During the first reporting period, we exchanged the three nanocellulose (NC) materials—CNC, CNF, and BNC—and tested their physical, chemical, toxicological, and antimicrobial properties. Since none of the unmodified materials showed antimicrobial activity, we enhanced them using three strategies: antimicrobial peptides (AMPs), 2-aminoimidazoles (2AIs), and nanopatterning.
During the second reporting period, we improved the antimicrobial properties of the NC. Enhancing NC with AMPs was achieved by simply mixing the NC with the AMPs. Higher concentrations could be retrieved by mixing in different steps (also known as ‘layering method’). Functionalization of the 2AIs was done in two ways. The first way involved mixing of TEMPO-oxidized NC with 2AIs, which lead to electrostatic interactions between the 2AIs and the NC. The second way was reductive amination, where binding sites were created on the NC and the 2AIs could be attached covalently. The first method is simpler and showed the strongest antimicrobial effects, so it was selected for further use.
The antimicrobial properties of the NC were further improved by creating nanopatterns in the NC. This was done using two method. First, spiky structures were introduced in the NC with ion milling. Using this method, ion beams physically push away atoms, creating NC spikes that can pierce the microbes. Second, wet nanoimprint lithography (wet-NIL) was used. Here, a pattern is introduced on the NC using a stamp. Ion milling was successful for all types of NC, whereas wet-NIL only worked well for CNC. However, wet-NIL offers better potential for up-scaling.
Next, different linker methods were tested to make durable NC coatings. Two systems showed the best stability and were applied through standardized spraying: one from our partner SuSoS based on their AziGrip technology and a polydopamine+boric acid linker. We achieved the best mechanical performance when spraying 10 layers.
We tested the modified NC for antimicrobial activity using standard tests and in-house assays that better reflect real-world conditions. AMP- and 2AI-modified NC showed strong antibacterial and antifungal activity in suspension. On coatings, 2AI remained consistently active; AMP coatings showed more variation and are still being optimized.
Based on performance and stability, four coating systems were selected for further validation. A first life cycle assessment was also completed, comparing the environmental impact of the different components of the coatings, identifying where some changes may be made to further improve their sustainability.
In the coming months, the selected coatings will undergo toxicity testing, and concentrations will be further optimized. Up-scaling of all components has started, and current production estimates indicate that we can supply the upcoming bus demonstration. Additional optimization will keep pushing the TRL of both AMPs and 2AIs forward.
