Primary AirâLiquid Interface Culture of Nasal ... - ACS Publications

Subscriber access provided by Temple University Libraries

Article

Primary Air Liquid Interface Culture of Nasal Epithelium for Nasal Drug Delivery Hui Xin Ong, Claire L. Jackson, Janice L. Cole, Peter M. Lackie, Daniela Traini, Paul M. Young, Jane Lucas, and Joy Conway Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00852 • Publication Date (Web): 25 May 2016 Downloaded from http://pubs.acs.org on June 4, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Primary Air Liquid Interface Culture of Nasal Epithelium for Nasal Drug Delivery Hui Xin Ong1,2,3,4*, Claire L. Jackson2,5, Janice L. Cole2,5, Peter M. Lackie2,5, Daniela Traini3,4, Paul M. Young3,4, Jane Lucas2,5 & Joy Conway1,2

1Faculty

of Health Sciences, Southampton University, Southampton SO16 6YD,

UK. 2NIHR

Southampton Respiratory Biomedical Research Unit, University of

Southampton and University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, UK. 3Respiratory

Technology, Woolcock Institite of Medical Research, NSW 2037,

Australia. 4Discipline 5

of Pharmacology, Sydney Medical School, NSW 2006, Australia.

Primary Ciliary Dyskinesia Centre, University Hospital Southampton NHS

Foundation Trust, Southampton SO16 6YD, UK.

*Corresponding author. 1Respiratory Technology, Woolcock Institute of Medical Research and Discipline of Pharmacology, Sydney Medical School, The University of Sydney, NSW 2037, Australia. Tel.: +61 2 91140373; E-mail: [email protected]

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 37

Abstract Graphic

Abstract Nasal drug administration is a promising alternative to oral and parenteral administration for both local and systemic delivery of drugs. The benefits include its non-invasive nature, rapid absorption and circumvention of first pass metabolism. Hence, the use of an in vitro model using human primary nasal epithelial cells could be key to understanding important functions and parameters of the respiratory epithelium. This model will enable investigators to address important and original research questions using a biologically relevant in vitro platform that mimics the in vivo nasal epithelial physiology. The purpose of this study was to establish, systematically characterise and validate the use of a primary human nasal epithelium model cultured at the air liquid interface for the study of inflammatory responses, drug transport and to simultaneously quantify drug effects on ciliary activity.

Keywords: nasal drug delivery, ciliary function, transport, tobramycin, epithelial cells


Page 3 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.0

Introduction

Nasal drug delivery provides a non-invasive delivery mode for both local and systemic treatment due to the large surface area in the nasal cavity (50–200 cm2, about four times that of the trachea)1, avoidance of first pass metabolism and relatively high blood flow, promoting rapid absorption 2. Examples of nasal drug delivery include localised treatment of rhinitis and allergies by corticosteroids, decongestants and antihistamines 3, while systemically active drugs available as nasal sprays include naloxone (treatment of opioid overdose) 4, benzodiazepine (treatment for intractable seizure)5 and ketorolac tromethamine (nonsteroidal anti-inflammatory for pain relief) 6 7. More recently, intranasal delivery has been shown to have the potential to deliver drugs directly to the brain through the olfactory epithelium, located on the upper part of the nasal cavity. This route could be used as an alternative to treat diseases of the central nervous system such as Parkinson’s and Alzheimer’s disease 8. However, there are several factors to be considered in formulating nasal products. These include possible detrimental effects of drug formulations on the nasal mucociliary clearance system or, the physical barrier of the epithelium and mucus. The limited administration volume possible (25-200 μL) and potential toxicity to the nasal epithelium also need to be taken into account.

Effective evaluation of nasal drug delivery systems requires a reliable in vitro or

in vivo model. In vivo animal models and ex vivo excised human or animal tissue models are currently used as standards to study nasal drug delivery due to methodological and ethical limitations associated with human samples 9.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 37

However, these models have several limitations in terms of species differentiation between animals and humans as well as ethical and regulatory consideration associated with experimental animal use. Consequently, research has been conducted to develop and validate human nasal epithelial cell culture systems that could accurately represent this barrier system to serve as an alternative to in vivo models. Recent studies on primary human nasal epithelium cell cultures have provided a valuable in vitro model for the study of key functions of the respiratory epithelium (Table 1). Respiratory epithelial cell lines, such as Calu-3 and RMPI 2650, are established for intranasal drug delivery investigations, reflecting their ease of culture, genetic homogeneity, highly reproducible results and unlimited lifespan. However, these models have different features; specifically they do not ciliate and differentiate appropriately compared to primary cultures 10. Primary nasal cells have the advantage of producing epithelial layers with heterogeneous cells reflecting the types found in normal nasal epithelium and multiple samples can reflect the genetic diversity of the subjects 11.

