**1. Introduction**

The main challenges for magnetic nanoparticles (MNPs) used in biomedicine are related to their tendency to agglomerate and lack of biocompatibility [1]. Regarding biocompatibility, iron oxide nanoparticles (NPs) maghemite (γ-Fe2O3) and magnetite (Fe3O4) are advantageous for in vivo applications. Unlike other materials with good magnetic behavior, iron cell homeostasis is well-controlled by absorption, excretion, and storage processes. Even excess iron is efficiently removed from the body [2]. Specially, different works show that the superparamagnetic behavior of magnetite NPs is closely related to their nanometric size [3]. This property is essential for the in vivo applications of the material as it ensures that no magnetization remains in the system after stopping the action of an external magnetic field. Moreover, uncoated magnetite NPs tend to decrease the surface

free energy by forming stable aggregates under physiological conditions [4]. In this regard, it has been observed that the coupling of different types of biomolecules to Fe3O<sup>4</sup> NPs increases the stability of the system while directing it towards the desired biological target. Thereby, magnetite has been coupled to polymers and different anticancer drugs, with prominent applications in biomedicine, nanoscience, and nanotechnology [5–7]. These conjugates are of interest mainly because the benefits of the Fe3O<sup>4</sup> NPs and biomolecules are combined in the same system.

Peptide–Fe3O<sup>4</sup> conjugates are promising for biotechnological applications [8–10], particularly because peptides from natural sources and synthetic derivatives have been identified as promising for the treatment of diseases, such as cancer [11,12]. From the conjugation of peptides to Fe3O<sup>4</sup> NPs, one could not only selectively transport drugs towards biological targets, but also treat different diseases using magnetic hyperthermia [13–16]. Despite the advances in the development of methodologies for the conjugation of peptides to magnetite NPs, the current protocols are still limited by long and complex reaction steps. In most cases, they are based on classical peptide coupling between peptides and MNPs functionalized with carboxylic acid groups [17]. In some studies, surface modification of MNPs has been performed with ligands suitable for their subsequent coupling to peptides through click chemistry [14]. This restricts and makes the application of peptide–Fe3O<sup>4</sup> conjugates in biomedical investigations more expensive.

Several procedures for the combination of complex molecules based on multicomponent reactions have been reported [18,19]. Multicomponent protocols have a high potential owing to their high chemical efficiency, simple execution, and structural diversity at low synthesis costs [20]. One of the most used is the Ugi four-component (U-4C) reaction, in which the condensation of an oxo derivative, primary amine, carboxylic acid, and isonitrile leads to the formation of a dipeptoid in a single reaction step. The N-substituted amide in peptoids derived from the U-4C reaction is a structural motif of biological relevance because it prevents proteolytic degradation and limits the conformational orientation of peptide structures [21]. However, to the best of our knowledge, this type of reaction has not been used for the conjugation of bioactive peptides to MNPs.

On the other hand, several studies demonstrated the important role of oxidative stress and presence of different reactive oxygen and nitrogen species (ROS and RNS, respectively) in numerous noncommunicable diseases, such as cardiovascular diseases, diabetes, atherosclerosis, arthritis, and cancer [22]. These diseases lead to approximately 41 million deaths per year, equivalent to 71% of all deaths globally [23]. Some peptides and biomimetic peptides inhibit the generation of ROS and other free radicals [24,25]. Therefore, the search for antioxidant peptide structures that can prevent oxidative stress and its associated adverse effects is of increasing interest. In this sense, González-Montoya et al. evaluated the anti-inflammatory, antioxidant, and antiproliferative activities of three peptide fractions obtained from germinated soybeans [26,27]. These peptide fractions exhibited exceptional antioxidant and antiproliferative activities in breast and cervical cancer cell lines. Motivated by these studies, we present the synthesis, characterization, and biological evaluation of conjugates of Fe3O<sup>4</sup> NPs with bioactive peptide fractions from germinated soybeans. To the best of our knowledge, these bioconjugates are reported for the first time. A multicomponent procedure is also used for the first time in the conjugation of bioactive peptide fractions from germinated soybeans to magnetite NPs.

