Exploring the Effects of Lignin Nanoparticles in Different Zebrafish Inflammatory Models

Purpose: Lignin is the most abundant source of aromatic biopolymers and has gained interest in industrial and biomedical applications due to the reported biocompatibility and defense provided against bacterial and fungal pathogens, besides antioxidant and UV-blocking properties. Especially in the form of nanoparticles (NPs), lignin may display also antioxidant and anti-inflammatory activities. Methods: To evaluate these characteristics, sonochemically nano-formulated pristine lignin (LigNPs) and enzymatically-phenolated one (PheLigNPs) were used to expose zebrafish embryos, without chorion, at different concentrations. Furthermore, two different zebrafish inflammation models were generated, by injecting Pseudomonas aeruginosa lipopolysaccharide (LPS) and by provoking a wound injury in the embryo caudal fin. The inflammatory process was investigated in both models by qPCR, analyzing the level of genes as il8, il6, il1β, tnfα, nfkbiaa, nfk2, and ccl34a.4, and by the evaluation of neutrophils recruitment, taking advantage of the Sudan Black staining, in the presence or not of LigNPs and PheLigNPs. Finally, the Wnt/β-catenin pathway, related to tissue regeneration, was investigated at the molecular level in embryos wounded and exposed to NPs. Results: The data obtained demonstrated that the lignin-based NPs showed the capacity to induce a positive response during an inflammatory event, increasing the recruitment of cytokines to accelerate their chemotactic function. Moreover, the LigNPs and PheLigNPs have a role in the resolution of wounds, favoring the regeneration process. Conclusion: In this paper, we used zebrafish embryos within 5 days post fertilization (hpf). Despite being an early-stage exemplary, the zebrafish embryos have proven their potential as predicting models. Further long-term experiments in adults will be needed to explore completely the biomedical capabilities of lignin NPs. The results underlined the safety of both NPs tested paved the way for further evaluations to exploit the anti-inflammatory and pro-healing properties of the lignin nanoparticles examined.