*2.4. Characterization of Peptide–Fe3O<sup>4</sup> Conjugates*

The main objective of this study was to develop a simple procedure for the conjugation of bioactive peptide fractions extracted from germinated soybeans to magnetite NPs. To this end, the U-4C reaction was chosen because it occurs in a single step and can be easily implemented [33,34]. The results for the remaining peptide concentrations are shown in Figure 5. There were no statistically significant differences in the percentage of free peptides at 4 and 8 h in either variant for the same peptide fraction. This suggests that the reaction ended after 4 h. However, the difference between the percentages of conjugation when the fraction >10 kDa was used (approximately 60%) and that at 5–10 kDa (approximately 92%) was remarkable. This was expected considering the smaller size of the peptides contained in the latter, which would facilitate the multicomponent process. On the other hand, the yields of the developed conjugation process can be considered good in all the cases, considering that the magnetic component of the reaction is solid-support-dispersed, while the other reactants are dissolved in the medium [35].

The conjugates were characterized using FTIR spectroscopy (Figure 6). In all the cases, the bands that confirmed the presence of carbonyl groups in each of the structures on the MNPs were observed at approximately 1620 cm−<sup>1</sup> . In addition, the band at 573 cm−<sup>1</sup> was retained, which corroborated the presence of Fe3O4.

method.

**Figure 5.** Percentages of unconjugated peptides to coated Fe3O4: (**a**) Fe3O4@citrate@ > 10 kDa, Fe3O4@citrate@ > 5–10 kDa; (**b**) Fe3O4@APTES@ 10 kDa, Fe3O4@APTES@ 5–10 kDa. The determinations were carried out in triplicate. The results are expressed as the means ± standard deviation. Different letters indicate statistical difference between the samples for each reaction time (*p* ≤ 0.05). **Figure 5.** Percentages of unconjugated peptides to coated Fe3O<sup>4</sup> : (**a**) Fe3O4@citrate@ > 10 kDa, Fe3O4@citrate@ > 5–10 kDa; (**b**) Fe3O4@APTES@ 10 kDa, Fe3O4@APTES@ 5–10 kDa. The determinations were carried out in triplicate. The results are expressed as the means ± standard deviation. Different letters indicate statistical difference between the samples for each reaction time (*p* ≤ 0.05). was retained, which corroborated the presence of Fe3O4.

**Table 1.** Determination of the degree of substitution on the MNPs using the bicinchoninate

**(mg Cu2+/g MNPs)**

The calculations for the subsequent synthesis step were carried out using the average

The main objective of this study was to develop a simple procedure for the conjugation of bioactive peptide fractions extracted from germinated soybeans to magnetite NPs. To this end, the U-4C reaction was chosen because it occurs in a single step and can be easily implemented [33,34]. The results for the remaining peptide concentrations are shown in Figure 5. There were no statistically significant differences in the percentage of free peptides at 4 and 8 h in either variant for the same peptide fraction. This suggests that the reaction ended after 4 h. However, the difference between the percentages of conjugation when the fraction >10 kDa was used (approximately 60%) and that at 5–10 kDa (approximately 92%) was remarkable. This was expected considering the smaller size of the peptides contained in the latter, which would facilitate the multicomponent process. On the other hand, the yields of the developed conjugation process can be considered good in all the cases, considering that the magnetic component of the reaction is solid-support-

Fe3O4@citrate 0 0 0 Fe3O4@citrate 1 14 0.44 Fe3O4@citrate 24 15 0.47 Fe3O4@APTES 0 0 0 Fe3O4@APTES 1 12 0.76 Fe3O4@APTES 24 12 0.76

**Amount (mmol of Free Groups/g MNPs)**

**Particle t (h) Cu2+ Adsorption Capacity**

value of the loading of functional groups on the Fe3O<sup>4</sup> surface.

dispersed, while the other reactants are dissolved in the medium [35].

*2.4. Characterization of Peptide–Fe3O<sup>4</sup> Conjugates*

**Figure 6.** FTIR spectra of the final conjugates: Fe3O4@citrate@ > 10 kDa (conjugate 1), Fe3O4@citrate@ 5–10 kDa (conjugate 2), Fe3O4@APTES@ > 10 kDa (conjugate 3), and Fe3O4@APTES@ 5–10 kDa (con-**Figure 6.** FTIR spectra of the final conjugates: Fe3O4@citrate@ > 10 kDa (conjugate 1), Fe3O4@citrate@ 5–10 kDa (conjugate 2), Fe3O4@APTES@ > 10 kDa (conjugate 3), and Fe3O4@APTES@ 5–10 kDa (conjugate 4).

