摘要：
本文设计合成了两种光敏的双亲性分子, 分别具有单齿和三齿的结构, 这两种双亲性分子可自组装成球形和楔形超分子纳米粒。超分子外部带有氨基的PEG片段可以通过静电作用与核酸结合。本文对超分子输送寡聚核苷酸进入细胞的效果进行了评价。结果表明, 具有三齿结构的分子与核酸形成的复合物通过内吞作用可以有效地将核酸输送进入细胞, 而单齿结构的复合物却几乎不具有此能力。给予光照后, 三齿的超分子发生醌甲基化重排而解离, 从而释放核酸。
中图分类号:
Supporting:
Materials and methods
Unless otherwise noted, all chemicals were obtained from commercial sources and used without further purification. All reactions were carried out under nitrogen environment unless otherwise noted. Tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile and triethylamine (TEA) were distilled over calcium hydride. Manual size-exclusion chromatography was performed on BIO RAD BioBeads S-X1 (200-400 mesh) in a long glass column (1.2 m) at atmospheric pressure.
1H NMR and
13C NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer. Chemical shifts are reported as δ in units of parts per million (ppm) relative to chloroform (
δ 7.26, s) or methanol (
δ 3.31, septet) and coupling constants are reported in Hertz. The size of nanoparticles in diameter was determined by dynamic light scattering (DLS) at 25 °C using a Zetasizer Nano-ZS from Malvern Instruments (Cumulant method). UV-Vis spectra were performed on DU800 UV-Vis spectrophotometer. Fluorescence spectra were obtained using Cary Eclipse ?uorometer. Scanning electron microscopy (SEM, Hitachi S-4800) was used to characterize nanoparticles. The toxicity of polymers evaluated by SRB assay in HeLa cells were detected with FlexStation 3 (Molecular Devices). B-100SP (UVP, LLC) (365 nm, 11 mW/cm
2) and UV-LED (365 nm, 7 mW/cm
2) were used as the light resource for solution and cell irradiation, respectively. Cell images were acquired on confocal laser scanning microscope (Nikon A1Rsi).
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Synthesis of 2. To a solution of hexaethyleneglycol (25 g, 89 mmol) and triethylamine (7 mL, 50 mmol) in THF (100 mL), p-toluenesulfonyl-chloride (11 g, 61 mmol) in THF (100 mL) was added. The reaction solution was stirred overnight and was then diluted with CH2Cl2. The organic layer was washed with HCl aqueous solution and brine, and was dried over anhydrous sodium sulfate. After filtration and evaporation of the solvents in vacuo, the residue was purified by silica gel column chromatography to give compound 2 (15.1 g, 35 mmol, 39.3%). 1H NMR (400 MHz, CDCl3) δ: 7.79 (d, J 8.0 Hz, 2H, Ar-CH), 7.34 (d, J 8.0 Hz, 2H, Ar-CH), 4.15 (t, J 4.8 Hz, 2H, OCH2CH2OSO2-Ar), 3.73–3.58 (m, 22H, OCH2CH2O), 2.86 (br, 1H, OH), 2.45 (s, 3H, Ar-CH3); 13C NMR (101 MHz, CDCl3) δ： 144.8, 133.0, 129.8, 128.0, 72.5, 70.7–70.3, 69.3, 68.7, 61.7, 21.6.
Synthesis of 3. Compound 2 (15.1 g, 35 mmol) and NaN3 (2.3 g, 35 mmol) in DMF (50 mL) were stirred overnight. Subsequently the solvent was evaporated in vacuo. The residue was redissolved in ethyl acetate, filtered over celite. The obtained solution was concentrated in vacuo to yield pure compound 3 (10 g, 32 mmol, 94%). 1H NMR (400 MHz, CDCl3) δ: 3.74–3.60 (m, 22H, OCH2CH2O), 3.38 (t, J 4.8 Hz, 2H, CH2N3); 13C NMR (101 MHz, CDCl3) δ: 72.7, 70.8–70.2, 61.9, 50.8.
