wuhan, China). The monofilament nylon threads were purchased from Meyue (Changsha, China). 2,3,5-Triphenyltetrazolium chloride staining solution (2%) was purchased from Solarbio (Beijing, China). ¹H NMR and ¹³C NMR spectra were recorded on a Bruker AV-400 or 600 MHz spectrometer. The NMR data were processed by software Mest Re-Nova (Ver.14.0.0.23239, Mestrelab Research S.L.). Chemicals shifts were referenced to the residue solvent peaks and given in ppm. High-resolution mass spectra (HRMS) were obtained using a Q-STAR Elite ESI-LC-MS/MS spectrometer. UV-Vis absorption spectra were acquired on a JASCO V-730 spectrophotometer. Fluorescence emission spectra were acquired on a PerkinElmer LS55 and Edinburgh FS5 fluorescence spectrophotometer. Samples for absorption and fluorescence emission measurements were contained in 1×1 cm quartz cuvettes (3.5 mL volume).

 Figure 6 

(A) Representative images of main organs with H&E staining after the tMCAO/R rats were treated with different drugs (Scale bar, 250 μm). (b) Representative DHE staining indicating the ROS level in neurons in penumbra of tMCAO/R rats with different treatments. DHE (red), DAPI (blue), (Scale bar: 100 μm). (d) Glial scars (GFAP) level in infarct sites of various groups 14 days after different treatments. GFAP (green), DAPI (blue), (Scale bar: 50 μm). The expression of proinflammatory cytokines including (c) TNF-α and (E) IL-1β decreased. Data are presented as means ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Synthesis and characterization of HSDF-NH₂, CODF-NH₂ and HSDG-NH₂

The synthetic route of HSDF-NH₂, CODF-NH₂ and HSDG-NH₂ were exhibited in supplementary information (Supplementary Data S1). The structure of donor was verified by ¹H NMR, ¹³C NMR and HRMS (Figure S10-S30).

General methods of UV-Vis absorption and fluorescence spectra

Unless otherwise noted, all the spectral measurements were carried out in 10 mM phosphate buffer (pH 7.4) according to the following procedure in triplicate. Typically, 35 mL of HSDF-NH₂ (10 μM) solution was prepared and added with CA (10 μg/mL) and H₂O₂ (final concentration of 100 μM). The resulting mixture was well shaken and placed at 37 ℃ before measurement.

Preparation of various solutions for selectivity analysis

A stock solution of 10 mM HSDF-NH₂ was prepared in DMSO. The fluorescence response of HSDF-NH₂ (10 μM) was performance in 3 mL PBS (10 mM pH = 7.40). The solutions containing different ROS and thiol nucleophiles (Cys, GSH, Na₂S) were prepared by dissolving the corresponding compounds in ultrapure water (final concentration of 100 μM). ClO^-, H₂O₂, ONOO^-, ¹O₂ and TBHP were generated according to the previous report [56]. •OH was generated from the reaction between Cu²⁺ and ascorbate [57]. Metal ions (Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Fe³⁺, Cu²⁺), glucose and thiol nucleophiles were also prepared in appropriate concentrations. The final concentration of metal ions is 30 μM, and the final concentration of glucose or amino acid is 1.5 mM. And CaCl₂, ZnSO₄, FeCl₂, FeCl₃, CuCl₂ were used for the metal ions solution preparation. Reaction time was set to be 120 min for all reagents.

HPLC measurement

HPLC was conducted using the Agilent 1260 Infinity II system. The analysis utilized a SuperLuC18-AQ5u column (4.6 mm × 250 mm, 5 μm) with a mobile phase composed of acetonitrile and 25 mM ammonium acetate buffer (8:2, v/v) at a flow rate of 1 mL/min and detection at 254 nm. The reaction mixture of HSF-NH₂ and H₂O₂ in PBS (10 mM, pH 7.4) served as the sample for measurement.

