*2.7. Stability of Peptide Ib*−*M1 and Ib*−*M1/Alg*−*Chi Bioconjugate*

The stability of the free peptide and the Ib−M1/Alg−Chi bioconjugate was determined under the conditions listed in Table 1. Subsequently, the antimicrobial activity against *E. coli* was determined, as mentioned in the previous section.

**Condition Features** pH 2 and 11 Temperature 4 and 100 ◦C Proteases Trypsin and Pepsin

**Table 1.** Conditions used to evaluate the stability of peptides Ib−M1 and Ib−M1/Alg-Chi.

#### 2.7.1. pH Stability

The peptide and the bioconjugate were dissolved in glycine buffer solution at pH 2 and pH 11 for stability tests. These solutions were left for 30 min at 37 ◦C under stirring at 120 rpm; then, the pH was adjusted to 7, and the antimicrobial activity was determined as indicated in Section 2.5 [36].

#### 2.7.2. Temperature Stability

For the stability tests at various temperatures, the Ib−M1 peptide and the Ib−M1/Alg-Chi bioconjugate were left for 90 min at the indicated temperatures. These tests were carried out on agitation plates. Then, antimicrobial activity was determined, as previously mentioned in Section 2.5 [37].

#### 2.7.3. Trypsin and Pepsin Stability

Ib−M1 peptide and Ib−M1/Alg−Chi bioconjugate were exposed to pepsin and trypsin in a ratio (enzyme: peptide) of 27:1 and 1:20, respectively, in a beaker. This solution was stirred at 120 rpm at 37 ◦C for 90 min, taking a sample every 30 min, starting with an initial sample at 0 min. For an activity with pepsin, it was necessary to adjust the pH of the solution to 2 in order to simulate gastric conditions and allow for the action of the enzyme [38]. Assays with bioconjugates were performed in the same way as for a free peptide, adjusting concentrations to maintain the enzyme: peptide ratio. Then, antimicrobial activity was determined, as previously mentioned in Section 2.5.

#### *2.8. Statistical Analysis*

*2.8. Statistical Analysis* 

Except where indicated otherwise, all results are representative of two experiments, and each experiment was replicated three times. Arithmetic means values ± standard deviations are reported for each case. All analyses and graphics were generated using Origin (Pro) version 2019 (OriginLab Corporation, Northampton, MA, USA). **3. Results**  *3.1. Preparation and Characterization of Alg−Chi NPs*  Figure 1 shows preparation scheme of Alg−Chi NPs. TPP was used as a crosslinking agent due to its high negative charge, allowing it to interact with the amino groups of

solution to 2 in order to simulate gastric conditions and allow for the action of the enzyme [38]. Assays with bioconjugates were performed in the same way as for a free peptide, adjusting concentrations to maintain the enzyme: peptide ratio. Then, antimicrobial activ-

Except where indicated otherwise, all results are representative of two experiments, and each experiment was replicated three times. Arithmetic means values ± standard deviations are reported for each case. All analyses and graphics were generated using Origin

#### **3. Results** chitosan. Similarly, the negative charge of the alginate interacts with the positive surface

#### *3.1. Preparation and Characterization of Alg*−*Chi NPs* charges of the chitosan-TPP aggregates, allowing for the formation of the Alg−Chi NPs. The obtained NPs present with high agglomeration, as evidenced by the SEM

*Polymers* **2022**, *14*, x FOR PEER REVIEW 5 of 15

ity was determined, as previously mentioned in Section 2.5.

(Pro) version 2019 (OriginLab Corporation, Northampton, MA, USA).

Figure 1 shows preparation scheme of Alg−Chi NPs. TPP was used as a crosslinking agent due to its high negative charge, allowing it to interact with the amino groups of chitosan. Similarly, the negative charge of the alginate interacts with the positive surface charges of the chitosan-TPP aggregates, allowing for the formation of the Alg−Chi NPs. micrograph presented in the inset of Figure 1. This aggregation is responsible for the size of the nanoparticles, which was determined to be 134.6 nm by DLS. Despite this aggregation, the dispersion index of the nanoparticle suspension corresponded to 0.177, which indicates that the obtained size distribution is homogeneous.

