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.