Methylene blue H₂S release assay

Each assay described was performed in triplicate in a one-dram vial containing 1.165 mL PBS buffer (pH = 7.4), 300 μL Zn(OAc)₂ solution (1% w/v), 20 μL HSDF-NH₂ solution (10 mM in DMSO), H₂O₂ solution, and 300 μL CA solution (0.5 mg/mL). The final concentrations were 133 μM HSDF-NH₂, 1 mM H₂O₂, and 100 μg/mL CA. At predetermined timepoints, 1.8 mL was removed from each reaction vial and diluted with 600 μL FeCl₃ solution (30 mM in 1.2 M HCl), followed by 600 μL N, N-dimethyl-p-phenylenediamine solution (20 mM in 7.2 M HCl). The MB reaction was conducted for 90 min, and the absorbance at 670 nm of the resulting solution was measured with an UV-Vis spectrometer. The concentration of H₂S in each sample was determined from a calibration curve of Na₂S, which was generated by plotting the H₂S concentration against the measured absorbance.

LogD measurement procedure

Initially, n-octanol served as a lipid solvent to dissolve HSDF-NH₂ of different concentrations, and the corresponding standard calibration curves were constructed using UV-visible spectroscopy. Subsequently, the PBS buffer solution (pH 7.4) was combined with n-octanol in a 1:1 volumetric ratio, followed by the introduction of a predetermined amount of the probe while agitating. After thorough mixing for 120 minutes, the mixture was left undisturbed to allow for phase separation of the n-octanol, and the UV absorbance was then measured. Subsequently, the concentration of the probe in the oily phase (C_oil) was determined using the established standard curve. The probe's concentration in the aqueous phase (C_water) was quantitatively deduced based on the principle of mass conservation. The partition coefficient (LogD) was calculated as the logarithm of the ratio of C_oil to C_water. The final LogD values were derived from the mean of triplicate measurements.

Cell culture and oxygen-glucose deprivation/reoxygenation (OGD/R) Model

PC-12 cell line was purchased from the iCell Bioscience Inc (Shanghai, Chian). The OGD/R model for PC-12 cells was developed using the following steps: The ischemia was simulated by replacing the high glucose RPMI-1640 medium with 12% FBS with a glucose-free RPMI-1640 medium. After incubating for 12 h at 37 ℃ in an anoxic environment, the cells were transferred back to a conventional incubator for 24 h with a fresh high glucose medium to mimic reperfusion. PC-12 cells maintained under normoxic conditions with high glucose RPMI-1640 medium served as control. Five groups of PC-12 cells were prepared: the normal (Con) group, the OGD/R model group, the HSDF-NH₂ pretreatment groups (HSDF-NH₂, 1-50 μM), the CODF-NH₂ group (10 μM, COD), and the propranolol pretreatment group (10 μM, EDR) as the positive control.

Cytotoxicity assay

The MTT assay was employed to assess the cytotoxicity of the compound HSDF-NH₂. Briefly, PC-12 cells were seeded in 96-well plates at a density of approximately 1 × 10⁴ cells per well and incubated for 24 h. Subsequently, fresh medium containing the test or control compounds at varying concentrations was added to the PC-12 cells. Following a 24 h incubation with the compounds, the cells were washed twice with PBS, and 100 μL of MTT solution (0.5 mg/mL) was added to each well. After an additional 4 h incubation at 37 ℃, the medium was removed and 100 μL of DMSO was added to dissolve the formazan crystals. The absorbance of each well was then measured at 570 nm using a plate reader.

ROS scavenging capability of HSDF-NH₂ in OGD/R model

To measure the ROS levels in cells subjected to OGD/R, DHE, which converts into a highly red fluorescent compound upon oxidation by ROS, was used to detect intracellular ROS formation. Briefly, PC-12 cells were plated in 24-well plates and subjected to OGD for 12 h. The cells were then cultured in complete medium containing various compounds for 24 h. After washing the cells with DPBS, they were incubated with medium containing DHE (10 μM) for 30 min. Fluorescence images were captured using a confocal laser scanning microscope (LSM800, Zeiss, Germany).

