TPGS Analog-Mediated Intracellular ROS-Amplifying Strategy Potentiates the In Vitro Anticancer Activity of a Dual-Thioketal-Linked Polymeric Drug Conjugate in A549 Lung Cancer Cells

Background/Objectives: Reactive oxygen species (ROS)-responsive polymeric drug conjugates (PDCs) can enable oxidative stress-triggered drug release, but their activation may be limited by heterogeneous or insufficient intracellular ROS. Herein, we synthesized a dual-thioketal-linked PDC bearing two ROS-cleavable thioketal (TK) units in series and combined it with D-α-Tocopheryl polyethylene glycol succinate analog (TPGSa) as a soluble ROS-modulating co-treatment. Methods: PDC was synthesized through stepwise construction of the TK linker and subsequent carbonate coupling with camptothecin (CPT). TPGSa was prepared by esterifying mPEG with tocopheryl succinate. PDC nanoassembly formation, colloidal stability, peroxide-induced structural changes, thiol generation, and CPT release behaviors were evaluated under oxidative conditions. Cytotoxicity was examined in A549 and BEAS-2B cells with intracellular ROS- and CPT-associated fluorescence. Results: PDC formed spherical nanoassemblies with a hydrodynamic diameter of 98.6 ± 2.6 nm and a zeta potential of −13.3 ± 1.2 mV. The PDC remained colloidally dispersed in 10% FBS-containing PBS and after lyophilized storage. Peroxide exposure produced concentration-dependent thiol generation, molecular size change, and CPT release. The PDC + TPGSa reduced A549 viability more than PDC alone, produced the most pronounced dead-cell staining, and yielded the highest intracellular ROS and CPT fluorescence signals. In contrast, BEAS-2B viability remained substantially higher under matched conditions. Conclusions: These findings support an A549-focused in vitro proof of concept in which TPGSa-associated redox perturbation is paired with a dual TK PDC to enhance CPT-associated cytotoxicity.