Furthermore, it has been suggested that nasal epithelial cells could be used as a substitute or surrogate to bronchial epithelial cells and since they have identical morphologies with similar expression of receptors and responses to cytokine stimulation 12. Although culturing epithelial cells from the human tracheobronchial region has been well documented, it is limited to the more invasive and complex bronchoscopy or availability of lung explants, while nasal epithelial cells can be easily obtained through less invasive superficial nasal


Page 5 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


brushes 11b. With this method, subjects can be brushed multiple times with no significant side effects 11a, 13. This also allows for direct comparisons of epithelium obtained from healthy and diseased populations, enabling characterisation of disease phenotypes and associated mechanisms in their epithelial cell function 11. Subsequently, novel nasal and inhaled therapeutics could be formulated to tailor for specific diseases and tested for its effectiveness on physiologically relevant conditions.

While the use of primary cell cultures to explore the potential for nasal drug delivery has previously been reported, there are inconsistencies between the studies regarding sampling technique, culture conditions, morphology and permeability of primary nasal epithelium (summarised in Table 1). The primary air liquid interface nasal epithelial cell model described in this study has not previously been characterised for drug transport and ciliary activity simultaneously, following drug administration. Important properties of cultured nasal epithelium include the formation of confluent cell layers with interconnecting tight junctions, formation of functional cilia, mucus secretion and a heterogeneous population of cells. Functional cilia on the nasal epithelial culture play an important role in physiological functions, such as mucociliary clearance, which is important to both nasal and pulmonary drug delivery 2.

Consequently, the aim of the study was to systematically determine the fundamental parameters for a nasal epithelial cell model to be used as a tool for the study of inflammatory responses, drug transport, and quantification of drug



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 37

effects on ciliary activity. The model was then validated using tobramycin and mannitol as model drugs; tobramycin for its potential as a topical antimicrobial drug in the treatment of bacterial rhinosinusitis 14 versus mannitol a positive control respectively 15.


Page 7 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


Study Ref.

Culture condition

Sampling technique

2

ALI LCC

Turbinectomy

3

LCC

16

Characterisation Mucus Morphology

TEER max (Ω cm2) 3453 (ALI) 1936(LCC)

Transport studies Mannitol Budesonide

Rt-PCR of mucin genes

Turbinectomy

3133 (P2) 2703 (P3) 1235 (P4)

Mannitol Budesonide

Mucin granules observed in TEM

LCC

Surgical biopsies

665

17

LCC

Surgical biopsies

1200

11b

ALI

Nasal brushing

-

Thyrothropinreleasing hormone Suforhodamine FIT-C dextran Atenolol Propranolol Talinolol Lidocaine Diazepam Fexofenadine Triprolidine -

-

-

Mucus stained with alcian blue on apical

Development of cilia in ALI with flattened cilia and microvilli observed in LCC under SEM Actin staining and TEM used to characterised tight junctions. Observations of microvilli on cell monolayer using SEM. Dome formation and actin staining to confirm tight junction formation. Observations of microvilli on cell monolayer using SEM TEM demonstrating monolayer of nasal epithelium with ciliated and non-ciliated cells.

Paraffin-embedded pseudostratified epithelium showed cuboidal structure,

Functional cilia -

-

Cilia beating observed under light microscope.

Cilia beat frequency measured in response to drugs used for transport studies. -

7

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

surface.

18

ALI

Surgical biopsies

650

Na-Flu

Present study

ALI

Nasal brushing

811

Na-Flu Tobramycin

mucus producing goblet cells and ciliated epithelial cells observed under light microscope. SEM showed epithelial differentiation into columnar/cuboidal ciliated and non-ciliated cells SEM showing SEM showed heterogeneous mucus secretion. cell population with mucus Mucus staining production, cilia and development and clear cellquantification on to-cell boundaries. apical surface of Expression of tight junction the epithelium and adherens junction using alcian blue proteins stained using immunochemistry.

Page 8 of 37

Cilia beat frequency decreased over time in culture. Simultaneous characterisation of cilia beat pattern and frequency in response to tobramycin and mannitol.

