Consolidated and next-generation models in DDS nanotoxicology assessment. Where do we stand

Nanomaterials have been widely used across medical and health sciences due to their unique physicochemical characteristics, versatile functionalisation, and their remarkable capacity to interact with (and often penetrate) biological tissues at the molecular and cellular level. These properties have made nanomaterials particularly attractive for drug delivery systems, improving targeted delivery and therapeutic efficacy. Nevertheless, the same features that make nanomaterials so valuable in biomedical contexts may also give rise to unforeseen biological interactions, including oxidative stress, inflammation, immune responses, and organ accumulation, thereby raising legitimate concerns about their potential toxicological implications. As the field of nanotechnology expands, the scientific community faces increasing pressure to thoroughly characterise the safety profiles of these materials. Despite significant progress, current toxicological data remain fragmented, inconsistent across studies, and insufficient to establish comprehensive risk assessments. Variability in particle size, shape, surface chemistry, solubility, and aggregation behavior can drastically alter biological responses, complicating efforts to generate broadly applicable safety guidelines. This review aims to provide an in-depth analysis of the methodologies currently employed for the evaluation of nanotoxicity, encompassing both in vitro and in vivo approaches. In vitro systems are discussed with particular emphasis on advanced cell culture models, including 3D spheroids, organoids, and microfluidic”organ-on-a-chip” platforms, which more accurately replicate human physiological conditions. It also discusses in vivo approaches using various animal models to investigate systemic effects, immune responses, and chronic exposure outcomes. In parallel, the review highlights only few emerging in silico tools, ranging from machine learning algorithms to quantitative structure-activity relationships, that promise to streamline hazard identification and reduce reliance on animal testing. Thus, the review traces the evolution of nanotoxicology by examining both the traditionally employed, well-established and validated experimental models, and the emerging methodologies that are progressively gaining increasing relevance within the research landscape. Finally, the review addresses the critical challenges associated with developing robust, standardized, and reproducible testing protocols. These include the need for harmonized characterization techniques, unified reporting standards, and validated reference materials, as well as a clearer regulatory framework that can keep pace with the rapid evolution of nanotechnologies.