A Molecular Trimming Strategy for Hypoxia‐Tolerant Photosensitizers With Enhanced cGAS‐STING Activation

ABSTRACT

The development of effective photosensitizers for photo‐immunotherapy is highly desirable yet remains challenging, particularly given the prevailing reliance on π‐conjugation extension in conventional molecular design. Herein, we propose a counterintuitive “π‐bridge trimming” strategy to construct high‐performance Ir(III) complexes photosensitizers. Unlike the conventional π‐extension approach, the three‐ring fused TTz‐Ir outperforms its π‐extended five‐ring fused analog TBTz‐Ir in multiple aspects, including molar absorptivity, solubility, photocatalytic activity, and photocytotoxicity. Mechanistic studies revealed that the superior performance of TTz‐Ir stems from its longer triplet‐state lifetime, more efficient charge separation, and transport favoring type I reactive oxygen species (ROS) generation. Upon light irradiation, TTz‐Ir not only produces ¹O₂ via energy transfer, but also efficiently generates type I ROS such as O₂ ^•−, H₂O₂, and •OH, primarily through oxygen reduction reaction (ORR) and water oxidation reaction (WOR) pathways, ensuring robust photocytotoxicity even under hypoxia. These ROS induces mitochondrial and nuclear DNA damage, leading to activation of the cGAS‐STING pathway and robust antitumor immunity. When encapsulated into DSPE‐PEG₂₀₀₀‐Biotin, TTz‐Ir NPs achieve effective tumor accumulation and significant tumor suppression in vivo. This work provides a novel molecular design paradigm and efficient metal complexes for photo‐immunotherapy.

This work presents a π‐bridge trimming strategy that yields an Ir(III) photosensitizer, TTz‐Ir, with enhanced photophysical properties and photocytotoxicity over its extended analog. With a longer triplet lifetime and more efficient charge separation, TTz‐Ir generates type I/II ROS under light, inducing DNA damage and cGAS‐STING pathway activation. It suppresses tumor growths and stimulates antitumor immunity in vivo.

1. Introduction

Immunotherapy has achieved notable success in clinical practice with the development of various immunogenic therapeutic agents [1, 2, 3, 4]. However, its efficacy is often constrained by the immunosuppressive tumor microenvironment. In recent years, the cyclic GMP–AMP synthase (cGAS)‐stimulator of interferon genes (STING) signaling pathway has emerged as a key mediator of immunogenic responses, offering considerable potential to enhance antitumor immunity. Known activators of the cGAS‐STING pathway fall into three main categories: (1) chemotherapeutic agents that cause DNA damage and cytoplasmic release; (2) non‐nucleotide STING agonists that bind directly to STING; and (3) nucleotide‐based STING agonists that also target STING directly [5, 6, 7]. Despite their promising effects, nucleotide‐based agonists often suffer from poor bioavailability, low stability, and limited tumor selectivity. Moreover, they are prone to lysosomal sequestration in cancer cells, which further restricts their clinical utility. Notably, mitochondrial DNA (mtDNA) is more susceptible to reactive oxygen species (ROS)‐induced damage than nuclear DNA due to the lack of protective histones and inefficient repair mechanisms. Studies have shown that endogenously generated mitochondrial ROS promote mtDNA leakage into the cytosol, activating the cGAS‐STING pathway and triggering antitumor immune responses [8, 9].

Capitalizing on this mechanism, photosensitizer‐based immunotherapy has recently gained attention as a promising modality that combines photodynamic therapy (PDT) [10, 11, 12, 13, 14, 15, 16, 17] with tumor immunotherapy [18, 19, 20, 21, 22]. This approach offers a potential route to efficient, low‐side‐effect, and durable cancer treatment [23, 24, 25, 26]. Among available photosensitizers, metal complexes are particularly attractive due to their high photostability, ease of synthesis, tunable photophysical properties, efficient intersystem crossing (ISC), and precise subcellular targeting [27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37]. Traditionally, extending the π‐conjugation length has been a central strategy for developing high‐performance metal complex photosensitizers [38, 39]. This approach effectively tunes key photophysical properties, such as absorption/emission wavelengths, and enhances excited‐state characteristics including triplet‐state lifetime and ISC efficiency, thereby improving the overall photosensitizing performance [40, 41, 42]. However, such π‐extension often leads to increased molecular size and hydrophobicity, reducing solubility, compromising cellular uptake, and diminishing subcellular targeting specificity. These drawbacks ultimately limit biological applicability.

To address these challenges, we developed a counterintuitive “π‐bridge trimming” strategy for designing high‐performance iridium (III) photosensitizers. Using this approach, we synthesized two fused‐ring Ir(III) complexes, TTz‐Ir and TBTz‐Ir, featuring three‐ring and five‐ring fused π‐bridges, respectively. Remarkably, the smaller TTz‐Ir outperformed TBTz‐Ir across multiple parameters, including molar absorptivity, solubility, photocatalytic activity, and photocytotoxicity. Mechanistic investigations revealed that TTz‐Ir possesses a longer triplet‐state lifetime, more efficient charge separation and transport for type I ROS generation. Upon photoactivation, TTz‐Ir not only produces ¹O₂ through energy transfer but also effectively generates type I ROS (O₂ ^{• −}, H₂O₂, and •OH), primarily via oxygen reduction reaction (ORR) and water oxidation reaction (WOR) pathways. At the cellular level, TTz‐Ir showed improved uptake and localized preferentially in mitochondria and lysosomes.

Light‐induced ROS production by TTz‐Ir disrupted cellular redox homeostasis, causing oxidative damage to mtDNA. The leaked mtDNA in the cytoplasm was sensed by cGAS, which produced cyclic GMP‐AMP (cGAMP), leading to STING activation and subsequent phosphorylation of TBK1 and IRF3. This signaling cascade stimulated the secretion of type I interferons (IFNs) and chemokines, promoting immune cell infiltration and robust antitumor immunity. To enhance tumor‐specific delivery, TTz‐Ir was encapsulated into nanoparticles using DSPE‐PEG₂₀₀₀‐Biotin. In vivo studies confirmed efficient tumor accumulation of TTz‐Ir NPs and light‐triggered activation of antitumor immunity, resulting in significant tumor suppression (Scheme 1). This work not only introduces a novel π‐bridge trimming strategy for molecular design but also demonstrates an effective Ir(III)‐based agent for photo‐immunotherapy via activation of the cGAS‐STING pathway.

SCHEME 1.

Schematic illustration of (a) π‐bridge trimming strategy for fused‐ring iridium(III) photosensitizers, (b) photoactive mechanisms, and (c) cGAS‐STING pathway activation.

