A colorimetric estimation of polyethyleneglycol-conjugated phospholipid in stealth liposomes

The paper describes a colorimetric method for estimation of polyethyleneglycol (PEG)-conjugated phospholipid in either form, free or bound to liposomes. It provides a rapid, highly reproducible, and sensitive tool to detect PEG-coupled phospholipid in amounts as low as 1 microg giving a linear response over a range of 1-100 microg. The method makes use of the biphasic system comprising aqueous ammonium ferrithiocyanate and chloroform, developed by Stewart for estimation of phospholipids. The same system was also applied for quantitation of PEG in PEG-protein conjugates in a recent report. The samples were digested with phospholipase-C, prior to analysis, in order to eliminate the contributions from liposomal phospholipids other than the PEG-conjugated phospholipid. The technique can give valuable information regarding the retention of PEG coating on the surface of the vesicles when employed in combination with markers for the aqueous compartment. In addition, it does not suffer from interference by proteins. This makes it particularly suitable for monitoring the pharmacokinetics of stealth liposomes, using PEG-phospholipid as a lipid probe. Since it does not involve handling hazardous radioisotopes, the suggested technique could even be utilized in clinical trials.