PEGylation of Bilirubin for Therapeutic Use

PEG-BR was prepared as previously described with modifications^{48 (link)}. 75 μmol of bilirubin and 33.75 μmol of EDC were added to 0.6 ml of DMSO containing 225 μl of TEA and 52 μmol of NHS for 10 min at RT. After then, 15 μmol of polyethylene glycol 20K-amine (PEG-NH2, Nanocs) and the mixture was mixed for 4 h under nitrogen gas. Subsequently, the mixture was added to 50 ml of chloroform and then the organic solvents were washed by 50 ml of 0.1M HCl twice, followed by washing with 50 ml of 0.1M NaHCO3 twice and finally washed with 50 ml of distilled water twice. The organic layer was evaporated, and 50 ml of methanol was added to the residue. After centrifugation at 3,000 x g for 10 min, the supernatant was collected and then evaporated. Dialysis was performed as described in the previous section. To yield PEGylated bilirubin (PEG-BR), lyophilization was performed. The final structure was confirmed by ¹H-NMR. ¹H-NMR spectra were recorded on a Varian 500MHz system; chemical shifts represent ppm downfield from tetramethylsilane.