Hybrid chalcogen bonds in prodrug nanoassemblies provides dual redox-responsivity in the tumor microenvironment

Homodimeric prodrug nanoassemblies (HPNAs) offer high drug loading and reduced excipient toxicity versus traditional carriers but are limited by poor self-assembly stability and insufficient, tumor-selective bioactivation. Prior work showed sulfur-containing linkers (e.g., trisulfide) can introduce near-90° bond angles that create steric hindrance to prevent over-aggregation, enhancing nanoparticle stability and providing strong reduction-responsivity to intracellular glutathione (GSH). Yet, tumors display heterogeneous and spatiotemporally variable redox states, with fluctuating levels of reactive oxygen species (ROS) and GSH, so single-mode redox-responsive systems may underperform. Given chalcogens’ periodic trends (Se, Te larger and more easily oxidized than S, with bond angles closer to 90°), the authors hypothesized that replacing the central sulfur in a trisulfide bridge with tellurium or selenium would yield hybrid chalcogen bonds (STeS and SSeS) that simultaneously enhance self-assembly and confer dual sensitivity to oxidation and reduction. The study designs and tests docetaxel (DTX) homodimeric prodrugs linked via STeS or SSeS (and SSS, SCS controls) to evaluate effects on self-assembly, redox-triggered activation, pharmacokinetics, biodistribution, and antitumor efficacy, aiming to address tumor redox heterogeneity.

Previous research established that sulfur-containing linkers, especially trisulfide bonds, improve the self-assembly and reduction-responsiveness of homodimeric prodrug nanoassemblies, prolonging circulation and enhancing tumor accumulation. However, most redox-responsive prodrug linkers have employed a single chalcogen element (S, Se, or Te), limiting adaptability to heterogeneous tumor redox states. Chalcogen bonding can drive self-assembly, with Te-based interactions stronger than Se or S, and Se/Te bonds tend toward ~90° bond angles that may increase steric hindrance beneficial for assembly. The tumor microenvironment exhibits variable ROS and GSH, motivating linkers with dual sensitivity. This work builds on those insights by introducing hybrid chalcogen bonds (STeS, SSeS) to couple enhanced assembly forces with oxidation and reduction responsivity in one linker.

Design and synthesis: Four DTX homodimeric prodrugs were synthesized using linkers containing sulfur–tellurium–sulfur (STeS), sulfur–selenium–sulfur (SSeS), trisulfide (SSS), or thioacetal (SCS) bridges. Hybrid linkers (3,3'-(telluriumdithio)- and 3,3'-(selenodithio)-dipropionic acids) and 3,3'-trithiodipropionic acid were prepared via literature methods. DTX dimers were formed by EDCI/DMAP-mediated esterification (DCM, nitrogen, 25 °C) and purified by reversed-phase HPLC. Structures were confirmed by 1H NMR and high-resolution FT-ICR MS; purity exceeded 99%. Nanoparticle preparation/characterization: Prodrug nanoassemblies were made by one-step nanoprecipitation in water from ethanol solutions of prodrugs with or without DSPE-PEG2k (20% w/w). PEGylated HPNAs were characterized by DLS (size, zeta), TEM (morphology), and drug loading; colloidal stability was assessed during storage (4 °C up to 35 days) and in PBS with 10% FBS. Non-PEGylated assembly stability was also compared. Computational analysis of self-assembly: Bond angles for S–Te–S, S–Se–S, S–S–S, and S–C–S in optimized structures were computed using xtb/gnf2 with gbsa water; conformer search by Molclus. Molecular docking and MD simulations (GAFF/UFF, TIP3P water box, Gromacs 2018) of 20–80 monomer systems, with and without DSPE-PEG2k and ethanol, evaluated binding energies, aggregation kinetics (radius of gyration), and electrostatic interactions. Hydrogen, hydrophobic, and chalcogen bonds were analyzed (Discovery Studio). Redox-triggered release and mechanistic studies: In vitro release used PBS pH 7.4 with 30% ethanol at 37 °C under oxidizing (H2O2: 1, 10, 50 mM), reducing (DTT: 0.01, 0.1, 1 mM; GSH 0.1 mM), or no-redox conditions, quantifying released DTX by HPLC. Redox intermediates were identified by triple quadrupole MS after incubation in 50 mM H2O2 or 50 mM DTT. Intracellular intermediates following cell treatment were extracted and analyzed by LC-MS/MS (triple quadrupole) to verify sulfoxide/sulfone formation and thiolysis products (e.g., DTX–SH, mixed disulfide/selenotrisulfide/tellurotrisulfide adducts with GSH/Cys). Cell studies: ROS and GSH heterogeneity were quantified in tumor (4T1, Hepa 1-6, B16F10) and normal (3T3) cells via DCFH-DA fluorescence and a glutathione assay kit. Cytotoxicity was assessed by MTT (48 h) to determine IC50 and selectivity index (3T3/tumor). Cellular uptake of coumarin-6–labeled HPNAs versus free coumarin-6 was imaged by CLSM and quantified by flow cytometry. Intracellular DTX release after HPNA incubation (48–72 h) was measured by UPLC-MS/MS. Apoptosis (Annexin V-FITC/PI) and microtubule polymerization inhibition (α-tubulin immunostaining) were evaluated. Pharmacokinetics and biodistribution: Chemical stability in rat plasma was tested over 24 h. PK in rats (IV, 4 mg/kg DTX equivalent) quantified plasma prodrugs, released DTX, and total DTX equivalents by UPLC-MS/MS. Tumor biodistribution used DiR-labeled HPNAs in 4T1 tumor-bearing BALB/c mice (IVIS at 4 and 12 h) and LC-MS/MS quantification of DTX in tumors at 1, 12, and 24 h after 20 mg/kg IV dosing. In vivo efficacy and safety: 4T1 tumor-bearing BALB/c mice received IV treatments at low (2 mg/kg), moderate (10 mg/kg), or higher tolerated doses (15, 20 mg/kg). Tumor growth, tumor burden, body weight, lung metastases (Bouin’s staining), TUNEL apoptosis, and Ki-67 proliferation in tumor sections were assessed. Safety evaluations included hematology, liver/kidney function (ALT, AST), and H&E of major organs. Statistical analyses used Student’s t-test and one-way ANOVA with specified significance thresholds.

