**4. Interaction of nanoparticles with cell interfaces**

although more than one mechanism can be used for the same nanoparticle [77, 78], and the formation of the protein corona may have a great influence in this choice [71]. The proteins adsorbed on the nanoparticle give it its biological identity, and they may present a sequence that is not recognized by the cell as relevant or needed, preventing its endocytosis [79]. It was verified that nanoparticles without a protein corona have higher rates of cellular uptake but can also cause more damage to the cell and alter the cellular metabolism and cell cycle [79, 80]. Nevertheless, if the sequence of proteins in the protein corona is identified as relevant, the endocytosis mechanisms are activated, and the nanoparticle is internalized [79]. When binding to the cellular membrane, the protein corona does not separate itself from the nanoparticle [81], nor does it detache when inside the cell, being internalized as a single complex [82]. After internalization, the nanoparticle's course must be followed by capturing its fluorescence, which can come from the nanoparticle itself or from a fluorescent dye added posteriorly. According to Guarnieri et al. [83], polystyrene nanoparticles follow a fairly diffuse pattern once inside the cell, which suggested no interaction between the nanoparticle and the cytosolic structures, in both situations with and without protein corona. This diffuse pattern can be explained by the nanoparticles being transported within the endocytic vesicles, whose movements are associated to the molecular motors, such as kinesin, myosin, and dynein [83, 84]. Therefore, Guarnieri et al. [83] report that, although the protein corona has some influence in the mechanisms of cellular uptake, it does not show an impact on the intracellular pathways taken by nanoparticles internalized by endocytosis. While leaving the cell, exocytosis mechanisms are activated, and they are dependent on proteins in the medium, because the proteins forming the corona interacted with biological systems inside the cells [76]. The exocytosis is also size, surface coating, and shape dependent, as smaller nanoparticles showed faster exocytosis rates and rod-shaped

**Figure 2.** Summary of the different kinds of endocytosis (information based on Conner and Schmid [71]).

36 Protein-Protein Interaction Assays

After internalization, it is important not only to guarantee the achievement of the nanoparticle's function but also to evaluate its effects on cellular organelles [86], as the toxicity cannot be too high, or it will ultimately exclude its use in nanomedicine. The understanding of the nanoparticles' interaction with each cellular organelle is still fairly underdeveloped, as researchers tend to overlook the possible connections between the nanoparticle's composition and the cellular response, focusing considerably more on its uptake [86]. Nonetheless, some studies have already been made to counteract this tendency, in order to give more information and also a better understanding of the nanoparticles' real impact in the cell. According to the experiment performed by Bertoli et al. [78], it is possible to separate the organelles retaining the nanoparticle through magnetism, if the particle is designed to have magnetic properties. Their experiment [78] was based on separating the nanoparticle from the cell after internalization, in order to identify the proteins adsorbed to it, and determining their origin, according to the characteristic proteins from each cellular organelle. The nanoparticles were verified to have the majority of proteins (over 44%) coming from the endocytic pathway, while fewer than 5% came from each of the different organelles studied, such as nucleus, mitochondria, or peroxisomes. However, some proteins can overlap by belonging to more than one organelle [87], acting like a contamination, as they can induce errors in the examination results. Nevertheless, it can be inferred that the majority

**Figure 3.** Summarization of the nanoparticle's cellular internalization. The nanoparticles interact with the cell, which can lead to cellular uptake or accumulation of the nanoparticle in the cell membrane. If uptake occurs, the cell engulfs the nanoparticle within endocytic vesicles, which transport the nanoparticle while inside the cell. According to the experiments performed by Bertoli et al. [79], the majority of nanoparticles does not leave the endocytic vesicles to interact directly with other organelles. The nanoparticles are exocytosed afterwards, through lysosomes, to be cleared by the liver or spleen.

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 intracellular target for the nanoparticle.

occurred in GG-coated nanoparticles, with higher levels of complement factors and opsonins, especially immunoglobulins, and low levels of lipoproteins. The presence of opsonins made the uptake more difficult, and when comparing with nanoparticles without protein corona, the differences in uptake were very significant, as nanoparticles without it entered

Protein Interactions and Nanomaterials: A Key Role of the Protein Corona in Nanobiocompatibility

http://dx.doi.org/10.5772/intechopen.75501

39

Another work that explored the composition of the protein corona was provided by Mahmoudi et al. [90], who verified the alteration of the proteins that formed the corona after submitting it to plasmonic heat induction. In this experiment, gold nanorods were used and immersed in fetal bovine serum (FBS) at different concentrations – 10% to mimic in vitro milieu and 100% that mimic in vivo milieu. The protein corona was then evaluated before and after exposure to plasmonic heat induction in both concentrations, and by LC-MS/MS, it was found that, at room temperature, at 10% FBS the most abundant proteins were apolipoprotein A-I precursor and the hemoglobin fetal subunit beta, while the least abundant was the apolipoprotein C-III precursor. At room temperature at 100% FBS, the protein corona was rich in the apolipoprotein A-II precursor and also in hemoglobin fetal subunit beta, while the most scarce was the apolipoprotein C-III precursor as well. After exposure to plasmonic heat induction, at 10% FBS the most abundant proteins became the α-2-HS-glycoprotein precursor and the apolipoprotein A-II precursor, while hemoglobin fetal subunit beta maintaining its numerousness and hemoglobin became the least frequent protein. At 100% FBS after plasmonic heat induction, α-2-HS-glycoprotein precursor also became the most frequent one, followed by hemoglobin fetal subunit beta as well, having a very significant decrease in the quantity of apolipoprotein A-II precursor but still having the apolipoprotein C-III precursor as the least prevalent of all the proteins evaluated. Accordingly with these results [90], it is then possible to conclude that the protein corona's composition is dependent on the medium where the nanoparticle is inserted, which, in this case, also translates to a difference between in vitro and in vivo applications of the nanoparticle, being demonstrated as well its dependency on a physical factor (temperature), which must be taken into account when dealing with hyperthermic nanoparticles as a therapeutic

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

the cells more easily.

method against tumors.

**6. Conclusions**

in nanomedicine.

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 internalization pathway of SiO2 fluorescent-labeled nanoparticles was characterized. The results 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 organelles (**Figure 3**).
