Enhanced ROS generation in AIE-active iridium(III) photosensitizers by cationization engineering for advanced photodynamic therapy

Abstract

Phosphorescent iridium(III) complexes have emerged as promising photosensitizers (PSs) for clinical photodynamic therapy (PDT) due to their notable antitumor efficacy. However, their practical application is hindered by weak emission in aggregated states and insufficient reactive oxygen species (ROS) generation. In this study, we present a straightforward cationization strategy aimed at simultaneously enhancing both the emission and ROS production of Ir(III)-based PSs. Two Ir(III) complexes PPI-C1 and PPI-C2 which feature an incremental number of hexafluorophosphate counterions were strategically designed through simple ligand engineering of the neutral precursor PPI-C0. Both experimental and theoretical analyses reveal that cationization effectively modulates the aggregate behavior and excited-state properties of these complexes, with PPI-C2 displaying a significantly improved AIE characteristic and effective intersystem crossing ability. As expected, the water-soluble PPI-C2 nanoparticles showed superior ROS production and good biocompatibility under light irradiation, leading to cell apoptosis and significant inhibition of tumor growth in vivo. This study will offer new insights into the design of effective AIE-active Ir(III)-based photosensitizers for PDT.

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Introduction

Photodynamic therapy (PDT), as an emerging phototherapy for cancer, can minimize the side effects on normal tissues through the simple operation of local delivery of photosensitizers (PSs) and appropriate light irradiation.^1–5 Compared with traditional chemotherapy and radiotherapy, PDT has received extensive attention due to its higher selectivity and fewer side effects.^6,7 Upon irradiation by an external light source, PSs in triplet states produce highly toxic reactive oxygen species (ROS) via electron transfer or energy transfer pathways, effectively killing tumor cells and causing irreversible damage to tumor tissues.^8,9 However, designing PSs with suitable triplet states and long triplet lifetimes remains a challenge.¹⁰ In recent years, the rapid development of luminescent transition metal complexes in biomedical fields has brought phosphorescent Ir(III) complexes to the forefront,^11–14 given their large Stokes shifts, long excited state lifetimes, and excellent photo-stability. These complexes have shown highly efficient anticancer effects,^15–19 operating through unique mechanisms such as catalyzing cellular redox reactions,^20,21 inhibiting angiogenesis,²² and targeting subcellular organelles such as mitochondria¹⁰ and lysosomes.^23,24 Despite their potential, Ir(III) complexes often face issues of high cytotoxicity and poor solubility in aqueous media.²⁵ Significant efforts, such as the encapsulation of water-soluble nanoparticles (NPs) with amphiphilic polymers and liposomes through nanoprecipitation and self-assembly, have been made to improve their solubility and biosafety.^26,27 However, aggregation-induced quenching (ACQ) can occur when PSs aggregate in tumor cells, leading to a reduction in triplet state generation and decreased ROS production, severely hampering their practical application in PDT.²⁸

Fortunately, photosensitizers with aggregation-induced emission (AIE) properties can effectively address this problem.^29,30 Unlike conventional PSs, AIE-active PSs exhibit brighter emission behavior in aggregated states.^31–34 Although some AIE-active Ir(III) complex PSs have been developed, most still suffer from weak ROS production due to a lack of strategy to enhance AIE activity and ROS generation ability. Traditionally, enhancing ROS generation in PSs involves constructing specific configurations such as donor–acceptor (D–A) and donor–π–acceptor (D–π–A) systems through precise molecular design and conjugation.^35,36 While these methods are effective, their synthesis processes are often costly and time-consuming. Therefore, there is a high demand for a simple approach to construct an AIE-active Ir(III) complex with highly efficient ROS generation.

