Quantitation of PEG on PEGylated Gold Nanoparticles Using Reversed Phase High Performance Liquid Chromatography and Charged Aerosol Detection: Version 1.0

This protocol describes a method for the quantitation of polyethylene glycol (PEG) in PEGylated colloidal gold nanoparticles using reversed phase high performance liquid chromatography (RP-HPLC) with charged aerosol detection (CAD). The method can be used to calculate the total PEG on the nanoparticle, as well as the bound and free unbound PEG fractions after a simple centrifugation step. This is a significant distinction as the bound PEG fraction affects biocompatibility, circulation time, and overall nanoparticle efficacy. PEG quantitation can be achieved through two methods, one involving dissolution of colloidal gold nanoparticles by potassium cyanide (KCN) and the other by displacement of PEG by dithiothreitol (DTT). The methods outlined herein were applied to 30 nm colloidal gold grafted with 20 kDa PEG, but they can be easily adapted to any size colloidal gold nanoparticle and PEG chain length. The method development was previously published in ref. (1) and the detailed protocol is published in ref. (2).