Materials and characterization
Aldehydes and ketone derivatives were got from Bidepharm. Diethylamine, nitromethane, ethanol, ammonium acetate, diisopropyl ethylamine were acquired from Macklin. Aldehydes and ketone derivatives were used directly. Other solvent were used without further purification. The nuclear paramagnetic resonance (Bruker Ultra Shield Plus) and Mass spectra (Bruker) were used for demonstrating the chemical structures of compounds. Transmission electron microscopy and dynamic light scattering (Nano ZS90) were used for confirming morphology and particle size of nanoparticles, respectively. Ultraviolet−visiblelight (UV-Vis) spectrophotometer (Cintra 2020) and spectrofluorometer (Horiba Fluoromax-4) are used to record the absorption emission spectra of samples. Confocal luminescence imaging was conducted by an Olympus IX81 laser scanning confocal microscope. Photothermal images were measured by NIR thermal imager (FLIR E40). The power density meter was obtained by VLP−2000 laser power meter.
Photothermal effect of samples
The temperature change of samples solution was monitored by infrared thermal imaging system. The temperature change of samples (300 μL) solution in different concentrations or light power was obtained. The temperature increase and decrease process of samples (20 μM) solution with irradiation (808 nm, 0.5 W cm-2) or not was obtained. Finally, samples photothermal conversion efficiency was got referring to previous work.19
PA signal of NIR998 NPs
Concentrations-dependent PA signal of NIR998 NPs was got via a point-to-point method. The PA signal of NIR998 NPs in different concentrations was got by monitoring region of interest, respectively, λEx = 808 nm.
Cytotoxicity assay
The dark toxicity assay of NIR998 NPs to SKOV3 cells was launched by the standared methyl thiazolyl tetrazolium experiments. SKOV3 cells were treated with NIR998 NPs in different concentrations for 24 h, respectively. Then, MTT (10 μL/well, 5 mg/mL) was added for further 4 h incubation. Finally, 150 μL of DMSO was added, and OD570 was measured by an enzyme-linked immunesorbent assay reader. Then cell viability was obtained referring to previous report.19 In addition, SKOV3 cells were treated with PBS and NIR998 NPs (20 μM), respectively. They were irradiated (808 nm) in various light power for 6 min. Then, they were treated following above operation.
In vitro Photothermal toxicity of NIR998 NPs
SKOV3 cells were treated with PBS (pH = 7.4), light irradiation (808 nm, 0.5 W cm-2, 6 min), NIR998 NPs (20 μM), NIR998 NPs plus irradiation, respectively. The redundant NIR998 NPs were washed with fresh complete medium. Propidium iodide (PI) and annexin V-FITC were used for differentiating dead cells and apoptotic cells for the following confocal microscopy and flow cytometry.
Animals and tumor model
Female mice were purchased from Medical Animal Laboratory Center of Guangdong (Permit number: 44007200079864). All in vivo experiments were performed with the approval of the Medical Department of Shenzhen University.
In vivo photothermal imaging exploration
All imaging and therapy exploration of mice are launched in line with standard principles and guidelines. SKOV3 tumor bearing nude mice with tumor volume about 100 mm3 were used for photothermal imaging of mice under irradiation (808 nm, 0.5 W cm-2, 6 min) before and after intravenous injection of NIR998 NPs (150 μL, 200 μM) with time.
In vivo photothermal efficacy
SKOV3 tumor-bearing mice with tumor volume about 100 mm3 were assigned into four groups. They were treated with NIR998 NPs (150 μL, 200 μM) + light irradiation (808 nm, 0.5 W cm-2, 6 min), NIR998 NPs, light irradiation, PBS, respectively. The first group served as experimental groups, and the rest groups worked as control groups. The tumors were irradiated after intravenous injection of NIR998 NPs for 6 h. The size of SKOV3 was obtained by V = LW2/2, L and W are length and width of tumors, respectively. Body weight and tumor size of mice were collected every two days for 30 days. At day 30, major organs and blood of mice were collected for hematoxylin-eosin staining and immunohistochemical analyses, respectively.
