PB (≥ 99), LCA (≥ 95%), low viscosity sodium alginate (SA; ≥ 99), Tween 80, gelatin, poloxamer, Polyvinyl alcohol (PVA), Poly(acrylic acid) (PAA), polystyrene sulfonate (PSO), Poly-L-ornithine (PLO) and silk fibroin protein were gained from Sigma-Aldrich (St Louis, MO, USA). 2,2′-Azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) was bought from Sapphire Bioscience (Redfern, NSW, Australia) and 20,70-Dichlorofluorescin diacetate (DCFH-DA) from Sigma-Aldrich as well. Dulbecco’s Eagle's Medium (DMEM) (Bioscience Gibco TM, Dublin, Ireland), foetal bovine serum (Thermofisher Scientific, Melbourne, Australia), MTT(3- (4, 5 - dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide) (Thermo Fisher Scientific MA, USA), (PBS), pH 7.4 (Thermofisher Scientific, Australia), DMSO (Sigma Chemical Co, USA), Hoechst 33258 (Thermofisher Scientific, Australia), propidium iodide (1 µg/ml) dye (Thermofisher Scientific, Australia), seahorse kit (Massachusetts, USA) and cytokine bead array assay (BD Bioscience, CBA Mouse, USA) were brought for cell work.
Preparations of Drug
Stock mixtures for the formulations were prepared in deionised water using PB (0.45% w/v), LCA (0.45% w/v), SA (1.7% w/v), tween 80 (1.7% v/v), gelatin (0.8% w/v), poloxamer (1% w/v), PVA (1.3% w/v), PAA (0.35% w/v), PSS (0.35% v/v), PLO (1.1% v/v) and silk fibroin protein (0.7% w/v). All formulations were mixed thoroughly for 10 hours at room temperature. The stock solutions were stored in the fridge and used within 24 hours of preparation.
Preparation of micro-nanocapsule
Three different formulations were prepared; F1= polymers only, F2= polymers and PB, F3= polymers, PB and LCA. The micro-nanocapsules were prepared by pumping in the formulation at a 5-10% flow rate in the BUCHI mini spray dryer B-290 with a 0.7 mm nozzle diameter (Meierseggstrasse, Flawil, Switzerland). Inlet and outlet temperatures at 150°C to 170°C and 60°C, respectively. The aspirator was set at 92-95%. All the formulations' dry powder was collected, weighed, and used for further analysis. Each experiment was analysed in triplicates (n = 3).
Toporphology, size and surface elemental analysis of micro-nanocapsules
The size and appearance of micro-nanocapsules were determined using a field emission scanning electron microscope (SEM; CLARA TESCAN, Brno, CZ). The mean particle size was determined by using the software Image J Analysis. The surface elemental analysis was characterised using energy-dispersive spectrometry (EDS, INCA X-Act; Oxford Instruments, UK). Prior to imaging, dried particles were mounted on an aluminium stub with double-sided adhesive carbon tape and coated under vacuum in an argon atmosphere with 5 nm platinum.
Rheological analysis, zeta-potential, surface tension and physicochemical compatibility
Rheological analyses such as viscosity, torque, shear stress and shear rate were measured for all the formulations using the viscometry (Visco-88 viscometer, Malvern Instruments, Malvern, UK) at various speeds. Particle electrokinetic stability was measured using the Zetasizer 3000 HS (Malvern Instruments, Malvern, UK). Tensiometer (Sigma 703, NSW, Australia) was used to analyse Surface tension.
Powder form compounds and the formulations' physical characterisation were analysed using the differential scanning calorimetry (DSC) (Netzsch DSC 3500 Sirius, JP) under a nitrogen atmosphere. The heat flow difference between reference and sample is determined, representing the sample’s caloric change. Briefly, dry samples (F1, F2, F3, individual and mixture of compounds) were sealed in an aluminium pan together with a reference (empty sealed pan) and heated range from 10°C to 300°C at a flow rate of 20 K/minute with auto cooling 35.
The chemical profile of compounds and formulations characterisation was performed by Fourier transform infrared spectroscopy (FT-IR) (Waltham, MA, USA) in a transmission frequency range of 450-4000 cm-1 35.
Drug content and release study
For drug content, 8 mg of F1 and F2 suspensions were stirred at 37°C, 100 rpm for 4 hours in 10 mL acetonitrile. 1 mL of the suspension was filtered and transferred to a glass vial and measured using high-pressure liquid chromatography (HPLC) using the method mentioned below. The amount of drug content and encapsulation efficiency was calculated as described previously 48.
For the PB release study, 100 mg of F2 and F3 micro-nano particles were suspended in 10 mL of perilymph-simulating fluid (pH 7.25 and Osmolarityms= 320 mOsm/L) at 37 °C for 4 hours, stirred at 100 rpm. The fluid was collected in a tube every 30 minutes, and sink conditions were maintained throughout the experiment. The tubes were centrifuged for 30 minutes at 11,000 xg and filtered using a 0.22-µm Millex syringe filter before being injected into HPLC to analyse the release parameters. For HPLC analysis, 20 μL samples were loaded with a low-pressure gradient in the Shimadzu Prominence HPLC system (Pump model: LC-20AT, UV detector: SPD-20A and injector model: SIL-20AC, Shimadzu Corporation, Kyoto, Japan) on a Phenomenex C18 reverse-phase column with an internal diameter of 4.5 mm and a length of 25 cm at a wavelength of 242 nm. The suspension and PB standard concentrations were run in acetonitrile: water (95:5% v/v) at the wavelength of 242nm with a flow rate of 1.5ml/min. PB standard concentrations were run from 0.04 to 12 mg/mL 34.
