Gold nanoparticle transport across tumour-associated biological barriers: <i>in vitro</i> models, imaging, and quantification

Gold nanoparticles have long been explored for their potential in medical diagnostics, drug delivery, and imaging, particularly in oncology. However, successful translation to clinical applications requires a deep understanding of their biomolecular interactions and transport mechanisms across cellular barriers and within cells. In this review, we examine the current understanding of the journey of gold nanoparticles from systemic administration to tumour infiltration. Specific focus is placed on the biological barriers crossed and the mechanisms involved in traversing those barriers, including active and selective transport pathways, like transcytosis, increasingly recognised as critical for nanoparticle translocation across endothelial and tumour barriers. We stratify the nanoparticle journey into smaller stages and critically discuss the most relevant in vitro models used to study each stage in isolation. Although traditional 2D cell cultures have provided some fundamental insights, more advanced tissue culture models outlined in this review offer enhanced physiological relevance. Monitoring nanoparticle behaviour within these models cannot be achieved without sophisticated imaging and quantification techniques. Herein, we have identified the most appropriate detection methods and their suitability for being used on each in vitro model for the detection of label-free gold nanoparticles. Using label-free nanoparticles preserves their native physicochemical properties and avoids potential artefacts introduced by fluorescent or radioactive tags, and conveniently, gold lends itself well to label-free detection due to its unique optical and electronic properties. By integrating insights from advanced in vitro modelling and cutting-edge detection strategies, this review highlights the current landscape and future directions for optimising the study of gold nanoparticle delivery across barriers in cancer nanomedicine.