AIE-active iridium(III) complex integrated with upconversion nanoparticles for NIR-irradiated photodynamic therapy

Abstract

The integration of an aggregation induced emission (AIE)-active Ir(III) complex and upconversion nanoparticles (UCNPs) has achieved a NIR-irradiated photosensitizer (PS), UCNPs@Ir-2-N. This PS has satisfactory biocompatibility, excellent phototoxicity, good accumulation in cells and high ¹O₂ generation ability, thereby effectively killing 4T1 mouse cancer cells in vitro. This work has potential for future photodynamic therapy (PDT) applications.

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Photodynamic therapy (PDT) is an efficient anticancer treatment of particular interest.^1–3 In the PDT process light irradiates photosensitizers (PSs) to generate highly cytotoxic reactive oxygen species (ROS) causing the ablation of cancer cells.^4–6 Traditional PSs suffer from several major drawbacks:^7–10 (i) low intersystem crossing (ISC) ability; (ii) poor photostability; (iii) aggregation-caused quenching (ACQ) of emission; (iv) unsatisfactory ROS production efficiency. Moreover, the short-wavelength irradiation light results in shallow tissue penetration and photodamage to cells and tissue.^11–13 Hence, the clinical application of PDT requires a solution to these problems.

Tang and co-authors have recently reported that aggregation-induced emission (AIE) luminogens show excellent aggregation-induced ROS generation activity.¹⁴ However, how to make the most of accelerating the ISC process to boost ROS generation still remains a key challenge. Inspired by the above facts, a few AIE-active Ir(III) complexes with efficient ISC processes, long excited-state lifetime, excellent photostability and tunable ligand modification have been developed as substitutes to traditional materials for efficient PDT.^7,15–18

Nevertheless, the absorption bands of Ir(III) complexes are usually in the UV to visible range and not in the near-infrared (NIR) region,^19,20 which limits their therapeutic effect in deep tissue. Upconversion nanoparticles (UCNPs) are desirable photoconversion materials for biosensing and biomedicine on account of their capability of transforming the NIR photons to UV/visible photons.^11,13,21 Two reports have corroborated that PSs combining cationic Ir(III) complexes and UCNPs can overcome the problems caused by short wavelength irradiation in PDT.^22,23 Possibly owing to the lack of a reliable molecular design strategy or facile synthetic routes, to the best of our knowledge, there have not been any reports of PSs based on AIE-active Ir(III) complexes and UCNPs.

Herein, AIE-active non-charged Ir(III) complexes integrated with UCNPs are employed as effective PSs for PDT for the first time. Taking advantage of the tunable ligand structure of Ir(III) complexes,²⁴ a Schiff base was introduced as the ancillary N^O ligand due to its simple synthesis, high yield and proven coordination to achieve AIE-active Ir(III) complexes,^16,25 giving Ir-1-N (structure in ESI†) and Ir-2-N (Scheme 1). Compared with Ir-1-N, the absorption of Ir-2-N was extensively enhanced through extending the π-conjugation of the C^N ligands. Furthermore, Ir-2-N exhibits bright luminescence in the aggregated state and high singlet oxygen (¹O₂) generation ability. The UCNPs@Ir-2-N nanoparticles (NPs) were formulated by encapsulating the Ir-2-N and UCNPs within D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) to obtain better stability and biocompatibility. UCNPs@Ir-2-N is the main focus of the current study.

Scheme 1 Structure of Ir-2-N, preparation of UCNPs@Ir-2-N and schematic of UCNPs@Ir-2-N as a PS for PDT.

The synthetic routes to Ir-1-N and Ir-2-N are shown in the ESI.† Their structures were validated by proton nuclear magnetic resonance (¹H NMR) spectroscopy (Fig. S1–S6, ESI†), ¹³C NMR spectroscopy (Fig. S7 and S8, ESI†) and high-resolution mass spectrometry (HRMS) (Fig. S9 and S10, ESI†). The UCNPs (NaYF₄@NaF:Yb,Tm@NaYF₄) were prepared by the chloride solvothermal method as reported before²⁶ (see ESI†).

