**6. Conclusions**

of nanoparticles does not leave the endocytic vehicles to associate itself with the different organelles that are present on the cell, which can become a problem, if there is a specific

In order to find the internalization's time distribution, Bertoli et al. [78] also performed a time-dependent experiment, evaluating the locations of the nanoparticles after submitting the cells to a short exposure. It was verified that, after only a few minutes, the presence of the nanoparticles was greater in early endocytosis' organelles, with the total absence of lysosomal markers. Still, a more prolonged exposure (24 h) revealed a stronger nanoparticle existence in lysosomes, without any proteins present in early endocytic organelles, indicating their concluding exocytosis from the cells. A similar experiment was previously performed by Shapero et al. [88], where by electron and fluorescence microscopy, the inter-

obtained also showed a greater number of nanoparticles in the early endocytic organelles after a short exposure, lessening those numbers as time passed and as the nanoparticle's location progressed to lysosomal structures, suggesting its clearance mechanism from the cell. Shapero et al. [88] also verified a nonexistent association between the nanoparticles and the cellular organelles, consolidating the theory that the great majority of nanoparticles does not leave or circulates outside the endocytic vehicles to interact with other organ-

**5. A selection of relevant proteins identified in the protein corona**

As mentioned before, the constitution of the protein corona is mainly dependent on the composition of the biological medium where it's inserted [19, 20], as different physiological environments have different proteins that compose them. The protein corona is also dependent on the time of exposure [11, 17, 20], chemical properties of the nanoparticle applied [23], and, in some cases, size and surface of the nanoparticle [23]. It is impossible to have a standard protein corona for a given nanoparticle, as each one will have a different composition [15]. The best approach possible then is to characterize the most occurring proteins, in order to predict the behavior of the nanoparticle when inside the physiological system. This issue of research is still emergent, with promising outcomes to better adjust the nanoparticle to its function and environment. An example of protein corona evaluation is the work presented by Mirshafiee et al. [89], who assessed the differences between nanoparticles with different coatings without a protein corona and the same nanoparticles with it adsorbed. Three different types of nanoparticles were used: the bare nanoparticle (with no coating), a nanoparticle with human serum albumin coating (HSA), and a nanoparticle with gamma-globulin coating (GG). The results indicated that there was a different protein corona associated to each of the various coatings, identifying the proteins by LC-MS/ MS. For instance, a greater number of lipoproteins and a low quantity of complement factors and immunoglobulins were found in HSA-coated nanoparticles, while the opposite

fluorescent-labeled nanoparticles was characterized. The results

intracellular target for the nanoparticle.

nalization pathway of SiO2

38 Protein-Protein Interaction Assays

elles (**Figure 3**).

The study of the protein corona is still in a very embryonic stage, with many problems and questions yet to be answered, such as the composition when formed in most nanoparticles, exact description of the uptake and clearance mechanisms, and extensive reports on the consequences of its formation. Some steps have been taken with the purpose of answering these questions, especially in resorting to bioinformatic approaches, allowing an easier and more efficient analysis and sharing of the data obtained. Nevertheless, these are without a doubt interesting research topics, leading the way to improve what is already a very auspicious field in nanomedicine.