Synthesis of 4. To a solution of 3 (9.2 g, 30 mmol) and triethylamine (8.4 mL, 60 mmol) in CH2Cl2 (50 mL), p-toluenesulfonyl chloride (7.4 g, 38 mmol) in CH2Cl2 (50 mL) was added dropwise. The reaction was stirred overnight. The reaction mixture was then washed with 1 N HCl aqueous solution and brine. Drying over sodium sulfate and solvent evaporation in vacuo gave the crude product. Further purification by silica gel column chromatography yielded the pure compound 4 (11.8 g, 26 mmol, 85%). 1H NMR (400 MHz, CDCl3) δ: 7.79 (d, J 8.0 Hz, 2H, Ar-CH), 7.34 (d, J 8.0 Hz, 2H, Ar-CH), 4.15 (t, J 4.8 Hz, 2H, OCH2CH2OSO2-Ar), 3.69–3.59 (m, 20H, OCH2CH2O), 3.37 (t, J 4.8 Hz, 2H, CH2N3), 2.46 (s, 3H, Ar-CH3); 13C NMR (101 MHz, CDCl3) δ: 144.9, 133.2, 129.9, 128.1, 70.9-68.8, 50.8, 21.7.
Synthesis of 5. A mixture of 4 (11.8 g, 26 mmol), methyl 3,4,5-trihydroxybenzoate (9.4 g, 8.4 mmol) and K2CO3 (11.5 g, 84 mmol) in dry DMF (50 mL) was stirred overnight at 70 °C. The reaction mixture was poured in water and extracted with CH2Cl2. The combined organic layers were washed with brine and dried over Na2SO4. After filtration and concentration in vacuo, the crude product was purified by silica gel column chromatography to afford the pure compound 5 (5.0 g, 4.8 mmol, 18.5%). 1H NMR (400 MHz, CDCl3) δ: 7.27 (s, 2H, Ar-H), 4.16 (m, 6H, OCH2CH2O-Ar), 3.86 (s, 3H, COOCH3), 3.76 (m, 6H, OCH2CH2O-Ar), 3.60 (m, 54H, OCH2CH2O), 3.35 (t, 6H, OCH2CH2N3); 13C NMR (101 MHz, CDCl3) δ: 166.6, 152.3, 142.6, 125.0, 109.1, 72.45, 70.9–68.9, 61.9, 52.2, 50.7.
Synthesis of 6. A solution of 5 (5.0 g, 4.8 mmol) and KOH (1.6 g, 28.1 mmol) in the mixed solvent of ethanol (40 mL) and water (40 mL) was heated under reflux overnight. Subsequently, the solution was acidified to pH = 2 with concentrated HCl aqueous solution, cooled and extracted with CH2Cl2. The CH2Cl2 layer was washed with brine. After drying over sodium sulfate and filtration, the CH2Cl2 layer was concentrated to afford the pure compound 6 (4.5 g, 4.3 mmol, 89%). 1H NMR (400 MHz, CDCl3) δ: 7.37 (s, 2H, Ar-H), 4.20 (m, 6H, OCH2CH2O-Ar), 3.85 (t, J 4.8 Hz, 4H, m-OCH2CH2O), 3.79 (t, J 4.9 Hz, 2H, p-OCH2CH2O), 3.73–3.62 (m, 54H, OCH2CH2O), 3.38 (t, J 5.0 Hz, 6H, OCH2CH2N3); 13C NMR (101 MHz, CDCl3) δ: 169.3, 152.24, 143.2, 124.5, 109.9, 72.5, 71.0-70.7, 70.1, 69.8, 69.1, 53.5, 50.8; MS (ESI): calculated. for [C43H75N9O20-H]- m/z = 1038.1 found m/z = 1036.8.
