Glutathione carbon dots as an intracellular reactive oxygen species scavenger for reducing cisplatin‐induced ototoxicity

It is widely recognized that platinum‐based chemotherapy, particularly cisplatin therapy, can cause ototoxicity. At present, there are no Food and Drug Administration‐approved drugs to prevent or alleviate ototoxicity. Ototoxicity is generally believed to be caused by excessive reactive oxygen species production in the inner ear. Accordingly, a variety of antioxidants have been developed to protect against ototoxicity. To improve the efficiency of drug delivery to the cochlea, here, we synthesized simple and easy‐to‐obtain glutathione carbon dots (GSH CDs) with ultra‐small dimensions. The experimental results revealed that the GSH CDs have strong free‐radical scavenging activity and can restore mitochondrial function, maintain hair cell stability, and protect hair cells from cisplatin‐induced oxidative stress. Thus, GSH CDs may serve as a new therapeutic agent for preventing cisplatin‐induced ototoxicity.

tive at controlling malignant tumors, they have several undesirable side effects including ototoxicity. 4At present, there are no clinically approved or effective approaches to prevent ototoxicity caused by platinumbased chemotherapy. 1,5The production of reactive oxygen species (ROS) in the inner ear is the primary cause of

S C H E M E 1
The synthesis process of GSH CDs and the schematic illustration of GSH CDs in the treatment of cisplatin-induced ototoxicity.
ototoxicity.0] To overcome these limitations, new antioxidants are urgently needed to treat cisplatin-induced hearing impairment.
0][21][22][23] Glutathione (GSH) is used to remove cellular free radicals, serving as an intracellular free radical scavenger.However, the effectiveness of GSH as a therapeutic agent is limited because of its low bioavailability. 24Therefore, GSH can be used as a CD precursor, combining the benefits of both CDs and GSH. 25 Meanwhile, several studies have demonstrated that CDs with excellent antioxidant properties, such as Se-doped CDs, can be used as antioxidant therapies and are capable of reducing H 2 O 2 -induced oxidative damage. 26However, few studies have used CDs to treat the oxidative injury caused by cisplatin.
In this study, we describe the microwave-based synthesis of GSH-citric acid CDs (GSH CDs) as a highly efficient antioxidant according to previous study. 25In vitro, GSH CDs were found to effectively reduce mitochondrial dysfunction and oxidative stress caused by cisplatin (Scheme 1).In addition, in animal experiments, GSH CDs significantly increased the number of hair cells (HCs) after cisplatin treatment.Together, these studies demonstrate that CDs are effective free-radical scavengers for the removal of ROS in oxidative stress-related diseases.The work presented here provides a useful approach to developing effective antioxidants to counteract ROS-induced diseases in the future.

Synthesis of GSH CDs
Citric acid (415 mg) and GSH (121.4 mg) were dissolved in 10 mL of ultrapure water, and a transparent solution was obtained.The solution was then microwaved in a 700 W microwave oven for 5 min until it became a brown clustered solid, indicating that GSH CDs had formed.Subsequently, the CDs were dispersed in ultrapure water, centrifuged for 10 min at 10,000 rpm to remove large particles, and then brown powder was obtained after freeze drying the GSH CDs solution.

Characterization of GSH CDs
High Resolution Transmission Electron Microscope (HRTEM, Talos F200X, Fei, NL) was used to examine the morphology of the CDs, while Fourier transform infrared spectroscopy (FT-IR, Nicolet iS50R, Thermo Scientific) and X-ray photoelectron spectroscopy (XPS; Axis Supra+, Kratos) were employed to examine their composition.The optical characteristics of the GSH CDs were investigated using UV-visible spectroscopy (Lambda 35, PerkinElmer) and a luminescence spectrometer (F-4600, Hitachi).Prepare the samples for HRTEM: 10 μL of GSH CDs solution was dropped onto an ultra-thin carbon supporting film, and left to dry overnight, and then examined with a transmission electron microscopy.Prepare the samples for FT-IR: a small drop of GSH CDs solution was placed on one of the KBr plates and then tested on the FT-IR machine.Prepare the samples for XPS: 1 mL of GSH CDs solution was freeze dried for 2 days to obtain brown solids powder.The powder was then carefully transferred to the tape and left to be used on the XPS machine.Prepare the samples for UV/vis and luminescence spectroscopy: the GSH CDs solution prepared above was diluted 10-fold into a quartz cuvette and analyzed on the equipment.

