*2.1.1. Isothermal titration calorimetry (ITC)*

Isothermal titration calorimetry is a method that allows the determination of thermodynamic parameters in a solution (binding affinity, binding stoichiometry, and binding enthalpy change [33]), in particular the ones coming from interactions between biological macromolecules [42]. This process is based on the changes in heat caused by the protein adsorption to the nanoparticle [43]. The main advantage of this method relies on the fact that it allows the characterization while still in the incubation medium [43], which consequently allows a greater optimization of the nanoparticle.

## *2.1.2. UV-visible spectrometry*

UV-visible spectrometry is a process based on the ratio between the passed light measured and the incident light in the UV-visible wavelength [33]. The presence of the protein corona induces changes on the absorption spectrum [17], which makes it an easy, fast, and applicable approach, as it requires no other chemicals or resources other than the protein corona itself [33]. However, it is an unreliable method, as the radiation energy reaching the sample is low [44]; it is very influenced by parameters such as size, temperature, pH [33], and equipment errors, which have a much bigger impact, as there is no other chemical or technique applied to lower the risk of incorrect results [44].
