*2.4. Transepithelial Electrical Resistance (TEER) Experiments*

Epithelial integrity was evaluated by measuring the transepithelial electrical resistance in CBFE41o- and FRT cells using the 24-transwell plates (24 Millicell plates PSHT010R1) and an electronic resistance system (Millicell ERS-2, EMD Millipore, Burlington, MA, USA). The medium was replaced every 3 days, and cells were used for TEER measurements after 7 days. Two days before the experiment, epithelia were incubated in their standard culture medium supplemented with 1 μM Vx-809 in the case of F508del- CBFE41o- and F508del-FRT. After 24 h, the medium was discarded, and epithelia were treated with Esc peptides, Esc-peptide-loaded PVA-PLGA NPs, or control bare PVA-PLGA NPs (in both the apical and basolateral compartments at different concentrations). After 24 h, the medium was replaced with a saline solution containing (in mM) 130 NaCl, 2.7 KCl, 1.5 KH2PO4, 1 CaCl2, 0.5 MgCl2, 10 glucose, and 10 Na-Hepes (pH, 7.4), which was added to both apical

and basolateral compartments of the permeable supports. A sterile electrode was applied onto the apical side of the transwell insert containing the cells with/without treatments, and TEER was measured in basal conditions by the epithelial voltmeter and then converted to transepithelial conductance (TEEC) using the formula TEEC = 1/TEER [30].

### *2.5. Scanning Electron Microscopy*

Primary cultured epithelial cells were seeded in 6.5 mm Transwell® with 0.4 μm-pore polyester membrane sterile insert (2 × <sup>10</sup><sup>5</sup> cells/insert/100 <sup>μ</sup>L) while 350 <sup>μ</sup>L of bronchial epithelial cell growth medium (Lonza, Basel, Switzerland) was added to the basolateral side of the transwell system (12 inserts in a 24 well plate). The apical medium was removed 3 days after seeding the cells on top of the inserts. The newly established air–liquid interface (ALI) cell culture system was cultured for an additional 8–10 days to achieve full confluency. Afterward, epithelia were washed apically with 100 μL of phosphate-buffered saline (PBS). A total of 100 μL of PBS supplemented or not with each Esc peptide at 20 μM was added on the apical compartment. Cells were then incubated for 5 h at 37 ◦C. Afterward, apical supernatant was removed, and samples were fixed with 2.5% glutaraldehyde in 0.01 M PBS for 60 min. The samples were then fixed in 1% osmium tetroxide (OsO4) for 60 min and extensively washed with the same buffer and dehydrated with a graded ethanol series. After dehydration in hexamethyldisilazane and sputter coating, the samples were examined using a scanning electron microscope (Philips XL 30 CP instrument).

### *2.6. Gene Expression*

All animal experiments were carried out based on a protocol (n. 20087639) approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh according to the National Institutes of Health (NIH) guide for the care and use of laboratory animals. Seven-week-old female wild-type CD1 mice were anesthetized by isoflurane inhalation and instilled intratracheally with 50 μL of peptide solution (0.1 mg/kg, i.e., 20 μM) or PVA-PLGA NP suspension (either bare NPs or NPs loaded with each peptide at 0.1 mg/kg), in PBS. Control mice were given 50 μL of PBS without peptide. After 24 h, mice were euthanized for lung tissue isolation to extract RNA for differential expression analysis (sequencing work was performed by Novogene US Marketing).

#### *2.7. Mouse Toxicity and Maximum Tolerated Dosage (MTD)*

Five-week-old female wild-type CD1 mice were anesthetized by isoflurane inhalation and instilled intratracheally with 1.5 mg/kg (corresponding to 300 μM) of Esc(1-21)-1c in the free or encapsulated form in 50 μL of PBS. Mice instilled with 50 μL of PBS or bare PVA-PLGA NPs were included for control. After 1 day and 14 days, mice were euthanized for tissue isolation and examined for histopathology. Tissues were fixed in situ with 4% paraformaldehyde for 10 min with open chest cavity. They were then embedded in paraffin, and 5 μm-thick tissue slices were prepared by staining in hematoxylin and eosin and analyzed for pathological severity. In parallel, another group of seven animals was anesthetized by isoflurane inhalation and instilled intratracheally with escalating concentrations up to 7 mg/kg Esc(1-21)-1c in 50 μL of PBS and monitored for survival to determine the MTDs.

### *2.8. Stability Measurements in Bronchoalveolar Lavage Fluid*

Bronchoalveolar lavage fluid (BAL) was collected from male and female C57BL/6 mice (4–5 months old) according to the procedure described in [31,32]. Mice were euthanized by means of CO2 narcosis; a small incision at the level of the neck was made and a blunt needle connected to a syringe was inserted into the trachea. Then, lungs were washed with 1 mL of sterile PBS. BAL samples from each mouse were pooled, centrifuged at 1000 rpm (corresponding to 0.1× *g*) for 5 min, and stored at −80 ◦C. The supernatant was used for stability tests.

Peptides were dissolved in 400 μL of PBS at a concentration of 300 μM, and added to an equal volume of BAL at a final concentration of 150 μM. At different time points, 150 μL aliquots were withdrawn and added to 500 μL of acetonitrile, and then centrifuged. The supernatants were diluted with 160 μL of 0.1% trifluoroacetic acid (TFA)—water and analyzed by RP-HPLC and mass spectrometry. Liquid chromatography was performed on a Phenomenex Jupiter C18 analytical column (300 Å, 5 μm, 250 × 4.6 mm) in a 30 min gradient, using 0.1% TFA in water as solvent A and acetonitrile as solvent B. Mass spectrometry analysis of diluted samples was performed with a Bruker Daltonic-ultraflex-matrix-assisted laser desorption ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometer.

#### *2.9. Statistical Analyses*

Quantitative data, collected from independent experiments, were expressed as the means ± standard errors of the means (S.E.M.). Statistical analysis was performed using one-way analysis of variance (ANOVA) with PRISM software version 8.0.1 (GraphPad, San Diego, CA, USA). Differences were considered statistically significant at a *p* value of <0.05. The levels of statistical significance are indicated in the legends of the figures.
