Chitosan nano-encapsulation enhances targeted delivery of cecropin-A (1–7)-Melittin peptide to combat intracellular multi-drug-resistant non-typhoidal Salmonella

Abstract

This study aimed to encapsulate the Cecropin A (1–7)–Melittin (CAMA) peptide within chitosan nanoparticles (CS NPs) and evaluate its antimicrobial activity against multi-drug-resistant (MDR) strains of non-typhoidal Salmonella, employing both in vitro and in vivo assays. The CAMA-loaded CS NPs, synthesized using the ionic gelation technique, exhibited a particle size of 219.31 ± 38.24 nm and a zeta potential of 8.2 ± 0.2 mV, as measured by dynamic light scattering. The encapsulation efficiency was 75.45 ± 2.5 %. Additionally, Fourier-transform infrared spectroscopy, scanning electron microscopy and transmission electron microscopy determined the functional groups, morphology and size of the CAMA-loaded CS NPs. The in vitro release kinetics revealed a pH-dependent release profile, with the highest cumulative release observed within 24 h at an alkaline pH (8.20), followed by physiological pH (7.40). Intracellular antimicrobial efficacy was assessed using HEp-2 cell lines, where CAMA-loaded CS NPs effectively cleared intracellular MDR Salmonella strains. Furthermore, CAMA-loaded CS NPs were found to be safe for use with sheep erythrocytes, HEp-2 and RAW 264.7 cell lines, and beneficial gut lactobacilli, and were stable in the presence of proteolytic enzymes and simulated biological fluids. In vivo assays in Galleria mellonella larvae demonstrated an improved survival rate, reduced bacterial count, and minimal cytotoxicity, as confirmed by the lactate dehydrogenase assay. These results correlated with histopathological examination, suggesting that CAMA-loaded CS NPs could be a promising therapeutic candidate for combating intracellular Salmonella.