In vitro H₂S release and fluorescence imaging in PC-12 cells

PC-12 cells were seeded and cultured according to the procedures outlined in section 2.7. The cells were initially treated with HSDF-NH₂ (10 μM) and Cy-NO₂ (10 μM) for 60 min. Following the removal of excess HSDF-NH₂ and Cy-NO₂, the cells were incubated in fresh medium containing either PBS (control), low-dose Rosup (A compound mixture of oxidative reagents with a concentration of 50 mg/ml, usually used as a positive control reagent for reactive oxygen species.) at 50 μg/mL (Rosup (L)), or high-dose Rosup at 100 μg/mL (Rosup (H)) for an additional 60 min. After fixation, the cells were observed and imaged using a confocal laser scanning microscope. The red channel of Cy-NO₂ was recorded at 700-730 nm with excitation at 673 nm, while the green channel of HSDF-NH₂ was recorded at 520-570 nm with excitation at 430 nm.

Cell apoptosis assay

The effect of HSDF-NH₂ on apoptosis in PC-12 cells was evaluated using an Annexin V-FITC apoptosis detection kit. Briefly, PC-12 cells were seeded in 24-well plates at a density of 1 × 10⁵ cells per well. Following treatment with or without HSDF-NH₂ or edaravone for 24 h, the cells were stained with Annexin V-fluorescein isothiocyanate (FITC) in binding buffer for 15 min at room temperature. The cells were then labeled with propidium iodide (PI), and apoptotic cells were assessed using a flow cytometer (BD FACSCanto).

Animals

The procedures of animal experiments in this study were approved by the Jinan University (Guangzhou, China). Adult male Sprague-Dawley rats (10-12 weeks old, 200-250 g) were purchased from the Guangdong medical laboratory animal center and housed in standard cages under standard conditions. All animals were acclimatized for one week before use.

Preparation of the rat tMCAO/R model

The rat transient middle cerebral artery occlusion/reperfusion (tMCAO/R) model was established using a previously reported monofilament method [53, 58]. Briefly, a silicone-coated nylon thread (0.32 ± 0.02 mm, catalog number M8507, Changsha Meyue) was inserted into the middle cerebral artery via the ipsilateral external carotid artery to induce occlusion. After 2 h, the thread was removed, and the external carotid artery was ligated to achieve left cerebral ischemia-reperfusion injury in rats. Throughout the procedure, health and humane care were rigorously maintained.

Evaluation of neurological scores

After 1 h of reperfusion, model rats were randomly assigned to three groups. These groups received intravenous injections of saline (Model), 4 mg/kg of HSDF-NH₂, and 4 mg/kg of CODF-NH₂, respectively. Neurological assessments [53] were conducted 24 h post-administration, employing a scoring system as follows: 0, indicating normal and active condition; 1, denoting inability to fully extend the right forepaw; 2, indicating circling towards the right side; 3, indicating inability to stand up and falling towards the right side; and 4, indicating absence of spontaneous movement.

Neuroprotection effect evaluated with TTC

Brains of MCAO/R rats were collected 72 h post-administration and sliced, immersed in 0.25% 2,3,5-Triphenyltetrazolium chloride (TTC) dye at 37 ℃ for 30 min [6]. The infarct area was quantified by ImageJ.

Behavior tests

The cylinder test, grid-walking test and adhesive removal test were carried out to evaluate the repair of sensorimotor functions. Before surgery, we pre-trained the rats for 3 days continuously. Behavioral tests were then performed at 3th, 5th, 7th, 9th and 13th day post stroke. For the cylinder test, rats were individually placed inside a transparent cylinder measuring 35 cm in height and 15 cm in diameter. The behavior of each rat was observed for 5 min, and the number of contacts made with the cylinder walls using the left forepaw (L), right forepaw (R), or both forepaws (B) was recorded. The asymmetric rate was calculated as (L - R)/(L + R + B) × 100 (%) [59]. For grid-walking task, slightly modified from previously reported literature [60], an elevated grid containing square (5× 5 cm²) wire mesh was employed. Every rat was placed onto the wire grid to move freely until reaching at least 100 steps with the left forelimb. The numbers of stepping errors and non-faults for both limbs were recorded. The result was then analyzed with the formula reported previously [61]. For the adhesive test, a 10 × 10 mm² sticker was placed onto the paralyzed forepaw of the rat. The rat was then returned to its cage. The time taken for the rat to first contact the sticker was recorded as the “time to touch”, and the duration required for the rat to successfully remove the sticker was recorded as the “time to remove” [6].