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Synthesis of 7. Aqueous solution of sodium borohydride (NaBH4) (1.3 g, 36 mmol) was added dropwise to the solution of 1-(2-nitrophenyl) ethanone (5 g, 30 mmol) in 1, 4-dioxane (30 mL) at 0 °C. The mixture was stirred in an ice bath for 1 h and at room temperature for an additional 30 min. Then, excess NaBH4 was quenched with acetone. After the removal of the solvents, the residue was dissolved in water and extracted with ethyl acetate. The organic layer was dried over sodium sulfate. After the removal of solvents, the obtained light yellow oil was used in next step without further puri?cation. Compound 7 (4.9 g, 29.4 mmol, 98%).
Synthesis of 8. To a solution of 7 (4.3 g, 25.9 mmol) and triphenylphosphine (10.1 g, 39 mmol) in THF, CBr4 (12.8 g, 39 mmol) in THF was added dropwise under ice bath. The reaction was stirred for 3 h at room temperature. The solvents were removed under reduced pressure. Purification by silica gel column chromatography yielded the pure compound 8 (5.2 g, 22.7 mmol, 87%). 1H NMR (400 MHz, CDCl3) δ: 7.90 (d, J 8.2 Hz, 1H, Ar-H), 7.81 (d, J 7.8 Hz, 1H, Ar-H), 7.62 (t, J 7.6 Hz, 1H, Ar-H), 7.41 (t, J 7.7 Hz, 1H, Ar-H), 5.78 (q, J 6.3 Hz, 1H, CHCH3), 2.07 (d, J 6.4 Hz, 3H, CHCH3); 13C NMR (101 MHz, CDCl3) δ:147.6, 137.8, 133.5, 130.0, 129.0, 124.4, 41.9, 27.2.
Synthesis of 13. To a cool 12% NaOH aqueous solution (70 mL, 0.21 mol), commercially available ethyl 4-hydroxybenzoate (15 g, 0.09 mol) was added, while being cooled to 0 ºC. Formaldehyde (37% in water, 60 mL, 2.1 mol) was added. The reaction was stirred at 55 ºC for 3 d and was monitored by TLC. After completion, the reaction was diluted with EtOAc and washed with NH4Cl. The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure after filtration. The crude product was recrystallized with EtOAc to give compound 13 (11.07 g, 48 mmol, 54%). 1H NMR (400 MHz, CD3OD) δ: 7.86 (s, 2H, Ar-H); 4.73 (s, 4H, CH2-OH); 4.28 (q, J 7.1 Hz, 2H, CH2CH3); 1.34 (t, J 7.1 Hz, 3H, CH2CH3); 13C NMR (101 MHz, CD3OD) δ: 168.3, 159.2, 129.6, 128.4, 122.5, 61.7, 61.7, 14.7.
Synthesis of 9. Compound 1-(1-bromoethyl)-2-nitrobenzene (3.6 g, 15 mmol) and sodium iodide (2.3 g, 15.3 mmol) were dissolved in acetone, the reaction was stirred at room temp for 2 h. After filtration, the filtrate was concentrated under reduced pressure. The residue, compound 13 (3.8 g, 17 mmol) and K2CO3 (2.4 g, 17 mmol) were dissolved in DMF. The reaction mixture was stirred at 50 ºC for 1 d. After completion, the reaction solution was diluted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous sodium sulfate and removed under reduced pressure after filtration. The crude product was purified by silica gel column chromatography to give compound 9 (2.52 g, 6.7 mmol, 44.6%). 1H NMR (400 MHz, CDCl3) δ: 8.05 (s, 2H, Ar-H), 8.00 (dd, J 7.9 Hz, 1.1 Hz, 1H, Ar-H), 7.94 (dd, J 8.2 Hz, 1.1 Hz, 1H, Ar-H), 7.72 (m, 1H, Ar-H), 7.48 (m, 1H, Ar-H), 5.80 (q, J 6.3 Hz, 1H, CHCH3), 4.57 (m, 4H, CH2-O), 4.33 (q, J 7.1 Hz, 2H, CH2CH3), 1.96 (br, 2H, OH), 1.68 (d, J 6.3 Hz, 2H, CH2CH3), 1.36 (t, J 7.1 Hz, 3H, CHCH3); 13C NMR (101 MHz, CDCl3) δ: 166.1, 158.0, 147.4, 138.4, 134.2, 133.9, 130.9, 128.8, 128.1, 126.7, 124.5, 78.9, 61.2, 61.0, 23.5, 14.5.