• Self-assembly and stability: Non-PEGylated DTX-STeS-DTX NPs showed the best colloidal stability (~200 nm stable for 10 days), while SCS-based NPs aggregated rapidly. PEGylated HPNAs had diameters ~90 nm, zeta potential ~−20 mV, drug loading >67%, spherical morphology, and stability at 4 °C for 35 days. In 10% FBS, DTX-STeS-DTX and DTX-SSeS-DTX NPs remained stable over 24 h, whereas SSS and SCS increased in size.
• Computational insights: Binding energies (more negative indicates stronger assembly): DTX-STeS-DTX −122.225 kcal/mol > DTX-SSeS-DTX −120.445 > DTX-SSS-DTX −119.802 > DTX-SCS-DTX −119.525. DTX-STeS-DTX assembled fastest (equilibrium ~9 ns) vs SSeS (10 ns), SSS (13 ns), SCS (18 ns). Te-based chalcogen bonding and reduced electrostatic repulsion strengthened assembly. Calculated bond angles: S–Te–S 90.94°, S–Se–S 101.88°, S–S–S 105.76°, S–C–S 111.62°, indicating STeS provides near-90° steric hindrance that balances strong intermolecular forces.
• Redox-triggered release: Baseline release <3% over 24 h without redox triggers. Oxidation-triggered release by H2O2 followed STeS > SSeS > SSS ≈ SCS across 1–50 mM, attributed to electron donation Te > Se > S > C, shifting electron density to S adjacent to ester and facilitating oxidation (confirmed intracellular sulfoxide/sulfone and Te/Se oxo-species). Reduction-triggered release by DTT/GSH followed STeS > SSeS > SSS >> SCS. Thiolysis favored lower electron density and available orbitals at Te > Se > S; carbon showed negligible reduction-responsivity. Intracellular intermediates (DTX–SH, DTX–SXS–GSH/Cys, GSH–SXS–GSH, Cys–SXS–Cys) were detected, corroborating mechanisms.
• Cell studies: Tumor cells had higher ROS and GSH than normal 3T3 cells. Cytotoxicity ranked DTX-STeS-DTX NPs > DTX-SSeS-DTX NPs > DTX-SSS-DTX NPs > DTX-SCS-DTX NPs, all less potent than Taxotere due to prodrug activation kinetics but with higher tumor selectivity indices. Apoptosis rates: Taxotere 52.2% > DTX-STeS-DTX NPs 43.15% > DTX-SSeS-DTX NPs 37.04% > DTX-SSS-DTX NPs 32.3% > DTX-SCS-DTX NPs 25.8%. DTX-STeS-DTX NPs showed strongest microtubule inhibition and fastest intracellular DTX release. HPNAs elevated intracellular ROS and decreased GSH/GSSG, promoting further activation.
• Pharmacokinetics: In plasma stability tests, intact prodrug remaining at 24 h: SCS > SSS > SSeS ≈ STeS (~20% for STeS/SSeS). In vivo, AUC of total equivalent DTX increased vs Taxotere by 31.98× (STeS), 31.61× (SSeS), 31.85× (SSS), and 127.35× (SCS). Despite lower chemical stability, STeS and SSeS achieved AUC similar to SSS, implying improved assembly compensated. Released DTX in blood was higher for STeS/SSeS, consistent with higher redox-responsivity.
• Biodistribution and tumor activation: DiR-labeled HPNAs accumulated in tumors more than DiR solution at 4 and 12 h. LC-MS showed Taxotere tumor DTX declined over time, whereas HPNAs’ tumor DTX increased, with STeS NPs achieving the highest tumor DTX at 24 h.
• Antitumor efficacy and safety: At 2 mg/kg, STeS, SSeS, and SSS NPs inhibited tumor growth and reduced lung metastases more than SCS and saline; Taxotere showed limited efficacy at this low dose. At 10 mg/kg, Taxotere and STeS NPs exhibited strong tumor inhibition; STeS NPs reduced Ki-67 and increased TUNEL apoptosis. At 15–20 mg/kg, STeS NPs achieved optimal tumor suppression without body weight loss. Taxotere caused weight loss even at 2 mg/kg, leukopenia/lymphopenia at 10 mg/kg, liver injury (ALT/AST elevation, histology). HPNAs, including STeS at 15 mg/kg, showed no significant hematological, biochemical, or histological toxicity.

