Alison Lansley is a Principal Lecturer in Pharmaceutics at the University of Brighton (UoB). She obtained her degree in Pharmacy and PhD in mucociliary clearance from the UoB and then undertook post-doctoral positions at UCLA (cellular control of ciliary activity) and King’s College London (the use of airway cell lines as drug absorption models of the lung) where she then took up a lectureship. Following a career break, she was awarded a Daphne Jackson Fellowship which she undertook at the University of Sussex before accepting her current position. Her research interests include the use of in vivo-reflective in vitro models to study the effect of mucus on drug/particle deposition behaviour and absorption in the respiratory tract, factors affecting mucociliary clearance and inhalation toxicity.
She has previously led the MPharm programme at the UoB and is a Senior Fellow of the Higher Education Academy, a Fellow of the Academy of Pharmaceutical Sciences (APS) and a member of the APS Biopharmaceutics Focus Group and the Nasal Research Focus Group. She teaches biopharmaceutics and formulation science to undergraduate and postgraduate students and has supervised PhD students and postdoctoral researchers with funding from research councils, charities, and industry. She has published papers and book chapters on pulmonary and nasal drug delivery.
The use of ciliary activity to determine the local toxicity of inhaled pharmaceuticals
The airway epithelium is the first tissue to encounter inhaled chemicals, pollutants and applied pharmaceuticals, so in vitro airway epithelial models have been considered as potential replacements for regulatory acute inhalation toxicity tests. Regulatory assessment of acute inhalation toxicity relies on in vivo testing. However, there is increasing interest in developing new approach methodologies (NAMs) to identify toxic effects with a view to the eventual replacement of animal tests.
The lining of the airways has evolved to provide a barrier to the entry of external substances. This is contributed to by mucociliary clearance (MCC) which is the movement of secreted mucus by hair-like cilia which constantly beat beneath the mucous layer. Inhaled particles and droplets tend to deposit on the mucus and are removed from the airways in a conveyor belt-like manner. MCC is an advantage when removing noxious substances such as inhaled pollutants but can be undesirable when the retention of pharmaceuticals is required for local activity e.g., delivery via the nose and lung to treat allergic rhinitis, asthma or COPD or absorption e.g., the systemic delivery of poorly absorbed drugs such as desmopressin and other biologics. However, substances toxic to MCC are also problematic as they make the individual more prone to respiratory infections. Therefore, measurement of ciliary beat frequency (CBF) is a useful endpoint in assessing the local toxicity of inhaled pharmaceuticals.
In order to study CBF without using animal tissue, primary cultures from human bronchial epithelial cells are required. Organotypic 3-D in vitro airway epithelial models are available commercially e.g. Mucilair , EpiAirway that are well characterised regarding their pseudostratified structure, barrier properties, cilia beating, etc. However, these are prohibitively expensive for routine use in acadaemia. Therefore, primary cultures from human bronchial epithelial cells (commercially supplied) will be cultured in-house on transparent permeable supports (Transwell Clear) at an air-liquid interface to form ciliated epithelia. This is technically demanding and, while the applicant has initiated this, further work is required to obtain fully differentiated cultures expressing beating cilia. Without this element, further work cannot proceed.
Once fully differentiated cultures have been achieved, CBF will be measured in the absence and presence of various drugs/excipients (surfactants, lipids, preservatives)/pollutants (ultrafine particles of AgCl, Fe(OH)3, polystyrene), or after the cells have been cultured in the presence of certain pharmaceuticals/pollutants (as above).
Being able to measure CBF will complement a suite of other techniques such as the measurement of mucus secretion, mucociliary clearance, airway epithelial permeability, cytotoxicity and therefore provide a strong technical base for future grant applications related to inhaled drug delivery or air pollution
This proposal has an element of career development and an element of service development.
Career development
I do not consider myself to be a typical ‘career development’ applicant. I have been a successful researcher with a record of external funding and a reputation in the field of nasal and pulmonary drug delivery (PubMed search: “Lansley AB” and “Batts AH” Publications (Scopus): 39; H-Index score (Scopus): 17). I also co-write a book chapter on Intranasal Drug Delivery in one of the the leading pharmaceutics textbook used by industrialists and academics (Aulton’s Pharmaceutics. The Design and Manufacture of Medicines. 6th edition. Eds. Aulton, M.E. and Taylor, K. Churchill Livingstone Elsevier, London).