Synthesis of 10. Compound 9 (3.8 g, 10 mmol) was dissolved in DMF and cooled to 0 ºC. Imidazole (1.5 g, 22 mmol) and TBDMS-Cl (3.3 g, 22 mmol) were then added. The reaction mixture was stirred at room temperature for 30 min. After completion, the reaction solution was diluted with ether and was then washed with NH4Cl aqueous solution. The organic layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 10 (6 g, 9.9 mmol, 98%). 1H NMR (400 MHz, CDCl3) δ: 8.12 (s, 2H, Ar-H), 8.02 (d, J 7.8 Hz, 1H, Ar-H), 7.95 (d, J1 8.1 Hz, J2 7.8 Hz, 1H, Ar-H), 7.71 (t, J 8.1 Hz, 1H, Ar-H), 7.47 (t, J 8.1 Hz, 1H, Ar-H), 5.60 (q, J 6.1 Hz, 1H, CHCH3), 4.59 (m, 4H, CH2-O), 4.32 (q, J 7.1 Hz, 2H, CH2CH3), 1.62 (d, J 6.2 Hz, 3H, CH2CH3), 1.35 (t, J 7.1 Hz, 3H, CHCH3), 0.91 (s, 18H, C(CH3)3); 0.04 (ds, 12H, Si-CH3); 13C NMR (101 MHz, CDCl3) δ:166.5, 155.8, 147.3, 138.9, 134.2, 133.7, 128.9,128.7, 128.3, 126.4, 124.5, 77.5, 60.8, 60.6, 26.0, 23.6, 18.5, 14.4, 5.21, 5.24.
Synthesis of 11. Compound 10 (6 g, 9.9 mmol) was dissolved in dry THF under nitrogen and cooled to 0 ºC. DIBAL-H was added dropwise and the reaction solution continued stirring for another 2 h. After completion, the reaction was quenched with NH4Cl aqueous solution and the solution was diluted with ether. Celite was added and the reaction mixture was stirred at room temperature for another 30 min. After filtration, the organic layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 11 (3.3 g, 6 mmol, 60%). 1H NMR (400 MHz, CDCl3) δ: 8.04 (dd, J 7.9 Hz, 1.3 Hz, 1H, Ar-H), 7.94 (dd, J1 8.2 Hz, J2 1.2 Hz, 1H, Ar-H), 7.71 (m, 1H, Ar-H), 7.46 (m, 1H, Ar-H), 7.40 (s, 2H, Ar-H), 5.47 (q, J 6.2 Hz, 1H, CHCH3), 4.55 (m, 6H, CH2-O), 1.61 (d, J 6.3 Hz, 3H, CHCH3), 0.89 (s, 18H, C(CH3)3), 0.03 (ds, 12H, Si-CH3); 13C NMR (101 MHz, CDCl3) δ: 151.6, 147.2, 139.4, 136.8, 134.2, 133.7, 128.5,128.3, 125.9, 124.4, 65.6, 60.6, 26.1, 23.6, 18.6, 5.2, 5.3.
