**3. Nanoparticle's intracellular trafficking**

After the nanoparticle's entry in the biological fluid, it is important to ensure its internalization into the cells and intracellular transport, as the formation of the protein corona influences directly the cellular uptake and may also have a significant role on the success of the nanoparticle or lack thereof [69]. Most of mammalian cells internalize the nanoparticles through pinocytosis, although big, specialized cells (such as macrophages) are able to do it by phagocytosis [70, 71], which is the uptake of large particles [70]. Some nanoparticles can also do it by passive penetration of the cellular membrane; however, if the nanoparticle is not small enough, it may deform the membrane [72] by forming holes or thinning it [73], increasing the cytotoxicity [71, 74]. Still, this mechanism is useful in drug delivery, as the nanoparticle travels directly to the cytosol, without making use of endocytic vesicles [74], promoting the reach of the intracellular targets [75]. Hence, it is necessary to take that into account, when designing the nanoparticle, as it may be possible to optimally design the surface of the selected nanoparticle for drug delivery and avoid the membrane's deformation [74]. As for the pinocytosis internalization, which is the cellular uptake of small particles (fluids and solutes), it has four different types of mechanisms [70] (**Figure 2**).

The physical properties of the nanoparticle such as size, net surface charge, and chemical composition determine which endocytosis process is chosen for the internalization [76],

nanoparticles showed more efficiency when compared to spherical nanoparticles [76, 85]. After performing its function within the cell, the nanoparticle is eventually cleared by the liver and spleen, where they can be kept for a long time, increasing the expected cytotoxic-

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

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

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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

ity of the nanoparticle [76].

liver or spleen.

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

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

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 nanoparticles showed more efficiency when compared to spherical nanoparticles [76, 85]. After performing its function within the cell, the nanoparticle is eventually cleared by the liver and spleen, where they can be kept for a long time, increasing the expected cytotoxicity of the nanoparticle [76].
