Almost all reagents and chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA). Some solvents such as dichloromethane (CH2Cl2), methanol (MeOH), or MgSO4, sodium azide (NaN3), sodium ascorbate (NaAsc), Copper (II) sulfate pentahydrate (CuSO4.5H2O) were purchased from Daejung chemical (Gyeonggi-do, South Korea), and used without further purification. Lactose-propargyl was synthesized in our previous literature17.
All compounds were characterized by 1H and 13C-NMR spectroscopy on a Bruker AM 250 spectrometer (Billerica, MA, USA). The impurity of the products was checked by thin-layer chromatography (TLC, silica gel 60 mesh). UV spectra were measured on a Shimadzu UV-1650PC spectrometer, and Fluorescence spectra were carried on a Hitachi F-7000 spectrometer. The size and morphology of BODIPY NPs were analyzed by using dynamic light scattering (DLS) on Malvern Zetasizer Nano ZS90 and transmission electron microscopy (TEM). We used machine JEOL- JEM 2100F at an accelerating voltage of 200 kV. The sample for TEM was prepared according to our reported literature41.
Synthesize of water-soluble BODIPY I, H, OMe, and NO2:
According to our reported literature17, the series of water-soluble BODIPY I, H, OMe, and NO2 were prepared using the same pathway. A representative routine is presented for the compound I. Briefly, BODIPY 2a (80 mg, 0.157 mmol), lactose propargyl (66 mg, 0.173 mmol), NaAsc (156 mg, 0.785 mol), and CuSO4.5H2O (79 mg, 0.316 mmol) were dissolved in the mixture of THF/water (15/5 mL, v/v). The resulting mixture was stirred for one day at room temperature, extracted with THF and water three times, and dried over MgSO4. After removing the solvent by a rotary evaporator, the crude product was purified by recrystallization using MeOH/diethyl ether to afford an orange solid (yield 76 mg, 52% yield). 1H NMR (300MHz, CD3OD, δ, ppm): δ 8.02 (s, 1H), 7.97- 7.95 (d, 2H), 7.17- 7.15 (d, 2H), 5.83 (s, 2H), 4.36- 4.33 (d, 2H), 3.87- 3.85 (d, 2H), 3.81- 3.80 (d, 2H), 3.76- 3.74 (d, 1H), 3.71- 3.69 (d, 1H), 3.57- 3.53 (d, 2H), 3.49- 3.48 (d, 2H), 3.31- 3.29 (d, 2H), 2.57 (s, 3H), 2.42-2.39 (q, 4H), 1.44 (s, 3H), 1.36 (s, 3H), 1.28 (s, 3H), 1.05- 1.00 (t, 3H). 13C NMR (75MHz, CD3OD, δ, ppm): δ 162.25, 143.55, 143.24, 142.81, 140.13, 138.60, 137.3, 135.75, 134.58, 134.16, 131.48, 131.32, 105.09, 103.29, 96.18, 80.57, 77.05, 76.45, 76.29, 74.75, 74.58, 72.52, 70.38, 63.00, 62.42, 61.82, 46.12, 17.57, 17.32, 14.87, 14.76, 13.2, 12.45, 11.92. HRMS (ESI): calculated for (C38H49BF2IN5O11): m/z: [M]: 928.2613; found: 928.2619.
Compound H: BODIPY H was synthesized according to the general procedure to afford the orange solid (67 mg, 57% yield). 1H NMR (300MHz, CD3OD, δ, ppm): δ 8.01 (s, 1H), 7.59- 7.57 (t, 3H), 7.37- 7.36 (d, 2H), 5.84 (s, 2H), 4.37- 4.33 (d, 2H), 3.87- 3.85 (d, 2H), 3.81- 3.80 (d, 2H), 3.76- 3.73 (d, 1H), 3.69- 3.66 (d, 1H), 3.59- 3.57 (d, 2H), 3.54- 3.52 (d, 2H), 3.49- 3.32 (d, 2H), 2.53 (s, 3H), 2.43-2.38 (q, 4H), 1.38 (s, 3H), 1.31 (s, 3H), 1.28 (s, 3H), 1.04- 0.99 (t, 3H). 13C NMR (75MHz, CD3OD, δ, ppm): δ 161.82, 144.19, 143.61, 143.46, 138.71, 136.45, 134.29, 131.7, 130.75, 130.61, 129.43, 105.16, 103.36, 80.45, 77.26, 77.15, 76.41, 74.6, 72.4, 70.26, 62.96, 62.53, 61.77, 61.66, 48.07, 17.68, 14.85, 14.5, 13.16, 12.24, 11.66. HRMS (ESI): calculated for (C38H55BF2IN5O12Na): m/z: [M+Na]+: 834.3713; found: 834.3714.