DPPH• scavenging activity
DPPH• was selected as the indicator to detect the ROS scavenging ability of the GSH CDs.In ethanol, DPPH has a purple color with a pronounced absorption peak at 520 nm.A free radical scavenger, also known as an antioxidant, binds to the electron of DPPH•, decreasing its absorbance at 520 nm and discoloring the solution.DPPH• ethanol solution (0.126 mmol/L) was blended with different concentrations (from 0 to 0.8 mg/mL) of GSH CDs and with pure water as the control.As the concentration of GSH CDs increased, UV-vis absorption spectrophotometry revealed a decrease in DPPH• at an absorption wavelength of 520 nm.The DPPH• elimination efficiency of the GSH CDs was estimated by measuring the decrease in absorbance at 520 nm, as follows: A 0 : The absorbance of the blank sample.
A s : The absorbance of the mixture of GSH CDs and DPPH•.

•OH scavenging activity
In the classical Fenton reaction, hydroxyl radical (•OH) reacts with salicylic acid to generate 2,3-dihydroxybenzoic acid, which exhibits a prominent absorption peak at 510 nm. 27As a result, the reduction in absorbance at 510 nm can be used to measure the elimination efficiency of •OH.Briefly, FeSO 4 (9 mmol/L) was mixed with salicylic acid (9 mmol/L) and GSH CDs at varying concentrations (0 to 0.8 mg/mL).In the control group, pure water replaced the GSH CDs solution.Then, 8.8 mmol/L H 2 O 2 was added.The •OH scavenging activity was determined by measuring the changes in the absorbance of the samples at 510 nm, according to the formula below: B 0 : The absorbance of the control.B s : The absorbance of the mixture of CDs and •OH solution.

Cell culture
9][30][31][32] OC-1 is an immortalized cochlear sensory epithelial cell line that was derived from the organ of Corti (OC) of rats on postnatal day 5.The human NPC cell lines CNE-2 and HNE-1 were kindly provided by Professor Hongling Jin (HUST, Wuhan).The SCC-7 cell lines were obtained from the Cell Resource Center, Peking Union Medical College (which is the headquarters of the National Science & Technology Infrastructure-National BioMedical Cell-Line Resource, NSTI-BMC).OC-1, CNE-2, and HNE-1 cell lines were cultivated in DMEM-complete medium (Gibco).SCC-7 cells were grown in RPMI-1640 complete medium (Gibco).All cells were incubated in a humidified incubator at 37 • C with 5% CO 2 , as in previous studies. 30,33Cells were subcultured with 0.25% trypsin (Gibco) at 80-90% confluence.

Intracellular uptake
To explore the cellular uptake of GSH CDs, the OC-1 cells were seeded into 12-well plates at a density of 5.0 × 10 5 cells per well.The cells were incubated with medium containing GSH CDs (0.8 mg/mL) for 24 h after overnight attachment.The cellular uptake efficiency was investigated by fluorescence microscopy.

Cytotoxicity studies
OC-1 cells were carefully seeded in a 96-well plate at a density of 5000 cells per well.After incubation at 37 • C for 24 h in a 5% CO 2 atmosphere, OC-1 cells were washed with PBS buffer three times, and then, different concentrations of GSH CDs (0 to 0.8 mg/mL, dispersed in fresh DMEM solution) were introduced for incubation at 37 • C for another 24 h.Afterward, OC-1 cells were rinsed three times with PBS buffer after removing the medium.The viabilities of the cells were investigated through a CCK-8 assay.

Viability of cells protected from cisplatin-induced cytotoxicity by GSH CDs
OC-1 cells were seeded in a 96-well plate at a density of 5000 cells per well and allowed to grow for 24 h.Next, OC-1 cells were initially cultured for 2 h in medium containing various concentrations of GSH CDs (0, 0.1, 0.2, 0.4, and 0.8 mg/mL).The cells were then treated with cisplatin (10 μM, Yunnan Plant Pharmaceutical Co., Ltd.) and cell viability was determined after 24 h of incubation.Cells without any treatment, those treated with only GSH CDs, and those treated with only cisplatin served as controls.Cell viability was measured by CCK-8.