Efficient intersystem crossing (ISC) is crucial for improving ROS production. Increasing ISC channels and/or reducing the energy gap (ΔE_ST) between singlet and triplet states effectively accelerate the ISC process. Electrostatic interactions are known to influence the packing structure in the aggregated state and modify the excited-state characteristics of the dyes.³⁷ With this in mind, we report a facile cationization strategy that requires only a simple modification of the ligand structure to introduce different numbers of counterions in Ir(III) complexes to adjust their ROS production and emission. We hypothesized that this approach would adjust the molecular stacking pattern and strengthen electrostatic interactions caused by the newly introduced counterions, via the accepted mechanism of restriction of intramolecular motions (RIM) after aggregate formation, thus providing more opportunities to control the photophysical properties.^38–40 Furthermore, the inhibited non-radiative transition of the AIE-active PSs, caused by restricted molecular motions in aggregated states, can lead to enhanced emission and allow more energy to transfer from singlet to triplet states via the ISC process, thereby achieving more efficient ROS production.^41,42

To verify our hypothesis, two cationic complexes, PPI-C1 and PPI-C2, were strategically designed and synthesized through the cationization engineering of the neutral Ir(III) complex PPI-C0 (Scheme 1). Interestingly, their AIE properties and ROS generation abilities increased progressively with the number of hexafluorophosphate counterions. The results demonstrated that cationization effectively regulated the excited states of PSs by the introduction of counterions, reducing the ΔE_ST value and increasing ISC channels in PPI-C2, thereby promoting ROS production. Water-soluble PPI-C2 nanoparticles exhibited ideal biocompatibility and produced various types of ROS under white light irradiation, inducing cell apoptosis. Additionally, PPI-C2 NPs effectively inhibited tumor growth in 4T1 tumor-bearing BALB/c mice with negligible systemic toxicity. This work demonstrates a feasible cationization molecular engineering strategy for designing high-performance phosphorescent Ir(III) complexes for PDT.

Scheme 1 Schematic of the cationization strategy to obtain AIE-active phosphorescent cationic Ir(III) complexes, and the subsequent application of PPI-C2 NPs in in vivo antitumor photodynamic therapy.

Results and discussion

Molecular design and synthesis

Phosphorescent Ir(III) complexes characterized by their octahedral configuration are readily internalized by cells, and their photophysical properties can be finely tuned through structural modification. In this work, the [Ir(C^N)₂(N^O)] framework was selected as the basis for designing the neutral precursor of the phosphorescent Ir(III) complex. The cationization engineering involved replacing the N^O ancillary ligand with an N^N ancillary ligand and the N-methylation of the carboline unit on the cyclometalated ligand to introduce controllable counterions. Specifically, 5-(4-(1-phenyl-1H-benzo[d]imidazol-2-yl)phenyl)-5H-pyrido[3,2-b]indole (PPI) was employed as the cyclometalated ligand, and (λ¹-oxidaneyl)(quinolin-2-yl)methanone (OQM) as the N^O ancillary ligand to form the neutral complex PPI-C0. Herein, we chose OQM as the ancillary ligand since quinoline-containing systems are attractive scaffolds for the development of medicinal and industrial chemistry, which show great potential for various studies, such as in biomedical applications.^43–45 Cationization led to changes in intermolecular interactions and excited-state properties, resulting in an overall improvement in emission efficiency and ROS generation, as evidenced by experimental and theoretical data. Detailed synthesis and structural characterization of complexes PPI-C0, PPI-C1 and PPI-C2 are confirmed and provided in Scheme S1 and Fig. S1–S11 in the ESI.†

