β-CD was used as a precursor for the nanoparticles. PAMAM-β-CD synthesis: β-CD (63.2 mg) and CDI (90.28 mg) were dissolved in DMSO (10 ml) with continuous stirring for 6 h, and the third-generation polyamide amine dendritic macromolecule (G3 PAMAM) (57.93 mg, 2.227 mol) dissolved in dimethyl sulfoxide (DMSO) was added dropwise to the solution with constant mechanical stirring (650 rpm) at room temperature for 3 days. The suspension was dialyzed against 0.01 M phosphate-buffered saline (PBS) using a 2-kDa dialysis bag for 24 h and purified using ultrapure water for 2 days to eliminate free chemical residues. The product was then freeze-dried, and the final white powder product (G3. NH₂-β-CD) was stored at −20°C.

Au @CD-PAMAM was produced by adding the G3. NH₂-β-CD solution into an aqueous solution of chloroauric acid (HAuCl₄) under continuous intense agitation for 30 min. Then, the aqueous solution of sodium borohydride (NaBH₄) was quickly added five times to continue the reaction for 4 h. Subsequently, the copolymers were purified in distilled water using a 2-kDa dialysis bag for 72 h and then isolated by freeze-drying.

Arg8 polypeptides (lipid concentration: 1.5 mg/ml), EDC (lipid concentration: 0.2 mg/ml), and NHS (lipid concentration: 0.4 mg/ml) were added into PBS (pH = 7.4) and stirred at room temperature for 4 h. The drug-loaded nanomaterial powder (Au@CD-PAMAM)was dissolved in PBS, and then the commixture of the two solutions was obtained and allowed to react overnight at roomtemperature. The mixture was freeze-dried to obtain the drug-loaded nanocompositematerial with targeted properties of Au @CD-PAMAM-Arg8(Au-DENPs). The free chemical residues were removed by dialysis using a 1-kDa MWCO membrane. Dexamethasone (Dex) dissolved in DMSO (concentration: 2 mg/ml) was added to the Au-DENPs. The mixed solution was stirred overnight at room temperature, filtrated with a 2-kDa MWCO membrane and freeze-dried to get Au @CD-PAMAM-Arg8/Dex (Au DENPs-Dex).

The ¹H NMR hydrogen spectrum of G3 NH₂-β-CD was recorded using a Bruker AV Ⅲ 500 MHz NMR spectrometer (Wang et al., 2021). The FT-IR spectra of the G3. NH₂-β-CD and Au @CD-PAMAM nanoparticles in the range of 4,000–500 cm⁻¹ were examined by the potassium bromide tablet method. The morphology of Au @PAMAM-β-CD-Arg8 was determined by cold-emission, high-resolution transmission electron microscopy (TEM, JEM-2010F, JEOL., Ltd.). The size distribution and zeta potentials of the targeted drug-loaded nanocomposite suspensions were measured by the dynamic light scattering (DLS) technique using a Nano-ZS 90 Zetasizer (Malvern, America) at 25°C. The zeta potential (ζ) was determined by using laser Doppler electrophoresis (LDE) (Qiu et al., 2018; Wang et al., 2021). In vitro CT images of each concentration of the NPs (0.005, 0.01, 0.02, 0.04, 0.08, and 0.16 M) were captured using a single-source CT imaging system (Aquilion ONE 320 CT, Canon Medical Systems). All CT scans were performed at 100 kV voltage, 80 mA current, and a slice thickness of 0.625 nm. CT images were recorded, and the HU values of each sample were obtained by using built-in software.

The loading content (%) and loading efficiency (%) of Au-DENPs-Dex were measured by high-performance liquid chromatography [HPLC (Thermo Fisher U3000)] at following time points: 0.5, 1, 3, 5, 8, 24, 48, and 72 h (Yoon et al., 2015). The test conditions were as follows: a series of Dex solutions with different concentrations (1,000, 500, 250, 125, 62.5, 31.25, 15.6, 7.8, and 3.9 μg/m L), a mobile phase of water and acetonitrile (60:40 v/v), sample injection volume of 20 μL, flow rate of 1.0 ml/min, column temperature of 30°C, and detection wavelength of 240 nm (Zhao et al., 2021).

