Synthesis of (M)PEG-grafted PEI

General procedure for carboxylic acid formation.

The methoxy-PEG-carboxylic acid was synthesized as follows (Figure 1). In a three-necked 250 mL flask under argon atmosphere was placed 1.20 g (50 mmol) of sodium hydride and 50 mL of dry tetrahydrofuran (THF) and then 2.75 g (5 mmol, 2.52 mL) of methoxy-PEG
(MPEG) was dropped in at room temperature and stirred for 4 h. 8.35 g (75 mmol, 8.31 mL) of ethyl bromoacetate was added to this mixture and stirred at room temperature for 48 h. The solution was evaporated under a reduced pressure to remove almost all of THF and then poured into hexane to precipitate ethyl MPEG acetate, which was recovered by centrifuge (3000 rpm). The precipitate was evaporated to remove residual ethyl bromoacetate. The ethyl MPEG acetate was dissolved in 50 mL of 1N sodium hydroxide. After overnight in reflux, pH of the mixture was adjusted to 2. The MPEG carboxylic acid was extracted into chloroform. The organic extract was purified by washing with water and evaporated to give the MPEG carboxylic acid.

General Procedure for Acid Chloride Formation.

For example, the MPEG acid chloride for 1 : 1 MPEG-grafted PEI was obtained as follows. Oxalyl chloride (0.64 mmol) was added dropwise to a solution of 70.0 mg (0.13 mmol) of MPEG and 2 drops of N,N-dimethylformamide (DMF) in 2 mL of dichloromethane (DCM) under Ar atmosphere. The reaction was allowed to reach completion for 2 h at room temperature. The excess oxalyl chloride was removed on the rotary evaporator by a stripping procedure with several portions of chloroform to give MPEG acid chloride as yellow
oil that was used without any further purification. Acid chlorides for other copolymers were synthesized similarly except in scale. PEG bis(carboxyl methyl) ether was also used to activate carboxyl group.

General Coupling Procedure. For example, as for 1:1 MPEG-grafted PEI, MPEG acid chloride synthesized above (0.13 mmol) diluted in a small amount of dry DCM and then was added drop by drop to the solution of 70 mg (0.0028 mmol) of PEI and 35 mL (0.26 mmol) of triethylamine (TEA) in 3 mL of dry DCM at room temperature under Ar atmosphere. The reaction was allowed overnight to reach comple-
tion. The DCM was evaporated and the yellowish viscous product was obtained. Figure 1 represented the synthesis scheme of cationic copolymer.

Purification of Grafted Copolymers.

The polymers were purified by dialysis against water using the Spectra/Por dialysis membrane (MWCO 3400, Spectrum, Los Angeles, CA).

After evaporation of DCM, the viscous product was solubilized in distilled water. Each product was dialyzed for 1 day and lyophilized before use for analysis and assay. As the PEG derivatives used were small in molecular weight, uncoupled ones were efficiently removed from the mixture.

Ratio of PEG and PEI. The ratio of PEG and PEI in the copolymer sample was determined from 1H NMR spectra using integral values obtained from the number of -CH2CH2O-protons of PEG and -CH2CH2N- protons of PEI. Prior to NMR measurements all copolymers were purified from admixtures of non-conjugated PEG by dialysis (MWCO 3400) with distilled water and freeze-drying.

Good transfection efficiency: PEG-PEI=1:1

Effect of Poly(ethylene glycol) Grafting on Polyethylenimine as a Gene Transfer Vector in vitro Bull. Korean Chem. Soc. 2001, Vol. 22, No. 1