ALI: air liquid interface, LCC: Liquid covered culture, Na-Flu: sodium fluorescein, P: Passage, Ref: references, SEM: Scanning electron microscope, TEM: Transmission electron microscope

Table 1: Summary of previous studies characterising primary nasal epithelia cell in terms of transepithelial electrical resistance (TEER), permeability studies, mucus production, morphology, cilia formation and function.

8

ACS Paragon Plus Environment

Page 9 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.0

Materials and methods

Local and national Research & Development and ethical approvals were obtained (Southampton and South West Hampshire Research Ethics Committee A CHI395, 07/Q1702/109). All subjects gave written consent for sampling and for use of their data.

2.1

Cell culture: Sampling, expansion and air-liquid interface cell culture

Nasal cells were obtained from healthy volunteers between the ages of 25 to 50, through nasal brushing biopsies and cultured according to methodologies as previously described.11 Up to four brushings were only performed on volunteers who were free of an upper respiratory tract infection for at least 4 weeks prior to sampling and did not have a diagnosis of respiratory disease. Briefly, a cytology brush (Olympus Keymed Ltd, 2 mm diameter, Southend, UK) was inserted into the patient’s naris and the inferior turbinate and the nasal epithelium were gently brushed yielding strips of ciliated nasal epithelium with each brushing containing up to 10,000 cells.

Prior to cell seeding, the plates, flasks and transwell inserts were coated with 0.5 mL, 1 mL and 0.1 mL of 0.3 mg/mL of bovine collagen (Nutacon, Netherlands) for 1 hour, respectively, and were air-dried. The strips of ciliated nasal epithelium were grown on collagen-coated tissue culture 12 well plates in bronchial epithelial growth medium (BEGM) (BEBM supplemented with SingleQuots, Lonza, USA) for approximately one week. The confluent unciliated basal cells



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 37

were then expanded onto collagen coated 25 cm2 flask with the BEGM replaced every 2-3 days. When cultures reached 70-80 % confluency, the basal cells were detached with 0.25 % trypsin-EDTA (Gibco Invitrogen, USA) and seeded (200,000-250,000 cells per well) onto collagen coated Transwell® (0.33 cm2) (Costar, Corning, USA). At this stage, the cells were fed with air liquid interface (ALI) medium made up of 1:1 BEGM:DMEM 4.5 g/L D-glucose including SingleQuots) until confluent. Subsequently, the air-liquid interface conditions were initiated 2 days post-seeding, with ALI medium supplemented with additional 100 nM all-trans retinoic acid (Sigma-Aldrich, UK) placed only in the basolateral chamber to allow for cell differentiation.2-3, 11a The medium was replaced three times a week and any apical surface liquid was removed.

2.2 Measurement of epithelial tight junction formation Transepithelial electrical resistance (TEER) was measured weekly using an epithelial voltohmmeter (EVOM, World Precision Instruments, USA) attached to STX-2 chopstick electrodes and corrected by subtracting the blank inserts and multiplied by the area of the transwell inserts 19. Prior to TEER measurements, 0.2 mL of medium was added to the apical chamber and allowed to equilibrate for 30 minutes.

Concurrently, epithelial permeability studies using fluorescein sodium (flu-Na, Sigma Aldrich, UK), a marker for paracellular permeability, were performed to evaluate barrier formation and tight junction functionality 19. Briefly, 250 μL of 2.5 mg/mL flu-Na solution were added on the apical chamber while 600 uL of 10 ACS Paragon Plus Environment

Page 11 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hanks Balance Salt Solution (HBSS, Gibco Invitrogen, USA) were added into the basolateral chamber. At pre-determined time points up to 4 hours, 100 μL samples were taken from the basolateral chamber and subsequently replaced with fresh buffer to maintain sink conditions. Samples were placed in a black, 96 well plate and fluorescence reading was subsequently measured using POLARstar Optima fluorescence plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission wavelengths settings of 485 and 520 nm respectively. The apparent permeability coefficient (Papp) of flu-Na across the nasal epithelial cells was calculated using the following equation: Papp = [V/(AC0)](dC/dt) where V is the volume of solution in the basolateral chamber, A is the surface area of the Transwell membrane (cm2), C0 is the initial concentration in the apical compartment (µg/mL) and dC/dt is the flux (rate of change in cumulative mass transport) of flu-Na.

