Aggregation of PEGylated liposomes driven by hydrophobic forces.

Polyethylene glycol (PEG) is widely used to sterically stabilize liposomes and improve the pharmacokinetic profile of drugs, peptides and nanoparticles. Here we report that ammonium sulfate (AS) can evoke the aggregation of PEGylated vesicles in a concentration-dependent manner. Liposomes with 5 mol% PEG were colloidally stable at 0.7 mM AS, but above this concentration they precipitated to form micron sized aggregates with irregular shape. While aggregation was reversible up to 0.9 M AS, above 1 M fusion occurred, which irreversibly distorted the size distribution of liposomes. Zeta potential of liposomes markedly increased from -71±2.53 mV to 2.02±0.45 mV upon raising the AS concentration from 0 to 0.1 M, but no considerable increase was seen during further AS addition, showing that the aggregation is independent of surface charge. There was no aggregation in the absence of the PEG chains, and increasing PEG molar % shifted the aggregation threshold to lower concentrations. Modifications of infrared spectrum of PEGylated vesicles suggests that AS dehydrates PEG chains. Other kosmotropic salts also led to aggregation, while chaotropic salts did not, which indicates a general kosmotropic phenomenon. The driving force behind aggregation is likely to be the hydrophobic effect due to salting out the polymer similarly to what happens during protein purification or Hydrophobic Interaction Chromatography. Since liposome aggregation and fusion may result in difficulties during formulation and adverse reaction upon application, the phenomena detailed in this paper may have both technological and therapeutical consequences.