New insights in the protection of wooden materials from fungal biodeterioration: chitosan-based nanoparticles loaded with R-(+)-pulegone

1. Introduction A large part of cultural heritage is wooden made and one of the main causes of wood decay is given by fungal attack. This research aims to develop a biocidal product to prevent wood biodeterioration by employing sustainable reagents and harmless synthesis procedures. To this aim, R-(+)-pulegone (Pu), which is a main component of many essential oils that showed biocidal activities towards several microorganisms [1,2], was selected to be loaded in chitosan nanoparticles (ChNPs) and tested against Aspergillus niger, considered one of the main responsible of soft-rot decay [3]. Ionic gelation is the method used to obtain the ChNPs and is based on electrostatic interactions between the positively charged ammino groups of chitosan, a polysaccharide known for its biocompatibility, biodegradability and biocidal properties, and the negatively charged groups of sodium tripolyphosphate (TPP) [4]. The ChNPs have been morphologically characterized and their biocidal potential was evaluated towards A. niger in vitro and on artificially colonized wooden samples. 2. Results and Discussion In the initial stages of the experiment, ChNPs loaded with Pu (Pu-ChNPs), at four Ch:Pu ratios (1:0; 1:0.25; 1:0.5; 1:1) were prepared following a two-step method for the formation of an oil-in-water emulsion and ionic gelation of chitosan with TPP. Dynamic Light Scattering (DLS) allowed to calculate the hydrodynamic diameter (HD) and the polydispersity index (PDI) of the nano-systems dispersed in aqueous suspensions, confirming the presence of nanoparticles with different sizes, depending on the concentration of Pu present. According to DLS, the best result was obtained for Pu-ChNPs with a Ch:Pu ratio 1:0.25 (HD: 198 ± 4 nm; PDI: 0.39 ± 0.05). Fourier-transform infrared spectroscopy (FTIR) was used to evaluate the encapsulation efficacy: variations of the spectral partner were observed and considered indicative of the efficient loading of Pu. Multiwell Assay (MA) allowed to assess the inhibitory effect of Pu, chitosan and the four Pu-ChNPs systems at six concentrations (from 3 to 0.093 mg/mL) towards A. niger. The biological assays gave encouraging results, since the treatments based on Pu-ChNPs resulted more effective at lower concentrations than the ones obtained by employing the substances alone, active at higher concentrations. Comparing the results from DLS and MA (inhibition at 0.75 mg/mL), the Pu-ChNPs system with a Ch:Pu ratio 1:0.25 was considered the best candidate for the following experimental steps. In this regard, the synthesis protocol of the NPs was optimized considering a) the initial concentration of chitosan, b) the molar ratio between chitosan and TPP, c) the stirring speed during the ionic gelation and d) the pH conditions. The new systems gained better results, as confirmed by the DLS where NPs with smaller sizes (HD: 73.73 ± 0.10 nm) and lower PDI (0.31 ± 0.04) were obtained. The morphology of the NPs was observed by Scanning Electron Microscope (SEM) imaging, where the NPs appeared to be regularly distributed, well-separated and spherically shaped. Two parallel experiments involved the treatment of wooden samples from three species (Quercus petraea, Abies alba and Fagus sylvatica) with an aqueous suspension of Pu-ChNPs (0.75 mg/mL), applied via spray. In the first case, the samples were preliminary treated with the solution and then inoculated with A.niger in laboratory conditions for two weeks. The treatments visually reduced the growth of the fungus, especially in the samples from F. sylvatica. In the second experiment, the same treatment was applied on wooden samples previously subjected to a natural biocolonization. A colorimetric monitoring confirmed the progressive toning of the samples towards their original color after the treatment with the NPs. This result can be considered an indicator of the biocidal effect of the treatments, as a further confirmation of the potential of Pu-ChNPs for future applications. 3. Conclusions R-(+)-pulegone was successfully loaded in chitosan spherical shaped NPs. The enhanced biocidal effects associated to the combination of chitosan and Pu towards A. niger could be explained taking into account that nano-encapsulation protects Pu from evaporation and oxidation, fostering its controlled release and improving its stability [5]. In this scenario, the promising results obtained suggest that such systems can represent a possible sustainable alternative to classical chemical biocides considered harmful for the environment and human health. Future applications will evaluate the combined biocidal and consolidant properties of Pu-ChNPs, in virtue of the recent applications of chitosan as a consolidant for archeological and waterlogged wood [6]. References [1] C. Genova, E. Fuentes, P.Sanmartin, G. Favero, B. Prieto, Coatings, 10(3), 2020, 295. [2] M. Božović, R. Ragno, Molecules, 22(2), 2017, 290. [3] S. A. M. Hamed, Int Biodeterior. Biodegrad., 78, 2013, 98-102. [4] L. Keawchaoon, & R. Yoksan, Colloids Surf. B, 84(1), 2011,163-171. [5] X. Wang, Y. Hu, Z. Zhang, B. Zhang, JCH, 53, 2022,206-211. [6] M. Christensen, E. Larnøy et al., J. Am. Inst. Conserv., 54(1), 2015, 3-13.