In vitro comparative evaluation of three CLD/siRNA nanoplexes prepared by different processes

In this study, a gemini-like cationic lipid （CLD） was used as the carrier to study the complexation features of CLD/ siRNA nanoplexes （CLD/siRNA NPs）. Three types of CLD/siRNA nanoplexes （named as AT NPs, HT NPs and MT NPs） were prepared by different processes： AT method （mixing siRNA solution with preformed CLD nanoparticles）, HT method （hydrating a CLD thin film with siRNA solution）, and MT method （dropping an ethanolic solution of CLD into siRNA solution under sonication）. The particle size, zeta potential, morphology, siRNA protection, cytotoxicity, cellular uptake, and targeted mRNA downregulation were studied. At the optimal N/P ratio of 10, the sizes of the three CLD/siRNA NPs were MT NPs （（222.3±19.1） nm）〉 HT NPs （（105.7±1.31） nm）〉AT NPs （（91.8±1.75） nm）. Different nanostructures were formed despite the fact that they were composed of the same components. Furthermore, the TEM images indicated that different morphologies were found in the three NPs, indicating that the nanoplexes were assembled by different mechanisms. Among the three NPs, the cell uptake capacity were as follows： AT NPs〉MT NPs〉HT NPs, whereas the silencing levels on epidermal growth factor receptor （EGFR） in HeLa cells were MT NPs〉AT NPs〉HT NPs. Based on the above results, we hypothesized that the different preparation processes resulted in nanostructures with varying biological effects. Therefore, we believe that structural optimization of siRNA nanoplexes is essential in achieving better siRNA encapsulation, protection, and gene silencing efficiency.

Novel cationic lipid with reduction-responsive cleavable hydrophobic tail for siRNA delivery

To achieve a higher transfection efficiency and lower toxicity, a novel herringbone-like cationic lipid（2 ss HLL） composed of hydrophilic aspartic acid linked with two reduction-responsive cleavable hydrophobic oleic acid tails was synthesized and assessed in this study. In our results, the cationic nanoplexes with a uniform spherical shape and a particle size of ~150 nm were successfully prepared by the electrostatic interaction between si RNAs and 2 ss HLL-based liposomes. From the results evaluated in Hep G2 cells, it was shown that the nanoplexes exhibited high cellular uptake of si RNA with a low cytotoxicity. Moreover, the significant down-regulation effects of 2 ss HLL/si EGFR nanoplexes on target m RNA were displayed by RT-PCR analysis, which were similar to those of Lipofectamine2000. It suggested that the enhanced si RNA gene silencing efficiency was probably attributed to the detachment of hydrophobic tail chains induced by reduction-responsive cleavage. This mechanism was also confirmed by the changes of size distribution and si RNA release of nanoplexes in the reductive environment and DTT-absence condition. Overall, we believed that the redox-active herringbone-like 2 ss HLL would be a potential nanocarrier towards si RNA delivery.