The photophysical properties of Ir-1-N and Ir-2-N were investigated (Fig. 1a and Fig. S11, Table S1, ESI†). In UV-vis absorption spectra, both of the Ir(III) complexes showed two absorption bands at 250–350 nm, corresponding to spin-allowed π–π* transitions at the ligand centers, and at 350–500 nm from metal-to-ligand charge transfer (¹MLCT), ligand-to-ligand charge transfer (¹LLCT), spin-forbidden metal-to-ligand charge-transfer (³MLCT) and spin-forbidden ligand-to-ligand charge-transfer (³LLCT).^19,27,28 The photoluminescence (PL) spectra show that Ir-1-N emits at λ_max 640 nm, and Ir-2-N at 660 nm. These results establish that the extended conjugation of the C^N ligands leads to a significant redshift in the PL of Ir-2-N and almost 1.5 times increase of the molar absorption coefficient of Ir-2-N relative to Ir-1-N. Moreover, these Ir(III) complexes are almost non-emissive in pure CH₃CN (Fig. 1b and Fig. S12, ESI†). On the contrary, the emission intensity of Ir-1-N and Ir-2-N was greatly enhanced when the water fraction reached respectively to 80% and 50% demonstrating their typical AIE feature. Ir-2-N showed a brighter luminescence indicating the effective restriction of nonradiative transition processes, as found in AIE luminogens.^29–31 Meanwhile, there are clear level-off tails at higher wavelengths in the UV-vis absorption spectra with the enhancement of water faction because of the Mie scattering effect from the aggregated suspensions³² (Fig. S13, ESI†). Transmission electron microscopy (TEM) provided further evidence for molecular aggregates of Ir-1-N and Ir-2-N in the 99% H₂O/CH₃CN mixture (Fig. S14, ESI†). Both complexes Ir-1-N and Ir-2-N exhibited a long excited-state lifetime of 0.56 μs and 0.62 μs, respectively, and high photoluminescence quantum yield (PLQY) of nearly 20% in the aggregated state (Table S1, ESI†).

Fig. 1 (a) The UV–vis absorption spectra in CH₃CN (solid line) and the PL spectra in the mixed solvent (CH₃CN : H₂O = 1 : 9 v/v) of Ir-1-N and Ir-2-N (10⁻⁵ M) (dashed line). (b) PL spectra of Ir-2-N in CH₃CN–H₂O mixtures (complex concentration = 1.0 × 10⁻⁵ M) with different water fractions (0–95% v/v) at room temperature. (c) The decay rates of ICG in the presence of Ir-1-N and Ir-2-N and their NPs upon exposure to light (425 nm, 20 mW cm⁻²). (d) Time-dependent ¹O₂ generation kinetics. A₀ = initial absorbance maximum of ICG. A = real-time absorbance maximum of ICG with various light exposure times.

In view of the promising optical properties of Ir-1-N and Ir-2-N, their PDT applications were investigated. Indocyanine green (ICG), for which the absorption band at 790 nm will decrease after encountering ¹O₂, was utilized as a ¹O₂ generation indicator.⁷ In Fig. S15 (ESI†), the absorption of ICG shows negligible change in the control groups. And there are also no changes in the absorption of the Ir(III) complexes upon irradiation (425 nm, 20 mW cm⁻²) implying their good photostability. In contrast, the absorption of ICG at 790 nm rapidly decreased in intensity under 425 nm light irradiation in the presence of 15 μg mL⁻¹ of Ir-2-N. However, irradiating the Ir-1-N solution at the same concentration did not bring a significant change of the spectra (Fig. S15c and d, ESI†). Fig. 1d illustrates that the ¹O₂ generation of the complexes follows first-order kinetics and the slope of Ir-2-N is much higher than that of Ir-1-N, suggesting the higher ¹O₂ generation ability of Ir-2-N, which could originate from the higher absorption coefficient of Ir-2-N.³³ The NPs of Ir-1-N and Ir-2-N (Ir-1-N NPs and Ir-2-N NPs) were self-assembled with poloxamer (F127) (ESI†), and their ¹O₂ generation ability was obviously enhanced compared to Ir-1-N and Ir-2-N (Fig. 1c, d and Fig. S16, ESI†). This enhancement is attributed to the AIE features of the Ir(III) complexes. Contemporaneously, Ir-2-N NPs exhibited higher ¹O₂ generation ability due to its stronger AIE features compared to Ir-1-N NPs, meaning Ir-2-N has the capacity to be utilized as a PS in the aggregated state. Therefore, Ir-2-N was used for subsequent experiments.

