Efficient siRNA delivery based on PEGylated and partially quaternized polyamine nanogels: enhanced gene silencing activity by the cooperative effect of tertiary and quaternary amino groups in the core.

For the development of an siRNA delivery system using polyion complexes (PICs) based on PEGylated nanogel consisting of a cross-linked poly[2-(N,N-diethylaminoethyl) methacrylate] (PEAMA) gel core and tethered poly(ethylene glycol) (PEG) chains, quaternary ammonium groups were introduced in the polyamine gel core to enhance the binding ability with siRNA and the stability of the PICs. Consequently, the quaternization of the polyamine core of the nanogel facilitated the binding ability with siRNA at a low N/P ratio, and the stability against polyanion displacement was enhanced as the degree of quaternization (DQ) of the nanogel increased. Although the installation of the positively charged quaternary ammonium moieties in the core of the nanogel resulted in the increment of the xi-potential of the PICs (e.g. + 23 mV for DQ=100%), the cytotoxicity was reduced with the increase of DQ presumably due to the hydrophilic character of the quaternary ammonium groups. The installation of quaternary ammonium groups in the core of the nanogel enhanced the endogenous gene silencing activity against the survivin gene in human hepatocarcinoma (HuH-7 cells), especially, the partly quaternized polyamine nanogel (DQ=10%) showed the highest gene silencing ability among the quaternized polyamine nanogels, including the tertiary amine nanogel. The cellular uptake analysis of the Rhodamine B-labeled Q-nanogel/fluorescein-labeled siRNA complex revealed that the quaternization of PEAMA moieties enhanced the cellular uptake level of fluorescein-labeled siRNA with the increase in DQ, whereas the cellular uptake of the Rhodamine B-labeled Q-nanogels was almost of the same level regardless of the DQ value, indicating that significant cellular uptake of the fluorescein-labeled siRNA is most likely due to the enhancement of the binding ability with siRNA in the serum-containing medium. Note that the endosomal escape efficiency was reduced with increase in the DQ value due to the decrease in the buffering capacity (tertiary amino groups) of the PEAMA core. On the basis of these results, the ratio of quaternary ammonium groups to tertiary amino groups in the core of the nanogel plays a pivotal role in the achievement of significant gene silencing through enhanced cellular uptake (quaternary ammonium groups) and subsequent endosomal escape (tertiary amino groups).