**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 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 the cells more easily.

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 method against tumors.