To overcome the drawbacks of the short wavelength absorption bands, Ir-2-N and UCNPs were integrated to improve the PS performance. Ir-2-N was encapsulated with UCNPs within TPGS to construct UCNPs@Ir-2-N (see ESI†) as PSs for the ensuing in vitro experiments. A scanning electron microscope (SEM) image showed a uniform morphology and size of the UCNPs (Fig. S17, ESI†). TPGS not only enables the integration of Ir-2-N and UCNPs, but also increases the biocompatibility of NPs.³⁴ The UV-vis absorption spectra of UCNPs@Ir-2-N showed a significant absorption at about λ_max 425 nm corresponding to that of Ir-2-N (Fig. 2a). Energy dispersive spectroscopy (EDS) confirmed the even distribution of the elements: F, Na, Y, Yb, Ir in UCNPs@Ir-2-N (Fig. 2e). The loading content of Ir-2-N in UCNPs@Ir-2-N was calculated to be 70% according to UV-vis spectral analysis (Fig. S18, ESI†). The hydrodynamic size and polydispersity index (PDI) of UCNPs@Ir-2-N were 113 nm and 0.256, respectively, detected by dynamic light scattering (DLS) (Fig. S19a, ESI†). The TEM images imply the average size of UCNPs@Ir-2-N is around 100 nm. Furthermore, one-week monitoring of the size and PDI of UCNPs@Ir-2-N revealed its excellent stability (Fig. S19b, ESI†) and hence potential applicability in PDT.

Fig. 2 (a) The UV-vis absorption spectra of Ir-2-N, UCNPs, UCNPs@TPGS and UCNPs@Ir-2-N. (b) The UV-vis absorption spectra of Ir-2-N (black line) and the PL spectra of UCNPs (red line). (c) The PL spectra of UCNPs and UCNPs@Ir-2-N. (d) The UV-vis absorption spectra of ICG (5 μg mL⁻¹) with UCNPs@Ir-2-N (15 μg mL⁻¹) under 980 nm irradiation. (e) The EDS mapping of UCNPs@Ir-2-N.

To ensure that the energy transfer between Ir-2-N and UCNPs can be realized and UCNPs@Ir-2-N could be irradiated by NIR light, the PL spectrum of UCNPs was measured under laser irradiation (980 nm). The absorption band of Ir-2-N is in the UV and blue regions overlapping with the emission bands of UCNPs (Fig. 2b). Next, the PL spectra of original UCNPs and UCNPs@Ir-2-N were compared. As shown in Fig. 2c and Fig. S18 (ESI†), the 345, 360, 450 and 470 nm bands nearly disappeared, due to strong absorption by Ir-2-Nvia ULRET (upconversion luminescence resonance energy transfer). This process is a prerequisite to use UCNPs@Ir-2-N as a PS for PDT upon NIR light irradiation.

Subsequently, the ¹O₂ generation ability of UCNPs@Ir-2-N was assessed through ICG as indicator. Compared with the control groups, the absorption bands of ICG decreased in UCNPs@Ir-2-N solution with increasing the irradiation time under 980 nm light (Fig. 2d and Fig. S21a, b, ESI†). The data reveal an effective process to generate ¹O₂: (i) UCNPs absorb the NIR light at 980 nm and emit the UV/blue light which is absorbed by Ir-2-N; (ii) Ir-2-N is then excited and undergoes energy transfer through O₂ to form ¹O₂. The absorption of UCNPs@Ir-2-N remains unchanged under irradiation (980 nm) over the same period (30 min) confirming its desirable photostability (Fig. S21c, ESI†). The ¹O₂ generation ability of UCNPs was tested as a control, which further confirmed that the good ¹O₂ generation ability of UCNPs@Ir-2-N was mainly from the Ir-2-N excited by UCNPs (Fig. S21d, ESI†). Hence, it has been demonstrated that the NIR-irradiated PSs obtained by the combination of UCNPs and Ir(III) complexes could ameliorate the problems coming from the short wavelength absorption of the Ir(III) complexes.

