【Angew.Chem.】深圳大学张平玉等|仅需3个环！一种攻克肿瘤低氧耐受、激活cGAS-STING免疫通路的光敏剂设计新策略#

Sccheme 1. Schematic illustration of (a) π-bridge trimming strategy for fused-ring iridium(III) photosensitizers, (b) photoactive mechanisms, and (c) cGAS-STING pathway activation.#

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.#

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.#

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.#

在细胞和体内实验中，TTz-Ir表现出优异的线粒体靶向性。光照激发的活性氧引起了严重的线粒体及核DNA损伤，促使DNA释放至细胞质中，从而强烈激活了cGAS-STING信号通路，显著上调了p-TBK1和p-IRF3的表达，并诱导一系列干扰素及趋化因子释放。为了提升体内靶向效率，研究者利用两亲性聚合物将其包裹为TTz-Ir纳米颗粒。小鼠模型实验证实，该纳米药物能在肿瘤部位高效富集，并在光照下引发强烈的全身性抗肿瘤免疫应答，显著增加肿瘤内CD8+ T细胞的浸润，最终实现对肿瘤生长的高效抑制。

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.#

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: λex = 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.#

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.#

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.#