*3.3. Esc(1-21)-1c Safety Profile in Lungs and Other Organs*

We previously demonstrated that i.t. instillation of 0.1 mg/kg of Esc peptides either in the free form or upon encapsulation into PVA-PLGA NPs did not alter the lung mucociliary clearance in healthy mice [27]. However, the in vivo toxicity of Esc-peptide-loaded PVA-PLGA NPs upon pulmonary administration is an unexplored area. Therefore, we investigated the lung safety of the most promising Esc peptide, Esc(1-21)-1c, for therapeutic development. To this purpose, Esc(1-21)-1c was i.t. instilled in CD1 mice either in the free or encapsulated form at 1.5 mg/kg (~300 μM), corresponding to a 15-fold-higher concentration than that used in previous in vivo efficacy studies (0.1 mg/kg) [27]. As highlighted by the histological analysis in Figure 2, we did not detect any inflammatory response (there were no infiltrates of inflammatory cells) or lung tissue damage either after 1 day or 14 days from peptide administration in the free or encapsulated form. In parallel, no toxicity was detected for the corresponding amount of bare PVA-PLGA NPs, in line with our previous work showing an invariant expression of inflammation-associated genes (including IL-6, IL-10, or the tumor necrosis factor-α and NF-κB) in the lungs of mice after instillation of PVA-PLGA NPs encapsulated or not with Esc peptides [27].

In addition, no visible tissue injury, necrosis, or alteration in cell density was observed for other organs, such as the liver and kidneys, in comparison to control samples (Figure 3).

**Figure 2.** Histological analysis of lungs in CD1 mice, after 1 day and 14 days from i.t. administration of Esc(1-21)-1c either in the free or encapsulated form at a dosage of 1.5 mg/kg. The amount of unloaded PVA-PLGA NPs was the same as that present in Esc(1-21)-1c-loaded NPs. The results were compared to those obtained with PBS-treated mice (control), (magnification 4×).

To ensure that the lung safety of Esc(1-21)-1c 24 h after its administration was not due to its complete degradation in the lung environment, we studied its biostability at 300 μM in the presence of mouse BAL. As pointed out by the mass spectra in Figure 4, when Esc(1-21)-1c was incubated with BAL, a peak of molecular mass of 2185 Da corresponding to the full-length peptide [22] was detected even after 24 h. On the contrary, the all-L isomer was significantly less stable, as highlighted by the appearance of multiple degradation products and the lack of the peak corresponding to the entire peptide sequence after 6 h. However, both spectra showed the appearance of a peak (2129 Da) at 6 h, which became prevalent at 24 h. This peak indicates the loss of a Gly residue from the peptide sequences.

**Figure 3.** Representative images of kidney (**A**) and liver (**B**) tissues in CD1 mice, after 1 day and 14 days from i.t. administration of Esc(1-21)-1c either in the free or encapsulated form at a dosage of 1.5 mg/kg compared to the corresponding bare PVA-PLGA NPs or PBS-treated animals (control). Organs were harvested, fixed, and stained for histological evaluation (magnification 4×).

### *3.4. Determination of Maximum Tolerated Dosage*

Development of peptides as anti-infective agents requires knowledge of their therapeutic index (TI), which is defined as the ratio between the maximum tolerated dosage, MTD (i.e., the highest concentration causing no obvious adverse effects and no mortality), and the therapeutic dosage mTd (i.e., the minimal dosage reducing bacterial burden by 2-log10 in the number of bacterial cells) [33]. Therefore, to identify the MTD of the most efficacious Esc(1-21)-1c, CD1 mice were i.t. instilled with increasing concentrations of the peptide, from 1.5 mg/kg to 7 mg/kg, and their survival was monitored for 14 days. Remarkably, the peptide was well-tolerated by the animals, which remained viable for the entire duration of the experiment and maintained the same range of motion both after a short time (1 h) and longer time (24 h) from its i.t. instillation at the highest dosage of 7 mg/kg. Notably, this concentration was 70-fold greater than the efficacious dose, indicating a TI of at least 70. When such a high concentration of Esc(1-21)-1c was used, the peptide was detectable in the

mouse BAL after 48 h of incubation with BAL (Figure S5). This finding indicates that the harmlessness of the peptide at the long term cannot be attributed to its degradation.

**Figure 4.** Mass spectra of Esc(1-21)-1c and the all-L Esc(1-21) after 6 and 24 h in BAL. Marked in red is the peak of molecular mass at 2185 corresponding to the full-length peptide. The arrow indicates the peak of molecular mass at 2185 found for Esc(1-21)-1c after 24 h of incubation with BAL.

Furthermore, histopathological examination of lung tissue did not reveal significant recruitment of inflammatory cells compared to PBS-treated mice within 48 h from i.t. instillation of Esc(1-21)-1c, in agreement with the absence of macroscopic damage to the lungs (Figure 5).

**Figure 5.** Histologic analysis of mouse lung tissues at 48 h after i.t. instillation of Esc(1-21)-1c at 7mg/kg. Lung tissues were harvested, fixed, and stained for histological evaluation without peptide treatment (**A**) or after peptide administration (**B**) (magnification 20×).

Similarly to what was found for the peptide dosage of 1.5 mg/kg, no alteration in the tissue structure was visible at the level of the liver, spleen, and kidneys after treatment with 7 mg/kg of Esc(1-21)-1c, compared to the control (Figure 6).

**Figure 6.** Representative images of liver, spleen, and kidneys from healthy mouse at 48 h after i.t. instillation of Esc(1-21)-1c at 7mg/kg. Tissues were harvested, fixed, and stained for histological evaluation without peptide treatment (control, left side) or after peptide administration (right side) (magnification 20×).