### **2. Results and Discussion**

A procedure for the conjugation of bioactive peptide fractions to magnetite NPs was developed (Scheme 1). The methodology comprised three fundamental parts: (1) synthesis of Fe3O<sup>4</sup> NPs, (2) functionalization of Fe3O<sup>4</sup> NPs in the form of either carboxylic acid or amine, and (3) conjugation using the U-4C reaction of the peptide fractions extracted from germinated soybeans to the functionalized Fe3O<sup>4</sup> NPs. Once the desired conjugates were obtained, the antioxidant activity of both the peptide–Fe3O<sup>4</sup> conjugates as well as the peptide fractions and coated magnetite were determined.

tide fractions and coated magnetite were determined.

**Scheme 1.** Experimental workflow. **Scheme 1.** Experimental workflow. netite were observed at 18.3, 30.2, 35.6, 43.3, 53.6, 57.3, 62.9, 74.5, and 90.6°. These peaks are indexed to the (111), (220), (311), (400), (422), (511), (440), (533), and (731) hkl planes of

### *2.1. Characterization of Fe3O<sup>4</sup> 2.1. Characterization of Fe3O<sup>4</sup>* the Joint Committee on Powder Diffraction Standards (JCPDS) 19629 magnetite standard

The X-ray diffraction (XRD) pattern of the synthesized magnetite is shown in Figure 1 (black profile). The main peaks associated with the characteristic spinel structure of magnetite were observed at 18.3, 30.2, 35.6, 43.3, 53.6, 57.3, 62.9, 74.5, and 90.6°. These peaks are indexed to the (111), (220), (311), (400), (422), (511), (440), (533), and (731) hkl planes of the Joint Committee on Powder Diffraction Standards (JCPDS) 19629 magnetite standard pattern, respectively. The estimated Fe3O<sup>4</sup> cell parameter was 8.3824 Å and the average crystallite size calculated using the Debye–Scherrer equation (Equation (1)) was 17 nm. This last value allowed confirming the nanometric size of the obtained magnetite. The X-ray diffraction (XRD) pattern of the synthesized magnetite is shown in Figure 1 (black profile). The main peaks associated with the characteristic spinel structure of magnetite were observed at 18.3, 30.2, 35.6, 43.3, 53.6, 57.3, 62.9, 74.5, and 90.6◦ . These peaks are indexed to the (111), (220), (311), (400), (422), (511), (440), (533), and (731) hkl planes of the Joint Committee on Powder Diffraction Standards (JCPDS) 19629 magnetite standard pattern, respectively. The estimated Fe3O<sup>4</sup> cell parameter was 8.3824 Å and the average crystallite size calculated using the Debye–Scherrer equation (Equation (1)) was 17 nm. This last value allowed confirming the nanometric size of the obtained magnetite. pattern, respectively. The estimated Fe3O<sup>4</sup> cell parameter was 8.3824 Å and the average crystallite size calculated using the Debye–Scherrer equation (Equation (1)) was 17 nm. This last value allowed confirming the nanometric size of the obtained magnetite.

germinated soybeans to the functionalized Fe3O<sup>4</sup> NPs. Once the desired conjugates were obtained, the antioxidant activity of both the peptide–Fe3O<sup>4</sup> conjugates as well as the pep-

germinated soybeans to the functionalized Fe3O<sup>4</sup> NPs. Once the desired conjugates were obtained, the antioxidant activity of both the peptide–Fe3O<sup>4</sup> conjugates as well as the pep-

*Molecules* **2021**, *26*, x FOR PEER REVIEW 3 of 15

tide fractions and coated magnetite were determined.

termined was 13 ± 5 nm. The fact that this value is similar to the calculated crystallite size suggests that the system was monocrystalline. In the STEM images, it is observed that uncoated Fe3O<sup>4</sup> NPs have spherical morphology and are agglomerated, most likely be-**Figure 1.** XRD patterns of the uncoated magnetite (Fe3O4) and magnetite samples coated with so-**Figure 1.** XRD patterns of the uncoated magnetite (Fe3O<sup>4</sup> ) and magnetite samples coated with sodium citrate (Fe3O4@citrate) and APTES (Fe3O4@APTES).

cause of their small sizes. dium citrate (Fe3O4@citrate) and APTES (Fe3O4@APTES). Additional information about the size and morphology of Fe3O<sup>4</sup> NPs was obtained using scanning transmission electron microscopy (STEM) (Figure 2). The particle size determined was 13 ± 5 nm. The fact that this value is similar to the calculated crystallite size Additional information about the size and morphology of Fe3O<sup>4</sup> NPs was obtained using scanning transmission electron microscopy (STEM) (Figure 2). The particle size determined was 13 ± 5 nm. The fact that this value is similar to the calculated crystallite size suggests that the system was monocrystalline. In the STEM images, it is observed that uncoated Fe3O<sup>4</sup> NPs have spherical morphology and are agglomerated, most likely because of their small sizes.

suggests that the system was monocrystalline. In the STEM images, it is observed that uncoated Fe3O<sup>4</sup> NPs have spherical morphology and are agglomerated, most likely be-

cause of their small sizes.

