Size characterisation of liposomes using asymmetrical flow field-flow fractionation : factors influencing fractionation and size determination

Abstract

Liposomes play an important role in medical and pharmaceutical science as e.g. nanoscale drug carriers. One of their most important features is their size and size distribution, influencing both their biodistribution and passive targeting abilities upon intravenous administration. To this day there is no single method for size characterisation of liposomes that fulfils all following criteria: being fast and easy to perform, covering the whole size range of liposomes from tens of nanometers up to tens of micrometers as well as determining size distributions rather than mean sizes in a non-destructive manner. <br>In this thesis a method for size characterisation of liposomes by asymmetrical flow field-flow fractionation coupled online with multi-angle light scattering (AF4-MALS) was established. Key factors influencing the fractionation behaviour of various liposome samples such as cross flow rate, focus flow rate, sample load, ionic strength of the carrier liquid and membrane effects were identified and characterised. Finally, size distributions obtained by AF4-MALS were compared to mean particle sizes and size distribution measured with other standard methods such as photon correlation spectroscopy (PCS), size exclusion chromatography in combination with PCS (SEC-PCS) and cryogenetic transmission electron microscopy. <br>Satisfactory fractionation of liposomes within reasonable experiment time was obtained by the use of programmed cross flow gradients. Both sample load mass and ionic strength of the carrier liquid were shown to extensively influence the elution behaviour of liposomes and may cause overloading of the channel for extreme values. Further, it was demonstrated that ionic strength of the carrier solution different to the ionic strength of the medium used during liposome preparation may change the vesicles in terms of osmotic shrinking/swelling during an AF4 run. The limit of detection was improved by the use of stained liposomes and for fitting of MALS data, the coated sphere model was found to describe liposomes best in terms of quality of fit. <br>The suitability of AF4-MALS for the size characterisation of liposomes was proven. Size characterization by AF4-MALS gave similar size distributions as the other standard methods tested, yet it was performed in less time and gave a better insight into the whole size distribution of the liposome samples used in this study.