**Figure 1.** Scheme: synthesis of Alg-Chit NPs by ionic gelation. Inset: scanning electron micrograph of Alg−Chi NPs at 40.000X and size distribution of Alg−Chi NPs. Alg−Chit NPs: alginate-chitosan nanoparticles. **Figure 1.** Scheme: synthesis of Alg-Chit NPs by ionic gelation. Inset: scanning electron micrograph of Alg−Chi NPs at 40.000X and size distribution of Alg−Chi NPs. Alg−Chit NPs: alginate-chitosan nanoparticles.

The Figure 2 shows the FTIR spectrum of Alg−Chi NPs; the stretching presented at 3267 cm−1 corresponds to the O-H and N-H groups of the polymers that are present in their structures. The band at 2916 cm−1 is attribuided to C-H strectching vibrations. The band at 1589 cm−1 corresponds to the carbonyl group of Alg [39], whereas the band at 1430 The obtained NPs present with high agglomeration, as evidenced by the SEM micrograph presented in the inset of Figure 1. This aggregation is responsible for the size of the nanoparticles, which was determined to be 134.6 nm by DLS. Despite this aggregation, the dispersion index of the nanoparticle suspension corresponded to 0.177, which indicates that the obtained size distribution is homogeneous.

The Figure 2 shows the FTIR spectrum of Alg−Chi NPs; the stretching presented at 3267 cm−<sup>1</sup> corresponds to the O-H and N-H groups of the polymers that are present in their structures. The band at 2916 cm−<sup>1</sup> is attribuided to C-H strectching vibrations. The band at 1589 cm−<sup>1</sup> corresponds to the carbonyl group of Alg [39], whereas the band at 1430 cm−<sup>1</sup> is assigned to the amino group of chitosan. The intense band that appears at 1040 cm−<sup>1</sup> is associated with molecules present in the polymers. Bands associated with TPP can also be seen in the spectrum; for example, the band at 1290 cm−<sup>1</sup> is associated with *p* = C strectching, that at 1084 cm−<sup>1</sup> is associated with symmetric and antisymmetric stretching vibrations in the PO<sup>2</sup> group, that at 1020 cm−<sup>1</sup> is associated with symmetric and

antisymmetric stretching vibrations in the PO<sup>3</sup> group, and that at 820 cm−<sup>1</sup> corresponds to antisymmetric stretching of the P-O-P bridge [40]. vibrations in the PO2 group, that at 1020 cm−1 is associated with symmetric and antisymmetric stretching vibrations in the PO3 group, and that at 820 cm−1 corresponds to antisymmetric stretching of the P-O-P bridge [40].

cm−1 is assigned to the amino group of chitosan. The intense band that appears at 1040 cm−1 is associated with molecules present in the polymers. Bands associated with TPP can also be seen in the spectrum; for example, the band at 1290 cm−1 is associated with *p* = C strectching, that at 1084 cm−1 is associated with symmetric and antisymmetric stretching

*Polymers* **2022**, *14*, x FOR PEER REVIEW 6 of 15

**Figure 2.** FTIR spectrum of Alg−Chi NPs. FTIR: Fourier transform infrared spectroscopy. **Figure 2.** FTIR spectrum of Alg−Chi NPs. FTIR: Fourier transform infrared spectroscopy.