NH<sup>4</sup>

**Normalized transmitance (a. u.)**

NH<sup>4</sup>

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**Figure 2.** Microscopy image of uncoated Fe3O<sup>4</sup> with the corresponding histogram. **Figure 2.** Microscopy image of uncoated Fe3O<sup>4</sup> with the corresponding histogram. sponding to the overlapping valence vibrations of the N–H and O–H bonds, was ob-

The synthesis of Fe3O<sup>4</sup> was also corroborated by infrared (IR) spectroscopy (Figure 3, black spectrum). From the assignment of the absorption bands observed in the IR spectra obtained, it was possible to analyze the respective functional groups of the molecules present in each of the synthesized materials. Thus, a broad band centered at 3252 cm−<sup>1</sup> sponding to the overlapping valence vibrations of the N–H and O–H bonds, was ob-The synthesis of Fe3O<sup>4</sup> was also corroborated by infrared (IR) spectroscopy (Figure 3, black spectrum). From the assignment of the absorption bands observed in the IR spectra obtained, it was possible to analyze the respective functional groups of the molecules present in each of the synthesized materials. Thus, a broad band centered at 3252 cm−<sup>1</sup> , corresponding to the overlapping valence vibrations of the N–H and O–H bonds, was observed. It was assigned to NH<sup>4</sup> <sup>+</sup> and OH<sup>−</sup> ions and adsorbed water molecules on the surface of MNPs. In addition, the band at 1628 cm−<sup>1</sup> can be attributed to the bending of the NH<sup>4</sup> <sup>+</sup> group on the magnetite surface. Finally, at 573 cm−<sup>1</sup> , a band assigned to the valence vibration of the Fe–O bond of Fe3O<sup>4</sup> was observed. served. It was assigned to NH<sup>4</sup> + and OH<sup>−</sup> ions and adsorbed water molecules on the surface of MNPs. In addition, the band at 1628 cm−1 can be attributed to the bending of the <sup>+</sup> group on the magnetite surface. Finally, at 573 cm−1, a band assigned to the valence vibration of the Fe–O bond of Fe3O<sup>4</sup> was observed.

, corre-

, corre-

**Figure 3.** IR spectra of the uncoated magnetite (Fe3O4) and magnetite samples coated with sodium **Figure 3.** IR spectra of the uncoated magnetite (Fe3O<sup>4</sup> ) and magnetite samples coated with sodium citrate (Fe3O4@citrate) and APTES (Fe3O4@APTES).

 Fe3O<sup>4</sup> Fe3O4@citrate Fe3O4@APTES citrate (Fe3O4@citrate) and APTES (Fe3O4@APTES). Finally, the hydrodynamic diameter of Fe3O<sup>4</sup> was determined using dynamic light scattering (DLS) measurements. The value obtained (180 ± 70 nm) was used as a reference for comparison to the coated magnetite samples.

**Figure 3.** IR spectra of the uncoated magnetite (Fe3O4) and magnetite samples coated with sodium

scattering (DLS) measurements. The value obtained (180 ± 70 nm) was used as a reference

Finally, the hydrodynamic diameter of Fe3O<sup>4</sup> was determined using dynamic light

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

Finally, the hydrodynamic diameter of Fe3O<sup>4</sup> was determined using dynamic light

As mentioned in the Introduction, though magnetite is a biocompatible material, its

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 addi-

tion, functionalization enables the conjugation with therapeutic biomolecules and contrib-

utes 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/function-

alization 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 sur-

face in the form of carboxylic acid and amine, respectively. Both functional groups are

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

**Wavenumber (cm−1)**

citrate (Fe3O4@citrate) and APTES (Fe3O4@APTES).

for comparison to the coated magnetite samples.

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