Preparation of dexamethasone nanosuspension
In this study, nanoparticles were prepared using fat and supercritical fluid (NUFS™) for nanoparticle fabrication, which is a novel technique for nanoparticle preparation (13). The preparation of dexamethasone nanoparticles consisted of the following two steps: polyvinylpyrrolidone (PVP), poloxamer, polyethylene glycol (PEG)-40 stearate and a saccharide were dissolved in deionized water at a ratio determined by the design of the experiment. The polymer solution was then mixed with the active ingredient, a fatty alcohol. The mixture was milled with a roller compactor, then dried under reduced pressure at room temperature. This solid dispersion was placed inside a pressure-resistant reactor (Bio-Synectics, Inc., BS-SF-1, Korea), and a continuous flow of liquid carbon dioxide was used to remove the fatty alcohol from the solid dispersion.
Characterization of dexamethasone nanocrystals
Particle size measurement
The hydrodynamic particle size of various nanoparticle formulations was measured using dynamic light scattering (DLS) (ELSX-1000, Otsuka Electronics Co., Ltd, Osaka, Japan) (27). After dispersion by sonication, dexamethasone suspensions were collected at predetermined time points (0, 1, 2, 6, and 8 h), and particle size was measured with DLS. For visual observation of the nanoparticles, a 120 kV bio-transmission electron microscope (JEM-1400 plus, JEOL, Tokyo, Japan) was employed according to the method used in previous studies (28).
In vitro dissolution studies
In vitro dissolution tests of the formulations were conducted with USP Dissolution Apparatus 2 (Vision Elite 8, Hanson, CA, USA) at 75 rpm. For each measurement, powder (100 mg as dexamethasone) was placed in 900 mL deionized water at 37 ± 0.5°C. Samples of 3 mL were collected at predetermined time points (3, 7, 15, 30, 60, and 120 min) and were replaced with an equal volume of fresh dissolution medium. The aliquot was filtered through a 0.2 µm membrane filter. The drug content was analyzed using an Alliance series high-performance liquid chromatography (HPLC) system (Waters Corporation, Milford, MA, USA) with ultraviolet (UV) detection at 249 nm. Twenty microliters of each sample was injected into an Epic C18 column (5 µm particle size, 4.6×250 mm) adjusted to 25°C at a flow rate of 0.8 mL/min. The mobile phase consisted of 0.01 M KH2PO4 buffer and acetonitrile (5:4% v/v).
In vitro safety and drug efficacy evaluation of dexamethasone nanosuspensions in HEI-OC1 cells
Culture of HEI-OC1 cells
The immortalized mouse organ of Corti cell line HEI-OC1 was used for in vitro tests (29). HEI-OC1 cells were grown and passaged in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 50 U/mL recombinant mouse interferon-γ, and 10 ng/mL ampicillin and then cultured in a humidified 10% CO2 environment at 33°C.
Cell Counting Kit (CCK) assay
The safety of the three dexamethasone nanosuspensions in HEI-OC1 cells was tested using a CCK-8 (Dojindo Molecular Technologies, Rockville, MD) assay. The cells were incubated with various concentrations of dexamethasone nanosuspensions (0.1–100 µg/ml) for 24 h. Cell viability was determined using a CCK-8 assay in accordance with the manufacturer’s protocol; optical densities were determined at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).
HEI-OC1 cells were treated with Dex-SP and dexamethasone nanosuspensions for 1 h and 6 h. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (50 mM Tris; 150 mM NaCl; 1 mM EDTA; 1% sodium deoxycholate (DOC); 1% Triton X-100; 0.1% SDS; and 1× protease, phosphatase-1, and phosphatase-2 inhibitor cocktails (Sigma Aldrich, Darmstadt, Germany)). The cell lysates were centrifuged at 13,200 rpm for 15 min at 4°C, and the supernatant was subjected to western blot analysis. Equal amounts of protein for each sample were electrophoresed and transferred onto nitrocellulose membranes. These membranes were incubated for 1 h with blocking solution (Translab, Daejeon, Republic of Korea) and then incubated overnight with primary antibodies against glucocorticoid receptor (Cell Signaling, Danvers, Massachusetts, US) and phospho-glucocorticoid receptor (Cell Signaling, Danvers, Massachusetts, US). Protein expression was detected by using a ChemiDoc XRS Image system (Bio-Rad, Hercules, California, United States).
In vivo evaluation of inner ear drug delivery efficiency and safety of dexamethasone nanosuspensions
Three forms of dexamethasone nanosuspensions or control drugs were injected into the middle ear cavity of animals, and the concentration of dexamethasone in the perilymph was investigated for up to 24 h. After comparing the drug concentration in the perilymph between the three nanosuspensions, we selected one dexamethasone suspension and further investigated its safety and efficacy in vivo.
Middle ear administration of drugs
Eight-week-old male BALB/c mice (Orient Bio, Seoul, Republic of Korea; weight 20–23 g) were used in the in vivo experiments. Drugs were injected into the middle ear using a surgical method described previously (6). Before surgery, the mice were anesthetized using a mixture of 30 mg/kg Zoletil (Virbac, Carros, France) and 10 mg/kg Rompun (Bayer, Leverkusen, Germany) and placed on a thermoregulated heating pad in the supine position; a midline incision was made, and the left bulla was exposed. A hole was created in the bulla using fine forceps, and a drug solution was injected into the bulla using an insulin syringe. To reflect clinical practice, we did not use gel foam to retain the drug in the middle ear. Next, Rimadyl (1.0 mg/kg; Pfizer, Walton Oaks, UK) was injected to relieve pain. Baytril (10 mg/kg; Orion, Hamburg, Germany) was intraperitoneally injected once daily as prophylaxis against middle ear infection.
