Materials, cell culture and animals
ATX, DMSO, dichloromethane, and cholate solution were purchased from Sigma-Aldrich (USA). CDDP was purchased from Aladdin (Shanghai, China). Methoxy (polyethylene glycol) (mPEG-PLA, MW 50 kDa) was purchased from Daigang Biomaterial Co, Ltd (Ji’nan, China). DMPC was purchased from Avanti Polar Lipids, Inc (USA). DAPI was purchased from Invitrogen (Carlsbad, CA, USA).
The House Ear Instituteorgan of Corti 1 (HEI-OC1) cell line (generated at the House Ear Institute, Los Angeles, CA, USA) was cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA) free of antibiotics at 33°C under 10% CO2.
Healthy male guinea pigs (4–6 weeks of age, weighing 220–250 g) without otitis media were purchased from the Shanghai Laboratory Animal Center (Shanghai, People’s Republic of China). All animal procedures were carried out in accordance with the Ethical Committee of Shanghai Jiao Tong University School of Medicine (Shanghai, China). Animal experiments complied with the 3R principles (reduction, replacement, and refinement).
Preparation of ATX-loaded lipid-polymer nanoparticles (ATX-LPN)
ATX-LPN were fabricated with a single emulsification solvent evaporation method. Briefly, mPEG-PLA and ATX (6:1, w/w) were mixed and completely dissolved in 1 ml of dichloromethane solution as the oil phase. DMPC was then dissolved in 3 ml of sodium cholate solution (1%, w/v) as the aqueous phase. The oil phase and aqueous phase were mixed well and sonicated at 390 W for 30 s (Scientz Biotechnology Co, Ltd, Ningbo City, People’s Republic of China). The resulting emulsion was further diluted in 36 ml of 0.5% sodium cholate solution, and the oil phase was removed by evaporation. The polymer core formed, and the lipid self-assembled around it. Finally, LPN were acquired by centrifugation (11,000×g, 30 min) and washed twice to remove excess emulsifier. ATX-LPN were frozen and stored at −20°C for further study.
Morphology and characterization of ATX-LPN
The particle size, zeta potential and polydispersity index (PDI) were determined with a Zetasizer Nano-ZS instrument (Particle Sizing Systems, Santa Barbara, CA, USA). Triplicate measurements for each sample were carried out. ATX-LPN were negatively stained with sodium phosphotungstate solution and scanned with a transmission electron microscope (Hitachi, Tokyo, Japan) to examine the morphology. The encapsulation efficiency (EE%) and drug loading (DL%) of the ATX-LPN were examined with LC-MS/MS, as described in a previous study. The methode in detail was described as the the following " LC-MS/MS method for ATX" section.
In vitro and in vivo release profiles
In vitro release profile analysis was performed in artificial perilymph (pH7.4) at 37°C in a water bath. ATX-LPN (0.2 mg/ml) suspension was placed in a centrifuge tube and diluted in artificial perilymph to 1 ml. At specific time points (0.5, 1, 6, 12 h, 1 d, 2 d, 5 d, 10 d, and 14 d), the tube was removed and centrifuged, and 900 μl supernatant was acquired and stored at −20°C for ATX detection. The tube was refilled with 900 μl of fresh artificial perilymph for the next release measurement.
The in vivo release profile was determined in guinea pigs divided into three groups: group I, intraperitoneal injection (IP) administration of 200 μg ATX; group II, RWM administration of 6 μg ATX; and group III, RWM administration of 6 μg ATX-LPN. The guinea pigs were decapitated at different time points, and the left cochlea was removed. The RWM was punctured with a glass electrode, approximately 7μl perilymphatic fluid was extracted with a 1 ml syringe, and the EP tube containing external lymphatic fluid was stored at −80°C. The obtained buffer was subjected to LC-MS/MS determine the ATX concentration, and the minimum detection limit was 1 ng/ml.\
LC-MS/MS method for ATX
The LC system comprised a Shimadzu (Shimadzu Co., Japan) liquid chromatography equipped with a binary pump (LC-30AD), an autosampler (SIL-30AC), a column oven (CTO-20A), a system controller (CBM-20A) and a degasser (DGU-20A). Mass spectrometric analysis was performed using an AB SCIEX API6500 triple-quadrupole (Ontario, Canada) instrument with an ESI interface. The data acquisition and control system were created by using Analyst 1.6.2 software from AB SCIEX (Ontario, Canada) . Chromatographic separation on a Waters X-Bridge BEH C18 (2.1×50 mm, 2.5 µm), mobile phase A is water (containing 1% formic acid), mobile phase B is ACN (containing 1% formic acid), The column was eluted at a flow rate of 0.6 ml/min in a gradient program consisting of 2% phase B (0-0.4 min), from 2 to 65% B (0.4-0.7 min), from 65 to 90% B (0.7-1.30 min ), 90% B (1.30-1.90 min), from 90 to 2% B (1.90-1.91 min ), 2% B (1.91-2.50 min). For Astaxanthin, the retention time for the analyte and IS (Diclofenac) were 1.82 min and 1.30 min respectively, injection volume is 5 µl.
