**6. Protein corona formation**

When nanomaterials are exposed to biological fluids a dynamic covering of various serum proteins and biomolecules attach to the nanomaterial and this structure is called protein corona (PC). The PC formation on NP can influence its toxicity and targeting behavior due to immunological effects [45].

PC has two different layers one is a hard corona which is the first tightly bound layer and another is soft corona which is the second layer which consists of proteins that are easily exchanged. The composition of corona can change any time by replacement of proteins, but the quantity will remain constant. Initial proteins that bind to the NP will be the most abundant in the blood or the biological fluid exposed to the NP later on the proteins are replaced according to the affinity. This is the 'Vroman Effect' which explains the change in the composition of the protein corona on NP surface in response to time. The proteins compete to get adsorbed onto NP surface. Different types of corona are formed based on the binding affinity and surface ligands on the engineered NP.

Engineered nanoparticles have high free energy and thus adsorb protein to a lowenergy state by increasing their dispersion. In less than 0.5 min of exposure to blood plasma, silicon NP adsorbed almost 300 proteins onto its surface [46] (**Figure 6**).

Toxicity of the NP are greatly influenced by the protein corona adsorbed. The fate of an engineered nanoparticle is different in cell lines and in vivo. According to the type of proteins adsorbed, the targeting of the NP and their cellular uptake also changes. There

#### **Figure 6.**

*Factors influencing protein corona formation on nanoparticles. Reproduced with permission from RSC with license id 1257299-1 [47].*

are chances of non-specific uptake by receptor-mediated endocytosis. Opsonization plays a major role in determining the fate of ENPs. Proteins act as opsonin and these NP are engulfed by macrophages, it can also lead to uncontrolled aggregation. The protein corona, when made by cell penetrating peptides and antibodies will result in active targeting. It is observed that certain protein corona like blood proteins reduce the toxicity of carbon nanotubes when compared to the fate in cell lines. Overall, the protein corona affects the cellular uptake, target organ and thus toxicity [47].
