Synthesis O-carboxymethyl chitosan

Preparation of O-carboxymethyl Chitosan (OCMCh)

Protocol 1

Carboxymethylation was carried out by stirring chitosan (5 g) in 20% NaOH (w/v, 100 mL) for 15 min. then monochloroacetic acid (15 g) was added dropwise to the reaction mixture and the reaction was continued for 2 h at 40 ± 2 °C with stirring. Then the reaction mixture was neutralized with 10% acetic acid, then was poured into an excess of 70% methanol. The produced carboxymethyl chitosan was filtered using a G 2 sintered funnel and was washed with methanol. The product was dried in a vacuum at 55 °C for 8 h to give 6.5 g of dried carboxymethyl chitosan. The degree of substitution of CMCh was determined to be 0.75 

Protocol 2

2 g of LMWSC (Low molecular weight water soluble chitosan) was immersed in 25 mL of 50% (w/v) NaOH solution and alkalized for 24 hours. The alkalized LMWSC was filtered
and then transferred into a flask, after which 5 g of monochloroacetic acid in isopropanol was added in a dropwise manner for 20 minutes. This flask was allowed to react for
a further 8 hours at room temperature (RT). The reactants were filtered to remove the solvent, and the filtrates were then dissolved in 100 mL of water, thereafter the pH was
adjusted to 7.0 using 2 N HCl. The product was precipitated by ethanol and the precipitates were harvested by filtration. Residual ethanol was removed by vacuum drying.

Protocol 3

Carboxymethyl chitosan was prepared by method as suggested by Sun et.al [6]. Chitosan (10g), sodium hydroxide (10g), isopropanol (50 ml) and water (50 ml) were added into a flask to swell and alkalize at 50 o C for 1 h. The monochloroacetic acid (15 g) was dissolved in isopropanol (20 ml), added into the reaction mixture drop-wise for 30 min and reacted for 4 h at the same temperature, then stopped by adding 70% ethyl alcohol (ethanol, 250 ml). The solid was filtered and rinsed in 70-90% ethyl alcohol, and vacuum dried at room temperature.  The product was Na salt CC (Na-CC). Na-CC (1 g) was suspended in 80% ethyl alcohol aqueous solution (100 ml), hydrochloric acid (10 ml, 37%) was added and stirred for 30 min. The solid was filtered and rinsed in 70-90% ethyl alcohol to neutral, vacum dried.

Phthaloylation of OCMCh

One gram of phthalic anhydride was dissolved in 10 mL of N,N-dimethylformamide containing 5% (v/v) water, followed by the addition of 0.36 g of OCMCh. The mixture was heated
under nitrogen at 120°C while gently stirring overnight. After reaction, the phthaloylated chitosan solution was cooled at RT and then left in ice-cold water. The precipitates were harvested by filtration and washed with methanol. Residual methanol was removed by vacuum-drying.

Preparation of phthaloylated OCMPEI

Phthaloylated OCMPEI copolymers with different degrees of grafting of 25 kDa bPEI to carboxyl groups of the phthaloylated OCMCh backbone were generated by chemical synthesis.
Briefly, phthaloylated OCMCh solution was ­activated by the addition of EDC and NHS at 1:2:2 molar ratios in 50 mM 4-(2-hydroxyethyl)-1-­piperazineethanesulfonic acid (HEPES, pH 7.0) buffer containing 40% (v/v) dimethyl sulfoxide (DMSO) for 1 hour at RT, after which a solution of 5%, 10%, 15%, or 20% bPEI (weight %) was added, gently stirred, and then incubated for 24 hours at RT. After reaction, the solution of phthaloylated OCMPEI was dialyzed against distilled water for 2 days; the resulting solution was freeze-dried.

Deprotection of phthaloylated OCMPEI

Phthaloylated OCMPEI (200 mg) was mixed in 50 mL distilled water with 30 mL of hydrazine monohydrate using gentle magnetic stirring overnight at 100°C; the mixture
solution was then poured into 500 mL of ice water. Excess hydrazine monohydrate in the mixture solution was removed by evaporating using a rotary evaporator. The final product
 was lyophilized under a vacuum to obtain the deprotected OCMPEI.