Zebrafish Embryos to Profile Nano(bio)materials: A Modular Platform for Developmental Toxicity, Neurotoxicity, and Inflammation‐Regeneration Assays

Zebrafish embryos provide a rapid, low‐cost in vivo platform to profile the developmental, neurotoxic, and immunomodulatory effects of engineered nano(bio)materials within a single vertebrate system. This article presents a modular workflow that integrates standardized fish embryo acute toxicity (FET) testing, chemically assisted dichlorination, inflammatory challenge, and regeneration assays to evaluate both bio‐based and synthetic nanoparticles. First, embryos are exposed to nanomaterials under FET conditions and subjected to quantitative morphometric analysis to establish sublethal concentration ranges and detect subtle growth defects. Sensitivity to nano(bio)material exposure is enhanced by a pronase‐based dechorionation procedure, which removes the chorion to promote direct particle–tissue contact while maintaining acceptable baseline viability. Immunoinflammatory responses are probed using two complementary modules: microinjection of Pseudomonas aeruginosa lipopolysaccharide (LPS) into the yolk sac to induce acute systemic inflammation and caudal fin wounding to elicit a localized inflammatory and regenerative response. Across these models, we combine qPCR panels of cytokine and Wnt/β‐catenin pathway genes with Sudan Black B staining of neutrophils and neutral red vital staining of macrophages, providing accessible cellular and molecular readouts in wild‐type embryos. Together, these protocols constitute a harmonized, technically accessible pipeline that enables comparative safety and bioactivity profiling of diverse nano(bio)materials and supports their assessment within safe‐and‐sustainable‐by‐design frameworks. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Nanoparticle preparation and suspension, and embryo dechorionation procedure (pronase solution) Basic Protocol 2 : Sudan Black B staining protocol Basic Protocol 3 : Neutral red staining protocol Basic Protocol 4 : LPS microinjection protocol Basic Protocol 5 : Wound inflammation model generation protocol