AIE Ir(III) complex conjugated with biotin as a photosensitizer for enhanced photodynamic anticancer therapy

Abstract

The first aggregation induced emission (AIE) Ir(III) complex bearing a biotin-functionalized ligand, Ir-Bio, is reported. Ir-Bio generates type I and type II reactive oxygen species and has an excellent photodynamic therapy effect in cancer cells.

---

Photodynamic therapy (PDT) is a non-invasive treatment that uses photosensitizers (PSs), light and oxygen (O₂) to selectively combat malignant tumors, vascular diseases and microbial infections.^1–3 PSs can operate through two main mechanisms: type I and type II. The type II mechanism involves sensitizing singlet oxygen (¹O₂) through an energy-transfer process from the excited triplet state of the PS to molecular oxygen in the ground state.^4,5 Regrettably, the highly hypoxic tumor microenvironment significantly limits the effect of type II PDT against tumors.⁶ The type I PDT mechanism is based on electron transfer reactions that generate a variety of reactive oxygen species (ROS), such as superoxide (˙O₂⁻) and hydroxyl (˙OH) radicals.^7,8 Type I PSs decrease reliance on O₂ and demonstrate significant promise in addressing tumor hypoxia.⁹

One of the major challenges of PDT technology is how to obtain sufficient quantities of the PSs in the target tissue for effective treatment.¹⁰ There remains an urgent need for PSs with specific targeting ability. Many Ir(III) complexes have attracted attention as candidate drugs for anticancer therapy due to their unique physiological properties and favorable photophysical attributes, including easy synthesis, structural modifications, strong photostability, and so on.^11–13 Iridium metal centers can be coordinated with different bidentate ligands through rational design strategies. Therefore, the auxiliary ligands and cyclometalated ligands can be tuned to have different biological functions to enrich the role of Ir(III) complexes in biological research and therapeutic applications. When the ligands are modified with targeting molecules, the Ir(III) complex will accumulate in large quantities in cancer cells, thus improving the antitumor properties. Cancer cells frequently exhibit high levels of tumor-specific receptors, including biotin receptors that facilitate accelerated growth, proliferation, and survival of the cells.^14–19 Purushothaman et al.²⁰ conjugated biotin as a targeting agent to the chlorophyll derivative TPP, which achieved selective targeting of the breast cancer cell line MCF-7. However, TPP derivatives may suffer aggregation-caused quenching (ACQ) of emission in aqueous media, reducing the ROS production. Thus, leveraging the excellent tumor-targeting ability of biotin and easy modifications of Ir(III) complexes should be an effective strategy to enhance PDT outcomes. However, to date the modification of Ir(III) complexes with biotin for this purpose has not been reported.

Most PSs for PDT are based on organic dyes, including boron dipyrromethene (BODIPY), porphyrin, and their derivatives which have a large planar π-electron system which favours ACQ.^21–23 The opposite phenomenon, namely aggregation-induced emission (AIE), can enhance fluorescence and ROS generation in an aggregate state through restriction of intramolecular motion (RIM) which prohibits the dissipation of energy.²⁴ PSs with AIE characteristics overcome the ACQ problem of traditional PSs and have the following advantages: (i) the intersystem crossing (ISC) ability of excited states is significantly improved, (ii) the luminescent quantum efficiency is enhanced, (III) there is high ROS generation. To date, a few AIE Ir(III) complexes have been reported as PSs, and obtaining AIE Ir(III) complexes as antitumor agents remains a challenge.^25,26

In the current work, biotin is linked to the auxiliary ligand to construct the neutral AIE Ir(III) complex Ir-Bio with the following desirable features: (i) the ability to generate type I ROS is obtained, (ii) the generation of type II ROS is greatly improved, (iii) the complex effectively accumulates in tumor cells. The PSs Ir1 (Fig. 1(A)) and Ir-Bio (Scheme 1) were obtained by a simple Schiff base reaction in high yields. With the introduction of the biotin-assisted ligand, the light absorption and singlet oxygen production capacity of Ir-Bio are significantly enhanced compared to Ir1. Ir-Bio readily accumulates in cells and has high ROS production capacity, which effectively improves the PDT antitumor effect. The work provides a new perspective to address the oxygen deficiency limitation of PSs in PDT, and to improve the uptake of PSs into cancer cells.