2.3 Visualisation of tight junction The presence of tight junctions was visualised by immunolabelling of tight junction proteins and SP5 laser scanning confocal microscopy. The nasal epithelial cells were washed with PBS and fixed using 3 % (v/v) paraformaldehyde in PBS. Cells were then washed with 10 mL of PBS and exposed to 1 %(v/v) Triton X 100 with 1 %(w/v) bovine serum albumin (BSA) in PBS for 60 minutes. The cells were washed again with PBS and then incubated with 50 mM ammonium acetate in PBS for 10 minutes. After further washing with PBS, cells were incubated at 37 oC with either mouse anti-ZO-1 (Zymed, Molecular Probes, Cambridge Biosciences, UK) (2 μg/mL in 1 % w/v BSA and 0.1



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 37

% v/v Triton X100 in PBS) or mouse anti-human E-cadherin (Takara, Japan) (10 μg/mL in 1 % w/v BSA and 0.1 % v/v Triton X100 in PBS) for 60 minutes. A further washing with 10mL PBS was performed and AlexaFlour 594 goat antimouse IgG (10 μg/mL in PBS) (Molecular Probes, Cambridge Biosciences, UK) was incubated for a further 60 minutes. After washing, the cells were counter stained with 1 µg/mL 4’-6-diamidino-2-phenylindone (DAPI) in water for 10 minutes. The epithelial cells on the Transwell membrane were cut from the plastic support and mounted on a microscope slide. Slides were stored at 4 °C prior to analysis. The cell layer was viewed using a Leica SP5 Laser Scanning Confocal Microscope (Leica Microsystem, Wetzlar, Germany).

2.4 Mucus production The mucus production from the nasal epithelial cells were characterised weekly by staining the glycoproteins present in the mucus secretions with alcian blue as previously described 20 to allow visualisation of mucus on the surface of the cells. Briefly, samples were washed with PBS and fixed with 4 % v/v paraformaldehyde (PFA) for 20 minutes. Then, cells were then rinsed twice 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 left for 15 min. The inserts were washed with PBS to remove extra stain and left to dry. Finally, cells were mounted onto glass slides with mounting medium and sealed. Samples were refrigerated at 4 °C prior to analysis. Images of each sample were taken using Olympus BX61 microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP71 camera. Data was processed to produce a semi- quantitative measure of mucus


Page 13 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


concentration based on the ratio of red, green, blue from mucus staining images as reported previously 21. Briefly, Apple Automator (v 2.0.4 Apple Inc., Cupertino, California, USA) was used to obtain TIFF images for analysis using ImageJ (v1.42q, NIH )22 with Colour Profile (Dimiter Prodanov; Leiden University Medical Center, Leiden, Netherlands). The 8-bit red, green, blue (RGB) value of each image was subsequently represented in a 3-dimensional colour space and the values generated used to quantify the intensity of the blue stained mucus. The ratio of red, green, blue (RGB ratio - RGBB) was calculated by dividing the mean RGB by the sum of the RGB values for each image (RGBR + RGBG + RGBB). The mean RGBB was used to quantify mucus production over the culture period.

2.5 Measurement of inflammatory responses The ability for the primary air liquid interface nasal cell model to produce inflammatory cytokines specifically interleukin (IL) -6, -8 and tumour necrosis factor-α (TNF-α) were evaluated after induction with lipopolysaccharide (LPS) from Pseudomonas aeruginosa (Sigma-Aldrich, Australia). Prior to LPS stimulation, medium was collected for analysis of basal cytokine concentrations. Then, LPS was added to the basaloteral chamber at concentrations of 1, 10 and 50 ug/mL to stimulate the nasal model and were incubated at 37°C, 5% CO2 for 24h to allow for the production of inflammatory cytokines. Samples of the culture medium were analysed for IL-6, IL-8 and TNF-α using an enzyme-linked immunosrobent assay (ELISA) Kit (BD OptEIA™, catalogue number 265KI, 2645KI and 2637KI, BD Biosciences, San Diego, California, USA) according to the manufacturer’s protocol. Experiments were performed following differentiation



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 37

of the nasal epithelium at approximately 6-7 weeks in culture. Stimulated cell samples were compared with basal concentrations from the same subjects.