### jugate 4). *2.5. Antioxidant Activities of the Conjugates*

*2.5. Antioxidant Activities of the Conjugates* The association between the generation of ROS and the development of chronic degenerative diseases has been reported [27]. Furthermore, in cellular metabolism, oxidative compounds are produced constantly, so it is important to counteract these oxidizing spices to maintain a balance of intra- and extracellular homeostasis. Hence, the antioxidant capacities of the conjugates obtained in this study were evaluated. The determina-The association between the generation of ROS and the development of chronic degenerative diseases has been reported [27]. Furthermore, in cellular metabolism, oxidative compounds are produced constantly, so it is important to counteract these oxidizing spices to maintain a balance of intra- and extracellular homeostasis. Hence, the antioxidant capacities of the conjugates obtained in this study were evaluated. The determinations were made by verifying their reducing power (RP) and hydroxyl radical (OH·) scavenger abilities. The results presented below (Figures 7 and 8) correspond to the tests carried out at a concentration of 15 mg/mL, at which a stronger effect was observed in the determinations.

tions were made by verifying their reducing power (RP) and hydroxyl radical (OH·) scavenger abilities. The results presented below (Figures 7 and 8) correspond to the tests car-

determinations. All the results were compared to those obtained for the nonconjugated

The RPs of the coated magnetite, as well as those of the conjugates and nonconjugated peptide fractions (Figure 7), were expressed as absorbances, where 1 represents the highest value. The RP of a sample refers to its ability to act as a proton acceptor (or electron donor) in an oxidation–reduction reaction and, therefore, the number of basic groups in the conjugates could be related to the obtained RP. In this sense, Fe3O4@APTES, with free amine groups on the magnetite surface, showed the highest RP value. Regarding the conjugates, those in which magnetite was conjugated to the fractions containing the peptides with masses >10 kDa (conjugates 1 and 3) had the highest RP values, with statistically significant differences between them and the conjugates 2 and 4. This result agrees with the study by González-Montoya et al. [27], who observed that the >10 kDa (RP = 0.7) fraction had a higher RP than the 5–10 kDa fraction (RP = 0.3). This could be attributed to the higher amount of basic amino acids in the >10 kDa fraction. A synergistic effect was observed when conjugating the peptide fractions to Fe3O4@APTES (conjugate 3 with respect to the >10 kDa peptide fraction), surely, due to the contribution of the amine groups that remained free, unreacted during the multicomponent reaction, on the surface of the magnetite coated with the APTES. It was also observed that the conjugation of the >10 kDa peptide fraction to magnetite NPs coated with sodium citrate (conjugate 1) slightly decreased the RP with respect to that of the peptide fraction, whilst the RP of the 5–10 kDa peptide fraction conjugated to Fe3O4@citrate (conjugate 2) did not exhibit statistically significant differences with respect to the nonconjugated fraction. In this way, conjugate 3

peptide fractions and for the coated magnetite (Fe3O4@APTES and Fe3O4@citrate).

All the results were compared to those obtained for the nonconjugated peptide fractions and for the coated magnetite (Fe3O4@APTES and Fe3O4@citrate). represents an opportunity for future evaluations of biological activities related with oxidative stress in chronic diseases.

