Evaluation of cytotoxicity

Figure 7 shows the distinction of the “live/dead” dye-stained SK-BR3 and FB cells cultured in the presence of GNP and GNP–Her after 3 days of incubation. Using this qualitative method, the living and the dead cells were stained green and red, respectively, and were visible by fluorescence microscopy.

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Figure 7A shows that all the FB cells remained viable after 3 days’ incubation, in the presence of NPs. On the other hand, after culturing for 3 days, in the presence of GNP–Her, most of the SK-BR3 cells had died, as shown in Figure 7B.

Figure 8 shows the viability of SK-BR3 cells cultured for 1 day and 3 days in the presence of GNP–Her, as determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay. After 1 day and 3 days of incubation, the level of SK-BR3 cell proliferation in the presence of GNP was similar to that of the cells cultured in the absence of NPs (polystyrene dish).

On the other hand, cell propagation in the presence of GNP–Her was significantly lower than that in the presence of GNP.

Therefore, the GNP conjugated with herceptin could increase the death of SK-BR3 cells considerably compared to the GNP without herceptin.

A probable explanation of this large decrease in cell viability in the case of GNP–Her is that intracellularly distributed herceptin shows significant apoptotic activity by networking with several transcription factors related to cell propagation.

Formerly, Bae et al30 stated that degradable heparin nanogels and heparin/chitosan polyelectrolyte nanocomplexes could effectively induce apoptosis via receptor-mediated endocytosis through specific herceptin–HER2–integrin interaction.

The endocytosed GNP–Her within the cells discharge free herceptin molecules in the cytoplasm by slicing the GNP–herceptin linkage under the reductive intracellular environment, which has a 300 times greater glutathione concentration than extracellular medium.28

Glutathione is the main abundant reducing agent in the cytoplasm, helping in the dissipation of herceptin from the GNP by breaking the MSA–herceptin linkage. Furthermore, NPs are taken up by the cells through endocytosis, which disrupts the cell membrane, or weak cell adhesive interactions with GNP may encourage apoptosis.

GNPs conjugated with herceptin may act as cellular markers and target the receptors expressed on the cell exterior, with cellular internalization.31

The receptors are highly controlled by the cell surface proteins, which facilitate the specific communication between cells and their extracellular environments, and they are usually confined on the plasma membrane.32