The precursor product ion pair was m/z 597.3→147.1 for Astaxanthin, m/z 296.2→214.2 for Diclofenac.
Standard curve preparation
Stock solution of Astaxanthin were prepared at 1 mg/mL in DMSO. The stock solution was diluted with MeOH to preparing serial working solution ( 200, 400, 600, 1000, 2000, 6000, 20000 ng/ml), an aliquote of 10 µl working solution was spiked into 190 µl guinea pig plasma: PBS 1:9 (V/V) to obtain calibration standard curve (10, 20, 30, 50, 100, 300, 1000 ng/ml ).
An aliquot of 3 µl sample was diluted with 12 µl guinea pig plasma : PBS 1: 9 (V/V). Then 75 µl IS (Diclofenac, 100 ng/ml in ACN) was added for protein precipitation. The mixture was vortexed for 10 min at 750 rpm and centrifuged at 6000 rpm for 10 min. An aliquot of 5µl supernatant was injected for LC-MS/MS analysis.
Cellular uptake by HEI-OC1 cells
To trace LPN uptake by HEI-OC1 cells, we encapsulated coumarin 6 in LPN and used DAPI to label nuclei. Briefly, HEI-OC1 cells were cultured on VWR Micro Cover Glasses in 24-well plates at a density of 5×104 cells/well. When the cells reached approximately 80% confluence, the medium was replaced with coumarin 6-labeled LPN and incubated for 1, 3, 6, and 12 h. After the coumarin 6-LPN were removed, and the wells were washed twice with PBS, the cells were fixed with 4% glutaraldehyde for 20 min, and the cell nuclei were stained with DAPI for 30 s. The cells were then observed under an LSM-510 CLSM (Carl Zeiss AG, Oberkochen, Germany) with a fluorescein isothiocyanate (FITC) filter (excitation, 488 nm; emission, 520 nm), and flow cytometry (BD LSR Fortessa, America) was performed.
The in vitro cytotoxicity of nanoparticles was assessed with CCK-8 assays. HEI-OC1 cells were seeded at a density of 5×103cells/well in a 96-well plate and allowed to attach overnight. Then they were divided into the following three groups of exposures: ATX or ATX-LPN at various concentrations (1, 5, 10, and 15 μg/ml); CDDP (10, 30, 60, and 100 μM); and co-treatment with CDDP (60 μM) and ATX/ATX-LPN (1, 5, 10, and 15 μg/ml). After a 24 h incubation, 10 μl CCK-8 reagent (Dojindo Molecular Technologies, Japan) was added to each well and reacted for 2 h at 37°C in 5% CO2. The absorbance was measured at 450 nm with a plate reader. The percentage cell viability was calculated by comparing cells treated with different formulations to the corresponding control cells.
The fluorescent probe 2, 7-dichlorofluorescein diacetate (DCFH-DA) was used to detect intracellular ROS production. HEI-OC1 cells, seeded at a density of 5×105 and cultured overnight, were treated with 60 μM CDDP alone or co-treated with 1, or 10 μg/ml ATX/ATX-LPN for 24 h, then incubated with 10 μM DCFH-DA (Abcam, MA, USA) in serum-free medium at 37°C for 30 min in the dark. Quantification of the fluorescence intensity (488 nm excitation/525 nm emission) was performed with a Zeiss confocal laser scanning microscope.
Detection of mitochondrial membrane potential
JC-1 (Thermo Fisher Scientific, MA, USA) and MitoTracker Green(Thermo Fisher Scientific, MA, USA) are widely used fluorescent dyes for monitoring mitochondrial membrane protentional (MMP). HEI-OC1 cells were cultured and treated with 60 μM CDDP alone or co-treated with 1 or 10 μg/ml ATX/ATX-LPN for 24 h, then stained with 10 μM JC-1 and 1 μM MitoTracker Green for 20 min and 45 min, respectively. JC-1 exhibits double fluorescence staining, either as red fluorescent J-aggregates (530 nm excitation/590 nm emission) at high potentials or as green fluorescent J-monomers (490 nm excitation/530 nm emission) at low potentials; a fluorescence change from red to green represents a decrease in MMP. MitoTracker Green, another fluorescent stain for mitochondria, labelsfunctioning mitochondria in living cells and exhibits green fluorescence (490 nm excitation/516 nm emission). Images were acquired with a Zeiss confocal laser scanning microscope.