**Figure 7.** Average results of the RPs of coated magnetite (Fe3O4@APTES and Fe3O4@citrate) and of the conjugates and nonconjugated peptide fractions at 15 mg/mL: Fe3O4@citrate@ > 10 kDa (conjugate 1), Fe3O4@citrate@ 5–10 kDa (conjugate 2), Fe3O4@APTES@ > 10 kDa (conjugate 3), Fe3O4@APTES@ 5–10 kDa (conjugate 4), and nonconjugated peptide fractions (>10 kDa and 5–10 kDa peptide fractions). The determinations were carried out in triplicate. The results are expressed as the means ± standard deviation. Different letters indicate statistical difference between the samples (*p* ≤ 0.05). **Figure 7.** Average results of the RPs of coated magnetite (Fe3O4@APTES and Fe3O4@citrate) and of the conjugates and nonconjugated peptide fractions at 15 mg/mL: Fe3O4@citrate@ > 10 kDa (conjugate 1), Fe3O4@citrate@ 5–10 kDa (conjugate 2), Fe3O4@APTES@ > 10 kDa (conjugate 3), Fe3O4@APTES@ 5–10 kDa (conjugate 4), and nonconjugated peptide fractions (>10 kDa and 5–10 kDa peptide fractions). The determinations were carried out in triplicate. The results are expressed as the means ± standard deviation. Different letters indicate statistical difference between the samples (*p* ≤ 0.05). *Molecules* **2021**, *26*, x FOR PEER REVIEW 10 of 15

**Figure 8.** Average results of the OH·scavenging activities of coated magnetite (Fe3O4@APTES and Fe3O4@citrate) and of the conjugates and nonconjugated peptide fractions at 15 mg/mL: Fe3O4@citrate@ > 10 kDa (conjugate 1), Fe3O4@citrate@ 5–10 kDa (conjugate 2), Fe3O4@APTES@ > 10 kDa (conjugate 3), Fe3O4@APTES@ 5–10 kDa (conjugate 4), and nonconjugated peptide fractions (>10 kDa and 5–10 kDa peptide fractions). The determinations were carried out in triplicate. The results are expressed as the means ± standard deviation. Different letters indicate statistical differences between the samples (*p* ≤ 0.05). **Figure 8.** Average results of the OH· scavenging activities of coated magnetite (Fe3O4@APTES and Fe3O4@citrate) and of the conjugates and nonconjugated peptide fractions at 15 mg/mL: Fe3O4@citrate@ > 10 kDa (conjugate 1), Fe3O4@citrate@ 5–10 kDa (conjugate 2), Fe3O4@APTES@ > 10 kDa (conjugate 3), Fe3O4@APTES@ 5–10 kDa (conjugate 4), and nonconjugated peptide fractions (>10 kDa and 5–10 kDa peptide fractions). The determinations were carried out in triplicate. The results are expressed as the means ± standard deviation. Different letters indicate statistical differences between the samples (*p* ≤ 0.05).

These results suggest that conjugates 2 and 3 could be a used as antioxidant conjugates and evaluated in the future in chronic diseases related with oxidative stress, such as

Ferrous chloride tetrahydrate (FeCl2·4H2O, PA), sodium citrate dihydrate (Na3C6H5O7·2H2O, ACS >99%), APTES (97%), paraformaldehyde, 1-pentynylisonitrile, trichloroacetic acid (TCA), phenanthroline, and ferrous sulphate (FeSO4) were purchased from Sigma-Aldrich, Mexico City, Mexico. Ferric chloride hexahydrate (FeCl3·6H2O, 99.0%) and the ammonium hydroxide solution (NH4OH, 28.4%) were purchased from J.T. Baker, Mexico City, Mexico. Hydrochloric acid (HCl, PA) and potassium ferricyanide (K3[Fe(CN)6]) were purchased from Fermont, Monterrey, Mexico, and hydrogen peroxide (H2O2) from Reasol, Mexico city, Mexico. The peptide fractions were supplied by Labora-

The NPs were synthesized using the coprecipitation method [37]. A mixture of acidic solutions (HCl, 2 M) of the FeCl2·2H2O (5 mL, 5 M) and FeCl3·6H2O (20 mL, 2.5 M) salts was added to an aqueous solution of NH<sup>3</sup> (250 mL, 0.7 M) contained in a three-neck balloon using a peristaltic pump. All the solutions were bubbled with N<sup>2</sup> (g) before the synthesis. The reaction mixture was kept at room temperature under an N<sup>2</sup> (g) atmosphere under mechanical stirring (500 rpm) and ultrasound-treated for 25 min. After the reaction time, the obtained MNPs were separated using a neodymium magnet. Subsequently, they

torio de Bioquímica de la Nutrición (ENCB-IPN), Mexico City, Mexico.

of many associated genes [36].