Determination of the mitochondrial transmembrane potential
The mitochondrial membrane potential (MMP) was estimated by monitoring JC-1 and TMRE fluorescence aggregates.OC-1 cells were seeded in a 96-well glass-bottomed plate at a density of 5000 cells per well and allowed to grow for 24 h.After different treatments, each group of cells was treated for 30 min at 37 • C in the dark with JC-1 (Beyotime Biotechnology, C2003S) or TMRE (Beyotime Biotechnology, C2001S) working solutions.Then, confocal fluorescence microscopy was performed to capture fluorescent images.

ROS detection
Intracellular ROS levels were determined using the DCFH-DA and DHE probes.A total of 5.0 × 10 5 OC-1 cells per well were seeded into a 12-well plate and incubated for 24 h.After different treatments, OC-1 cells were washed with prewarmed PBS and stained for 30 min with either the DHE (10 μM) or DCFH-DA (10 μM) working solution at 37 • C in the dark.Intracellular ROS levels were analyzed qualitatively and quantitatively using fluorescence microscopy and flow cytometry, respectively.

Caspase-mediated apoptosis assay
To measure caspase-3 expression in live cells, a caspase-3 activity test kit (Beyotime) was employed.The experiment was carried out exactly as instructed by the manufacturer.Briefly, OC-1 cells were seeded in a 96-well glass-bottomed plate at a density of 5000 cells per well and allowed to grow for 24 h.After different treatments, OC-1 cells were rinsed with PBS before being stained with 5 μM caspase-3 at 37 • C for 30 min in the dark.Photographs were then taken using fluorescence microscopy.

Apoptosis assays
A total of 5.0 × 10 5 OC-1 cells per well were seeded into a 12-well plate and incubated for 24 h.Next, the treated cells were collected and resuspended after incubation with 10 μM cisplatin alone or co-treatment with 0.8 mg/mL GSH CDs for 24 h.Then, apoptosis was quantified by flow cytometry.A TUNEL Flow Cytometry Apoptosis Kit (Elabscience) and Annexin V-FITC/PI Kit (Elabscience) were used to detect apoptosis.

TUNEL assay
A total of 5.0 × 10 5 OC-1 cells per well were seeded into a 12-well plate and incubated for 24 h.The TUNEL assay was processed using a One-Step TUNEL In Situ Apoptosis Kit (Elabscience).Briefly, OC-1 cells and cochleae were fixed, permeabilized, and washed, and then incubated in TUNEL working solution at 37 • C for 1 h protected from light.

Western blot analysis
OC-1 cells were seeded in a 6-well plate at a density of 1.0 × 10 6 cells per well and allowed to grow for 24 h.The OC-1 cells were submitted to the following different treatments: (1) Control; (2) GSH CDs: treated with 0.8 mg/mL GSH CDs only; (3) Cis; treated with 10 μM cisplatin only; (4) Cis + GSH: OC-1 cells were initially cultured for 2 h in medium containing GSH CDs (0.8 mg/mL) and then treated with 10 μM cisplatin.After lysis with RIPA lysate reagent, the total cellular proteins were extracted, and the protein extracts were electrophoresed, electrotransferred to PVDF membranes, blocked, and incubated overnight at 4 • C with the specified primary antibodies.The next day, the membranes were incubated with secondary antibodies for 1 h at room temperature and then visualized by chemiluminescence (Biosharp).GAPDH and β-actin served as the internal controls.The intensity of the bands in each western blot was semiquantified using Image J software.

Cochlear explants
The cochleae of C57BL/6 mouse pups of both sexes were dissected on postnatal day (P) 3 and the surrounding tissues and bones were washed with HBSS.The basal membrane was isolated and incubated overnight at 37 • C in DMEM/F12 medium (Gibco) supplemented with N2/B27 (Gibco) to stabilize them for each treatment.The cochlear explants were pretreated for 2 h with GSH CDs (0.8 mg/mL) and subsequently co-treated for 24 h with 10 μM cisplatin.Control cultures were treated with cisplatin alone for 24 h or not treated at all.Myosin 7a-labeled HCs with normal nuclei were considered to be surviving HCs.

Immunofluorescence
Samples that have been permeabilized were blocked with donkey serum (Biosharp), and then incubated with primary antibodies overnight at 4 • C. Anti-myosin 7a antibodies (1:300 dilution; Proteus Biosciences) and anticleaved caspase-3 antibodies were utilized (1:500 dilution; Servicebio).Next, the samples were incubated for 1 h with fluorescent secondary antibodies before being labeled with DAPI for 10 min.