However, while undertaking significant teaching-focused administrative roles e.g., MPharm Course leader (600 students; ~40 staff) my research lost its momentum (three PhD students completed but there was insufficient time to apply for further funding and write all the papers). This was recognised by my employer, the University of Brighton, and in 2020-21 I was given a sabbatical from teaching to bring about a step change in my research.
During my sabbatical, I wrote four papers and recommissioned the ciliary beat frequency (CBF) -image analysis equipment that is to be used in the proposed project. The CBF-image analysis apparatus has previously been used to study the effect of endothelin and various nasal formulations on CBF (Pawsey et al. Pulm Pharmacol Ther 2011: 24: 602-9: Ayoub PhD Thesis 2015). It was updated in 2015 at a total cost of £12,205 (£8,510 for a high speed digital charged-coupled device (CCD) camera, image analysis software image analysis system and £3,695 for 37oC incubation chamber). As part of its recent re-commissioning, software has been written in-house for data acquisition and image analysis. Preliminary data has shown that the system works as expected.
Unfortunately, my sabbatical coincided with the COVID-19 pandemic, and I was unable to achieve all that I had hoped due to lockdown. In particular, I was unable to undertake training with a local company to learn how to culture human bronchial epithelial cells such that they will express beating cilia. The company has generously shared its standard operating procedures with me and offered support. Therefore, part of the DDL Career Development Grant will be spent on developing these organotypic 3-D in vitro airway epithelial models. Commercially obtained human bronchial epithelial cells (already purchased) will be cultured on Transwell Clear polyester inserts using Promocell medium. Both myself and a technician at the University of Brighton will work on gaining these skills which will then be taught to any research students/fellows that may subsequently use the CBF equipment.
Once beating cilia are expressed, the membrane bearing the cells will be mounted in a custom-built perfusion chamber. Ciliary activity is temperature-dependent, so the chamber will be placed on the temperature-controlled stage of the microscope and maintained within a temperature-controlled chamber at 37oC. The cilia will be observed using phase contrast microscopy at x40 magnification using a microscope mounted on an anti-vibration table while the tissue is perfused with warmed (37oC) medium at a controlled flow rate in the absence and presence of pharmaceuticals/pollutants, or after having been cultured in the presence of certain pharmaceuticals/pollutants (UFPs) (see project summary). Images of ciliary activity will be detected with a CCD camera (340 fps; resolution 2048 x 1088) and images will be recorded and analysed using the programs developed in-house.
Data obtained will be published and form the basis of future funding applications.
Collaboration
I am a member of an international, multidisciplinary consortium of researchers (both industrial e.g., Nemura, Proveris, Aptar and academic e.g., KCL, Universities of Kiel, Brussels, Parma). Our aim is to raise the awareness of nasal drug delivery and obtain funding for this type of research (Nasal Research Focus Group (NRFG)). My expertise in ciliary activity is unique to this group and having a means of measuring CBF would enhance our success in funding applications. Successful applications will permit the future development of research students and fellows in this area.
By attending national and international meetings such as DDL and RDD and presenting the project results, I would be able to enhance my research profile, and chances of collaboration. This would enable me to build wider and stronger networks with other academics and industrialists which would enhance future funding application. Part of the DDL Career Development Grant would be spent on networking.
Service Development
There are relatively few academic laboratories in the UK able to undertake studies of ciliary activity. In addition, there is increasing interest in developing new approach methodologies (NAMs) for acute inhalation toxicity testing of chemicals and pharmaceuticals (drugs, excipients, nanoparticles). Measurement of CBF, alongside a suite of other techniques such as the measurement of mucus secretion, barrier function e.g., transepithelial electrical resistance, cytotoxicity (MTT and/or LDH assays), cytokine release, etc., would provide a better understanding of the toxicological impact of inhaled substances.
I would be open to collaborations with academic and industrial partners.