Rationally designed Ru(ii)-metallacycle chemo-phototheranostic that emits beyond 1000 nm

Ruthenium complexes are emerging as potential complements to platinum drugs. They also show promise as photo-diagnostic and therapeutic agents. However, most ruthenium species studied to date as potential drugs are characterized by short excitation/emission wavelengths. This limits their applicability for deep-tissue fluorescence imaging and light-based therapeutic treatments. Here, we report a Ru(ii) metallacycle (Ru1100) that emits at ≥1000 nm. This system possesses excellent deep-tissue penetration capability (∼7 mm) and displays good chemo-phototherapeutic performance. In vitro studies revealed that Ru1100 benefits from good cellular uptake and produces a strong anticancer response against several cancer cell lines, including a cisplatin-resistant A549 cell line (IC50 = 1.6 μM vs. 51.4 μM for cisplatin). On the basis of in vitro studies, it is concluded that Ru1100 exerts its anticancer action by regulating cell cycle progression and triggering cancer cell apoptosis. In vivo studies involving the use of a nanoparticle formulation served to confirm that Ru1100 allows for high-performance NIR-II fluorescence imaging-guided precise chemo-phototherapy in the case of A549 tumour mouse xenografts with no obvious side effects. This work thus provides a paradigm for the development of long-wavelength emissive supramolecular theranostic agents based on ruthenium.


In vitro photostability test
The photostability of Ru1100 was investigated in PBS, with 1 cm quartz cuvettes containing 10 μM Ru1100 (200 μL) being illuminated using a continuous laser (808 nm, 0.8 W/cm 2 ) for 60 min. The absorbance of the Ru1100 solution was recorded every 10 min.

Photothermal and photothermal stability tests in vitro
Ru1100 solutions of varying concentrations (0, 2.5, 5 and 10 μM) were irradiated with a 808 nm laser (0.8 W/cm 2 ) for 5 min. In addition, the 5 μM Ru1100 solution was irradiated with the 808 nm laser at different power densities (0.6, 0.8, 1 and 1.2 W/cm 2 ) for 5 min. An infrared thermal imaging camera was used to record the solution temperature in 1.5 mL Eppendorf tubes.
To test further the photothermal stability of Ru1100, solutions of Ru1100 at a concentration of 5 μM were irradiated with an 808 nm pulsed laser (0.8 W/cm 2 ) for 5 min and then allowed to cool naturally for 10 min. The solution temperature was recorded over the course of seven heating-cooling cycles using an infrared thermal imaging camera.

Measurement of the photothermal conversion efficiency (η)
The photothermal conversion efficiency (η) of Ru1100 was calculated using the following equations where Tmax (or Tsur) is the equilibrium temperature (or ambient temperature), I is the incident laser power (I= 0.8 W/cm 2 ), A808 is the absorbance at 808 nm, and $ is the system time constant of the sample.
8. Octanol/water partition coefficient (log Po/w) The partition-coefficient of each compound expressed as was determined by the "shake-flask" method. S3 Water and octanol were mixed and shaken thoroughly to reach equilibrium, which resulted in the separation of two layers, i.e., water saturated with octanol and octanol saturated with water. The two layers were separated, and Ru1100 was dissolved using water that was previously saturated with octanol (3 mL). The same volume of an octanol phase previously saturated with water was then added to the solution. The mixture was shaken at room temperature for 24 hours. The concentration of Ru1100 and the Ru(II) precursor 2 were determined by UV-VIS spectroscopy using the extinction coefficients of the complexes in water saturated with octanol. The evaluation was repeated three times.

