James Smith Lab

Our current laboratory research

Mapping surface heterogeneity using the AFM

For a number of years, we have been interested in using the AFM to chart maps showing local variations in physical properties, such as adhesion, stiffness (Young's Modulus) and friction. These can be obtained by pulling the cantilever away from or pushing it into a surface or by monitoring the cantilever's twisting motion during scanning. The related forces are quantified using suitable calibration methods, and software written by ourselves. This approach is an alternative use of the AFM, which is typically used for high-resolution imaging. We have used such mapping methods to investigate adhesion properties of polymer blends and low-surface-energy (non-stick) polymers, filler inclusions in polymer composites, the effects of magnetic fields and biocides on bacterial biofilms and frictional variations in surface substructures of the human hair cuticle. We are further developing mapping methdods to investigate other properties of biomedical interest.

Drug delivery systems for boron neutron-capture therapy (BNCT)

The PhD research work, carried out by Temidayo (Temmy) Olusanya supervised by Dr John Tsibouklis and Dr James Smith, is aimed towards the development of active anti-tumour agents for use in Boron Neutron-Capture Therapy (BNCT). This technique works by irradiation with neutrons where 10B, located in close-proximity to cancer cells, is converted to 7Li + (-particles that locally target cancer cells.

Two types of delivery systems, both incorporating the boron-containing agent ortho-carborane, are being investigated:

Liposomes for delivery across the blood-brain-barrier to target brain tumours (due to their high cell membrane potential)
Spray-dried particles to target liver/kidney cancers.

Spray drying produces micrometre-sized particles from a feed solution. Co-spray drying of insoluble o-carborane with polyvinylpyrrolidone (PVP) is being developed as a one-step method of enhancing the bioavailability of o-carborane. We have successfully incorporated o-carboranes into liposomes and produced PVP microparticle formulations for potential use as BNCT agents. Future studies will focus of further their characterisation and in vitro and in vivo performance.

AFM nanoindentation studies of cancer cells

In a collaborative project with the neurooncology group, we have used AFM to compare the elastic properties (Young’s Modulus) of cells from glioblastoma multiforme, an aggressive glioma, to suitable control cells, under physiological conditions. Such measurements can be obtained by indenting the AFM cantilever into cell surfaces and using Hertzian mechanics to obtain Young’s Modulus. The cell cytoskeleton, consisting of F-actin, intermediate filaments and microtubules, is essential for cell division, movement and intracellular transport. This architecture is often impaired in cancer cells, resulting in a reduction in Young’s Modulus, which is thought to play a key role in invasion and metastasis. It has also been suggested that an assessment of elastic properties may be of clinical diagnostic value in addition to providing mechanistic insights. For example, an increase in metastatic efficiency has been linked with further reductions in mechanical properties. Our nanoindentation studies are also investigating the effects of silencing certain genes in glioma cells on Young’s Modulus.

AFM characterisation of drug delivery systems for nanomedicine

Nanoparticles and carbon nanotubes (single-walled and multi-walled) are proving to be useful materials as drug delivery vehicles – the field of ‘nanomedicine’. In the case of nanoparticles, their small size permits passage across the blood-brain-barrier, a natural barrier that prevents potentially useful therapeutic drugs from entering the brain. Carbon nanotubes, although of only a few nanometres in thickness, may be many micrometres long. Their hydrophobic nature can be modified through covalent and non-covalent attachment of various molecules, such as surfactants and polymers that aid their dispersion in aqueous systems. Dispersions of carbon nanotubes stabilised with chitosan (a natural polysaccharide) derivates could potentially be used as a drug delivery system in gene therapy. We have used AFM to characterise these materials and have measured thicknesses of coatings on carbon nanotubes. Different aspects of work are carried out in collaboration with Drs Tsibouklis, Barbu and Roldo, and with Dr Dimitris Fatouros from Aristotle University of Thessaloniki, Greece.

AFM studies of amyloids in age-related disease states

Serum Amyloid P component (SAP) is a 125 kDa, pentameric, highly conserved, human serum glyco-protein that circulates in all individuals at a concentration of 20-30 mg/l. Along with its presence in serum, SAP also makes up 14% w/w of in vivo amyloid deposits found in diseases, such as the systemic amyloidosis, Creutzfeldt Jakob disease, Alzheimer’s and type II diabetes. Small molecules that deplete SAP are currently undergoing clinical trials for the treatment of a variety of amyloid related disorders. Despite the considerable progress in elucidating SAP’s pathological role, understanding the normal role of this protein is much less advanced. Binding interactions with other molecules have been detected that imply important functions consistent with its evolutionary conservation, but the details of these interactions are far from clear. For example SAP is the only plasma protein bound when whole plasma is passed over an immobilised DNA column. SAP also binds avidly to nucleosomes and long chromatin, which it solubilises by displacing H1-type histones. SAP and histone H1 both recognise the same linker DNA regions on long chromatin despite substantial differences in their tertiary and quaternary structure.

In a collaborative project with Drs Simon Kolstoe and Darren Gowers, we are seeking to understand how SAP recognises and interacts with large macromolecular structures, such as DNA and amyloid fibres; this will be of value in SAP depletion therapy. Investigations are being carried out using AFM imaging, in conjunction with electrophoretic mobility shift assays (EMSAs), to determine binding constants, and X-ray crystallography and related biophysical techniques to elucidate molecular structure and thermodynamics.