Phalloidin staining
The samples were fixed and permeabilized, and then incubated with FITC-phalloidin (1:300 dilution; Servicebio) at 37 • C for 1 h to visualize F-actin fluorescence.

Auditory functional tests
Cisplatin-induced hearing loss was induced as described previously. 33Wild-type male C57BL/6 mice with normal hearing were randomly divided into four groups: (1) Control: mice treated with saline only; (2) GSH CDs: the mice were injected transtympanically with GSH CDs at 100 mg/mL for 5 μL.(3) Cis; cisplatin (30 mg/kg) was i.p. injected to mice at P28 based on previous reports 33 ; 4][35] The mice were transtympanically injected with GSH CDs at 100 mg/mL for 5 μL, followed by 30 mg/kg cisplatin (i.p.) 2 h later.Auditory Brainstem Response (ABR) was measured after 14 days.During the ABR recordings, the mice were anesthetized and heated at 38

Biocompatibility assay
The mice were euthanized 14 days after administration and the cochleae, along with the major organs, were excised for hematoxylin and eosin (H&E) staining.After fixing in 4% paraformaldehyde, cochleae from the control and GSH CDs groups were decalcified for 48 h.The OC, spiral ligament, stria vascularis, tectorial membrane (TM), Reissner's membrane (RM), and spiral ganglion cells (SGCs) were dissected.Approximately 0.3 mL serum was collected from each mouse for biochemistry examination.Serum alanine aminotransferase (ALT), alanine transaminase (AST), urea nitrogen (BUN), and urea nitrogen (CRE) levels were used to measure liver and kidney function using a fully automated biochemistry analyzer (Chemray 800, Raytor).

Statistical analysis
GraphPad Prism was used to perform one-or two-way ANOVA for data analysis.The significance level was set at p < .05.

GSH CDs characterization
As shown in Figure 1A, citric acid and GSH were used to synthesize GSH CDs.The TEM image revealed that the CDs were about 5 nanometers in size with a uniform spherical shape and good dispersion.The well-defined crystal lattice (0.22 nm) of the GSH CDs can be clearly observed in the high-resolution TEM images (Figure 1B).Hydrodynamic particle size analysis after 24 h shows that the GSH CDs are stable in water, PBS (pH 7.4 and 6.5), and DMEM (Supporting Information Figure S1).It also suggests that GSH CDs can remain stable in the acidic microenvironment of tumors.Furthermore, the GSH CDs were characterized by their FT-IR spectra to reveal their chemical composition and function on the surface.As shown in Figure 1C

Photoluminescence properties and In Vitro cytotoxicity of GSH CDs
The optical properties of the GSH CDs were measured using a UV/vis spectrophotometer and fluorescence spectrometer, respectively.As shown in Figure 1I, the GSH CDs exhibited blue fluorescence under UV light and colorless transparency under sunlight.Based on the UV/vis absorption spectrum, CDs exhibited a broad band of absorption from 200 to 500 nm.GSH CDs exhibited absorption peaks at 240 and 340 nm, representing the π → π* and n → π* electronic transitions of typical aromatic systems and surface carbonyl groups, respectively.GSH CDs exhibited a peak in fluorescence excitation at 400 nm, with the intensity decreasing gradually with increasing excitation wavelength, as observed by fluorescence spectrometry (from 350 to 450 nm) (Figure 1J).Next, the CCK-8 assay was used to determine the cytotoxicity of the GSH CDs.When the concentration of CDs ranged from 0 to 26.82 mg/mL, the viability of the cells exceeded 80%, indicating no cytotoxicity under these conditions (Figure 1K).

Antioxidant activity and intracellular ROS-scavenging effect
The free radical scavenging activity of GSH CDs was assessed using DPPH•.DPPH• can form a DPPH 2 complex in the presence of hydrogen radicals, which reduces the absorption peak at 520 nm after receiving a hydrogen radical.GSH CDs exhibited elimination efficiency of more than 60% after incubation with DPPH• in a dosedependent manner, as shown in Figure 2A & B, and Supporting Information Figure S2A.The elimination efficiency of GSH CDs toward DPPH• increased steadily as the concentration of GSH CDs increased (from 0 to 0.8 mg/mL).The scavenging capacity of GSH CDs toward •OH was also analyzed.As shown in Figure 2C and Supporting Information Figure S2B, CDs effectively eliminated •OH.These results indicate that the GSH CDs act as an effective antioxidant by scavenging free radicals such as DPPH• and •OH.To determine whether GSH CDs can protect OC-1 cells, we induced oxidative stress with H 2 O 2 .OC-1 cells were cultured with 250 μm H 2 O 2 , which reduced the cell viability by approximately 50% (Supporting Information Figure S2C).However, the addition of GSH CDs to the culture medium significantly enhanced the cell viability; this can be attributed to the decreased production of intracellular ROS (Figure 2D).