Cellular uptake mechanism studies
Cellular imaging was carried out to examine the cellular uptake mechanism. S4 A549 cells were seeded on 35 mm confocal dishes (Corning) at a density of 1 × 10 4 cells/mL and allowed to adhere overnight. The culture medium was refreshed with PBS. For the temperaturedependent uptake study, A549 cells were incubated with 5 μM Ru1100 (1% DMSO, v%) for 6 h at 37 °C and 4 °C, respectively. For the cellular uptake inhibition study, triethylamine (1 mM) was used as anion channel inhibitor. Methyl-β-cyclodextrin (50 mM) and sucrose (5 μM) were adopted as caveolin-mediated and clathrin-mediated inhibitors, respectively, while NH4Cl (50 mM) and chloroquine (100 μM) were used as endocytic inhibitors. A549 cells were pretreated with these protein inhibitors for 40 min at 37 °C, respectively. PBS was then used to wash the cells and the cells were further incubated solely with 5 μM Ru1100 (1% DMSO, v%) for 6 h at S6 37°C. All of the cells were then washed with PBS three times and subjected to confocal microscopy.

Cell viability studies
In vitro cytotoxicity of Ru1100 was determined by means of MTT assays using several human cell lines, including A549, Hela, HepG-2, A549/DDP and 16HBE. For instance, A549 cells were incubated on a 96-well plate in a DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% CO2 humidified atmosphere for 24 h with 0.5 × 10 4 cells seeded per well. Cells were then cultured in the medium supplemented with Ru1100 at various concentrations for 12 h. The "dark groups" containing Ru1100 were incubated in the dark for 48 h. For the "light groups", after incubation with Ru1100 for 12 h, the cells were exposed to 808 nm irradiation for 5 min and then allowed to incubate for an additional 12 h in the dark. The addition of 10 μL of MTT (BioFrox, China) as a 0.5 mg/mL solution to each well was followed by incubation for 4 h at 37°C to allow the formation of formazan crystals. Then, the supernatant was removed and the products were lysed with 200 μL of DMSO. The absorbance value was recorded at 570 nm using a microplate reader. The absorbance of the untreated cells was used as a control and its absorbance was used as the reference value for calculating 100% cellular viability.
13. Wound healing assay A549 cells were seeded in 12-well plates at a density of 1 × 10 5 cells/well. The cells were treated with culture medium (negative control, NC), 5-ALA, [Ru(bpy)3] 2+ , cisplatin, or Ru1100 (5 μM), respectively. Then, the medium was replaced with PBS, and a scratch was made with a sterile pipet tip (200 μL) in all groups. The detached cells were removed. Laser irradiation was performed in the laser-only group, Ru1100 + laser group and 5-ALA + laser group. Images were taken immediately after irradiation and 24 h later using a fluorescence microscope. The area of the scratch was analyzed using the ImageJ software.
14. Matrigel invasion assay S7 Matrigel (Becton Dickinson, Bedford, MA) was added into Transwell inserts (Jet Biofil, China) for solidification in a 24-well plate. Cells were collected and resuspended in serum-free medium and then transferred into the upper chambers, 1 × 10 6 cells for each Transwell insert.
The lower wells were supplemented with 600 μL of the complete medium containing Ru1100 (5 μM). Photo-irradiation was performed in the PBS + laser group and Ru1100 + laser group.
