Editing Preparation of Good Quality Chitosan

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Deacetylation of Chitin to Chitosan (Autoclave Method)<br> 

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Deacetylation of chitin under autoclaving conditions (15 psi/121 °C) was evaluated for the preparation of chitosan under different NaOH concentrations and reaction times. Deacetylation was effectively achieved by treatment of chitin under elevated temperature and pressure with 45% NaOH for 30 min and a solids/solvent ratio of 1:15. Treated chitosan showed similar nitrogen content (7.42%), degree of deacetylation (90.4%), and molecular mass (1560 kDa) but significantly higher viscosity values (2025 cP) compared with those (7.40%, 87.6%, 1304 kDa, and 143 cP, respectively) of a commercial chitosan. Reduction of the solids/solvent ratio from 1:15 to 1:10 did not affect degree of deacetylation, viscosity, and molecular mass of chitosan. 

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<br>Chitin (3 g) was mixed with various concentrations of NaOH (40%, 50%, and 60%,<br>w/v) at a ratio of 1:10 (w/v) and was left to steep in NaOH solution for two days. After<br>that, the samples were treated under a pressure of 101.3 kPa at 121°C for 30 minutes in<br>an autoclave (Hirayama, HVE-50) (Hong et al. 2000). After autoclaving, the solid<br>products were washed to neutrality under running tap water, rinsed with distilled water,<br>and filtered to remove excess NaOH. The obtained product was dried for 24 h at 60 o C,<br>after which it was further treated with 2% acetic acid solution (1:15 ratio, w/v) under<br>stirring for 10 minutes which dissolved the chitosan, leaving the remaining chitin as an<br>insoluble solid. The mixture was filtered using a sieve and chitosan collected in a beaker.<br> 

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by extraction with acetone and dried for 2 hr at ambient temperature, followed by<br>bleaching with 0.315&nbsp;% (v/v) sodium hypochloride solution (containing 5.25% available<br>chlorine) for 5 min with a solid to solvent ratio of 1:10 (w/v), based on dry shell. But,<br>the color of chitin products varied from cream white to intermediate pink color (No et<br>al., 1989). Without prior acetone extraction, bleaching for more than 1 hr was needed to<br>obtain a commercially acceptable white product.<br> 

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Deacetylation is the process to convert chitin to<br>chitosan by removal of acetyl group. It is generally achieved by treatment with<br>concentrated sodium or potassium hydroxide solution (40-50%) usually at 100oC or<br>higher for 30 min or longer to remove some or all of the acetyl groups from the polymer<br>(No and Meyers, 1989). The N-acetyl groups cannot be removed by acidic reagents<br>without hydrolysis of the polysaccharide, thus, alkaline methods must be employed for<br>N-deacetylation (Muzzarelli, 1977).<br> 

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Depolymerization of chitosan<br>Chitosan was selectively depolymerized following the<br>method developed by Huang et al. 26 Briefly, 100 mL of a 2%<br>(w/v) chitosan (400,000 g/mol) solution in 6% (v/v) acetic<br>acid solution was prepared. Then the dissolved chitosan<br>was depolymerized at room temperature under stirring with<br>10 mL of NaNO 2 solutions in water at different concen-<br>trations (7.0, 2.7, and 1.6 g/L), to obtain the desired final<br>molecular weight of 20,000, 50,000, and 100,000 g/mol,<br>respectively. After 1 hour of reaction, precipitated chitosan<br>was obtained by raising the pH to 9.0 by addition of 4N NaOH. The white-yellowish solid was filtrated, washed thor-<br>oughly with acetone, and re-dissolved in a minimum volume<br>of acetic acid 0.1 N (40 mL).<br>Purification was carried out by subsequent dialyses<br>against purified water (Sigma dialysis tubes, molecular<br>weight cutoff, 12 kDa). The dialyzed product was lyophilized<br>using a LyoTrap plus Freeze dryer (LTE Scientific, Oldham,<br>UK), and the yellowish lyophilized product was then stored<br>at 4°C until use.<br>

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Thiolated chitosan was prepared with covalent attachment of<br>reduced glutathione to different molecular weight of chito-<br>san according to the slightly modified method described by<br>Atyabi et al and Kafedjiiski et al as follows: 5,27<br>One gram of chitosan was dispersed in 50 mL demineral-<br>ized water by stirring, then 10 mL HCl (1 mol/L) was added<br>and finally dissolved by the addition of demineralized water<br>to obtain a 1% (w/v) polymer solution. The pH was adjusted<br>to 6.0 by the addition of NaOH (5 mol/L). Afterwards,<br>5 g of reduced glutathione in 10 mL demineralized water<br>was added to the above solution under continuous stirring.<br>EDC and NHS were then added in a final concentration of<br>200 mmol/L each. The pH was readjusted to 6.0 by adding<br>NaOH (5 mol/L). The reaction mixture was incubated for<br>15 hours at room temperature under constant stirring. To<br>eliminate unbound reagents, the resulting polymer conjugate<br>was dialyzed using dialysis tubing (molecular weight cutoff<br>12 kDa), first against 5 mmol/L HCl, twice against 5 mmol/L<br>HCl containing 1% NaCl, and finally twice against 1 mmol/L<br>HCl. Controls were prepared in the same way but omitting<br>EDC and NHS during the coupling reaction. Finally, the<br>frozen aqueous polymer solutions were lyophilized at −50°C<br>and 0.01 mbar (LyoTrap plus; LTE Scientific, UK) and stored<br>at 4°C until further use<br>

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Preparation of thiolated<br>chitosan-pMMA NPs<br>The NPs were prepared by using a modif ied radical<br>polymerization method. Conjugated chitosan (37.5 mg) with different theoretical molecular weight (20, 50, and 100 kDa)<br>was dissolved in 4 mL nitric acid (0.2 mol/L) in a two-necked<br>flask at 40°C, under gentle stirring and nitrogen bubbling.<br>After 10 minutes, under vigorous magnetic stirring, a solu-<br>tion of 0.08 mol/L cerium IV ammonium nitrate (CAN) in<br>0.2 mol/L nitric acid and MMA was added to obtain a 5-mL<br>solution. Nitrogen bubbling was continued for an additional<br>10 minutes. The reaction was allowed to continue at 40°C<br>under gentle stirring for 40 minutes. After cooling to room<br>temperature, the pH of the obtained suspension was adjusted<br>to 4.5 by addition of sodium hydroxide (1 N) drop-wise. The<br>NP suspension was then purified by dialysis (Sigma dialysis<br>tubes, molecular weight cutoff, 12 kDa) against 1 L of acetic<br>acid solution (16 μmol/L) in demineralized water, twice for<br>90 minutes and once overnight. The frozen NP suspensions<br>were lyophilized at −50°C and 0.01 mbar and stored at 4°C<br>until further use.<br>

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