2.6 Scanning electron microscopy (SEM) Scanning electron microscopy was performed to characterise the morphology of the epithelial cells grown on permeable supports at weeks 4, 5 and 7. The cell layers were washed three times with phosphate buffered saline solution (PBS) and fixed with 4 % v/v paraformaldehyde and 3 % v/v glutaraldehyde in 0.1 M piperazine-N,N’-bis(ethanesulfonic acid) buffer (PIPES buffer, Sigma, UK). The cells were stored at 4 °C for up to 4 weeks. Cells were then incubated in 1 % v/v osmium tetroxide in PIPES buffer for 1 hour and subsequently dehydrated in through serial ethanol solution (30, 50, 70, 95, 100 % v/v). The epithelial cells on the transwell membrane were cut from the permeable support before transferring to a critical point dryer (Balzers CPD 030, Leica Microsystem, Wetzlar, Germany). Samples are then mounted onto aluminium stubs with adhesive carbon tape and coated with gold and palladium in a sputter coater (Polaron E5100, Quarum Technologies, Sussex, UK). Finally, the samples were then examined under vacuum conditions by the scanning electron microscope (Quanta 200, FEI, USA) at an accelerating voltage of 10 kV and 6000X magnification.

2.7 High speed video analysis of ciliary function Prior to cell imaging, 200 μL of BEBM medium was added to both the apical and basolateral chamber of the transwell and cells equilibrated for 30 minutes. The 14 ACS Paragon Plus Environment

Page 15 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ciliated nasal epithelial cells at week 7 on the transwell insert were imaged with a x60 objective of a light microscope (Olympus IX71 inverted microscope) within an environmental controlled chamber heated to 37 °C (Solent Scientific, UK). Ciliary activity was recorded using a digital high-speed camera (Photron FASTCAM MC2) at a rate of 250 frames per second (fps) at times 0, 1, 2 and 24 hours. Analysis of cilia beat frequency (CBF) was quantified using a fast Fourier transform (FFT) algorithm (Image J plugin, P. Lackie, Southampton, UK) to measure the mean CBF in areas of cilia beating on the epithelium 23. A minimum of six measurements of various ciliated areas on the nasal epithelium was quantified. Cilia beat pattern (CBP) on the other hand was observed using an x100 objective at 24 hours where the transwell membrane was cut from the plastic insert and mounted onto a glass slide with 0.5 mm CoverWell imaging chamber (Sigma Aldrich, UK). Qualitative analysis of the cilia movement was performed by replaying the video at 30-60 frames per second. This provides information such as synchronisation and radial beat motion that is observed as radial forward power stroke of the cilia followed by a slow, slightly sideways and finally a backward recovery stroke.

To investigate the effects of drugs on cilia function, solutions of 200 μL tobramycin (Sigma-Aldrich, UK) and mannitol (Sigma-Aldrich, UK) (0.01 and 1 mM) were prepared in bronchial epithelial basal medium (BEBM, Lonza,USA) and introduced onto the apical chamber. The ciliary activity was recorded and quantified as previously described at 0, 1, 2 and 24 hours. Any changes to the CBP were assessed at the end of the experiments and flu-Na permeability was



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 37

also performed for 1 hour to measure the barrier’s integrity of the nasal epithelial cell after the experiments.

2.8 Drug Testing Simultaneously, transport studies of 0.01 and 1 mM tobramycin across the nasal epithelial cells were performed. Samples from the basolateral chamber were taken every 30 minutes for 3 hours and finally at 24 hours and was replaced with fresh medium. The concentrations of tobramycin in the samples were analysed using a validated method by high performance liquid chromatography (HPLC). Prior to HPLC, samples were subjected to tobramycin chemical derivatisation due to low UV absorptivity and detection sensitivity of tobramycin. For derivatisation, 50 μL of each sample were transferred into a HPLC vial and 125 μL of 2,4 dinitrofluorobenzene solution (10 mg/mL in absolute ethanol and 125 μL of TRIS solution (15 mg/mL Tris (hydroxymethyl) aminomethane stock solution in water diluted to 20 % v/v in dimethyl sulfoxide) were added. After shaking, the vials were placed in a heated water bath at 60 oC for 50 minutes. The vials were then removed and allowed to cool to room temperature for 10 – 15 minutes. Subsequently, 325 μL of acetonitrile were added to the vials and mixed again. Finally, the derivatised samples of tobramycin-(2,4-dinitrophenyl)524 were analysed using a Shimadzu Prominence UFLC system equipped with the SPD-20A UV-Vis detector, LC-20AT liquid chromatograph and SIL-20A HT Autosampler (Shimadzu Corporation, Japan) and Waters Resolve reverse phase C18 column (5 µm, 150 X 3.9 mm). The mobile phase is a mixture of acetonitile and 2.5 g/L Tris (hydroxymethyl) aminomethane at a ratio of 20:80 (v/v) with


Page 17 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20 mL of 1 N sulphuric acid. HPLC was set according to the following conditions: detection wavelength 365 nm, flow rate of 1.0 mL/min and an injection volume of 100 µL. Linearity was obtained between 0.1 and 100 ug/mL (R2=0.99) with a retention time of 4.1 minutes.

