Tse, Man Tsuey; (2007) Development of Microparticulate DNA Vaccines for Pulmonary Delivery. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

DNA vaccines have emerged to be an alternative to conventional vaccines due to their increased safety compared with recombinant protein vaccines and live/attenuated vaccines. Pulmonary delivery of vaccines could offer additional advantages, because from an immunological point of view, there is a large presence of macrophages for antigen/DNA uptake and it also part of the mucosal immune system, which is important for effective immune protection. Encapsulating the DNA into a particulate delivery system would be advantageous for protection against degrading nucleases. The aim of this project was to produce microparticles encapsulating plasmid DNA with a high loading efficiency, appropriate size suitable for pulmonary delivery and with the maintenance of plasmid DNA conformation. Microparticles were produced using poly(DL-lactide-co-glycolide) as the polymer due to its well-known biodegradable and biocompatibility characteristics, and polyvinyl alcohol (PVA) as the main stabiliser. Plasmid DNA encoding for luciferase protein was used as a model plasmid for studies. Water-in-oil-in-water (w1/o/w2) was used as the method for making particles. Factorial experimental design was used to rapidly optimize a formulation that had the desired properties. Further studies examined the effect of adding various excipients (0.1M NaHCO3, 1% m/m Na2HPO 4) on the loading, diameters, morphology and charge on the microparticles. Analytical (including TGA, NMR and DSC) and cell culture experiments - using a human alveolar cell line (A549) and mouse macrophage/monocyte cell line (J774A.1) - were carried out to analyse their physical and biological properties. The factorial experimental design aided in choosing a platform formulation, with particle diameters of 2-5 μm and loading of 66-72% m/m. Particles without added excipients had low plasmid DNA loading and the DNA had lost most of its original conformation. However, addition of buffers into the aqueous phases, particularly 1% m/v NaH2PO4, reduced the loss of conformation and enhanced loading efficiency. Physicochemical studies gave mixed results on the formulations tested in terms of thermal stability, DCM and PVA levels and performance as a dry powder. Results demonstrated that microparticles made without buffer were more thermally stable, had lower residual DCM and PVA levels in contrast to those made with 1% m/v Na2HPO4 and 0.1M NaHCO3 . The microparticles did not aerosolise well as a dry powder - even with addition of surfactants such as lecithin and DPPC - with diameters between 69-95 μm being measured, which is in contrast to the diameters measured in a 'wet' state (2-5 μm). Cell culture studies demonstrated that entrapped DNA (which was extracted out from the particles and delivered to cells using a commercial agent) was still biologically active, but the A549 cell line was much easier to transfect than the J774A.1 cells. However, the microparticles themselves were poor delivery vehicles of DNA in these models. Overall, these experiments demonstrated the range of factors that need to be considered when trying to create suitable microparticulate carriers for pulmonary delivery of DNA vaccines.

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