Estimation of intracellular uptake

The quantity of GNP–Her taken up by SK-BR3 cells was estimated by ICP–MS and the outcomes are presented in Figure 9. After herceptin conjugation, the NPs’ interaction with SK-BR cells was amplified significantly, in contrast to that of the original NPs, and increased to 1.4 pg per cell within the first day of culture, much higher than that of the original NPs.33

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The peak internalization of GNP–Her occurred at 4.6 pg per cell after 3 days of culture. The cause for the uptake enhancement might be ligand–receptor interactions on the cell membrane.34

Moreover, the greater uptake of herceptin-modified GNPs indicates that the alteration not only enabled the NPs to target specific cells but also improved the cell internalization yield.

Similar results obtained from (carboxymethyl)chitosan-modified superparamagnetic iron oxide nanoparticles were shown by Shi et al35 in their reports; the quantity of NPs internalized in human muscle-derived cells is much more than that of superparamagnetic iron oxide nanoparticles, as observed by ICP–MS.

The interaction between herceptin and SK-BR3 receptors expressed on the membrane might have contributed to the enhancement of GNP–Her internalization based on receptor-mediated endocytosis.36

The internalization of GNP–Her into SK-BR3 cells was also confirmed by CLSM. Figure 10 shows that herceptin is an effective antibody, binding specifically to the HER2-bearing breast tumor cells.

The internalization of GNP–Her into SK-BR3 cells was confirmed by CLSM to characterize the delivery of GNP–Her to the cytoplasm of the SK-BR3 cells.

The fluorescence image was derived from the nucleus of the SK-BR3 cells (DAPI, blue) and the internalized GNP–Her (red). The cells were cultured in the presence of GNP–Her at various incubation times. Weak GNP–Her conjugates were observed in the fluorescence image (red color) after 1 hour (Figure 10A) and slightly higher fluorescence was observed after 3 hours (Figure 10B). Intense fluorescence was noted after 6 hours (Figure 10C).

On the other hand, the blue fluorescence derived from the nuclei stained with DAPI was strong after 1 hour of incubation, but it decreased with increasing incubation time and almost disappeared after 6 hours of incubation.

In particular, the interaction of SK-BR3 with GNP–Her began after 1 hour of incubation and was accelerated and saturated after 6 hours. The CLSM images suggest that the NP-mediated delivery of monoclonal antibodies was achieved efficiently, resulting in cell death.36

The mechanism of internalization involves endocytosis, followed by the release of herceptin-conjugated NPs to the cytoplasm.29,37

This suggests that the growth signal of SK-BR3 cells is withdrawn completely by the specific binding of herceptin to the receptor on SK-BR3 membrane, resulting in cell death.35,36