Ethical approval
All animal experiments reported herein were performed following the guidelines and were approved by Peking University Institutional Animal Care and Use Committee (LA2021316). Patient-derived xenograft models were conducted in accordance with established guidelines, having obtained written consent from the respective patients, and received approval from the Second Xiangya Hospital of Central South University (2020591). The mice were euthanized through CO2 inhalation when the tumor volume surpassed 1500 mm³ or when the mice showed signs of serious weight loss, dreadful weakness, or Unhealed ulcers, indicating they were moribund. During treatment of the PDX model of osteosarcoma, since the individuality of the mice, the tumor volume of one mouse reached 1500 mm3 by the end of the treatment, and this mouse was not euthanized in time to obtain reliable and intuitive experimental results. However, the status of the mice was closely monitored during the subsequent treatment and no significant weight loss or extreme weakness was detected. In addition, the veterinarian was notified of the situation, and approval was obtained from the Ethics Committee.
Animal experiment
BALB/c mice (4–5 weeks old, female) and BALB/c nude mice (4–5 weeks old, female) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). All mice were housed under a 12 h light/dark cycle, with the temperature consistently maintained between 18 °C and 23 °C and the humidity kept at ~50%.
Sex of the animal models
To the best of our knowledge, the occurrence of osteosarcoma is not related to the sex of the patient. Therefore, in this work, the biological properties of the compounds were studied only in female mice.
Material
Potassium iodide, 2-hydrazinylpyridine, hydroquinone, 3-bromo-1-propanol, ruthenium (III) chloride hydrate, γ-terpinene, ammonium hexafluorophosphate, 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluorodecane-1,10-diol, 1,2,4,5-cyclohexanetetracarboxylic dianhydride, mPEG5000-OH, pyridine hydrofluoride, acetone, and mercaptoacetic acid were obtained from Aladdin (Shanghai, P. R. China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Energy Chemical (Shanghai, China). Fetal bovine serum (FBS) was obtained from MeilunBio. RPMI-1640 medium, high-glucose DMEM were obtained from Thermo Fisher Scientific Inc. trypsin-EDTA (0.25%) and L-Glutamine-Penicillin-Streptomycin solution were obtained from Wuhan Servicebio Technology Co., Ltd.
Instrumentation
1H NMR spectra were measured by a 400 MHz NMR spectrometer (Bruker) at room temperature. Inductively coupled plasma mass spectrometer (Agilent technologies 7700 series, USA) was used for quantitative analysis of the total ruthenium contents from different samples and cells. The morphology and size of nanoparticles were obtained by transmission electron microscopy (TEM) carried out with a HT7700 and JEM-F200 electron microscopy. Localization NPs were performed using a confocal laser scanning microscopy (LSM-880, ZEISS). Size and zeta potential measurements were conducted on a Malvern Zetasizer (Nano ZS, UK). High resolution mass spectrometry (HR-MS) was conducted by Agilent 1290 UPLC/6540 Q-TOF. MTT assay was conducted using a microplate reader (SpectraMax). Ultra performance liquid chromatography (UPLC) analysis was performed on an Agilent 1290 series instrument. Graphite furnace atomic absorption spectrometry (GF-AAS, PerkinElmer PinAAcle D900 series, USA) was used for quantitative analysis of the total ruthenium contents from different samples and cells.
Theoretical calculations
The geometry of a metal complex was determined using density-functional theory calculations with the Gaussian software package. (Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A. V.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J. Gaussian 16 Rev. C.01, Wallingford, CT, 2016) The metal atom was described using the Los Alamos (LANL2) effective core potential with the corresponding triple-zeta basis set while all other atoms were described with the Pople double-zeta basis set with a single set of polarization functions on non-hydrogen atoms (6-31 G(d)). The geometry of the calculated structures was verified by comparison with crystal structures of structurally related compounds from the CCDC. Solvent effects were included using a polarizable continuum model (PCM). The structures of all calculated molecules correspond to ground state minima on the ground state potential energy surfaces with no imaginary frequencies present.
Synthesis of 4-(pyridin-2-yldiazenyl)phenol (compound 1)
The 4-(pyridin-2-yldiazenyl)phenol was synthetized using a similar procedure as previously reported45. 1,4-benzenediol (493 mg, 4.56 mmol) was dissolved in a mixture of water (50 mL) and 60% perchloric acid (3.6 mL). A solution of 2-hydrazine pyridine (504 mg, 4.62 mmol) in water (8 mL) was dropwise added. The solution was stirred at room temperature for 1 h. Subsequently, an orange precipitate was formed that collected by filtration. The solid was washed with water (30 mL) and diethyl ether (30 mL). The compound was dried under high vacuum. Yield: 800 mg, 88%. 1H NMR (400 MHz, DMSO-d6) δ = 10.49 (s, 1H), 8.67 (dd, J = 5.2, 1.8 Hz, 1H), 8.00 (td, J = 7.7, 1.9 Hz, 1H), 7.92 – 7.83 (m, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.51 (ddd, J = 7.3, 4.8, 1.1 Hz, 1H), 7.06 – 6.89 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ = 163.5, 162.4, 149.6, 149.6, 145.7, 139.3, 139.2, 126.0, 125.4, 116.6, 113.4. The analytical data was found to be in agreement with the previous literature45.
Synthesis of 3-(4-(pyridin-2-yldiazenyl)phenoxy)propan-1-ol (compound 2)
The 3-(4-(pyridin-2-yldiazenyl)phenoxy)propan-1-ol was synthetized using a similar procedure as previously reported46. 4-(pyridin-2-yldiazenyl)phenol (200 mg, 1 mmol) was dissolved in a mixture of DMF (10 mL), potassium carbonate (207 mg, 1.5 mmol), and potassium iodide(183 mg, 1.1 mmol). The bromopropanol (207 mg, 1.5 mmol) was added to the solution. The solution was stirred at 80°C for 24 h. After this time, the color of solution became blood red, and distilled water (200 ml) was added. The red solution was extracted three times with dichloromethane. The organic solvent was collected and concentrated. The products were obtained by silica gel column chromatography. 1H NMR (400 MHz, Chloroform-d) δ = 8.65 (dd, J = 4.8, 2.6 Hz, 1H), 8.04–7.93 (m, 2H), 7.83 (td, J = 7.7, 2.2 Hz, 1H), 7.74 (dd, J = 8.1, 2.6 Hz, 1H), 7.32 (p, J = 3.3 Hz, 1H), 7.02–6.91 (m, 2H), 4.15 (dt, J = 8.6, 3.9 Hz, 2H), 3.83 (dt, J = 8.5, 3.7 Hz, 2H), 2.03 (dt, J = 8.4, 3.9 Hz, 2H). 13C NMR (100 MHz, Chloroform-d) δ = 163.0, 162.5, 149.3, 146.8, 138.4, 125.7, 124.7, 115.0, 114.8, 65.6, 59.4, 32.0. The analytical data was found to be in agreement with the previous literature46.
Synthesis of dichloro(p-cymene)ruthenium(II) dimer (compound 3)
The dichloro(p-cymene)ruthenium(II) was synthetized using a similar procedure as previously reported47. The ruthenium chloride hydrate (128 mg, 0.57 mmol) was dissolved in ethanol anhydrous (6.2 mL). γ-terpinene (425 μL, 2.65 mmol) was added into solution. The obtained solution was stirred at 80 °C for 2 h. Subsequently, the solution was cooled to room temperature. An orange precipitate formed that collected by filtration. The products were dried under high vacuum. Yield: 800 mg, 90%. 1H NMR (400 MHz, DMSO-d6) δ = 5.82 (d, J = 6.1 Hz, 2H), 5.78 (d, J = 6.2 Hz, 2H), 2.84 (hept, J = 7.0 Hz, 1H), 2.09 (s, 3H), 1.20 (d, J = 6.9 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ = 106.9, 100.6, 86.8, 86.0, 30.5, 22.0, 21.9, 18.4, 18.3. The analytical data obtained were found to be consistent with the previous literature47.
Synthesis of diiodo(p-cymene)ruthenium(II) dimer (compound 4)
The diiodo(p-cymene)ruthenium(II) dimer was synthetized using a similar procedure as previously reported12. The dichloro(p-cymene)ruthenium(II) (1.06 mmol, 650 mg) was dissolved in water (250 mL). The solution was heated reflux for 1 h, filtered instantly. Potassium iodide (4.45 g, 26.8 mmol) was added to the filtrate under stirring conditions. The brown-red precipitate formed that collected by filtration. The solid was washed with ethanol (30 mL) and ether (30 mL). A purplish-red solid product was obtained. Yield: 941 mg, 91%. 1H NMR (400 MHz, DMSO-d6) δ = 5.85 (q, J = 6.3 Hz, 4H), 3.15 (hept, J = 6.9 Hz, 1H), 2.39 (s, 3H), 1.21 (d, J = 6.9 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ = 110.6, 102.5, 88.3, 86.5, 31.8, 22.5, 20.5, 20.4. The analytical data obtained were found to be consistent with the previous literature12.
Synthesis of compound Ru(II)-OH
The diiodo(p-cymene)ruthenium(II) dimer (54.8 mg, 0.05 mmol) was dissolved in methanol (20 mL) under 40 °C. A solution of 3-(4-(pyridin-2-yldiazenyl)phenoxy)propan-1-ol (26 mg, 0.1 mmol) in methanol (10 mL) was dropwise added. The reaction solution gradually becomes black, stirred at room temperature for 3 h, the solvent is concentrated to 10 mL by distillation under reduced pressure. Ammonium hexafluorophosphate (83 mg, 0.5 mmol) was added to the solution, which was then stirred for 1 h. The brown-black solid product was obtained through silica gel column chromatography. Yield: 44 mg, 57%. 1H NMR (400 MHz, Chloroform-d) δ = 9.30 (d, J = 5.7 Hz, 1H), 8.50 (d, J = 7.9 Hz, 1H), 8.21 – 8.07 (m, 3H), 7.69 (t, J = 6.8 Hz, 1H), 7.09 (dd, J = 9.3, 4.7 Hz, 2H), 6.17 (d, J = 6.3 Hz, 1H), 5.85 (dd, J = 15.4, 6.5 Hz, 2H), 5.69 (d, J = 6.3 Hz, 1H), 4.37 – 4.28 (m, 2H), 3.92 (s, 2H), 2.50 (s, 3H), 2.33 – 2.21 (m, 1H), 2.14 (p, J = 6.1 Hz, 2H), 1.08 (d, J = 6.9 Hz, 3H), 0.99 (d, J = 6.9 Hz, 3H).13C NMR (100 MHz, DMSO-d6) δ = 164.6, 163.5, 156.2, 151.5, 141.8, 128.5, 127.9, 127.7, 115.5, 111.7, 108.7, 91.6, 90.7, 89. 6, 66.3, 57.5, 22.2, 21.7, 20.9. HR-ESI (positive ion mode): 620.0349 m/z [M]+; calcd for C24H29IN3O2Ru+ 620.0343 m/z [M]+.
Synthesis of the 2,2’-(propane-2,2-diylbis(sulfanediyl))bis(ethan-1-ol)
The 2,2’-(propane-2,2-diylbis(sulfanediyl))bis(ethan-1-ol) was synthetized using a similar procedure as previously reported48. Mercaptoacetic acid (19.82 g, 215.15 mmol) and trifluoroacetic acid (TFA, 100 µL) were dissolved in acetone (5.00 g, 86.09 mmol). The reaction was carried out at 25 °C for 24 h. Then, acetonitrile was added at 85 °C until the components were dissolved completely. The resulting acetonitrile solution was slowly cooled to give 2,2’-(propane-2,2-diylbis(sulfanediyl))diacetic acid.
Lithium aluminum hydride (7.50 g, 197.63 mmol) was added into a solution of 2,2’-(propane-2,2-diylbis(sulfanediyl))diacetic acid (11.10 g, 49.55 mmol) in dry tetrahydrofuran (200 mL). The mixture was stirred at room temperature for 12 h. Subsequently, the reaction system was diluted with ethyl acetate (200 mL × 3), followed by washing with water (100 mL × 3) and saturated sodium chloride solution (100 mL × 3). The organic layer was dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and purified by silica gel column chromatography to give light-yellow oil pruducts 2,2’-(propane-2,2-diylbis(sulfanediyl))bis(ethan-1-ol). Yield: 4.25 g, 43.8%. 1H NMR (400 MHz, DMSO-d6) δ = 4.78 (s, 2H), 3.52 (t, J = 7.0 Hz, 4H), 2.66 (t, J = 7.1 Hz, 4H), 1.53 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ = 61.2, 55.8, 33.2, 31.4. The analytical data obtained were found to be consistent with the existing literature48.
Synthesis of ROS-sensitive polymers (P1)
To a solution of 1 h,1 h,10 h,10h-perfluoro-1,10-decanediol (0.62 g, 1.35 mmol) and ROS sensitive molecular (2,2’-(propane-2,2-diylbis(sulfanediyl))bis(ehan-1-ol)) (0.26 g, 1.35 mmol) in anhydrous DMF (5 mL), 1,2,4,5-cyclohexanetetracarboxylic dianhydride (0.63 g, 2.83 mmol) was quickly added. Following 8 h of magnetic stirring at 25 °C, mPEG5000-OH (1.35 g, 0.27 mmol) was incorporated into the reaction system. The stirring continued for an additional 12 h at 50 °C, after which the mixture was introduced to 10 mL of deionized water while being sonicated. Subsequently, the solution was subjected to dialysis using a dialysis bag with a molecular weight cutoff of 8000–14,000 Da. After 72 h, the resulting solution was freeze-dried by using low-temperature freeze dryers, yielding P1(1.23 g) as a yellow powder. 1H NMR (400 MHz, DMSO-d6) was shown in Supplementary Fig. 16.
Synthesis of ROS-sensitive polymers conjugated with Ru(II)-OH (P2)
4-Dimethylaminopyridine (DMAP, 126 mg) was added at room temperature to a solution of P1 (300 mg) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 200 mg). Following an additional 1 h of stirring at 25 °C, Ru(II)-OH (50 mg) was introduced into the reaction system. After an additional 12 h of stirring at room temperature, the solution was introduced into 10 mL of deionized water while undergoing sonication. This was followed by dialysis using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. Following a 72 h dialysis period, the solution was freeze-dried by using low-temperature freeze dryers, resulting in P2 (287 mg), which appeared as a brown powder. 1H NMR (400 MHz, DMSO-d6) was shown in Supplementary Fig. 18.
Synthesis of ROS-sensitive polymers (P3)
To a solution of ROS sensitive compound (2,2’-(propane-2,2-diylbis(sulfanediyl))bis(ehan-1-ol)) (0.26 g, 1.35 mmol) in anhydrous DMF (5 ml), 1,2,4,5-cyclohexanetetracarboxylic dianhydride (0.31 g, 1.41 mmol) was quickly added into the solution. After undergoing magnetic stirring for 8 h at room temperature, mPEG5000-OH (0.68 g, 0.13 mmol) was introduced into the reaction system. The stirring process continued for another 12 h at 50 °C, after which the mixture was introduced into 10 mL of deionized water while being sonicated. Following this, the solution was subjected to dialysis using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. After 72 h of dialysis, the resulting solution was freeze-dried by using low-temperature freeze dryers, yielding P3 (0.62 g), which was obtained as a white powder. 1H NMR, 19F NMR (400 MHz, DMSO-d6) was shown in Supplementary Fig. 19.
Synthesis of ROS-sensitive polymers conjugated with Ru(II)-OH (P4)
At room temperature, 4-Dimethylaminopyridine (DMAP, 126 mg) was introduced to a solution of Ru(II)-OH (50 mg) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 200 mg). After stirring for another 1 h at 25 °C, P3 (300 mg) was introduced to the reaction mixture. After an 1 h of magnetic stirring at room temperature, P3 (300 mg) was introduced into the reaction system. The stirring continued for another 12 h at room temperature, after which the mixture was transferred into 10 mL of deionized water while being sonicated. Subsequently, the solution underwent dialysis using a dialysis bag with a molecular weight cutoff of 8000–14,000 Da. Following 72 h of dialysis, the resulting solution was by using low-temperature freeze dryers, which yielded P4 (301 mg), presented as a brown powder. 1H NMR (400 MHz, DMSO-d6) was shown in Supplementary Fig. 21.
General procedure for experimental methods
NMR analysis
The conversion of GSH into GSSG was monitored by NMR (Bruker Avance 400). 1H-NMR spectra of GSH (50 mM) in the presence of Ru(II)-OH (2 mM) were directly conducted after preparation and after incubation for 12 h.
Mass spectrometry
The conversion of GSH into GSSG was monitored by mass spectrometry (Agilent 1290 UPLC/6540 Q-TOF). High resolution mass spectrometry of GSH (50 mM) in the presence of Ru(II)-OH (2 mM) were directly conducted after preparation and after incubation for 12 h. Each experiment was replicated twice.
Ultra performance liquid chromatography (UPLC) analysis
The conversion of GSH into GSSG was monitored by UPLC (Agilent 1290 Infinity II) equipped with a Poroshell 120 EC-C18 column (4.6 × 150 mm, 4 μm, 120 Å) at a flow rate of 1.0 mL/min at 37 °C. The UPLC mobile phase consisted of a linear gradient of the two mixtures of 0.1% TFA in water and 0.1% TFA in acetonitrile (ACN). The chromatogram was recorded via UV absorption at 210 nm.
Catalytic activity of Ru(II)-OH
For kinetic analysis, the redox reaction of glutathione (GSH) was conducted at 37 °C. Ru(II)-OH with a concentration of 50 µM was incubated with different concentrations of GSH substrates (250 µM, 500 µM, 1000 µM and 2000 µM). The GSH and GSSG contents were analyzed after incubation for 0, 1, 2, 3, 4, 5, 6, 8, 10 and 12 h. The kinetic data were analyzed by using the GraphPad Prism software with a simple pseudo first-order reaction model to estimate the apparent kinetic parameters.
where “S” is the substrate (GSH), v is the reaction rate, k1 is the apparent first-order rate constant k1 was calculated based on the slope of fitted v-[S] curve.
To calculate the turnover number (TON) and the turnover frequency (TOF), 2000 µM GSH substrates were incubated with 50 µM Ru(II)-OH. The reaction mixtures were subjected to UPLC assay after 12 h incubation at 37 °C. The ratios of starting materials and the calculated TON and TOF in the reactions.
Preparation of nanoparticles
P2 (100 mg) or P4 (100 mg) was dissolved in 1 mL DMSO, and the solution were then slowly added dropwise to 10 mL of water. Unloaded drugs and organic solvent were removed through dialysis against deionized water for 48 h. The obtained NP2 and NP4 were stored at 4 °C for subsequent use. The concentration of Ru(II)-OH in nanoparticles were assessed via ICP-MS.
Dyes (Cy5.5, Cy7.5, or Nile Red) and P4 (100 mg) were co-dissolved in 1 mL DMSO, and the solution were then slowly added dropwise to 10 mL of water. Unloaded dyes and organic solvent were removed through dialysis against deionized water for 48 h. The obtained NP4-Cy5.5, NP4-Cy7.5, and NP4-NR were stored at 4 °C for subsequent use.
General characterization of nanoparticles
The size distribution of NP2 and NP4 was detected via a dynamic light scattering device (Malvern Zetasizer Nano, UK). The morphology and shape of NP2 and NP4 was measured with a TEM.
Ru release of NP4
To examine the release profile of the nanoparticles, NP4 (Ru concentration: 800 μM, 3 mL) was placed in a dialysis bag with a molecular weight cutoff of 3500 Da. This dialysis bag was then immersed in either 200 mL of phosphate-buffered saline (PBS) or a 200 mL aqueous solution of hydrogen peroxide (H2O2) at a concentration of 10 mM, all while being maintained in a shaking culture incubator at 37 °C. At specified time intervals (0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h), the sample solution (1.5 mL) was withdrawn from the dialysate, after which an equal volume of fresh corresponding solution (1.5 mL) was promptly added back into the dialysate to maintain the overall volume. All collected samples were subsequently analyzed using ICP-MS. The ratio of ruthenium released from the nanoparticles was calculated as the percentage of cumulative ruthenium in the dialysate relative to the total amount of ruthenium present in the nanoparticles.
Measurement of oxygen loading capacity of NP4
PBS, NP2, and NP4 were firstly bubbled with the flowing N2 to exclude the content of O2. Afterward, the PBS, NP2, and NP4 solutions were bubbled with the flowing O2, where the O2 concentration was monitored using a portable dissolved oxygen meter (Hanna S20 Instruments HI-2004-02 Edge® Dissolved Oxygen Meter) for 5 min. The values were recorded automatically every 30 s.
Cell culture
All cell lines (K7M2 (CRL-2836), 143B (CRL-8303), HOS (CRL-1543)) were obtained from the American Type Culture Collection (ATCC). These cell lines are not on the list of known misidentified cell line maintained by the International Cell Line Authentication Committee. The 143B and HOS cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% P/S. The K7M2 cells were maintained in DMEM, also supplemented with 10% FBS and 1% P/S. Mycoplasma tests were performed monthly and were found to be negative.
Cell uptake of nanoparticles
The cellular uptake of nanoparticles was monitored by CLSM. Briefly, Poly-lysine cell crawls were placed in a 12-well plate. Following this, cell media containing 143B cells at a density of 3 × 105 were added and incubated overnight in a cell culture incubator. Subsequently, the cells were incubated with Cy5.5-labeled NP4 for varying time intervals of 1 h, 4 h, or 7 h, respectively. After being washed with cold PBS, the cells were fixed with paraformaldehyde. Cell nuclei were stained with DAPI. Subsequently, images were collected with CLSM (LSM-800, ZEISS, Germany) (DAPI, λex = 405 nm, λem = 460 nm, Cy5.5, λex = 673 nm, λem = 692 nm).
Flow cytometry was further applied to observe the cellular uptake of nanoparticles. 143B cells were plated in 12-well plates at a density of 3 × 105 cells/well and subsequently cultured for 12 h. Cell were then treated with Cy5.5-labeled NP4 for 1 h, 4 h, or 7 h, respectively, washed with PBS, and analyzed by flow cytometry (Beckman Coulter, U.S.A.).
The uptake and apoptosis test in 3D tumor spheroids
To establish 3D tumor spheroids, 50 μL of a 1% agarose gel solution (Aladdin) was added into each well of a 96-well plate. Subsequently, 200 μL media containing 143B cells at a density of 2×103 was added to each well. At day 7, the formation of cell spheres was completed. For analysis of cellular uptake of NP4, the 3D spheroids were treated with Cy5.5-labeled NP4 for 12 h. After washing with cold PBS, the cellular uptake of NP4 was measured via CLSM. For the apoptosis test, the spheroids were treated with PBS, Ru(II)-OH, NP2, or NP4 for 24 h, respectively (Ru 10 μM). After being washed with cold PBS, the spheroids were stained with Calcein AM/PI Cell Viability Kit (Beyotime). Subsequently, images were collected with CLSM.
Cell viability assays
To evaluate cell viability, 143B and K7M2 cells were seeded in 96-well plates at a density of 5 × 103 cells per well and cultured for 24 h. Subsequently, the cells were treated with PBS, Ru(II)-OH, NP2, or NP4 at various final concentrations ranging from 0.025 μM to 15 μM for 48 h. Subsequently, cells were then incubated with 10% MTT for 4 h before measurement at 570 nm.
Apoptosis analysis
Cellular apoptosis was assessed with an Annexin V-FITC apoptosis detection kit (Beyotime) according to the manufacturer’s instructions. In brief, 143B cells were seeded on 12-well plates at 3 × 105 cells per well. After 12 h incubation, cells were treated with PBS, Ru(II)-OH, NP2, or NP4 for 24 h (Ru 10 μM), washed with PBS and incubated with Annexin/PI reagent in the dark for 15 min at 25 °C. Thereafter, the cells were immediately detected by flow cytometer.
Intracellular hypoxia detection
Hypoxyprobe™-1 (Hypoxyprobe Inc, Burlington, USA) is a substituted 2-nitroimidazole compound known as pimonidazole. Since it can bind to cells if the pO2 levels are less than 10 mm Hg, Hypoxyprobe™-1 is used as a probe for intracellular hypoxia detection. The 143B cells were cultured to 80% confluence, and incubated with 200 μM hypoxyprobe and PBS, Ru(II)-OH, NP2, or NP4 (Ru 10 μM) under hypoxia condition for 12 h. Following incubation, the 143B cells were fixed and blocked with 3% bovine serum albumin (BSA) and then treated with anti-hypoxyprobe antibody and DAPI. A blank control group was subjected to the same protocol. All treated 143B cells were subsequently analyzed using CLSM.
Measurement of ROS production
To measure intracellular ROS, the 143B cells were plated in 12-well plates at a density of 3 × 105 cells per well. Following a 12 h incubation period, the 143B cells were incubated with PBS, Ru(II)-OH, NP2, or NP4 (10 μM Ru) for 6 h. The cells were subsequently treated with DCFH-DA (10 μM, Bio-lab) for 1 h in the dark, and washed three times with PBS. The fluorescence was then analyzed using flow cytometer and CLSM.
Measurement of GSH/GSSG level
For GSH/GSSG level, 143B cells were plated in 12-well plates. Subsequently, the 143B cells were incubated with PBS, Ru(II)-OH, NP2, and NP4 (Ru 10 μM) for 6 h. The GSH and GSSG amount was evaluated using GSH/GSSG assay kit.
Intracellular lipid peroxide (LPO) detection
To detect the intracellular LPO content, 143B cells were incubated with PBS, Ru(II)-OH, NP2, and NP4 (Ru 10 μM) for 6 h. Subsequently, the cells were washed with PBS and stained by C11-BODIPY581/591 (5 μM) for 20 min. Then the cells were analyzed by CLSM and flow cytometer.
Intracellular hydrogen peroxide (H2O2) detection
To detect the intracellular H2O2 content, 143B cells were incubated with PBS, Ru(II)-OH, NP2, and NP4 (Ru 10 μM) for 6 h. Subsequently, the cells were washed with PBS and stained by ROS GreenTM H2O2 Probe. Then the cells were analyzed by CLSM.
Measurement of intracellular and cell supernatant H2O2 levels
For intracellular and cell supernatant H2O2 levels, 143B cells were plated in 6-well plates. Subsequently, the 143B cells were incubated with PBS, Ru(II)-OH, NP2, and NP4 (Ru 10 μM) for 6 h. The intracellular and cell supernatant H2O2 amount was evaluated using H2O2 assay kit.
Analysis of calcium ion (Ca2+) levels in macrophages
To detect the intracellular Ca2+ content, 143B cells were incubated with PBS, Ru(II)-OH, NP2, and NP4 (Ru 10 μM) for 6 h. The medium was replaced, and after 8 h the cell supernatant was collected and co-incubated with RAW264.7 cells for 4 h. Subsequently, the RAW264.7 cells were washed with PBS and stained by Ca2+ Probe Fluo-4 AM for 20 min. Then the cells were analyzed by CLSM.
Western blot assay
K7M2 cells were incubated with the PBS, Ru(II)-OH, NP2, or NP4 (10 μM Ru) for 12 h at 37 °C, and the supernatant was subsequently co-incubated with RAW264.7 cells. Then, the RAW264.7 cells were lysed using RIPA lysis buffer supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF). The cells were lysed by ultrasound for 20 min at 4 °C, followed by centrifugation at 12,000 rpm for 15 min to collect the supernatant. The protein concentration was quantified using a BCA kit, and then SDS-PAGE buffer was added. Following this, the protein lysates were denatured in a metal bath at 95 °C and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for separation. The separated proteins were then transferred onto a PVDF membrane (Merck Millipore) and blocked using skim milk for 2 h at room temperature. After blocking, the membrane was incubated overnight at 4 °C with the specified primary antibodies, including TRPM2 antibody at a dilution of 1:1000 and GAPDH antibody at a dilution of 1:3000. The PVDF membrane was subsequently washed (5 min × 5) with tris-buffered saline with tween (TBST) buffer and then incubated with the appropriate secondary antibody (Peroxidase-conjugated goat anti-rabbit IgG (H + L), at a dilution of 1:5000) in a 5% bovine serum albumin solution for 1 h at 25 °C. After further washing with TBST buffer, the membrane was photographed using the chemiluminescent imaging system.
Biosafety assay of NP4
The biosafety of Ru(II)-OH and NP4 (Ru 800 μΜ, 200 μL) were evaluated in healthy female BABL/c mice. Twelve mice were randomly divided into 4 groups and injected into the tail vein with PBS, Ru(II)-OH, NP2, and NP4 (2 mg Ru/kg) on the days 1, 4, and 7. On day 14, the mice were euthanized, and a histological analysis of the major organs was performed using H&E staining.
Pharmacokinetic assay of NP4
The pharmacokinetics of Ru(II)-OH and NP4 (Ru 800 μΜ, 200 μL) were evaluated in healthy female BABL/c mice (5 weeks). Six mice were randomly divided into 2 groups and injected into the tail vein with Ru(II)-OH and NP4, respectively, and 10 μL of blood was collected from the tail tip at 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h. Finally, the blood samples were nitrated with nitric acid, and the ruthenium content was determined by ICP-MS.
Establishment of K7M2-Luc orthotopic osteosarcoma mouse model and therapeutic effect evaluation
Tumor model establishment
To establish orthotopic model, K7M2 cells (3 × 106 cells/well) were dispersed in PBS buffer and implanted into right tibia of BALB/c mice.
Biodistribution study
The biodistribution of NP4 in orthotopic osteosarcoma mouse model was investigated using in vivo imaging system (IVIS). Ten 5-week-old female BALB/c mice bearing tumors were randomly assigned into two groups. When the tumor volume reached approximately 200 mm³, the mice were intravenously administered with Cy7.5-labeled NP4 at a dosage of 2 mg/kg. Following the injection, in vivo imaging was conducted using an IVIS Spectrum (PerkinElmer) at various time points: 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h post-injection, utilizing an excitation wavelength of 780 nm and a fluorescence emission wavelength of 840 nm. At 48 h post-injection, the mice were euthanized to collect tumors and major organs for subsequent ex vivo imaging.
The biodistribution of NP4 was studied by using ICP-MS. Six 5-week-old female BALB/c mice bearing tumors were randomly assigned into two groups. When the tumor size reached approximately 200 mm³, the mice received intravenous injections of Ru(II)-OH and NP4 at a dosage of 2 mg Ru/kg. After 48 h, the mice were sacrificed, and the heart, liver, spleen, lung, kidney, and tumor tissues were subsequently isolated, weighed, and nitrated for further analysis. Finally, the ruthenium content in each organ and tumor tissue was quantified by ICP-MS.
Therapeutic effect evaluation
Twenty tumor-bearing female BALB/c mice, each aged 5 weeks, were randomly assigned into four groups, with each group comprising five mice. When the size of tumor reached 100 mm3, the mice were intravenous injected with PBS, Ru(II)-OH, NP2, and NP4 at the dose of 2 mg Ru/kg. The tumor volume was monitored through imaging using an IVIS Spectrum system (PerkinElmer) at 0-day, 4-day, 8-day, and 12-day postinjection, respectively (100 µL luciferin—per mouse, 15 mg ml−1).
Flow cytometry analysis of the animal tissue
Female BALB/c mice (5 weeks old) were implanted with K7M2 cells (3 × 106) in the right tibia. Once the tumor volume reached approximately 100 mm3, twelve mice were assigned at random (n = 3) and received the same treatment as previously described. Mice were sacrificed at day 12. The harvested tumors and draining lymph nodes were utilized to prepare single-cell suspensions. The cells from tumor tissue were stained with anti-CD3-PE, anti-CD4-APC, and anti-CD8-FITC antibodies for 30 min, and further analyzed the ratio of different types T cells by flow cytometry. the cells from tumor and draining lymph nodes tissue were stained with anti-CD11c-PE, anti-CD80-FITC, and anti-CD86-APC antibodies for 30 min, and further analyzed the ratio of mature dendritic cells (DCs). All antibodies used in this study were purchased from Dakewe Biotech Co., Ltd. Data acquisition and processing for flow cytometry were conducted using CytExpert software.
Establishment of 143B subcutaneous osteosarcoma mouse model and therapeutic effect evaluation
Tumor model establishment and biodistribution study of NP4 in vivo
Three female BALB/c nude mice were subcutaneously inoculated with 143B human osteosarcoma cells (3 × 106) on the right buttock. The tumor-bearing mice were intravenously administered Cy7.5-labeled NP4 at a dosage of 2 mg/kg (Ru) once the tumor volume reached approximately 200 mm³. Following the injection, imaging was performed using an IVIS Spectrum system (PerkinElmer) at various time points: 1 h, 4 h, 7 h, 12 h, 24 h, 36 h, 48 h, and 60 h post-injection, with an excitation wavelength of 780 nm and a fluorescence emission wavelength of 840 nm. At 60 h post-injection, the mice were euthanized in order to collect tumor tissues and major organs for subsequent ex vivo imaging.
In vivo antitumor efficacy assessment
When the tumor volumes reached approximately 100 mm³, twenty female BALB/c nude mice bearing 143B tumors were randomly assigned into four groups. Subsequently, they received intravenous injections of PBS, Ru(II)-OH, NP2, or NP4 (at a dosage of 2 mg/kg Ru) on days 0, 3, 6, and 9, respectively. The tumor volume and body weight of all the mice were recorded every two days. The tumor volume (in mm³) was calculated using the formula V = (a × b²)/2, where “a” represents the length and “b” denotes the width of the tumor.
Establishment of PDXOS model and therapeutic effect evaluation
The osteosarcoma patient-derived xenograft animal model (PDX) was established in female BALB/c nude mice. The tumor tissue of osteosarcoma was obtained, cut into small fragment, and transplanted subcutaneously into BALB/c nude mice.
When the tumor volumes reached approximately 100 mm3, twenty female BALB/c nude mice bearing 143B tumors were randomly assigned into four groups. The mice were injected intravenously with PBS, Ru(II)-OH, NP2, NP4 (2 mg/kg Ru) on days 1, 4, 7, 10, respectively. The tumor volume and body weight of all the mice were recorded every two days. The tumor volume (in mm³) was calculated using the formula V = (a × b²)/2, where “a” represents the length of the tumor and “b” denotes its width.
Histopathological analysis
The solid tumor and major organs were harvested from tumor-bearing mice for histological observation by standard H&E and immunofluorescence staining. For H&E staining, the excised tumor and organs were fixed in 4% paraformaldehyde solution, embedded in paraffin, sectioned, and stained with H&E. The nuclei were counterstained using DAPI (Thermo Fisher Scientific), after which the stained sections were imaged with a confocal microscope (LSM880, Zeiss).
Statistical analysis
All statistical analyses were conducted by utilizing GraphPad Prism 10 (GraphPad Software). Data are expressed as mean ± standard deviation (SD) from at least three independent experiments unless otherwise specified in the figure legend. Statistical significance between pairs of groups was assessed using an unpaired two-sided t-test, one-way ANOVA followed by the Dunnett multiple comparison test, or two-way ANOVA with the Tukey multiple comparison test, as indicated. The number of replicates performed is detailed in each figure legend, where applicable.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.