GSH CDs attenuate cisplatin-induced cytotoxicity
Before the protective effect studies, the internalization of GSH CDs by OC-1 cells was investigated to understand the role of CDs better.As shown in Supporting Information Figure S3, it can be observed that OC-1 cells treated with GSH CDs showed blue fluorescence under the radiation of 405 nm, indicating that GSH CDs can be internalized by OC-1 cells.To determine the optimal dose of cisplatin to induce cell damage, OC-1 cells were incubated for 24 h in media containing a range of different cisplatin concentrations (0, 2.5, 5, 10, 20, 40, and 80 μM).The cell viability was 99.87 ± 7.66, 95.93 ± 1.36, 79.29 ± 5.38, 58.53 ± 0.55, 39.66 ± 2.54, 30.36 ± 2.22, and 25.71 ± 3.93% at each cisplatin concentration, respectively (Figure 2E).The 10 μM cisplatin treatment for 24 h was chosen as a suitable condition for OC-1 cell damage due to the nearly 50% reduction in surviving cells.GSH CDs were then examined against cisplatin (10 μM) injury at different concentrations (0.1, 0.2, 0.4, and 0.8 mg/mL) to determine if they produced cytoprotective effects.The cell survival rates were 58.72 ± 1.73, 62.78 ± 3.37, 76.11 ± 1.92, and 90.27 ± 6.73% at the different GSH CDs pretreatment concentrations (from 0.1 to 0.8 mg/mL), and these rates were significantly higher than those in the untreated group (49.53 ± 1.68%) (Figure 2F).Together, these results indicate that OC-1 cells were less susceptible to cisplatin-induced cytotoxicity when treated with GSH CDs.

The killing effects of cisplatin on tumor cell lines are not affected by GSH CDs
Currently, there is no consensus on the effects of administration of antioxidants with cisplatin chemotherapy.However, accumulating evidence suggests that antioxidant supplements may reduce chemotherapy side-effects.Although GSH CDs were found to protect against cisplatin ototoxicity, it was essential to determine whether they inhibited the chemotherapeutic effect of cisplatin.To this end, three head and neck cancer cell lines (SCC-7, CNE-2, and HNE-1) were used to study the effects of GSH CDs on the killing ability of cisplatin.A CCK-8 assay was used to assess the viability of cancer cells after 24 h of treatment with GSH CDs and cisplatin.The findings revealed that GSH CDs did not prevent cisplatin-induced cell death (Supporting Information Figure S4).7][38] These enzymes are necessary for the intracellular transport of GSH, and they are abundant in essential organs of the body (e.g., kidneys), making it easier for GSH to enter cells and play a role in free radical scavenging.Thus, the exogenous addition of GSH has no influence on the efficacy of cisplatin in the treatment of malignancies.Overall, GSH CDs did not interfere with the efficacy of cisplatin to kill tumor cells.

GSH CDs attenuate cisplatininduced oxidative stress and mitochondrial dysfunction
Previous research has shown that cisplatin-induced apoptosis of HCs is associated with increased ROS accumulation. 39Therefore, we evaluated intracellular ROS levels in OC-1 cells using DHE and DCFH probes after cisplatin treatment.Cisplatin damage significantly increased the fluorescence intensity of DHE and DCFH-DA staining (Figure 3A & B and Supporting Information Figure S5), suggesting a high degree of ROS generation.Cisplatin activates intrinsic apoptotic pathways in auditory sensory HCs, with mitochondria acting as the principal initiators.The first symptom of mitochondrial malfunction is a decrease in the MMP, which is an important measure of mitochondrial function.Thus, the effect of GSH CDs on mitochondria-dependent apoptosis was investigated.First, the MMP was measured by JC-1 and TMRM mitochondrial staining.JC-1 aggregated on the mitochondrial membrane of OC-1 cells in the control group, resulting in a prominent red fluorescence signal (Figure 3C and Supporting Information Figure S6A).OC-1 cells exhibited a substantial green fluorescence signal after cisplatin treatment, indicating the formation of the JC-1 monomer and depolarization of the mitochondrial membrane. 40,41Cells pretreated with GSH CDs, on the other hand, displayed a considerable increase in red fluorescence, indicating that GSH CDs repaired cisplatininduced mitochondrial damage.Additionally, the amount of TMRM fluorescence was significantly reduced in cisplatin-damaged cells, also indicating a reduction in the MMP (Figure 3D and Supporting Information Figure S6B).In contrast, GSH CDs prevented cisplatin-induced mitochondrial fission.3][44][45] As a result, GSH CDs can significantly inhibit cisplatin-induced mitochondrial damage, indicating that GSH CDs exert their otoprotective effect through mitochondria-mediated pathways.

