2.1 Materials
Lidocaine base, sodium fluorescein (flu-Na), Hanks’ Balanced Salt Solution (HBSS) were purchased from Sigma Aldrich (Sydney, Australia). Ethanol (100%) and acetonitrile were purchased from Chem-Supply Pty Ltd (Australia) and PTFE 0.45 µm filter was purchased from FilterBio® (China). All cell culture reagents including Dulbecco’s modified eagle’s medium (DMEM), Minimum Essential Medium Eagle (MEM), phosphate-buffered saline (PBS), foetal bovine serum (FBS), trypsin-EDTA solution (2.5 g/l trypsin, 0.5 g/l EDTA), L-glutamine solution, non-essential amino acids were obtained from Invitrogen (Sydney, Australia). Water was purified by reverse osmosis (MilliQ, Millipore, France). All solvents used were of analytical grade.
2.2 Cell lines
Detroit 562 (Immortalised Epithelial Human Pharyngeal cells- carcinoma derived) were purchased from the American Type Culture Collection (VA, USA). All cells were maintained in minimum essential cell growth medium (MEM; Invitrogen) supplemented with FBS (10%) and L-glutamine (1%) and incubated at 37°C under 5% CO2. All experiments were conducted between passage numbers 51–62 for the Detroit 562 cell line.
2.3 Air-Liquid Interface (ALI) culture of Detroit 562 cells
To establish an in vitro ALI model of Detroit 562 cells, Transwell cell culture inserts (0.33 cm2, polyester terephthalate (PET) membrane, 0.4 µm pore size) (Corning Costar, USA) were used as previously described [22]. To determine the appropriate seeding density for Detroit 562 cell line, three different seeding densities were chosen: 30,000 cells/well (c/w) (0.9 × 105 cells/cm2), 60,000 c/w (1.8 × 105 cells/cm2) and 80,000 c/w (2.4 × 105 cells/cm2). Briefly, Detroit 562 cells were seeded within the apical chamber in MEM media supplemented with 10% v/v FBS and 1% L-glutamine and the same media was added to the basolateral chamber. The cells were incubated at 37°C with 5% CO2 for 24 hours until confluency was achieved. To initiate ALI conditions, media in the apical chamber was removed after 24 hours indicating day 0 and the cells were maintained under ALI conditions for 21 days. Differentiation media in the basolateral chamber was replaced every 2 days. All experiments were performed on day 7, 14, 18 and 21 post ALI induction.
2.4 Transepithelial Electrical Resistance (TEER)
Transepithelial electrical resistance (TEER) was measured as described previously. Briefly, pre-warmed media was added to the apical chamber and allowed to equilibrate for 30 mins at 37°C under 5% CO2. TEER was measured using EVOM2® epithelial voltohmmeter (World Precision Instruments, USA) attached to STX-2 chopstick electrodes for the ALI cultures, corrected by subtracting the blank inserts, and multiplied by the area of the Transwell inserts (0.33 cm2) and six measurements were taken per Transwell.
2.5 Sodium fluorescein permeability assay
Tight junction functionality and paracellular permeability of Detroit 562 ALI cultures were determined using the sodium fluorescein permeability assay. Sodium fluorescein (2.5 mg/mL), (Sigma Aldrich, Sydney, Australia) was added to the apical chamber and pre-warmed Hanks' Balanced Salt Solution (HBSS) was added to the basolateral chamber. Transwells were incubated for 4 hours at 37°C with 5% CO2, with basolateral samples (100 µL) collected and replaced with fresh HBSS after every 30 minutes for the first 2 hours and then every hour for the final 2 hours to measure the rate of transport (flux) of the sodium fluorescein from the apical chamber to the basolateral chamber. For analysis, the collected basolateral samples were diluted (1:20) and fluorescence was measured using the SpectraMax M2 plate reader (excitation: 485 nm; emission: 538 nm). The permeation coefficient (Papp) was calculated according to Eq. 1.
