Preparation of PEG with hydrazine and FA conjugation

The method of PEG attachment to basic hydrazine was conducted as described in a previous study (6). Dried PEG (20 g) was dissolved in 60 ml anhydrous methylene chloride, and added into 40 ml anhydrous methylene chloride solution containing 6.05 g nitrophenyl chloroformate and 12.5 ml anhydrous triethylamine. This was performed drop by drop in an ice-water bath, the resulting molar ratio of PEG, nitrophenyl chloroformate and triethylamine was 1:3:9. Spin steaming was used to remove the majority of the dichloromethane solvent following a 12-h reaction in the dark. Anhydrous ether was added to precipitate the PEG activated by nitrophenyl chloroformate, this was then vacuum dried.

The PEG activated by nitrophenyl chloroformate was dissolved in 100 ml dichloromethane, and 29.1 ml water and 29.1 ml hydrazine solution was added, the final molar ratio of PEG and hydrazine was 1:30. Spin steaming was performed to remove the majority of the dichloromethane solvent following the reaction at room temperature. Anhydrous ether was added to precipitate the PEG modified by hydrazine and this was vacuum dried.

FA-PEG-NHNH₂ was prepared as previously described (7). FA (0.88 g) was dissolved in anhydrous DMSO,0.84 g DCC and 0.44 g NHS were added, and the reaction proceeded at room temperature for 4 h with continuous mixing. Double hydrazine (4 g) was added to the PEG [PEG2000(NHNH₂)₂], continuously mixed and the reaction proceeded for 8 h. Following the reaction, 120 ml double-distilled water (ddH₂O) was added, the undissolved solid was removed by filtering and the product (FA-PEG-NHNH₂) was freeze-dried.