Materials and characterization
Aldehydes and ketone derivatives were got from Bidepharm. Diethylamine, nitromethane, ethanol, ammonium acetate, and diisopropyl ethylamine were acquired from Macklin. Aldehydes and ketone derivatives were used directly. Other solvents were used without further purification. Nuclear paramagnetic resonance (Bruker Ultra Shield Plus) and mass spectra (Bruker) were used to reveal the chemical structures of compounds. TEM and dynamic light scattering (Nano ZS90) were used for confirming morphology and particle size of nanoparticles, respectively. An ultraviolet−visible light (UV-Vis) spectrophotometer (Cintra 2020) and spectrofluorometer (Horiba Fluoromax-4) were used to record the absorption emission spectra of the samples. Confocal luminescence imaging was conducted using an Olympus IX81 laser scanning confocal microscope. Photothermal images were measured with an NIR thermal imager (FLIR E40). The power density was measured with a VLP−2000 laser power meter.
Photothermal effect of samples
Temperature changes in the sample solutions were monitored with an infrared thermal imaging system. Temperature changes of the sample solution (300 μL) in different concentrations using different levels of light power were obtained. The processes of temperature increase and decrease in the sample solution (20 μM) with irradiation (808 nm, 0.5 W cm−2) and without irradiation were obtained. Finally, the photothermal conversion efficiency of the samples was obtained following the methods of a previous study.23
PA signal of NIR998 NPs
A concentration-dependent PA signal of NIR998 NPs was obtained using a point-to-point method. The PA signal of NIR998 NPs in different concentrations was obtained by monitoring the region of interest, λEx = 808 nm.
Cytotoxicity assay
The dark toxicity assay of NIR998 NPs and SKOV3 cells was launched using the standard methyl thiazolyl tetrazolium experiments. SKOV3 cells were treated with NIR998 NPs in different concentrations for 24 h. Then, MTT (10 μL/well, 5 mg/mL) was added for further 4 h incubation. Then, 150 μL DMSO was added, and OD570 was measured with an enzyme-linked immunosorbent assay reader. Next, the cell viability was obtained following the method of a previous report.23 In addition, SKOV3 cells were treated with PBS or NIR998 NPs (20 μM). They were irradiated (808 nm) in various light power conditions for 6 min. Finally, they were treated following the steps used for the 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); or NIR998 NPs plus irradiation. The redundant NIR998 NPs were washed with fresh complete medium. Propidium iodide (PI) and annexin V-FITC were used to differentiate between dead cells and apoptotic cells for the subsequent confocal microscopy and flow cytometry analyses.
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 approval from the Medical Department of Shenzhen University.
In vivo photothermal imaging exploration
All imaging and therapy exploration of mice were launched in line with standard principles and guidelines. SKOV3-tumor-bearing nude mice with tumor volume of approximately 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).
Pharmacokinetics study
The pharmacokinetics study was conducted by monitoring the NIR absorption of NIR998 NPs (150 μL, 200 μM) in serum with time after their intravenous injection in mice. The absorption of free NIR998 obtained from major organs and tumors was used to assess the biodistribution and accumulation of NIR998 NPs after intravenous injection (200 μM, 150 μL) for 6 h. The major organs and tumors were collected, broken, digested, ultrasonic destroyed after nanoparticles injection in different time. Then, the free molecule was extracted to test their near-infrared absorption.
In vivo photothermal efficacy
SKOV3 tumor-bearing mice with a tumor volume about 100 mm3 were assigned into four groups. They were treated with I) NIR998 NPs (150 μL, 200 μM) + light irradiation (808 nm, 0.5 W cm−2, 6 min); II) NIR998 NPs; III) light irradiation; or IV) PBS. 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, where L and W are the tumor length and width, respectively. The body weight and tumor size of mice were collected every two days for 30 days. At day 30, the major organs and blood of mice were collected for H&E 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.
A concentration-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.