*2.4. Preparation of Ib*−*M1/Alg*−*Chi Bioconjugate*

The Ib−M1 peptide in the Alg−Chi NPs was immobilized according to the methodology of Ropero-Vega [33]. Briefly, the carboxyl group in the Glu-1 residue of the peptide was activated. To this end, 44.2 mg of TBTU and 24.2 µL of DIPEA were added to a 2000 µM solution of Ib−M1 in Tris HCl buffer (10 mM pH 7.4), allowing the reaction to take place for 20 min under constant stirring. Subsequently, this reaction mixture was added to 0.4 mg/mL of nanoparticles and left under constant stirring for 2 h, allowing for the formation reaction of the peptide bond between the activated carboxyl group and the amino groups of the nanoparticles. Finally, the Ib−M1/Alg−Chi bioconjugate was separated by centrifugation at 15,000 rpm for 45 min at 18 ◦C.

To determine the amount of immobilized peptide, the concentration of peptide in the supernatant was determined using the Bradford method [34] with a Quick Start™ Bradford protein assay kit from BioRad at 595 nm. Before the preparation of the immobilized peptide, the free peptide solution was measured under the same conditions. With these data and using the following formula, it was possible to determine the amount of peptide immobilized in the nanoparticles forming the Ib-M/Alg−Chi bioconjugate:

$$\% \text{\textbullet millimeter} = \frac{\text{Free peptide abs} - \text{supermutant abs}}{\text{Free peptide abs}} \ast 100$$

where Free peptide abs is the absorbance of the peptide activated with TBTU and DIPEA before being combined with the nanoparticles for immobilization, and supernatant abs is the absorbance of the supernatant from the first centrifugation [33].

#### *2.5. Antimicrobial Activity against E. coli ATCC 25922*

*Escherichia coli* strain ATCC 25922 was cyropreserved at −80 ◦C in Luria–Bertani broth (LBB) with 15% glycerol. For the reactivation of the microorganism, 50 µL of cryopreserved material was added to 5 mL of LB and incubated at 35 ± 2 ◦C for 18 to 24 h before each assay.

The minimum inhibitory concentration (MIC) was determined using the microdilution method as described in the Clinical and Laboratory Standards Institute protocol M07A9 [35]. Briefly, twofold serial dilutions of the Ib−M1 peptide (100 and 0.78 µM concentration) were incubated in 96-well, round-bottom plates at 200 µL/well final volume for 24 h at 37 ◦C with shaking at 150 rpm with 5 <sup>×</sup> <sup>10</sup><sup>5</sup> colony forming units per mL (CFU/mL) of bacterial inoculum. The bacterial suspension was adjusted from a concentration of 1 <sup>×</sup> <sup>10</sup><sup>6</sup> CFU/mL. Absorbance at 595 nm was determined every hour for 8 h, with a final measurement at 24 h with a microplate reader kit (Bio-Rad, iMark). Mueller–Hinton (MH) broth and *E. coli* in MH broth were taken as negative and growth positive controls, respectively. The MIC was

defined as the lowest peptide concentration that inhibited bacterial growth after 24 h. Data represent at least two independent experiments.
