AbstractMany drugs have complex chemical properties which limit solubility in common solvents like water. Alternative formulations such as polymeric micelles formed by self-assembly of amphiphilic macromolecules may alleviate problems associated with bioavailability of poorly water-soluble drugs. The mechanisms underlying uptake and trafficking of polymeric micelles and the extent to which they are transported across epithelia remain largely unknown. This thesis provides insight into the nature of micellar drug delivery by focussing on the interactions of poloxamer P407 micelles and a model cargo with kidney and placental epithelia.
Fluorescent (RITC) poloxamer P407 micelles encapsulating DiO as a model fluorescent cargo were synthesised and characterised. The resultant micelles had a hydrodynamic diameter of 12nm and a critical micelle temperature of 25°C that was influenced by the presence of DiO. Fluorimetric analysis confirmed FRET between DiO and RITC that could be used to monitor cargo loading and release.
The kinetics of uptake and subcellular distribution of fluorescent micelles in epithelial cell lines (dog kidney MDCK and human placental BeWo b30 cells) was examined by confocal microscopy. Immunofluorescence identified punctate structures accumulating fluorescent micelles as late endosomes/lysosomes. Inhibition of receptor-mediated endocytosis partially blocked micelle uptake supporting the role of endocytosis in the process but suggesting the possible operation of alternative uptake pathways.
MDCK and BeWo b30 cells were grown on permeable Transwell supports and their development into polarised epithelia with significant barrier function confirmed by microscopy and electrical resistance measurements. Fluorescent micelles were taken up by both polarised cell barrier models but in MDCKs the extent of endocytosis appeared reduced compared to non-polarised cells. A minor proportion of apically applied DiO-loaded micelles was transported across both cell barriers. BeWo cells were also cultured under flow to mimic physiological conditions. Preliminary data indicated flow affected morphological and functional differentiation and accelerated micelle uptake.
|Date of Award||28 Nov 2019|
|Supervisor||Mark A Jepson (Supervisor) & Margaret Saunders (Supervisor)|