Surfactant-shell lipid nanocapsules (LNCs) are promising skin delivery systems. They are composed of an oily core with a stabilising shell of surfactant and phosphatidylcholine. LNCs' hydrodynamic diameter can be easily tuned by varying the surfactant content in the formulation. Hydrophilic surfactants incorporated into LNCs have shown toxicity in mammalian cells. To date, the toxicity of all published surfactant-shelled LNCs produced by the novel PIT method has been investigated using hydrophilic surfactants, with no studies examining the impact of incorporating hydrophobic surfactants on LNCs' in vitro behaviour. Span 80 is a hydrophobic surfactant and has been extensively used in manufacturing various ranges of nanoparticles. The present study formulated Span 80-containing LNCs to evaluate their in vitro behaviour in the B16F10 melanoma cell line. LNC-100-S8 of Kolliphor HS 15/Span 80 (65/35 w/w%) and original LNC100-0 LNCs of Kolliphor HS 15 with a hydrodynamic diameter of 100 nm were prepared using a phase inversion method. A salt aggregation test confirmed increased surface hydrophobicity of LNC100-S8 compared to LNC100-0. Cytotoxicity assays demonstrated that LNC100-S8 had a three-fold lower cytotoxicity than LNC100-0 (IC80 = 11757 μg/mL vs 3184 μg/mL). Flow cytometry analysis indicated significantly higher cellular uptake of LNC100-S8 compared to LNC100-0, with 1.52-fold, 1.46-fold, and 1.67-fold increase at 1 h, 3 h, and 24 h, respectively (p <
0.05). Mechanistic investigations revealed that LNC100-S8 uptake predominantly occurs via phosphoinositide 3-kinase (PI3K)-regulated macropinocytosis and actin-dependent endocytosis, whereas LNC100-0 also utilises Na+/H + exchanger-mediated macropinocytosis. Furthermore, protein corona analysis demonstrated increased interactions between LNC100-S8 and B16F10-conditioned media proteins, leading to bimodal size distribution and elevated polydispersity index (>
0.3), which may influence their endocytic pathways. Overall, Our findings revealed the high promise of our Span 80-containing LNCs as a drug delivery system with enhanced cellular uptake and biocompatibility in the B16F10 melanoma cells compared to conventional LNCs composed of Kolliphor HS15 surfactant, highlighting their potential uses in topical delivery to melanoma and other skin diseases.