Optical properties and light-triggered ROS generation

The photophysical properties of Ir(III) complexes were first analyzed, as depicted in Fig. 1A. All complexes exhibited strong absorption below 325 nm, attributed to spin-allowed ligand-centered ¹π–π* transitions.⁴⁶ The absorption bands of PPI-C0 and PPI-C2 extend up to 465 nm, whereas that of PPI-C1 extends to 430 nm. These absorption bands are primarily attributed to a mixture of MLCT (metal-to-ligand charge transfer), LLCT (ligand-to-ligand charge transfer), and π–π* (LC) transitions.⁴⁷ The emission spectra of all complexes in their aggregated states, measured in a THF/H₂O system, are shown in Fig. 1B, displaying emission colors ranging from yellow to orange. The Ir(III) complexes exhibited significant Stokes shifts of approximately 187 nm for PPI-C0, 164 nm for PPI-C1 and 215 nm for PPI-C2 in THF solution, accompanied by phosphorescence lifetimes of 0.22 μs for PPI-C0, 0.14 μs for PPI-C1, and 0.20 μs for PPI-C2 in aggregated states. These characteristics are conducive to high-sensitivity imaging. The absolute photoluminescence quantum yields of PPI-C0, PPI-C1 and PPI-C2 were evaluated to be 4.3%, 43.2% and 1.3%, respectively. To further explore their aggregation-induced emission properties, emission spectra of PPI-C0, PPI-C1 and PPI-C2 were recorded in THF/H₂O mixtures with varying water fractions (f_w). As illustrated in Fig. 1C, D and Fig. S12,† the PL intensity of neutral PPI-C0 exhibited a decreasing trend in the aggregated state along with increasing water fraction, which indicated a typical ACQ phenomenon. In contrast, PPI-C1 and PPI-C2 showed AIE characteristics with enhanced PL intensity in aggregated states compared with THF solution. Hence, we hypothesized that cationization effectively suppresses molecular motions through the RIM mechanism, thereby enhancing AIE properties and resulting in brighter emission in the aggregated state. To comprehensively examine the effect of cationization on excited states, density functional theory (DFT) calculations were performed, as shown in Fig. 1E and F. The highest occupied molecular orbital (HOMO) of PPI-C0 is predominantly localized on the central Ir(III) atom and the cyclometalated ligand, while the lowest unoccupied molecular orbital (LUMO) is primarily associated with the ancillary ligand. In PPI-C1, the electron cloud distribution of the LUMO and HOMO remains similar to that of PPI-C0. PPI-C2 shows similar HOMO distributions to PPI-C0 but with significant changes in the LUMO, which is mainly localized in the cyclometalated ligand. Time-dependent DFT (TD-DFT) results suggest that the lowest triplet state (T₁) of the complexes originates from the excitation of the HOMO → LUMO, with contributions from both metal-to-ligand charge-transfer (³MLCT) and ligand-to-ligand charge transfer (³LLCT) characters (Table S1†). The T₁ states of all complexes were calculated using the spin-unrestricted UB3LYP method and are shown in Fig. 1F. After full-geometry relaxation, the predicted T₁ spin density showed similar distributions of components [Ir: 0.62, C^N ligands: 0.27, N^O ligand: 1.11 for PPI-C0; Ir: 0.43, C^N ligands: 0.56, N^N ligand: 1.01 for PPI-C1; Ir: 0.64, C^N ligands: 0.22, N^O ligand: 1.14 for PPI-C2], which confirmed their mixed charge transfer (CT) characters of the lowest excited-states. The CT nature of their emitting triplet states is in agreement with their emission profiles. Additionally, the calculated HOMO–LUMO energy gaps were 3.36 eV for PPI-C0, 3.35 eV for PPI-C1, and 3.10 eV for PPI-C2. The reduced HOMO–LUMO energy gap in PPI-C1 and PPI-C2 compared to that of neutral PPI-C0 indicates that cationization significantly influences the excited state properties, potentially enhancing photosensitization capabilities.

Fig. 1 (A) Normalized absorption of PPI-C0, PPI-C1 and PPI-C2 in THF. (B) Normalized PL spectra of PPI-C0, PPI-C1 and PPI-C2 in THF/H₂O mixtures (f_w = 90%); λ_ex = 415 nm for PPI-C0 and PPI-C1, and λ_ex = 375 nm for PPI-C2. (C) PL spectra of PPI-C2 in THF/H₂O mixtures with different f_w. (D) PL intensity (I/I₀) of PPI-C0, PPI-C1 and PPI-C2 in THF/H₂O mixtures (f_w = 0% and 90%). Concentration: 5 × 10⁻⁵ M. (E) Calculated HOMO and LUMO for PPI-C0, PPI-C1 and PPI-C2. (F) The spin density distributions of PPI-C0, PPI-C1 and PPI-C2 in T₁ states.