Synthesis of 12. Compound 11 (3.3 g, 6 mmol) was dissolved in MeOH and amberlyst 15 (0.35 g) was then added. The reaction was stirred at room temperature for 2 h. After completion, the amberlyst 15 was filtered out and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 12 (1.3 g, 3.9 mmol, 39.3%). 1H NMR (400 MHz, CD3OD) δ: 8.08 (d, J 7.5 Hz, 1H, Ar-H), 7.91 (dd, J 8.2 Hz, 1H, Ar-H), 7.77 (t, J 7.6 Hz, 1H, Ar-H), 7.53 (t, J 7.5 Hz, 1H, Ar-H), 7.39 (s, 2H, Ar-H), 5.57 (q, J 6.3 Hz, 1H, CHCH3), 4.48 (m, 6H, CH2-O), 1.62 (d, J 6.3 Hz, 3H, CHCH3); 13C NMR (101 MHz, CD3OD) δ: 153.8, 149.0, 139.8, 138.5, 135.4, 134.6, 129.7,129.5, 128.3, 125.0, 79.3, 64.9, 60.5, 23.2; MS (ESI): cald. for [C17H19NO6+Na]+ m/z = 356.3 found m/z = 356.4.
Synthesis of 14. Compound 6 (0.5 g, 0.48 mmol) was dissolved in dry dichloromethane containing a catalytic amount of DMF, and oxalyl chloride (100 μL, 1 mmol) was then added. The reaction solution was stirred overnight at room temperature and was then concentrated in vacuo. The resulting residue was redissolved in dry dichloromethane and cooled to 0 ºC. Compound 12 (30 mg, 0.09 mmol) and triethtylamine (0.3 ml, 2.1 mmol) were added. The reaction was stirred overnight at room temp. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to give compound 14 (80 mg, 0.023 mmol, 26%). 1H NMR (400 MHz, CDCl3) δ: 8.07 (d, J 7.0 Hz, 1H, Ar-H), 7.88 (dd, J1 8.2 Hz, J2 1.1 Hz, 1H, Ar-H), 7.66 (m, 1H, Ar-H), 7.56 (s, 1H, Ar-H), 7.41 (m, 1H, Ar-H), 7.29 (s, 2H, Ar-H), 7.27 (s, 4H, Ar-H), 5.85 (q, J 6.1 Hz, 1H, CHCH3), 5.21 (m, 6H, CH2-O), 4.15 (m, 18H, Ar-OCH2CH2O), 3.77 (m, 18H, Ar-OCH2CH2O), 3.72–3.63 (m, 162H, OCH2CH2O), 3.36 (m, 18H, OCH2CH2N3), 1.70 (d, J 6.2 Hz, 3H, CHCH3); 13C NMR (101 MHz, CDCl3) δ: 165.8, 155.2, 152.3, 146.7, 142.8, 138.3, 134.0, 132.4, 131.7, 129.8, 128.6, 128.1, 124.7, 124.5, 124.4, 109.1, 78.7, 72.4, 70.7-68.9, 62.2, 50.6, 29.6, 23.1. FTIR: ν 2105 cm-1 (N3)
Synthesis of TD. To a solution of 14 (0.12 g, 0.035 mmol) in THF (50 mL), triphenylphosphine (0.1 g, 0.38 mmol) was added at room temperature. The reaction solution was stirred for 16 h. The solution was then treated with water (30 μL, 1.8 mmol), and continued stirring for another 3 h. After the removal of the solvent, the resulting crude product was purified on a size exclusion chromatography (CH2Cl2) to give compound TD (0.1 g, 0.031 mmol, 88%). 1H NMR (400 MHz, CDCl3) δ: 8.08 (d, J 8.0 Hz, 1H, Ar-H), 7.89 (dd, J1 8.2 Hz, J2 1.0 Hz, 1H, Ar-H), 7.67 (m, 1H, Ar-H), 7.56 (s, 1H, Ar-H), 7.41 (m, 1H, Ar-H), 7.34–7.28 (m, 6H, Ar-H), 5.85 (q, J 6.2 Hz, 1H, CHCH3), 5.22 (m, 6H, CH2-O), 4.15 (m, 18H, Ar-OCH2CH2O), 3.77 (m, 18H, Ar-OCH2CH2O), 3.73–3.49 (m, 162H, OCH2CH2O), 2.86 (br, 18H, OCH2CH2N3), 1.71 (d, J 6.2 Hz, 3H, CHCH3); 13C NMR (101 MHz, CDCl3) δ: 165.8, 165.8, 155.2, 152.3, 146.7, 142.8, 138.3, 134.0, 132.4, 131.7, 129.8, 128.6, 128.1, 124.7, 124.5, 124.4, 109.1, 91.3, 78.7, 77.4, 73.2, 72.4, 71.1, 70.7–68.9, 66.1, 62.2, 53.5, 45.4, 41.7, 32.1, 29.6, 25.0, 23.0. MS (MALDI): calculated for [C146H256N10O63+Na]+ m/z = 3157.7; found m/z = 3158.2.
