The peptide dendrimer KK-46 enhances transfection efficiency
A novel formulation KK-46 based on a peptide dendrimer was developed for safe and efficient nucleic acid delivery. In order to evaluate the transfection activity of KK-46 (Fig. 2e), Hep-2 cells were transfected with mixtures of pGL3 Luciferase Reporter Vectors (Promega)/ peptide dendrimer KK-46 at weight ratios of 5:1,12.5:1, 20:1, 25:1, 50:1, 100:1 (KK-46:pGL3). The commercial transfection reagent Lipofectamine 3000 was used as positive control. As shown in Figure 2e the weight ratios 20:1 and 25:1 yielded the highest levels of luciferase activity compared to ratio 5:1 (P<0.01) and for the 25:1 ratio it was even significantly higher than with commercial Lipofectamine 3000 (P<0.05). The half maximal inhibitory concentration (IC50) of KK-46 measured by MTT test for Vero cells was 548±23 μg/mL (data not shown). Thus, the optimal concentration of KK46 at 20-25 μg/mL was much lower than IC50. A testing of KK46 regarding its ability to block the RBD-ACE2 interaction showed no inhibition up to 100 μg/mL (data not shown).
Modified siR-7-EM shows increased nuclease resistance
Introduction of LNA into classic antisense oligos has been shown to increase their stability(32). Therefore, we designed LNA-modified siR-7-EM to test if these modifications could improve the properties of siR-7 for in vivo application. Stability of unmodified siR-7 and LNA-modified siR-7-EM was evaluatedin vitro by incubation in 50% mouse serum for 264h at 37°C. As shown in Figure 3, unmodified siR-7 was markedly degraded after 48h of incubation. In contrast, LNA-modified siR-7-EM did not show signs of degradation even after 264h of incubation. Moreover, we found that the half-life (t1/2) of siR-7 and siR-7-EM were 72.4 and 256.8 h, respectively (Fig. 3a). These experiments showed that incorporation of LNA molecules in siR-7 contributes to increased half-life and thus may have a positive influence on overall biostability.