MRI imaging in vivo

The tMCAO/R rats with different treatment were anaesthetized with isoflurane. To monitor the infarct area, T2-weighted coronal images of the brain were recorded with 9.4 T MR scanner for small animal imaging system (BioSpec 94/30 USR, Burke, Germany) on day 4 post reperfusion. The acquisition parameters for T2-weighted MRI imaging: TR = 2500.0 ms, TE = 33.0 ms, Slice thickness = 0.8 mm. Images were analyzed using RadiAnt DICOM Viever software (Medixant, Poznan, Poland).

Fluorescence imaging of H₂S release in tMCAO/R rat models

To study the metabolism and biodistribution of HSDF-NH₂ in rats with tMCAO/R, rats received a single intravenous injection of HSDF-NH₂ (4 mg/kg) at 1 h post ligation. Rats in control group were injected with saline. At 2 h post injection, rats were euthanized and major organs including heart, liver, spleen, lung, and kidneys were collected for ex vivo imaging (IVIS Spectrum PerkinElmer, U.S.A).

To evaluate the release of H₂S in living rats with tMCAO/R by in vivo imaging, rats received a single intravenous injection of HSDF-NH₂ (4 mg/kg) at 1 h post ligation. At each defined time point (0, 0.5, 1, 2, 6, 12 and 24 h post injection), rats were euthanized and brains were isolated for ex vivo imaging using the IVIS Spectrum (PerkinElmer, U.S.A) in a fluorescence mode.

In vivo biocompatibility evaluation of HSDF-NH₂

On day 14 post-administration, comprehensive blood panel analyses and serum biochemistry tests were performed using collected blood samples. These tests included measurements of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Additionally, major organs—such as the heart, liver, spleen, lungs, and kidneys—were harvested from the rats for subsequent histological analysis.

Enzyme-linked immunosorbent assay (ELISA)

To assess the expression of inflammation-related cytokines in the ischemic hemisphere, brain tissues were promptly collected and homogenized in cold PBS. Following centrifugation at 12000g for 15 min at 4°C, the content of TNF-α and IL-1β in the samples were quantified using an ELISA kit following standard protocols.

Statistical analysis

All results were reported as means ± standard deviation (SD). Significance was determined by the student's t-test or one-way analysis of variance (ANOVA) using GraphPad Prism (version 8). The statistical significance was considered when the P value was less than 0.05.

Abbreviations

H₂S: hydrogen sulfide; BBB: blood-brain barrier; ROS: reactive oxygen species; I/R: ischemia-reperfusion; rt-PA: recombinant tissue plasminogen activator; SOD: superoxide dismutase; GSH-PX: glutathione peroxidase; MDA: malondialdehyde; LR: Lawesson's Reagent; COS: carbonyl sulfide; CA: carbonic anhydrase; OGD/R: oxygen-glucose deprivation/reoxygenation; FITC: fluorescein isothiocyanate; PI: propidium iodide; tMCAO/R: transient middle cerebral artery occlusion/reperfusion; TTC: 2,3,5-Triphenyltetrazolium chloride; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ELISA: Enzyme-linked immunosorbent assay; HRMS: high-resolution mass spectrometry; DHE: dihydroethidium; ECA: External carotid artery; ICA: Internal carotid artery; PPA: Paramedian pontine arteries; PCA: Posterior cerebral artery; MCA: Middle cerebral artery; ACA: Anterior cerebral artery; logD: oil-water distribution coefficient.

Supplementary Material

Supplementary methods, figures and table.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 82071367), Science and Technology Program of Guangzhou, China (No. 2023A03J0525), Natural Science Foundation of Guangdong Province of China (Nos. 2021A1515220047 & 2023A1515011135), Clinical Characteristic Technology Project of Guangzhou Municipal Health Commission in Guangzhou Area (No. 2023C-TS41).

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Competing Interests

The authors have declared that no competing interest exists.

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Author contact

Corresponding author: Ping Hu; Mailing address: Jinan University, No. 855, East Xingye Avenue, Panyu Districst, Guangzhou 510632, China. Tel.: +86-18581483142; E-mail: inzahucom.

Received 2024-7-14
Accepted 2024-10-3
Published 2024-11-4

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