GSH CDs attenuate cisplatininduced OC-1 cells apoptosis
As revealed in Figure 4A-D, cleaved caspase-3, cleaved caspase-9, and Bax protein expression levels were dramatically enhanced by cisplatin treatment but significantly suppressed by pretreatment with GSH CDs.Flow cytometry was used to assess cell apoptosis.The proportion of apoptotic cells rose dramatically following cisplatin treatment (30.27 ± 1.47%), as compared to the control and GSH CDs-only groups (5.83 ± 0.62 and 5.73 ± 0.48%, respectively) (see Figure 4E & F).However, there was a significant decrease in apoptotic cells after pretreatment with GSH CDs (13.50 ± 1.58%), as compared with the cisplatin group.In addition, we monitored the production of caspase-3 in living cells using a Caspase-3 Live Apoptosis Assay Kit.In comparison to the control and GSH CDs-only groups, the cisplatin-treated group had a significantly higher number of caspase-3-positive cells.In contrast, the cisplatin plus GSH CDs group showed considerably fewer caspase-3-positive cells than the cisplatin alone group (Figure 5A).The analysis of apoptosis according to the TUNEL and flow cytometry assays was in agreement with the caspase-3 detection results.The TUNEL immunostaining signals in OC-1 cells and the apoptotic cell proportions were significantly reduced in the cisplatin plus GSH CDs group (Figure 5B & C).Upon overproduction of ROS, mitochondrial depolarization occurs, triggering caspase-3 activation, and ultimately, HC apoptosis.These findings demonstrate that GSH CDs inhibit apoptotic proteins, enhance the MMP, and decrease ROS levels in OC-1 cells, indicating that they protect against cisplatin-induced cell death.

GSH CDs protect mouse cochlear explants against cisplatin-induced HC loss
First, we investigated whether HCs cultured from cochlear explants could be protected from cisplatin-induced damage by GSH CDs. Figure 6A depicts the administration of cochlear explants.Cochlear explants were administered as shown in Figure 6A.The cochlear explants were pretreated for 2 h with GSH CDs (0.8 mg/mL) and subsequently cotreated for 24 h with 10 μM cisplatin.Control cultures were treated with cisplatin alone for 24 h or not treated at all.In comparison with the cisplatin-only group, GSH CDs significantly increased HC survival (Figure 6B), revealing a protective effect of GSH CDs.There was no damage to the HCs in the cochlear explants treated with GSH CDs alone.Moreover, a quantitative analysis of HC numbers was carried out after each set of treatments for the base, midline, and apical parts of the cochlear explants.As shown in Figure 6C, the cisplatin alone-treated group had an average of 39.3 ± 7.0, 55.3 ± 5.0, and 74.5 ± 6.4 HCs per 200 μm in the basal, middle, and apical turns, ).These results demonstrate that GSH CDs protect HCs from death caused by cisplatin.Next, we stained cultured cochleae with cleaved caspase-3 and TUNEL to determine if GSH CDs reduced apoptosis.The results revealed that the number of double-positive cleaved caspase-3/phalloidin cells was increased significantly in the cisplatin alone group (Figure 7 and Supporting Information Figure S7A).A similar increase in double-positive TUNEL/Phalloidin cells was observed in the group treated with cisplatin (Figure 8 and Supporting Information S7B).When compared to the cisplatin alone group, the GSH CDs pretreatment group exhibited a significant reduction in the number of cleaved caspase-3/phalloidin and TUNEL/phalloidin double-positive cells.These findings demonstrate that pretreatment with GSH CDs can inhibit the apoptotic cascade triggered by cisplatin.

