Khatri, Lazeena; (2001) Protein entrapment in liposomes for nebulisation. Doctoral thesis (Ph.D.), University College London.

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Abstract

This thesis focuses on the potential use of nebulisation to deliver therapeutic proteins. Liposomes were investigated as a means to protect unstable proteins against degradation during nebulisation. Lactate dehydrogenase (LDH) and mushroom tyrosinase (TYR) were chosen as model proteins and the effects of jet and ultrasonic nebulisation on protein solutions and liposomal protein formulations were examined. The effects of nebulisation on protein activity and concentration, size distribution of the aerosols and temperature of nebuliser fluid were investigated. The duration of nebulisation time and the mass output of the nebulisers were measured and compared for all nebulisers and protein formulations. A twin impinger was utilised to determine the delivery of total and active protein. Inter and intra-nebuliser variability was also investigated. LDH was found to be more sensitive to degradation during nebulisation than TYR. The Omron U1 ultrasonic nebuliser was found to be the nebuliser of choice because TYR was not degraded and LDH showed less degradation using this nebuliser than the others. Liposomal entrapment protected against protein degradation during nebulisation. High sensitivity differential scanning calorimetry (HSDSC) and a Langmuir trough were used to establish how the proteins interacted with the liposomes and to explore why this protection occurred. The interaction between proteins and liposomes was electrostatic and no evidence of penetration of the proteins into the bilayer was observed. This interaction was believed to stabilise the protein by protecting against shear forces which occurred during nebulisation. Also by encapsulation of protein within liposomes, unfolding of protein structure and consequent denaturation of protein at air/water interfaces of aerosolised droplets was hindered. This work has demonstrated the ability of liposomes to protect proteins during nebulisation. The considerable differences in the characteristics of aerosols produced by different nebulisers highlight that manufacturers should specify the appropriate nebuliser to be used with a particular protein formulation.

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