Fig. 1 (A) Ultraviolet (UV)-vis absorption spectra of Ir1/Ir-Bio (10⁻⁵ M) in CH₃CN/H₂O (v : v = 1 : 9); (B) fluorescence emission spectra, λ_ex = 400 nm; (C) fluorescence emission spectra of Ir-Bio in CH₃CN/H₂O mixed solution (10⁻⁵ M), λ_ex = 400 nm; (D) the change of the emission intensity of Ir-Bio in CH₃CN/H₂O with the change of the water concentration.

Scheme 1 Structural formula of Ir-Bio and its PDT process.

The synthetic routes to the ligands L1, L2, and the complexes Ir1 and Ir-Bio (Schemes S1–S5) and corresponding characterization data are provided in the SI with ¹H NMR and mass spectra (Fig. S1–S6). The UV-Vis absorption spectra of Ir1 and Ir-Bio at room temperature are shown in Fig. 1(A). Both complexes show strong absorption peaks in the ultraviolet region, and the bands in the range of 250–350 nm are attributed to the π → π* transition centered on the ligands. The relatively weak absorption bands in the visible region can be attributed to metal-to-ligand charge transfer transitions, spin-permitted ligand-to-ligand charge transfer transitions, spin-barred metal-to-ligand charge transfer transitions, and spin-barred ligand-to-ligand charge transfer transitions. As shown in Table S1, the absorptive capacity of Ir-Bio (ε 58 571 and 12 063 m⁻¹ cm⁻¹) was significantly increased compared to Ir1 due to the introduction of biotin, indicating high efficiency in photon absorption and utilization. The emission spectra of Ir1 and Ir-Bio were similar, with λ_max at 610 nm (Fig. 1(B)).

The AIE properties of Ir1 and Ir-Bio were investigated in CH₃CN–H₂O mixtures, where the water content varied from 0 to 99%. As shown in Fig. 1(C), (D) and Fig. S7, Ir1 and Ir-Bio barely emit in pure CH₃CN due to the non-radiative transition process enhanced by molecular vibrations and rotations. As the water content increased to 95%, Ir1 shows a strong red emission (Fig. S7). Ir-Bio showed strongest red emission when the water content increased to 99%. These results establish that Ir1 and Ir-Bio have typical AIE characteristics which will be beneficial to enhance ROS production and therefore improve the effect of PDT.

Efficient ¹O₂ generation is very important for PSs to improve the PDT effect. The ¹O₂ production of Ir1 and Ir-Bio in DMSO : H₂O (v : v = 1 : 99) was evaluated by monitoring the absorbance changes of indocyanine green (ICG) at 790 nm. For ICG alone, the absorption intensity basically did not change during 210 s of illumination (Fig. 2(A)). Also, the spectra of ICG in the presence of Ir1 or Ir-Bio showed little change without light (Fig. S8). In the cell experiments, PSs need to be irradiated for a relatively long time, and PSs with good light stability are therefore advantageous for PDT. The UV-vis absorption spectra of Ir1 and Ir-Bio did not change during 210 s of illumination which proved their good photostability (Fig. S9). When irradiated with a 405 nm LED lamp for 210 s, the ICG absorption at 790 nm significantly decreased (by >80%) in the presence of Ir-Bio compared with Ir1 (only about 20%) (Fig. 2(B)–(D)). This data establishes the excellent ¹O₂ generation ability of Ir-Bio. As shown in Fig. 2(E), these data for Ir1 and Ir-Bio conform to the first-order kinetic equation. The higher the slope of the straight line, the stronger the ¹O₂ generation capacity. The Ir-Bio slope (0.00757) is 8.5 times that of Ir1 (0.000888). Using Rose Bengal (RB singlet oxygen production efficiency of 73%) as a reference, the ¹O₂ quantum yields of Ir1 and Ir-Bio were 17% and 88%, respectively.

Fig. 2 (A) UV absorption spectra of ICG at different illumination times (405 nm, 20 mW cm⁻²); (B) ICG + Ir1 + light; (C) ICG + Ir-Bio + light; (D) the attenuation curve of ICG at 790 nm when Ir1 or Ir-Bio (18 μM) is present under different illumination times, data in DMSO : H₂O (v : v = 1 : 99); (E) ¹O₂ kinetic generation curve of Ir1/Ir-Bio; (F) EPR signals of Ir1 and Ir-Bio (0.1 mM) and DMPO (100 mM) under light and dark conditions in DMSO.