2. Results and Discussion

2.1. Design, Synthesis, and Characterization

Among all the endeavors to modulate the photophysical properties of photosensitizers, the introduction of an electron donor–acceptor (D–A) architecture is widely regarded as a cutting‐edge strategy. This approach is highly effective for achieving long wavelength absorption and emission, as well as facilitating efficient ROS generation. Besides, the rational design of the ligands is crucial as it affects not only the final molecular topology structures but also photophysical properties of complexes. With those in mind, we adopted a simple D–A–D typed molecule skeleton to construct the π‐conjugated ligands, TTz and TBTz. The propeller‐shaped triphenylamine (TPA) units were utilized as electron donors. 2,2'‐Bithiazole or 2,2'‐bibenzo[d]thiazole was used as the acceptor core and chelation sites, which can bind with metal moiety. The target Ir(III) complexes TTz‐Ir and TBTz‐Ir could be obtained upon the ligands TTz and TBTz chelation with the Ir(III) precursor [Ir(ppy)₂Cl]₂, respectively (Scheme S1). The introduction of iridium atom not only changes the molecular configuration of the ligands, but also endows the complexes with significantly improved photophysical properties compared with the ligands, such as red‐shifted absorption/emission wavelength and enhanced photosensitizing performance, etc. Notably, the iridium atom is connected with the ligands TTz or TBTz by coordination bonds, which can form three or five‐ring fused π‐bridges in complexes, resulting in extended π‐conjugation.

The chemical structures of the intermediates and final products were confirmed by NMR spectra and mass spectra (MS) (Figures S1–S9). High performance liquid chromatography (HPLC) analysis revealed that the purities of TTz‐Ir and TBTz‐Ir were 98.4% and 99.6%, respectively (Figure S10). TTz‐Ir and TBTz‐Ir can dissolve in common organic solvents such as dimethyl sulfoxide (DMSO), dichloromethane (DCM), and chloroform, etc. However, TBTz‐Ir tends to aggregate and precipitate as the concentration increases, particularly in polar organic solvents like methanol (Figure S11). The result indicates that TBTz‐Ir has lower solubility compared to TTz‐Ir. The lower solubility of TBTz‐Ir may be attributed to its larger π‐conjugation backbone, which facilitates molecular aggregation in polar solvents and thereby reduces its solubility.

We further investigated the molecular conformation and packing mode in the crystalline state through single‐crystal structure analysis. Single crystals of TTz, TBTz, and TTz‐Ir [43, 44, 45] were obtained by slow solvent evaporation from a mixture of DCM and petroleum ether (Figure 1a). Despite our best efforts, suitable single crystals of TBTz‐Ir could not be obtained. The crystallographic data and collection parameters for x‐ray diffraction are summarized in Tables S1 and S2. The molecular structures and packing modes of TTz, TBTz, and TTz‐Ir are depicted in Figures S12 and S13. The analysis revealed that TTz and TBTz molecules preferentially adopt a slip‐stacking arrangement, with the thiazole or benzo[d]thiazole moieties displaying a head‐to‐tail connection configuration. In contrast, the crystal structure of TTz‐Ir results in a head‐to‐head connection configuration of the thiazole moieties and a buckled TTz backbone. Additionally, TTz‐Ir exhibits a looser packing mode with larger intermolecular distances compared to TTz, due to increased steric hindrance.

FIGURE 1.

(a) X‐ray single crystal structures of TBTz, TTz, and TTz‐Ir. C atoms are in gray, Ir atoms are in red, N atoms are in blue, H atoms are in white, and S atoms are in yellow. Counterions and solvent molecules are omitted for clarity. (b) Optimized ground‐state (S₀) geometries of two complexes. (c) Calculated HOMO and LUMO of the two complexes. (d) Electron‐hole distribution of two complexes excited at the S₁ state. Green and blue indicate the electrons and holes, respectively. (e) Quantitative analysis the charge transfer amounts of TTz‐Ir and TBTz‐Ir. The triphenylamine (TPA) units are shown in green, the 2,2'‐bithiazole or 2,2'‐bibenzo[d]thiazole unit in red, and the phenylpyridine‐‑iridium fragment in purple. (f) Normalized absorption and (g) emission spectra of two complexes in DMSO. (h) Luminescence decay curves of the two complexes in DMSO.

2.2. Theoretical Calculations and Photophysical Properties

Based on Gaussian 16 program, density functional theory (DFT) and time‐dependent density functional theory (TD‐DFT) theoretical methods were used for ground state and excited state optimizations and computations of the complexes in DMSO (Tables S3–S5). The influence of molecular geometry was investigated. The optimized ground‐state (S₀) geometry of TTz‐Ir shows dihedral angles of approximately 15° between the 2,2'‐bithiazole core and the TPA units. In contrast, when the core is replaced by a larger and more sterically hindered 2,2'‐bibenzo[d]thiazole unit in TBTz‐Ir, the corresponding dihedral angles increase to about 27°, indicating a more distorted molecular configuration in TBTz‐Ir (Figure 1b). As shown in Figure 1c, the highest occupied molecular orbitals (HOMO) of both complexes are delocalized over the entire ligand frameworks (TTz and TBTz), whereas the lowest unoccupied molecular orbitals (LUMO) are primarily located on the central acceptor core. Accordingly, the calculated energy bandgaps decrease from 2.63 eV for TTz‐Ir to 2.43 eV for TBTz‐Ir, suggesting that extending the π‐conjugation effectively narrows the bandgap. The electron transitions corresponding to S₀→S₁ in both complexes were visualized using electron‐hole distribution maps (Figure 1d), with green and blue representing electron density and hole distribution, respectively. In TTz‐Ir and TBTz‐Ir, electrons are mainly localized on the central 2,2'‐bithiazole or 2,2'‐bibenzo[d]thiazole acceptors, while holes are predominantly distributed over the conjugated backbones of the TTz and TBTz ligands. As shown in Figure 1e, electron transfer data reveal that the excited state is dominated by pronounced intraligand charge‐transfer (ILCT) from the TPA donors to the 2,2'‐bithiazole or 2,2'‐bibenzo[d]thiazole acceptors, with transfer values of 0.5626 e⁻ for TTz‐Ir and 0.7296 e⁻ for TBTz‐Ir. In addition, a minor redistribution of charge involving the metal center is observed, comprising a small forward transfer from TPA to the phenylpyridine‐iridium fragment (0.0181 e⁻ and 0.0220 e⁻) and a slight reverse transfer from the 2,2'‐bithiazole or 2,2'‐bibenzo[d]thiazole to phenylpyridine‐iridium (‐0.0085 e⁻ and ‐0.0134 e⁻). These weaker contributions indicate the participation of both metal‐to‐ligand (MLCT) and ligand‐to‐metal charge transfer (LMCT) character in the excited state. Overall, the excited state is primarily ILCT‐driven, with subsidiary participation from metal‐centered charge‐transfer pathways. In addition, the singlet triplet excited state energy gaps (ΔE _st) of TTz‐Ir and TBTz‐Ir were determined to be 0.55 and 0.31 eV, respectively (Figure S14), indicating the potential for effective generation of ROS.

