In vitro cytotoxicity and transfection assay
In vitro cytotoxicity of blank nanoparticles and siRNA-FAM/PTX encapsulated nanoparticles were compared after 48 h. As it was seen in Fig. 8A, the siRNA/PTX-free nanoparticles (NPsA, NPsB, and NPsAB) showed good biocompatibility, and no remarkable cytotoxicity was observed after 48 h of incubation with MCF-7 cells. However, its toxicity significantly increased after encapsulation of siRNA/PTX in the nanoparticles (NPsA/siRNA/PTX, NPsB/siRNA/PTX, and NPsAB/siRNA/PTX). As it can be seen in Fig. 8B, siRNA has no toxicity effect on MCF-7 cells. Therefore, it can be concluded that the decrease in cell viability might be due to the effect of PTX on MCF-7 cells. The IC50 value of NPsAB/siRNA/PTX was significantly lower than NPsA/siRNA/PTX and NPsB/siRNA/PTX after 48 h of incubation. Several studies have demonstrated the effect of folic acid and glucose conjugated nanoparticles to increase drug transfer efficiency (Jain, Rathi, Jain, Das, & Godugu, 2012; Macheda, Rogers, & Best, 2005; Zhang et al., 2013).
However, this improvement could be continuing until the saturation of folic acid receptors on the surface of cancer cells by nanoparticles. In other word, after saturating the folic acid receptors by nanoparticles, nonspecific uptake of nanoparticles into healthy tissues increases. The higher cytotoxicity of free PTX may be due to unrestricted and rapid diffusion of PTX into MCF-7 cells, while the nanoparticles are internalized into cancer cells via the receptor-mediated endocytosis pathway (S. Wang et al., 2013). In addition, sustained release of PTX from these nanoparticles due to the presence of polylactic acid in their structure may be another reason for low cytotoxicity of the nanoparticles compared to free PTX.
Fluorescence microscopy was used to evaluate the ability of the nanoparticles for co-delivery of nucleic acid and drug into MCF-7 cells.
Fig. 8 C-E shows fluorescence images of MCF-7 cells after 48 h of incubation with NPsA/siRNA/PTX, NPsB/siRNA/PTX and NPsB/siRNA/PTX nanoparticles. The fluorescence emission observed within the cells indicated the ability of the nanoparticles to transfer siRNA-FAM into MCF-7 cells. Moreover, the circular morphology of MCF-7 cells after incubation with the nanoparticles may be happened since the induction of apoptosis in MCF-7 cells byNPsA/siRNA/PTX, NPsB/siRNA/PTX and NPsB/siRNA/PTX nanoparticles.