Although previous studies have demonstrated that PEI induces cytotoxicity [54, 55], our results (shown in Figure 2) revealed that in the range of concentrations used for siRNA transfection, PEI, and the rest of the tested materials did not promote cell death (at N/P ratios up to 60 viability of the cells was close to that of untreated ones) in both CHO-K1 and HeLa cells lines. However, above an N/P ratio of 200 all materials tested caused cell death (Figure 2). At an N/P = 200, the toxicity of all materials are indistinguishable from that of PEI. Figure 2 Effect of nanoparticle/siRNA (N/P)

ratio on metabolic activity in CHO-K1 ((a) and (b)) and HeLa ((c) and (d)) cell lines, as a function of polymer/siRNA Inhibitors,research,lifescience,medical (N/P) ratios. The cell viability was determined by MTS assay and was shown as the mean. Error bars … These results suggest that the dose-dependent and the observed differences Inhibitors,research,lifescience,medical in siRNA transfection efficiency among the nanoparticle vehicles (highlighted in Figure 1), are unrelated to cell viability. Furthermore, contrary to previous studies, siRNA was not toxic at the concentrations used Inhibitors,research,lifescience,medical in this study [56]. Next, we investigated the effects of the particles and polymers under study on the cell membrane

integrity (cytotoxicity) using the LDH assay (see Section 2). These experiments were carried out under similar conditions as the MTS assay, where CHO-K1 and HeLa cells were exposed Inhibitors,research,lifescience,medical to various N/P ratios of the NPs complexes. As shown in Figures 3(a) and 3(b), up to the N/P ratios of about 40 wherein optimum siRNA transfection was observed, PEI induced the most membrane damage to CHO-K1 cells. The remainder of the NPs possessed cytotoxicity ranging Inhibitors,research,lifescience,medical from 20 to 40%. Notably, PHMBG-M/SiO2-magnetofection

versus PHMBG-M/SiO2 showed an increase in cytotoxicity from 30 to 80% when the N/P ratio was increased from 10 to 20 due to the influence of the external magnetic field (Figure 3(b)). However, the external magnetic field did not significantly affect the cytotoxicity of PEI-M/SiO2. These results suggest that PEI’s siRNA transfection efficiency (Figure Sclareol 1(a)) could be due to disruption of the membrane (cytotoxicity). As shown in Figure 3(a), attaching cytotoxic PEI to the magnetic NPs reduced its cytotoxicity. At the highest N/P ratios employed, PEI and PEI-M/SiO2 with or CP-673451 in vivo without the external magnetic field significantly enhanced the membrane damage in CHO-K1cells, showing dose-dependent LDH release (Figure 3(a)). No NP dose dependence was observed on membrane permeability of CHO-K1 cells with PHMBG and PHMBG-M/SiO2 (except for PHMBG-M/SiO2-magnetofection, as previously mentioned—Figure 3(b)). In contrast, for HeLa cells all materials used in the study (with and without an external magnetic field) showed dose-dependent LDH release (Figures 3(c) and 3(d)).