Analyzing the influence of PEG molecular weight on the separation of PEGylated gold nanoparticles by asymmetric-flow field-flow fractionation

Polyethylene glycol (PEG) is an important tool for increasing the biocompatibility of nanoparticle therapeutics. Understanding how these potential nanomedicines will react after they have been introduced into the bloodstream is a critical component of the preclinical evaluation process. Hence, it is paramount that better methods for separating, characterizing, and analyzing these complex and polydisperse formulations are developed. We present a method for separating nominal 30-nm gold nanoparticles coated with various molecular weight PEG moieties that uses only phosphate-buffered saline as the mobile phase, without the need for stabilizing surfactants. The optimized asymmetric-flow field-flow fractionation technique using in-line multiangle light scattering, dynamic light scattering, refractive index, and UV–vis detectors allowed successful separation and detection of a mixture of nanoparticles coated with 2-, 5-, 10-, and 20-kDa PEG. The particles coated with the larger PEG species (10 and 20 kDa) were eluted at times significantly earlier than predicted by field-flow fractionation theory. This was attributed to a lower-density PEG shell for the higher molecular weight PEGylated nanoparticles, which allows a more fluid PEG surface that can be greater influenced by external forces. Hence, the apparent particle hydrodynamic size may fluctuate significantly depending on the overall density of the stabilizing surface coating when an external force is applied. This has considerable implications for PEGylated nanoparticles intended for in vivo application, as nanoparticle size is important for determining circulation times, accumulation sites, and routes of excretion, and highlights the importance and value of the use of secondary size detectors when one is working with complex samples in asymmetric-flow field-flow fractionation.