**3. Nanoparticle Interactions**

Although NPs in biological systems are surrounded by large quantities of biomolecules, depending on the different factors that characterize the biological environment. The NP promotes multiple and different interactions. Multifunctional NPs as nanomedicines (see Figure 2) are embedded in human proximal fluids, inside cells, and inside culture media among others [29]. This implies a huge variety of different microenvironments with additional challenges for the design and development of NPs suitable to be functional in all kinds of conditions. However, depending on the medium conditions like pH [30], ionic strength, oxygen levels, organic matter, etc., NPs present different forms or stages, such as ionized particles [31], which form aggregates or combine into complex aggregates or even interact with other nanomaterials [32]. This is especially relevant because it may be the origin of a heterogeneous morphology, which might be correlated with a lack of stability and immuno-biocompatibility of these nanomaterials [33].

**Figure 2.** Schematic representation of multi-functional nanoparticles.

NP aggregation and agglomeration have been recognized to a ffect cellular uptake and even induce potential toxicity based on the nanoparticle composition and the cell type [34]. Aggregation and agglomeration e ffects are often used in nanotechnology, but both terms are commonly mistaken. Aggregation indicates strongly bonded or fused particles and it occurs when the Van der Waals attractive forces between particles are greater than the electrostatic repulsive forces produced by the nanostructure surface [34,35]. On the other hand, agglomeration indicates more weakly bonded particles and it does not require a definite pattern, shape, and size [35]. Pellegrino F. et at. studied the agglomeration and aggregation influence on the optical properties of TiO2 NPs demonstrating that this e ffect can lead to an incorrect assessment of the photoactivity [36]. Zook M.J. et al. [37] developed a bottom-up-based method to produce controllable, reproducible, and stable NP agglomerates in an aqueous medium. They used this method to show how silver NP agglomeration a ffects hemolytic activity.

The main factors that will determine the type of interactions between NPs are: the complementarity between nanomaterials and their distance and geometry [38]. In addition, it is also essential to know what the main interactions drivers are in an NP assembly. For example, Van der Waals forces form nanocrystal superlattice membranes, electrostatic interactions obtain colloidal dimers, and magnetic interactions where iron oxide NPs coated with azobenzene-terminated catechol ligands self-assemble by UV-light-induced, or even molecular force [38].

An example that demonstrates the importance of the complementarity between the materials and the influence of the forces used in such an interaction is one discussed by Pileni and co-workers. They stress the di fference of using octanoic and dodecanoic acids as organic ligands in magnemite NPs in the absence (only with dipolar forces between the magnetic nanoparticles) and the presence of Van der Waals interactions, when the distance is small [39,40].

On the other hand, an interaction between molecules on surfaces is highly dependent on surface functionalization (Figure 3). This implies the presence of reactive chemical moieties on the surface being homo-functional or hetero-functional depending on whether there is only one chemical group on the surface or whether di fferent chemical reactive groups co-exist [41].

**Figure 3.** Schematic representation of the strategy to couple nanoparticles and biomolecules or other nanoparticles.

Due to their composition and structure, the surface might not allow di fferent types of interactions. Thus, for example, circulatory cells are covered by a lipid bilayer with proteins and polysaccharides that, depending on the NP exposed groups, will favor one type of interaction mechanism [42]. Another example includes the proteins a ffected by their molecular weight, charge (greater adsorption near the isoelectric pH), or its stability that influences the number of binding points [43]. A soft protein layer has a low structural stability and a greater number of active centers to interact with, besides other influencing physicochemical factors on the surface (i.e., humectability). The hydrophobicity/hydrophilic surface ratio influences protein reactivity and/or its adsorption properties. Another remarkable feature is the size, including those with a size comparable to that of the NP, which will be more easily adsorbed.

Lastly, it is not only necessary to consider the concentration or size of NPs, but also the species and quantity of resulting products from chemical interactions between NPs.

### *3.1. Interaction Mechanisms Between Nanoparticles and Biomolecules*

There is a wide-open variety of biomolecules, which could interact directly onto the NPs surface or through other biomolecules coating the NPs surface (Figure 2). These layers of coating biomolecules are directly related with the type of organism, biological fluid, cells, etc., among the physicochemical conditions of the media and NP surface, nature, and structure of biomolecules.

