Optical assay interference and temporal redox retention in liposome-associated Trolox under mixed-metal stress

Absorbance-based antioxidant assays are widely used to characterize liposomal formulations, yet lipid vesicles scatter light and can contribute signal unrelated to redox activity. Using Trolox as a model antioxidant, we examined how liposome association alters the measured redox response over time, and how carrier-dependent optical interference affects its interpretation under a mixed-metal stress challenge. Free Trolox, empty liposomes and liposome-associated Trolox were followed over 0-48 h without and with a commercial 23-element metal mixture (10-1000 µg/L total), using dithiothreitol (DTT), Cupric Reducing Antioxidant Capacity (CUPRAC), Trolox Equivalent Antioxidant Capacity (TEAC) and Folin-Ciocalteu assays with parallel turbidity, plus a composition-matched physical mixture in an exploratory Escherichia coli panel. Empty liposomes, containing no antioxidant, gave TEAC and Folin signals exceeding free Trolox and shared the same principal-component region as liposome-associated Trolox, so the combined axis reflects a redox-optical response rather than antioxidant capacity. DTT provided the clearest separation between the free-Trolox response and the empty-carrier background (free/empty ≈ 3.3), although it remained associated with turbidity. Without metal, the liposome-associated form declined more slowly on DTT (retention 0.89 versus 0.62). The metal mixture reduced the DTT response to near-background levels at every tested non-zero dose and exerted a dominant negative effect across the multichannel model. At 1000 µg/L, liposome-associated Trolox and the physical mixture were indistinguishable, showing no association-specific benefit. Empty-carrier, turbidity and composition-matched controls are essential to avoid overestimating liposomal antioxidant activity.