*3.2. Preparation of the Ib−M1/Alg−Chi Bioconjugate 3.2. Preparation of the Ib*−*M1/Alg*−*Chi Bioconjugate*

Figure 3A shows the reaction scheme for the immobilization of the Ib−M2 peptide on the surface of Alg−Chi NPs. Figure 3A shows the reaction scheme for the immobilization of the Ib−M2 peptide on the surface of Alg−Chi NPs. *Polymers* **2022**, *14*, x FOR PEER REVIEW 7 of 15

An amide bond was formed between the carboxyl group of Glu−1 of the peptide and

**Figure 3.** (**A**) Reaction scheme into amino groups of chitosan of Alg−Chi NPs and the carboxyl group of Glu residue of Ib−M1. (**B**) Monitoring of immobilization of the peptide by UV–Vis. Alg−Chit NPs: alginate-chitosan nanoparticles. **Figure 3.** (**A**) Reaction scheme into amino groups of chitosan of Alg−Chi NPs and the carboxyl group of Glu residue of Ib−M1. (**B**) Monitoring of immobilization of the peptide by UV–Vis. Alg−Chit NPs: alginate-chitosan nanoparticles.

**Figure 4.** Growth kinetics of *E. coli* (ATCC 25922) in the presence of free Ib−M1 peptide at 25 µM ( ■), 12.5 µM (▲), 6.25 µM (▼), and 3.12 µM (◆), with streptomycin as reference antibiotic at 6.25 µM (⬤). Growth of *E. coli* without the addition of compounds (⬟) was included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation. The Ib-M1 concentrations evaluated ranged from 100 µM to 0.78 µM; and are plotted in the Supplementary Figures (Figure S1A,B).

The antimicrobial activity of peptide Ib−M1 against *E. coli* 25922 was determined. To this end, the MIC was determined using the microdilution method, with streptomycin as the reference antibiotic. Figure 4 shows the growth kinetics of free Ib−M1 in the presence

*3.3. Antimicrobial Activity against E. coli* 

An amide bond was formed between the carboxyl group of Glu−1 of the peptide and the amino groups on the surface of the NPs. Immobilization was monitored by determining free amino groups in the suspension using the Bradford method. As shown in Figure 3B, after 2 h of reaction, 37% immobilization of the peptide on the Alg−Chi NPs was reached. **Figure 3.** (**A**) Reaction scheme into amino groups of chitosan of Alg−Chi NPs and the carboxyl group of Glu residue of Ib−M1. (**B**) Monitoring of immobilization of the peptide by UV–Vis. Alg−Chit NPs: alginate-chitosan nanoparticles.

*Polymers* **2022**, *14*, x FOR PEER REVIEW 7 of 15

#### *3.3. Antimicrobial Activity against E. coli 3.3. Antimicrobial Activity against E. coli*

The antimicrobial activity of peptide Ib−M1 against *E. coli* 25922 was determined. To this end, the MIC was determined using the microdilution method, with streptomycin as the reference antibiotic. Figure 4 shows the growth kinetics of free Ib−M1 in the presence of streptomycin at 6.25 µM and peptide Ib−M1 at concentrations between 25 and 3.12 µM. The antimicrobial activity of peptide Ib−M1 against *E. coli* 25922 was determined. To this end, the MIC was determined using the microdilution method, with streptomycin as the reference antibiotic. Figure 4 shows the growth kinetics of free Ib−M1 in the presence of streptomycin at 6.25 µM and peptide Ib−M1 at concentrations between 25 and 3.12 µM.

**Figure 4.** Growth kinetics of *E. coli* (ATCC 25922) in the presence of free Ib−M1 peptide at 25 µM ( ■), 12.5 µM (▲), 6.25 µM (▼), and 3.12 µM (◆), with streptomycin as reference antibiotic at 6.25 µM (⬤). Growth of *E. coli* without the addition of compounds (⬟) was included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation. The Ib-M1 concentrations evaluated ranged from 100 µM to 0.78 µM; and are plotted in the Supplementary Figures (Figure S1A,B). **Figure 4.** Growth kinetics of *E. coli* (ATCC 25922) in the presence of free Ib−M1 peptide at 25 µM (), 12.5 µM (N), 6.25 µM (H), and 3.12 µM (), with streptomycin as reference antibiotic at 6.25 µM (•). Growth of *E. coli* without the addition of compounds ( ⬟ ) was included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation. The Ib-M1 concentrations evaluated ranged from 100 µM to 0.78 µM; and are plotted in the Supplementary Figures (Figure S1A,B).