GSH CDs partly repaired cisplatininduced hearing loss in the ABR test
The antioxidant effects of GSH CDs were observed as described earlier.To further investigate the effects of GSH CDs on hearing function in vivo, ABR testing was performed 14 days after administration (Figure 9A).The cisplatin-only treatment group exhibited a significant increase in the hearing threshold at all test frequencies, as shown in Figure 9B, whereas pretreatment with GSH CDs reduced the threshold elevation.Immunofluorescence staining further confirmed that pretreatment with GSH CDs significantly reduced the loss of HCs (Figure 9C).

3.10
In vivo biocompatibility evaluation H&E staining was carried out on the heart, liver, spleen, lung, kidney, and cochlear tissues to assess the systemic biosafety of GSH CDs.In comparison to the saline group, the GSH CDs group showed no visible morphological or tissue damage.Furthermore, the structure and morphology of the OC were well preserved, with no differences observed between the GSH CDs group and the control group.Neither the control group nor the experimental groups showed degeneration or myelin sheath detachment in SGCs (Figure 10A).][48][49][50] Therefore, we tested the liver and kidney functions of mice.Both liver and kidney functionality were within the normal range, demonstrating that GSH CDs had no adverse effects on either organs (Figure 10B).Thus, GSH CDs exhibited good biocompatibility and caused no apparent histological changes in the main organs or inner ear.

CONCLUSION
In summary, this study describes a nanoparticle-based delivery system for loading GSH CDs to alleviate cisplatininduced ototoxicity.The synthesized GSH CDs had a uniform spherical shape and exhibited satisfactory chemical stability and biocompatibility.The GSH CDs were found to significantly suppress ROS generation, MMP collapse, and cisplatin-induced apoptosis of HCs, ultimately reducing HC loss.Thus, GSH CDs may be used to alleviate hearing loss caused by cisplatin, although the detailed mechanisms need to be further investigated.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

F I G U R E 1
Characterization of GSH CDs.A Schematic illustration of preparation progress of GSH CDs.B TEM image of GHS CDs.C FTIR spectrum of GSH CDs.D XPS spectrum of GSH CDs and high-resolution XPS spectra of E C 1s, F N 1s, G O 1s, and H S 2p.I UV-vis absorption spectra of GSH CDs (inset shows the aqueous solution of GSH CDs under sunlight and UV irradiation).J Photoluminescence emission and excitation spectra of the GSH CDs.K In vitro cytotoxicity of GSH CDs.
, the absorption bands at approximately 1410 and 1188 cm −1 represent the C = N and C = S stretching vibrations, respectively.The N-H and O-H vibrations are represented by the wide peak between 3700 and 3000 cm −1 .Moreover, the peaks at approximately 2600and 626 cm −1 are attributed to the S-H and C-S vibrations, respectively.To further investigate the surface functionality and elemental states of the GSH CDs, XPS was performed.There were four broad peaks in the XPS survey spectrum at around 285.0, 400.7, 532.7, and 164.2 eV, belonging to the C 1s, N 1s, O 1s, and S 2p core levels, respectively (Figure1D-H).The CDs were found to contain 62.6% (C), 33.4% (O), 3.1% (N), and 1.0% (S).C constituted the majority of the GSH CDs, and trace amounts of S were also present.The above results indicate that the GSH CDs are rich in unsaturated double bonds and thiol groups, which endows them with super reducibility.

F I G U R E 2
Antioxidant activity and intracellular ROS-scavenging effect.A The DPPH• scavenging activity.B Absorbance spectra of DPPH• after the addition of different concentrations (from 0 to 0.8 mg/mL) of GSH CDs.C The •OH elimination efficiency of GSH CDs.D The protective effect of GSH CDs on OC-1 cells from oxidative stress.Oxidative stress was induced by exposing the OC-1 cells to 250 μm H 2 O 2 .The data shown are mean values (n = 3).*p < .05,**p < .01.E The cell viability of OC-1 cells after being treated with different concentrations of cisplatin for 24 h.F The protective effect of GSH CDs on OC-1 cells from cisplatin-induced cytotoxicity.

