Formulation and aerosol delivery of nano-sized biomaterials
thesisposted on 28.06.2018, 10:04 by Eugene R. Arulmuthu
The aim of this research has been to study and develop the engineering principles associated with the impact of formulation and device parameters on the safe delivery of nano-sized biomaterials such as plasmid DNA. In the present investigation, Omron U22 and U03 mesh nebulisers operating at frequencies of ~175 kHz and ~65 kHz respectively were used. Since the U22 device is a recently introduced mesh nebuliser for respiratory drug delivery, detailed characterisation, experimentation, modelling and analysis was carried out for this device. Plasmids of size 5.7, 8.7, 13 and 20 kb were purified from Escherichia coli cells and used for nebulisation experiments. Experiments on the nebulisation of plasmid DNA using the U22 device in a bio-safety cabinet showed no damage to the sc structure of the 5.7 kb plasmid, but almost complete damage to the 20 kb plasmid in the condensed aerosols collected using a fabricated aerosol collection apparatus. The damage to the sc structure of plasmid DNA was analysed using gel electrophoresis, PicoGreen assay and atomic force microscope (AFM). Engineering analysis was performed using computational fluid dynamics (CFD) modelling to determine the shear and elongational strain rates in the mesh nozzle of nebuliser. The estimated maximum hydrodynamic force on plasmid DNA based on the Ryskin equation was calculated in picoNewton (PN) from the actual molecular size of the sc structure and predicted strain rates. Optimisation of the formulation and device parameters were carried out using Design of Experiments (DOE) to predict damage to the sc structure. Formulation of the 20 kb plasmid with polyethyleneimine (PEI) resulted in safe aerosol delivery using the mesh nebuliser. In vitro transfection studies in suspension-adapted Chinese Hamster Ovary (CHO-S) cells resulted in successful integration of Green Fluorescent Protein (GFP) from the 5.7 kb plasmid after nebulisation. The commercially available U22 mesh nebuliser promises to be a useful pulmonary device for the successful delivery of plasmid DNA for non-viral gene therapy. Realisation of this promise however will require both innovations in the design of experiments, formulation and methods of studying plasmid DNA damage as demonstrated in this thesis.
- Mechanical, Electrical and Manufacturing Engineering