As shown in Figure 4, the minimum inhibitory concentration of Ib−M1 was 12.5 µM, which is concentration at which the growth of *E. coli* is inhibited for up to 24 h.

The MIC of the Ib−M1/Alg−Chi bioconjugate was determined by incubation with *E. coli* 25922 at an immobilized peptide concentration of between 12.5 and 0.78 µM. Figure 5 shows the growth kinetics of the bacteria in the presence of the Ib−M1/Alg−Chi bioconjugate and Alg−Chi NPs at a concentration of 0.4 mg/mL.

As shown in Figure 5, the peptide activity was maintained when it was immobilized on the nanoparticles. In addition, a synergistic effect was observed in the combination of the Alg−Chi NPs with the peptide. Figure 5 shows that Ib−M1 immobilized at a concentration of 6.25 µM inhibited approximately 35% of the growth of the microorganism after 24 h of incubation with the free peptide in the same concentration (Figure 4). On the other hand, the nanoparticles did not affect the growth of the bacteria. Therefore, Alg and chitosan did not show antimicrobial activity against *E. coli*.

1

As shown in Figure 4, the minimum inhibitory concentration of Ib−M1 was 12.5 µM,

The MIC of the Ib−M1/Alg−Chi bioconjugate was determined by incubation with *E. coli* 25922 at an immobilized peptide concentration of between 12.5 and 0.78 µM. Figure 5 shows the growth kinetics of the bacteria in the presence of the Ib−M1/Alg−Chi bioconju-

which is concentration at which the growth of *E. coli* is inhibited for up to 24 h.

gate and Alg−Chi NPs at a concentration of 0.4 mg/mL.

**Figure 5.** Growth kinetics of *E. coli* (ATCC 25922) in the presence of the Ib−M1/Alg−Chi bioconjugate at 12.5 µM (▲), 6.25 µM (▼), and 3.12 µM (◆). Results from *E. coli* with Alg−Chi NPs at 0.4 mg/mL (⬤) and *E. coli* without the addition of compounds (⬟) are included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation. As shown in Figure 5, the peptide activity was maintained when it was immobilized **Figure 5.** Growth kinetics of *E. coli* (ATCC 25922) in the presence of the Ib−M1/Alg−Chi bioconjugate at 12.5 µM (N), 6.25 µM (H), and 3.12 µM (). Results from *E. coli* with Alg−Chi NPs at 0.4 mg/mL (•) and *E. coli* without the addition of compounds ( ⬟ ) are included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation.

#### on the nanoparticles. In addition, a synergistic effect was observed in the combination of *3.4. Cytotoxicity*

3.5.1. pH Stability

the Alg−Chi NPs with the peptide. Figure 5 shows that Ib−M1 immobilized at a concentration of 6.25 µM inhibited approximately 35% of the growth of the microorganism after 24 h of incubation with the free peptide in the same concentration (Figure 4). On the other hand, the nanoparticles did not affect the growth of the bacteria. Therefore, Alg and chi-Figure 6 shows the percentage of cytotoxicity of Ib−M1 and Ib−M1/Alg-Chit at the MIC determined for *E. coli* strain 25922 (12.5 µM); the cytotoxicity of the Alg−Chi NPs (0.4 mg/mL) was also recorded. *Polymers* **2022**, *14*, x FOR PEER REVIEW 9 of 15

tosan did not show antimicrobial activity against *E. coli*.

**Figure 6.** Cytotoxic activity of the free peptide Ib−M1 (12.5 µM), the bioconjugate Ib−M1/Alg−Chi (12.5 µM) and Alg−Chi NPs (0.4 mg/mL) in Vero cells. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation. Cells maintained in culture medium without any treatment were used as a negative control (— Control; not shown). *p* < 0.0001: \*\*\*\*; *p* ≤ 0.0001: \*\*\*. *3.5. Stability of Ib−M1 and Ib−M1/Alg−Chit*  **Figure 6.** Cytotoxic activity of the free peptide Ib−M1 (12.5 µM), the bioconjugate Ib−M1/Alg−Chi (12.5 µM) and Alg−Chi NPs (0.4 mg/mL) in Vero cells. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation. Cells maintained in culture medium without any treatment were used as a negative control (— Control; not shown). *p* < 0.0001: \*\*\*\*; *p* ≤ 0.0001: \*\*\*.