Encouraged by the ¹O₂ generation ability of NIR-irradiated UCNPs@Ir-2-N, their ability to kill cancer cells through PDT was investigated. In vitro photo-cytotoxicity experiments using UCNPs@Ir-2-N with concentrations from 0 to 15 μg mL⁻¹ (in terms of the calculated concentration of Ir-2-N in NPs, see ESI† for details) were evaluated against 4T1 mouse breast cancer cells via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Fig. 3a). Without NIR irradiation, the >85% viability of 4T1 cells indicated UCNPs@Ir-2-N possess a satisfactory biocompatibility. After exposure to 980 nm laser irradiation, the viability of UCNPs@Ir-2-N-treated cells decreased to 9%, pointing to the considerable cytotoxicity of UCNPs@Ir-2-N (IC₅₀ value = 3.68 μg mL⁻¹). The same concentration of UCNPs was used to conduct MTT assays as a control experiment (Fig. S22, ESI†). The data suggested that only UCNPs have negligible phototoxicity to 4T1 cells. To further visually evaluate the therapeutic effect of UCNPs@Ir-2-N, a Calcein AM and propidium iodide (PI) co-staining assay was performed to stain viable and dead cells, respectively. There were negligible dead cells in control groups and the groups in dark conditions (Fig. 3b). In contrast, a majority of cells were dead in the group with UCNPs@Ir-2-N under 980 nm irradiation. This was in good accordance with the MTT assays. The data demonstrated that UCNPs@Ir-2-N could be employed as an NIR-irradiated PS and applied in efficient PDT.

Fig. 3 (a) Relative viability of 4T1 cells after 24 h co-incubation with UCNPs@Ir-2-N under darkness and under irradiation (980 nm, 0.6 mW cm⁻²). (b) Confocal fluorescence images of 4T1 cells co-stained with calcein-AM (live cells, green fluorescence) and propidium iodide (dead cells, red fluorescence) after treatment with UCNPs@Ir-2-N (15 μg mL⁻¹). (c) Cellular uptake of UCNPs@Ir-2-N (15 μg mL⁻¹) detected by CLSM at different periods of time. (d) Confocal fluorescence images for the detection of ¹O₂ generation in 4T1 cells treated with UCNPs@Ir-2-N under irridiation (980 nm, 0.6 W cm⁻²).

The cellular uptake experiments further confirmed that UCNPs@Ir-2-N could be effectively ingested by 4T1 cells according to confocal laser scanning microscope (CLSM) imaging (Fig. 3c). The blue fluorescence signal (from the UCNPs in UCNPs@Ir-2-N) gradually enhanced with incubation time, implying a time-dependent uptake pathway and the efficient accumulation of UCNPs@Ir-2-N in cells. Furthermore, the in situ¹O₂ generation capability was evaluated using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). As expected, UCNPs@Ir-2-N-treated cells exhibited strong green fluorescence under 980 nm irradiation, but almost no fluorescence was detected under dark conditions (Fig. 3d). The results are consistent with the fact that Ir-2-N could absorb the UV/blue light from NIR-irradiated UCNPs through ULRET and then generate ¹O₂. All the results illustrate the feasible strategy that UCNPs and AIE-active Ir-2-N are encapsulated together to achieve effective energy transfer, and hence exert their respective advantages to construct NIR-irradiated PSs, UCNPs@Ir-2-N which generate toxic ROS and thereby kill the cancer cells. This approach has effectively overcome the problems of short-wavelength absorption of Ir(III) complexes.

