**1. Introduction**

Owing to their biocompatibility, biodegradability, bioavailability, and low-toxicity properties, biopolymers, such as alginate and chitosan, are widely used in the biomedical and pharmaceutical industries [1]. They have a wide range of medical applications in tissue engineering, implants, and drug delivery [2–5]. Additionally, they have reactive functional groups that allow for the conjugation of peptides and proteins. [5–10].

Different strategies have been used to explore the preparation of polymeric nanoparticles, including coprecipitation, chemical crosslinking, thermodecomposition, coacervation, emulsification, and ionic gelation [11–14]. The latter is widely used for the preparation of nanoparticles of alginate and chitosan with the aim of bioconjugating them with antimicrobial peptides [7,15].

Antimicrobial peptides (AMPs) are small molecules composed of a length of 12 to 50 amino acids and are usually positively charged and amphiphilic [16]. They are among the body's first line of defense against the inactivation of pathogens, such as Gram-negative

**Citation:** Osorio-Alvarado, C.E.; Ropero-Vega, J.L.; Farfán-García, A.E.; Flórez-Castillo, J.M. Immobilization Systems of Antimicrobial Peptide Ib−M1 in Polymeric Nanoparticles Based on Alginate and Chitosan. *Polymers* **2022**, *14*, 3149. https://doi.org/10.3390/ polym14153149

Academic Editors: Lorenzo Antonio Picos Corrales, Angel Licea-Claverie and Grégorio Crini

Received: 24 June 2022 Accepted: 29 July 2022 Published: 2 August 2022

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and Gram-positive bacteria, fungi, viruses, and parasites. The positive charge of AMPs allows for initial binding to the membrane via electrostatic interaction [17].

Despite their high antimicrobial activity, most AMPs are not widely used in clinical settings due to limitations such as toxicity and stability. Thus, AMPs present considerable challenges when considering the type of administration. For example, in oral administration, the pH and proteases in the gastrointestinal tract can inhibit the action of AMPs by hydrolysis or denaturation [18]. The conjugation of AMPs with nanoparticles (NPs) has been proposed to increase the local concentration of the peptide and improve its antimicrobial activity [19–22]. Alginate and chitosan nanoparticles (Alg−Chi NPs) have been used to achieve such strategies, as they preserve their structure and can therefore enhance bioactivity [14].

Chitosan is a linear polysaccharide composed of D-glucosamine and N-acetyl-Dglucosamine. It is a deacetylated form of chitin, the structural component of the exoskeletons of crustaceans [23]. The amino (-NH2) and hydroxyl (-OH) groups of the polymer guarantee high reactivity and charge, allowing for its union with different biomolecules [23]. In addition, alginate is a non-toxic polysaccharide composed of one to four linked β-Dmannuronate and α-l-guluronate blocks and can form chitosan-crosslinked gels. Owing to its properties, it is qualified as one of the best drug delivery systems and bioapplications, particularly in the immobilization of peptides, affording bioconjugates with equal or improved biological activity [24–26].

The Ib−M1 peptide is part of the group of Ib-M peptides synthesized by Flórez-Castillo et al. [27], which exhibit very good antimicrobial activity at low inhibitory concentrations against clinical and reference strains of *Escherichia coli* [27–29]. Ib−M1 peptides have a net charge of +6 and an isoelectric point of 12.5. Ib-M peptides are promising for clinical applications [28–30].

In this study, we immobilized the Ib−M1 peptide on polymeric nanoparticles of alginate and chitosan to maintain the antibacterial activity of this peptide and increase its stability with respect to proteases, changes in pH, and temperature. *E. coli* ATCC 25922 was used as a reference microorganism to evaluate antibacterial activity. In this experimental study, we report, for the first time, the effect of the immobilization of the Ib−M1 peptide on Alg−Chi nanoparticles to establish the scope of its clinical use.

#### **2. Materials and Methods**

#### *2.1. Materials*

Peptide Ib−M1 (sequence EWGRRMMGRGPGRRMMRWWR-NH2 [27]) was purchased through the commercial company Biomatik USA (Wilmington, DE, USA). Chitosan (Sigma-Aldrich, St. Louis, MO, USA, ≥75%), alginate (Sigma-Aldrich, St. Louis, MO, USA), sodium tripolyphosphate (TPP, Sigma-Aldrich, St. Louis, MO, USA), acetic acid (CH3COOH, Merck KGaA, Darmstadt, Germany), 2-(1H-Benzotriazole-1-yl)-1,1,3,3 tetraethylammonium tetrafluoroborate (TBTU, Sigma-Aldrich, St. Louis, MO, USA), N, N-diisopropylethylamine (DIPEA, Sigma-Aldrich, St. Louis, MO, USA); *Escherichia coli* strain ATCC 25922, Vero ATCC CCL-81 cell line, Müller–Hinton broth (MH—Scharlau), Luria–Bertani broth (LB Broth, Oxoid, Basingstoke, England), trypsin, phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), bromide-3 (4,5-dimethylthiazole- 2-yl) 2,5-diphenyltetrazolium (MTT), streptomycin, Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum.