F I G U R E 3
Intracellular ROS and mitochondrial membrane potential detection.A Measurement of intracellular reactive oxygen species (ROS) production.The intracellular ROS generation detected by the DHE probe after various treatments.Blue: DAPI; Red: DHE.Scale bar: 100 μm.B Quantitative changes in the DHE were determined by flow cytometry.C The MMP was measured by JC-1 and TMRM mitochondrial staining.Representative fluorescence images of OC-1 cells stained with JC-1 and D TMRM.Scale bar: 50 μm.

F I G U R E 4
Effects of GSH CDs on cisplatin-induced apoptosis in OC-1 cells.OC-1 cells were initially cultured for 2 h in medium containing GSH CDs (0.8 mg/mL) and then treated with 10 μM cisplatin.A Representative western blot images of Bax, cleaved caspase-9, and cleaved caspase-3 in OC-1 cells from different treatments.Quantification of B Bax, C cleaved caspase-9, and D cleaved caspase-3 expression levels.*p < .05,**p < .01,## p < .01.E Flow cytometry was used to measure the rate of apoptosis after different treatments.Annexin V-FITC/PI double staining by flow cytometry.F Quantification of the early and late apoptotic cells.*p < .05,**p < .01,## p < .01.

F I G U R E 5
Caspase 3 and TUNEL staining.A Representative images of Caspase 3 staining in the control, GSH CDs only, cisplatin only, and cisplatin plus GSH CDs groups.Scale bar: 50 μm.Apoptosis analysis by B TUNEL immunostaining and C flow cytometry assay.Scale bar: 100 μm.

F I G U R E 6
Effects of GSH CDs on cisplatin-induced cochlear hair cell loss in vitro.A The experimental workflow.B Representative images of hair cell staining labeled with myosin 7a in the control, GSH CDs, Cisplatin (Cis), and GSH CDs + cisplatin cotreatment.The number of cochlear HCs in the basal, middle, and apical turns was counted every 200 μm.Since HCs in the apical turn are resistant to cisplatin, the number of apoplastic HCs was only counted in the middle turn of each explant.Scale bars indicate 20 μm.C Quantification of myosin 7a-positive hair cells in the apical, middle, and basal turns of different groups.Values were represented as the mean ± SD. * p < .05,* * p < .01,## p < .01.

F I G U R E 7
Immunofluorescence staining with cleaved caspase 3 (red) and phalloidin (green) in the middle turns of the cochleae from the control, GSH CDs, Cisplatin (Cis), and GSH CDs plus cisplatin groups.Scale bars = 20 μm.respectively, while the GSH CDs-pretreated group had an increased number of HCs per 200 m in the basal, middle, and apical turns of the cochlear explants (base: 71.3 ± 5.1, middle: 83.0 ± 4.6, and apical: 93.0 ± 8.5

F I G U R E 8
Immunofluorescence staining for TUNEL (red) and phalloidin (green) in the middle turns of the cochleae from the control, GSH CDs, Cisplatin (Cis), and GSH CDs plus cisplatin groups.Scale bars = 20 μm.F I G U R E 9 GSH CDs partially restored cisplatin-induced hearing loss in ABR tests.A The experimental workflow.B ABR thresholds were analyzed in mice after different treatments.Control group: mice treated with saline only; GSH CDs group: the mice were injected transtympanically with GSH CDs; Cis group: cisplatin (30 mg/kg) was i.p. injected to mice at P28; Cis + GSH group: the mice were transtympanically injected with GSH CDs, followed by 30 mg/kg cisplatin (i.p.) 2 h later.C Hair cell quantification.Scale bars = 20 μm.Values were represented as the mean ± SD (n = 3).* p < .05,* * p < .01,## p < .01.F I G U R E 1 0 Safety evaluation.A Representative H&E staining of heart, liver, spleen, lung, kidney, and cochlear tissues from control and GSH CDs groups.Scale bar = 50 μm.B Blood biochemical assessment.Serum alanine aminotransferase (ALT), alanine transaminase (AST), urea nitrogen (BUN), and urea nitrogen (CRE) levels were used to measure liver and kidney function.
., G.F., and N.W. contributed equally to this work.Y.T., S.T., and H.X. codesigned the experiment.Y.T., G.F., N.W., and B.L. participated in the experiments.Y.T. wrote the original manuscript.H.S., Q.W, and W.Z. reviewed the manuscript.A C K N O W L E D G M E N T SThe authors thank the Analytical and Testing Centre of HUST for XPS, FTIR, and HRTEM measurements.
This work was supported by the National Natural Science Foundation of China (Nos.81871473, 82071057, and 82000988).