Eq 1.\({P}_{app}=\frac{dQ}{dT\bullet {C}_{0}\bullet A}\)
where dQ/dT represents the flux of sodium fluorescein (µg/s) across the membrane, C0 is the initial donor concentration (µg/mL), and A is the surface area (cm2).
2.6 Live and Dead Cell Staining
LIVE/DEAD® Viability/Cytotoxicity Kit (Molecular Probes) and the Hoechst stain (Sigma Aldrich) were used to stain the ALI cultured cell layers. The assay was performed as per the manufacturer's instructions. Briefly, the cell layer was washed 3 times with pre-warmed PBS and 2 µM calcein AM and 4 µM ethidium homodimer-1 (EthD-1) were added to the apical compartment. Cells were incubated with the solution for 30 minutes in the dark at room temperature. Cells were incubated with Hoechst (1:10000) for 10 minutes to stain the nucleus. The Transwell membranes containing the cell layer were excised and mounted on a glass microscope slide for analysis. Cells were imaged using a Nikon ECLIPSE Ti inverted microscope controlled by the NIS Elements software (Nikon) and equipped with the APO Fluor 20X air objective. Images were captured using a CoolSNAP ES2 high-resolution digital camera (Photometrics). 10 images of different fields of view were taken per Transwell membrane.
2.7 Mucus production
Mucus production of the ALI cultures was characterized by staining the glycoproteins (mucins) with alcian blue (Sigma Aldrich) as previously described [4, 23]. Briefly, cell layers were washed twice with prewarmed PBS and fixed using 4% paraformaldehyde (v/v) for 15 minutes. Subsequently, the cells were washed thrice with PBS and Alcian blue (1% w/v Alcian blue in 3% v/v acetic acid/water at pH 2.5) was added to the apical chamber and incubated for 20 minutes. The cell layer was washed up to 10 times with PBS to remove excess Alcian blue and allowed to air-dry for 3 hours at room temperature. The Transwell membranes containing the cell layer were excised and mounted on a glass microscope slide for analysis. Mucus staining was imaged using an Olympus BX61 microscope (Olympus) equipped with an Olympus DP71 camera and a 20X air objective. 10 different fields of view were captured per Transwell membrane. Images were analyzed using Image J software (NIH) and the mean RGB values (Red, Green, Blue) were measured for each image. The ratio of blue to all other colours (RGBB ratio) was calculated by dividing the mean RGBB by the sum of the mean RGB values (RGBR + RGBG + RGBB) for each image.
2.8 Evaluation of cytokine production and inflammatory responses
Lipopolysaccharide (LPS) from E.coli (Sigma-Aldrich) was resuspended at 10 µg/mL in differentiation media (MEM with 10% FBS and 1% L-glutamine) and added to the basolateral chamber to stimulate the cells to model bacterial infection. The cells were stimulated with polyinosinic-polycytidylic acid (Poly (I:C)) (10 µg/mL, Sigma Aldrich) by resuspending in the differentiation media to model viral infection. Cells were then incubated at 37°C under 5% CO2 for 24 and 48 hours and untreated cells served as the control. After treatment, samples were collected from the basolateral culture medium for subsequent analysis of IL-6, IL-8 and IL-1β cytokine production using an enzyme-linked immunosorbent assay (ELISA) kit (BD OptEIA, BD Biosciences) according to the manufacturer’s instructions.
2.9 Immunofluorescence
The presence of tight junctions in the ALI cultures was visualized by immunolabeling tight junction proteins Zonula Occludens-1 (ZO-1). Cell layers were washed 3 times with PBS and fixed with 4% paraformaldehyde (v/v) for 15 min. The cell layers were then washed 3 times with PBS and permeabilised following a 10 min incubation with 0.2% Triton X-100 (v/v) and then blocked and quenched with 10% normal goat serum (v/v) (Invitrogen) and 0.3 M glycine (Sigma Aldrich) respectively, and incubated for 1 h at room temperature. The primary antibody, ZO-1 rabbit polyclonal (Abcam) (1:200) was incubated overnight at 4°C. The next day, the cell layers were washed 3 times with PBS and incubated with goat anti-rabbit Alexa Fluor® 488 (Life Technologies) (1:500) for 2 h at room temperature. Cell layers were then counterstained with DAPI (Sigma Aldrich) (1:10000) and incubated for 30 min at room temperature. Finally, Transwell membranes containing the cell layer were excised and mounted using FluoroSave mounting media (Millipore) on a glass microscope slide for analysis.