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Synthesis of 16. 2-nitro-benzaldehyde (1.51 g, 10 mmol) was dissolved in dry CH3CN under nitrogen. TMSCN (2 mL, 12 mmol) was added dropwise and the reaction was stirred overnight. After completion the solvent was removed under reduced pressure. Then concentrated HCl aqueous solution (15 mL) was added into the residue. The reaction was refluxed at 110 ºC for 30 min. After completion, the reaction solution was diluted with ethyl acetate, and the organic layer was washed with brine and dried by anhydrous sodium sulfate. After the removal of solvents under reduced pressure, the resulting crude product was recrystallized with petroleum in ethyl acetate to give compound 16 (1.51 g, 7.6 mmol, 76%). 1H NMR (400 MHz, MeOD) δ: 7.97 (dd, J1 8.1 Hz, J2 1.1 Hz, 1H, Ar-H), 7.81 (d, J 6.9 Hz, 1H, Ar-H), 7.67 (td, J1 7.7 Hz, J2 1.1 Hz, 1H, Ar-H), 7.55–7.51 (m, 1H, Ar-H), 5.85 (s, 1H, CH); 13C NMR (101 MHz, MeOD) δ: 174.4, 149.5, 135.7, 134.3, 130.1, 130.0, 125.6, 70.4.
Synthesis of 17. Compound 16 (0.21 g, 1 mmol) and dioctadecylamine (0.5 g, 0.93 mmol) were dissolved in the mixed solvent of DMF (3 mL) and THF (20 mL) under nitrogen. BOP (0.48 g, 1.1 mmol) and TEA (0.3 mL, 2.1 mmol) were then added and the reaction was stirred at room temperature overnight. After completion, the reaction solution was diluted with ethyl acetate, and the organic layer was washed with brine for 8 times and dried over anhydrous sodium sulfate. After the removal of solvents under reduced pressure, the resulting crude product was purified by silica gel column chromatography to give compound 17 (0.42 g, 0.56 mmol, 56%). 1H NMR (400 MHz,CDCl3) δ: 7.90 (dd, 1H, J1 8.1 Hz, J2 1.2 Hz, Ar-H), 7.56 (td, J1 7.7 Hz, J2 1.2 Hz, 1H, Ar-H), 7.46 (m, 1H, Ar-H), 7.37 (m, 1H, Ar-H), 5.94 (s, 1H, CH), 4.76 (br, 1H, OH), 3.51 (m, 1H, N-CH2), 3.20 (m, 1H, N-CH2), 3.06 (m, 1H, N-CH2), 2.94 (m, 1H, N-CH2), 1.56–1.11 (m, 60H, CH2CH2), 0.86 (t, J 6.6 Hz, 3H, CH2CH3); 13C NMR (101 MHz, CDCl3) δ: 170.9, 149.1, 134.4, 133.7, 129.5, 129.1, 124.9, 65.8, 46.8, 46.6, 32.1, 29.8–26.6, 22.8, 14.3.