Most PSs rely on oxygen to produce singlet oxygen, which is cytotoxic, to achieve cell damage. The oxygen consumption during PDT will further aggravate the oxygen deficiency in the tumor. Therefore, type I PSs that can produce superoxide anion radicals (˙O₂⁻) have a broad application in PDT. As shown in Fig. 2(F), Ir1 (0.1 mM) and 2,2-dimethyl-1-oxido-3,4-dihydropyrrol-1-ium (DMPO as a spin-trapping agent for free radicals) (100 mM) showed no EPR signal in DMSO solution under either dark or light conditions, indicating that Ir1 could not produce superoxide anion radicals. In the dark, Ir-Bio (0.1 mM) and DMPO (100 mM) also did not give any EPR signal. However, when the Ir-Bio and DMPO solution was illuminated, the characteristic superoxide anion radical signal was generated. Therefore, Ir-Bio can realize a type I PDT process and effectively overcome the limitation of photosensitizer oxygen deficiency in PDT, which opens a new platform for the application of transition metal Ir(III) complex PSs in PDT. In DMSO : H₂O solution (1 : 99 v : v) Ir1/DMPO and Ir-Bio/DMPO gave no ˙OH EPR signal under light (405 nm, 20 mW cm⁻¹) or dark, indicating that ˙OH is not generated in aqueous conditions.

Since Ir1 and Ir-Bio showed excellent ROS production ability in solution, we studied their PDT in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Fig. 3, HeLa cell survival exceeded 80% in dark conditions, even with photosensitizer concentrations up to 50 μM, indicating relatively low dark toxicity of Ir1 and Ir-Bio. However, Ir1 and Ir-Bio showed concentration-dependent phototoxicity with increasing concentration when irradiated by an LED lamp with emission wavelength of 405 nm. When Ir1 concentration reached 50 μM, the cell survival rate was only about 20%. When the Ir-Bio concentration was only 25 μM, the cell survival rate was already reduced to less than 10%. Since Ir1 does not produce superoxide anions, both the Ir1 dark group and the Ir1 light group did not exhibit significant cytotoxicity under hypoxic conditions. Therefore, the phototoxicity of Ir1 under normoxic conditions should be primarily attributed to the type II photodynamic process. The cell survival rate in the irradiated group with Ir-Bio at concentration of 50 μM was less than 40%. This result proved that Ir-Bio could still effectively kill HeLa cells under hypoxic conditions (Fig. S10). Therefore, Ir-Bio has better potential than Ir1 as a PS for cancer treatment.

Fig. 3 Survival rate of (A) Ir1 and (B) Ir-Bio-pretreated HeLa cells under dark (D) and illuminated (L) conditions (405 nm, 20 mW cm⁻²).

We further detected and verified ROS production in HeLa cells by using the indicator 2′,7′-dichlorofluorescein diacetate (DCFH-DA). As shown in Fig. 4(A), (B) and Fig. S11 in the presence of the PSs Ir1 and Ir-Bio, the intracellular cells showed obvious green fluorescence after light exposure, indicating that DCFH-DA was oxidized to green-emitting DCF by ROS produced by the intracellular photosensitizers. According to the above data Ir1 and Ir-Bio photosensitizers can effectively produce ROS in cells under light.

Fig. 4 The intercellular ¹O₂ capacity was evaluated by DCFH-DA fluorescence intensity after (A) Ir1 and (B) Ir-Bio under darkness and illumination (405 nm, 20 mW cm⁻²); (C) Ir1 was used; (D) fluorescent images of HeLa cells incubated with Ir-Bio (50 μM).