The optical properties of TTz‐Ir and TBTz‐Ir were studied by UV–vis absorption and photoluminescence (PL) spectroscopies. As shown in Figures 1f and S15, both compounds exhibited broad absorption bands in common organic solvents, including THF, DCM, DMSO, CH₃CN, and MeOH. For example, in DMSO, TTz‐Ir and TBTz‐Ir showed absorption maxima at 515 and 513 nm, respectively. The optical data are summarized in Tables S6 and S7. The molar extinction coefficients of TTz‐Ir and TBTz‐Ir at their absorption maxima in CH₂Cl₂ were determined to be 4.27 × 10⁴ and 3.15 × 10⁴ L mol⁻¹ cm⁻¹, respectively (Figure S16). The lower extinction coefficient of TBTz‐Ir may be attributed to its more distorted molecular geometry (Figure 1b). As illustrated in Figure 1g, the emission maxima of TTz‐Ir and TBTz‐Ir in DMSO were located at 613 nm and 748 nm, respectively. Notably, benefiting from the extended π‐conjugation and more distorted molecular configuration, TBTz‐Ir exhibited a much larger Stokes shift (235 nm) than that of TTz‐Ir (98 nm), suggesting stronger excited‐state structural relaxation and more pronounced intramolecular charge transfer (ICT) character. In contrast, both compounds showed very weak photoluminescence in THF, CH₃CN and MeOH despite clear absorption, indicating efficient non‐radiative decay pathways in these solvents (Figure S15). Collectively, TBTz‐Ir is more promising for near‐infrared (NIR) emission applications, though its solvent‐dependent emission requires careful selection of the solvent medium. The absorption curves of TTz‐Ir and TBTz‐Ir showed negligible changes after continuous 550 nm light irradiation for 30 min, indicating their excellent photostability (Figure S17). Moreover, the luminescence lifetimes of TTz‐Ir and TBTz‐Ir were determined to be 2.93 and 2.77 ns, respectively (Figures 1h, S18, and Table S7).

2.3. Photodynamic/Photocatalytic Properties and Mechanisms

The ROS generation upon light irradiation is essential for PDT. Initially, the ¹O₂ production of TTz‐Ir and TBTz‐Ir was assessed under 550 nm light irradiation using 9,10‐anthracenediyl‐bis(methylene) dimalonic acid (ABDA) as an indicator, which undergoes absorbance decay in the presence of ¹O₂ [46, 47]. Under identical conditions, both complexes demonstrated superior ¹O₂ production efficiency compared to the conventional photosensitizer [Ru(bpy)₃]^{2 +} (Figures 2a and S19). Furthermore, the ¹O₂ quantum yields (Φ _Δ) of TBTz‐Ir and TTz‐Ir were determined to be as high as 0.60 and 0.32, respectively (Figure S20), using [Ru(bpy)₃]^{2 +} (Φ _Δ = 0.22) as a reference.

FIGURE 2.

(a) Decomposition rates of ABDA (100 µM, for ¹O₂ detection) at 378 nm in the presence of TBTz‐Ir or TTz‐Ir (10 µM) under 550 nm light irradiation for different time intervals. (b) Relative changes (I/I ₀‐1 at 526 nm) in PL intensity of DHR123 (10 µM, for O₂ ^•− detection) in the presence of TBTz‐Ir or TTz‐Ir (20 µM) upon 550 nm light irradiation for different intervals. (c) Plots of ΔAbs (A–A₀ at 415 nm) of ABTS (80 µM, for •OH detection) in the presence of TBTz‐Ir or TTz‐Ir (10 µM) under 550 nm light irradiation for different time intervals with or without pyruvic acid (50 mM) as the electron acceptor. (d) ¹H NMR spectrum of pyruvic acid (0.5 mM) in D₂O containing TTz‐Ir (0.1 mM) under 550 nm light irradiation for different time intervals. Lac: lactic acid. (e) Time profiles of H₂O₂ generation catalyzed by TTz‐Ir (100 µM) and TBTz‐Ir (100 µM) in the dark or under light irradiation, as measured by iodometry. (f) H₂O₂ generation rates catalyzed by TTz‐Ir (100 µM) and TBTz‐Ir (100 µM) under light irradiation for 2 h under different atmospheric conditions, as measured by iodometry. (g) H₂O₂ generation rates catalyzed by TTz‐Ir (100 µM) and TBTz‐Ir (100 µM) under light irradiation for 2 h with or without different hole sacrificial agents, as measured by iodometry. (h) H₂O₂ generation rates catalyzed by TTz‐Ir (100 µM) and TBTz‐Ir (100 µM) under light irradiation for 2 h with or without pyruvic acid as an electron sacrificial agent under air or N₂ atmosphere, as measured by iodometry. (i) H₂O₂ generation rates catalyzed by TTz‐Ir (100 µM) and TBTz‐Ir (100 µM) under light irradiation for 2 h with or without different scavengers, as measured by iodometry. TBA: tert‑butyl alcohol, BQ: benzoquinone.

Superoxide anion (O₂ ^•−) formation was evaluated using dihydrorhodamine 123 (DHR123) as a probe, which emits strong fluorescence upon reaction with O₂ ^•− [46]. As shown in Figures 2b and S21, irradiation of DHR123 alone produced no signal enhancement, while both TTz‐Ir and TBTz‐Ir induced a marked increase in fluorescence after 14 min of 550 nm light exposure, confirming efficient O₂ ^•− generation via a type I pathway. Notably, TTz‐Ir exhibited stronger O₂ ^•− production than TBTz‐Ir.

Hydroxyl radical (•OH) generation was monitored using 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS), which shows increased absorbance upon oxidation by •OH [46, 48]. As shown in Figures 2c and S22, under 550 nm light irradiation, ABTS alone showed negligible change, whereas solutions containing TTz‐Ir or TBTz‐Ir displayed a clear absorbance increase after 5 min of irradiation. TTz‐Ir produced stronger •OH generation than TBTz‐Ir under identical conditions, indicating its higher efficiency in •OH production. The addition of pyruvic acid (Pyr) further enhanced •OH generation for both complexes upon light irradiation (Figures 2c and S23), suggesting Pyr acts as an electron acceptor [48]. The photoreduction of Pyr was corroborated by ¹H NMR spectroscopy. Under light irradiation, the proton signal of Pyr at 2.37 ppm decreased gradually, while signals corresponding to lactic acid (Lac) emerged at 2.09 ppm, confirming the conversion of pyruvic acid to lactic acid in the presence of the complexes (Figures 2d and S24).

To further elucidate the mechanism of ROS generation, we evaluated the photocatalytic hydrogen peroxide (H₂O₂) production capabilities of TTz‐Ir and TBTz‐Ir. The concentration of H₂O₂ was determined by iodometry (Figure S25) [49, 50]. Initial experiments were performed in pure water under air, without any sacrificial agent, under irradiation with 550 nm light. As shown in Figures 2e and S26, no H₂O₂ was produced in the dark, confirming that light is essential for the catalytic reaction. Upon irradiation, however, both complexes exhibited a significant increase in H₂O₂ generation. The nearly linear time‑dependent production curves indicate a steady and continuous H₂O₂ generation rate for both catalysts. After 2  h of irradiation, the accumulated H₂O₂ concentrations were 24.9 µM for TTz‑Ir and 10.4 µM for TBTz‑Ir, corresponding to average production rates of 215.9 and 82.9 µmol g^{− 1} h^{− 1}, respectively. The superior performance of TTz‑Ir under identical conditions demonstrates its higher photocatalytic activity.

