Size distribution of spontaneously formed liposomes by the alcohol injection method

A dynamic light scattering study of the size distribution of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes formed by the injection method is presented. By this method, an aliquot of methanol stock solution containing the surfactant is injected into water. The main aim of the, present work was to determine under which conditions a mono-modal and, narrow size distribution could be obtained. The influence of several parameters on the size distribution was investigated. Firstly, we examined the influence of the POPC concentration in the initial stock methanol solution, when the POPC concentration in the final aqueous solution remains constant; secondly, the influence of POPC concentration in the aqueous phase, while the lipid concentration in the stock methanol remains constant. In both cases narrow monomodal size distributions of liposomes, centered between 40 and 70 nm, are obtained at low concentrations of POPC, in the stock methanol solution (less than or equal to25 mM) as well as in the final aqueous solution. However, at a higher lipid concentration range a more complex size distribution is observed, and an additional component larger than 500 nm is detected. The effect of dilution was also studied. It can be shown that dilution of the more concentrated POPC solutions determines the disappearance of the larger component, which is then ascribed to aggregates or clusters of smaller liposomes. Interestingly, by concentrating the diluted, monomodal liposomal suspensions, the monomodal distribution is maintained. Concentrated liposome preparations can therefore be obtained having only one population of liposomes centered around 40-70 nm radii. Finally, the effect of aging was studied. It appears that the larger components in the light scattering results are not time-stable, whereas the smaller liposome species are. It is shown that these features are also present when alcohols different from methanol are used.