Synthesis of 18. Compound 6 (0.4 g, 0.39 mmol) was dissolved in dry dichloromethane containing a catalytic amount of DMF, oxalyl chloride (100 μL, 1 mmol) was then added. The reaction was stirred overnight at room temperature. Then the reaction solution was concentrated in vacuo. The resulting residue was redissolved in dry dichloromethane and cooled to 0 ºC. Compound 17 (0.3 g, 0.4 mmol) and triethylamine (0.3 mL, 2.1 mmol) were then added. The reaction was stirred overnight at room temperature. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to give compound 18 (0.48 g, 0.30 mmol, 73%). 1H NMR (400 MHz, CDCl3) δ: 8.12 (d, 1H, Ar-H), 7.67 (d, 2H, Ar-H), 7.52 (m, 1H, Ar-H), 7.33 (s, 2H, Ar-H), 7.26 (s, 1H, CH), 4.21 (t, 2H, p-OCH2CH2O), 4.15 (t, 4H, m-OCH2CH2O), 3.84 (t, 2H, p-OCH2CH2O), 3.78 (t, 4H, m-OCH2CH2O), 3.72–3.65 (m, 54H, OCH2CH2O), 3.37 (t, 2H, OCH2CH2N3), 3.59–3.54, 3.34–3.14 (m, 4H, CH2CH2), 1.61–1.19 (m, 60H, CH2CH2), 0.86 (t, 3H, CH2CH3); 13C NMR (101 MHz, CDCl3) δ: 166.4, 164.8, 152.6, 148.1, 143.5, 134.0, 131.8, 129.6, 129.2, 125.2, 123.6, 109.5, 72.6, 70.9-69.1, 50.8, 48.1, 46.7, 32.0, 29.8-27.0, 22.8, 14.2. FTIR: ν = 2104 cm-1 (N3)
Synthesis of MD. To a solution of
18 (0.15 g, 0.087 mmol) in THF (50 mL), triphenylphosphine (0.1 g, 0.38 mmol) was added at room temperature. The reaction solution was stirred for 16 h. The solution was treated with water (30 μL, 1.8 mmol), and after stirring for an additional 3 h, the solvent was removed. The crude product was chromatographed on a size exclusion chromatography (CH
2Cl
2) to give compound
MD ( 0.12 g, 0.073 mmol, 84%).
1H NMR (400 MHz, CDCl
3)
δ: 8.11 (d, 1H, Ar-
H), 7.67 (d, 2H, Ar-
H), 7.54 (m, 1H, Ar-
H), 7.33 (s, 2H, Ar-
H), 7.24 (s, 1H,
CH), 4.22 (t, 2H,
p-O
CH2CH
2O), 4.16 (t, 4H, m-O
CH2CH
2O), 3.85 (t, 4H,
p-O
CH
2CH2O), 3.80 (t, 2H, m-OCH
2CH2O), 3.72–3.53 (m, 54H, O
CH2CH2O), 3.37–3.18 (m, 4H, ), 2.82 (br, 12H,
CH2CH2) 1.61–1.20 (m, 60H,
CH2CH2), 0.86 (t, 6H, CH
2C
H3);
13C NMR (101 MHz,CDCl
3)
δ: 166.4, 164.8, 152.5, 148.2, 143.3, 133.9, 131.5, 129.7, 129.3, 125.2, 123.7, 109.4, 72.6, 70.8-69.0, , 48.1, 46.7, 41.5, 39.6, 32.0, 29.8–29.3, 22.8, 14.2; MS (ESI): calculated. for [C
87H
159N
5O
23+3H]
3+ m/z = 548.8, found
m/z = 549.0.