Although HEIOC1 cells were undifferentiated, both hair cell and support cell markers were expressed on them (Tan et al., 2020). The HEIOC1 cells were cultured in the high-glucose Dulbecco’s modified Eagle’s medium (high-glucose DMEM; Sigma-Aldrich, Saint Louis, United States) mixed with 10% fetal bovine serum (FBS; Gibco, BRL) and 1% penicillin-SM (Sigma, Saint Louis, United States). The cells were cultured in a humidified 10% CO₂ environment at 37°C. The mouse fibroblasts L929 cells were maintained in the DMEM complete medium supplemented with 10% FBS and 1% penicillin-SM at 33°C in a humidified incubator with 10% CO₂.

The cytotoxicity of Au @CD-PAMAM against HEIOC1 cells and L929 cells was quantitatively detected by the CCK-8 method. L929 cells and HEIOC1 cells at a density of 5,000 cells/well were inoculated into a 96-well plate and placed in a CO₂ incubator for overnight culture and wall sticking. A medium with different concentrations of Au @CD-PAMAM (concentrations of Au @CD-PAMAM in HEIOC1 cells: 1, 5, 10, 50, 100, 200, 400, 600, and 800 ug/mL and concentrations of Au @CD-PAMAM in L929 cells: 50,100,200,400,600, and 800 ug/mL) and 0.01 M PBS was used as the control. Cells were detected by the CCK-8 method after culture for 24 h (Su et al., 2019). The microplate reader (Bio-Rad Laboratories, Hercules, CA) was used to determine the optical densities of the samples at 450 nm.

Cell uptake and intracellular localization of FITC-labeled Au-DENPs-Dex (0.6 mg/ml) in HEIOC1 cell were investigated after 0.5, 2, and 6 h of treatment using confocal microscopy (LSM880 Live Configuration Variotwo VRGB; Zeiss, Jena, Germany). The HEIOC1 cells were treated with FITC-labeled Au-DENPs-Dex at the maximum safe dose, determined by the cytotoxicity test. The cells were fixed with 4% paraformaldehyde (PA) solution after incubation and stained with DAPI (Sigma, Saint Louis, United States). A confocal microscope was used to observe the treated cells (Zambito et al., 2021).

Cell uptake of the Au-DENPs complex with and without Arg8 functionalization was analyzed to confirm Arg8’s targeting ability. HEIOC1 cells were cultured overnight in a 96-well plate, and then FITC-labeled Au@PAMAM-β-CD-Arg8, FITC-labeled Au@PAMAM-β-CD (0.2 mg/ml), and 0.01 M PBS (pH = 7.4) were added and incubated for 0.5, 2, and 6 h. Cellular uptake efficiency in each group was quantified by flow cytometry. The untreated cells were used as negative controls. The corresponding fluorescence intensity (three parallel for each well) was quantified using Flow Jo 7.6.1 software.

Surgery: male C57/BL6 mice aged 2 months were anesthetized with intraperitoneal (I. p.) injection pentobarbitone (with 0.5% pentobarbital sodium at the dosage of 0.02 ml/g body weight) and placed on a temperature-controlled heating mat to hold body temperature at 37°C in the supine position. An incision along the posterior auricular sulcus was made, and the left-side of the otic vesicle was exposed, in which a small hole was made with an insulin syringe. Next, the maximum safe dose of Au-DENPs-Dex (0.4 mg/ml), FITC-labeled Au-DENPs (80 ug/mL), or free Dex (5 mg/ml) was injected into the tympanum until the cavity was filled. Bone wax was used to plug the hole after injection (JOHON, Shen Zheng City, China), and the wound was sutured. After surgery, baytril (1 mg/kg; Sigma-Aldrich, Saint Louis, MO, United States) was used to treat middle ear infection by intraperitoneal injection once daily (Yang et al., 2018).

After 0.5, 2, 3, 24, and 72 h of injection of Au-DENPs-Dex (0.4 mg/ml, 100 µL) and free Dex (5 mg/ml, 100 µL) into the middle ear cavity (n = 5/group, control group: n = 5), immunohistochemical staining with an antibody-targeting Dex was conducted. The ABC HRP Kit and a peroxidase substrate (Vector, Burlingame, CA) were used to detect nanoparticle distribution in the inner ear at different time points (Riquelme et al., 2010).

