**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**

**Floriana Cappiello 1,†, Bruno Casciaro 1,†, Maria Rosa Loffredo 1, Elena Puglisi 1, Qiao Lin 2, Dandan Yang 2, Gemma Conte 3, Ivana d'Angelo 4, Francesca Ungaro 3, Loretta Ferrera 5, Raffaella Barbieri 6, Laura Cresti 7, Alessandro Pini 7, Yuanpu Peter Di 2,\* and Maria Luisa Mangoni 1,\***


**Abstract:** In recent years, we have discovered Esc(1-21) and its diastereomer (Esc peptides) as valuable candidates for the treatment of Pseudomonas lung infection, especially in patients with cystic fibrosis (CF). Furthermore, engineered poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) were revealed to be a promising pulmonary delivery system of antimicrobial peptides. However, the "ad hoc" development of novel therapeutics requires consideration of their stability, tolerability, and safety. Hence, by means of electrophysiology experiments and preclinical studies on healthy mice, we demonstrated that neither Esc peptides or Esc-peptide-loaded PLGA NPs significantly affect the integrity of the lung epithelium, nor change the global gene expression profile of lungs of treated animals compared to those of vehicle-treated animals. Noteworthy, the Esc diastereomer endowed with the highest antimicrobial activity did not provoke any pulmonary pro-inflammatory response, even at a concentration 15-fold higher than the efficacy dosage 24 h after administration in the free or encapsulated form. The therapeutic index was ≥70, and the peptide was found to remain available in the bronchoalveolar lavage of mice, after two days of incubation. Overall, these studies should open an avenue for a new up-and-coming pharmacological approach, likely based on inhalable peptide-loaded NPs, to address CF lung disease.

**Keywords:** antimicrobial peptides; cystic fibrosis; lung infection; biodegradable nanocarrier; therapeutic index; transepithelial resistance; lung gene expression; mouse bronchoalveolar lavage
