Development of environmentally-friendly biocidal systems based on chitosan-nanoparticles loaded with R-(+)-pulegone to protect wood from fungal attack

As society is increasingly aware to environmental issues, research into new sustainable strategies is becoming a central topic in the field of Cultural Heritage conservation. This study is aimed to develop a novel biocidal system for the conservation of wooden artworks, by employing reagents, products and synthesis procedures consistent with Green Chemistry principles. To this aim, R-(+)-pulegone (Pu), which is a natural derivative having assessed biocidal properties against several microorganisms [1], also responsible of biodegradation of wood, was selected to be loaded in chitosan nanoparticles (ChNPs) and applied on wooden sample artificially aged and colonized with Aspergillus niger, causing soft-rot on wooden artefacts. Nanoencapsulation was performed with a simple and harmless synthesis method, based on ionic gelation of chitosan, which is a versatile polysaccharide derived from chitin and is attracting attention for its non-toxicity, biocompatibility, biodegradability, biocidal properties as well as its consolidant action, recently studied for the conservation of archeological and waterlogged wood [2]. The inhibition properties and the concentrations of pure chitosan and pulegone against A. niger were established in vitro through a Multiwell Assay (MA) and chitosan nanoparticles loaded with pulegone (ChNPs-Pu) at different Ch:Pu ratios (1:0; 1:0.25; 1:0.5; 1:1) were obtained following experimental procedures described in other studies [3], with some modifications to obtain ChNPs-Pu having suitable characteristics for the application on wooden samples. The morphological characteristics of the NPs and their stability on time were considered and characterized through different analytical methods, included dynamic and dielectrophoretic light scattering (DLS), UV-Visible spectrophotometry, Fourier Transformed Infra-Red (FTIR) spectroscopy and SEM analysis. The biocidal and inhibitory potentials of ChNPs at different R-(+)-pulegone concentrations were evaluated against A. niger at different experimental conditions. After the successful results obtained with MA, we are experimenting the spray application of the ChNPs-Pu on wooden samples with variable degree and type of biological colonization. Antimicrobial effect of ChNPs will be evaluated by several methodologies, such as colorimetric assays, measurements A. niger colonization and relative vitality. The valuable achievements obtained suggest that adopting natural compounds represent a promising strategy to preserve the Cultural Heritage from biocolonization. [1] M. Božović, R. Ragno, Molecules, 22(2), 2017, 290. [2] M. Christensen, E. Larnøy et al., J. Am. Inst. Conserv., 54(1), 2015, 3-13. [3] X. Wang, Y. Hu et al., JCH, 53, 2022, 206-2011.