Quick nuclear transportation of siRNA and in vivo hepatic ApoB gene silencing with galactose-bearing polymeric carrier.

Since previous studies have linked the genetic mutations of Apolipoprotein B (ApoB) to the low density lipoprotein (LDL) cholesterol levels, it can be believed that the knockdown of ApoB by siRNA silencing is a useful method to reduce the cardiovascular disease. However, the spontaneous uptake of siRNA is hindered, and thus vectors are necessary to aid its transfer into the cells. Among the synthetic non-viral vectors, cationic polymers are extensively investigated as possible candidates for efficient and specific gene delivery, because they can be easily modified to get different set of properties. Therefore, in this work a set of random copolymers with different molecular weight and composition were synthesized. These vectors present 2-(dimethylamino)ethyl methacrylate, as cationic monomer, and galactose units as liver-targeting moieties. From in vitro experiments, copolymers with monomer ratio and molecular weight about 0.1 and 80kDa, respectively, showed adequate transfection capabilities and displaying good cell viability, independently of the nature of the saccharides units. However, in the in vivo experiments in C57BL/6 high-fat-fed mice, a better blood compatibility and protection against degradation leading to better transfection by the random copolymers bearing galactose units was confirmed.

Effects of CC chemokine receptor 5 (CCR5) inhibitors on the dynamics of CCR5 and CC-chemokine-CCR5 interactions.

BACKGROUND: This study aimed to examine how CC chemokine receptor 5 (CCR5) inhibitors (aplaviroc [APL], TAK779 and maraviroc [MVC]) interact with CCR5 and affect its dynamics and physiological CC-chemokine-CCR5 interactions. METHODS: A yellow fluorescent protein (YFP)-tagged CCR5-expressing U373-MAGI cell line was generated and a stable CCR5-expressing clonal population, (YFP)CCR5-UM16, was prepared. (YFP)CCR5-UM16 cells were exposed to RANTES, macrophage inflammatory protein (MIP)-1alpha or MIP-1beta (all 100 ng/ml) with or without CCR5 inhibitors and (YFP)CCR5 internalization was visualized with real-time by laser scanning confocal microscopy. The mobility of (YFP)CCR5 was also examined in the presence of CCR5 inhibitors with fluorescence recovery after photobleaching (FRAP) imaging. RESULTS: Following the addition of each CC chemokine, intracellular fluorescence intensity increased whereas membranous fluorescence decreased, signifying (YFP)CCR5 internalization. All three CCR5 inhibitors failed to induce (YFP)CCR5 internalization. All three CCR5 inhibitors blocked the CC-chemokine-induced internalization at a high concentration of 1 microM; however, the ratio of APL concentration that blocked RANTES-induced internalization by 50% over APL concentration that blocked HIV type-1 (HIV-1) replication by 50% was 16.4, indicating that APL permits CC-chemokine-induced internalization to a much greater extent compared with TAK779 and MVC, having ratios of 1.1 and 0.9, respectively. The examination of (YFP)CCR5 mobility with FRAP imaging revealed that (YFP)CCR5 continuously underwent rapid redistribution, which none of the three inhibitors blocked. CONCLUSIONS: The finding that APL moderately blocked the RANTES-triggered (YFP)CCR5 internalization despite the highly potent anti-HIV-1 activity of APL strongly suggests that development of CCR5 inhibitors, which do not overly inhibit physiological CC-chemokine-CCR5 interactions, is practically feasible.

Liver-targeted siRNA delivery by polyethylenimine (PEI)-pullulan carrier.

Recently, small interfering RNA (siRNA)-based therapeutics have been used to treat diseases. Efficient and stable siRNA delivery into disease cells is important in the use of this agent for treatment. In the present study, pullulan was introduced into polyethylenimine (PEI) for liver targeting. PEI/siRNA or pullulan-containing PEI/siRNA complexes were delivered into mice through the tail vein either by a hydrodynamics- or non-hydrodynamics-based injection. The incidence of mortality was found to increase with an increase in the nitrogen/phosphorus (N/P) ratio of PEI/siRNA complexes. Moreover, the hydrodynamics-based injection increased mice mortality. Introduction of pullulan into PEI dramatically reduced mouse death after systemic injection. After systemic injection, the PEI/fluorescein-labeled siRNA complex increased the level of fluorescence in the lung and the PEI-pullulan/siRNA complex led to an increased fluorescence level in the liver. These results suggest that the PEI-pullulan polymer may be a useful, low toxic means for efficient delivery of siRNA into the liver.