2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCF-DA), a sensitive green-emission indicator for total ROS generation, was utilized to assess their ROS generation efficiency. As shown in Fig. 2A and S13,†PPI-C0, PPI-C1 and PPI-C2 all demonstrated notable ROS generation compared to the blank control group (DCFH + Light), with PPI-C2 exhibiting superior ROS production and reaching a plateau after only 15 seconds of irradiation. To further identify the types of produced ROS, 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA), hydroxyphenyl fluorescein (HPF) and dihydrorhodamine 123 (DHR123) were used as indicators for ¹O₂, ˙OH and ˙O₂⁻, respectively. For type II ROS, the absorption peak of ABDA at 378 nm decreased significantly within 2.5 minutes under white light irradiation, indicating efficient ¹O₂ production. Among them, cationic PPI-C2 exhibited a markedly enhanced ¹O₂ generation capacity (Fig. 2B and S14†). Similar trends were also observed for type I ROS, ˙OH and ˙O₂⁻ (Fig. 2C, D and S15, 16†). Additionally, the generation of ˙O₂⁻ was confirmed using ascorbic acid (Vc) as an inhibitor. As shown in Fig. S16E,† the addition of Vc reduced the emission intensity of the DHR123 solution treated with three complexes under the same conditions, confirming effective free radical production. In addition, electron spin resonance (ESR) testing was carried out to further verify the ROS production of PPI-C2 with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) and 4-amino-2,2,6,6-tetramethylpiperidine (TEMP) as spin-trap agents, respectively.⁴⁸ As shown in Fig. 2E, significant ESR signals were detected after white light irradiation for 5 min, verifying the generation of ˙O₂⁻, ˙OH and ¹O₂, whereas no signal was captured in the control experiments. The results clearly indicate that cationic PPI-C2 exhibit superior type I and II ROS generation capabilities under light irradiation, validating the effectiveness of the cationization engineering strategy in enhancing the photosensitization ability of Ir(III) complexes. Moreover, to elucidate the intrinsic relationship between cationization and ROS production, the energy gaps between singlet and triplet states (ΔE_ST) were calculated. The values were evaluated to be 0.09 eV and 0.042 eV for PPI-C0 and PPI-C1, respectively, and 0.002 eV and 0.108 eV for PPI-C2, indicating more intersystem crossing (ISC) pathways (Fig. 2F–H). The calculated spin orbital coupling (SOC) constants (ξ) also demonstrated the effectiveness of these ISC channels. The reduced ΔE_ST values and increased ISC channels are advantageous for ROS production, contributing to higher PDT performance.⁴⁹

Fig. 2 Time-course plot of (A) DCFH PL intensity enhancement; (B) ABDA decomposition; (C) HPF PL intensity enhancement and (D) DHR123 PL intensity enhancement in the presence of PPI-C0, PPI-C1 and PPI-C2, respectively, under white-light (400–700 nm, 100 mW cm⁻²) irradiation. Concentration: complexes (2 μM), DCFH-DA (5 μM), ABDA (20 μM), DHR123 (5 μM), and HPF (5 μM). (E) ESR signals of BMPO and TEMP in the presence of PPI-C2 after white-light (400–700 nm, 100 mW cm⁻²) irradiation for 5 min. Concentration: complexes (1 mM), BMPO (100 mM), and TEMP (100 mM). (F and G) Calculated energy gap between S_n and T_n for PPI-C0, PPI-C1 and PPI-C2 from TD-DFT.

To further improve their biocompatibility, nanoparticles (NPs) with good stability were fabricated by encapsulating the Ir(III) complexes in an amphiphilic polymer, DSPE-PEG₂₀₀₀. Dynamic light scattering (DLS) measurements revealed that the average hydrodynamic diameters of the NPs in water were 136.1 nm for PPI-C0, 105.6 nm for PPI-C1, and 116.5 nm for PPI-C2, respectively. Moreover, the emission properties of PPI-C1 and PPI-C2 NPs did not undergo quenching, and the corresponding PL intensity was higher than that of aggregated states under the same concentration (Fig. S18†). Transmission electron microscopy (TEM) imaging confirmed that the NPs exhibited a well-dispersed and spherical morphology, as shown in Fig. S17.† The appropriate size distribution facilitated the efficient internalization of NPs by cells, enabling effective tumor cell destruction via photodynamic therapy (PDT). Hence, ROS production of PPI-C0, PPI-C1 and PPI-C2 NPs was then investigated using the same indicators in aqueous solutions with satisfactory results (Fig. S19–22†).

