Fourier Transform Infrared Spectroscopic Determination of the Hydrolysis of Poly(ethylene glycol)− Phosphatidylethanolamine-Containing Liposomes

Abstract

For poly(ethylene glycol)-derivatized liposomes (PEG−liposomes), the chemical stability of both the phospholipid ester groups and the urethane anchor between the PEG chain and the phospholipid are two important factors determining their long-term stability. High-performance liquid chromatography (HPLC) quantification of PEG-derivatized phospholipids such as PEG−distearoylphosphatidylethanolamine (PEG−(DS)PE) is hampered by the molecular mass distribution of the PEG polymer. We describe a novel Fourier transform infrared (FTIR) method to monitor the hydrolysis of (PEG−)phospholipids. The integrated intensities of the CO stretching bands of the various components of hydrolyzed (PEG−)phospholipid samples are determined by curve fitting. Separation of intact lipids and hydrolysis products is not required. To validate the method, the hydrolysis of dipalmitoylphosphatidylcholine/distearoylphosphatidylethanolamine (DPPC/DSPE) liposomes was monitored both by HPLC and FTIR. The results from both methods are in excellent agreement. Under the experimental conditions (pH 9, 70 °C), the hydrolysis rate constant of esters in liposomes was hardly affected by the presence of hydrophilic PEG chains at the surface of the liposomes. The stability of the PEG−phospholipid linkage could also be assessed. Although the urethane group is probably located in a more hydrophilic environment than the ester groups, hydrolysis of the PEG−PE anchor is still much slower than that of the esters.