In vitro methodologies to evaluate nanocarriers for cancer treatment: where are we?

Cancer treatment continues to face significant challenges, including lack of selectivity, systemic toxicity, and the adaptive resistance mechanisms of cancer cells, which limit the effectiveness of conventional therapies. Nanomedicine offers a promising solution, using nanocarriers for targeted drug delivery, improved bioavailability, and reduced off-target effects due to properties such as the enhanced permeability and retention (EPR) effect and advanced surface modifications. Additionally, the integration of theranostic capabilities allows for real-time monitoring of treatment efficacy. However, the clinical translation of nanocarriers remains restricted due to the limitations of existing predictive models. Traditional two-dimensional (2D) in vitro models often fail to replicate the complexity of the human tumor microenvironment (TME), leading to discrepancies between preclinical and clinical outcomes. More sophisticated models have been developed to address these challenges, including three-dimensional (3D) tumor spheroids, organoids, and microfluidic tumor-on-a-chip (ToC) systems. These models offer a more accurate representation of the TME, enabling better assessment of nanoparticle penetration, retention, and therapeutic effects, while also reducing reliance on animal models. This review provides a comprehensive analysis of the in vitro models used to evaluate the anti-tumoral effects of nanocarriers alongside the methodologies employed to assess their safety and efficacy. Specifically, we explore the evolution from 2D monolayer cultures to advanced 3D systems, including tumor spheroids, organoids, and ToC platforms, and delve into the main methodologies employed to evaluate nanoparticle behavior, including cellular uptake mechanisms, cytotoxicity assays (e.g., MTT, WST-1/WST-8, LDH, Live/Dead assays), cell death mechanisms (e.g., apoptosis, necrosis, and autophagy), signaling pathways exploring gene expression analysis (qRT-PCR, RNA-seq, microarrays) and protein expression analysis (western blot, immunochemistry, mass spectrometry), oxidative stress evaluation, and cell migration/invasion assays (e.g., scratch/wound-healing, transwell, and microfluidic chip-based models). Furthermore, we cover clonogenic assays for assessing long-term cell survival, and cytoskeleton evaluation to understand how nanoparticles affect cell structure. By highlighting the advantages and limitations of these models and methodologies, this review aims to guide researchers in selecting the most appropriate experimental approaches, ultimately supporting the development of more effective nanomedicine-based cancer therapies.