How Key in vivo Models Can Advance Anticancer Nanotherapeutics.

Nanomedicine offers powerful opportunities for targeted drug delivery and cancer therapy, yet clinical translation remains limited by the complex and dynamic interactions of nanoparticles within biological systems. This review integrates current evidence on how nanoparticle size, shape, and surface properties govern cellular uptake and processing, systemic biodistribution, immune interactions, and therapeutic performance across in vitro, ex vivo, and in vivo models. By comparatively analyzing findings across two-dimensional, three-dimensional, xenograft, and immunocompetent tumor models, this review identifies model-dependent determinants of nanoparticle performance that influence translational outcomes. Foundational studies in two-dimensional monolayers established relationships between physicochemical attributes, endocytic pathways, vesicular trafficking, and exocytosis. Three-dimensional spheroids introduce extracellular matrix density and cellular packing constraints that limit nanoparticle movement and alter uptake trends observed in monolayers. In vivo, xenograft models emphasize the influence of vascular permeability and stromal architecture but often overestimate delivery due to exaggerated enhanced permeability and retention effects and the lack of adaptive immunity. Immunocompetent tumor models capture complement activation, opsonization, macrophage-mediated clearance, dynamic protein corona evolution, and cytokine-driven vascular changes. These immune-mediated processes reshape biodistribution patterns and often diminish ligand-mediated targeting benefits. They operate alongside systemic factors such as renal and hepatosplenic filtration, biotransformation, and species-specific differences in vascular structure and immune composition, influencing nanoparticle pharmacokinetics and therapeutic response. Together, these findings underscore that successful nanomedicine design requires integrating material engineering with an understanding of immune surveillance, vascular biology, tumor microenvironment heterogeneity, and whole-body transport dynamics. Future progress will depend on developing nanoparticles that maintain functional stability in immune-intact hosts, minimize premature clearance, and achieve sustained intratumoral delivery. These advances must be supported by predictive, physiologically relevant models that bridge gaps between in vitro results, preclinical outcomes, and clinical translation.