*4.3. NP Biosafety: A Critical Aspect in Nanomedicine and Immunotherapy*

To guarantee the effective and safe use of nanomaterials used in Nanomedicine, it is necessary to characterise the interaction between a material and the biological system involved. These are biocompatibility studies that have to be performed with a focus on the environment in which the biomaterial will be administered [242]. To ensure the drug delivery, it is necessary to evaluate this biocompatibility, ensuring the safe release of the medicine and minimizing its toxicity. For example, NPs that have not been modified on their surface are absorbed by phagocytic cells, which can lead to undesirable interactions with the immune system, decreasing the bioavailability of the drug [243].

Biocompatibility is defined as "the ability of a material to function with an appropriate host response in a specific situation" [244]. The employed material has to fulfil its intended functions, the reaction induced has to be appropriate to the intended application, and the nature of the reaction to a material and its suitability may be different in different contexts. The high degree of compatibility is achieved when a material interacts with the body without causing toxic, immunogenic or carcinogenic responses [243]. Importantly, biocompatibility is anatomically dependent, so a biomaterial can cause adverse effects in one type of tissue and will not cause the same response in another [245,246]. The half-life of exposure is also determinant and, therefore, the clearance of each NPs as well. Biocompatibility is subjective, as it is based on the risk-benefit ratio.

Currently, a few studies on biological processes in response to foreign materials or on the nature of the methods available for biocompatibility, so there is a strong need to evaluate the biocompatibility of each material individually and specifically for each tissue and application [243]. An example of this type of studies is the analysis realized by Mikhail V. Zyuzin et al., who proved the immunocompatibility of polyelectrolyte capsules synthesized by layer-by-layer deposition through the incubation of different cell lines with this capsules [247].

NPs are delivered into the bloodstream and are therefore exposed to many biomolecules that will give rise to a protein crown around them [248]. This causes NPs to undergo changes in their physicochemical properties, and therefore it is necessary to study NP–protein interactions in nanomedicine. Proteins in the biological environment are adsorbed to NPs by affinity and protein–protein interactions [2]. The first proteins to bind are those found in high concentrations, although they have low affinity, and are subsequently replaced by proteins with higher affinity that are found in lower concentrations. This phenomenon is called the Vroman effect [249]. The protein crown is classified into hard or soft crown depending on the duration of protein replacement. The hard crown is formed by high affinity proteins with a long exchange time and is the innermost layer. The soft crown is made up of proteins with low affinity and a rapid exchange of proteins [250].

Both the characteristics of NPs and protein concentration, and the biological environment will determine the formation of the protein crown. Therefore, it is important to understand the relationship between the properties of nanomaterials and the biological environment to understand the behaviour and viability of the NPs used.
