A COLORIMETRIC ESTIMATION OF POLYETHYLENEGLYCOL-CONJUGATED PHOSPHOLIPID IN STEALTH LIPOSOMES

The paper describes a colorimetric method for estimation of polyethyle neglycol (PEG)-conjugated phospholipid in either form, free or bound t o liposomes. It provides a rapid, highly reproducible, and sensitive t ool to detect PEG-coupled phospholipid in amounts as low as 1 mu g giv ing a linear response over a range of 1-100 mu g. The method makes use of the biphasic system comprising aqueous ammonium ferrithiocyanate a nd chloroform, developed by Stewart for estimation of phospholipids. T he same system was also applied for quantitation of PEG in PEG-protein conjugates in a recent report. The samples were digested with phospho lipase-C, prior to analysis, in order to eliminate the contributions f rom 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 combina tion with markers for the aqueous compartment. In addition, it does no t suffer from interference by proteins. This makes it particularly sui table 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 utilize d in clinical trials. (C) 1997 Academic Press.