Subsequently, the influence of reaction atmosphere and sacrificial agents was investigated. Under a N₂ atmosphere, both complexes exhibited very low H₂O₂ yields, highlighting the essential role of O₂. Yields increased significantly in air and further increased under a pure O₂ atmosphere (Figures 2f and S27), confirming that H₂O₂ production is primarily driven by the oxygen reduction reaction (ORR) [51, 52]. Notably, a detectable amount of H₂O₂ was still generated by TTz‑Ir even under N₂, suggesting that this complex can also promote H₂O₂ formation via the WOR pathway [52, 53]. When the hole scavenger was changed from isopropanol (IPA) to benzyl alcohol, the H₂O₂ production of both complexes increased beyond that observed in pure water (Figures 2g and S28). This improvement is likely due to benzyl alcohol being more readily oxidized by photogenerated holes than its aliphatic counterparts [53]. With benzyl alcohol as the hole sacrificial agent, the H₂O₂ yield rate of TTz‑Ir reached 402.5 µmol g^{− 1} h^{− 1}, which was 2.2 times higher than that of TBTz‑Ir (Figures 2g and S28). The significant increase in H₂O₂ yield upon addition of benzyl alcohol confirms that effective hole scavenging suppresses charge‑carrier recombination and supplies protons (H⁺) necessary for ORR [52, 54]. Furthermore, compared with the results obtained in pure water under a N₂ atmosphere, the addition of Pyr significantly enhanced the H₂O₂ generation of both complexes under both normoxic and hypoxic conditions (Figures 2h and S28). This enhancement aligns with the role of Pyr as an electron sacrificial agent that facilitates the WOR pathway.

As indicated above, aside from the minor WOR contribution observed under N₂, H₂O₂ production is predominantly governed by ORR. The ORR pathway can proceed via two principal routes: (i) a direct two‑electron transfer and (ii) an indirect stepwise single‑electron transfer [51, 52, 55].

Direct (one‑step) 2e⁻ ORR:

$${\mathrm{O}}_2+2{\mathrm{e}}^{-}+2{\mathrm{H}}^{+}\to {\mathrm{H}}_2{\mathrm{O}}_2$$

Indirect (stepwise) 1e⁻ ORR:

$${\mathrm{O}}_2+{\mathrm{e}}^{-}\to {\mathrm{O}}_2^{·-}$$

$${\mathrm{O}}_2^{·-}+{\mathrm{e}}^{-}+2{\mathrm{H}}^{+}\to {\mathrm{H}}_2{\mathrm{O}}_2$$

For the stepwise route, O₂ ^•− is a critical intermediate. Addition of the O₂ ^•− scavenger benzoquinone (BQ) sharply decreased the H₂O₂ generation rate for both complexes, although the rate remained higher than that in pure water under N₂ (Figures 2f–i and S28) [52]. In contrast, introduction of the •OH scavenger tert‑butyl alcohol (TBA) caused no significant change in H₂O₂ yield, indicating that •OH is not a primary reactive intermediate in this photocatalytic system (Figures 2i and S28) [52]. These results suggest that both the indirect (stepwise) and direct (2e⁻) ORR pathways contribute to H₂O₂ formation, with the direct pathway being dominant.

2.4. Excited‐State Properties

Nanosecond transient absorption (TA) spectroscopy was performed for both complexes in de‐aerated DMSO to elucidate their excited‐state dynamics. For TTz‐Ir, the TA spectra exhibit two ground‐state bleach (GSB) features between 300–350 nm and 450–600 nm (Figure 3a), coinciding with the absorption bands in the ground‐state spectrum (Figure 1f). In addition, two excited‐state absorption (ESA) bands are observed at approximately 380 and 750 nm. It is noteworthy that the band at 750 nm is well established to originate from the one‐electron oxidized triphenylamine radical cation [56], whereas the band at 380 nm is tentatively attributed to the one‐electron reduced thiazole moiety. These observations indicate that the triplet intra‐ligand charge‐transfer (³ILCT) transition proceeds from the TPA moiety to the thiazole moiety, in agreement with the TD‐DFT calculation results (Table S4). A very similar behavior was observed in the TA spectra of TBTz‐Ir in de‐aerated DMSO. Two GSB features at approximately 360 and 540 nm and ESA signals centered at 410 and 750 nm were detected, indicating the occurrence of a similar ³ILCT process (Figure 3b).

FIGURE 3.

Transient absorption spectra of (a) TTz‐Ir and b) TBTz‐Ir in deaerated DMSO at 20 °C following direct excitation at 605 nm, recorded at different delay times as indicated in the inset. Kinetic decays from transient absorption spectra of TTz‐Ir and TBTz‐Ir in (c), (d) deaerated and (e), (f) O₂‐saturated DMSO. Excited‐state absorption (black and red lines) and ground‐state bleach (blue line) were monitored at 380/520/750 nm (de‐aerated) and 390/515/750 nm (O₂‐saturated) for TTz‐Ir, and at 410/550/750 nm (de‐aerated) and 400/550/750 nm (O₂‐saturated) for TBTz‐Ir. Corresponding lifetimes (τ) are 144/96 ns for TTz‐Ir and 49/21 ns for TBTz‐Ir under deaerated and O₂‐saturated conditions, respectively. KA: kinetic absorption. Comparison of excited‐state lifetimes of (g) TTz‐Ir and (h) TBTz‐Ir in de‐aerated (black trace) and O₂‐saturated DMSO (red trace), respectively.

To further understand the difference in photocatalytic performance between TTz‐Ir and TBTz‐Ir from the experimental results, the kinetic decays of their ESA were measured to determine their triplet excited‐state lifetimes. For both complexes, the excited‐state absorption and GSB recoveries are consistent with monoexponential decay kinetics, yielding excited‐state lifetimes (τ) of 144 ns for TTz‐Ir and 49 ns for TBTz‐Ir, respectively (Figures 3c,d, S29, and S30). The longer lifetime of TTz‐Ir generally accounts for its superior photocatalytic activity. In contrast, the experimental results of ¹O₂ generation (Figure 2a) show a reversed trend, with TBTz‐Ir exhibiting higher efficiency than TTz‐Ir. To further explore this phenomenon, the dynamic behavior and excited‐state lifetimes of both complexes under O₂‐quenching conditions in DMSO were investigated. The TA spectra reveal the same spectral features as those obtained under inert conditions (Figures 3e,f, S31, and S32). For TTz‐Ir, the excited‐state lifetime decreases from 144 ns to 96 ns, corresponding to a 33.3% quenching efficiency. In contrast, TBTz‐Ir exhibits a more pronounced shortening of the lifetime from 49 ns to 21 ns, with a quenching efficiency of 57%, thereby resulting in its higher ¹O₂ generation efficiency (Figures 3g,h, S33, and S34). Interestingly, based on the TD‐DFT calculations, the lowest triplet energies of TTz‐Ir and TBTz‐Ir are 1.56 and 1.70 eV, respectively, indicating a higher energy‐transfer driving force for TBTz‐Ir, which is likely responsible for its more efficient energy transfer to O₂ (Figure S14).

2.5. Photoelectrochemical Properties and Photoactive Mechanisms

To further understand the performance difference between TTz‑Ir and TBTz‑Ir, their photo‑induced charge‑transfer characteristics were investigated. Efficient type I photoactivity depends on effective charge separation and transport. Transient photocurrent measurements revealed that TTz‑Ir exhibits a higher photocurrent density over multiple light on‐‑off cycles than TBTz‑Ir (Figure S35), indicating superior charge‑separation efficiency [57]. Electrochemical impedance spectroscopy (EIS) further showed that TTz‑Ir films display a smaller semicircle in Nyquist plots (Figure 4a), corresponding to lower charge‑transfer resistance and more efficient charge transport [58]. Together, these data rationalize the enhanced type I photoreactivity of TTz‑Ir.

