Vllasaliu, Driton (2010) In vitro investigation into strategies for mucosal delivery of proteins. PhD thesis, University of Nottingham.
Mucosal surfaces offer a potential for non-invasive delivery of proteins. The role of these surfaces, however, is to limit the movement of material from the external environment (mucosal lumen) into systemic circulation. Mucosal absorption of protein therapeutics is constrained through several physiological barriers such as mucus and mucociliary clearance, protease enzymes, epithelial tight junctions (TJs) and cellular membranes. This work explores different strategies with the view to improving the transport of macromolecules (proteins and protein drug models) across polarised epithelial cell layers in vitro, which could potentially be a reflection of improved mucosal absorption and bioavailability in vivo. The Calu-3 cell line was used in this work to produce such layers, serving as an in vitro model of the airway epithelium. Following growth of Calu-3 cells on filters under air-interface culture conditions polarised layers of closely packed cells were formed. The cell layers exhibited a TEER ≥500 Ωcm2 and cells showed structural features similar to the native epithelium, including the TJs, the microvilli and the secretory granules. Cell layers presented a barrier to the permeability of FITC-dextrans (FDs, paracellular markers) and nanoparticles (NPs).
The first class of tested compounds, namely alkylglycoside (AG) surfactants, exhibited severe toxicity at concentrations considerably lower than those used in the literature. Data indicated that the cellular toxicity of AGs possibly results from a membrane effect. Investigation of calcium depletion as a proposed strategy to improve mucosal absorption of protein therapeutics, revealed that calcium depletion on the apical side produced limited TJ opening, as demonstrated by reversible decrease in transepithelial electrical resistance (TEER) and modest enhancement of permeability of macromolecules. Although combined apical and basolateral calcium exhaustion produced significant effects on TJs, this scenario becomes irrelevant in an in vivo situation. Application of chitosan in the form of solution and NPs to Calu-3 layers demonstrated that chitosan NPs formulated by the ionic gelation method, exhibit a similar TJ-opening effect to solution. This was shown by similarities in measurable indicators of TJ opening such as reduction in TEER and enhancement of dextran permeability across the cell layers. Furthermore, chitosan NP and solution exhibited similar effects on the TJ protein, Zonula Occludens-1. These results therefore indicated that chitosan NPs could potentially be used to carry and protect fragile therapeutic proteins to the mucosal surface(s) of interest and at the same time promote their absorption through TJ opening.
TJ opening as a strategy to improve mucosal absorption of macromolecular therapeutics is rather inefficient for larger proteins such as antibodies, or for nano- sized drug carriers following their mucosal administration; this led to investigation of the IgG/neonatal Fc receptor (FcRn) transcytotic pathway. Immunostaining data demonstrated that Calu-3 cells express FcRn. IgG was shown to traverse the Calu-3 layers and studies characterizing this transport indicated FcRn involvement in this process. Confocal microscopy revealed that IgG- or Fc-adsorbed NPs were taken up by Calu-3 cells. Adsorption of Fc on the surface of NPs was seen to promote their cellular uptake and transport across the cell layers. Characterisation of cell uptake and transport of Fc-NPs revealed data that strongly suggested FcRn involvement in these processes.
|Item Type:||Thesis (PhD)|
|Faculties/Schools:||UK Campuses > Faculty of Science > School of Pharmacy|
|Deposited By:||Dr Driton Vllasaliu|
|Deposited On:||24 Aug 2010 13:49|
|Last Modified:||24 Aug 2010 13:49|
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