Liposomal steroid preparation

Liposomes were prepared as previously described³⁰. In brief, dipalmitoyl phosphatidyl choline (DPPC), cholesterol, and PEG2000 distearoyl phosphatidylethanolamine (PEG-DSPE) were added in a 62%, 33%, and 5% molar ratio. Steroids were dissolved in water for injection while the lipids were dissolved in absolute ethanol at 65 °C. The alcoholic lipid solution was injected in the aqueous steroid solution and mixed under heating to 65 °C, forming a multilamellar vesicle dispersion. This dispersion was downsized to the desired particle size of approximately 100 nm in diameter by repeated homogenization cycles using an Avestin C55 high-shear homogenizer (Avestin, Mannheim, Germany). Unencapsulated steroids were removed by ultrafiltration using membranes with a molecular weight cut off of 30 kDa and replaced with clean dispersion buffer. Finally, the liposomal dispersion was sterile filtered, collected in vials and stored between 2 and 8 °C. Cyanine 5.5 and Fluorescein isothiocyanate (FITC) liposomes were prepared identically with the addition of 0.2% of DSPE-CY5.5 or FITC as described by Lobatto et al.³¹. The characteristics (Table 1) and drug release profile of this formulation in aqueous medium and plasma has previously been published. In such media, they show good drug retention properties, which is essential to ensure transport and delivery of the liposomal encapsulated drug at the target cells (e.g. macrophages) in the inflamed site^26,27,29. The formulation studied here is the same as the formulation developed and evaluated by Lobatto et al. With this formulation, neither in vitro (buffer, 37 °C) nor in vivo (in the blood circulation) release of encapsulated drug from the liposomes was observed³¹. However, despite the complete stability of the liposomes in vitro and in the circulation, low levels of free drug were detected in plasma, which are due to liposome clearance from blood and subsequent drug release by liver and spleen macrophages back to the circulation²⁶.

Characteristics of liposomes.