To observe the live and dead states of cells after the PDT treatment, calcein-AM (live cells, green fluorescence) and propidium iodide (PI) (dead cells, red fluorescence) were co-stained with HeLa cells to determine the cell death through different fluorescence changes. As shown in Fig. 4(C), (D) and Fig. S12, in the absence of the photosensitizers, green fluorescence was emitted in the cells, and basically no red fluorescence was observed, indicating good cell growth. However, upon illumination at λ_em 405 nm, the cells showed strong red fluorescence, and basically no green fluorescence. The HeLa cells co-incubated with Ir-Bio emitted stronger red fluorescence which indicated that Ir-Bio was more enriched in HeLa cells to show better phototoxicity due to the presence of the biotin unit. The results of the live/dead cell staining are consistent with the MTT assay, indicating that Ir-Bio is highly enriched and has high ROS production ability in cancer cells, which provides a new molecular strategy to solve the difficulty of transition metal complex photosensitivities in hypoxic PDT.

The uptake of PSs in cells is an important factor affecting the efficacy of tumor therapy. As shown in Fig. 5 and Fig. S13, both Ir1 and Ir-Bio showed time-dependent enhancement of red fluorescence in HeLa cells through confocal laser scanning microscopy (CLSM), indicating that the uptake capacity of both PSs increased gradually over time. In addition, compared with Ir1, it was obvious that HeLa cells incubated with Ir-Bio emitted much stronger red fluorescence for the same incubation time. Furthermore, the uptake ability of A549 cells for the two photosensitizers (Ir1 and Ir-Bio) was measured (Fig. S14 and S15).

Fig. 5 CLSM images of HeLa cells incubated with Ir1 or Ir-Bio for 2 h, 6 h and 12 h.

After 6 hours of co-incubation with Ir-Bio, red fluorescence was emitted within the cells indicating that Ir-Bio was effectively internalized by the cells due to stronger uptake of biotin-conjugated ligands, compared with Ir1, which would contribute to better PDT killing of cancer cells by Ir-Bio. Furthermore, to verify that the uptake is receptor-specifically mediated, we performed a binding assay by incubating HeLa cells with free biotin. After incubating with free biotin for one hour, Ir-Bio was added for further incubation. It is notable that no red fluorescence was observed even after 6 hours of co-incubation. This is attributed to the complete binding of the free biotin to the over-expressed biotin receptor on the surface of the HLla cells (Fig. S16 and S17).

In summary, the significance of this work stems from the rational design of an Ir(III) complex bearing biotin functionality on the ancillary ligand. Specifically: (i) Ir-Bio has an excellent AIE effect; (ii) Ir-Bio achieves type I and type II PDT, and (III) the biotin ligand is beneficial to internalization of the Ir(III) complex by HeLa cells. Overall, this work provides an effective and versatile strategy for improving the PDT effect of Ir(III) complex photosensitizers under hypoxic conditions. Future work will address ligand design for obtaining longer wavelength absorption in Ir-Bio complex PSs.

This work was supported by NSFC (no. 52473167), the Key Scientific and Technological Project of Jilin Province (20240402036GH), the Development and Reform Commission of Jilin Province (2024C017-4). M. R. B. thanks EPSRC (UK) grant EP/L02621X/1 for funding.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data associated with this article are available in the manuscript and supplementary information (SI). Supplementary information: experimental details, supporting figures and tables. See DOI: https://doi.org/10.1039/d5cc04806k.