Table S1. The size, PDI and zeta-potential of ODN/NP-TD complexes at different N/P ratios and TD blank nanoparticle in aqueous solution (The concentration of ODN was fixed to be 600 nM)
Table S2. The size, PDI and zeta-potential of ODN/NP-MD complexes at different N/P ratios and MD blank nanoparticle in aqueous solution (The concentration of ODN was fixed to be 400 nM)
Figure S1. SEM images of I) NP-MD/ODN complexes and II) NP-TD/ODN complexes. (A: N/P=50, B: N/P=150, ODN = 600 nM). Scale bar = 1 μm.
Figure S2. Fluorescence emission spectra (excitation wavelength: 550 nm) of Nile red-loaded supramolecular assembled nanoparticle of MD (0.4 mg/mL) in aqueous solution at different light irradiation (365 nm, 11 mW cm-2) time. Inset: Plots of normalized fluorescence intensity (630 nm) vs. irradiation time of MD particle solution.
Figure S3. Fluorescence emission spectra (excitation wavelength: 570 nm) of NR-loaded nanoparticles of TD (0.25 mg/mL) in aqueous solutions at different light irradiation (365 nm, 11 mW cm-2) time. Inset: Plots of normalized fluorescence intensity (650 nm) vs irradiation time of particle solution.
Figure S4. Fluorescence emission spectra of Nile red in TD self-assembled particle solution at an excitation wavelength of 570 nm at different days. The concentration of TD molecule was 0.15 mg /mL.
Figure S5. Fluorescence emission spectra of Nile red in MD self-assembled particle solution at an excitation wavelength of 550 nm at different days. The concentration of MD molecule was 0.25 mg/mL.
Figure S6. Confocal microscopy image of live MA-231 cells incubated with FAM-ODN/NP-TD complex (N/P = 100, FAM-ODN = 100 nM) for 2 h, washed and further incubated 15 h before imaging. Cell nuclei and endosomes/lysosomes were stained with Hoechst 33342 (blue) and LysoTracker Red DND-99 (red) respectively. Fluorescence was measured in three channels using 405, 488, and 561 nm laser excitation. A: FAM-ODN; B: Lysotracker Red; C: Hoechst; D: Merged. Scale bar = 50 μm
Figure S7. Confocal microscopy image of live HeLa cells incubated with FAM-ODN/NP-MD complex (N/P=100, FAM-ODN=100 nM) for 2 h, washed and further incubated 15 h before imaging. Cell nuclei and endosomes/lysosomes were stained with Hoechst 33342 (blue) and LysoTracker Red DND-99 (red) respectively. Fluorescence was measured in three channels using 405, 488, and 561 nm laser excitation. A: FAM-ODN; B: Lysotracker Red; C: Hoechst; D: Merged. Scale bar = 20 μm.
Figure S8. Confocal microscopy image of live HeLa cells incubated with Nile red-loaded NP-MD complex (dialysis in aqueous solution with 5% DMSO for 24 h) for 2h, washed and further incubated 15 h before imaging. Cell nuclei were stained with Hoechst 33342. Fluorescence measured in two channels using 405 and 561 nm excitation. Scale bar = 20 μm.
Figure S9. Confocal microscopy image of live HeLa cells incubated with FAM-ODN/NP-TD complex (N/P=100, FAM-ODN=100 nM) for 2 h, washed and further incubated 15 hours before imaging without UV treated (A: Hoechst B: bright field) and with UV treated for 2 min (C: Hoechst; D: bright field). Cell nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm.
NMR, MS and FT-IR spectra of synthetic compounds
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MS of compound 6
MS of compound 13
王鹏, 纪玉卓, 王志轩, 汤新景. 超分子组装用于寡聚核苷酸的输送及其在细胞中的光诱导释放[J]. 中国药学(英文版), 2014, 23(7): 454-462.
Peng Wang, Yuzhuo Ji, Zhixuan Wang, Xinjing Tang. Supramolecular self-assembly for delivery of oligonucleotides and its phototriggered unpacking in living cells[J]. Journal of Chinese Pharmaceutical Sciences, 2014, 23(7): 454-462.
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