Mechanism of in vitro cytotoxicity of antimicrobial peptides in combination with stabilizing excipients

Abstract

Emerging antibiotic resistance poses a critical public health threat, particularly the alarming increase of resistant bacteria commonly associated with gastrointestinal (GI) infections. Antimicrobial peptides (AMPs) are a diverse class of peptides produced by many organisms as a first line defence mechanism against microbial threats and show promising potential as alternatives to conventional antibiotics. This class includes melittin and mastoparan, well studied cationic α-helical toxins isolated from bee and wasp venom respectively. Nisin Z, on the other hand, is also an AMP and classified as a cationic bacteriocin produced by bacterial strains of Lactococcus lactis. Previous research on the antibacterial effects of these peptides against GI pathogens, such as Staphylococcus aureus and Escherichia coli, support the therapeutic application as pharmaceuticals. However, clinical advancement of antimicrobial peptides is limited by the associated toxicity towards mammalian cells and the lack of sufficient data on this cytotoxicity. Furthermore, most peptide formulations include excipients to enhance absorption or increase the stability of the peptide and these excipients also have the risk of interacting with the peptide in such a way as to affect the cytotoxicity thereof. Therefore, the aim of this study was to investigate and characterise the mechanisms of in vitro cytotoxicity of two venom peptides, melittin and mastoparan, and the bacteriocin peptide, nisin Z, toward the human hepatocellular liver carcinoma cell line (HepG2) and human epithelial colorectal adenocarcinoma cell line (Caco-2). In addition, this study aims to evaluate and describe the varying cytotoxicity of AMPs in combination with peptide stabilising excipients (L-glutamic acid, chitosan and polysorbate 80) when compared to individual peptide toxicity. Cytotoxicity was investigated and determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and the lactate dehydrogenase (LDH) assay. Neutral red staining was additionally employed to visually illustrate the varying cytotoxic effect of combination treatments compared to viable cells. It was determined that treatments with melittin:excipient combinations resulted in lower cytotoxicity towards HepG2 and Caco-2 cells relative to melittin alone treatment of 1 μM. Caco-2 cells treated with mastoparan:L-glutamic acid combinations resulted in higher cytotoxicity when compared to both individual mastoparan and L-glutamic acid treatments with 61.80±4.97%, 54.02±5.79% and 53.89±6.65% at 40 μM: 0.75 mg/ml, 40 μM: 1.5 mg/ml and 40 μM: 3 mg/ml respectively. Chitosan in combination with mastoparan similarly displayed cytotoxicity towards Caco-2 cells with respective values of 54.41±3.95%, 57.17±4.28% and 55.71±7.18% at 40 μM: 5 mg/ml, 40 μM: 10 mg/ml and 40 μM: 20 mg/ml treatments. Nisin Z in combination with polysorbate 80 displayed high cytotoxicity in both HepG2 and Caco2 cells. The cytotoxicity was determined as 76.14±2.15%, 72.78±6.08% and 59.14±11.07% at 370 μM: 2 mg/ml, 370 μM: 4 mg/ml and 370 μM: 8 mg/ml, respectively towards HepG2 cells and 72.90±6.70%, 80.49±3.92% and 87.73±3.03% for 370 μM: 2 mg/ml, 370 μM: 4 mg/ml and 370 μM: 8 mg/ml, respectively towards Caco-2 cells. The LDH assay suggested that melittin and mastoparan induce necrotic cell death in HepG2 and Caco-2 cells. Mastoparan in combination with L-glutamic acid and chitosan is furthermore suggested to cause necrosis in Caco-2 cells, whereas nisin Z:polysorbate 80 combinations induced cell death possibly by means of apoptosis in both cell lines. It was concluded that peptide stabilising excipients in combination with melittin decreases the individual cytotoxicity of melittin. L-glutamic acid and chitosan, individually, in combination with mastoparan induced a higher cytotoxic effect than mastoparan alone towards Caco-2 cells. Finally, polysorbate 80 in combination with nisin Z was the most cytotoxic combination that displayed high cell death in both cell lines. Determining the cytotoxicity and additionally the antibacterial effect of AMPs in combination with peptide stabilising excipients can impact the clinical advancement and application of these novel antibiotics in the treatment of threatening GI infections.