Download Stabilization of poly-L-lysine-based cancer

Stabilization of poly-L-lysine-based cancer-specific vectors via
disulfide bond formation
Kai LI1, Guoxi ZHAO1 ,Takeshi MORI1,2,3, Akihiro Kishimura 1,2,3,Yoshiki KATAYAMA1,2,3,
1
Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 2The Center for Future
Chemistry, Kyushu University, 3International Research Center for Molecular Systems, Kyushu University
Introduction
We have proposed a novel gene targeting strategy, called “drug or gene delivery system
responding to cellular signals (D-RECS)”. The systems can activate transgene in response to
malfunctions of signal transduction occurring inside diseased cells. In the precious study we
succeed in activating gene expression in response to several protein kinase1). However, the
polyplex formed from polymeric carrier and plasmid DNA unfavorably dissociates in blood
circulation. A promising approach to stabilize the polyplex will be reversibly cross-linking of
the polyplex with disulfide bonds. Here a new gene carrier, poly-l-lysine (PLL) is modified with
substrate peptide that can be phosphorylated by PKC and is also modified with
15-Carboxy-1-pentadecanethiol (15-CPDT) that can form disulfide bond to increase the stability
of polyplex in blood circulation.
Experiment
PLL (Mw.15000-30000) was simply modified by peptide and 15-CPDT using HOBt and
HBTU. After that, the protection group was removed by TFA. Four kinds of polymers with
constant peptide modification ratio (5 mol%) and different 15-CPDT modification ratios (0, 5,
10 and 20 mol%) were prepred. The stability of complex that polymer and plasmid DNA formed
(N/P=10) was evaluated by gel retardation analysis.
Result and Discussion
The release of pDNA from the by oxidation of
disulfide bond was examined.
Figure 1 shows the gel retardation assay of
polyplexes fromed from each polymer. Free pDNA
become weaker with the increase of 15-CDPT
contents. On the contrary, the free pDNA bands were
observed for reductive condition containing 20 mM
DTT (lane 7-9). These results indicated that the
disulfide cross-linking can stabilize the polyplexes
and the polyplex can be weakened at the reductive
condition
1) Riki Toita, et al, J. Am. Chem. Soc., 134, 1540-15417(2012)