Cells were imaged using a confocal microscope (Nikon Eclipse Ti) equipped with a Plan Apo VC 60 × oil objective. Images were taken using the resonant scanner at a step size of 0.31 µm, 512 × 512 pixels with an average line scan of 16. For cells immunolabelled with FITC-tagged secondary antibodies, the 488 nm laser was set to 3.5% with a smart gain of 55 V and an offset of − 3%. For nuclei excitation, the 405 nm laser was again set to 3% with the smart gain and offset set to 50 V and − 1% respectively.
2.10 Lidocaine transport study
To investigate whether the developed ALI model of the Detroit 562 cell line could be used to study drug transport, a study was conducted using Lidocaine as a model drug. Lidocaine transport across the Detroit 562 ALI cultures was conducted on day 18 post ALI formation. Lidocaine was dissolved in ethanol to produce the stock solution and then further diluted in HBSS to prepare a 20 µg/mL Lidocaine (0.1% ethanol in final Lidocaine solution) to be used for the transport study. Lidocaine solution was added to the apical chamber and HBSS was added to the basolateral chamber. Samples (100 µL) were taken from the basolateral chamber every 30 min for the first 2 h and then every hour for the final 2 h, with samples being replaced by fresh, warm HBSS. After the 4 h assay, the apical chamber was washed twice with HBSS to collect any residual drug using a pipette (denoted as On) and the cell layer was then scraped from the insert membrane and lysed using CelLytic™ buffer (Invitrogen) to quantify the amount of drug inside the cells (denoted as Cellular). TEER measurements were performed before and after the transport, study to check whether drug deposition altered the epithelial barrier integrity of Detroit 562 ALI culture models. All the samples were subsequently analysed using High-Performance Liquid Chromatography (HPLC) using the quantification method described in the next section.
2.11 HPLC quantification method for Lidocaine
All Lidocaine samples were analysed using a High-Performance Liquid Chromatography (HPLC) system equipped with SPD-20A UV–Vis detector, an LC-20AT liquid chromatograph, a SIL-20A HT autosampler (Shimadzu) and a Kinetex C-18 column (250 × 4.6 mm, 5 µm, Phenomenex, Torrance, USA), according to a validated method. The mobile phase was a mixture of acetonitrile: phosphate buffer (26:74 (v/v) with pH 5.5 adjusted using sodium hydroxide (Sigma Aldrich). Samples were analysed at 230 nm at a flow rate of 1.0 mL/minute and an injection volume of 10 µL. Linearity was obtained between 0.2 and 100 µg/mL (R2 = 0.99) with a retention time of 8 min.
2.12 Development of a 3D printed throat model incorporated with cells
To evaluate drug deposition and transport of inhaled drugs targeted at the oropharyngeal region, a realistic and more physiologically relevant throat model was designed and developed to include the integration of cells for enhanced in vitro-in vivo correlation. A computer-aided (CAD) design of the medium-sized Virginia Commonwealth University (VCU) throat model was prepared by AutoCAD® (version 23, USA). The design was modified to connect two separate lower and upper pieces and insertion of two Snapwell inserts (denoted as Upper and Lower snapwells respectively) in which cells grown in ALI conditions could be incorporated for subsequent deposition and transport studies (Fig. 1). The prepared 3D design was then 3D printed using clear photopolymer resin (FLGPCL02, Formlabs Inc., USA) by stereolithography (SLA), using Form 2 (Formlabs Inc., USA).