Compound OMe: BODIPY OMe was synthesized according to the general procedure to afford the orange solid (55 mg, 47 % yield). 1H NMR (300MHz, CD3OD, δ, ppm): δ 8.0 (s, 1H), 7.23- 7.2 (d, 2H), 7.13- 7.1 (d, 2H), 5.87 (s, 2H), 4.37- 4.34 (d, 2H), 3.89 (s, 3H), 3.77- 3.71 (q, 3H), 3.6- 3.5 (m, 6H), 3 42- 3.39 (d, 1H), 2.56 (s, 3H), 2.4-2.38 (q, 4H), 1.43 (s, 3H), 1.36 (s, 3H), 1.28 (s, 3H), 1.01- 0.95 (t, 3H). 13C NMR (75MHz, CD3OD, δ, ppm): δ 162.15, 144.48, 143.03, 138.59, 136.87, 134.71, 134.18, 132.17, 130.55, 128.29, 116.06, 105.04, 103.33, 80.37, 77.08, 76.53, 76.22, 74.82, 74.64, 72.64, 70.21, 63.04, 62.43, 61.75, 55.8, 46.14, 17.73, 17.44, 14.87, 14.26, 13.28, 12.53, 11.56. HRMS (ESI): calculated for (C39H52BF2N5O12Na): m/z: [M+Na]+: 854.3571; found: 854.3572.
Compound NO2: BODIPY NO2 was synthesized according to the general procedure to afford the orange solid (80 mg, 61 % yield). 1H NMR (300MHz, CD3OD, δ, ppm): δ 8.47- 8.44 (d, 2H), 8.01 (s, 1H), 7.69- 7.66 (d, 2H), 5.83 (s, 2H), 4.37- 4.34 (d, 2H), 3.86- 3.32 (d, 2H), 3.79- 3.75 (d, 2H), 3.6- 3.5 (m, 6H), 3.31- 3.29 (d, 2H), 2.59 (s, 3H), 2.41- 2.39 (q, 4H), 1.39 (s, 3H), 1.31 (s, 3H), 1.29 (s, 3H), 1.04- 0.98 (t, 3H). 13C NMR (75MHz, CD3OD, δ, ppm): δ 163.11, 149.96, 143.16, 141.37, 138.46, 137.78, 134.63, 133.69, 131.64, 130.96, 125.76, 105.14, 103.6, 80.37, 77.35, 76.4, 76.24, 74.84, 74.35, 72.54, 70.36, 63.11, 62.44, 61.76, 46.06, 19.39, 17.86, 14.86, 14.46, 13.09, 12.63, 11.71. HRMS (ESI): calculated for (C38H49BF2N6O13Na): m/z: [M+Na]+ : 869.3316; found: 869.3318.
Measurement of photophysical properties
The Φf were measured by a comparative method using the standard reference with the already-known value of Φf as follows:
The subscript of S and R represent sample and reference, respectively. N is the refractive index of solvent. A and D are the absorbance and the integrated fluorescence area, respectively. Solutions should be optically dilute to avoid inner filter effects. The Rhodamine 6G was used as the reference sample, which possessed a known quantum yield of 0.9435.
Detection of singlet oxygen quantum yields
The quantum yields of singlet oxygen (ΦΔ) of I, H, OMe, and NO2 were studied using diphenylisobenzofuran (DBPF) as a chemical quencher36. Briefly, a mixture of the BODIPY dye (absorption ~0.06 at 515 nm in EtOH) and the DPBF (absorption ~1.0 at 424 nm in EtOH) was irradiated with green laser light (λmax = 520 nm). The photooxidation of DPBF was monitored between 0~70 min depending on the efficiency of the BODIPY. The singlet oxygen quantum yield was calculated using hematoporphyrin (HP) as the reference with a yield of 0.53 in ethanol according to the following equation:
Where k is the slope of the photodegradation rate of DPBF, S means the sample, R represents the reference, and is the reference's singlet oxygen quantum yield.
