A supramolecular photosensitizer derived from an Arene-Ru(II) complex self-assembly for NIR activated photodynamic and photothermal therapy

Abstract

Effective photosensitizers are of particular importance for the widespread clinical utilization of phototherapy. However, conventional photosensitizers are usually plagued by short-wavelength absorption, inadequate photostability, low reactive oxygen species (ROS) quantum yields, and aggregation-caused ROS quenching. Here, we report a near-infrared (NIR)-supramolecular photosensitizer (RuDA) via self-assembly of an organometallic Ru(II)-arene complex in aqueous solution. RuDA can generate singlet oxygen (¹O₂) only in aggregate state, showing distinct aggregation-induced ¹O₂ generation behavior due to the greatly increased singlet-triplet intersystem crossing process. Upon 808 nm laser irradiation, RuDA with excellent photostability displays efficient ¹O₂ and heat generation in a ¹O₂ quantum yield of 16.4% (FDA-approved indocyanine green: Φ_Δ = 0.2%) together with high photothermal conversion efficiency of 24.2% (commercial gold nanorods: 21.0%, gold nanoshells: 13.0%). In addition, RuDA-NPs with good biocompatibility can be preferably accumulated at tumor sites, inducing significant tumor regression with a 95.2% tumor volume reduction in vivo during photodynamic therapy. This aggregation enhanced photodynamic therapy provides a strategy for the design of photosensitizers with promising photophysical and photochemical characteristics.

Fig. 1. Schematic illustration of RuDA-NPs for phototherapy.

A Schematic illustration of the photophysical mechanism of RuDA in monomer and aggregate forms for cancer phototherapy, B synthesis of RuDA-NPs, and C RuDA-NPs for NIR-activated PDT and PTT.

Fig. 2. Photo-physicochemical properties of RuDA.

A Chemical structure of RuDA. B Absorption spectra of RuDA in mixtures of DMF and water at various ratios. C Time-dependent variation in the normalized absorbance values of RuDA (800 nm) and ICG (779 nm) under 808 nm laser irradiation at 0.5 W cm⁻². D Photodegradation of ABDA indicative of ¹O₂ generation induced by RuDA in DMF/H₂O mixtures with different water fractions under 808 nm laser irradiation at 0.5 W cm⁻².

Fig. 3. Quantum-chemical calculations of RuDA.

A Calculated HOMOs and LUMOs of RuDA in monomeric and dimeric forms. B Singlet and triplet energy levels of of RuDA in monomer and dimer, respectively. C Calculated energy levels and possible ISC channels of RuDA in monomeric C and dimeric D forms. The arrows refer to the possible ISC channels.

Fig. 4. Photoelectrochemical properties of RuDA and RuET.

A Chemical structure of RuET. B Absorption spectra of RuET in mixtures of DMF and water at various ratios. C EIS Nyquist plots of RuDA and RuET. D Photocurrent responses of RuDA and RuET under 808 nm laser irradiation.

Fig. 5. The characterization and photothermal properties of RuDA-NPs.

A DLS analysis and TEM image (inset) of RuDA-NPs. B Thermal images of RuDA-NPs at different concentrations under 808 nm (0.5 W cm^-2) laser irradiation. C Photothermal conversion profile of RuDA-NPs at different concentrations, which is the quantitative data of B. D Temperature elevation of RuDA-NPs and ICG during five cycles of heating-cooling processes.

Fig. 6. In vitro phototherapy efficiencies of RuDA and RuDA-NPs.

A RuDA- and B RuDA-NPs-dose dependent cell viabilities in MDA-MB-231 cells in the presence or absence of Vc (0.5 mM), respectively. Error bars, mean ± SD (n = 3). Unpaired, two-sided t tests *p < 0.05, **p < 0.01, and ***p < 0.001. C Live/dead cell staining assays using calcein AM and propidium iodide as fluorescence probes. Scale bars: 30μm. A representative image of three biological replicates from each group is shown. D Confocal fluorescence images of ROS generation in MDA-MB-231 cells under different treatment conditions. Green fluorescence from DCF indicates the presence of ROS. Irradiation was performed using 808 nm laser at 0.5 W cm⁻² for 10 min (300 J cm⁻²). Scale bars: 30 μm. A representative image of three biological replicates from each group is shown. E Flow cytometry analysis for apoptosis of MDA-MB-231 cells treated with RuDA-NPs (50 μM) or RuDA (50 μM) in the presence and absence of Vc (0.5 mM), and irradiated with or without 808 nm laser (0.5 W cm⁻²) for 10 min. A representative image of three biological replicates from each group is shown. F Cellular Nrf-2, HSP70, and HO-1 expressions of MDA-MB-231 cells treated with RuDA-NPs (50 μM) with or without 808 nm laser irradiation (0.5 W cm⁻², 10 min, 300 J cm⁻²). A representative image of two biological replicates from each group is shown.

Fig. 7. Ex vivo biodistribution and in vivo PA imaging.

A Ex vivo tissue distribution of RuDA-NPs in mice determined by the content of Ru (% injected dose (ID) of Ru per gram of tissues) at different post injection time. The data represent the mean ± SD (n = 3). Unpaired, two-sided t tests *p < 0.05, **p < 0.01, and ***p < 0.001. B In vivo PA images of tumor sites under excitation at 808 nm after intravenous injection of RuDA-NPs (10 μmol kg⁻¹) at different time points. C Ru excreted out of the mice body via urine and feces after intravenous administration of RuDA-NPs (10 μmol kg⁻¹) at different time intervals. The data represent the mean ± SD (n = 3).

Fig. 8. In vivo phototherapy efficacies of RuDA and RuDA-NPs on tumors.

A IR thermal images of MDA-MB-231 tumor-bearing mice irradiated with 808 nm laser for different time at 8 h post injection. A representative image of four biological replicates from each group is shown. B Relative tumor volume and C average tumor weights for different groups of mice during the therapeutic process. D Body weight curves of different groups of mice. Irradiation was performed with 808 nm laser at 0.5 W cm⁻² for 10 min (300 J cm⁻²). Error bars, mean ± SD (n = 3). Unpaired, two-sided t tests *p < 0.05, **p < 0.01, and ***p < 0.001. E H&E staining images of major organs and tumors from different treatment groups, including Saline, Saline + Laser, RuDA, RuDA + Laser, RuDA-NPs, and RuDA-NPs + Laser groups. Scale bars: 60 μm.