Non-toxicity of Plant Candicidal Peptides for Mammalian Cell Lines and Galleria mellonella Model to Improving Selectivity for Clinical Use

Antimicrobial peptides (AMPs) are promising candidates for the development of new drugs. However, thorough studies on the toxicity of these molecules are scarce, which is a gap, as host toxicity is one of the main reasons for nonapproval of the drug by regulatory agencies. This work aimed to evaluate the toxicity of three AMPs isolated from Capsicum annuum leaves, named CaCPin-II, CaCDef-like and CaCLTP2. The AMP toxicological profile was evaluated by in vitro cytotoxicity against mammalian cells and systemic in vivo toxicity using Galleria mellonella larvae as study model. AMP cytotoxicity was evaluated in a broad panel of human cell lines, namely, vascular endothelium, cervical adenocarcinoma, prostatic epithelium, mammary epithelium and fibroblasts, and in murine macrophages. Cell viability was evaluated through metabolic activity, a gold standard method for assessing viability due to the speed, robustness and reliability of the results. To elucidate the toxicity mechanism of the peptides, their ability to bind to the cell surface and to permeabilize membranes was evaluated by measuring the zeta potential and the absorption of the SYTOX® Green fluorescent probe, respectively. The AMPs did not decrease cell viability or permeabilize the membranes of the cell lines at the tested concentrations. Only CaCLTP2 had the ability to interact with the cell surface, but it was not able to permeabilize them. The in vivo systemic toxicity was evaluated by the survival rate of the G. mellonella larvae inoculated with peptides. CaCPin-II showed in vivo toxicity, as the larval survival rate after the test was 60% lower than that of the controls. The results suggest that these peptides have potential as antimicrobial agents because they have low or no toxicity to mammalian cells and can serve as a framework for drug development.