In vitro PDT performance under white light irradiation and cell imaging

To evaluate the photodynamic therapy performance of the complexes at the cellular level, HeLa cells were incubated with the NPs for 8 hours, followed by storage in darkness or irradiation with white light for 15 minutes. The cytotoxicity of the three water-soluble NPs under different treatments was assessed using a CCK-8 assay. As shown in Fig. 3A and S23,† all NPs demonstrated excellent biocompatibility with negligible dark toxicity. Upon white light irradiation, neutral PPI-C0 exhibited weak ROS generation capability, resulting in minimal cell killing even at a high concentration of 100 μg mL⁻¹. In contrast, cationic PPI-C1 and PPI-C2 NPs showed significant phototoxicity, with the cell survival rate reduced to approximately 20% at the concentrations of 5 μg mL⁻¹ and 100 μg mL⁻¹, respectively. To further visualize PDT effectiveness, live/dead cell staining assays were performed using fluorescein diacetate/propidium iodide (FDA/PI) as indicators to differentiate dead and viable cells.⁵⁰ As illustrated in Fig. S24,† bright red fluorescence was observed in “PPI-C2 NPs + L” groups, indicating the significant cell death caused by the PDT effect. In contrast, no obvious cell death was observed in PBS or PPI-C0 NP treated groups after light irradiation, consistent with the CCK-8 assay results. Additionally, apoptosis analysis was performed by the fluorescence-activated cell sorting (FACS) technique using an Annexin V-FITC/PI detection kit to further explore the mechanism of cell death.⁵¹ The results showed that approximately 69.3% of cancer cells were induced to undergo apoptosis in the “PPI-C2 NPs + L” NP group, whereas apoptosis or necrosis was not obvious in the control groups and neutral “PPI-C0 NPs + L” group (Fig. 3D and S25†). These results perfectly confirm the excellent PDT efficacy of the cationization strategy. Furthermore, the limited action region (20–40 nm) and short lifetime (<40 ns) of ROS in practical therapy applications has limited the therapeutic effect.⁵² Organelle-targeting was considered as a desirable strategy to solve the drawbacks. Given the excellent emission properties in the aggregated state and suitable particle size distribution, the NPs were incubated with HeLa cells and co-stained with commercial fluorescent probes Mito-tracker Green and Lyso-Tracker Green to investigate their organelle-targeting behaviors via confocal laser scanning microscopy (CLSM). As shown in Fig. 3C and S26,†PPI-C0 and PPI-C2 NPs were predominantly localized in lysosomes, with Pearson's correlation coefficients of 0.87 and 0.79, respectively. PPI-C1 NPs showed extensive distribution in mitochondria, with a Pearson's correlation coefficient of 0.95. Additionally, ROS production at the cellular level was also evaluated using DCFH, SOSG, HPF, and DHE as indicators. As shown in Fig. 3B and S27, 28,† all NPs showed different degrees of ROS production upon light irradiation compared to the control group of PBS or dark conditions. The neutral PPI-C0 NPs exhibited weak ROS production across all types of ROS. In contrast, cationic PPI-C1 and PPI-C2 NPs demonstrated excellent type I and II ROS generation abilities, consistent with the in vitro test results.

Fig. 3 (A) Viability of HeLa cells incubated with PPI-C2 NPs at different concentrations with/without white-light (400–700 nm, 100 mW cm⁻²) irradiation for 15 min. (B) Intracellular ROS generation by PPI-C2 NPs upon white light (400–700 nm, 100 mW cm⁻²) irradiation inside HeLa cells: DCFH-DA for total ROS and SOSG for ¹O₂, HPF for ˙OH, and DHE for ˙O₂⁻. Scale bar = 40 μm. Concentration: NPs (30 μg mL⁻¹), DCFH (10 μM), SOSG (10 μM), DHE (10 μM), and HPF (20 μM). (C) CLSM images of HeLa cells incubated with PPI-C2 NPs for 8 h, followed by co-staining with Lyso-tracker green for 5 min. Scale bar = 20 μm. Concentration: NPs (10 μg mL⁻¹) and Lyso-tracker green (25 nM). (D) Live/dead cell staining assays using FDA and PI as indicators treated with PPI-C2 NPs or PBS upon white light (400–700 nm, 100 mW cm⁻²) irradiation for 15 min. Concentration: NPs (100 μg mL⁻¹), FDA (2 μg mL⁻¹), and PI (2 μg mL⁻¹). Scale bar = 40 μm. (E) Apoptosis analysis of HeLa cells after different treatments with various formulations by FACS.