Synthesis of NIR-II fluorophores
Synthesis and Characterization of NIR998. 1-3 (0.31 g, 0.40 mmol) dissolved into the mixture of diisopropylethylamine (4 mL) and dry CH2Cl2 (20 mL) at 0 oC. Then, BF3·OEt2 (6.40 mmol) was dropped. They reacted for 8 h at 0 oC. The final reaction solution was quenched by methanol. The precipitate was obtained by vacuum filter. The final blue solid NIR998 (0.29 g, Yield: 91 %) was acquired by column chromatography. 1H NMR (400 MHz, CDCl3) δ(ppm) = 8.08 (d, J = 7.2 Hz, 4H), 7.96 (d, J = 8.4 Hz, 2H), 7.80 (s, 2H), 6.85 (s, 2H), 6.72 (d, J = 7.6 Hz, 4H), 6.65 (d, J = 8.4 Hz, 2H), 3.50 – 3.31 (m, 16H), 2.85 (t, J = 5.2 Hz, 4H), 2.01 (t, J = 4.8 Hz, 4H), 1.23 – 1.18 (m, 18H). 13C NMR (100 MHz, CDCl3) δ(ppm) = 154.34, 147.83, 144.40, 139.94, 130.34, 128.90, 127.87, 121.05, 120.12, 118.23, 113.02, 110.03, 109.23, 47.67, 44.38, 43.40, 27.36, 21.21, 11.75, 10.08. MALDI-TOF-MS m/z: 804.992.
Synthesis and Characterization of NIR1028. 2-3 (0.31 g, 0.40 mmol) dissolved into the mixture of diisopropylethylamine (4 mL) and dry CH2Cl2 (20 mL) at 0 oC. Then BF3·OEt2 (6.40 mmol) was dropped. They reacted for 8 h at 0 oC. The final reaction solution was quenched by methanol. The precipitate was obtained by vacuum filter. The final blue solid NIR1028 (0.31 g, Yield: 93 %) was acquired by column chromatography. 1H NMR (400 MHz, CDCl3) δ(ppm) = 8.05 (d, J = 9.0 Hz, 4H), 7.59 (s, 4H), 6.81 (s, 2H), 6.71 (d, J = 9.0 Hz, 4H), 3.43 (q, J = 6.6 Hz, 8H), 3.24 (t, J = 5.4 Hz, 8H), 2.78 (t, J = 6.6 Hz, 8H), 2.02 – 1.97 (m, 8H), 1.21 (t, J = 7.2 Hz, 12H). 13C NMR (100 MHz, CDCl3) δ(ppm) = 155.15, 148.81, 144.87, 143.31, 141.31, 131.36, 128.18 , 121.23, 121.02, 119.35, 114.37, 111.03, 50.12, 44.42, 28.01, 21.96, 12.78. MALDI-TOF-MS m/z: 829.173.
Synthesis and Characterization of NIR980. 3-3 (0.29 g, 0.40 mmol) dissolved into the mixture of diisopropylethylamine (4 mL) and dry CH2Cl2 (20 mL) at 0 oC. Then BF3·OEt2 (6.40 mmol) was dropped. They reacted for 8 h at 0 oC. The final reaction solution was quenched by methanol. The precipitate was obtained by vacuum filter. The final blue solid NIR980 (0.28 g, Yield: 90 %) was acquired by column chromatography. 1H NMR (400 MHz, CDCl3) δ(ppm) = 8.19 – 8.09 (m, 4H), 7.68 – 7.60 (m, 2H), 7.44 – 7.33 (m, 4H), 7.18 – 7.13 (m, 4H), 7.00 – 6.96 (m, 2H), 6.83 – 6.73 (m, 4H), 4.44 (q, J = 13.2, 4H), 3.48 (q, J = 15.6, 8H), 1.43 (t, J = 6.4 Hz, 6H), 1.26 (t, J = 7.2 Hz, 12H). 13C NMR (100 MHz, CDCl3) δ(ppm) = 155.08, 154.11, 150.05, 148.64, 131.08, 128.47, 122.19, 120.154, 115.21, 116.23, 115.21, 110.13, 107.36, 47.61, 44.60, 15.60, 12.81. MALDI-TOF-MS m/z: 773.271.
