*3.2. In Vivo Studies: Effect of Free or Encapsulated Esc Peptides on Global Pulmonary Genetic Changes*

To go in-depth into the potential clinical application of inhalable PVA-PLGA NPs as delivery systems of Esc peptides, we determined their pulmonary safety profile in healthy mice, when used either in their bare form or loaded with the Esc peptides. For a complete overview of the global genetic changes in the lungs of animals after 24 h exposure to peptide-loaded PVA-PLGA NPs compared to the free peptides or the vehicle PBS, we performed transcriptome studies by RNA-seq. To this aim, the standard immunocompetent outbred mouse strain CD-1 was used. Due to its ability to reflect the natural response of a non-immunocompromised human being, this is a common animal model for toxicity analysis. Esc-peptide-loaded PVA-PLGA NPs as well as free Esc peptides at a dosage of 0.1 mg/kg (~20 μM), which have already found to be active in vivo by provoking a 2-log10 reduction in lung bacterial burden [26], were intratracheally administered.

Only a minimal number of lung genes (up to 6 out of 25,000) were up- or downregulated by more than two-fold in animals treated with Esc peptides or with Esc-peptideloaded PVA-PLGA NPs versus PBS-treated animals. The identified genes are shown in Table 1; the corresponding volcano plots of the differential gene expression are reported in Figure S4. Among them were a member of the non-protein-coding small nucleolar RNA gene family (*Snhg11)*, three pseudogenes (i.e., *Eif4a-ps4, Gm10320*, and *Gm14150*), and two genes predicted to be structural constituents of ribosomes (i.e., *Rpl26* and *Rpl30*) or encoding a component of the cytoskeletal motor protein dynein (*Dynlt1f*) or alpha-synuclein (*Snca*), while the other genes were found to be involved in the positive regulation of protein phosphorylation (*Chil1*) or the signaling receptor-binding activity (*H2-T22*). Remarkably, none of these genes appear to be involved in toxicity-related processes.

**Table 1.** List of up- or downregulated genes in lungs of animals treated with Esc(1-21)-1c, Esc-peptideloaded PVA-PLGA NPs, compared to those of PBS-treated animals.


Up: upregulated; down: downregulated. Log2 fold change is also indicated.
