FluoSphere PEGylation and characterization

Carboxylate-modified FluoSphere^TM NPs (FNP, 100 nm) were covalently modified with mPEG-Amine (2 kDa MW) by carbodiimide chemistry as previously described by Nance et al.⁵⁴. FNPs were washed to remove sodium azide using 0.5 ml Amicon-Ultra centrifugation filters (10k MWCO). mPEG-amine (5x molar excess) was added and allowed to stir for 15 min. Next, 6.5 mg N-Hydroxysuccinimide (NHS) dissolved in 6 ml borate buffer (200 mM, pH8) was added, followed by 15.4 mg 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). After 3 hours, the reaction was quenched with excess glycine (100 mM) for 30 min. Unreacted PEG, EDC, NHS and glycine were removed by dialysis (100k MWCO) for 24 hours. FNPs were washed and collected by centrifugation using Amicon-Ultra centrifugation filters (100k MWCO) and resuspended to 20 mg/ml in 1x PBS for storage at 4 °C.

FNP size and zeta potential was measured in 1 mM KCl before and after PEGylation using a NanoBrook 90Plus Zeta (Brookhaven Instruments, Holtsville, NY USA). Pegylated FS stability in 10% FBS at 37 °C was measure at 1, 2, 4, 12, 24 hours and 1 week. 1 mg of lyophilized sample was dissolved in 0.6 mL of chloroform-d (CDCl3). H-NMR spectra of the samples were obtained using a 300 MHz Bruker Avance NMR spectrometer at standard room temperature.