After 3 and 24 h of intratympanic injection and 3 h of tail intravenous injection of FITC-labeled Au-DENPs (0.01 M PBS was used as control), the cochlear basilar membranes of the group subjected to intratympanic injection for 3 h (n = 2/group) were incubated with Alexa Fluor 660-conjugated phalloidin to label actin, which is heavily expressed in the stereocilia and the cuticular plate of outer hair cells (OHCs), inner hair cells (IHCs), and pillar cells (PCs) and DAPI (Sigma-Aldrich, Saint Louis, United States). The other membranes were incubated with anti-β-tubulin Ⅲ antibody (Abcam, United Kingdom) to label never fibers (NFs) and spiral ganglion neurons (SGNs) and then immersed in a solution consisting of secondary antibody conjugated with Alexa Fluor 555 (1:200, Solarbio, Beijing City, China) normal goat serum, Triton X-100, and 0.01 M PBS as well as with DAPI. Then, the membranes were studied under a confocal microscope (Zeiss LSM-880) (Ding et al., 2011; Ding et al., 2012; Wang et al., 2017; Zhang J. et al., 2018) to determine the distribution of FITC-loaded nanoparticles in the inner ear after the two different injection methods.

A total of sixteen mice, aged 2 months, were injected with 100 μL of FITC-labeled Au-DENPs (2 mg/kg) into the tympanum, posterior ear, muscle, and tail vein. After 0.5, 1, 3, 5, and 7 h, fluorescent optical imaging was performed by using a small animal imaging system (FX Pro Bruker) to evaluate the enrichment of FITC-labeled Au-DENPs in vivo following the different injection methods. The mice were anesthetized and placed on the imaging stage. The excitation wavelength is 495 nm, and the emission wavelength was 519 nm. Acquisition and image analysis were performed using Living 302 Image 4.4.1 software.

The mice were injected with 100 μL of Au-DENPs (2 mg/kg) via the left tympanum and tail vein. The animals were euthanized after 5 h and the main organs (heart, liver, spleen, lung, kidney, and bilateral cochlear) were excised and subjected to induced coupled plasma atomic emission spectroscopy (ICP-AES) to explore the distribution of the nanoparticles in vivo.

A total of twelve male C57/BL6 mice, aged 2 months, were divided into four groups (n = 3/group). The control group mice did not undergo surgery or treatment, and the other three groups received Au-DENPs-Dex, Au-DENPs, or Au @CD-PAMAM treatment via caudal vein injection (0.6 mg/ml; 100 uL). The mice were euthanized 2 weeks later, and H&E staining was conducted to observe the cytotoxicity of the nanoparticles in the main organs (heart, liver, spleen, lung, and kidney).

The HEIOC1 cells were treated with the fresh complete medium with various concentrations of SM (0, 1, 2, 5, 10, 15, 20, 30, and 40 mg/ml) to determine the optimal dosing concentration of SM. In another 96-well plate, the HEIOC1 cells were dealt with the best dosing concentration of SM after pretreatment with six different concentrations (1, 5, 10, 15, 20, and 30 μg/ml) of Au-DENPs-Dex and Dex alone; 0.01 M PBS was used as the control group (Zhao et al., 2021). Then, the cytotoxicity of SM to HEIOC1 and the efficacy of free Dex and Au-DENPs-Dex in treating SM ototoxicity were quantitatively detected by the CCK-8 method to verify drug delivery efficiency of nanoparticles.

Compared with auditory brainstem response (ABR) in vivo, compound action potential (CAP) in electrocochleography (ECochG) was good in reflecting the functional status of the peripheral auditory neurons and cochlear sensory epithelium following SM damage. The mice were anesthetized 3 days after tympanic cavity injection of Au-DENPs-Dex mixed with SM, mixture of SM and Dex, SM (concentration of Au-DENPs-Dex or Dex: 30 μg/ml; SM: 37.5 mg/ml, 100 µL), or 0.01 M PBS. After exposing the left facial nerve to set a silver wire into the facial nerve canal for CAP recording, the reference electrode and ground electrode were inserted subcutaneously at the earlobe of the test ear and the opposite earlobe, respectively (Li et al., 2021). The data were collected by using an analyzer (Neurosoft LLC, Russia). ECochG click was generated in response to tones (1, 2, 3, 4, 6, 8, 16, and 32 kHz) between 100 and 20 dB in 5 dB increments.

After CAP recording, the mice were euthanized, and the basement membranes were stained with anti-β-tubulin Ⅲ antibody, secondary antibody conjugated with Alexa Fluor 488, Alexa Fluor 660-conjugated phalloidin, and DAPI to evaluate the damage caused to hair cells, spiral ganglion cells, nerve fibers behind the inner hair cells, and ganglion cells following treatment of SM, Au-DENPs-Dex, and Dex alone (Ding et al., 2011; Zhang J. et al., 2018).

