## 3 Results and Discussion
### Smaller Particle Conglomerates Are More Likely to Be Internalized by the Cells  
Owing to the ambiguities surrounding the exact location and geometries of nanoparticle clusters overlapping with cellular boundaries, all measurements used to test the proposed hypotheses were performed on fluorescent optical images of cells in interaction with HAp nanoparticles. The results of the testing of the relationship between the cellular uptake of the nanoparticle agglomerates and their sizes demonstrated a clear tendency for the smaller clusters to undergo a more abundant intracellular localization than the larger ones. Specifically, as seen in Figure 5a, the average size of intracellularly located nanoparticle agglomerates was significantly lower than that of the extracellularly located ones. Meanwhile, the cell granularity following the overnight treatment with HAp nanoparticles was in excess of 98% (Figure 5b), indicating that the great majority of cells did uptake large enough amounts of nanoparticles to display significantly higher granularities than the untreated cells. The literature data overwhelmingly agree with this trend, as indicated by the results of the meta-analysis presented in Figure 5c and showing that in all but one report analyzed, the smaller particles were taken up more efficiently than the larger ones. By this, in both the experimental and the literature analyses, it is meant that the smaller particles outnumber the larger ones inside the cells, not that their absolute amounts are larger, given than a single large nanoparticle cluster can outweigh thousands or millions of smaller ones, which would present a misleading metric. In addition to this, a number of studies have demonstrated diametrically opposite effects of the particle size on the uptake efficiency in different cell lines. In one of them, for example, the uptake increased with the PLGA particle size in RAW264.7 macrophages, and decreased in human epidermoid cancer KB cells [52]. Likewise, in another study, the uptake percent of rhodamine B labeled carboxymethyl chitosan nanoparticles increased with the particle size in murine macrophages and decreased in L02 and SMMC-7721 cells [53]. Other studies [54; 55] have confirmed that macrophages, the comparatively large cells specializing in particle uptake, usually deviate from the trend observed, both here and in the literature, to apply to fibroblasts, osteoblasts, epithelial cells, MSCs, and multiple other cell types, for which reason they, alongside particles lower than 20 nm in size, were excluded from the literature meta-analysis reported in Figure 5c. This does not go against the definite presence of a lower critical limit for nanoparticle uptake [56], for which reason cells often wait for nanoparticles adhering onto their membranes to cluster together before they uptake them within a single endocytic vesicle [57]. Typically, thus, around the nanoparticle size of 50 nm the maximum uptake efficiency is reached, below which it begins to drop with a further reduction in the particle size [58; 59]. Here it should be pointed out that the particle uptake and colloidal stability are tightly related in a sense that smaller particles that tend to be more stable in suspension tend also to come into less contact with the cells seeded at the bottom of the cell culture plates compared to the larger particles, which are more prone to sedimentation. However, since this effect favors the uptake of larger clusters of nanoparticles, which, as per these results, get uptaken less than the finer ones, it can be concluded that cells do definitely prefer to uptake the smaller particles. In any case, 2D culture is not always a good proxy for 3D in vivo scenarios, for which reason deviations from the trend observed may occur in real-life conditions. Figure 5: The significantly lower average size of the surface projection of optically imaged intracellular (IC) HAP nanoparticle agglomerates in interaction with hMSCs (n = 40) compared to that of the extracellular (EC) nanoparticle agglomerates (**a**).