Comparative Biofunctionality Assessment of Lignin and Lignin/Chitosan Nanoparticles: Impact of Chitosan Co-Assembly on Cytotoxicity, Cytocompatibility, Radical-Scavenging Activity, and Antimicrobial Performance

<b>Background/Objectives:</b> The aim of the present study was to conduct a systematic in vitro assessment of the biofunctionalities of newly synthesized lignin (LNPs) and lignin-chitosan nanoparticles (LCNPs) via a comparative in vitro estimation of their cytotoxicity, cytocompatability potential, radical-scavenging activity, and antimicrobial performance, thereby establishing a benchmark for their sustainable design and biomedical applications. <b>Methods:</b> LNPs and LCNPs were synthesized via "green" self-assembly and co-assembly methods. <b>Results:</b> In vitro cytotoxicity studies on L929 fibroblasts and HaCaT keratinocytes demonstrated higher long-term viability for LCNPs (half-maximal inhibitory concentration IC₅₀ = 3.05 mg/mL at 72 h) compared with LNPs (IC₅₀ = 1.37 mg/mL), while both formulations maintained >76% viability at a concentration of 0.5 mg/mL. Electron Paramagnetic Resonance (EPR) and spectrophotometric antioxidant assays displayed strong radical scavenging activity, with LNPs excelling in ^●OH, NO, and ABTS scavenging and LCNPs exhibiting enhanced lipid peroxidation and superoxide inhibition potential. Antimicrobial testing revealed minimal inhibitory concentration (MIC) reductions of the nanoparticles up to 8-13-fold compared to lignin solutions, with LCNPs showing higher activity against Gram-positive and Gram-negative microbial strains. <b>Conclusions:</b> These results highlight LCNPs as biocompatible, antioxidant, and antimicrobial nanoplatforms with potential for regenerative medicine, oxidative stress mitigation, and infection control.