Editing Synthesis Zwitterionic Chitosan

Chitosan glutamate (Protasan UP G113; MW: 200 kDa; degree of<br>deacetylation: 75–90%) was purchased from Novamatrix (Nor-<br>way), low-molecular-weight chitosan (LMCS; MW: 15 kDa;<br>degree of deacetylation: 87%) from Polysciences, and glycol<br>chitosan (MW: 82 kDa; degree of deacetylation: 83%) from Wako<br>USA (Richmond, VA).<br> 

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LMCS, 15 kDa from polysciences: Degree of deacetylation: 87% Insoluble at pH7.4<br> 

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Synthesis of ZWC and other chitosans<br>ZWC was synthesized as previously described [22]. Briefly,<br>LMCS was first dissolved in 1% acetic acid to obtain an acetate<br>salt form. LMCS acetate 200 mg was dissolved in 30 mL of<br>deionized water. '''Succinic anhydride '''was added as solid to the<br>LMCS solution under vigorous stirring varying the quantities<br>according to the desired molar feed ratio of anhydride to amine<br>(An/Am ratio). The pH of the reaction mixture was maintained at<br>6–6.5 and subsequently increased to 8–9 with 1 N NaHCO 3. After<br>an overnight reaction at room temperature under stirring, the<br>reaction mixture was dialyzed against water (molecular weight<br>cutoff: 3500) maintaining the pH at 8–9 with 1 N NaOH. The<br>purified ZWC was freeze-dried and stored at 220uC. Unless<br>specified otherwise, ZWC prepared with An/Am ratio of 0.7 was<br>used in most experiments.<br> 

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Synthesis of succinic anhydride-conjugated low molecular weight chitosan (SALM-CS) 

Chitosan was first dissolved in 1% acetic acid and centrifuged at 4000 rcf for 20 min, and the clear supernatant was collected and freeze-dried to obtain an acetate salt form of chitosan. Chitosan acetate 200 mg was dissolved in 30 mL deionized (DI) water. Succinic anhydride was added as solid to the chitosan solution in varying quantities according to the desired molar feed ratio of anhydride to amine (An/Am ratio), over 5 min under vigorous stirring. For example, for An/Am ratio of 0.7, 140 mg of succinic anhydride was added to 400 mg chitosan. After 1 hour of reaction, the pH of the reaction mixture was adjusted to 8-9 with 0.2 N NaHCO3. After overnight reaction at room temperature under stirring, the reaction mixture was dialyzed against water (molecular weight cut-off: 3500), whose pH was adjusted to 10-11 with 1 N NaOH. The purified '''succinic anhydride-conjugated low molecular weight chitosan (SALM-CS)''' was freeze-dried and stored at -20°C. 1H NMR spectra of SALM-CS dissolved in 2% CD3COOD/D2O were obtained on a Bruker ARX (300 MHz). The proton signals were recorded at 70°C. δ (ppm): 5.41 (m), 5.13 (m), 4.41-4.20 (br), 3.71 (t), 3.16 (t), 2.58 (s)<br> 

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Chitosan (15 kDa) was purchased from Polysciences, Inc. (Warrington, PA). Polyamidoamine (PAMAM) dendrimer generation 5 (G5) was purchased from Sigma-Aldrich (St. Louis, MO). Fluorescence dyes Flamma FPR-552, FPR-581, and FNG-456 were a gift from BioActs (DKC Corp., Incheon, Korea). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and all other cell culture supplies were purchased from Invitrogen (Eugene, OR) unless specified otherwise.<br>Synthesis of Zwitterionic Chitosan (ZWC)<br>Zwitterionic chitosan (ZWC) was synthesized by following previously established methods.32 In short, chitosan acetate was resuspended in deionized (DI) water, and succinic anhydride was added to the chitosan mixture while stirring. After overnight reaction, the solution was dialyzed (molecular-weight cut off: 3500 Da) against water maintaining pH 10–11, and the purified ZWC was lyophilized. ZWC was re-suspended in deionized water (DI water) and reacted with 30% H2O2 under vigorous stirring for 1h at room temperature to produce a lower molecular-weight ZWC. The reaction was quenched by the addition of methanol, and the resulting solution was purified by dialysis. The purified product was lyophilized and stored in −20°C. The low molecular-weight ZWC was used throughout this study.<br>

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