According to the literature, the most relevant interacting biomolecules to the NP surfaces are proteins and nucleic acids [44]. Proteins have many di fferent binding sites (as amino acidic key structures and/or post-translational modifications) through specific or non-specific adsorption [43,45]. In addition, the proteins are critical on the immune-biocompatibility of the nanomaterials. Nucleic acids have many di fferent applications as a consequence of its physicochemical stability, mechanical rigidity, easy accessibility, and its high specificity of base pairing, which results in a suitable receptor for molecular nano-construction [46].

Regarding interactions with human biomolecules, two factors must be considered in the description of the interaction [23]. The first one is that NPs in biological systems are surrounded by multiple potentially interacting biomolecules that may modify and saturate their surface. Therefore, custom modified NPs are the ones that may interact specifically with the biomolecules of interest later on. The second factor is NP entering pathways into the human body. This depends on the way it can influence the force of the interaction. For example, NPs entering by inhalation strongly interact with the pulmonary system (proteins and phospholipids).

Two immobilization mechanisms have been studied through an interaction with di fferent types of biomolecules [45]: by simple absorption or by chemical linkages. The immobilization of enzymes on NPs through adsorption is a very useful method because it takes place through non-covalent forces (hydrogen bonding, ionic interactions, and Van der Waal forces), mainly through negatively charged phosphate groups and hydrophobic moieties not disturbing the initial structure of the enzyme or its active site. Immobilization through chemical linkages may lead to the immobilization of biomolecules on a biocompatible matrix, such as within phospholipid bilayers, not interacting with the native structure of the biomolecule and altering its biological activity.

We also find two other types of interaction mechanisms with cells: ligand-receptor interaction and chemical conjugation [47]. An example of the first interaction method is the NP surface functionalization with a receptor, such as streptavidin-biotin. Its non-covalent interaction results in a greater bond strength, which provides resistance to pH, temperature variations, and denaturants. In addition, they have a greater binding a ffinity to cells. Chemical conjugation simply consists of the coupling of functional groups (such as thiol groups) to the NP surface, which favors subsequent binding to the cell and, in turn, reduces the toxicity of this interaction. A disadvantage of this method is that, in terms of biomedical applications, the covalent binding of the drug to the NP restricts its e fficient release, which limits its e ffectiveness.

### *3.2. Nanoparticle Design: Influence on Interaction Mechanisms*

NPs undergo di fferent changes in a concrete environment such as the generation of a coating protein corona once plasma proteins are adsorbed on its surface. Therefore, it is necessary to study the NP states and characteristics prior to interaction assays [48].

Many NP-based investigations focus on issues a ffecting NP characteristics and, subsequently, their impact on cellular internalization and biodistribution. Centi J. et al. [49] and Tatini J. et al. [50] talk about the interest of gold nanorods (GNRs) in the biomedical field. GNRs are gold NPs that are elongated along one direction with characteristic optical properties, which depend on the particle size and shape [51]. They are attractive in biomedical optics because of their special and intense absorption band near infrared light (650–1000 nm). Other important features of GNRs include their coating, which are crucial for their biological applications, i.e., conjugation with PEG. In addition, their shape and size are critical for modulating cellular penetration, intracellular localization, and bio-distribution. GNRs may become coated with, which may modify their conformation and cause a loss of their biological activity. Bovine Serum Albumin has been chosen as a protein target to investigate NPs coating with polyethylene glycol (NP-PEG) exposition to biological fluids because it is the most abundant protein in the blood and can transport metal compounds. The Tatini J. et al article proposes CA-125 as the

molecular target cancer antigen to model "in vitro" some of the most critical issues that arise from the interactions between GNRs and the bloodstream using an analytical approach.

The physicochemical properties of NPs (Figure 4, some already commented) represent their identity and influence on the synthetic moieties incorporated [52] among all including size, shape, surface, coating and morphology, surface charge, solubility, chemical composition, crystalline structure, and, lastly, the agglomeration status. These properties will also play a characteristic role in relevant mechanisms such as cellular biocompatibility studies.

**Figure 4.** Schematic illustration of the main physicochemical properties of nanoparticles governing interaction mechanisms in biological systems.
