Li, Huitong
(2021)
Non-covalent polymer and drug complexes as targeted delivery system for pulmonary delivery.
PhD thesis, University of Nottingham.
Abstract
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 (<5 µm) for lower airway deposition. in vivo lung tolerance studies of copolymer-protein complexes demonstrated that repeated administration of formulated dry powders over a 3 week period in healthy BALB/c mice induced no significant toxicity or indications of lung inflammation, as assessed by cell population count and quantification of IL-1β, IL-6, and TNF-α pro-inflammatory III markers. Importantly, the in vivo data appear to suggest that B12 targeted polymer complexes administered as dry powder enhance lung retention of their protein payload, relative to protein alone and non-targeted counterparts.
Next, to test the versatility of this formulation approach, we sought to investigate the inclusion of different targeting ligands detailed in Chapter 4 and 5. Studies have suggested a potential use of another essential vitamin – biotin – as an endocytosis ligand. Biotin receptor was found overexpressed in cancer cells, relatively more than folate or vitamin B12 receptors, and investigated as ligand for targeted delivery of nano-systems and prodrugs. Furthermore, this vitamin is a promoter of cell growth in many cell types which does not limit this delivery system only to target cancer cells. To this aim we synthesised biotin-terminal PEG2k-b-poly(glutamic acid) linear copolymers using commercially available biotin-PEG2k-amine suitable to act as a macroinitiator for N-carboxyanhydrides (NCA) ring opening polymerisation using the same strategy utilised previously for the B12 targeted B12-PEG-b-pGA polymer synthesis. These also form complexes in sub-200 nm size with a model protein lysozyme. Flow cytometry data demonstrated statistically significant increased cellular internalisation by biotin receptor positive A549 cells of biotin-PEG2k-b-GA10/30:lysozyme nanocomplexes relative to untargeted mPEG2k-b-GA30:lysozyme control complexes or ‘free’ Cy5-lysozyme. By fluorescently labelling lysozyme and tagging IV biotin targeted copolymers after cellular internalisation with Alexa Fluor™ 488 conjugated streptavidin, co-localisation analysis of the two fluorescently labelled polymer and protein using Pearson’s correlation analysis was performed on several confocal images. An average r value of ~ 0.5 was obtained, suggesting that at 1 h after the complexes were applied to, and internalised by A549 cells, lysozyme and biotin-PEG2k-b-GA10 copolymers were co-localised.
These promising preliminary results encouraged us for further in vitro investigation in understanding the cellular internalisation mechanism that might be responsible for targeted polymer-protein complex uptake. Therefore, work was carried out to probe the mechanisms of entry into the cells of targeted biotin-PEG2k-b pGA:lysozyme nanocomplex, and whether it significantly differs to free lysozyme or non-targeted mPEG2k-b-GA30:lysozyme nanocomplexes. In this respect, a panel of pharmacological inhibitors was used to investigate which endocytic mechanisms were responsible for cellular uptake of our targeted nanoparticles. Dynamin which plays a vital role in vesicular trafficking processes by facilitating vesicle pinching of membranes of cells and organelles was found to play a major role in the uptake of all 3 samples, with lysozyme predominantly taken up via the clathrin mediated pathway while mPEG2k-b-GA30:lysozyme and biotin-PEG2k-b GA10/30:lysozyme preferred the caveolae-mediated pathway; V inhibiting the caveolae pathway had higher impact on the uptake of biotin-PEG2k-b-GA10/30:lysozyme nanocomplexes.
The use of targeted PEG-b-pGA copolymer delivery systems to successfully complex and deliver model protein lysozyme to epithelial cells in vitro and in vivo pave the way for further investigations, wherein the PEG-b-pGA copolymer can be used to deliver protein or peptide therapeutics and maintain or improve the therapeutic effect. Furthermore, it was envisaged that the PEG-b pGA copolymer delivery system can potentially be applied not only to proteins but also small molecule therapeutics formulated in nano sized entities, which became the focus of the final chapter of this thesis.
Finally, in Chapter 6, antibiotic cationic peptide colistin was successfully formulated into nanocomplexes with mPEG2k-b-GA30 copolymer and complexation was confirmed by agarose electrophoresis and transmission electron microscopy. Colistin:mPEG2k-b-GA30 r = 1:2 nanocomplexes possess enhanced bacteriostatic effect in non-pathogenic E. coli XL1 and bactericidal activity in pathogenic E. coli E2348/69 compared to free colistin applied at the same concentration. It should be noted that colistin:mPEG2k-b-GA30 r = 1:2 formulation showed enhanced bactericidal effect in EPEC E2348/69 infected of HeLa cells, compared to free colistin by killing extra 2 logs of EPEC after 2 h VI treatment. This result strongly indicates that PEG-b-pGA delivery system can be utilised widely aiding the delivery of proteins and small molecule drugs.
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