​As a result of decades of misuse of antibiotics, the number of bacteria resistant to commercially available antibiotics is increasing worldwide and is one of the major challenges facing global health. A unique drug-delivery system has been tested in vitro to understand its effect on bacteria.

Antimicrobial peptides (AMPs) are host defense peptides present as part of the innate immune system in virtually all life forms and are being considered a potential treatment for bacterial infections. However, as AMPs often exhibit antibacterial activity across a broad spectrum, they have a limited capacity to induce any significant resistance, as a result of millions of years of co-evolution alongside bacteria. 

LL-37 is an important AMP found in humans as part of the innate immune system and recent studies have shown that this peptide can be incorporated into cubic liquid crystalline nanoparticles known as cubosomes. Cubosomes have already shown great promise as a delivery system for pharmaceuticals. Their well-organised internal structure consisting of alternating hydrophilic and hydrophobic domains means they are compatible with both water soluble, insoluble and amphipathic molecules. These cubosomes are able to protect LL-37 from enzymatic degradation by human and bacterial elastases, which has previously limited its therapeutic use. However, the bactericidal mechanisms of cubosomes loaded with LL-37 is not fully understood.

A team of researchers from Sweden and Denmark used the CRISP reflectometer at ISIS alongside quartz crystal microbalance with dissipation monitoring (QCM-D) and a combination of other analytical tools, to investigate the cubosome-membrane interaction and study the interaction of a LL-37 loaded cubosomes with bacteria.

“This shows how much there is to learn about the interactions between delivery systems for pharmaceuticals, such as cubosomes, at simplified bacterial model membranes," says Maja Hellsing. “We are keen to carry on our research with hope of better understanding the bactericidal mechanisms of cubosomes loaded with antimicrobial peptides. Working with the CRISP reflectometer at ISIS has been very valuable to our research"

The data from the neutron reflectivity experiments, combined with the QCM-D and previous studies, suggests that LL-37 penetrates the outer leaflet of the bilayer membrane of a simplified bacterial model, inducing the formation of defects in the membrane. By comparing the effects of unencapsulated LL-37, cubosomes loaded with LL-37 and unloaded cubosomes, the researchers propose that the bactericidal effects of the LL-37-loaded cubosomes is due to a physical interaction between the cubosome and LL-37 with the bacterial membrane, and not solely the release of the peptide.

The experiments at ISIS allowed the researchers to differentiate between 3 different interactions: (a) peptide adsorption to the bilayer membrane, (b) adsorption of peptide-loaded particles, or (c) no absorption but with removal of lipids. This cannot be directly monitored using other techniques.

The findings of this research suggest that LL-37 loaded cubosomes form an antibacterial unit, potentially altering gram-negative bacteria in a lethal way, whilst protecting the LL-37 from enzymatic degradation. Although further in vivo studies are needed to evaluate the clinical relevance of cubosomes as a drug delivery system, the results from this study suggest that the cubosome-bacteria interaction is of a significant importance for the killing of bacteria and the antibiotic crisis.

Further information:

The full paper can be found at DOI: 10.1021/acsami.9b01826​

Other highlights from CRISP can be found here.