**3. Materials and Methods** 

*3.2. Synthesis of Fe3O<sup>4</sup> NPs*

*3.1. Materials*

The RPs of the coated magnetite, as well as those of the conjugates and nonconjugated peptide fractions (Figure 7), were expressed as absorbances, where 1 represents the highest value. The RP of a sample refers to its ability to act as a proton acceptor (or electron donor) in an oxidation–reduction reaction and, therefore, the number of basic groups in the conjugates could be related to the obtained RP. In this sense, Fe3O4@APTES, with free amine groups on the magnetite surface, showed the highest RP value. Regarding the conjugates, those in which magnetite was conjugated to the fractions containing the peptides with masses >10 kDa (conjugates 1 and 3) had the highest RP values, with statistically significant differences between them and the conjugates 2 and 4. This result agrees with the study by González-Montoya et al. [27], who observed that the >10 kDa (RP = 0.7) fraction had a higher RP than the 5–10 kDa fraction (RP = 0.3). This could be attributed to the higher amount of basic amino acids in the >10 kDa fraction. A synergistic effect was observed when conjugating the peptide fractions to Fe3O4@APTES (conjugate 3 with respect to the >10 kDa peptide fraction), surely, due to the contribution of the amine groups that remained free, unreacted during the multicomponent reaction, on the surface of the magnetite coated with the APTES. It was also observed that the conjugation of the >10 kDa peptide fraction to magnetite NPs coated with sodium citrate (conjugate 1) slightly decreased the RP with respect to that of the peptide fraction, whilst the RP of the 5–10 kDa peptide fraction conjugated to Fe3O4@citrate (conjugate 2) did not exhibit statistically significant differences with respect to the nonconjugated fraction. In this way, conjugate 3 represents an opportunity for future evaluations of biological activities related with oxidative stress in chronic diseases.

The hydroxyl radical (OH·) is one of the most reactive free radicals and is generated by the Fenton reaction in cells [27]. This radical can be transformed into a superoxide anion and hydrogen peroxide in the presence of metal ions such as copper and iron. The OH· scavenging activities of the coated magnetite (Fe3O4@APTES and Fe3O4@citrate), as well as those of the conjugates and nonconjugated peptide fractions, are shown in Figure 8. According to the data, the nature of the groups exposed to the medium on each sample determines the properties of the compound. In the case of conjugates with the fraction > 10 kDa (conjugates 1 and 3), the activity is higher when it is used as an acidic component of the multicomponent reaction (conjugate 3). Therefore, in the final compound, the peptides would mostly expose their amine groups (which are apparently involved in the measured activity) to the environment. In the conjugates with the 5–10 kDa fraction (conjugates 2 and 4), the opposite effect was observed. The peptides contained in this fraction exerted a stronger OH· scavenging effect by exposing their acidic groups (conjugate 2). Remarkably, the OH· scavenging activities of conjugates 2 and 3 were significantly higher than that of the nonconjugated fractions. This result could be due to a synergistic effect of the respective carboxylic acid (from Fe3O4@citrate) and amine (from Fe3O4@APTES) groups in the corresponding final conjugates.

These results suggest that conjugates 2 and 3 could be a used as antioxidant conjugates and evaluated in the future in chronic diseases related with oxidative stress, such as cancer since it has been reported that antioxidant activity can modulate the activity of key proteins involved in the control of cell cycle progression and may influence the expression of many associated genes [36].

### **3. Materials and Methods**

### *3.1. Materials*

Ferrous chloride tetrahydrate (FeCl2·4H2O, PA), sodium citrate dihydrate (Na3C6H5O7·2H2O, ACS >99%), APTES (97%), paraformaldehyde, 1-pentynylisonitrile, trichloroacetic acid (TCA), phenanthroline, and ferrous sulphate (FeSO4) were purchased from Sigma-Aldrich, Mexico City, Mexico. Ferric chloride hexahydrate (FeCl3·6H2O, 99.0%) and the ammonium hydroxide solution (NH4OH, 28.4%) were purchased from J.T. Baker, Mexico City, Mexico. Hydrochloric acid (HCl, PA) and potassium ferricyanide (K3[Fe(CN)6]) were purchased from Fermont, Monterrey, Mexico, and hydrogen peroxide (H2O2) from Reasol, Mexico city, Mexico. The peptide fractions were supplied by Laboratorio de Bioquímica de la Nutrición (ENCB-IPN), Mexico City, Mexico.
