Hypothesis: Liposomes made of single-chain amphiphiles and a large amount of sterols display several advantages including a limited permeability. In the present paper, we examine the possibility to prepare such non-phospholipid liposomes with interfacial polyethylene glycol (PEG) in order to improve their circulation in the blood stream. Cholesterol (Chol) was chosen as the PEG anchor. Experiments: The phase behavior of mixtures of palmitic acid (PA) and cholesterol including various proportions of PEGylated cholesterol (PEG-Chol) was characterized. In conditions leading to the formation of fluid bilayers, properties of the resulting liposomes were assessed. Findings: Up to 20 mol% of PEGylated cholesterol could be introduced without significant perturbations in fluid bilayers made of PA and cholesterol. With 10 mol% PEG-Chol, PA/Chol/PEG-Chol liposomes showed a very limited permeability to calcein and doxorubicin. Doxorubicin could be actively loaded in PA/Chol/PEG-Chol liposomes with a high drug loading efficiency and a high drug to lipid ratio. Pharmaco-kinetic experiments in rats indicated that interfacial PEG reduced the clearance of PA/Chol liposomes compared to the naked ones. However the lifetime of these non-phospholipid liposomes in the blood circulation was considerably shorter than that observed for control PEGylated phospholipid liposomes, a phenomenon associated with the negative interfacial charge of the PA/Chol/PEG-Chol liposomes. (C) 2014 Elsevier Inc. All rights reserved.