Non-covalent polymer and drug complexes as targeted delivery system for pulmonary delivery

Pulmonary delivery is increasingly seen as an attractive, non invasive route for the delivery of forthcoming protein therapeutics. However, the lung presents specific barriers for macromolecular/protein therapeutics. Susceptibility to enzymatic degradation, poor absorption across epithelial barrier, as well as lung induced immunogenicity are significant challenges to be addressed if this approach is to be adopted as a viable alternative for protein delivery. In this context, nanomedicine and, in particular, polymer therapeutics (PT) is emerging as a powerful alternative strategy to overcome these limitations. To this aim, in the present PhD project we utilised non-covalent polymer-protein nanocomplexes to allow reversible complexations of protein formulation and investigate their potential to protect and release them at the target site following pulmonary administration. It has been recognised that nanocarriers conjugated with specific ligands can increase the interaction with epithelial cells which makes ligand targeted delivery a very attractive area for in-depth research. In Chapter 3, we describe protein complexes with a new ‘complexing excipient’ - vitamin B12-targeted poly(ethylene glycol)-block poly(glutamic acid) (PEG-b-pGA) copolymer. Native B12 was first activated at the 5’-hydroxyl group of the ribose moiety using 1,1’ carbonyldiimidazole (CDI) followed by the addition of α II dibenzocyclooctyne-amine giving α-dibenzocyclooctyne-vitamin B12. The product was precipitated, purified by preparative TLC and characterised by mass spectrometry ready to react with azide terminated linear PEG-b-poly(glutamic acid) via copper-free azide alkyne cycloaddition to afford the desired targeted B12-PEG-b-pGA copolymers. The synthesis of azide-terminated linear PEG-b-pGA started from a commercially available azide-PEG-amine 3500 precursor acting as a macroinitiator for NCA polymerisation. Following click reaction, a purification step by dialysis yielded the desired B12-PEG3k-b-pGA copolymer. These form complexes in sub 200 nm size with a model protein interacting via ionic interaction, suitable for cellular targeting and intracellular delivery. Initially we confirmed expression of vitamin B12-internalisation receptor (CD320) by Calu-3 cells of the in vitro lung epithelial model used and demonstrated enhanced B12 receptor-mediated cellular internalisation of B12-targeted complexes, relative to non-targeted counterparts or protein alone. The nanocomplexes were spray-dried into inhalable particles with aerodynamic diameter within the suitable range (