1

**Figure 7.** Effect of pH conditions on the antimicrobial activity of peptide Ib−M1 (**A**) and bioconjugate Ib−M1/Alg−Chi (**B**). Results of *E. coli* without the addition of compounds (⬟) are included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and ex-

pressed in terms of arithmetic average plus standard deviation.

Ib−M1/Alg−Chi biconjugate was not affected under alkaline and acid conditions.

The free peptide at a concentration of 12.5 µM (MIC) exhibited an approximate cytotoxicity of 5%, showing a statistically significant difference with respect to the Ib−M1/Alg−Chi bioconjugate, with a *p* value < 0.0001 (42.19 ± 10.13). The cytotoxicity value of the Alg−Chi NPs also showed a statistically significant difference (*p* ≤ 0.0001) with respect to the free Ib−M1 peptide. **Figure 6.** Cytotoxic activity of the free peptide Ib−M1 (12.5 µM), the bioconjugate Ib−M1/Alg−Chi (12.5 µM) and Alg−Chi NPs (0.4 mg/mL) in Vero cells. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation. Cells maintained in culture medium without any treatment were used as a negative control (— Control; not shown). *p* < 0.0001: \*\*\*\*; *p* ≤ 0.0001: \*\*\*.

#### *3.5. Stability of Ib*−*M1 and Ib*−*M1/Alg*−*Chit* 3.5.1. pH Stability *3.5. Stability of Ib−M1 and Ib−M1/Alg−Chit*  3.5.1. pH Stability

Figure 7 shows the results of the antimicrobial activity with prior exposure to different pH conditions. As shown in Figure 5, the activity of the Ib−M1 peptide and the Ib−M1/Alg−Chi biconjugate was not affected under alkaline and acid conditions. Figure 7 shows the results of the antimicrobial activity with prior exposure to different pH conditions. As shown in Figure 5, the activity of the Ib−M1 peptide and the Ib−M1/Alg−Chi biconjugate was not affected under alkaline and acid conditions.

*Polymers* **2022**, *14*, x FOR PEER REVIEW 9 of 15

**Figure 7.** Effect of pH conditions on the antimicrobial activity of peptide Ib−M1 (**A**) and bioconjugate Ib−M1/Alg−Chi (**B**). Results of *E. coli* without the addition of compounds (⬟) are included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation. **Figure 7.** Effect of pH conditions on the antimicrobial activity of peptide Ib−M1 (**A**) and bioconjugate Ib−M1/Alg−Chi (**B**). Results of *E. coli* without the addition of compounds ( ⬟ ) are included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation. *Polymers* **2022**, *14*, x FOR PEER REVIEW 10 of 15

#### 3.5.2. Thermal Stability 3.5.2. Thermal Stability

3.5.3. Proteolytic Stability

The influence of the temperatures used in the tests on Ib−M1 and Ib−M1/Alg−Chi are shown in Figure 8. There was no decrease in the activity of the compounds, which indicates good stability of the peptide and the bioconjugate during thermal treatments. The influence of the temperatures used in the tests on Ib−M1 and Ib−M1/Alg−Chi are shown in Figure 8. There was no decrease in the activity of the compounds, which indicates good stability of the peptide and the bioconjugate during thermal treatments.

**Figure 8.** Effect of heat treatments on the antimicrobial activity of the Ib−M1 peptide (**A**) and the Ib−M1/Alg−Chi bioconjugate (**B**). Results of *E. coli* without the addition of compounds (⬟) are included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation. **Figure 8.** Effect of heat treatments on the antimicrobial activity of the Ib−M1 peptide (**A**) and the Ib−M1/Alg−Chi bioconjugate (**B**). Results of *E. coli* without the addition of compounds ( ⬟ ) are included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic average plus standard deviation.