After 12 h, the cells that did not invade the lower surface of the transwell inserts were cleaned with a cotton swab, while the cells invading the inserts were fixed with 4% paraformaldehyde and subjected to crystal violet staining after being washed with PBS three times. Transwell inserts were visualized by light microscopy. Then, 33% acetic acid was added into the well to dissolve the crystal violet. Cell invasion ratios were calculated according to the absorbance of 33% acetic acid at 590 nm.
15. Annexin V-FITC/propidium iodide double staining assay A549 cells were seeded in 12-well plates at a density of 1 × 10 5 cells/well. The dark groups containing Ru1100 were incubated in the dark for 24 h. For the light groups, after incubation with Ru1100 for 12 h, the cells were exposed to 808 nm irradiation for 5 min and then allowed to incubate for another 12 h in the dark. Cells in the control group were treated with aculture medium. The cells were further live stained with annexin V-FITC and propidium iodide (PI, Beyotime Biotechnology, China) following the protocols of the manufacturer. Cells were imaged before and after being subject to 5 minutes of laser irradiation (808 nm, 0.8 W/cm 2 ).
Finally, the samples prepared in this way were analyzed via flow cytometry (CytoFLEX, Beckman Coulter).
16. Lysosomes disruption assay A549 cells were subject to different treatments: 1) untreated; 2) irradiated with 808 nm laser irradiation (0.8 W/cm 2 ) for 5 min; 3) incubated with 5 μM Ru1100 for 24 h; 4) incubated with 5 μM Ru1100 for 24 h and then irradiated with 808 nm laser (0.8 W/cm 2 ). After treatment, cells were incubated with acridine orange (Macklin, China) at a concentration of 5 μM for 20 min and subjected to confocal luminescence imaging. Confocal luminescence imaging was S8 performed with excitation at 488 nm and monitoring at 505−545 nm for the green channel or 617-640 nm for the red channel.
17. Analysis of mitochondrial membrane potential (MMP) MMP was assessed by means of JC-1 staining. A549 cells were seeded onto corning confocal dishes at a density of 1 × 10 4 cells/mL and allowed to adhere overnight. The cells were then treated with culture medium (control) or 5 μM Ru1100 (1% DMSO, v%), respectively. The cells were incubated at 37 ℃ for 2 h in the dark and then washed with PBS. The cells were then cultured with JC-1 (Solarbio, China) (5 μM) in PBS at room temperature for 20 min in the dark.
Fluorescent images were captured by CLSM before and after 808 nm laser irradiation (0.8 W/cm 2 ). The excitation wavelength for the JC-1 monomer was 488 nm, and the emission filter was adjusted to around 529 nm for the JC-1 monomer (green). For the JC-1 aggregate, and excitation of 543 nm was used, and the emission was collected around 590 nm (red).
18. Caspase-3/7 activation assay A549 cells were seeded in white-walled nontransparent-bottomed 96-well microculture plates at a density of 1.5 × 10 4 cells/well and allowed to incubate overnight to adhere. The cells were then treated with culture medium (negative control, NC), 5-ALA, [Ru(bpy)3] 2+ , cisplatin, or Ru1100, respectively. The cells were incubated for 12 h in the dark and divided into two equal groups. The dark group was incubated for an additional 12 h and treated with acaspase-3/7 activity kit (Beyotime Biotechnology, China) according to the manufacturer's protocol. The other group was exposed to laser irradiation (808 nm, 0.8 W/cm 2 ) for 5 min, and incubated for an additional 12 h in the dark. The caspase-3/7 activity was determined using an analogous method.

