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 & micro;g/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 approximate to 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 & micro;g/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.
A reduction-sensitive cationic bolaamphiphile bearing N-methylpyridinium terminal groups and a central disulfide bridge was synthesized and shown to self-assemble in water into stable vesicles with diameters of 200-250 nm. The vesicles undergo redox-triggered disassembly in the presence of tris(2-carboxyethyl)phosphine (TCEP), and analogous behavior was also observed using glutathione (GSH), supporting their responsiveness to reductive environments. Their potential as nanocarriers was evaluated using the photosensitizer Rose Bengal (RB) and the chemotherapeutic agents paclitaxel (PTX) and camptothecin (CPT) in A549, HT29, and MCF7 cancer cell lines. The vesicles significantly enhanced the cellular uptake of RB, as demonstrated by flow cytometry and confocal microscopy. Colocalization analysis with MitoTracker Green further supported preferential mitochondrial localization of RB fluorescence. This was accompanied by a pronounced increase in photodynamic activity upon light irradiation in A549 and MCF7 cells, where apoptosis was identified as the dominant cell-death pathway. In addition, vesicle-based formulations of PTX and CPT improved their cytotoxicity across all tested cell lines, with a particularly strong effect observed in HT29 cells under the experimental conditions employed. Overall, these pyridinium-based bolaamphiphilic vesicles represent a versatile redox-responsive delivery platform capable of enhancing intracellular delivery and therapeutic performance of both photosensitizers and poorly water-soluble anticancer drugs.