The effect of the proteolytic activity of trypsin and pepsin on the free peptide and the

The free peptide Ib−M1, as well as the immobilized peptide Ib−M1/Alg-Chi, showed

in the microbial population was observed at 24 h with the Ib−M1/Alg−Chi bioconjugate, and the growth of the microorganism slowed down within the first 8 h for the same wells, whereas the stability of the free peptide against pepsin was lost once it was exposed to this protease. On the contrary, the Ib−M1/Alg−Chi bioconjugate maintained its antimicro-

**Figure 9.** Antimicrobial activity of the peptide and the bioconjugate with prior exposure to trypsin (**A**) and pepsin (**B**). Results of *E. coli* without the addition of compounds (⬟) are included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and ex-

1

1

bioconjugate is shown Figure 9A and B, respectively.

bial activity after being exposed to pepsin.

pressed in terms of arithmetic mean plus standard deviation.

#### 3.5.3. Proteolytic Stability whereas the stability of the free peptide against pepsin was lost once it was exposed to

3.5.3. Proteolytic Stability

3.5.2. Thermal Stability

The effect of the proteolytic activity of trypsin and pepsin on the free peptide and the bioconjugate is shown Figure 9A,B, respectively. this protease. On the contrary, the Ib−M1/Alg−Chi bioconjugate maintained its antimicrobial activity after being exposed to pepsin.

**Figure 8.** Effect of heat treatments on the antimicrobial activity of the Ib−M1 peptide (**A**) and the Ib−M1/Alg−Chi bioconjugate (**B**). Results of *E. coli* without the addition of compounds (⬟) are included for comparison purposes. Each result was evaluated in triplicate in two independent exper-

The effect of the proteolytic activity of trypsin and pepsin on the free peptide and the

The free peptide Ib−M1, as well as the immobilized peptide Ib−M1/Alg-Chi, showed instability against the proteolytic action of trypsin, with proliferation of the microorganism at 24 h and optical densities like that of the growth control group. However, a decrease in the microbial population was observed at 24 h with the Ib−M1/Alg−Chi bioconjugate, and the growth of the microorganism slowed down within the first 8 h for the same wells,

iments and expressed in terms of arithmetic average plus standard deviation.

bioconjugate is shown Figure 9A and B, respectively.

*Polymers* **2022**, *14*, x FOR PEER REVIEW 10 of 15

The influence of the temperatures used in the tests on Ib−M1 and Ib−M1/Alg−Chi are shown in Figure 8. There was no decrease in the activity of the compounds, which indicates good stability of the peptide and the bioconjugate during thermal treatments.

**Figure 9.** Antimicrobial activity of the peptide and the bioconjugate with prior exposure to trypsin (**A**) and pepsin (**B**). Results of *E. coli* without the addition of compounds (⬟) are included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic mean plus standard deviation. **Figure 9.** Antimicrobial activity of the peptide and the bioconjugate with prior exposure to trypsin (**A**) and pepsin (**B**). Results of *E. coli* without the addition of compounds ( ⬟ ) are included for comparison purposes. Each result was evaluated in triplicate in two independent experiments and expressed in terms of arithmetic mean plus standard deviation.

The free peptide Ib−M1, as well as the immobilized peptide Ib−M1/Alg-Chi, showed instability against the proteolytic action of trypsin, with proliferation of the microorganism at 24 h and optical densities like that of the growth control group. However, a decrease in the microbial population was observed at 24 h with the Ib−M1/Alg−Chi bioconjugate, and the growth of the microorganism slowed down within the first 8 h for the same wells, whereas the stability of the free peptide against pepsin was lost once it was exposed to this protease. On the contrary, the Ib−M1/Alg−Chi bioconjugate maintained its antimicrobial activity after being exposed to pepsin.