Flow cytometry was carried out for quantitative evaluation of apoptosis. HEI-OC1 cells were seeded in six-well culture plates at a density of 5 × 105 per well for 24 h before the assay. After incubation with 60 μM CDDP alone or co-treatment with 1 or 10 μg/ml ATX/ATX-LPN for 24 h, treated cells were trypsinized, collected, washed with PBS, and resuspended. The cells were then incubated with FITC-conjugated Annexin V (BD biosciences) and PI (BD biosciences) for 15 min at room temperature in the dark. Annexin V-FITC/PI positive cells were analyzed by fluorescence-activated cell sorting (FACS) (Becton Dickinson).
To determine levels of proteins in the apoptosis pathway, we treated HEI-OC1 cells with 60 μM CDDP and 1 or 10 μg/ml ATX/ATX-LPN as described above, and then harvested the cells for western blot analyses. The protein extracts were subjected to 10% SDS-PAGE and electrotransferred to PVDF membranes. The membranes were then blocked for 1h in quick-blocker at room temperature and incubated overnight in a cold chamber (4°C) with specific primary antibodies. After incubation, the membranes were washed with TBS and then incubated with HRP conjugated secondary antibody for 1 h at room temperature, washed repeatedly, and visualized with an enhanced chemiluminescence kit (Thermo Fisher Scientific). GAPDH and α-tubulin served as internal standards. The relative intensity was quantified by Quantity One.The following primary antibodies were used: anti-cleaved-caspase 3, anti-cleaved-caspase 9, anti-p53, anti-BCL-2, anti-P-P38, anti-T-P38 and anti-cytochrome 3 (Cell Signaling Technology, CST), anti-P-JNK (Abcam), and anti-T-JNK(Abcam).
Evaluation of hair cell damage on zebrafish
This experiment was carried out by Hunter Biotechnology,Inc (Hangzhou, China). Briefly, zebrafish larvae (5 days post-fertilization, n = 30 per concentration) were pretreated with various concentrations (1, 10 and 50 µg/ml) of ATX (1, 10 and 50 µg/ml), ATX-LPN (1, 10 and 50 µg/mL) or GSH (154 μg/mL) for 4 h, then exposed to CDDP(60 μM) for 24 h. After being washed three times, larvae were stained with 2-[4-(dimethylamino)styryl]-1-ethylpyridinium iodide) (Sigma) to evaluate the hair cell damage of neuromasts. Then ten zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope, and the images were analyzed in Image-J. The fluorescence intensity (S) of neuromast hair cells in the zebrafish body was calculated. The below formula was used to evaluate the damage to hair cells: protective effect on hair cell damage (%) ×100%
Administration of ATX-LPN on RWM
RMW administration was described in our previous study , Briefly, guinea pigs were anesthetized with an intraperitoneal (IP) injection of 1% pentobarbital sodium (35 mg/kg). During the procedure, the animals were placed on a hearing pad (38℃). A subcutaneous injection of 1% lidocaine was performed to reduce pain. A postauricular incision was made and the muscle was dissociated via blunt dissection and retracted until exposure of the auditory bulla. A hole of 2-3 mm in diameter was drilled on the bulla to provide direct visualization of the round window niche. A ATX or ATX-LPN solution (6μl) was applied onto the RWM using a microsyringe. The guinea pigs were fixed at this position for 30 minutes. The bulla opening was sealed with dental cement and the incision was closed with sutures.
Evaluation of hearing loss and hair cell damage in guinea pigs
Auditory brainstem responses (ABR) were measured in anesthetized animals before and 3 days after the drug administration. Guinea pigs were placed on a heating pad maintained at 37°C in a sound-proof booth. Pure tone stimuli of 1.4, 2, 2.8, 4, 5.6, 8, 11.3, 16, 22.6, or 32 kHz between 0 and 90 dB SPL in 5 dB steps were delivered through an open-field microphone. Subcutaneous electrodes collecting signals were inserted at the pinna (recording electrode), vertex (reference electrode), and rump (ground electrode). The acoustic signals were generated with Tucker-Davis Technologies (Alachua, FL, USA) hardware. Thresholds were defined as the lowest stimulus level at which a response was observed.
Guinea pig cochleae from the three groups were perfused with 4% paraformaldehyde for 2 h for fixation and decalcified in 0.12 mM EDTA at room temperature for 1 week. The cochleae were then microdissected into the apex, middle and basal turns and subjected to immunohistostaining of FITC-conjugated phalloidin (green) and caspase-3 (red), indicating stereocilia and apoptosis. Specimens were mounted on slides with anti-fade mounting medium and imaged with a Zeiss confocal microscope. The percentage of survival OHCs at the basal, middle and apex turns was determined for areas located at 4, 11, and 32 kHz.
All statistical analysis was performed in GraphPad Prism. Variables are expressed as means ± standard error of mean (SEM). Student's t-test and two-way ANOVA with Bonferroni correction were used for statistical analysis. Values of P<0.05 were considered significant.