## 5 Conclusions  
This study highlights the potential of KHS-Cnd as an anti-biofilm and anti-virulence molecule against _P. aeruginosa_ clinical strains obtained from CF patients. For a possible therapeutical application, the anti-virulence activity of KHS-Cnd against the preformed biofilms might be its most interesting feature. However, some limitations should be considered. The anti-biofilm activity of KHS-Cnd seems to act at relatively high concentrations, where a toxicity of about 50% was observed on the A549 cells after 24 h of treatment (Figure 6). Despite this, morphological alterations and hemolytic activity on primary cell lines were not observed for the peptide [21].  
Given the importance of identifying new anti-biofilm agents that counteract _P. aeruginosa_ related infections, our data look promising, as we found a peptide scaffold that could be used as a starting point for drug development through the design of new peptide mutants with optimized and improved biological performance. In this scenario, derived peptides, particularly shorter forms of native ones, which retain the anti-biofilm properties and display an enhanced activity compared to the original peptide, could be selected; moreover, we can quote some examples of derived peptides that also exert a reduced toxicity on eukaryotic cells [47]. Alternatively, a prodrug strategy to limit cytotoxicity but exploit anti-biofilm activity can be explored. This could be obtained by the conjugation of a drug moiety to AMP (forming AMP prodrug or pro-AMP), with consequent reversible peptide inactivation up to reaching the release site, thus confining the potential cytotoxic in vivo effect to the site of infection. This approach has been successfully applied in vivo to the AMP mellitin, which exhibited high in vitro toxicity [48]. Moreover, new pro-AMPs have been also investigated for their use in the treatment of _P. aeruginosa_ infection in CF patients [49]. In addition, we need to consider that AMPs could also be used in synergistic combinations with known antimicrobial agents to counteract antimicrobial resistance; in this case, the dosage of AMP added to the antimicrobial should be relatively low, thus avoiding cytotoxicity.  
Aside from the improvement in the biological activity, the set-up of delivery systems using liposomes or nanoparticles (NPs) is fundamental to enhance the bioavailability of peptides within the _P. aeruginosa_ biofilm and throughout the thick mucus clogging the tracheobronchial tree of patients with CF or COPD. In this challenging context, the use of robust pro-AMPs or AMPs conjugated with nanocarriers represents a resolutive tool to improve peptide stability and selective targeting; this strategy reduces both toxicity and the overall dose, and allows for a controlled local release of the AMP. Once the pharmacodynamic behavior of the AMP, encapsulated within NPs, has been deeply investigated, dosing regimens can be rationally optimized: this aspect also needs to be addressed to guarantee a clinical impact of projects focused on AMPs, especially for those such as KHS-Cnd, which counteract biofilms in vitro, but at rather high concentrations [29].  
M.A., E.I., F.B., M.R., F.P., O.D., E.V.F., V.T.G.A., R.P. and L.S. are responsible for conceptualization, methodology, software, validation, formal analysis, investigation, resources, data curation, writing, visualization, supervision, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Bambino Gesu Hospital (protocol code 1016 02/08/2017).  
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.  
Not applicable.  
The authors declare no conflict of interest.