*3.1. In Vitro Effect of Esc Peptides and Esc-Peptide-Loaded PVA-PLGA NPs on F508del-CFTR-Expressing Epithelium*

Esc-peptide-loaded PVA-PLGA NPs, comprising a PLGA core to efficiently entrap and slowly release the peptide cargo and a PVA shell providing for mucus-/biofilmpenetrating properties, were produced as previously reported [27]. In the optimized formulation conditions, NPs display a hydrodynamic diameter lower than 300 nm, a low polydispersity index, a slight negative ζ-potential, a complete peptide entrapment (entrapment efficiency always around 100%), and a typical biphasic in vitro release profile of the entrapped Esc peptide, lasting for 3 days [27]. Conceiving PVA-PLGA NPs for inhalation [27], their effect on the lung epithelium integrity was initially tested, either in the free or loaded form, by measuring the transepithelial electrical conductance after 24 h of treatment. Bronchial epithelial cells expressing a functional copy of CFTR or its mutated F508del form (wt-CBFE41o- and F508del-CBFE41o-, respectively) were employed. Peptides in the soluble free form were also included for comparison. They were used at two different concentrations, i.e., 10 and 20 μM. Note that 10 μM corresponds to the best (minimal) concentration able to display a CFTR potentiator activity in bronchial epithelial cells [25], while 20 μM was the optimal peptide concentration showing pulmonary antimicrobial efficacy [26]. As reported in Figure 1, negligible changes in the transepithelial electrical conductance were attained when the epithelium was treated with the Esc-peptideloaded PVA-PLGA NPs in comparison to untreated samples or samples treated with the free-peptide counterparts. This indicates that neither PVA-PLGA NPs or Esc peptides are harmful to epithelial cells expressing either wild-type or mutated CFTR nor cause paracellular leakage of ions, meaning that cell junctions remain well-tightened.

Remarkably, similar results were obtained for the FRT expressing F508del-CFTR, which have been extensively used for studies on CFTR protein (Figure S2).

Note that epithelial cells may be able to repair the damage induced by the administration of exogenous compounds in the long term (24 h). However, the harmless effect of Esc peptides on both normal and CF lung epithelia was also confirmed at a shorter time (5 h) by scanning electron microscopy (SEM). As shown in Figure S3, treatment of both normal and CF primary bronchial epithelial cells (grown in ALI to better mimic the human airway conditions and to drive differentiation towards a mucociliary phenotype) with 20 μM Esc peptides did not provoke any significant morphological change in the epithelial surface.

**Figure 1.** Effect of bare or Esc-peptide-loaded PVA-PLGA NPs on transepithelial conductance measured in F508del (upper panel) and wt (lower panel) CFBE41o- after 24 h of incubation compared to the free Esc peptides at two different concentrations. The controls were untreated cells. Data are expressed as mean ± S.E.M. from three independent experiments. No statistical difference was found.
