Now, researchers in Australia, Singapore and the US have devised a simple, single-step process to make such plant secondary metabolites (PSMs) much more effective. Treating PSMs with plasma increases their potency against Staphylococcus aureus, a common cause of infections in humans. The method could greatly extend the range of antibiotic substances available to clinicians, and help stem the rise of drug-resistant species (Nano Futures 1 025002).

The huge diversity of PSMs in nature represents a resource of great potential, but to use these substances at safe concentrations frequently requires them to be potentiated first by either heating or chemical modification. Neither method is ideal, since PSMs potentiated by heating lose their enhanced effect when they cool, while chemical techniques are complex and not easy to make applicable to different molecules.

Another way to boost a PSM’s effectiveness is by exposing it to plasma, as this generates reactive species such as hydroxyl radicals, ozone and hydrogen peroxide. As explained by Kateryna Bazaka, a researcher at Queensland University of Technology, James Cook University, and the Commonwealth Scientific and Industrial Research Organization (CSIRO) in Australia, even otherwise inert chemicals can be potentiated in this way.

"Our team and others have shown that plasma treatment of water renders it biologically active, with activity against cancer cells and pathogenic bacteria retained for some time after potentiation," Bazaka told our sister site nanotechweb.org. "When this plasma-activated water was used in conjunction with other drugs, the treatment outcome was enhanced, even though in those experiments the drugs were not treated by plasma directly."

Writing in Nano Futures, Bazaka and a multi-institution collaboration report a new approach that applies this plasma treatment to the potentially therapeutic macromolecules themselves. Bazaka describes the motivation behind the work: "Recently, our group showed that plasma treatment of amino acids changes their structure. It gave us the idea that direct treatment of biologically-active molecules, such as PSMs, may also induce some beneficial changes in these molecules, enhancing their activity without using chemicals or heat-based treatment."

Effective against a common cause of infection

To test their technique, the team exposed solutions of two different PSMs – terpinene-4-ol and γ-terpinene – to an argon plasma at ambient pressure. The plasma-potentiated solutions were then applied to S. aureus as a free-floating suspension and as a biofilm.

Terpinene-4-ol, familiar for its presence in tea tree oil, is known to have an antibacterial effect in its pristine form. Unmodified γ–terpinene, on the other hand, has only limited efficacy against S. aureus. This difference in activity was reflected in the experiment: treatment with terpinene-4-ol resulted in a slight decrease in biofilm thickness and suspended colony-forming cells, whereas the result of γ–terpinene exposure was similar to that of the control experiment.

For both PSMs, however, the result of plasma activation was profound, with biofilm thickness, free-floating cell viability, and individual cell morphology all affected. Although terpinene-4-ol was still the more potent chemical after activation, even the otherwise ineffective γ–terpinene caused significant changes to the biofilms and suspended bacteria.

General technique

Bazaka and her collaborators expect plasma treatment to be just as effective for other PSMs as it is for the two terpinenes studied so far. What's more, says Bazaka, "depending on the chemistry of the original molecule, the activity of the product will be different, which is an excellent opportunity to optimize against a specific pathogen. These differences can be exploited by using mixtures of natural or artificial active molecules that target different structural elements or processes within cells, thus making them more efficient at killing pathogens, and reducing their ability to develop resistance."

The reactive oxygenated species generated by the plasma persist for up to 24 hours, and the simplicity of the approach and the affordability of the equipment make plasma activation a widely accessible technique. "In fact," says Bazaka, "the device used in this study is certified in Europe to be used by medical and veterinary practitioners for wound treatment. It can be easily scaled up."

Beyond its obvious clinical applications, Bazaka suggests that plasma activation might find uses in biofabrication and chemical synthesis "in the formation of novel oligomeric and polymeric materials, and transformation of low-value and waste materials into more useful chemicals, for example." For now, however, the team will investigate how treatment time and storage time influence the effectiveness of the method, and how plasma-potentiated PSMs affect healthy and cancerous human cells.

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