Synthesis and Characterization of NIR1030. 4-3 (0.27 g, 0.40 mmol) dissolved into the mixture of diisopropylethylamine (4 mL) and dry CH2Cl2 (20 mL) at 0 oC. Then BF3·OEt2 (6.40 mmol) was dropped. They reacted for 8 h at 0 oC. The final reaction solution was quenched by methanol. The precipitate was obtained by vacuum filter. The final blue solid NIR1030 (0.28 g, Yield: 96 %) was acquired by column chromatography. 1H NMR (400 MHz, CDCl3) δ(ppm) = 8.18 (d, J = 3.6 Hz, 4H), 7.77 – 7.66 (m, 4H), 7.57 (d, J = 8.0 Hz, 2H), 7.38 – 7.28 (m, 6H), 6.76 (s, 4H), 3.48 (q, J = 12.8Hz, 8H), 1.26 (t, J = 7.2 Hz, 12H). 13C NMR (100 MHz, CDCl3) δ(ppm) = 156.08, 155.11, 150.65, 149.64, 132.08, 129.47, 125.24, 123.19, 121.54, 118.39, 116.23, 111.43, 111.13, 108.36, 44.60, 12.81. MALDI-TOF-MS m/z: 719.242.
Synthesis and Characterization of NIR1028-S. 5-3 (0.31 g, 0.40 mmol) dissolved into the mixture of diisopropylethylamine (4 mL) and dry CH2Cl2 (20 mL) at 0 oC. Then BF3·OEt2 (6.40 mmol) was dropped. They reacted for 8 h at 0 oC. The final reaction solution was quenched by methanol. The precipitate was obtained by vacuum filter. The final blue solid NIR1028-S (0.31 g, Yield: 93 %) was acquired by column chromatography. 1H NMR (400 MHz, CDCl3) δ(ppm) = 8.32 (s, 2H), 8.16 – 8.12 (m, 4H), 7.89 – 7.85 (m, 4H), 7.43 – 7.35 (m, 4H), 7.10 (s, 2H), 6.77 – 6.73 (m, 4H), 3.46 (q, J = 7.2 Hz, 8H), 1.24 (t, J = 7.2 Hz, 12H). 13C NMR (100 MHz, CDCl3) δ(ppm) = 156.00, 149.68, 145.01, 141.01, 140.48, 135.49, 134.43, 132.02, 125.45, 125.05, 124.64, 124.08, 122.21, 118.28, 117.07, 111.43, 44.61, 12.80. MALDI-TOF-MS m/z: 750.264.
The preparation of NIR998 NPs (ICG NPs). 20 mg DSPE−mPEG5000 dissolved into 8 mL deionized water under sonication (120 W, 2 min). 1.5 mg NIR998 (ICG) in 4 mL THF was dropped in above mixture quickly under sonication (120 W, 2 min). The prepared solution was stirred by blowing its surface with argon at 50 ºC overnight. The blue solution was washed with PBS (pH = 7.4) by a centrifugal-filter for three times. The final concentrated blue solution was used for next experiments.
Concentrations-dependent absorption of NIR998 in DMSO was acquired. The linear relationship between concentration and the absorption of NIR998 at 859 nm (maximal absorption) in DMSO was then obtained. NIR998 NPs solution (150 μL) was dried. Then, their absorption in DMSO was got. Finally, according to above linear relationship, the concentration of NIR998 NPs solution was obtained as about 400 μM.