As shown in Figure 8A, an increase in SM concentration led to a gradual decrease in cell activity. An SM concentration of 30 mg/ml was used as an ototoxicity model to explore the therapeutic effects of treatments using Au-DENPs-Dex and dexamethasone only. As the concentration of Au-DENPs-Dex nanoparticles accumulated, the cell viability gradually increased. The cell viability of the Au-DENPs-Dex nanoparticles group, at the same concentration, was higher than that of both Dex only and 0.01 M PBS groups (Figure 8B). It indicated that the therapeutic effect of our nanoparticles is superior to that of direct drug administration at the cellular level.

After SM treatment, overproduction of ROS contributed to the production of pro-inflammatory cytokines, which severely exacerbated inflammation in the cochlea (Ding et al., 1993; Wrzesniok et al., 2013; Cui et al., 2015). The administration of Dex can reduce pro-inflammatory cytokines (Shan et al., 2012; Xiong et al., 2019). To further explore the drug delivery efficiency of nanoparticles, CAP thresholds recorded from the facial nerve for 1, 2, 3, 4, 6, 8, 16, and 32 kHz were performed 3 days after surgery, to investigate hearing function in vivo. There was a significant effect from SM alone, SM mixed with Dex, SM mixed with Au-DENPs-Dex treatment, and control groups on CAP thresholds [F (3, 64) = 188.7, p < 0.0001]. Hearing losses in the SM and SM mixed with Dex groups were significant compared to the control and SM mixed with Au-DENPs-Dex groups. (Figures 9A,B). Almost no hearing loss was reported in the SM mixed with Au-DENPs-Dex group. CAP thresholds in the SM group (ranging from 93.3 dB SPL at 1 kHz to 43.3 dB at 32 kHz) increased more than those in the SM mixed with Au-DENPs-Dex group (ranging 58.3 dB SPL at 1 kHz to 23.3 dB SPL at 32 kHz). This shows that Au-DENPs-Dex is effective for reducing SM-induced injury. The therapeutic effect of nanoparticles is greater than that of direct administration.

Confocal microscopy images of nerve endings below the inner hair cells (Figure 9C), spiral ganglion cells (Figure 9D), and hair cells (Figure 9E) were used to explore the therapeutic effect of Au-DENPs-Dex and Dex on SM ototoxicity. In the SM mixed with Au-DENPs-Dex and control groups, a thick bundle of NF can be seen running medial and parallel to the row of IHC (Figures 9C l,p), and thin fascicles of NF project out radially from the thick fiber bundle in the direction of the OHC. Many blue-labeled nuclei were observed in the outer sulcus cell (OSC) area radial to the OHC (Figures 9C l,p), while the longitudinal NF bundle was medial to the row of IHC in the SM group.

The longitudinally oriented fascicle of NF medial to the IHC in the SM (Figure 9C, a) and SM mixed with Dex groups (Figure 9C, e) was severely damaged and the thickness was less than that in the control (Figure 9C, m) and SM mixed with Au-DENPs-Dex groups (Figure 9C i). SGNs were described as large, round, blue-labeled nuclei surrounded with the green cytoplasm marked with β-tubulin Ⅲ; numerous diminutive blue-labeled nuclei appeared in the circumambient support cells labeled with phalloidin (Figures 9D a,e,i,m). However, mostly β-tubulin-labeled SGNs were eliminated (Figures 9D d,h), and the soma and nuclei were shrunken in the SM or SM mixed with Dex groups (Figures 9D d,h) compared to the control and SM mixed with Au-DENPs-Dex groups (Figures 9D p,l). In addition, there was no significant difference in hair cells of each group; stereocilia and hair cells in all frequency regions of the organ of Corti in each group exhibited almost no damage and appeared similar to those of the control (Figure 9E).

We observed an apparent recovery in the morphology of nerve fibers, nerve endings below the inner hair cell, and SGN in the SM mixed with Au-DENPs-Dex group compared to the SM and SM mixed with free Dex groups (Figure 9), supporting the concept that acute cochlear damage caused by low concentrations of streptomycin mainly occurred in the nerve fibers, nerve endings below the inner hair cell, and SGN. Destruction of the efferent endings by SM leads to an increase in the auditory threshold. It is evident that Au-DENPs-Dex is significant for SM ototoxicity. These results indicate that the systemic toxicity of nanomaterials is negligible and has a potential clinical value in SM-induced ototoxic injury or other inner ear disease.