2.9 Data analysis All results are expressed as mean ± standard deviation of at least 4 separate determinants. To determine significance between groups and control, unpaired 2-tailed t-tests were performed (quoted at the level of p20% of the nasal epithelium was observed. Figure 6A shows the frequency map of an area of differentiated nasal epithelial cells that displayed different patches of ciliated cells with different frequencies of cilia beating. The mean CBF measured in 6 areas of 7 different cultures was 11.58 ± 1.52 Hz and there was no significant difference in the mean and range of the CBF in each culture up to 24 hours (Figure 6B). This was within the normal range of 8.8 -14.09 Hz for epithelium from healthy individuals 23. The cilia beat pattern was also observed to be normal with coordinated and synchronised cilia movements (see supplementary information).


Page 27 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6: (A) A typical frequency map of baseline cilia beat frequency (CBF) on differentiated nasal epithelium after 5 weeks in culture (grey) with a magnification of 60X and (B) mean CBF of nasal epithelial over 24 hours incubation with BEBM medium (n≥6, mean ± S.D.)

To further validate this model as a biological platform to test potential nasal formulations, tobramycin and mannitol (positive control) were used as model drugs. Additionally, future studies will focus on correlating the ciliary activity in response to these drugs to in vivo airway mucociliary clearance. Analyses of the nasal CBF and frequency maps in response the different concentrations (0.01 and 1 mM) of both drugs are shown in Figure 7. The dose selection was based on



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 37

preliminary calculations of the minimum inhibitory concentration of tobramycin towards Pseudomonas aeruginosa pathogen, as well as the relevant doses used in recent clinical trial14a and in vivo animal studies37 of tobramycin nasal spray adjusted to the surface area of the transwell. No statistical difference was found with tobramycin at the different concentrations over the time scale studied. This was in agreement with a previous study investigating the effectiveness of a range of antibiotic classes including macrolides, aminoglycosides and polypeptide antibiotic on nasal epithelial cells exposed to bacteria toxins 38. The cilio-toxic effects of bacteria toxins are known to contribute to the pathogenesis of nasal infections. Although the antibiotics did not significantly influence the ciliary activity of normal epithelium, they were able to restore the CBF completely by treating the underlying bacterial infection.

As for mannitol, the ciliary activity was found to be concentration dependent with no significant difference at the lower concentration of 0.01 mM. On the other hand, the higher concentration of mannitol (1 mM) was shown to significantly increase CBF compared to baseline after 1 hour of treatment and returned to baseline levels after 24 hours. The changes in CBF was reflected in the frequency maps of the epithelium (Figure 7B and D), where the change in colour intensity from blue-green to orange-red in response to 1 mM mannitol was observed, indicating an enhancement of ciliary activity. The return to baseline CBF could be due to the absorption of mannitol across the epithelium into the basolateral chamber through the paracellular route reducing the apical concentrations over time. The increase in CBF in response to hyperosmolar stimulation causes further dilution of the mucus layer on the epithelial cells 28 ACS Paragon Plus Environment

Page 29 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


enhancing the CBF, which was in good agreement to previous findings 15, 39. However, one might hypothesise that the changes in CBF would be more significant in vivo in response to hyperosmolar agents delivered by nasal spray solutions or dry powder formulations. The non-homogenous deposition of the aerosols onto the epithelium will create a high localised concentration that would induce a larger transepithelial water flux from the cells and subsequently lead to an increase in the airway surface liquid and diluting the mucus layer 19. Further studies to establish a deposition method onto the air liquid interface nasal epithelial cell model using a pharmacopoeia method for nasal drug delivery are currently in progress.

Figure 7: Effects of different concentrations (0.01 and 1 mM) of tobramycin and mannitol on cilia beat frequency (CBF) on epithelial cells after 5 weeks in culture at different time points compared to control. Typical frequency maps at 1-hour 29 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 37

post-treatment of (B) control, (C) 1 mM tobramycin and (D) 1 mM mannitol. *Significantly different to baseline CBF values, p

Primary AirâLiquid Interface Culture of Nasal ... - ACS Publications

Recommend Documents

Primary AirâLiquid Interface Culture of Nasal ... - ACS Publications