In summary, two AIE-active Ir(III) complexes (Ir-1-N and Ir-2-N) were synthesized through rational molecular design. Extending the conjugation within the C^N ligands of Ir-2-N resulted in a redshift of its PL spectra and enhanced absorption. Moreover, Ir-2-N showed high ¹O₂ generation ability and its AIE properties lead to increased ¹O₂ generation in the aggregated state. UCNPs were applied as photoconversion materials to obtain UCNPs@Ir-2-N, in which ULRET between AIE-active Ir-2-N and UCNPs play a very important role under NIR irradiation. In vitro, NIR-irradiated UCNPs@Ir-2-N has satisfactory biocompatibility, excellent phototoxicity, good accumulation in cells and superior ¹O₂ generation ability. To the best of our knowledge, this is the first report of PSs that combine AIE-active Ir(III) complexes with UCNPs. The work represents a significant development for PSs based on readily-available, high-performance transition metal complexes.

This work was funded by NSFC (Grants 52073045, 51773195, 62075217), the Key Scientific and Technological Project of Jilin Province (20190701010GH), Jilin Provincial Department of Science and Technology (Grant 20210101148JC), the Development and Reform Commission of Jilin Province (2020C035-5), and Changchun Science and Technology Bureau (CC202110378310002101). M. R. B. thanks EPSRC (UK) grant EP/L02621X/1 for funding. D. Zhu is grateful for the support from the Key Laboratory of Nanobiosensing and Nanobioanalysis at the Universities of Jilin Province. The authors acknowledge the support from the Jilin Provincial Department of Education.

Conflicts of interest

There are no conflicts to declare.

