**1. Introduction**

The rising appearance of drug-resistant microorganisms has undermined the success achieved in the last century in the field of medicine via the utilization of antibiotics [1–4]. Antimicrobial peptides (AMPs) with a rapid-membrane-perturbing activity make bacteria less prone to develop resistance and constitute a promising source of new effective therapeutics [5–7]. Because of their biocompatibility, seldom accumulation in body tissues, and the harmlessness of their degradation products, peptides have become a unique class of therapeutic agents [8]; to date, more than 80 peptides have reached the global market [9].

**Citation:** Cappiello, F.; Casciaro, B.; Loffredo, M.R.; Puglisi, E.; Lin, Q.; Yang, D.; Conte, G.; d'Angelo, I.; Ungaro, F.; Ferrera, L.; et al. Pulmonary Safety Profile of Esc Peptides and Esc-Peptide-Loaded Poly(lactide-co-glycolide) Nanoparticles: A Promising Therapeutic Approach for Local Treatment of Lung Infectious Diseases. *Pharmaceutics* **2022**, *14*, 2297. https://doi.org/10.3390/ pharmaceutics14112297

Academic Editors: Scavello Francesco, Jean-Eric Ghia and Amiche Mohamed

Received: 16 September 2022 Accepted: 21 October 2022 Published: 26 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In parallel, extensive research has been carried out in terms of the discovery, production, and optimization of AMPs, and an average of 70 AMPs are in preclinical/clinical evaluation [10,11]. AMPs are generally produced by all living species as key effectors of the innate immune system [12]; amphibian skin represents an invaluable wealthy storehouse of such molecules [13]. Studies conducted in our laboratory have led to the discovery of a frog-skin-derived membrane-active AMP, i.e., Esc(1-21), with bactericidal activity against both the planktonic and biofilm forms of the human pathogen *Pseudomonas aeruginosa* [14]. Nowadays, lung colonization by the sessile form of this bacterium holds a challenging threat, mostly in patients affected by cystic fibrosis (CF) [15,16], a genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which controls chloride and bicarbonate transport mainly at the apical membrane of secretory epithelia, including those at the airways [17,18]. The most common mutation is the deletion of phenylalanine 508 (F508-del CFTR), which causes the production of an incorrectly folded protein that is rapidly degraded [19]. Furthermore, the small fraction of F508del-CFTR that reaches the plasma membrane [20] exhibits a defect in channel gating. As a result, the outflow of anions is inhibited, leading to increased water absorption by epithelial cells and the formation of a sticky airway mucus where inhaled microbes accumulate, giving rise to the onset of chronic pulmonary infection with serious respiratory dysfunctions.

During the last several years, by changing the stereochemical configuration of only two L-amino acids of Esc(1-21), i.e., Leu<sup>14</sup> and Ser17, to the corresponding D-enantiomers, we discovered that the resulting diastereomer Esc(1-21)-1c is: (i) more resistant to enzymatic degradation [21] and (ii) more efficient in restoring bronchial epithelium integrity [22,23]. This last feature is not shown by any traditional antibiotic and is expected to accelerate the healing of a damaged lung epithelium, especially in CF lungs, where wound-healing processes are highly compromised [24]. Interestingly, we also discovered an unprecedented property of AMPs, which is the ability of both Esc(1-21) and Esc(1-21)-1c (Esc peptides) to act as potentiators of the CFTR with ion conductance defects [25].

However, the "ad hoc" development of novel therapeutics based on peptide antibiotics and their translation from basic research to the clinic requires consideration of several underexplored aspects associated with the effective application of AMPs. These include studies on: (i) peptides' stability under conditions that reflect the physiology of the target site (e.g., the lung); (ii) tolerability for the assessment of a therapeutic window; (iii) identification and validation of an efficient delivery system of AMPs without eliciting undesirable local and/or systemic side effects. Previous studies highlighted Esc(1-21)-1c as the most efficient Esc peptide in reducing the lung bacterial burden in a mouse model of acute Pseudomonas lung infection, upon a single intratracheal (i.t.) instillation at a very low dosage (0.1 mg/kg, corresponding to 20 μM) with comparable efficacy to colistin, the last-resort antibiotic for the treatment of infections [26]. Nevertheless, studies aimed at evaluating Esc peptides' local toxicity or tolerability at higher therapeutic dosages are missing. Recently, we also demonstrated how polyvinyl-alcohol (PVA)-engineered poly(lactide-co-glycolide) (PLGA) nanoparticles (PVA-PLGA NPs) represent an enticing nanoformulation for pulmonary delivery of AMPs, able to (i) assist AMP diffusion through biological barriers, such as the mucus (which becomes a thick layer in CF) and (ii) prolong AMP antibacterial efficacy against Pseudomonas-induced lung infection [27]. Conceiving PVA-PLGA NPs as a promising inhalable formulation for the treatment of lung infections, and investigating their pulmonary safety, in terms of gene expression and tissue integrity compared to the free peptides, would be highly recommended [28].

Hence, in line with the above, we initially performed studies aimed at identifying the effect(s) of peptide-based PLGA formulation in comparison to the soluble free form of Esc peptides on both (i) the integrity of CFTR-expressing epithelium and (ii) the pulmonary host response after i.t. administration in healthy mice. Afterward, we assessed the maximum tolerated dosage and stability in the bronchoalveolar lavage for the Esc isoform endowed with the highest in vivo antimicrobial effectiveness, Esc(1-21)-1c.
