Maguire, Leigh Anthony; (2003) Processing and characterisation of liposomes for use in gene delivery. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In recent years liposomes have received much attention for use in gene therapy and DNA vaccines. This project aims to investigate issues involved with the processing of liposome based gene delivery vectors and in doing so propose a controllable, reliable and scaleable method for processing such a vector. With this in mind a high velocity jet homogeniser was investigated for use in the preparation of small unilamellar vesicles (SUV). The device was able to produce SUV with a distribution having a mean of approximately 140nm following a single pass at 103.4 mNm-2: this size is suitable for sterilisation by filtration and in vivo gene delivery. By increasing either the number of passes through the device or the operating pressure it was possible to control the final size of the SUV between 80 and 140nm. The liposomes produced were used to encapsulate plasmid DNA that had been condensed with poly-L-lysine (PLL). Charge ratio was determined to be a key processing criteria with a liposome: PLL/DNA charge ratio of 4 or above being required to form stable complexes. The liposomes encapsulated between 85 and 95% of the PLL/DNA with smaller liposomes being more effective. Targeting moieties are often attached to the surface of liposomes in order to improve their delivery. In order to investigate the effects of such molecules upon processing of liposomes and PLL/DNA/liposome complexes a Fab' antibody fragment was attached to the liposome. The presence of the Fab' resulted in increased size and reduced negative charge. These effects were shown to result in reduced PLL/DNA encapsulation and stability. Polyethylene glycol (PEG) can be used to improve the stability of complexes. The effects of PEG with a molecular weight of 2000 KDa upon processing included improved stability of PLL/DNA/liposome complexes and successfully shielded the zeta potential of the particles. However its presence also resulted in a reduced PLL/DNA encapsulation efficiency.

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