Perilymph sampling and measurement of dexamethasone concentration
Perilymph was sampled from the lateral semicircular canal (SCC) under the same anesthetic regimen. During perilymph sampling, an incision was made behind the left ear of the animal to prevent contamination with perilymph, and the drug was injected into the middle ear cavity. Then, the animal's pinna was retracted anteriorly, and the muscle of the temporal bone was bluntly dissected to expose the lateral and posterior SCC. A small hole was made in the middle portion of the lateral SCC with a 26 G needle. After we observed perilymph leakage through the hole, approximately 3 µL of perilymph was sampled into a calibrated capillary tube. Each perilymph sample was transferred to an Eppendorf tube and centrifuged for 5 min at 13,200 rpm. Then, 2 µL of the supernatant was collected and diluted 25-fold in 50% methanol to a volume of 50 µL, and liquid chromatography-mass spectrometry (LC/MS; 1290 Infinity II/Qtrap 6500; Sciex, Washington, DC, USA) was performed on the diluted perilymph sample (30).
Preparation of cochlear homogenates and measurement of dexamethasone concentrations
In order to determine the concentration of dexamethasone absorbed into the tissues of the cochlea, the concentration of dexamethasone in cochlear homogenate was analyzed by LC/MS (25). The cochlea was harvested from animals, then immersed in isotonic phosphate buffered saline (PBS) and washed to remove any drug residue from the outside of the cochlea. Additionally, the round window membrane was opened, and the inside of the cochlea was washed with PBS to remove the perilymph. Then, the cochlea was homogenized using Tissue Lyser II (Qiagen, Hilden, Germany) with RIPA buffer. The lysates were centrifuged at 13,200 rpm for 15 min at 4°C, and the supernatants were collected.
In order to compare the concentration of dexamethasone in the cochlear tissue of each individual animal, the protein concentration of the supernatants was adjusted to an equal value, as in western blotting. A dilution of 30 µL of supernatant containing approximately 25 µg of protein was prepared and then combined with 30 µL of methanol to make a sample of 60 µL, and the concentration of dexamethasone in the sample was examined by LC/MS. The results were expressed as the amount of dexamethasone per 25 µg of protein in the cochlear tissue.
To compare the efficacy of dexamethasone nanosuspensions and Dex-SP in the cochlea, we compared the quantities of phosphorylated glucocorticoid receptors (P-GR) in cochlear tissue. Western blotting was performed in cochlear homogenates using the same method described for the in vitro into.
Evaluation of the efficacy of dexamethasone nanosuspensions in a mouse model of ototoxicity
Eight-week-old male BALB/c mice (Orient Bio, Seoul, Republic of Korea; weight 20–23 g) were divided into three groups. The animals in three groups received middle ear drugs 2 h prior to the induction of ototoxicity, as described above; these animals then received dexamethasone nanosuspension, Dex-SP, or distilled water (deaf-sham group). Ototoxicity was induced by the intraperitoneal injection of kanamycin (1000 mg/kg; Sigma-Aldrich, St. Louis, MO) and intraperitoneal injection of furosemide (180 mg/kg; Sigma-Aldrich) 30 min later. The mice were subjected to ABR testing and whole-mount staining of the organ of Corti 2 week later.
Two weeks after injection of the drug into the middle ear cavity, the animals' hearing was evaluated by using the auditory brainstem response (ABR); afterward, the cochlea and bulla were collected, and histological damage was observed with an optical microscope. Cochleae and bullae were fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) and decalcified in 5% ethylenediaminetetraacetic acid (EDTA, 0.3 M). Cochlea sections on slides were stained with hematoxylin (YD Diagnostics Corp., Gyeonggi-do, Republic of Korea) and eosin (BBC Biochemical, McKinney, TX, United States). Images were examined using a light microscope system (Nikon Eclipse E400, Tokyo, Japan).
Immunofluorescence staining for dexamethasone was performed to examine the absorption of dexamethasone in the cochlear tissue (5). Anti-dexamethasone antibody (Abcam, UK) was used as the primary antibody, and the tissue was stained using the Vectastain Elite ABC HRP Kit and the Vector NovaRED Peroxidase Substrate (Vector Laboratories, Burlingame, CA). Images were captured using a light microscope system (Nikon Eclipse E400, Tokyo, Japan).
ABR test in vivo
ABRs were recorded 2 weeks after the operation. The animals were anesthetized with a mixture of 10 mg/kg Rompun (Bayer, Leverkusen, Germany) and 30 mg/kg Zoletil (Virbac, Carros, France). The active electrode was placed approximately at the vertex of the skull, the reference electrode was inserted under the pinna of the left ear, and the ground electrode was inserted under the contralateral ear. ABR thresholds were measured in response to frequencies from 4 to 32 kHz as well as clicks; only the operated ear was tested. TDT System-3 (Tucker Davies Technologies, Gainesville, FL, USA) hardware and software were used to record the ABRs. A computer-generated tone pip was used for stimulation. Tone bursts at frequencies of 4, 8, 16, 32 kHz with a duration of 4 ms and a rise-fall time of 1 ms were used, along with clicks. The sound intensity was varied in 5-dB intervals for the clicks near the threshold and in 10-dB intervals for the tone bursts. The ABRs were analyzed using a custom program (BioSig RP, ver. 4.4.1). Threshold differences among mouse groups were statistically compared.