FIGURE 4.

(a) The electrochemical impedance spectroscopy (EIS) of TBTz‐Ir and TTz‐Ir. (b) XPS valence band spectrum of TBTz‐Ir and TTz‐Ir; the work function (Φ) is 4.50 eV. (c) Mott–Schottky plot of TBTz‐Ir and TTz‐Ir at 1500, 2000, and 2500 Hz obtained in the dark; TBTz‐Ir or TTz‐Ir on ITO electrode as a working electrode, Ag/AgCl as a reference electrode, platinum wire as a counter electrode, and 1.0 M Na₂SO₄ as a supporting electrolyte. (d) Band structures diagram of two complexes. (e) Schematic illustration of the proposed type I (electron‐transfer) and type II (energy‐transfer) mechanisms for TTz‐Ir. ILCT: intra‐ligand charge transfer.

The redox characteristics of the complexes were analyzed using energy‑band theory, as the formation of type I ROS is governed by their valence‑band (VB) and conduction‑band (CB) potentials [48]. The VB potentials were derived from x‐ray photoelectron spectroscopy (XPS) valence band spectra. Figure 4b shows valence band maxima at 1.53 eV for TTz‐Ir and 1.45 eV for TBTz‐Ir, corresponding to VB potentials of 1.59 V and 1.51 V (vs. NHE), respectively. Mott–Schottky measurements were conducted to determine the flat‐band potentials [48]. The positive slopes observed in the Mott–Schottky plots confirm the n‐type semiconductor behavior of both complexes (Figure 4c). The measured flat‐band potentials versus Ag/AgCl were ‐0.63 V for TTz‐Ir and ‐0.54 V for TBTz‐Ir, which closely approximate their CB potentials. When converted to the normal hydrogen electrode (NHE) scale, these values correspond to CB potentials of ‐0.43 V and ‐0.34 V, respectively. As summarized in Figure 4d, the VB potentials of both complexes are more positive than the thermodynamic potential for water oxidation to H₂O₂ (WOR), while their CB potentials are suitably positioned to enable the reduction of oxygen to H₂O₂ via either the direct 2e⁻ ORR (0.28 V vs. NHE, pH = 7) or the stepwise 1e⁻ ORR (‑0.33 V vs. NHE) and to promote the reduction of pyruvate to lactate [48, 53]. From a thermodynamic perspective, these results confirm that both complexes can catalyze three key photochemical processes: water oxidation to H₂O₂, oxygen reduction to H₂O₂, and pyruvic acid reduction to lactic acid.

Based on the comprehensive analysis of photoexcited states and experimental data, a mechanistic framework outlining the type I (electron‐transfer) and type II (energy‐transfer) pathways of TTz‐Ir is depicted in Figure 4e. Upon photoexcitation, the ground‐state TTz‐Ir undergoes ILCT from the electron‐donating TPA unit to the electron‐accepting 2,2'‐bithiazole moiety. This process generates a triplet excited state (PS*), which resembles a charge‐separated species (^•− TTz‐Ir ^{• +}), with ISC enhanced by the heavy‐atom effect of iridium. In the type II pathway, the triplet excited state of TTz‐Ir directly transfers energy to ground‐state O₂, generating ¹O₂ and returning the complex to its ground state. In the type I pathway, under aerobic conditions, the strongly reduced (thiazole)^{• −} site can reduce molecular oxygen (O₂) to generate O₂ ^•−, which serves as a key intermediate in the stepwise ORR leading to H₂O₂ formation. Simultaneously, the highly oxidized TTz‐Ir ^{• +} species can be regenerated to its ground state through the one‐electron oxidation of water (WOR), producing H₂O₂ and completing the catalytic cycle. Alternatively, O₂ ^•− can undergo disproportionation to yield highly reactive •OH with concomitant release of O₂ [59, 60]. Furthermore, under the action of a photosensitizer, H₂O₂ can be decomposed to produce •OH via a photoinduced electron transfer process [61]. Under anaerobic conditions, the electron‐rich (thiazole)^{• −} site transfers an electron to a substrate such as Pyr, resulting in its reduction to Lac. In parallel, the oxidized TTz‐Ir ^{• +} is reduced back to the ground state via water oxidation, again generating H₂O₂. This integrated mechanism explains the generation of multiple ROS (¹O₂, O₂ ^•−, •OH, and H₂O₂) and accounts for the observed photocatalytic activities across different atmospheric conditions, consistent with the measured photoelectrochemical properties and thermodynamic band positions of TTz‐Ir.

2.6. In Vitro Photocytotoxicity and Intracellular ROS Evaluation

First, the photo‐ and dark‐cytotoxicities of TTz‐Ir and TBTz‐Ir against various cell lines were evaluated by MTT assay. As shown in Figure 5a,b and Table 1, and Figures S36–S38, after 4 h of drug exposure, the half‐maximal inhibitory concentration (IC₅₀) values of both TTz‐Ir and TBTz‐Ir exceeded 50 µM in all tested cancer cell lines (4T1, MDA‐MB‐231, and MCF‐7) under both normoxic and hypoxic conditions, indicating low dark cytotoxicity. Furthermore, both compounds also exhibited low dark cytotoxicity toward normal BALB/c 3T3 cells, suggesting excellent biosafety (Figure S39). In contrast, upon light irradiation, the viabilities of 4T1, MDA‐MB‐231, and MCF‐7 cells significantly decreased, demonstrating potent PDT efficacy (Figure S36–S38). Taking 4T1 cells as an example, the light‐induced IC₅₀ values of TTz‐Ir under normoxia and hypoxia were determined to be 4.2 µM and 9.8 µM, respectively. Under the same conditions, the (IC₅₀)_Light values of TBTz‐Ir were 15.9 µM and 22.5 µM, respectively. Protoporphyrin IX (PPIX) was used as a control, with (IC₅₀)_Dark values of 25.5 µM (normoxia) and 34.6 µM (hypoxia), and (IC₅₀)_Light values of 5.6 µM and 8.9 µM, respectively (Figure S40). Another control, 5‐aminolevulinic acid (5‐ALA), also showed low cytotoxicity under the same drug incubation time (Figure S41). Collectively, these results indicate that TTz‐Ir exhibits higher photo‐cytotoxicity than TBTz‐Ir under both normoxic and hypoxic conditions.

FIGURE 5.