Cell cycle analysis
A549 cells (1 × 10 5 ) were seeded in 6-well plates and incubated overnight. Ru1100 (5 μM) were added into different groups. The dark groups containing Ru1100 were incubated in the dark for 24 h. For the light groups, after incubation with Ru1100 for 12 h, cells were exposed S9 to 808 nm irradiation for 5 min and then allowed to incubate for another 12 h in the dark. After treatment, cells were lysed by RNaseA (100 μg/mL, 37℃, 20 min) and stained with PI (100 μg/mL, r.t. 15 min) and analyzed via flow cytometry (CytoFLEX, Beckman Coulter).

Synthesis of Ru1100 NPs
Ru1100 NPs were prepared using the matrix-encapsulation method. DSPE-PEG5000 (9 mg in double distilled water) and Ru1100 (1 mg in THF) were stirred at room temperature overnight. After removing the THF by bubling with nitrogen gas, the resulting mixed solution was centrifuged for 20 min at a speed of 3,000 rpm using a 50 kDa centrifugal filter to remove the residual DSPE-PEG5000 and any free Ru1100. Filtering through a 0.22 μm polyester sulfone filter yielded Ru1100 NPs as an aqueous solution.

Hamolysis assay
Fresh blood was obtained in heparinized tubes from mice and subjected to centrifugation at 3000 rpm for 10 min. where ODsample, ODsaline and ODwater are the absorbances of the sample, positive control and negative control, respectively.

In vitro NIR-II cell imaging
NIR-II images of the cells were taken at an exposure time of 100 ms using a NIR-II fluorescence microscope. Excitation was effected at 808 nm using a diode laser with an 80 μm S10 diameter spot focused by a 100 × objective lens (Olympus). The resulting NIR-II fluorescence (FL) was collected using a liquid-nitrogen-cooled, 320 × 256 pixels, two-dimensional InGaAs camera (Princeton Instruments) with sensitivity over the 800 to 1700 nm spectral region. The excitation light was filtered out using a 900 nm long-pass filter and an 1100 nm long-pass filter (both Thorlabs). The NIR FL images were taken at a fixed exposure time of 300 ms. For bright field white-light images, a fiber optic illuminator (Fiber-Lite) was used to illuminate the sample in the trans-illumination mode. Images were recorded using the same filters at a fixed exposure time of 2 ms.

In vivo NIR-II fluorescence imaging
For brain vessel imaging, a Ru1100 NPs solution (200 μL, 1mg Ru/kg) was injected into the vein of C57BL/6 mice (n = 4) tail. After injection, the brain vessel system was visualized using the NIR-II imaging apparatus. For tumour imaging, A549 tumour-bearing mice were mounted on the imaging stage beneath the laser. NIR-II fluorescence images were collected using a NIR-II imaging system which was purchased from Suzhou NIR-Optics Technologies CO., Ltd.
Photoexcitation was provided by an 808 nm diode laser. The laser power density was 28mW/cm 2 during imaging.

In vivo antitumour activity
Tumour volume and body weight were measured for animals in all experiments. Tumour volume was determined by measuring the tumour in two dimensions with calipers and calculated using the formula tumour volume = (length × width 2 )/2. The mice were divided into four groups randomly (n = 4) when the mean tumour volume reached about 100 mm 3 and this day was set as day 0. Mice were administrated intravenously with PBS, PBS + laser, cisplatin (at a dose of 1 mg Pt per kg body weight), Ru1100 NPs (at a dose of 1 mg Ru per kg body weight), Ru1100 NPs + laser. Tumour volumes and body weights were measured every 3 days in the case of the A549 tumour-bearing mice. The tumour inhibition study was stopped on the S11
After stirring at ambient temperature for 24 h, the solution was concentrated to 0.5 mL.The self-assembly products were isolated via precipitation by adding diethyl ether into the   Table S1. Absorption and emission data for Ru1100 in different solvents [a] Maximum absorbance of Ru1100. [b] The maximal emission of Ru1100. [c] The relative fluorescence quantum yield relative to IR-26 (0.1% in dichloromethane) used as a reference standard. Figure S12. Absorption spectra of Ru1100 in water recorded at various times. S19 Figure S13. 1 H NMR spectra of Ru1100 at 0 h and after allowing to sit for 24 h in DMSO-d6 and D2O (v/v = 4/1).     Hoechst with the corresponding correlation coefficients. Scale bars = 10 μm. Figure S22. Cell uptake mechanistic study of Ru1100 (5 μM) in the presence of different inhibitors/conditions. Scale bars = 20 μm. Table. S2 Cytotoxicity (IC50, μM) of cisplatin, 5-ALA and Ru1100 toward various cell lines.
Synthesis of compound 1c:

S36
Compound 1d (850 mg, 2.15 mmol) was dissolved in 1-butanol (20 mL). Ammonium acetate (2.49 g, 32.25 mmol) was added and the reaction was stirred at 115 °C for 12 h. The reaction was cooled to room temperature and the volume was taken to 5 mL under reduced pressure.