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Title: Chitosan nanoparticles for siRNA delivery to the lungs using pressurised metered dose inhalers and nebulisers
Author: Sharma, Ketan
ISNI:       0000 0004 2733 0130
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2012
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Purpose: To produce and characterise high molecular weight chitosan (HMC; glutamate derivative) and low molecular weight chitosan (LMC) nanoparticles associated with different grades of PEG (PEG600, PEG1000 and PEG5000) and chitosan-grafted PEG (LMC-g-PEG5000) nanoparticles as carriers for pulmonary delivery of siRNA using pMDIs and nebulisers. Materials and Methods: Nanoparticles encapsulating siRNA were prepared using an ionic crosslinking technique at a chitosan to siRNA weight/weight ratio of 10:1, 30:1 and 50:1. They were characterised for their functional groups, physicochemical properties and siRNA encapsulation efficiency using FTIR, Malvern Zetasizer and PicoGreen reagent respectively. Formulation physical stability was studied in propellants (HFA-227 and HFA-134a) using visual and infrared (Turbiscan) techniques. Gel electrophoresis was used to assess the association of siRNA with nanoparticles, including after aerosolisation. The aerosolisation properties of the formulations were tested using cascade impaction techniques and laser diffraction. Cell viability, cellular uptake and gene silencing were performed using A-549/H-292 cells. Results: Nanoparticles were positively charged with mean diameters less than 250 nm, with and without PEG, at all chitosan to siRNA ratios. FTIR confirmed PEG association and PEG grafting to chitosan. Nanoparticles were non-aggregated at the pH of the airways (6.5) and showed high siRNA loading efficiency (>89% PEG associated and 57% PEG grafted nanoparticles). PEG1000 associated nanoparticles displayed acceptable stability (~10 min) as dispersions with both grades of chitosan in HFA-227, whereas other formulations either rapidly creamed or sedimented. Complete binding of siRNA was observed when the w/w ratio approached 50:1 for PEG associated particles. Nebulisation studies showed ~50-70% fine particle fraction (FPF) for all formulations. Following actuation from pMDIs, the FPF for HMC-PEG1000 nanoparticles, was 34 (± 1.4) % whereas for LMC-PEG1000 nanoparticles, the FPF was 38 (±2.7) % (without siRNA) and 25% (with siRNA). The volume median diameter (VMD) of LMC-PEG1000 particles; post-actuation was 1.43 (±0.08) μm (without siRNA) and 4.41 μm (with siRNA). Cell viability was high (>80%) at the highest chitosan concentration, with PEG1000 associated nanoparticles. LMC-g-PEG5000 nanoparticles were taken up within A-549 cells and showed gene silencing activity (58.20 ±18.6 %), however gene silencing with other formulations could not be demonstrated due to poor RNA recovery. Conclusion: The results suggest that PEG-1000 associated chitosan nanoparticles have potential for delivery of siRNA to the lungs using a pMDI, whereas LMC-g-PEG5000 was potentially suitable as a siRNA carrier for use with nebulisers.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available