Generation of cellular ROS under irradiation, photo‐cytotoxicity, and antitumor effect of the TTz‐Ir in vitro. The viabilities of 4T1 cells treated with different concentrations of (a) TBTz‐Ir or (b) TTz‐Ir in the dark or upon light irradiation under normoxia. The data are shown as mean ± SD (n = 3). (c) ICP‐MS quantification of Ir content in whole cells, mitochondria, and lysosomes of 4T1 cells. Cells were first uniformly divided into three parallel aliquots, then incubated with TTz‐Ir or TBTz‐Ir (10 µM) for 4 h. After incubation, whole cells were collected from one aliquot, while mitochondria and lysosomes were isolated from the other two aliquots using different commercial organelle extraction kits. The data are shown as mean ± SD (n = 3). (d) Confocal microscopy images of ¹O₂ detected by SOSG (10 µM) and •OH detected by HPF (10 µM) in 4T1 cells treated with TBTz‐Ir and TTz‐Ir (10 µM). SOSG: λ_ex = 488 nm, λ _em = 530 ± 30 nm. HPF: λ _ex = 488 nm, λ _em = 550 ± 50 nm. Scale bar = 20 µm. (e) Colony formation assays of A549 (upper) and 4T1 (lower) cells treated with different concentrations of TTz‐Ir under light conditions. (f) Transwell assay of A549 cells treated with different concentrations of TTz‐Ir for 72 h under light conditions. (g) Wound healing assay images of A549 cells treated with different concentrations of TTz‐Ir under light conditions. (h) Immunoblot analysis of the indicated proteins in A549 (upper) and 4T1 (lower) cells treated with different concentrations of TTz‐Ir under light conditions. The uncropped original image of this blot is available in the Supporting Information. (i) Confocal microscopy images of the living cells treated with TTz‐Ir (10 µM, 4 h), co‐stained with DCFH‐DA (10 µM, 30 min) and Mito‐Tracker Deep Red (MTDR, 200 nM, 30 min). DCFH‐DA: λ _ex = 488 nm, λ _em = 530 ± 30 nm; MTDR: λ _ex = 633 nm, λ _em = 670 ± 30 nm; Scale bar = 20 µm. Incubation time: 4 h. Light irradiation: 550 nm, 13.2 mW cm⁻², 0.5 h.

TABLE 1.

IC_{50, Dark} and IC_{50, Light} (µM) values of TBTz‐Ir and TTz‐Ir toward different cell lines.

Cell lines		TBTz‐Ir			TTz‐Ir
		Dark a	Light b	PI c	Dark a	Light b	PI c
4T1	Normoxia	> 50	15.9 ± 0.06	> 3.1	> 50	4.2 ± 0.05	> 11.9
	Hypoxia	> 50	22.5 ± 0.04	> 2.2	> 50	9.8 ± 0.01	> 5.1
MDA‐MB‐231	Normoxia	> 50	17.8 ± 0.04	> 2.8	> 50	6.1 ± 0.03	> 8.2
	Hypoxia	> 50	22.4 ± 0.05	> 2.2	> 50	15.7 ± 0.18	> 3.2
MCF‐7	Normoxia	> 50	25.6 ± 0.12	> 2.0	> 50	2.5 ± 0.01	> 20.0
	Hypoxia	> 50	30.0 ± 0.02	> 1.7	> 50	7.1 ± 0.07	> 7.0
BALB/c 3T3	Normoxia	> 50	N.D.	—	> 50	N.D.	—

^a 4 h drug exposure in the dark.

^b 4 h drug exposure, then removed the medium and added fresh medium, followed by light irradiation for 0.5 h (TBTz‐Ir and TTz‐Ir: 550 nm, 13.2 mW cm⁻²) and then 44 h incubation in the dark.

^c The photo index (PI) is defined as IC_{50, Dark}/IC_{50, Light}. N.D. = not detection.

We speculated that the lower cytotoxicity of TBTz‐Ir compared to TTz‐Ir could be partially attributed to its lower solubility and weaker cellular uptake ability as well as lower •OH production. To investigate this, first, in solution, we experimentally calculated the lipid–water partition coefficient (Table S7). The log P value of TTz‐Ir was higher compared to that of TBTz‐Ir, indicating that TTz‐Ir has better drug absorption and can more easily cross the cell membrane to enter cells. Then, inductively coupled plasma mass spectrometry (ICP‐MS) was used to measure the cellular uptake of TTz‐Ir and TBTz‐Ir by 4T1 cells (Figure 5c). After incubating 4T1 cells with TTz‐Ir or TBTz‐Ir (10 µM) for 4 h, TTz‐Ir exhibited much higher cellular uptake than TBTz‐Ir (7.8 vs. 4.7 µg/10⁸ cells). The results confirmed that the cellular uptake ability of 4T1 cells for TTz‐Ir was much stronger than that for TBTz‐Ir.

The intracellular distribution of TBTz‐Ir and TTz‐Ir was evaluated by co‐staining with commercial mitochondrial dye Mito‐Tracker Green (MTG) and lysosomal dye Lyso‐Tracker Green (LTG). As shown in Figures S42–S43, after 4 h of co‐incubation, both complexes predominantly localized to mitochondria with minimal lysosomal co‐localization, demonstrating excellent mitochondrial targeting. Notably, TTz‐Ir showed stronger and more continuous mitochondrial co‐localization than TBTz‐Ir, suggesting greater mitochondrial accumulation, likely due to its enhanced cellular uptake. To quantitatively validate this, organelle‐specific ICP‐MS was performed (Figure 5c). Both complexes predominantly accumulated in mitochondria (TTz‐Ir: 6.1; TBTz‐Ir: 3.1 µg/10⁸ cells) with only minor lysosomal distribution (TTz‐Ir: 1.3; TBTz‐Ir: 0.9 µg/10⁸ cells). These quantitative data confirm the results observed by confocal microscopy.

To verify the (photo‐)toxic effects of the two complexes on 4T1 cells, we used calcein acetoxymethyl ester (calcein‐AM) and propidium iodide (PI) staining to distinguish between living cells (green) and dead cells (red). As shown in Figure S44, the control groups exhibited strong green fluorescence (indicating living cells) with no red fluorescence (indicating dead cells) under both normoxic and hypoxic conditions. In contrast, TTz‐Ir + Light group showed significant red fluorescence, indicating that TTz‐Ir with light irradiation can cause a large number of cell deaths. However, TBTz‐Ir + Light group showed only a small amount of red fluorescence, indicating that TTz‐Ir showed the better photocytotoxicity to cancer cells than TBTz‐Ir.

We further investigated the phototoxic mechanisms of TTz‐Ir and TBTz‐Ir on 4T1 cells under both normoxic and hypoxic conditions. To monitor the production of total ROS, ¹O₂ and •OH in 4T1 cells, 2,7‐dichlorodihydrofluorescein diacetate (DCFH‐DA), singlet oxygen sensor green (SOSG), and hydroxyphenyl fluorescein (HPF) were used as probes, respectively (Figures 5d and S45) [62]. Under normoxic conditions, the TTz‐Ir + Light and TBTz‐Ir + Light groups exhibited bright green fluorescence for all three probes (DCFH‐DA, SOSG and HPF), while no significant fluorescence was observed in the control groups. This indicated that both TTz‐Ir and TBTz‐Ir efficiently generate ¹O₂ and •OH in 4T1 cells upon light irradiation. However, under hypoxic conditions, the green fluorescence signals associated with SOSG in the TTz‐Ir + Light and TBTz‐Ir + Light groups were notably weaker, suggesting a significant reduction in ¹O₂ generation in the absence of O₂. In contrast, the fluorescence signals associated with DCFH‐DA and HPF in the TTz‐Ir + Light group showed little variation compared to normoxic conditions, indicating that the generation of •OH by TTz‐Ir is less dependent on O₂. Notably, the green fluorescence signals for all three probes in the TTz‐Ir + Light group were stronger than those in the TBTz‐Ir + Light group under both normoxic and hypoxic conditions (Figure S45). This suggests that TTz‐Ir has a superior ability to generate intracellular ROS, likely due to its higher solubility and stronger cellular uptake ability.