In vitro study
HEI-OC1 (House Ear Institute-Organ of Corti 1) cells were obtained from UCLA medical centre. Cells were cultured in permissive conditions (33°C and 10% CO2) in high glucose in DMEM without antibiotics, with 10% foetal bovine serum. At every 24 hours interval, media was changed and when the cells population reached around 80% confluence, a sub-culture was performed as described75,76.
For each assay, cells were collected and adjusted at 2.0 × 105 cells/mL concentrations and left overnight in incubation for the attachment on the cell-well plate. The experiment started by exposing the cells to a chemical AAPH stressor in various concentrations. The cells' viability was significantly decreased with the increasing concentration of AAPH. AAPH at a 12.5 millimole (mM) rendered a ~50% cell viability (IC50) and this concentration was selected for this study. Cells were then treated with differently prepared micro-nanocapsules (F1, F2 and F3) for 48 hours and compared within the different groups and controls (C1= untreated cells and C2 = cells treated only with AAPH; negative control).
Cell viability assay
Cell viability analysis was performed via MTT assay. The MTT stock solution (5mg/ml) was prepared in PBS. For this assay, cells were collected and adjusted to the required concentration (2.0 × 105 cells/mL). Cells were treated with AAPH and co-treated with micro-nanocapsules. After 48 hours of treatment, MTT assay was performed as mentioned previously with slight modification 76. Briefly, after 48 hours, cell media was removed, washed with PBS, and 30 μL of MTT reagent was added with serum-free DMEM media and incubated for 3 hours. After 3 hours, DMSO was added to stop the reaction; the multi-well plate was then kept on a plate shaker for another 10-20 minutes to ensure the reaction was completed. PerkinElmer Multimode Plate Readers (Waltham, Massachusetts, USA) set at 550 nm were used to measure the absorbance, and the result was expressed as a normalised percentage of viable cells compared to the control culture cells.
For live/dead cell staining, the cell was seeded on 24 well plates and left overnight to allow attachment to the substrate in an incubator, and then cells were stressed with the addition of AAPH. After 48 hours of the treatment, the cells were washed with PBS, treated with the mixture of Hoechst 33258 (5 µg/ml) and propidium iodide (1 µg/ml) dye, and incubated for another 10 minutes. Fluorescent images were captured by Olympus IX51 inverted fluorescence microscope (Tokyo, JP). For the analysis of sufficient cell numbers to allow the presented data to have statistical significance, images from four adjacent fields were taken per well, and the percentage of the dead and live/cell was calculated. Cells were considered dead if they emitted the fluorescence signal only propidium iodide.
Mitochondrial function was analysed by measuring oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and proton production rate (PPR) using real-time Seahorse Flux Analyser XF 96 (Seahorse Bioscience, USA) with our well-established method 33. Briefly, the first injection was with media without glucose, with subsequent ATP synthase inhibitor oligomycin (1.5 µM) and FCCP (1 µM) (carbonyl cyanide-p-trifluoromethoxy phenylhydrazone) and the final injection was complex I + II inhibitors Rotenone + antimycin A (0.5 µM) 77. The result was automatically generated and analysed by the wave software.
Cytokine bead array assay
Cytokine release (TNF- α, Tumor necrosis factor-alpha and IFN-γ, interferon-gamma) from 48 hour treated HEI-OC1 cells was performed via the cytokine bead array assay BD Bioscience cytometry bead using the BD FACSCanto II cell analyser 9BD Bioscience, USA) as per protocol provided. Data were analysed by using the FlowJO software (FlowJo, Ashland, OR, USA).
Cellular anti-oxidant assay
This assay measures the anti-oxidant effect on live cells. It is the standard approach to measure intracellular ROS via the use of DCFH-DA. For this assay, after 48 hours of the AAPH treatment and co-treatment with micro-nanocapsules, cells were washed with PBS, treated with 50 µM fluorescence label probe DCFH-DA and incubated for 30 minutes. DCFH-DA stock solution (25 mM) was prepared in methanol, aliquoted and stored at -20°C. DCFH-DA is esterified into DCFH when it diffuses through the cell membrane into the cytosol, which is oxidised by intracellular ROS to form fluorescing DCF. The higher the fluorescence activity, the greater the DCFH-DA oxidation which was measured at 485 nm absorption and 538 nm emission using Enspire Multimode Plate Reader 34,78,79.
All the experiments were analysed thrice; data were shown as the mean ± standard deviation (SD) or standard error of deviation (SED) and analysed using one-way analysis of variance (ANOVA) followed by Tukey's posthoc analysis or by using Pearson t-test using Graphpad Prism version 8.1.2. Statistical significance value was set at p < 0.01 or p < 0.05.