Quantum chemical calculations
Molecular structure optimizations for BODIPY derivatives were carried out using density functional theory (DFT), and their electronic states were calculated using time-dependent DFT (TD-DFT). For H, OMe, and NO2, the b3lyp functional of the Gaussian 16 program package and 6-31G(d) basis sets were chosen. For I, due to the heavy iodine atom, lanl2dz basis sets were chosen instead. All calculations were carried out in water solvent environment. The lactose-tethered triazole moiety attached to the a-position of the BODIPY core was confirmed to have a negligible influence on calculation results (Figure S2) and therefore was replaced by a hydrogen atom for simplicity.
Preparation of BODIPY nanoparticles (NPs)
The stock solution of each BODIPY dyes in THF (0.5 mg.ml-1) was prepared, then 50 µL of stock solution was slowly added to 5 mL of water. The mixture was stirred overnight to evaporate all of THF naturally to yield the designed nanoparticles for further experiment.
Cells and cell culture
HeLa (human cervix adenocarcinoma), Huh7 (human liver carcinoma) cells were obtained from the Korean Cell Line Bank. The cells were maintained in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37 ºC in a humidified 5% CO2 incubator.
Cell proliferation assay
Cell proliferation was studied using CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, HeLa and Huh7 (3 × 103 cells/well) were seeded in 96-well plates. After the cells were maintained for 24 h, cells were treated with BODIPY I, H, OMe, and NO2 at different concentrations (0, 0.25, 0.5, and 1 µM) for 24 h. Following 24 h of incubation, 20 µL MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reagent were added to each well and incubated for 4 h at 37°C. The absorbance was determined at 490 nm using an ELISA plate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Assessment of cellular uptake and cellular imaging
To confirm the cellular uptake and cellular imaging by using the BODIPY I, H, OMe, and NO2, Huh-7 cells were incubated with 2 µM of BODIPY I, H, OMe, and NO2 for 2 h and washed three times with DPBS. After then, the cells were subsequently counterstained with Hoechst 33342 for 10 min. After washing three times with DPBS, the morphologies of Huh-7 cells were taken by an automated live-cell imager (Lionheart FX, BioTek Instruments, Inc., VT, USA) with 40 x objective lens and fluorescence optics (excitation at 520 nm for BODIPY I, H, OMe, and NO2 and at 377 nm for Hoechst 33342, and emission at 535 nm for BODIPY I, H, OMe, and NO2 and at 447 nm for Hoechst 33342). Cellular images were analyzed using Gen5TM imager software (Ver.3.04, BioTek Instruments, Inc., VT, USA).
Photodynamic anticancer activity assessment.
The HeLa and Huh7cells were seeded at 3 × 103 cells/well in a 96-well plate and incubated at 37 °C in 5 % CO2. After 24 h, the cells were incubated again with various concentrations of BODIPY I, H, OMe, and NO2 (0, 0.25, 0.5, and 1 µM) at 37 °C in 5 % CO2 for 2 h under dark conditions. After 2 h incubation for uptaking the BODIPY I, H, OMe, and NO2 into the cells, the media in all plates were changed with RPMI 1640 media without phenol red. Irradiation of cells was performed with a green light-emitting diode (LED) using about 9 mW (530 nm, for 20 min, 80%). After irradiation, the cells were incubated for an additional 24 h, and the cell proliferation was measured using CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay as the same method for the cytotoxicity described above.
Cellular uptake using flow cytometry
The cells (HeLa and Huh7) were seeded at 1×105 cells/well in 6-well plate. After 24 h incubation at 37℃ in 5 % CO2, the BODIPY I, H, OMe, and NO2 (2 μM) were treated with cells for 2 h. Then, the cell were washed with phosphate buffered saline and analyzed using flow cytometry FC500 (Beckman coulter, CA, USA).
All results are expressed as the means ± standard deviations and were compared by one-way analysis of variance (ANOVA followed by Tukey’s analysis with Prism GraphPad 6 software (San Diego, CA, USA). A significance level was set at p<0.05.