**4. Discussion**

Polystyrene μPs were successfully biofunctionalized with an anti-HER2 antibody or with a secondary antibody, as demonstrated by the detection of fluorescence under a microscope and by changes in their ζ-potential. The reduction in μPs electronegativity could help in their interaction with the plasma membrane, which contains negatively charged saccharides. In fact, it has been described that positively or slightly negatively charged particle surfaces favor cell intake [14,33–35].

The cell lines used in this study are isogenic breast epithelial cell lines. D492 was established by isolating suprabasal cells from a reduction mammoplasty and subsequently immortalizing them using the *E6* and *E7* oncogenes from the human papilloma virus [30,31]. D492 has stem cell properties evaluated by the ability to generate luminal and myoepithelial cells and, in 3D culture, to form branching ductal–alveolar-like structures. D492HER2 was generated by overexpressing the HER2 oncogene in D492 [32]. Interestingly, D492 showed higher percentages of μPs internalization than D492HER2. A possible explanation could be the differences in phenotype between these cells. D492HER2 cells undergo an epithelial to mesenchymal transition (EMT) and express mesenchymal markers, which could reduce their ability to internalize μPs [31,32]. Higher rates of μP internalization have also been described in MCF10A breast epithelial normal cells than in SKBR3 breast epithelial tumoral cells, which also overexpress HER2 [14].

The molecules used to bio-functionalize the μPs also influenced the internalization rates. For both cell lines, the percentage of cells with internalized μP-antiH was higher than that of cells with internalized μP-secAb. This result was expected, since we hypothesized that a specific recognition between the cells and the μPs, such as that afforded by antiH biofunctionalization, could result in an increase in μP internalization. Also as expected, the increase in the percentages of μPs internalization obtained with antiH biofunctionalization was higher for D492HER2 cells, which overexpress HER2, than for D492 cells, which express HER2 at much lower levels [32]. These results clearly indicate that when μPs are biofunctionalized with molecules that specifically interact with plasma membrane receptors, their internalization can be improved, and the level of improvement is related to the number of receptors that are present on the cell surface.

Finally, in relation to the culture conditions, our results indicate that the percentage of cells with internalized μPs clearly decreased in fluidic conditions compared with static conditions. This was probably because the establishment of prior transient contacts between the plasma membrane and the μPs may be more difficult in fluidic conditions or in high shear stress conditions [36,37]. One can hypothesize that in the case of dynamic cultures, specific targeting may be helpful to improve the internalization rates, as it may facilitate the frequency and strength of μPs-cell contacts. Our results agree with this hypothesis because, for both cell lines, the increase of μPs-antiH internalization was higher in fluidic than in static cocultures when compared with μPs-secAb internalization, which opens the door to scale-up studies.

The present results, together with previous research, emphasize the relevance of cell type, specific targeting, and culture conditions such as mono- or cocultures and static or fluidic, to the potential application of NPs and μPs for drug delivery to cancer cells.