#### **4. Discussion**

Results obtained in this work are consistent with those reported by Bagre et al. [41]. Using SEM, they observed that alginate and chitosan nanoparticles obtained by ionic gelation were agglomerated. Furthermore, they observed particle sizes of 213 ± 3.8 nm, whereas in the present study, we obtained particle sizes of 150 ± 56 nm with a polydispersity value of 0.177, which indicates particles of homogeneous size. This aggregation is due to the tight crosslinking caused by the TPP with the chitosan molecules [41].

We prepared bioconjugates according to the methodology described by Ropero-Vega et al. [33], who used iron oxide nanoparticles coated with chitosan to immobilize the Ib-M2 peptide, obtaining a value of 30% of the immobilized peptide. In the present study, we achieved 37% immobilization, a value close to that reported by Ropero-Vega et al. The above shows that this method allows for the immobilization of Ib-M peptides on chitosan, which could expand the applications to obtaining antibacterial surfaces based on this polymer.

*E. coli* (ATCC 25922) was used as a reference microorganism for antimicrobial activity and was inhibited for the free peptide Ib−M1 at 12.5 µM. The result shows promising characteristics with respect to the clinical use of peptides to treat infectious diseases caused by microbial agents such as *E. coli.* Prada et al. [28] used the Ib−M1 peptide as an antimicrobial from different species of *E. coli* as ATCC reference strains and clinical isolates with MIC values of 4.7 and 1.6 µM, respectively. In addition, Flórez-Castillo et al. [30] showed the antimicrobial action of Ib−M1 against fourteen strains of pathogenic *E. coli*. The characteristics of the Ib-M peptide have been associated mainly with its positive charge and increased hydrophobicity as a result of the presence of arginine and tryptophan residues in the sequence [27].

1

The antimicrobial activity of the Ib−M1 peptide was not affected by immobilization in the Alg−Chi NPs. The above was evidenced by the MIC being maintained in the bioconjugate. These immobilization strategies have been used in various ways and have the advantage of preserving the properties of the compound of interest. Additionally, it was found that Alg−Chi NPs do not show antibacterial activity against *E. coli* at the evaluated concentrations, showing that the activity of the bioconjugate is due only to the presence of the peptide. This result is significant because it shows that the immobilization of the Ib−M1 peptide does not affect its activity. In addition, the activity of the peptide immobilized at 0.5 × CMI (6.25 µM) prolonged the latent phase of the bacteria for at least 8 h and reduced its final concentration by up to 40% at 24 h relative to the growth control. These properties are similar to those reported in previous studies, wherein Ib-M2 was immobilized on iron oxide nanoparticles and its antibacterial activity was maintained against *E. coli* O157:H7 [33].

The cytotoxic effect of Ib−M1 and Ib−M1/Alg−Chi on Vero cells was evaluated by MTT assay. Our studies showed that Ib−M1 at the concentration of the CMI (12.5 µM) did not result in significant percentages of cytotoxicity, whereas the cytotoxicity generated by the bioconjugate showed a considerable increase, as well as the nanoparticles without the peptide. This result is consistent with that reported by Prada-Prada et al. [28], who observed that the Ib−M1 peptide presented cytotoxic concentration 50 (CC50) in Vero cells at 395.2 µM. These results differ from those reported in previous studies with Alg−Chi polymers in the Vero cell line, as we observed 10% cytotoxicity (cell viability of 90%) [42]. Alginate and chitosan have been used in studies aimed at drug delivery and as healing polymers for skin wounds owing to their biocompatible properties, with low cytotoxicity in Vero cell lines and BHK-21 cells [43].

The stability tests at pH 2 and 11 during the 30 min of treatment and the thermal tests (4 and 100 ◦C) revealed that the Ib−M1 peptide and the Ib−M1/Alg−Chi bioconjugate maintained stable antimicrobial activity under these conditions. In contrast, Fahimirad et al. [44], evaluated the free antimicrobial peptide LL37 and observed that acidic pH (2) inhibits antibacterial activity. However, at neutral and basic pH, its activity is maintained, and when boiled for longer than 10 min, it loses stability, increasing the MIC. However, these characteristics improved once the LL37 peptide was immobilized in Chi NPs, maintaining its antimicrobial activity in terms of MIC under the evaluated pH and temperature conditions. These findings are also consistent with those reported by Yu et al., who showed higher stability of the antimicrobial peptide microcin J25 (MccJ25) when conjugated with chitosan nanoparticles [45]. Therefore, the stability of the Ib−M1 peptide is another characteristic that makes it promising for use as a new antimicrobial compound.

