**4. Discussion**

The urgent health concern related to antibiotic resistance has driven a renewed interest in the clinical development of peptides compared to the previous two decades. Interestingly, besides having direct antimicrobial activity, some AMPs also display additional biological functions involved in the modulation of host immunity, including enhanced chemotaxis of immune cells, activation of immune cell differentiation, stimulation of wound healing, and angiogenesis, scavenging of bacterial endotoxins [34]. The broad spectrum and rapid bactericidal activity of AMPs combined with the low risk of inducing resistance makes them a valid option for alternative antimicrobial compounds. However, to the best of our knowledge, there are no AMPs in clinical trials for the development of new inhalable drugs against lung infections.

Here, to emphasize the attractive properties of Esc peptides for the treatment of lung diseases, especially in CF, likely via pulmonary delivery upon incorporation into PVA-PLGA NPs, we investigated their effect on the lung epithelial integrity and the host immune response in terms of gene expression and tissue damage at the target district. Remarkably, we discovered that both Esc peptides and PVA-PLGA (drug carrier) are harmless to epithelial cells. This was evidenced by the invariant transepithelial conductance in bronchial epithelia upon treatment, as measured by TEER experiments, which appear to be more sensitive than typical cytotoxicity tests by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Nonetheless, one of the most critical limitations of AMPs, particularly when embedded into a delivery system, is the lack of potential toxicity data in animal models. There are currently 15 FDA-approved PLA-/PLGA-based drug products available on the US market [35], and several studies on the usage of PLGA-based nanocarriers for pulmonary drug delivery are in progress [36,37]. Together with chitosan, PLGA is the most investigated polymer for the development of inhaled formulations

aimed at extending the pulmonary exposure and pharmacological effect of encapsulated drugs [38]. This is because of its biodegradability [39], which can be modulated by varying the lactide/glycolide ratio, molecular weight, chemical structure, and biocompatibility [28]. We formerly highlighted how incorporating AMPs into PVA-PLGA NPs and their pulmonary administration represent a suitable approach to assist peptide diffusion through an artificial lung mucus, eradicate biofilm, and potentiate and extend the in vivo antimicrobial efficacy of Esc peptides in the lung [27]. The work from Haque and colleagues [40] reported that PLGA NPs do not appear to be absorbed into the lungs after pulmonary administration but rather are degraded into lower-molecular-weight constituents that are subsequently absorbed. PLGA NPs have also been used (i) to release loaded AMPs to the wounds to accelerate healing processes [41] and (ii) to deliver drugs in tumor combination therapy [42].

Nevertheless, a limited number of studies have been performed to confirm the safety of PLGA NPs in the short and long term, especially in the lung compartment [43–46].

Our previous work highlighted that PVA-PLGA NPs do not induce any recruitment of inflammatory cells into the lung alveoli of healthy mice nor changes in the expression of inflammation-associated genes 36 h after pulmonary administration [27]. Here, the transcriptomic analysis provided us with the first demonstration that Esc peptides, either in the free or encapsulated form in PVA-PLGA NPs, promote a response closer to the gene expression pattern of the PBS-treated host.

To the best of our knowledge, this is the first case showing the effect of peptide-loaded PVA-PLGA NPs on the global genetic profile of targeted tissues (i.e., lungs) upon administration in the conductive airways of mice, in comparison to the vehicle-treated animals. Interestingly, only 2 genes out of 25,000 were found to be up-/downregulated by more than 2-fold by the Esc-peptide-loaded PVA-PLGA versus the PBS-treated mice (i.e., *H2-T22* and the pseudogene *Gm10320* for Esc(1-21)-loaded NPs and *Snca* and the pseudogene *Gm14150* for Esc(1-21)-1c-loaded NPs). Moreover, as pointed out by histological analysis of lungs tissue, no sign of inflammation/damage was detected by NPs when used in the free or loaded form with Esc(1-21)-1c at a concentration 15-fold higher than the therapeutic dosage of 0.1 mg/kg [47–49], either after 1 day or 14 days from i.t. administration. Together, these data have contributed to further supporting the harmlessness of PVA-PLGA NPs in the respiratory tract.

The other important finding of this work is the discovery of the safety profile of Esc(1-21)-1c and its tolerability by animals, at a concentration 70-fold higher than the efficacy dose, without eliciting any detectable damage to the lung, spleen, liver, and kidney, unlike what has been described for the AMP D-BMAP18, which was found to cause lung edema when used at doses of 1 and 2 mg/kg [50]. In addition, we proved that the absence of toxicity was not due to the complete degradation of the peptide, which instead remained available in the BAL within the first 48 h. This is in contrast with the low biostability of other investigated AMPs for treatment of lung infections, such as (i) all-L-BMAP18 AMPs, which were degraded by pulmonary proteases in murine BAL fluids within the first 20 min of exposure [51] and did not display any in vivo antibacterial activity, and (ii) P-113, which showed a half-life of a few minutes in undiluted sputum [52].

#### **5. Conclusions**

In conclusion, we have filled the gap of some underexplored but relevant aspects for the translation of AMPs into new inhalable medicines by providing evidence of (i) the pulmonary safety profile of Esc peptides and PVA-PLGA NPs as a valuable nanoparticulate system for AMP delivery to the lungs; (ii) a prolonged biostability of Esc(1-21)-1c in the mouse BAL together, and (iii) its promising therapeutic index.

Overall, in addition to expanding our knowledge on the safety profile of Esc peptides for the development of new drugs to treat *P. aeruginosa* lung infection, our studies should open the avenue for a new up-and-coming pharmacological approach, likely based on inhalable peptide-loaded NPs, to address CF lung disease.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/pharmaceutics14112297/s1. Figure S1: HPLC and mass spectra of Esc peptides; Figure S2: Effect of bare or Esc-peptide-loaded PVA-PLGA NPs on F508del-FRT epithelium integrity after 24 h of incubation; Figure S3: SEM images of bronchial epithelia; Figure S4: Effect of free or encapsulated peptides on lung gene expression; Figure S5: Mass spectra of Esc(1-21)-1c after 48 h of incubation with mouse BAL.

**Author Contributions:** Conceptualization, B.C., Y.P.D., and M.L.M.; formal analysis, F.C., B.C., and L.F.; investigation, F.C., B.C., M.R.L., E.P., Q.L., D.Y., G.C., L.C., and R.B.; funding acquisition M.L.M.; resources, I.d., F.U., A.P., Y.P.D., and M.L.M.; supervision, Y.P.D. and M.L.M.; writing—original draft, F.C., B.C., and M.L.M.; writing—review and editing, Y.P.D. and M.L.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** Fondazione Italiana per la Ricerca sulla Fibrosi Cistica (Project FFC 8/2019) Delegazione FFC di Imola e Romagna con Gruppo di sostegno FFC di Faenza.

**Institutional Review Board Statement:** 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.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article or Supplementary Material.

**Acknowledgments:** M.L.M. thanks Luis Galietta (Telethon Institute of Genetics and Medicine, Pozzuoli, Italy) for providing cell lines.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