These results described above demonstrate that the rationally designed TTz‐Ir exhibits superior photosensitizing capability and phototoxicity. Therefore, we selected TTz‐Ir as the representative compound for subsequent experiments. Colony formation assays showed that TTz‐Ir + Light effectively inhibited colony formation in both A549 and 4T1 cells in a dose‐dependent manner (Figure 5e). We further evaluated the effects of TTz‐Ir + Light on invasion and migration using transwell and wound healing assays. The results demonstrated that TTz‐Ir + Light not only significantly inhibited A549 cell invasion but also dose‐dependently suppressed A549 cell migration (Figure 5f,g). Additionally, we assessed the expression levels of apoptosis‐related proteins in A549 and 4T1 cells (Figure 5h and S46). The results indicated that these proteins were upregulated to varying degrees, further supporting the potential of TTz‐Ir + Light to inhibit tumor growth, invasion and promoting tumor cell apoptosis.

2.7. Intracellular PDT‐Induced Activation of cGAS‐STING Pathway

Studies have shown that PDT can generate ROS that induce endoplasmic reticulum stress and danger signal release, triggering the immunogenic cell death (ICD) and thus activating the associated antitumor immune response [63, 64]. The above experiments proved that TTz‐Ir can induce the production of a large amount of ROS through light irradiation at 550 nm. Due to the good localization of TTz‐Ir in mitochondria, we speculated that TTz‐Ir + Light could generate a large amount of ROS in mitochondria. We used DCFH‐DA probe to detect ROS and Mito tracker deep red (MTDR) probe to detect mitochondria (Figure 5i). The results showed that TTz‐Ir + Light group generated a large amount of mitochondrial ROS (mtROS), indicating that the green fluorescent ROS and red fluorescent mitochondria in the TTz‐Ir + Light group were significantly co‐localized. Therefore, illumination of TTz‐Ir generated mtROS, causing mitochondrial oxidative stress and release of mtDNA into the cytoplasm.

ROSs are capable of inducing elevated levels of intracellular oxidative stress, thereby mediating cellular stress responses such as endoplasmic reticulum stress and autophagy, which are critical early steps in the development of ICD. ICD is linked to several biochemical markers, including the exposure of calreticulin (CRT) on the surface of dying cells, and the secretion of large amounts of adenosine triphosphate (ATP). Accordingly, we first assessed whether TTz‐Ir promotes ATP release, as reflected by increased ATP levels in the surrounding medium. A chemiluminescence‐based kit was used to quantify ATP in the cell culture supernatants across different treatment conditions (Figure S47). The results indicated that ATP release from 4T1 cells significantly increased by 5.5‐fold following treatment with TTz‐Ir and 550 nm light irradiation. As illustrated in Figure S48, after treatment with TTz‐Ir and 550 nm light irradiation, the green signal indicating CRT expression on the cell surface was confirmed by IF staining.

During the production of large amounts of ROS and ICD, intracellular DNA is released from organelles such as the nucleus or mitochondria into the cytoplasm [65]. The released DNA activates the cGAS‐STING pathway, which in turn induces immune response and promotes antitumor immunity (Figure 6a). To investigate whether TTz‐Ir + Light can activate the cGAS‐STING pathway, we examined 𝛾H2AX, a DNA damage marker, in 4T1 and A549 cells by Western blot [66, 67]. The results showed that TTz‐Ir + Light significantly increased 𝛾H2AX expression (Figures 6b and S49–S51). Immunofluorescence (IF) staining also revealed that TTz‐Ir + Light enhanced 𝛾H2AX fluorescence compared to other treatment groups (Figure 6c). To further confirm the activation of the cGAS‐STING pathway, we analyzed the expression of the key indicators of the cGAS‐STING pathway activation in cells by Western blot. The results showed that TTz‐Ir + Light significantly upregulated phospho‐TBK1 (p‐TBK1) expression in both A549 and 4T1 cells (Figures 6d, S50, and S52). IF staining further validated the upregulation of p‐TBK1 and p‐IRF3 expression (Figures 6e and S53). Moreover, RT‐qPCR analysis revealed that TTz‐Ir + Light treatment led to the highest levels of IFN‐𝛽, CCL5, CXCL9, and CXCL10 expression (Figures 6f and S54), indicating the release of these IFNs and chemokines following cGAS‐STING pathway activation.

FIGURE 6.

Intracellular PDT‐induced cGAS‐STING pathway. (a) Schematic illustration of the cGAS‐STING pathway. (b) Immunoblot analysis of the γH2AX in 4T1 cells treated with TTz‐Ir (10 µM) for 48 h. The uncropped original image of this blot is available in the Supporting Information. (c) Fluorescence images of γH2AX in 4T1 cells treated with TTz‐Ir (10 µM) for 48 h. γH2AX: λ _ex = 488 nm; λ _em = 530 ± 30 nm. DAPI: λ _ex = 405 nm, λ _em = 430 ± 20 nm. Scale bar = 20 µm. (d) Immunoblot analysis of the indicated proteins in 4T1 cells treated with TTz‐Ir (10 µM) for 48 h. The uncropped original image of this blot is available in the Supporting Information. (e) Fluorescence images of p‐TBK1 in 4T1 cells treated with TTz‐Ir (10 µM) for 48 h. p‐TBK1: λ _ex = 488 nm; λ _em = 530 ± 30 nm. DAPI: λ _ex = 405 nm, λ _em = 430 ± 20 nm. Scale bar = 20 µm. (f) RT‐qPCR of Ifnb1, Cxcl9, Cxcl10, and Ccl5 levels in 4T1 cells treated with TTz‐Ir (10 µM) for 48 h. Light irradiation: 550 nm, 13.2 mW cm⁻², 0.5 h. The data are shown as mean ± SD (n = 3). p‐values are calculated using unpaired Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001.

In conclusion, TTz‐Ir combined with light induces mitochondrial and nuclear DNA damage, resulting in cytosolic dsDNA accumulation and activation of the cGAS‐STING pathway, which may subsequently elicit a robust systemic antitumor immune response and contribute to a favorable therapeutic outcome.

