*2.2. Characterization of the Functionalized Fe3O<sup>4</sup>*

As mentioned in the Introduction, though magnetite is a biocompatible material, its stabilization in a physiological environment is only possible after coating [2,4]. In addition, functionalization enables the conjugation with therapeutic biomolecules and contributes to achieving effective directionality of magnetite nanoparticles towards the site of interest inside the body [7]. In this work, it was decided to carry out the coating/functionalization of the Fe3O<sup>4</sup> NPs in a single step. Thus, from the use of sodium citrate and 3-aminopropyltriethoxysilane (APTES), it was possible to functionalize the magnetite surface in the form of carboxylic acid and amine, respectively. Both functional groups are required for subsequent conjugation to the peptide fractions from the Ugi 4C reaction. Each of the variants made is described below.

## 2.2.1. Characterization of Fe3O4@citrate

Carboxylate is one of the functional groups that most strongly binds to the surface of magnetite NPs [28]. Sodium citrate (or the corresponding acid), a small biocompatible molecule, has three carboxylate groups in its structure. Only one or two of the carboxylate functions of sodium citrate have been found to coordinate to the magnetite surface, allowing at least one of them to be exposed to the medium [29]. This effect is very convenient for the application of the magnetic system since it protects the nanoparticulate nucleus from the possible formation of aggregates while providing it with hydrophilicity and functionalizing it for subsequent derivatizations and couplings. Here, after the synthesis of Fe3O<sup>4</sup> NPs coated with sodium citrate (Fe3O4@citrate), the resulting product was characterized.

The Fourier-transform infrared (FTIR) spectroscopy analysis of the MNPs coated with sodium citrate suggested that the desired product was obtained (Figure 3, blue spectrum). The three bands at 3435, 1629, and 1398 cm−<sup>1</sup> indicate carboxylate in the sample. These signals correspond to the valence vibrations of the O–H, C=O, and COO– bonds, respectively. The band corresponding to Fe3O<sup>4</sup> remained at 537 cm−<sup>1</sup> . In addition, the XRD patterns of the starting magnetite (Figure 1, black profile) and that coated with citrate (Figure 1, blue profile) were similar, which suggests that the crystallinity of the material was not affected by the coating. However, the calculated cell parameter was 8.3643 Å. The difference with respect to the cell parameter of the uncoated magnetite indicates a strong interaction between citrate atoms and magnetite, which results in the contraction of the unit cell of the starting material [30]. The average crystallite size determined using the Debye–Scherrer equation (Equation (1)) was 16 nm. This value is similar to the particle size estimated using STEM (13 ± 3 nm). The similarity between these results suggests that the compound was monocrystalline. Figure 4a shows a microscopy image and obtained size distribution with the corresponding histogram. The obtained NPs had spherical morphology with some aggregation because of their small sizes.

Another evidence of the presence of citrate on the surface of the MNPs is the increase in the hydrodynamic diameter (284 ± 140 nm) relative to that of the uncoated MNPs. As some of the polar groups of citrates (carboxylate and hydroxyl) are dangling towards the surrounding medium, a larger hydration layer is generated. Finally, the weight percentage of the coating material on magnetite, with respect to the uncoated Fe3O4, was determined using thermogravimetric analysis (TGA). The weight loss was 2.21%, confirming that magnetite was coated with an organic material.

tograms.

acterized.

phology with some aggregation because of their small sizes.

required for subsequent conjugation to the peptide fractions from the Ugi 4C reaction.

Carboxylate is one of the functional groups that most strongly binds to the surface of

The Fourier-transform infrared (FTIR) spectroscopy analysis of the MNPs coated

with sodium citrate suggested that the desired product was obtained (Figure 3, blue spectrum). The three bands at 3435, 1629, and 1398 cm−1 indicate carboxylate in the sample. These signals correspond to the valence vibrations of the O–H, C=O, and COO– bonds, respectively. The band corresponding to Fe3O<sup>4</sup> remained at 537 cm−1. In addition, the XRD patterns of the starting magnetite (Figure 1, black profile) and that coated with citrate (Figure 1, blue profile) were similar, which suggests that the crystallinity of the material was not affected by the coating. However, the calculated cell parameter was 8.3643 Å . The difference with respect to the cell parameter of the uncoated magnetite indicates a strong interaction between citrate atoms and magnetite, which results in the contraction of the unit cell of the starting material [30]. The average crystallite size determined using the Debye–Scherrer equation (Equation (1)) was 16 nm. This value is similar to the particle size estimated using STEM (13 ± 3 nm). The similarity between these results suggests that the compound was monocrystalline. Figure 4a shows a microscopy image and obtained

magnetite NPs [28]. Sodium citrate (or the corresponding acid), a small biocompatible molecule, has three carboxylate groups in its structure. Only one or two of the carboxylate functions of sodium citrate have been found to coordinate to the magnetite surface, allowing at least one of them to be exposed to the medium [29]. This effect is very convenient for the application of the magnetic system since it protects the nanoparticulate nucleus from the possible formation of aggregates while providing it with hydrophilicity and functionalizing it for subsequent derivatizations and couplings. Here, after the synthesis of Fe3O<sup>4</sup> NPs coated with sodium citrate (Fe3O4@citrate), the resulting product was char-

Each of the variants made is described below.

2.2.1. Characterization of Fe3O4@citrate

**Figure 4.** Microscopy images of (**a**) Fe3O4@citrate and (**b**) Fe3O4@APTES with the corresponding his-**Figure 4.** Microscopy images of (**a**) Fe3O4@citrate and (**b**) Fe3O4@APTES with the corresponding histograms.
