Abstract
The spread of bacterial resistance against conventional antibiotics generates a great need for the discovery of novel antimicrobials. Polypeptide antibiotics constitute a promising class of antimicrobial agents that favour attack on bacterial membranes. However, efficient measurement platforms for evaluating their mechanisms of action in a systematic manner are lacking. Here we report an integrated lab-on-a-chip multilayer microfluidic platform to quantify the membranolytic efficacy of such antibiotics. The platform is a biomimetic vesicle-based screening assay, which generates giant unilamellar vesicles (GUVs) in physiologically relevant buffers on demand. Hundreds of these GUVs are individually immobilised downstream in physical traps connected to separate perfusion inlets that facilitate controlled antibiotic delivery. Antibiotic efficacy is expressed as a function of the time needed for an encapsulated dye to leak out of the GUVs as a result of antibiotic treatment. This proof-of-principle study probes the dose response of an archetypal polypeptide antibiotic cecropin B on GUVs mimicking bacterial membranes. The results of the study provide a foundation for engineering quantitative, high-throughput microfluidics devices for screening antibiotics.
Highlights
Phospholipid membranes provide bacterial cells with a permeability barrier that prevents conventional antibiotics from reaching their intracellular targets
In order to optimise the output of a single experiment and minimise the consumption of valuable peptides required for characterisation, we developed a complete lab-on-a-chip platform that integrates the octanol-assisted liposome assembly (OLA) vesicle formation component with a parallelised vesicle capture and drug perfusion system (Fig. 1)
We have designed a fully integrated microfluidic platform to test the efficacy of antimicrobial peptides (AMPs) on biomimetic vesicle membranes
Summary
Phospholipid membranes provide bacterial cells with a permeability barrier that prevents conventional antibiotics from reaching their intracellular targets. Imaging techniques can reveal useful mechanistic information of the targeted activity at a single membrane level, which bulk culture assays, such as MIC assays, cannot capture In this regard, highly controlled in vitro optical methods may provide drug developers with a highly efficient and reliable procedure for probing the antimicrobial activity of emerging antibiotics. The ability to investigate populations containing thousands of GUVs at the single-vesicle level is a considerable improvement on earlier techniques where studies were conducted on tens of GUVs per experiment in open chambers. Such throughput allows the acquisition of statistically significant results that portray the stochastic nature of lipid–peptide interactions. The validation is aimed at qualifying our approach as an AMP characterisation assay in the pre-clinical development of membrane active antibiotics
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