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

Abstract

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.

Introduction

Camptothecin (CPT) is a potent topoisomerase I inhibitor and a clinically validated scaffold for cancer chemotherapy, but its direct therapeutic application is limited by poor aqueous solubility and pH-dependent conversion of the active lactone to the inactive carboxylate form under physiological conditions [1,2]. Polymeric drug conjugate (PDC) can improve the aqueous handling of CPT, reduce premature drug leakage, and provide a chemical framework for stimulus-responsive release [3,4]. Conjugation through the 20-hydroxyl group of CPT is widely used to mask the parent drug behind a cleavable linkage, which makes the linker a central determinant for conjugate stability and drug liberation [5].

Reactive oxygen species (ROS)-responsive linkers are attractive because many cancer cells exhibit elevated oxidative stress relative to non-cancerous cells, providing a redox difference that has been explored for anticancer drug release [6,7,8,9,10,11,12,13]. Among ROS-cleavable motifs, thioketal (TK) linkers have been widely used owing to their ROS-mediated oxidative cleavage under ROS-rich conditions while remaining comparatively stable under non-oxidative physiological conditions [14,15,16]. Existing TK-based delivery designs generally fall into two structural categories: single-TK polymer–drug conjugates, which place one oxidative cleavage site at the polymer–drug junction [17,18], and multi-TK polymer backbones, in which repeated TK cleavage destabilizes or degrades the carrier framework [14,19]. These arrangements leave a distinct linker-level design opportunity: positioning multiple TK units in series at the polymer–drug linker to increase the ROS-labile motifs at the drug release junction.

A key limitation of ROS-responsive PDCs is that their activation depends on the level, localization, and lifetime of intracellular ROS. Endogenous antioxidant systems can buffer ROS, and ROS-rich cancer cells may still provide insufficient or transient oxidative input for timely linker cleavage [7,9,20]. In particular, endogenous ROS may be insufficient in some cancer cell types, particularly those with modest basal oxidative stress, to sustain timely and efficient cleavage of ROS-responsive linkers. This trigger-limited behavior has motivated pairing an ROS-responsive PDC with a separately administered redox-modulating component.

D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is an amphiphilic vitamin E derivative widely used as a solubilizer, formulation stabilizer, and P-glycoprotein modulator [21,22]. Beyond these formulation roles, TPGS and related tocopheryl succinate-derived amphiphiles have been associated with mitochondrial perturbation, increased intracellular ROS signals, and the potentiation of anticancer drugs in selected models [7,23,24,25]. We therefore prepared mPEG2k-tocopheryl succinate (TPGSa), a PEG2000-based TPGS analog, for use as a co-administered redox-modulating component for the ROS-responsive PDC system. The TPGSa retains the tocopheryl succinate motif with TPGS-inspired mitochondrial perturbation and ROS modulation, while its mPEG2k segment provides hydrophilicity, supports aqueous solubility, and matches the PDC composition.

Based on this rationale, we hypothesized that TPGSa-associated redox perturbation would support oxidative activation of a dual TK PDC and thereby increase CPT-associated cytotoxicity in A549 cells. The linear PDC was designed to self-assemble into PEG-shielded CPT-containing nanoassemblies. Cleavage of the mPEG-proximal TK was proposed to weaken the PEG-shielded architecture, whereas cleavage of the CPT-proximal TK was proposed to generate a thiol-containing carbonate species capable of subsequent CPT liberation through a self-immolative process [26].

Herein, we report the synthesis, physicochemical characterization, peroxide responsiveness, and in vitro biological evaluation of the PDC in combination with TPGSa. A549 lung adenocarcinoma cells served as the cancer model and BEAS-2B bronchial epithelial cells as a non-cancerous comparison model. The study is positioned as an A549-focused in vitro proof of concept rather than as evidence of dual-TK superiority or in vivo therapeutic performance. The proposed overall design and schematic illustration are summarized in Figure 1.

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Materials

mPEG (Mw 2000 g/mol), 4-dimethylaminopyridine (DMAP), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 3-mercaptopropionic acid, triphosgene, and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). CPT and α-tocopherol succinate were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Cell Counting Kit-8 (CCK-8) was obtained from Dongin LS (Seoul, Republic of Korea). Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), and penicillin–streptomycin solution were purchased from Welgene Inc. (Gyeongsan, Republic of Korea). The LIVE/DEAD Viability/Cytotoxicity Kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). A549 human lung adenocarcinoma cells and BEAS-2B human bronchial epithelial cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All solvents were of reagent grade and were used without further purification unless otherwise stated.

Kim, H.-C.; Lee, K.-M.; Hwang, Y.J.; Lee, J.; Han, H.S. 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. Pharmaceutics 2026, 18, 886. https://doi.org/10.3390/pharmaceutics18070886

See also the interesting video on Vitamin E TPGS: