Acceptor engineering of metallacycles with high phototoxicity indices for safe and effective photodynamic therapy

Although metallacycle-based photosensitizers have attracted increasing attention in biomedicine, their clinical application has been hindered by their inherent dark toxicity and unsatisfactory phototherapeutic efficiency. Herein, we employ a π-expansion strategy for ruthenium acceptors to develop a series of Ru(ii) metallacycles (Ru1–Ru4), while simultaneously reducing dark toxicity and enhancing phototoxicity, thus obtaining a high phototoxicity index (PI). These metallacycles enable deep-tissue (∼7 mm) fluorescence imaging and reactive oxygen species (ROS) production and exhibit remarkable anti-tumor activity even under hypoxic conditions. Notably, Ru4 has the lowest dark toxicity, highest ROS generation ability and an optimal PI (∼146). Theoretical calculations verify that Ru4 exhibits the largest steric bulk and the lowest singlet–triplet energy gap (ΔEST, 0.62 eV). In vivo studies confirm that Ru4 allows for effective and safe phototherapy against A549 tumors. This work thus is expected to open a new avenue for the design of high-performance metal-based photosensitizers for potential clinical applications.


Synthesis of compound 4a S1
Compound 2a (4.0 g, 19.88 mmol) and compound 3a (2.98 g, 19.88 mmol) were dissolved in EtOH (60 mL). Potassium hydroxide (5.5 g, 99.36 mmol) was then added and the reaction mixture was stirred at room temperature for 24 h. The EtOH were removed under reduced pressure and the residue was diluted with brine and extracted with EA. The combined organic fractions were dried over anhydrous Na 2 SO 4 .

Synthesis of compound Ru2
At a 1:1 molar ratio, the L (10.0 mg, 0.0095 mmol) and A2 (9.09 mg, 0.0095 mmol) were placed in an 8 mL of vial, followed by the addition of CHCl 3 /CH 3 OH= 2/1 (10 mL). After stirring at ambient temperature for 24 h, the solution was concentrated to 0.5 mL. The self-assembly products were isolated

Synthesis of compound Ru3
At a 1:1 molar ratio, the L (5.0 mg, 0.0047 mmol) and A3 (4.78 mg, 0.0047 mmol) were placed in an 20 mL of vial, followed by the addition of CHCl 3 /CH 3 OH= 2/1 (8 mL). 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 concentrated solution, washing twice with diethyl ether and drying under vacuum to obtain product Ru3 (9.5 mg, 98% yield). 1 H NMR (400 MHz, CD 3 CN) δ 8.68 (s,

Synthesis of compound M S3
1,4-Dibromobenzene (2.0 g, 8.5 mmol), imidazole (2.4 g, 35.6 mmol), K 2 CO 3 (3.75 g, 27.2 mmol) and CuSO 4 (0.027 g, 0.17 mmol) were stirred and heated at 180 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and water was added to wash the mixture. The crude product was dissolved in ethanol (30 mL). The organic layer was separated. The organic layer was evaporated to dryness to give the crude product. The residue was recrystallized from water and methanol to give a white solid (1.5 g, 86% yield

Absorption and photoluminescence excitation (PLE) spectral studies
The UV-Vis-NIR absorbance spectra of Ru1-Ru4 were recorded on a PerkinElmer Lambda 25 UV-Vis spectrophotometer. PLE spectra of Ru1-Ru4 solutions were obtained using an Applied Nano Fluorescence spectrometer.

The FL penetration depths measurement
To detect the tissue penetration depth response of Ru metallacycles, 1% intralipid was used to mimic biological tissue (5 mL 20% intralipid were mixed with 100 mL water). Glass capillary tubes were filled with either Ru1-Ru4 (200 μM) or Ru-M1 (200 μM) and taped to the bottom of a cylindrical dish. The dish was filled with different volumes of intralipid that approximates the wavelength dependence of light scattering in biological tissues. The depth of the capillary tubes was calculated from the area of the dish.
The fluorescence images were then obtained by using 808 nm laser illumination for Ru1-Ru4 or Ru-M1 images were analyzed by ImageJ software.

The ROS penetration depths measurement
To examine the tissue penetration depth response of Ru1-Ru4, biological tissue was simulated using 1% intralipid (5 mL of 20% adipose lactide mixed with 100 mL of water). A 96 well plates were filled with a solution of DCFH treated with Ru1-Ru4 (10 μM) or Ru-M1 (10 μM). Petri dishes were filled with different volumes of fat, which approximated the wavelength dependence of light scattering in biological tissue. The depth of the capillary is calculated from the area of the petri dish. Place the culture dish on the top of 96 well plate, and then irradiated with 808 nm laser for 3 min to obtain fluorescence image, and the images were analyzed by ImageJ software.

Superoxide anion radical (O 2 −• ) detection
The light triggered O 2 •− generation was measured. Detection of O 2 •− was performed by taking advantage of the interaction between dihydroethidine and DNA. Dihydroethidium (DHE) was utilized as O 2 •− specific probe because it could intercalate in DNA and emit red fluorescence at the participation of O 2 •− . Briefly, photosensitizer (Ru1-Ru4, 10 M) and DHE (50 μM) were dissolved in a water solution that contained 250 g/mL of ctDNA. A mixture only containing DHE and ctDNA was used as the control. The mixtures were irradiated with the 808 nm laser (1 W/cm 2 ). After different irradiation times, fluorescence spectra (excited at 490 nm) of these mixtures were recorded using a Edinburgh FL900/FS900 spectro fluorometer.

Detection of intracellular O 2 •− generation.
About 1 × 10 5 A549 cells in Dulbecco's modified Eagle's medium (DMEM) were seeded in confocal dishes (Corning) and incubated overnight at 37 o C under a humidified 5% CO 2 atmosphere. The intracellular O 2 •− was examined by using DHE as a fluorescence probe. A549 cells were incubated with Ru4 (10 μM) for 2 h followed by incubation with 10 μM DHE for 30 min. After then washed with PBS for two times, cells were irradiated with a 808 nm laser 1 W/cm 2 for 5 min). Then, the fluorescence was immediately observed using a Leica laser fluorescent confocal microscope (CLSM) with the excitation wavelength of 488 nm, and emission collection wavelength was 570 nm to 630 nm.

Theoretical calculation.
Equilibrium geometries at the ground state were optimized using the density functional theory (DFT) method with the B3LYP functional through Gaussian 16. The 6-31G(d) basis set was used for H, C, N, and S atoms, and the Los Alamos National Laboratories (LANL2DZ) basis set and pseudopotential were used for Ru. The vibrational frequencies of all the optimized structures were calculated to ensure that the optimized structures of molecules have no imaginary vibrational frequencies. The minimized structures were used for the orbital generation and analyzed. The bending angle is defined by dihedral angle∠N1−N2−B1−N3, which represents the angle between the phenylpyridine modified groups on both sides of the aza-BODIPY core.

15
A549 cells were seeded onto 35 mm confocal dishes at a density of 1 × 10 4 cells/mL and allowed to adhere overnight. The cells were loaded with Ru4 (10 μM, 1% DMSO, v%) for 6 h at 37 °C in the dark.

The cells were then incubated with LysoTracker ® Green (100 nM), MitoTracker ® Deep Red (500 nM) and
Hoechst 33342 (5 μg/mL) (Thermo Fisher Scientific (USA)) for 45 min at 37 °C in the dark. The cells were washed with PBS three times at the end of every period. Confocal images were taken using a laser scanning confocal microscope (LSM 710, Carl Zeiss, Germany). For the LysoTracker ® Green channel, the excitation wavelength was 488 nm, and the signal was collected between 515-535 nm. For the MitoTracker ® Deep Red channel, the probe was excited at 644 nm, and the emission filter was between 665-700 nm. For the Hoechst 33342 channel, the excitation wavelength was 405 nm, and the fluorescence signal was recorded at 430-460 nm. For Ru4, an excitation wavelength of 808 nm was used, and the emission filter was between 1000-1100 nm.

Elemental imaging by LA-ICP-MS.
A549 cells were seeded in 24-well cultureplates with cell climbing slices for overnight and then incubated with Ru4 (10 µM) for 6 h. After washed by PBS w/o Ca/Mg, cells were fixed with 70% coldalcohol solution and washed by water. Then, cells were allowed to adhere on slidesto prepare LA-ICP-MS test. A laser spot size of 3 µm diameter, 10 µm s −1 scanspeed, 100 Hz repetition frequency, and laser fluence of ~3 J cm −2 were utilized to perform the test. ICP-MS parameters were as follows: radio frequency power of 1500 W, nebulizer gas flow of 1.25 L min −1 , auxiliary gas flow of 1.2 L min −1 , andplasma gas flow of 15 L min −1 . The monitored isotope 102 Ru was measured incounting mode. Images integration was performed by the IGOR-based lolite software.

Cellular uptake of the ruthenium content measured by ICP-MS
A549 cells were seeded at a density of 1 × 10 6 cells in 6-well cell culture plates. The cells were left to grow for 24 h in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO 2 humidified atmosphere. After 12 h, Ru4 (10 μM) was added into the wells and the cells were incubated for 1 h, 3 h, 6 h, and 12 h, respectively. Following incubation, the cells were washed, digested and collected, and the Ru content in the cells determined by ICP-MS. All experiments were 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 at a density of 1 × 10 4 cells/mL and allowed to adhere overnight. The culture medium was refreshed with PBS. For the temperature-dependent uptake study, A549 cells were incubated 16 with 10 μM Ru4 (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 NH 4 Cl (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 10 μM Ru4 (1% DMSO, v%) for 6 h at 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 Ru1-Ru4, A1-A4, and L was determined by means of MTT assays using several human cell lines, including A549 and 16HBE. For instance, A549 cells were seeded onto 96-well plates at 0.5 × 10 4 cells per well and incubated at 37ºC for 24 h. For normoxic photo-cytotoxicity evaluation, different concentrations of Ru1-Ru4 in DMEM medium were added to the wells of normoxic cells, respectively. For hypoxic photo-cytotoxicity evaluation, different concentrations of Ru1-Ru4 in DMEM medium were added to the wells of cells, and placed in closed anoxic sealed bags (O 2 content < 1%). Then, the cells were further incubated for 6 h under normoxic or hypoxic conditions, respectively. Subsequently, the cells were subjected to 808 nm laser (1 W/cm 2 , 5 min) under respective conditions. Then the cells were further incubated for 12 h at 37 ºC. 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.

Annexin V-FITC staining assay
A549 cells were seeded in 12-well plates at a density of 1 × 10 6 cells/well. The dark groups containing Ru4 were incubated in the dark for 24 h. For the light groups, after incubation with Ru4 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 following the protocols of the manufacturer. Cells were imaged before and after being subject to 5 min of laser irradiation (808 nm, 1 W/cm 2 ). Finally, the samples prepared in this way were analyzed via flow cytometry (CytoFLEX, Beckman Coulter).
Confocal luminescence imaging was performed with excitation at 488 nm and monitoring at 505−545 nm for the green channel or 617-640 nm for the red channel.

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 10 μM Ru4 (1% DMSO, v%), respectively. The cells were incubated at 37ºC 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 (1 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).

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), cisplatin, or Ru4, 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, 1 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. Ru4 (10 μM) were added into different groups. The dark groups containing Ru4 were incubated in the dark for 24 h. For the light groups, after incubation with Ru4 for 12 h, cells were exposed 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, 37ºC, 20 min) and stained with PI (100 μg/mL, r.t. 15 min) and analyzed via flow cytometry (CytoFLEX, Beckman Coulter).

In vitro NIR-II cell imaging
NIR-II fluorescence images of A549 cells were taken at an exposure time of 100 ms using a NIR-II fluorescence microscope. Excitation was affected at 808 nm using a diode laser with an 80 μm 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 100 ms.

Animals and tumor model
All animal experimental procedures were conducted in agreement with the guidelines of the Institutional Animal Care and Use Committee (CCNU-IACUC-2011-058). A549 cells (1 × 10 6 in 100 μL of PBS) were injected into female nude mice (Suzhou Belda Bio-Pharmaceutical Co.). When tumor volume reached to ~100 mm 3 , the nude mice bearing xenograft A549 tumors were subjected to further experiments.

In vivo antitumor activity
Tumor volume and body weight were measured for animals in all experiments. Tumor volume was determined by measuring the tumor in two dimensions with calipers and calculated using the formula tumor volume = (length × width 2 )/2. The mice were divided into four groups randomly (n = 5) when the mean tumor volume reached about 100 mm 3 and this day was set as day 0. Mice were administrated intratumorally with PBS (group 1), PBS plus laser (group 2), cisplatin (1 mg Ru/kg) (group 3), Ru4 (1 mg Ru/kg) (group 4) and Ru4 (1 mg Ru/kg) plus laser (group 5). Tumor volumes and body weights were measured every 2 days for A549 tumor-bearing mice. 12 h after intratumoral injection, the tumors (group 2 and 5) were illustrated with 808 nm laser (1 W cm -2 , 10 min).

Histological examination
Upon completion of the PDT treatment, the mice were sacrificed. The tumors and organs including heart, liver, spleen, lung, kidney, intestine and brain were resected, immersed in 4% paraformaldehyde and stored at 4 °C for seven days. The sections of the tumors and organs were obtained as paraffin-embedded samples and stained with hematoxylin and eosin (H&E). Deep blue-purple hematoxylin and pink Eosin 19 stained nucleic acids and proteins, respectively. A Carl Zeiss Axio Imager Z2 microscope was used to observe the tissue structure and cell state of the sections. Figure S1. Density functional theory (DFT) calculation of L.                            43 Figure S43. 1 H NMR spectrum (400 MHz, CDCl 3 , 298 K) of 2a. Figure S44. 13 C NMR spectrum (100 MHz, CDCl 3 , 298 K) of 2a.