In vivo photodynamic therapy

Subsequently, benefiting from the superior photophysical and ROS generation capabilities, PPI-C2 NPs were utilized to evaluate their in vivo PDT antitumor effect on 4T1 tumor-bearing BALB/c mice. Prior to the experiment, biosafety assessments were performed and the results revealed no significant toxicity with hematological parameters remaining within the normal ranges (Fig. S29†). The mice were then randomly divided into four groups (“PBS”, “PBS + L”, “PPI-C2” and “PPI-C2 + L”) and treatments were started when the tumor volume reached approximately 100 mm³. Following intratumoral injection of either PPI-C2 NPs or PBS, tumors were irradiated with white light for 30 minutes. During the 14 days’ treatments, the tumor volume and body weight of the mice were recorded every 2 days (Fig. 4A). The relative growth curves are presented in Fig. 4B and C. Tumors in the three control groups exhibited rapid growth throughout the treatment period, whereas tumors in the “PPI-C2 + L” group showed significantly slower growth, indicating effective inhibition (Fig. 4D). H&E staining of tumor sections from the “PPI-C2 + L” group revealed pronounced nuclear loss and atrophy compared to the control groups, demonstrating a potent PDT effect (Fig. 4E). Furthermore, no abnormal changes in body weight were observed in mice from all groups during the treatment period, indicating negligible acute toxicity of PPI-C2 NPs for in vivo applications. The H&E-stained slices of the major organs, including the heart, liver, spleen, lungs, and kidneys, from four groups after treatment showed normal tissue morphology without any changes, which further demonstrated the excellent biosafety of PPI-C2 NPs for PDT applications.

Fig. 4 (A) Schematic of the time schedule for treatment. (B). Tumor growth curves of 4T1 tumor-bearing mice with various treatments after intratumoral injection of 100 μL PBS or PPI-C2 NPs (500 μg mL⁻¹). Irradiation was performed under 100 mW cm⁻² irradiation for 30 min. (C) Relative body weight change curves of mice during different treatments. (D) Photograph of tumors of each group harvested at day 14 with relevant treatments. (E) H&E staining analyses of major organs and tumor tissues in different groups after 14 days of treatment. Scale bar: 50 μm.

Conclusions

In summary, we have demonstrated a feasible cationization strategy for the rational design of a highly efficient AIE-active Ir(III) photosensitizer. This strategy effectively modulates the excited-state properties and intermolecular interactions, resulting in enhanced emission in aggregated states and a concurrent reduction in ΔE_ST, which facilitate improved intersystem crossing processes and significantly improve ROS generation capacity. The water-soluble cationic PPI-C2 NPs exhibited efficient cellular internalization and robust ROS production under light irradiation. In vivo experiments confirmed that PPI-C2 NPs show excellent biosafety and substantial tumor growth inhibition in 4T1 tumor-bearing BALB/c mice. This study presents a feasible approach for designing phosphorescent Ir(III) complexes with enhanced AIE activity and improved photosensitizing ability.

Author contributions

Shanshan Huang: data curation, formal analysis, validation, visualization, and writing – original draft. Yuancheng Li, Xiaohan Xie and Jialin Tong: data curation, formal analysis, investigation, software, validation, and visualization. Guo-Gang Shan, Chao Qin, Xiyan Xiao, Qianruo Wang, Yuanyuan Li and Hualei Wang: project administration, resources, supervision, and writing – review & editing.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the funding from the Natural Science Foundation of Jilin Province (No. 20220101191JC) and National Natural Science Foundation of China (No. 22175033).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4qi02477j

‡ These authors contributed equally to this work.

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