Notes and references

1. Z. Zhou, J. Song, L. Nie and X. Chen, Chem. Soc. Rev., 2016, 45, 6597–6626.
2. X. Zhong, X. Wang, J. Li, J. Hu, L. Cheng and X. Yang, Coord. Chem. Rev., 2021, 437, 213828.
3. Y. Wan, L.-H. Fu, C. Li, J. Lin and P. Huang, Adv. Mater., 2021, 33, 2103978.
4. G. Li, M. Wu, Y. Xu, Q. Wang, J. Liu, X. Zhou, H. Ji, Q. Tang, X. Gu, S. Liu, Y. Qin, L. Wu and Q. Zhao, Coord. Chem. Rev., 2023, 478, 214979.
5. D. Zhu, Y. Duo, M. Suo, Y. Zhao, L. Xia, Z. Zheng, Y. Li and B. Z. Tang, Angew. Chem., Int. Ed., 2020, 59, 13836.
6. J.-N. Liu, W. Bu and J. Shi, Chem. Rev., 2017, 117, 6160.
7. K. Chen, P. He, Z. Wang and B. Z. Tang, ACS Nano, 2021, 15, 7735.
8. R. Lin, J. Liu, W. Xu, Z. Liu, X. He, C. Zheng, M. Kang, X. Li, Z. Zhang, H. T. Feng, J. W. Y. Lam, D. Wang, M. Chen and B. Z. Tang, Adv. Mater., 2023, 35, e2303212.
9. M. Li, J. Xia, R. Tian, J. Wang, J. Fan, J. Du, S. Long, X. Song, J. W. Foley and X. Peng, J. Am. Chem. Soc., 2018, 140, 14851.
10. J. Li, L. Zeng, K. Xiong, T. W. Rees, C. Jin, W. Wu, Y. Chen, L. Jia and H. Chao, Chem. Commun., 2019, 55, 10972–10975.
11. D.-Y. Zhang, Y. Zheng, H. Zhang, J.-H. Sun, C.-P. Tan, L. He, W. Zhang, L.-N. Ji and Z.-W. Mao, Adv. Sci., 2018, 5, 1800581.
12. 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.
13. P. Zhang, C. K. Chiu, H. Huang, Y. P. Lam, A. Habtemariam, T. Malcomson, M. J. Paterson, G. J. Clarkson, P. B. O’Connor, H. Chao and P. J. Sadler, Angew. Chem., Int. Ed., 2017, 56, 14898.
14. M. Li, J. Xia, R. Tian, J. Wang, J. Fan, J. Du, S. Long, X. Song, J. W. Foley and X. Peng, J. Am. Chem. Soc., 2018, 140, 14851–14859.
15. K. H. Gebremedhin, M. Li, F. Gao, B. Gurram, J. Fan, J. Wang, Y. Li and X. Peng, Dyes Pigm., 2019, 170, 107617.
16. D. Li, X.-Z. Wang, L.-F. Yang, S.-C. Li, Q.-Y. Hu, X. Li, B.-Y. Zheng, M.-R. Ke and J.-D. Huang, ACS Appl. Mater. Interfaces, 2019, 11, 36435–36443.
17. X. Pei, F. Huo, Y. Yue, T. Chen and C. Yin, Sens. Actuators, B, 2020, 304, 127431.
18. K. Li, L. Qiu, Q. Liu, G. Lv, X. Zhao, S. Wang and J. Lin, J. Photochem. Photobiol., B, 2017, 174, 243–250.
19. C. Wang, Y. Xiu, Y. Zhang, Y. Wang, J. Xu, W. Yu and D. Xing, Nanoscale, 2025, 17, 1812–1873.
20. B. Purushothaman, J. Choi, S. Park, J. Lee, A. A. S. Samson, S. Hong and J. M. Song, J. Mater. Chem. B, 2019, 7, 65.
21. H. He, S. Ji, Y. He, A. Zhu, Y. Zou, Y. Deng, H. Ke, H. Yang, Y. Zhao, Z. Guo and H. Chen, Adv. Mater., 2017, 29, 1606690.
22. B. Yu, S. Goel, D. Ni, P. A. Ellison, C. M. Siamof, D. Jiang, L. Cheng, L. Kang, F. Yu, Z. Liu, T. E. Barnhart, Q. He, H. Zhang and W. Cai, Adv. Mater., 2018, 30, 1704934.
23. B. Yu, H. Wei, Q. He, C. A. Ferreira, C. J. Kutyreff, D. Ni, Z. T. Rosenkrans, L. Cheng, F. Yu, J. W. Engle, X. Lan and W. B. Cai, Angew. Chem., Int. Ed., 2018, 57, 218.
24. L. Liu, C. Li, J. Gong, Y. Zhang, W. Ji, L. Feng, G. Jiang, J. Wang and B. Tang, Angew. Chem., Int. Ed., 2023, 62, e202307776.
25. L. Zhang, Y. Li, W. Che, D. Zhu, G. Li, Z. Xie, N. Song, S. Liu, B. Tang, X. Liu, Z. Su and M. R. Bryce, Adv. Sci., 2019, 6, 1802050.
26. S. Liu, Y. Pei, Y. Sun, Z. Wang, H. Chen, D. Zhu, M. R. Bryce, B. Tang and Y. Chang, Aggregate, 2024, 5, e547.

---

Footnote

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

---