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Fowler, R.C. (2013) Crossing mucosal barriers for non-invasive protein delivery: a vitamin B12-mediated approach. PhD thesis, University of Nottingham.

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Abstract

Mucosal delivery of biotherapeutics as a non-invasive means of delivery could potentially be enhanced using nanoscale therapeutic carriers. However, nanoparticles do not readily cross the mucosal barriers, with the epithelium severely restricting their translocation into the systemic circulation. Translocation of nanocarriers across the mucosae may be improved by employing ligands capable of exploiting receptor-mediated cell uptake processes.

This work explores the potential of vitamin B12 transport pathway for mucosal delivery of B12-decorated model nanoparticles and investigates the cell trafficking pathways involved in these processes. Cyanocobalamin (vitamin B12) was chemically modified to produce the α-ω-aminohexylcarbamate B12 derivative – as a suitable bioconjugate – which was then conjugated to fluorescent, carboxy-functional nanoparticles (<200 nm). These systems were applied to intestinal Caco-2 monolayers, expressing the relevant proteins involved in B12 trafficking and endocytic processes.

Vitamin B12-conjugated nanoparticles demonstrated notably increased cell uptake and transport capacities in Caco-2 monolayers, compared to their unconjugated counterparts. Importantly, the cell uptake of B12-conjugated nanoparticles occurred via a pathway that was different to that used by both soluble B12 and unmodified nanoparticles. B12-conjugated nanoparticles circumnavigated the lysosomal compartment and were transported by a route perturbed by caveolae-specific inhibitors, unlike the clathrin-mediated trafficking of soluble vitamin B12. These previously unreported observations are important and have potential implications in the field of bioconjugate and nanocarrier-mediated drug delivery.

Epithelial cell uptake and transport of B12-conjugated nanoparticles was also investigated in airway-derived Calu-3 cells, shown to express the B12-intrinsic factor receptor, cubilin. B12-nanoparticles showed markedly larger cell uptake and transport capacities in Calu-3 layers, with B12-conjugation dramatically influencing the intracellular trafficking of the particles in a similar way to Caco-2 cells. The B12 endocytotic machinery therefore shows potential for delivery of nanocarrier-associated therapeutics across the airways.

Present work also aimed to establish methods for the production of stable nano-sized protein crystals displaying a slow drug release profile, based on evidence that protein therapeutics which are formulated in this manner, offer beneficial drug-delivery properties and can be targeted using biological ligands. Nano- and micron-sized insulin crystals were prepared by an adaptation of the batch crystallisation approach. The crystals were stabilised using a chemical crosslinker, namely β-[Tris(hydroxymethyl) phosphino] propionic acid (THPP). The resulting insulin crystals were generally stable in the absence of crystalisation buffer, displayed a slow-release profile, with the released insulin retaining its biological activity. This study therefore shows that formulating protein bioactives in this form is possible and may provide a promising strategy to develop biotherapeutics with improved drug delivery properties.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Stolnik-Trenkic, S. Alexander, C. Garnett, M.C.
Subjects:	R Medicine > RM Therapeutics. Pharmacology R Medicine > RS Pharmacy and materia medica
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	13029
Depositing User:	EP, Services
Date Deposited:	14 Nov 2013 10:43
Last Modified:	15 Dec 2017 15:45
URI:	https://eprints.nottingham.ac.uk/id/eprint/13029

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