Synthesis of Chitosan-Tat
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.