Abstract
Dual centrifugation (DC) is a novel in-vial homogenization technique for the preparation of liposomes in small batch sizes under gentle and sterile conditions which allows encapsulation efficiencies (EE) for water soluble compounds of >50%. Since liposome size, size distribution (PDI), and EE depend on the lipid concentration used in the DC process, a screening method to find optimal lipid concentrations for a defined lipid composition was developed. Four lipid mixtures consisting of cholesterol, hydrogenated or non-hydrogenated egg PC, and/or PEG-DSPE were screened and suitable concentration ranges could be identified for optimal DC homogenization. In addition to the very fast and parallel liposome preparation of up to 40 samples, the screening process was further accelerated by the finding that DC generates homogeneously mixed liposomes from a macroscopic lipid mixture without the need to initially prepare a molecularly mixed lipid film from an organic solution of all components. This much simpler procedure even works for cholesterol containing lipid blends, which could be explained by a nano-milling of the cholesterol crystals during DC homogenization. Furthermore, EE determination was performed by time-resolved fluorescence measurements of calcein-loaded liposomes without removing the non-entrapped calcein. The new strategy allows the rapid characterization of a certain lipid composition for the preparation of liposomes within a working day.
Highlights
Dual centrifugation (DC) was first described by Massing et al in 2007 [1] as a powerful method for the preparation of vesicular phospholipid gels (VPGs) and, after simple dilution of a VPG, of a liposomal dispersion
To produce a liposome dispersion from a VPG made in calcein buffer, the latter has to be diluted by, for example, a factor of 5000, which results in a dispersion containing two populations of calcein
We showed a decrease in the rnal vesicles in oligovesicular or oligolamellar liposomes reduce the slope of me sizes decrease with increasing ratios of EPC/Chol liposomes with increasing amounts ofof
Summary
Dual centrifugation (DC) was first described by Massing et al in 2007 [1] as a powerful method for the preparation of vesicular phospholipid gels (VPGs) and, after simple dilution of a VPG, of a liposomal dispersion. DC homogenization of viscous lipid/buffer mixtures along with ceramic beads in small disposable vials is achieved by the additional, fast turning of the sample vial around its own axis (axis: 90◦ with respect to the longitudinal direction of the vial) during normal centrifugation. This results in a frequent and very powerful movement of the viscous sample material from top to bottom of the vial and vice versa. This very powerful movement of the potentially highly viscous samples is based on the high centrifugal acceleration of up to 1000× g caused by the rotation around the primary axis, and is more than one magnitude higher than the sample acceleration that can be reached by the most powerful lab shakers [2].
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