Immobilization of the peptide did not prevent the effect of trypsin; therefore, this protease can break peptide bonds, affecting the free and immobilized peptide activity. Trypsin is cleaved into amino acids adjacent to Arg and Lys [46]. The structure of the Ib−M1 peptide is rich in arginine; about 35% of the polypeptide chain is composed of this amino acid. The peptide is easily hydrolyzed upon exposure to trypsin, even when the peptide is immobilized in the NPs, generating oligopeptide chains with no activity against *E. coli*.

Pepsin is a protease that preferentially cleaves peptide bonds of amino acids, such as Leu, Ile, Phe, Val, and Trp [47]. Ib−M1 contains three Trp residues (W) susceptible to hydrolysis by pepsin. Therefore, the activity of the Ib−M1 peptide was affected in the presence of this protease. However, this effect was reduced in the Ib−M1/Alg−Chi bioconjugate. It is possible that the immobilization of the peptide makes it difficult for pepsin to access the Trp residues of the peptide.

#### **5. Conclusions**

Alg−Chi NPs was synthesized by the ionic gelation method. However, due to the properties of the polymers, agglomerates with average sizes of 150 nm were observed. Bioconjugates were obtained by forming a bond between the amino group of chitosan and the carboxyl group of glutamic acid of Ib−M1.

Peptide Ib−M1 was found to be resistant to temperature and pH conditions. Furthermore, its immobilization in nanoparticles did not affect its activity against *E. coli.* On the contrary, it maintained its characteristics and achieved stability when the peptide was exposed to pepsin, maintaining its antimicrobial properties at MIC concentrations.

The Ib−M1 peptide exhibited low cytotoxic percentages against Vero cells, again showing this compound's favorable characteristics for biomedical applications. On the other hand, cytotoxicity percentages of 40% were observed for the Ib−M1/Alg−Chi bioconjugate. These values are attributed to Alg−Chi NPs and can be explained by their size. Therefore, further studies are necessary to reduce aggregation and obtain smaller particle sizes.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym14153149/s1. Figure S1A: Growth kinetics of *E. coli* ATTCC 25922 in the presence of peptide Ib−M1 at concentrations of 100 µM, 50 µM, 25 µM, and 12.5 µM, with streptomycin used as reference antibiotic at 6.25 µM and *E. coli* as growth control. Figure S1B. Growth kinetics of *E. coli* ATTCC 25922 in the presence of peptide Ib−M1 at concentrations of 6.25 µM, 3.12 µM, 1.56 µM, and 0.78 µM, with streptomycin used as reference antibiotic at 6.25 µM and *E. coli* as growth control.

**Author Contributions:** Conceptualization, J.M.F.-C.; data curation, C.E.O.-A. and J.L.R.-V.; formal analysis, C.E.O.-A., J.L.R.-V., A.E.F.-G. and J.M.F.-C.; funding acquisition, A.E.F.-G. and J.M.F.-C.; investigation, C.E.O.-A., J.L.R.-V. and J.M.F.-C.; methodology, C.E.O.-A., J.L.R.-V. and J.M.F.-C.; project administration, A.E.F.-G.; resources, A.E.F.-G.; supervision, J.M.F.-C.; writing—original draft, C.E.O.- A.; writing—review and editing, J.L.R.-V., A.E.F.-G. and J.M.F.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by grant number 129980763392 CT-760-2018 of Ministerio de Ciencia, Tecnología e Innovación (MinCiencias), Call for projects on Science, Technology and Innovation in Health 807-2018, Colombia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

