G.J. Lee, K. Ryu, T.I. Kim
Seoul National University,
Keywords: bioreducible polymer, crosslink, cytotoxicity, gene delivery system, polypropylenimine
Summary:Polypropylenimine (PPI) dendrimers and their derivatives have been used for gene delivery because of their properties such as cationic charge, equivalent surface functionalities, and internal cavities [1–4]. However, high generation PPIs made high cytotoxicity induced by the interaction with cell membranes; it limits their applications in vivo . Generally, polymeric gene carriers with high molecular weights showed high transfection efficiency but also high cytotoxicity. Therefore, many studies also reported that polymeric gene delivery carriers composed of low molecular weight polymers with biodegradable linkages showed high transfection efficiency and low cytotoxicity due to enhanced gene condensation and intracellular degradation . Especially, bioreducible crosslinkers have been used for bioreducible polymeric gene carriers. Bioreducible polymeric gene carriers, which have internal disulfide bonds, possess many advantages for gene delivery systems, including selective degradability and release control of condensed genes from polyplexes in reducing environment [7–9] such as cytoplasm containing glutathione with high concentration (0.5–10 mM) . They also have been reported that they have low cytotoxicity by avoiding accumulation and undesirable interaction of toxic cationic molecules via degradation in cytoplasm. From now on, a variety of crosslinked polymeric gene carrier possessing bioreducible linkages have been reported. However, crosslinked PPI dendrimers with bioreducible linkages have not been reported for gene delivery systems to our knowledge so far. Therefore, in this work, we synthesized high molecular weight bioreducible polymers (PPI–CBAs) by crosslinking low molecular weight PPI dendrimers with bioreducible CBA using Michael-type polyaddition in order to utilize the advantages of PPI dendrimers, achieving high transfection efficiency and maintaining low cytotoxicity. The physicochemical properties of the polymers were characterized and their potency for gene delivery systems was evaluated. The polymers could form nano-sized polyplexes, which show high stability even in reducing condition probably because of the re-crosslinking in the polymers, unlike linear bioreducible polymers. They displayed lower transfection efficiency than PEI25k in the absence of serum but high transfection efficiency comparable to PEI25k in the presence of serum. Actually, interesting results about the degradation behaviors and cytotoxicity of PPI–CBAs were observed and they were further studied. The cytotoxicity of the polymers is increased with the increase in concentration and crosslinking degrees of them. The decrease of glutathione (GSH) levels and the following increase of reactive oxygen species (ROS) levels are also observed with the increase in concentration and crosslinking degrees of the polymers. PPI-CBA polyplexes also could induce the similar effects with less degree. These results suggest that the cytotoxicity of bioreducible polymers may be closely related with the polymer structures and that reduction of intracellular GSH quantity due to degradation of re-crosslinked bioreducible polymers and resulting increase of intracellular ROS quantity may cause cytotoxicity by oxidative stress.