Quantifying PEG Density on Nanoparticles

The PEG density on nanoparticle surface (no. of PEG chains/100 nm²) and Γ/Γ*, where Γ is the PEG surface coverage over the total surface area (Γ*), were calculated from the ¹H integrals of the ethylene oxide peak of PEG, using a previously described method (36 (link)). Briefly, nanoparticles were lyophilized, weighed, and dissolved in CDCl₃ containing 0.1% (v/v) trimethylsilane as an internal standard. Nuclear magnetic resonance (NMR) spectra were obtained at 500 MHz using an Agilent DD2 500 MHz Spectrometer. A calibration curve was obtained by plotting the ¹H NMR integrals of various concentrations of 5 kDa PEG (~ 3.6 parts per million) in CDCl₃ solvent containing 0.1% (v/v) trimethylsilane. The average PEG density (no. of PEG chains/100 nm²) on the nanoparticle surface was calculated by taking the total quantity of PEG detected by NMR and the total nanoparticle surface area. The nanoparticle surface area was calculated, assuming that the particles are made of individual particles of diameter equal to that measured by the Zetasizer and using a PLGA density of 1.34 g/cm³.