2.13 In vitro aerosol deposition using USP-IP and the 3D printed throat models
Deposition profiles of the Lidocaine spray targeted to the throat were determined using the European Pharmacopeia Apparatus E, Next Generation Impactor (NGI) (Copley Instruments Ltd) fitted with the USP stainless steel 90° induction port (US-IP model), as specified in European Pharmacopoeia (Ph. Eur. 8th Edition, monograph 2.9.18). To optimise the conditions for studying the throat deposition of Lidocaine spray, the experiment was performed at two different angles of spraying (45° and 90°) with 3 different flow rates of 0, 15 and 30 L/min representing no airflow, light breathing and normal breathing condition respectively. Briefly, the NGI was connected to a high-capacity vacuum pump, and the flow rate was set using a flow meter (Model 4040, TSI Precision Measurement instruments). Lidocaine throat spray was primed by firing 5 shots to waste and weighed before each shot. The distance between the spray nozzle and the throat was measured at 7 cm for each NGI experiment to ensure that the spray is aimed primarily toward the throat and not in the oral cavity. The Lidocaine throat spray was attached to the impactor via an airtight adaptor with an actuation time of 4 s. Following the completion of the delivered dose, all components of the NGI (actuator, adaptor, IP, stages 1–7 and micro-orifice collector (MOC)) were washed with acetonitrile: phosphate buffer (26:74 (v/v), transferred to volumetric flasks and sonicated for 10 mins. Samples were then filtered (0.45 µm, PTFE) and Lidocaine was quantified using HPLC. Subsequently, the flow rate that resulted in maximum Lidocaine deposition in the throat region was used to determine drug deposition using our novel 3-D printed VCU model at both 45 and 90° angles. All the samples were subsequently analysed using a High-Performance Liquid Chromatography (HPLC) quantification method for Lidocaine described in the previous section.
2.14 Transport of Lidocaine throat spray using the 3D printed VCU model integrated with Detroit 562 ALI culture
To investigate whether the developed novel 3D printed VCU model integrated with the ALI model of Detroit 562 cells grown on Snapwells could be used to study drug transport of Lidocaine throat spray, a transport experiment was conducted over a 4-h period. Prior to conducting the transport study, optimization of the number of shots of the throat spray on the cellular layers of the Detroit 562 ALI culture was performed at the optimised flow rate of 30L/min and an angle of 45° using 1 shot and 3 shots of Lidocaine spray. Using the optimised conditions, deposition, and transport of lidocaine spray across the Detroit 562 cells grown in ALI conditions on snapwell inserts placed within the 3D-printed VCU throat model were studied. The snapwells with the Detroit 562 cells were placed in the lower and upper part of the 3D throat model and hence referred to as Lower and Upper snapwells respectively. Lidocaine throat spray was attached to the impactor via an airtight adaptor and one shot was fired into the 3D throat at an angle of 45°. After deposition, the snapwell inserts were removed from the 3D printed throat and transferred into culture plates with 2 mL of fresh HBSS added into the basal chamber. Samples (200 µL) were taken from the basolateral chamber every 30 min for the first 2 h and then every hour for the final 2 h, with samples being replaced by fresh, warm HBSS. The same method of transport study was followed as described in the previous section to determine the amount of drug transported during and after the 4h period (Transported) and to evaluate the amount of drug present inside the cells (IN) and remaining on the cells (ON). Additionally, to determine whether drug deposition and transport study altered the epithelial barrier integrity of the Detroit 562 cells, sodium fluorescein permeability assay was conducted on untreated cells that served as control and on treated cells following Lidocaine deposition and post 4 h transport study as described earlier in the previous section.
2.15 Statistical analysis
All results are expressed as mean ± standard error of the mean (SEM) of at least three biological replicates. Statistical software, GraphPad Prism (version 8.2.1) was used to test for significance using One-Way or Two-Way ANOVA for each experiment. Significance was determined as p < 0.05.