Antimicrobial surfaces by the application of natural active substances

Bacteria and fungi preferably live in biofilms. These consist of cooperatively acting microorganisms surrounded by a self-produced layer of slime, to protect from external attacks. They are omnipresent at natural and technical boundaries in humid or wet environments. Biofilms in the wrong place or those containing pathogenic organisms can represent a health hazard, cause damage to materials, or greatly increase the energy demand in plant operation. This is why biocides are used in many areas. Their approval and application is controlled by a series of laws, ordinances and European regulations. With increasing statutory restrictions for their use comes an increase in the need for biocide-free antimicrobially active equipment for technical surfaces. At the Fraunhofer IGB, materials scientists, micro-, molecular- and cell-biologists work together, across divisions, to develop suitable systems.

In order to prevent biofilms we examine the effects of naturally occurring, antimicrobially active compounds such as plant extracts, cationic peptides and enzymes. Because technical application requires that active agents can be applied in a suitable form, we are developing layer systems for longer-term and targeted release and, particularly in the case of biomolecules, for the long-term maintenance of their function. The type of application of the active agents depends on the form and geometry of the surface and the agent to be immobilized.

One option for the immobilization of active biomolecules is to embed them into a polymer matrix applied as a coating to a component. It releases the active agent over a determined period of time. We created active layers using lysozyme, DNase and LL-37. Lysozymes are innate immune system enzymes and damage the bacterial cell wall. The human antimicrobial peptide LL-37 is also produced by the immune system and destroys the cell walls of numerous Gram-positive and Gram-negative bacteria. It is also very resistant to proteolysis. The enzyme DNase cleaves DNA. As a great proportion of biofilms consists of the liberated DNA of dead microorganisms, DNase is able to reduce the biofilm.

The polymer matrix was constructed from short-chain poly(ethylene glycol) diacrylates by UV polymerization. This was done placing the active agent and additives into the aqueous polymer solution, applying it to the desired surface and curing for 3 seconds. The UV light crosslinks the polymer chains and the active agent is thereby embedded into the hydrogel. We showed that the curing time used was sufficient for the formation of crosslinking and that the biomolecule is not significantly altered in its secondary structure or activity.

The release characteristics were studied at room temperature and in a phosphate-buffered saline solution, which was regularly changed. The left figure shows the quantity of lysozyme released over 100 days, depending on the network density. The largest amount is released within the first days but output was still detected after even 100 days. At this point, 50 percent of the total lysozyme applied had been released. The release itself can be adjusted via the prepared network density.

The systems described were investigated for their antimicrobial, bacteriostatic action with E. coli, P. aeruginosa and P. pseudoalcaligenes. Their effect was examined in planktonic and biofilm cells. The left figure shows the statistically significant reduction in the metabolic activity of the microorganisms studied, in comparison to the reference biofilm, by the prepared systems. A statistically significant reduction in the planktonic cell count was also achieved for hydrogels with lysozyme and LL-37.

We were able to show that natural antimicrobial agents can be embedded in deposit layers, while retaining their function and their release can be controlled through the design of this layer. The prepared systems had an antimicrobial action in the microorganisms studied. Natural antimicrobial materials usually have a narrow spectrum of action, so technical applications may require combinations of agents for the prevention of the undesirable microorganisms present. The deposit layer must thereby be matched to the agent to be released in each case.

[1] Weber, C.; Burger-Kentischer, A.; Müller, M.; Trick, I.; Hirth, T. (2011) Biofilmvermeidung durch natürliche Wirkstoffe – gezielte und langfristige Freisetzung durch ein PEG-basiertes Depotsystem, Biomaterialien12: 2