**3. Results**

### *3.1. Microparticles Characterization after Biofunctionalization*

Biofunctionalization did not affect the size of μPs, as can be observed in TEM images (Figure 1A). Moreover, biofunctionalization was confirmed in two ways, microscopically and by analyzing the ζ-potential. As expected, under fluorescence microscopy, μP-secAb emitted far-red fluorescence, and μP-antiH emitted green fluorescence after incubation with an Alexa® 488-conjugated secondary antibody (Figure 1B). Biofunctionalization was also confirmed by changes in the μP surface charge. Non-biofunctionalized polystyrene carboxylate μPs (μP-COOH) showed a ζ-potential value of −32.3 mV, whereas μP-secAb and μP-antiH increased their ζ-potential to smaller negative values of −11.23 mV and −11.5 mV, respectively (Figure 1C).

**Figure 1.** Characterization of microparticles (μP) biofunctionalization. (**A**) Transmission electronic microscopy (TEM) images of microparticles before (COOH) and after biofunctionalization (μP-secAb and μP-antiH). (**B**) Images of microparticles biofunctionalized with a secondary antibody (μP-secAb) or an anti-HER2 antibody (μP-antiH) in bright-field (upper panels) and fluorescence (lower panels) microscopy. (**C**) Zeta potential before (COOH) and after biofunctionalization (μP-secAb and μP-antiH).

### *3.2. Microparticles Internalization by Cells*

The internalization of μPs by cells was evaluated through the orthogonal images captured by a CLSM in both static and fluidic culture conditions (examples in Figures 2 and 3, respectively). Staining of actin filaments was useful to visualize the cell perimeter and, together with the orthogonal projections, allowed us to clearly distinguish between internalized and non-internalized μPs. From these images, the number of cells with at least one internalized μP was scored.

**Figure 2.** Immunofluorescence analysis by confocal laser scanning microscope (CLSM) of cells cultured in static conditions. Confocal images of D492 and D492HER2 cells cocultured in static conditions and incubated with microparticles biofunctionalized with a non-specific secondary antibody (μP-secAb) or a specific anti-HER2 antibody (μP-antiH). Cells, constitutively expressing green fluorescent protein (GFP, green), were incubated with Alexa Fluor® 546 Phalloidin (red) to label actin microfilaments and Alexa Fluor® 405 conjugate secondary antibody (blue) to label HER2 in the plasma membrane. The arrows point to some examples of μPs located inside the cells.

**Figure 3.** Immunofluorescence analysis by CLSM of cells cultured in fluidic conditions. Confocal images of D492 and D492HER2 cells cocultured in fluidic conditions and incubated with microparticles biofunctionalized with a non-specific secondary antibody (μP-secAb) or a specific anti-HER2 antibody (μP-antiH). The cells, constitutively expressing GFP (green), were incubated with Alexa Fluor® 546 Phalloidin (red) to label actin microfilaments and Alexa Fluor® 405 conjugate secondary antibody (blue) to label HER2 in the plasma membrane. The arrows point to some examples of μPs located inside the cells.

As can be seen in Figure 4, in all conditions, the percentage of D492 cells with internalized microparticles was always higher than that of D492HER2 cells, indicating that D492 cells have an inherent superior capacity to internalize microparticles. Regarding the importance of specific biofunctionalization in μPs recognition and intake by the cells, the internalization related to non-specific binding due to the intrinsic cell endocytic capacity, was represented by the percentage of cells with internalized μPs biofunctionalized with the non-specific antibody (μPs-secAb). In contrast, the internalization related to the specific recognition of μPs by the cells was represented by the increase in the percentage of cells with internalized μPs when these were specifically functionalized

(μP-antiH) to recognize a cell membrane receptor (HER2). For both cell lines, the biofunctionalization with a specific targeting antibody (antiH) resulted in higher internalization percentages than the biofunctionalization with a non-specific targeting antibody (secAb). The differences between static monoculture and coculture conditions were not significant. As expected, fluidic culture conditions globally decreased internalization, but again internalization was higher for microparticles which were specifically biofunctionalized (μP-antiH) than for those that were biofunctionalized with a non-specific antibody (μP-secAb). The increase in the percentage of cells with internalized μP-antiH was also higher for cells with HER2 overexpression (D492HER2) than for cells without HER2 overexpression (D492) (Figure 4). Remarkably, the increase in the percentage of cells with internalized μP-antiH with regard to μP-secAb internalization was higher in fluidic conditions than in static conditions, especially in D492HER2 cells (164% versus 77–99% in D492HER2 cells, and 100% versus 22–35% in D492 cells).

**Figure 4.** Microparticles internalization by monocultured or cocultured D492 and D492HER2 cells in static and fluidic conditions. Percentages of cells with internalized microparticles biofunctionalized with a non-specific secondary antibody (μP-secAb) or a specific anti-HER2 antibody (μP-antiH). Statistically significant differences are indicated with different letters on top of the bars. An increase of μPs internalization, as a percentage, is indicated in blue together with blue arrows.
