Paul.xu: Created page with " Synthesis of chitosan/DOX/TAT conjugates
Chitosan solution [m.w. 2000, 1 mg/mL in PBS (pH 7.4, 1.05
mM KH 2 PO 4 , 155 mM NaCl and 2.9 mM Na 2 HPO 4 �7H 2 O)]
wa..."

2014-07-21T03:08:09Z

Created page with " Synthesis of chitosan/DOX/TAT conjugates Chitosan solution [m.w. 2000, 1 mg/mL in PBS (pH 7.4, 1.05 mM KH 2 PO 4 , 155 mM NaCl and 2.9 mM Na 2 HPO 4 �7H 2 O)] wa..."

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

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Synthesis of Chitosan-Tat - Revision history

Paul.xu: Created page with " Synthesis of chitosan/DOX/TAT conjugatesChitosan solution [m.w. 2000, 1 mg/mL in PBS (pH 7.4, 1.05mM KH 2 PO 4 , 155 mM NaCl and 2.9 mM Na 2 HPO 4 �7H 2 O)]wa..."

Paul.xu: Created page with " Synthesis of chitosan/DOX/TAT conjugates
Chitosan solution [m.w. 2000, 1 mg/mL in PBS (pH 7.4, 1.05
mM KH 2 PO 4 , 155 mM NaCl and 2.9 mM Na 2 HPO 4 �7H 2 O)]
wa..."