Editing Synthesis of Chitosan-Tat

&nbsp;Synthesis of chitosan/DOX/TAT conjugates<br>Chitosan solution [m.w. 2000, 1 mg/mL in PBS (pH 7.4, 1.05<br>mM KH 2 PO 4 , 155 mM NaCl and 2.9 mM Na 2 HPO 4 �7H 2 O)]<br>was allowed to react with N-succinimidyl S-acetylthiopropio-<br>nate (SATP, 50 mM in PBS; Pierce) and was then reacted<br>with 50 mM hydroxylamine solution (10 mM EDTA in PBS)<br>to generate the sulfhydryl group in chitosan. The unreacted<br>SATP was removed by ultrafiltration using a 1.0-K molecular<br>weight cutoff membrane. The sulfhydryl content was assayed<br>using Ellman’s Reagent (Pierce) according to the manufac-<br>turer’s instructions. The chitosan/DOX conjugate was gener-<br>ated by linking the sulfhydryl groups in chitosan and DOX<br>with a heterobifunctional crosslinker, succinimidyl 4-(N-mal-<br>eimidomethyl) cyclohexane-1-carboxylate (SMCC; 50 mM in<br>PBS; Pierce). A volume of 20 lL of triethylamine and 300 lL<br>of SMCC (18 mg/ml in DMSO) were added to the DOX so-<br>lution (0.2 mg/mL in PBS, pH 8). The mixture was incubated<br>at room temperature for 2 h. After adjusting the pH of the<br>mixture to 5.5, 2 mL of sulfhydryl chitosan solution was<br>added (1.5 mg/mL in PBS). The mixture was incubated at<br>room temperature for 2 h and then kept at 4 � C overnight.<br>The free DOX, SMCC and triethylamine were removed from<br>the chitosan/DOX solution via ultrafiltration using a 1.0-K<br>molecular weight cutoff membrane. After adjusting the pH of<br>the chitosan/DOX solution to 5.5, the TAT peptide (1.0 mg/<br>0.2 mL PBS) was added. The mixture was incubated at room<br>temperature for 2 h and then kept at 4 � C overnight. The chi-<br>tosan/DOX/TAT conjugates were separated from free chito-<br>san, chitosan/DOX and TAT by passing the mixture through<br>a HiTrap heparin affinity column connected to a FPLC with<br>a gradient elution containing a 2.0 M NaCl solution. 23 Elu-<br>tion profiles were determined by absorbance at 280 nm. The<br>column was eluted at a flow rate of 1 mL/min, while the<br>NaCl gradient was increased at a rate of 50 mM/min. Salts<br>in the collected chitosan/DOX/TAT conjugate fraction were<br>removed by passing the sample through a desalting column<br>and washing with PBS. Successful production of the chitosan/<br>DOX/TAT conjugate was confirmed by SDS-PAGE.





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