Notes and references

1. D. W. Felsher, Nat. Rev. Cancer, 2003, 3, 375–380.
2. T. Yan, L. Ren, F. Li, F. Tian, C. Jiang, Q. Wang, X. Song and S. Zhang, Chem. Commun., 2022, 58, 1617–1620.
3. X. Zhao, J. Liu, J. Fan, H. Chao and X. Peng, Chem. Soc. Rev., 2021, 50, 4185–4219.
4. L. Huang, S. Zhao, J. Wu, L. Yu, N. Singh, K. Yang, M. Lan, P. Wang and J. S. Kim, Coord. Chem. Rev., 2021, 438, 213888.
5. Z. Zhang, M. Kang, H. Tan, N. Song, M. Li, P. Xiao, D. Yan, L. Zhang, D. Wang and B. Z. Tang, Chem. Soc. Rev., 2022, 51, 1983–2030.
6. G. Jin, G. Feng, W. Qin, B. Z. Tang, B. Liu and K. Li, Chem. Commun., 2016, 52, 2752–2755.
7. L. Zhang, Y. Li, W. Che, D. Zhu, G. Li, Z. Xie, N. Song, S. Liu, B. Z. Tang, X. Liu, Z. Su and M. R. Bryce, Adv. Sci., 2019, 6, 1802050.
8. M. Liu, Y. Chen, Y. Guo, H. Yuan, T. Cui, S. Yao, S. Jin, H. Fan, C. Wang, R. Xie, W. He and Z. Guo, Nat. Commun., 2022, 13, 2179.
9. Y. Hong, J. W. Lam and B. Z. Tang, Chem. Commun., 2009, 4332–4353.
10. J. Zhang, F. Fang, B. Liu, J. H. Tan, W. C. Chen, Z. Zhu, Y. Yuan, Y. Wan, X. Cui, S. Li, Q. X. Tong, J. Zhao, X. M. Meng and C. S. Lee, ACS Appl. Mater. Interfaces, 2019, 11, 41051–41061.
11. Z. Wang, B. Liu, Q. Sun, L. Feng, F. He, P. Yang, S. Gai, Z. Quan and J. Lin, ACS Nano, 2021, 15, 12342–12357.
12. P. S. Chelushkin, J. R. Shakirova, I. S. Kritchenkov, V. A. Baigildin and S. P. Tunik, Dalton Trans., 2022, 51, 1257–1280.
13. Y. Wang, Y. Li, Z. Zhang, L. Wang, D. Wang and B. Z. Tang, Adv. Mater., 2021, 33, 2103748.
14. Y. Li, R. Zhang, Q. Wan, R. Hu, Y. Ma, Z. Wang, J. Hou, W. Zhang and B. Z. Tang, Adv. Sci., 2021, 8, 2102561.
15. J. Liu, C. Jin, B. Yuan, X. Liu, Y. Chen, L. Ji and H. Chao, Chem. Commun., 2017, 53, 2052–2055.
16. L. Li, L. Zhang, X. Tong, Y. Li, Z. Yang, D. Zhu, Z. Su and Z. Xie, Dalton Trans., 2020, 49, 15332–15338.
17. D. Ding and B. Z. Tang, Adv. Healthcare Mater., 2021, 10, 2102499.
18. X. Cai, K. N. Wang, W. Ma, Y. Yang, G. Chen, H. Fu, C. Cui, Z. Yu and X. Wang, J. Nanobiotechnol., 2021, 19, 254.
19. Y. You and W. Nam, Chem. Soc. Rev., 2012, 41, 7061–7084.
20. G. Li, D. Zhu, X. Wang, Z. Su and M. R. Bryce, Chem. Soc. Rev., 2020, 49, 765–838.
21. Y. Feng, H. Chen, Y. Wu, I. Que, F. Tamburini, F. Baldazzi, Y. Chang and H. Zhang, Biomaterials, 2020, 230, 119637.
22. J. Zhao, S. Sun, X. Li, W. Zhang and S. Gou, ACS Appl. Bio Mater., 2020, 3, 252–262.
23. J. Zhao, X. Zhang, L. Fang, C. Gao, C. Xu and S. Gou, Small, 2020, 16, 2000363.
24. T. S. Teets, ChemPhotoChem, 2018, 2, 380–381.
25. P. Ghosh, S. K. Dey, M. H. Ara, K. Karim and A. B. M. N. Islam, Egypt. J. Chem., 2019, 62, 523–547.
26. H.-S. Qian and Y. Zhang, Langmuir, 2008, 24, 12123–12125.
27. C. You, D. Liu, J. Yu, H. Tan, M. Zhu, B. Zhang, Y. Liu, Y. Wang and W. Zhu, Adv. Opt. Mater., 2020, 8, 2000154.
28. H. F. Li, X. Q. Liu, C. Lyu, J. Gorbaciova, L. L. Wen, G. G. Shan, P. B. Wyatt, H. Q. Ye and W. P. Gillin, Light: Sci. Appl., 2020, 9, 32.
29. J. Luo, Z. Xie, J. W. Lam, L. Cheng, H. Chen, C. Qiu, H. S. Kwok, X. Zhan, Y. Liu, D. Zhu and B. Z. Tang, Chem. Commun., 2001, 1740–1741.
30. J. Mei, Y. Hong, J. W. Lam, A. Qin, Y. Tang and B. Z. Tang, Adv. Mater., 2014, 26, 5429–5479.
31. F. Würthner, Angew. Chem., Int. Ed., 2020, 59, 14192–14196.
32. B. Xu, W. Li, J. He, S. Wu, Q. Zhu, Z. Yang, Y. C. Wu, Y. Zhang, C. Jin, P. Y. Lu, Z. Chi, S. Liu, J. Xu and M. R. Bryce, Chem. Sci., 2016, 7, 5307–5312.
33. X. Zheng, L. Wang, S. Liu, W. Zhang, F. Liu and Z. Xie, Adv. Funct. Mater., 2018, 28, 1706507.
34. T. Y. Lu, W. F. Lu, Y. H. Wang, M. Y. Liao, Y. Wei, Y. J. Fan, E. Y. Chuang and J. Yu, ACS Appl. Mater. Interfaces, 2021, 13, 38074–38089.

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Footnotes

† Electronic supplementary information (ESI) available: Experimental details, supporting figures and tables. See DOI: https://doi.org/10.1039/d2cc03622c

‡ The authors contributed equally to the preparation of this work.

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