Orthogonal viability assays and high-content phenotyping identify enhanced cytotoxicity of SLN-pDNA complexes and heightened sensitivity in PC-3 cells.

Solid lipid nanoparticles (SLNs) have emerged as a versatile platform for the intracellular delivery of therapeutic molecules being widely used in drug and gene delivery owing to their biocompatibility, scalability, and long-term stability. Despite their favorable safety profile, SLN-cell interactions are not necessarily biologically inert, particularly when SLNs are complexed with biomolecules such as plasmid DNA (pDNA). Here, we systematically evaluate the sublethal cytotoxic effects of SLNs and SLN-pDNA complexes (SLNplexes) across a range of concentrations using complementary viability and imaging-based assays. We show that indirect metabolic assays, including MTT and calcein-AM fluorescence, can underestimate cytotoxic responses, whereas image-based approaches, such as live-dead assays and cell counting using acridine orange, reveal subtle but reproducible effects that persist at low exposure levels. Notably, SLN-pDNA complexes were associated with greater cytotoxicity than blank SLNs in a cell-type-dependent manner, with a more pronounced impact on PC-3 prostate cancer cells compared with non-tumoral PNT1A cells. Together, our findings demonstrate that SLN biocompatibility is context dependent and underscore the importance of orthogonal, multiparametric strategies for accurately assessing nanoparticle-induced sublethal toxicity in nanomedicine development.