The study addresses the dual challenges of homodimeric prodrug nanoassemblies—assembly stability and tumor-selective activation—by introducing hybrid chalcogen bonds. STeS linkers provide stronger chalcogen bonding and near-orthogonal bond geometry that together enhance nanoparticle assembly without over-aggregation, yielding stable, PEGylated particles with prolonged circulation and tumor accumulation. Concurrently, STeS exhibits ultrahigh dual sensitivity to both oxidation and reduction, enabling activation across heterogeneous tumor redox landscapes. Mechanistic analyses (MS-identified oxidative and thiolytic intermediates, cellular ROS/GSH modulation) confirm on-demand bioactivation, translating into efficient intracellular DTX release, robust apoptosis, and microtubule disruption. In vivo, these properties balance plasma stability with assembly stability to maintain exposure, increase tumor DTX levels over time, and improve antitumor efficacy while mitigating systemic toxicities associated with conventional Taxotere. Collectively, hybrid chalcogen bonding functions as a “double control switch,” offering a generalizable strategy to overcome tumor redox heterogeneity in prodrug-based chemotherapy.

Hybrid chalcogen bonds, especially STeS, simultaneously enhance self-assembly and confer dual redox-responsivity in DTX homodimeric prodrug nanoassemblies. This design achieves stable, high-loading nanoparticles with prolonged circulation, efficient tumor accumulation, and on-demand activation in heterogeneous redox environments, improving antitumor efficacy and safety versus standard Taxotere. The work expands the design paradigm for redox-responsive prodrugs from single-element to hybrid chalcogen linkers. Future directions include translating STeS-based HPNAs toward clinical evaluation: determining industrially viable dosage forms (e.g., lyophilized vs solution), ensuring chemical/physical stability under manufacturing and sterilization conditions, and closely monitoring selenium/tellurium-related toxicities in preclinical and clinical studies. Exploration with other drugs and tumor models may broaden applicability.

The authors note translational considerations rather than experimental shortcomings: the final clinical dosage form (lyophilized powder or solution) must be defined; chemical and physical stability must meet manufacturing and sterilization requirements; and potential selenium/tellurium-related toxicities warrant close monitoring in clinical trials.