New approach methodologies in nanomedicine: bridging the gap between in silico, in vitro, and in vivo systems.

The rise of nanomedicine has led to the regulatory approval of more than 80 nanoparticle (NP)-based therapeutics over the past two decades. Further advancement in the field requires a deeper understanding of the nano-bio interface of NPs, including their uptake, delivery, efficacy, toxicity, and adverse effects. Despite the heavy reliance on conventional cell cultures for NP screening, these models do not recapitulate the in vivo microenvironment, complexity, and physiological responses; hence, follow-up animal studies are required. Nonetheless, because of species-specific differences and ethical concerns around animal studies, developing alternative platforms has become critical. To address these issues, 3D cell culture models such as organoids, spheroids, and organ-on-chip systems have emerged to bridge the gap between in vitro and in vivo systems. Because they better represent cell interactions, gene expression, nutrient gradients, and tissue-specific functions, 3D models also align with the U.S. FDA's promotion of new approach methodologies (NAMs) to reduce animal testing. This review covers examples of organic and inorganic NPs tested in 3D models and discusses organ-specific NAMs, considering their advantages and limitations. The review also highlights how 3D models improve NP evaluation for drug delivery, toxicity, and targeted therapy, and provides a coherent NAMs toolbox for developing in silico methods for NP assessment. Future perspectives are introduced to underscore the importance of integrating in silico NAMs and 3D models to complement in vivo models for advancing precision medicine and improving the translational potential of complex drug products containing nanotechnology.