A Simple Ionic-Gelation Method for Chitosan Nanoparticle Synthesis and Standardized Protocols for Biological Safety Assessment: Antibacterial Activity, Phytotoxicity, and Biocompatibility

Chitosan nanoparticles (Ch NPs) are versatile nanomaterials with expanding agricultural and biomedical applications, highlighting the need for reproducible, low-cost, and scalable synthesis methods to ensure their safe and widespread use in biological systems. This study presents a simple ionic-gelation protocol using a serological pipette–needle dropwise system that minimizes reagent waste and requires no sophisticated equipment. The synthesized Ch NPs were characterized by UV–Vis spectroscopy, ESEM, TEM, EDS, DLS, XRD, and FTIR, confirming nanoscale size, strong positive surface charge, and characteristic chitosan–TPP interactions. To establish a standardized biological safety assessment framework, three representative bioassays were implemented across microbial, plant, and mammalian systems. Antibacterial testing against Xanthomonas oryzae pv. oryzae (Xoo) using a resazurin-based microdilution assay revealed a minimum inhibitory concentration (MIC) of 128 µg/mL, whereas bulk chitosan showed no inhibition up to 512 µg/mL. Phytotoxicity and seed germination assays on rice (Oryza ‘KDML105’) demonstrated no inhibitory effects on germination, with over 90% germination by day 3 and significantly enhanced seedling growth parameters (p < 0.05) at 64–128 µg/mL, indicating non-phytotoxicity. MTT assays confirmed that Ch NPs were non-toxic to both human skin cell lines (HDF and HaCaT) across 2.5–160 µg/mL, showing enhanced cell viability in HDF cells at specific concentrations and stable viability in HaCaT cells, indicating overall biocompatibility. Importantly, all bioassays were conducted under aligned concentration ranges to enable cross-system comparison and reproducibility. This integrated workflow links nanoparticle synthesis with a standardized, multi-system evaluation strategy, supporting the safe application of Ch NPs in biological systems.