*2.1. Cellular Internalization*

Nanomedicines based on NPs must cross the cell membrane by different mechanisms (phagocytosis, micropinocytosis, clathrin-dependent endocytosis, caveolae-dependent endocytosis, or by direct penetration) to produce an effect inside the cells [16] because, usually, cell membranes are impermeable to NP diffusion. Moreover, non-specific internalization mechanisms may induce toxicity. Since a lack of biocompatibility is desirable, it becomes a suitable mechanism for internalization [17].

As expected, the NP size directly affects the internalization process. In fact, NPs in the range of 10–100 nm achieve higher cellular uptake and, on the other hand, small ones imply a higher energy cost to the cells [18]. Usually, NPs larger than 100 nm are internalized by specialized phagocytic cells (such as macrophages, dendritic cells) which allows targeted design.

Furthermore, the optimal size for internalization inside the cells is strongly linked to the NPs surface chemistry. In general, Van der Waals or electrostatic forces are critical in the NP interactions with biomolecules and cells. In fact, several studies show correlation between zeta potential and endocytosis/exocytosis mechanisms [19]. Then, specific cellular internalization could be targeted to favor specific interactions (i.e., employing affinity ligands) as opposed to nonspecific interactions (i.e., hydrophobic). In this sense, antibody-coated NPs present an internalization potential in targeted cells four to eight folds higher than positively or negatively charged NPs without the affinity component [20]. Besides the use of antibodies for targeting delivery, non-specific interactions through chemical moieties are always present and influence target affinities, which must be always taken into consideration.

Protein adsorption also depends on the NP shape and, consequently, affects the cellular uptake. It seems that spherical and highly homogeneous NP conjugates have better cellular uptake than amorphous and non-geometrically symmetric nanoconjugates [21]. Moreover, several authors claim that shape could be employed to prevent non-specific cellular internalization in the targeted cells [19].