2.8. In Vivo Phototherapeutic Efficacy

To enhance tumor targeting capability, TTz‐Ir molecules were encapsulated using the amphiphilic polymer DSPE‐PEG₂₀₀₀‐Biotin as a coating matrix, thereby promoting their accumulation in tumor sites during in vivo experiments (Figure S55a). This approach leverages a dual‐mechanism strategy for tumor targeting: first, the polymer‐encapsulated nanoparticles are delivered to the tumor vasculature via circulation; second, biotin (vitamin B7), an essential micronutrient, is actively taken up by cells through the sodium‐dependent multivitamin transporter (SMVT), which is overexpressed in many tumor types compared to normal tissues [68, 69]. The resulting nanoparticles, designated as TTz‐Ir NPs, were characterized by DLS, showing a hydrodynamic diameter of 125.6 nm (Figure S55b). Transmission electron microscopy (TEM) further confirmed that the NPs possess a uniform spherical morphology with an average diameter of approximately 90 nm (Figure S55b). In addition, the zeta potential of TTz‐Ir NPs was determined to be −29.3 mV (Figure S56). As shown by the DLS results and UV‐vis absorption spectra, no significant changes in particle size or absorption characteristics were observed after 7 days of storage in pure water at room temperature (Figure S57). These results indicate that TTz‐Ir NPs possess excellent colloidal stability. The cytotoxicity of the nanoparticles was evaluated using the MTT assay. As shown in Figure S58 and Table S8, the empty DSPE‐PEG₂₀₀₀‐Biotin carrier showed no cytotoxicity (IC₅₀ > 50 µM), confirming its biological inertness. In contrast, TTz‐Ir NPs exhibited potent light‐triggered cytotoxicity, with an IC₅₀ of 5.0 µM under light irradiation versus > 50 µM in the dark. For comparison, cisplatin showed an IC₅₀ of 6.4 µM under dark conditions. These results demonstrate that TTz‐Ir NPs are efficiently taken up by 4T1 cells within 4 h and exhibit superior light‐controllable cytotoxicity with a high phototoxicity index, offering a safer and more spatiotemporally controllable therapeutic strategy compared to cisplatin. To further support the selection of the injection‐to‐irradiation interval, an in vivo biodistribution study was conducted. As shown in Figure S59, mice were intravenously injected with free TTz‐Ir or TTz‐Ir NPs, and Ir content in tumors and major organs was measured by ICP‐MS at 1, 4, and 8 h post‐injection. Encapsulation of TTz‐Ir with DSPE‐PEG₂₀₀₀‐Biotin significantly enhanced tumor accumulation compared to free TTz‐Ir. For TTz‐Ir NPs, no significant difference in tumor enrichment was observed between the 4 h and 8 h time points, indicating that 4 h is sufficient for optimal tumor targeting. Therefore, a 4 h post‐injection interval was selected for TTz‐Ir NPs before light irradiation therapy.

To assess the therapeutic efficacy of TTz‐Ir in vivo, a mouse model bearing 4T1 tumors was established using female BALB/c mice. The mice were randomly assigned to four groups (control, TTz‐Ir NPs, light, and TTz‐Ir NPs + light). One week after subcutaneous inoculation of 4T1 cells into BALB/c mice, TTz‐Ir NPs were administered via tail vein injection, followed by irradiation with a 550 nm light array for 30 min. The detailed drug administration protocol is shown in Figure 7a. These results demonstrated that tumor growth in the TTz‐Ir NPs + light group was significantly inhibited (Figure 7b–d). Furthermore, no significant changes in body weight were observed (Figure 7e), suggesting that systemic toxicity was negligible at the tested dose. The hematoxylin and eosin (H&E) staining of major organs from all treatment groups did not reveal any severe structural or pathological changes (Figure S60).

FIGURE 7.

In vivo phototherapeutic efficacy of TTz‐Ir NPs. (a) Schematic illustrating the administration of TTz‐Ir NPs (1.0 mg/kg) for tumor treatment in 4T1 tumor‐bearing BALB/c mice. b) Photographs of tumor tissues at the end of treatment. (c), (d) Tumor volumes during treatment and tumor weight of mice at the end of treatment. The data are shown as mean ± SD (n = 6). p‐values are calculated using unpaired Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001. (e) The weight of mice during treatment. The data are shown as mean ± SD (n = 6). (f) Microscopy images of H&E and TUNEL stained tumor sections. Scale bar = 100 µm. (g) Quantitative analysis of CD8⁺ T cells in 4T1 tumors. The data are shown as mean ± SD (n = 3). P‐values are calculated using unpaired Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001.

These findings demonstrated that TTz‐Ir NPs exhibited good in vivo biosafety. Moreover, TUNEL staining indicated significant DNA damage in the tumors of mice treated with TTz‐Ir NPs + light (Figure 7f). Enzyme‐linked immunosorbent assay (ELISA) revealed that TTz‐Ir NPs + light treatment significantly increased the levels of TNF‐α, IFN‐γ, and IL‐10 in the peripheral blood of mice (Figure S61). Immunohistochemistry (IHC) results also showed an increased infiltration rate of CD8⁺ T cells in the tumors of the TTz‐Ir NPs + light group (Figures 7g and S62). These results indicated that TTz‐Ir NPs + light induced a robust cancer immune response in vivo.

3. Conclusion

In summary, we have developed a counterintuitive “π‐bridge trimming” strategy for designing high‐performance Ir(III) photosensitizers. Through this innovative method, we synthesized two fused‐ring Ir(III) complexes, TTz‐Ir and TBTz‐Ir, with three‐ring and five‐ring fused π‐bridges, respectively. Strikingly, the smaller and “trimmed” TTz‐Ir outperformed its extended analog, TBTz‐Ir, in multiple key aspects critical for PDT, including superior molar absorptivity, enhanced solubility, improved photocatalytic activity, and significantly greater photocytotoxicity. Mechanistic studies revealed that the superior performance of TTz‐Ir stems from its longer triplet‐state lifetime, more efficient photo‐induced charge separation and transport favoring type I ROS generation, and enhanced cellular uptake. Upon light irradiation, TTz‐Ir not only produces ¹O₂ via energy transfer, but also efficiently generates type I ROS such as O₂ ^•−, H₂O₂, and •OH, primarily through ORR and WOR pathways, ensuring robust photocytotoxicity even under hypoxic conditions. At the cellular level, TTz‐Ir preferentially localizes in mitochondria and lysosomes. Light‐induced ROS production disrupts cellular redox homeostasis, leading to oxidative damage to both mitochondrial and nuclear DNA. The resulting damaged DNA is recognized by cGAS, which in turn activates the cGAS‐STING signaling pathway. This activation ultimately initiates antitumor immune responses. To increase tumor targeting ability and enrichment, DSPE‐PEG₂₀₀₀‐Biotin was used to encapsulate TTz‐Ir molecules in vivo experiments. The resulting TTz‐Ir NPs had good biocompatibility and accumulated effectively at the tumor site and activated antitumor immune responses upon light irradiation, effectively inhibiting tumor growth. This work introduces a novel “π‐bridge trimming” design principle for photosensitizers. By moderately shortening the conjugation length, this strategy crucially improves solubility, cellular uptake, and charge separation efficiency, leading to enhanced hypoxia tolerance. This simple yet effective molecular engineering approach not only facilitates the development of phototheranostic agents for the hypoxic tumor microenvironment but also yields a potent iridium(III)‐based photosensitizer for photo‐immunotherapy.

Author Contributions

D. Li, F. Chen, B.‐Z. Chu, H.‐Y. Huang, and P.‐Y. Zhang designed the study. D. Li, F. Chen, Y. Tu, J.‐Y. Jiang, Y.‐J. Zhu, S. Feng, and X. Lin synthesized and characterized the complexes. D.‐L. Wang completed the theoretical calculation of the complexes. T. Jin, O. S. Wenger measured and discussed the transient absorption spectroscopy. F. Chen, Y. Tu, and G. Q. Wen performed the experiments on mice. All authors analyzed the data and wrote the paper.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File:Additional experimental details, materials, and methods, including crystallographic data, NMR, and mass spectra of the complexes, absorption/emission and HPLC spectra, and calculation data, etc. CCDC 2426715, 2426716, 2426717.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.